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1. (WO2018227426) METHODS AND SYSTEMS FOR SAMPLE ANALYSIS
Document

Description

Title of Invention 0001   0002   0003   0004   0005   0006   0007   0008   0009   0010   0011   0012   0013   0014   0015   0016   0017   0018   0019   0020   0021   0022   0023   0024   0025   0026   0027   0028   0029   0030   0031   0032   0033   0034   0035   0036   0037   0038   0039   0040   0041   0042   0043   0044   0045   0046   0047   0048   0049   0050   0051   0052   0053   0054   0055   0056   0057   0058   0059   0060   0061   0062   0063   0064   0065   0066   0067   0068   0069   0070   0071   0072   0073   0074   0075   0076   0077   0078   0079   0080   0081   0082   0083   0084   0085   0086   0087   0088   0089   0090   0091   0092   0093   0094   0095   0096   0097   0098   0099   0100   0101   0102   0103   0104   0105   0106   0107   0108   0109   0110   0111   0112   0113   0114   0115   0116   0117   0118   0119   0120   0121   0122   0123   0124   0125   0126   0127   0128   0129   0130   0131   0132   0133   0134   0135   0136   0137   0138   0139   0140   0141   0142   0143   0144   0145   0146   0147   0148   0149   0150   0151   0152   0153   0154   0155   0156   0157   0158   0159   0160   0161   0162   0163   0164   0165   0166   0167   0168   0169   0170   0171   0172   0173   0174   0175   0176   0177   0178   0179   0180   0181   0182   0183   0184   0185   0186   0187   0188   0189   0190   0191   0192   0193   0194   0195   0196   0197   0198   0199   0200   0201   0202   0203   0204   0205   0206   0207   0208   0209   0210   0211   0212   0213   0214   0215   0216   0217   0218   0219   0220   0221   0222   0223   0224   0225   0226   0227   0228   0229   0230   0231   0232   0233   0234   0235   0236   0237   0238   0239   0240   0241   0242   0243   0244   0245   0246   0247   0248   0249   0250   0251   0252   0253   0254   0255   0256   0257   0258   0259   0260   0261   0262   0263   0264   0265   0266   0267   0268   0269   0270   0271   0272   0273   0274   0275   0276   0277   0278   0279   0280   0281   0282   0283   0284   0285   0286   0287   0288   0289   0290   0291   0292   0293   0294   0295   0296   0297   0298   0299   0300   0301   0302   0303   0304   0305   0306   0307   0308   0309   0310   0311   0312   0313   0314   0315   0316   0317   0318   0319   0320   0321   0322   0323   0324   0325   0326   0327   0328   0329   0330   0331   0332   0333   0334   0335   0336   0337   0338   0339   0340   0341   0342   0343   0344   0345   0346   0347   0348   0349   0350   0351   0352   0353   0354   0355   0356   0357   0358   0359   0360   0361   0362   0363   0364   0365   0366   0367   0368   0369   0370   0371   0372   0373   0374   0375   0376   0377   0378   0379   0380   0381   0382   0383   0384   0385   0386   0387   0388   0389   0390   0391   0392   0393   0394   0395   0396   0397   0398   0399   0400   0401   0402   0403   0404   0405   0406   0407   0408   0409   0410   0411   0412   0413   0414   0415   0416   0417   0418   0419   0420   0421   0422   0423   0424   0425   0426   0427   0428   0429   0430   0431   0432   0433   0434   0435   0436   0437   0438   0439   0440  

Claims

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Drawings

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Description

Title of Invention : METHODS AND SYSTEMS FOR SAMPLE ANALYSIS

BACKGROUND

[0001]
Gel electrophoresis is a technique used to separate and analyze biological samples, such as DNA, RNA, and proteins, based on their mass and/or electrical charge. Sample molecules, such as DNA, can migrate through a separation matrix under the influence of an electrical. Samples can be loaded into the separation matrix and placed inside an electrophoresis chamber, which can be connected to a power source to generate the electric field for driving the electrophoresis.
[0002]
The samples are usually loaded to the separation matrix by opening the containers containing the samples to remove the samples, for example, by pipetting, and then aspirating the samples into the separation matrix. This process is not performed in a closed system, and contamination of the samples may result. The contamination of the samples is especially troublesome when the samples are subsequently subject to an assay in which the signal from trace amount of impurity may be greatly propagated, for example, a polymerase chain reaction (PCR) . Moreover, if the samples to be assayed are pathogenic, the risk of biohazard is greatly pronounced by transferring them in an open system.
[0003]
SUMMARY
[0004]
The present disclosure provides methods, apparatus and systems for loading a biological sample from its container to the electrophoretic apparatus in a closed system such that the risks of contamination of the sample and biohazard caused by leakage of the sample are greatly reduced.
[0005]
In one aspect, the present disclosure involves a system for sample analysis. The system may comprise a sample vessel comprising (i) a container that retains a biological sample and (ii) a penetratable membrane at an end of said container that seals said container in the absence of an object inserted through said penetratable membrane; a separation medium that subjects said biological sample or a portion thereof to separation upon application of a flow-inducing field across said separation medium, which separation is on the basis of mass or charge; a flow channel in fluid communication with said separation medium, wherein during use, at least a portion of said flow channel traverses said penetratable membrane to bring said container in fluid communication with said separation medium; and a controller comprising one or more computer processors that are individually or collectively programmed to (i) pierce said penetratable membrane to bring said container in fluid communication with said separation medium through said flow channel, (ii) subject said biological sample to flow from said container through said flow channel to said separation medium, and (iii) apply said flow-inducing field across said separation medium under conditions that are sufficient to direct said biological sample through said separation medium to separate said biological sample or portion thereof on the basis of mass or charge.
[0006]
In some embodiments, said one or more computer processors are programmed to apply positive or negative pressure to subject said biological sample to flow from said container through said flow channel to said separation medium.
[0007]
In some embodiments, the system further comprises a holder for receiving and securably holding said sample vessel.
[0008]
In some embodiments, at least a portion of said flow channel is secured in a housing, and wherein during use, said holder is brought in contact with said housing.
[0009]
In some embodiments, during use, (1) said holder is brought in contact with or in proximity to said housing in a first position in which said penetratable membrane is not pierced, and (2) said penetratable membrane is pierced by directing said housing or said holder to a second position.
[0010]
In some embodiments, said flow channel is brought in fluid communication with said separation medium by directing said housing to a third position.
[0011]
In some embodiments, said housing further comprises a first covering element that covers an end of said flow channel when said housing is in said first position, wherein said first covering element allows said end of said flow channel to emerge from said first covering element when said housing or said holder is in said second position such that said end of said flow channel traverses said penetratable membrane.
[0012]
In some embodiments, said housing further comprises a second covering element that covers another end of said flow channel when (a) said housing is in said first position or (b) said housing or said holder is in said second position, wherein said second covering element allows said another end of said flow channel to emerge from said second covering element when said housing is in a third position such that said another end of said flow channel pierces an additional penetratable membrane separating said flow channel from said separation medium.
[0013]
In some embodiments, the system further comprises a cannula that pierces said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[0014]
In some embodiments, said cannula comprises at least a portion of said flow channel extending therethrough.
[0015]
In some embodiments, said cannula includes a tip at an end thereof, which tip pierces said penetratable membrane to bring said flow channel in fluid communication with said container.
[0016]
In some embodiments, said separation medium is a polymeric separation medium.
[0017]
In some embodiments, said separation medium comprises an agarose gel.
[0018]
In some embodiments, said flow-inducing field is an electric field.
[0019]
In some embodiments, the system further comprises at least two electrodes on ends of said separation medium, wherein said at least two electrodes provide said electric field.
[0020]
In some embodiments, said flow-inducing field is a pressure field.
[0021]
In some embodiments, the system further comprises an actuator that pierces said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[0022]
In some embodiments, the system further comprises a plurality of sample vessels, wherein a given sample vessel of said plurality of sample vessels comprises said container and said penetratable membrane.
[0023]
In some embodiments, the system further comprises a compartment having said separation medium.
[0024]
In some embodiments, the system further comprises a unique identifier on said compartment.
[0025]
In some embodiments, said unique identifier is a barcode.
[0026]
In some embodiments, said unique identifier is a radio-frequency identification tag.
[0027]
In some embodiments, said penetratable membrane comprises a slit.
[0028]
In some embodiments, said penetratable membrane is resealable.
[0029]
In some embodiments, said penetratable membrane is formed of a polymeric material.
[0030]
In some embodiments, the system further comprises a detector that detects said biological sample or portion thereof.
[0031]
In some embodiments, said detector detects said biological sample or portion thereof in said separation medium.
[0032]
In some embodiments, said one or more computer processors are individually or collectively programmed to bring said flow channel in fluid communication with said separation medium.
[0033]
In some embodiments, said flow channel is brought in fluid communication with said separation medium upon said flow channel piercing an additional penetratable membrane separating said flow channel from said separation medium.
[0034]
In some embodiments, said housing comprises a securing element that prevents relative movement between said flow channel and said securing element.
[0035]
In some embodiments, said securing element does not cover ends of said flow channel.
[0036]
In another aspect, the present disclosure involves a method for sample analysis. The method may comprise (a) receiving a sample vessel comprising (i) a container that retains a biological sample and (ii) a penetratable membrane at an end of said container that seals said container in the absence of an object inserted through said penetratable membrane; (b) piercing said penetratable membrane to bring said biological sample in fluid communication with a flow channel, wherein at least a portion of said flow channel traverses said penetratable membrane, wherein said flow channel is in fluid communication with a separation medium that subjects said biological sample or a portion thereof to separation upon application of a flow-inducing field across said separation medium, which separation is on the basis of mass or charge;
[0037]
(c) subjecting said biological sample to flow from said container through said flow channel to said separation medium; and (d) applying said flow-inducing field across said separation medium under conditions that are sufficient to direct said biological sample through said separation medium to separate said biological sample or portion thereof on the basis of mass or charge.
[0038]
In some embodiments, in (c) , said biological sample is subjected to flow from said container through said flow channel to said separation medium upon application of positive pressure or negative pressure.
[0039]
In some embodiments, said positive pressure is applied by squeezing the container.
[0040]
In some embodiments, in (a) , said sample vessel is received in a holder that receives and securably holds said sample vessel.
[0041]
In some embodiments, at least a portion of said flow channel is secured in a housing, and wherein (b) comprises bringing said holder in contact with said housing.
[0042]
In some embodiments, (b) comprises (1) bringing said holder in contact with or in proximity to said housing at a first position in which said penetratable membrane is not pierced, and (2) piercing said penetratable membrane by directing said housing or said holder to a second position.
[0043]
In some embodiments, the method further comprises bringing said flow channel in fluid communication with said separation medium by directing said housing to a third position.
[0044]
In some embodiments, said housing further comprises a first covering element that covers an end of said flow channel when said housing is in said first position, wherein said first covering element allows said end of said flow channel to emerge from said first covering element when said housing or said holder is in said second position such that said end of said flow channel traverses said penetratable membrane.
[0045]
In some embodiments, said housing further comprises a second covering element that covers another end of said flow channel when (i) said housing is in said first position or (ii) said housing or said holder is in said second position, wherein said second covering element allows said another end of said flow channel to emerge from said second covering element when said housing is in a third position such that said another end of said flow channel pierces an additional penetratable membrane separating said flow channel from said separation medium.
[0046]
In some embodiments, (b) comprises using a cannula to pierce said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[0047]
In some embodiments, said cannula comprises at least a portion of said flow channel extending therethrough.
[0048]
In some embodiments, said cannula includes a tip at an end thereof, which tip pierces said penetratable membrane to bring said flow channel in fluid communication with said container.
[0049]
In some embodiments, said separation medium is a polymeric separation medium.
[0050]
In some embodiments, said separation comprises an agarose gel.
[0051]
In some embodiments, said flow-inducing field is an electric field.
[0052]
In some embodiments, the method further comprises at least two electrodes on ends of said separation medium, wherein said at least two electrodes provide said electric field.
[0053]
In some embodiments, said flow-inducing field is a pressure field.
[0054]
In some embodiments, the method further comprises using an actuator to pierce said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[0055]
In some embodiments, the method further comprises a plurality of sample vessels, wherein a given sample vessel of said plurality of sample vessels comprises said container and said penetratable membrane.
[0056]
In some embodiments, the method further comprises a compartment having said separation medium.
[0057]
In some embodiments, the method further comprises a unique identifier on said compartment.
[0058]
In some embodiments, said unique identifier is a barcode.
[0059]
In some embodiments, said unique identifier is a radio-frequency identification tag.
[0060]
In some embodiments, said penetratable membrane comprises a slit.
[0061]
In some embodiments, said penetratable membrane is resealable.
[0062]
In some embodiments, said penetratable membrane is formed of a polymeric material.
[0063]
In some embodiments, the method further comprises using a detector to detect said biological sample or portion thereof.
[0064]
In some embodiments, said detector detects said biological sample or portion thereof in said separation medium.
[0065]
In some embodiments, (c) further comprises bringing said flow channel in fluid communication with said separation medium.
[0066]
In some embodiments, said flow channel is brought in fluid communication with said separation medium upon said flow channel piercing an additional penetratable membrane separating said flow channel from said separation medium.
[0067]
In some embodiments, said housing comprises a securing element that prevents relative movement between said flow channel and said securing element.
[0068]
In some embodiments, said securing element does not cover ends of said flow channel.
[0069]
A non-transitory computer-readable medium comprising machine-executable code that, upon execution by one or more computer processors, implements a method for sample analysis, the method comprising: (a) receiving a sample vessel comprising (i) a container that retains a biological sample and (ii) a penetratable membrane at an end of said container that seals said container in the absence of an object inserted through said penetratable membrane; (b) piercing said penetratable membrane to bring said biological sample in fluid communication with a flow channel, wherein at least a portion of said flow channel traverses said penetratable membrane, wherein said flow channel is in fluid communication with a separation medium that subjects said biological sample or a portion thereof to separation upon application of a flow-inducing field across said separation medium, which separation is on the basis of mass or charge;
[0070]
(c) subjecting said biological sample to flow from said container through said flow channel to said separation medium; and (d) applying said flow-inducing field across said separation medium under conditions that are sufficient to direct said biological sample through said separation medium to separate said biological sample or portion thereof on the basis of mass or charge.
[0071]
Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
[0072]
INCORPORATION BY REFERENCE
[0073]
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0074]
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “Fig. ” herein) , of which:
[0075]
FIG. 1 shows an exemplary three-quarters view schematic of a multi-lane electrophoresis apparatus.
[0076]
FIG. 2A shows an exemplary top-down view schematic of a multi-lane electrophoresis matrix.
[0077]
FIG. 2B shows an exemplary top-down view schematic of a multi-lane electrophoresis matrix with multiple sets of electrodes and connectors between lanes.
[0078]
FIG. 2C shows an exemplary top-down view schematic of a multi-lane electrophoresis matrix with multiple sets of electrodes and connectors between lanes.
[0079]
FIG. 3 shows an exemplary side view schematic of a multi-lane electrophoresis apparatus.
[0080]
FIG. 4 shows an exemplary schematic of a lane electrophoresis apparatus with a gel casting frame and comb.
[0081]
FIG. 5 shows an exemplary schematic of a gel casting frame and comb.
[0082]
FIG. 6 shows an exemplary schematic of a lane electrophoresis apparatus with a gel casting frame and an auto pipette with pipette tips for delivering fluid.
[0083]
FIG. 7 shows an exemplary schematic of a circuit composition for an electrophoresis apparatus.
[0084]
FIG. 8 shows an exemplary schematic of a high voltage circuit.
[0085]
FIG. 9 shows an exemplary schematic of an electrophoresis apparatus with an illuminating area, voltage control and display, and imaging equipment.
[0086]
FIG. 10 shows an exemplary schematic of an electrophoresis apparatus with electrodes.
[0087]
FIG. 11 shows one schematic diagram of a sample analysis system comprising a sample vessel, a flow channel, and a separation medium.
[0088]
FIG. 12 shows another schematic diagram of a sample analysis system comprising a sample vessel, a flow channel, and a separation medium.
[0089]
FIG. 13 is an exemplary side view of a sample vessel.
[0090]
FIGs. 14A-B are schematic diagrams for a process for bringing a container in fluid communication with a separation medium through a flow channel.
[0091]
FIGs. 15A-D are schematic diagrams for another process for bringing a container in fluid communication with a separation medium through a flow channel.
[0092]
FIG. 16 is a schematic diagram for a process of subjecting a biological sample to flow from a container through a flow channel to a separation medium.
[0093]
FIGs. 17A-B are schematic diagrams for a process of applying a flow-inducing field across a separation medium to direct a biological sample through a separation medium.
[0094]
FIG. 18 is a side view of a holder with a sample vessel held therein.
[0095]
FIGs. 19A-C are schematic diagrams depicting the relative movement between a holder and a housing which brings about the piercing of a penetratable membrane by a flow channel.
[0096]
FIGs. 20A-F illustrate several configurations between the housing and the holder.
[0097]
FIGs. 21A-C are schematic diagrams depicting the relative movement between a holder and a housing which brings about the piercing of a penetratable membrane by a flow channel.
[0098]
FIGs. 22A-C illustrate an exemplary process in which a housing is brought to a third position to allow a flow channel to pierce an additional penetrable membrane.
[0099]
FIGs. 23A-F illustrate exemplary configurations in which a first covering element covers and seals an end of the flow channel and exemplary processes for using these configurations in accordance with the present disclosure.
[0100]
FIGs. 24A-F illustrate exemplary configurations in which a second covering element covers and seals another end of the flow channel and exemplary processes for using these configurations in accordance with the present disclosure.
[0101]
FIG. 25 illustrates a configuration comprising both a first and a second covering elements and a securing element.
[0102]
FIG. 26 illustrates a computer control system that is programmed or otherwise configured to implement methods provided herein.
[0103]
FIG. 27 illustrates an example of the system of sample analysis in accordance with the present disclosure.
[0104]
FIGs. 28A-C illustrates various components of the system as illustrated in FIG. 27.
[0105]
FIGs. 29A-G illustrates an exemplary process by which a method for sample analysis is performed in accordance with the present disclosure.

DETAILED DESCRIPTION

[0106]
While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
[0107]
As used in the specification and claims, the singular form “a” , “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a molecule” includes a plurality of molecules, including mixtures thereof.
[0108]
The term “about” or “nearly” as used herein refers to within +/-10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%of the designated amount.
[0109]
As used herein, the terms “amplifying” and “amplification” are used interchangeably and generally refer to generating one or more copies or “amplified product” of a nucleic acid. The term “DNA amplification” generally refers to generating one or more copies of a DNA molecule or “amplified DNA product” . The term “reverse transcription amplification” generally refers to the generation of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template via the action of a reverse transcriptase.
[0110]
As used herein, the term “nucleic acid” generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides (dNTPs) or ribonucleotides (rNTPs) , or analogs thereof. Nucleic acids may have any three dimensional structure, and may perform any function, known or unknown. Non-limiting examples of nucleic acids include DNA, RNA, coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be made before or after assembly of the nucleic acid. The sequence of nucleotides of a nucleic acid may be interrupted by non-nucleotide components. A nucleic acid may be further modified after polymerization, such as by conjugation or binding with a reporter agent.
[0111]
As used herein, the term “primer extension reaction” generally refers to the denaturing of a double-stranded nucleic acid, binding of a primer to one or both strands of the denatured nucleic acid, followed by elongation of the primer (s) .
[0112]
As used herein, the term “reaction mixture” generally refers to a composition comprising reagents necessary to complete nucleic acid amplification (e.g., DNA amplification, RNA amplification) , with non-limiting examples of such reagents that include primer sets having specificity for target RNA or target DNA, DNA produced from reverse transcription of RNA, a DNA polymerase, a reverse transcriptase (e.g., for reverse transcription of RNA) , suitable buffers (including zwitterionic buffers) , co-factors (e.g., divalent and monovalent cations) , dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG) ) , etc) . In some cases, reaction mixtures can also comprise one or more reporter agents.
[0113]
As used herein, a “reporter agent” generally refers to a composition that yields a detectable signal, the presence or absence of which can be used to detect the presence of amplified product.
[0114]
As used herein, the term “target nucleic acid” generally refers to a nucleic acid molecule in a starting population of nucleic acid molecules having a nucleotide sequence whose presence, amount, and/or sequence, or changes in one or more of these, are desired to be determined. A target nucleic acid may be any type of nucleic acid, including DNA, RNA, and analogues thereof. As used herein, a “target ribonucleic acid (RNA) ” generally refers to a target nucleic acid that is RNA. As used herein, a “target deoxyribonucleic acid (DNA) ” generally refers to a target nucleic acid that is DNA.
[0115]
As used herein, the term “subject, ” generally refers to an entity or a medium that has testable or detectable genetic information. A subject can be a person or individual. A subject can be a vertebrate, such as, for example, a mammal. Non-limiting examples of mammals include murines, simians, humans, farm animals, sport animals, and pets. Other examples of subjects include, for example, food, plant, soil, and water.
[0116]
As used herein, the term “electrophoresis” involves the migration of species in a sample through a separation matrix or medium, such as a gel, in the presence of an electric field. The physical properties of the matrix and of the sample species can affect the rate of migration, allowing separation of different species within a sample. Relevant physical properties of sample species include size, electrical charge, and conformation. Electrophoresis can be conducted within an apparatus, which can provide a matrix (e.g., a gel) , buffer solution, and electrodes for generating an electric field.
[0117]
System for Sample Analysis
[0118]
In one aspect, the present disclosure provides a system for sample analysis.
[0119]
The system may comprise a sample vessel. In some embodiments, the system for sample analysis as described herein may contain a plurality of sample vessels. In some embodiments where there is a plurality of sample vessels, the sample vessels may be arranged in a rack or an array. In some embodiments, the sample vessels may be held in a microplate before transferring to the system. In some embodiments, the microplate may be separable to strips or single wells to allow flexible loading of the sample vessels.
[0120]
In some embodiments, the plurality of sample vessels are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 sample vessels.
[0121]
The sample vessel may comprise a container and a penetratable membrane at an end of the container. In some embodiments, where the system comprises a plurality of sample vessels, a given sample vessel of the plurality of sample vessels may comprise the container and the penetratable membrane.
[0122]
The container as described herein may be a vial, test tube, Eppendorf tube, PCR tube, flask, bottle, syringe and/or other container elements, into which a sample can be placed. In some embodiments of the present disclosure, the container is a PCR tube. Other details of containers may be found elsewhere herein.
[0123]
The penetratable membrane as described herein may be any membrane intended for separation purpose. The penetratable membrane can be soft or rigid. The penetratable membrane can be a synthetic membrane. In some embodiments, the penetratable membrane may be a polymeric membrane, a ceramic membrane, a liquid membrane, a metallic membrane, a wooden membrane, or the like. In some embodiments of the present disclosure, the penetratable membrane may be a polymeric membrane. The polymeric membrane may be formed of a polymeric material. Non-limiting examples of polymeric material that may be used to form the polymeric membrane include cellulose acetate (CA) , nitrocellulose (CN) , cellulose esters (CE) , polysulfone (PS) , polyether sulfone (PES) , polyacrilonitrile (PAN) , polyamide, polyimide, polyethylene (PE) , polypropylene (PP) , polytetrafluoroethylene (PTFE) , polyvinylidene fluoride (PVDF) , polyvinylchloride (PVC) , and mixtures and copolymers thereof.
[0124]
The penetratable membrane may be of any form or shape, as long as it is capable of sealing the container in the absence of an object inserted through the penetratable membrane. Non-limiting examples of the forms that the penetratable membrane can assume include caps, tops, screw caps, friction fits, lids, plugs, hinged caps and the like.
[0125]
The penetratable membrane may seal the container in the absence of an object inserted through the penetratable membrane. In some embodiments, the penetratable membrane may be an integral part of the container in a configuration that seals the container. In some embodiments, the penetratable membrane may be deployed in an open or closed configuration relative to the container, where when the penetratable membrane is deployed in the closed configuration, it seals the container. In some embodiments, the penetratable membrane may be a different entity from the container. In the latter case, the penetratable membrane may seal the container by any applicable element, for example, by sealing tape, by pressure sensitive adhesive, by glass glue, or the like.
[0126]
In some embodiments, the penetratable membrane may be resealable after, for example, insertion of an object through the penetratable membrane. The resealing of the penetratable membrane may be effected by any applicable element, for example, by sealing tape, by pressure sensitive adhesive, by glass glue, or the like.
[0127]
In some embodiments, the container may retain a sample, in some cases a biological sample. The sample may be any sample analysis of which is of interest to the user of the system or method of the present disclosure. Other details of samples may be found elsewhere herein.
[0128]
In some cases, the penetratable membrane may comprise a slit. In some cases, the penetratable membrane may be intact before being penetrated. The system may further comprise a separation medium. The separation medium may be any separation medium capable of separating biological samples. Non-limiting examples of a separation medium that can be used in the present disclosure include gel, chromatographic column, paper, ceramic, capillary column, and the like.
[0129]
In one aspect, the separation medium may be a polymeric separation medium. Non-limiting examples of a polymeric separation medium include agarose, acrylamide, starch, resin, silica, and the like. In some embodiments of the present disclosure, the polymeric separation medium may be an agarose gel.
[0130]
The biological sample or a portion thereof may be subject to separation upon application of a flow-inducing field across the separation medium. The flow-inducing field may be any field that drives relative movement among different components of the biological sample or a portion thereof. Non-limiting examples of a flow-inducing field include an electrical field, a magnetic field, an electromagnetic field, a gravity field, pressure field, and the like.
[0131]
Under the influence of the flow-inducing field, different components of the biological sample or a portion thereof may be separated due to difference in their physical or chemical properties. The different components may be separated due to difference in any one or more of the properties selected from the group consisting of mass, charge, geometry, affinity to an agent, hydrophobicity, hydrophilicity, electro-osmotic properties, or any combination thereof.
[0132]
In some aspects, the flow-inducing field may be an electric field. The electric field may be generated by electrodes. In an aspect, the electric field may be generated by at least two electrodes. In an aspect, the system may further comprise two electrodes on ends of the separation medium, wherein the at least two electrodes may provide the electric field. In some aspects, the system may comprise an apparatus for gel electrophoresis. Other details of electrophoresis suitable for methods and systems of the present disclosure may be found elsewhere herein.
[0133]
In some embodiments of the present disclosure, the separation may be on the basis of mass or charge. In some embodiments, the separation may be based on a ratio of mass to charge. Alternatively, the flow-inducing field may be a pressure field. For example, the pressure field may be an osmotic pressure field, such as in forward osmosis or reverse osmosis. In some embodiments, the forward osmosis mechanism is employed to allow the separation medium to induce the flow. In some embodiments, the reverse osmosis mechanism is employed to allow the separation medium to induce the flow.
[0134]
In some embodiments, the flow-inducing field may employ both the electric field and the pressure field, such as in electro-osmosis. In such cases, the system comprises an apparatus capable of conducting an electro-osmotic separation, such as a capillary electrophoretic apparatus.
[0135]
The system may further comprise a flow channel. The flow channel may be in fluid communication with the separation medium. Alternatively, the flow channel may not be in fluid communication with the separation medium before use, and the flow channel may only be brought into fluid communication with the separation medium during use. In some cases, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium.
[0136]
The additional penetratable membrane, other than being a separate entity from the aforesaid penetratable membrane, may be subject to any description or limitation on the aforesaid penetratable membrane. For example, the additional penetratable membrane may seal the separation medium in the absence of an object inserted through the penetratable membrane. In some cases, the additional penetratable membrane may comprise a slit. In some cases, the additional penetratable membrane may be resealable. The additional penetratable membrane may be formed of a polymeric material.
[0137]
FIG. 11 and FIG. 12 show two schematic diagrams of a sample vessel 1101, a flow channel 1102, and a separation medium 1103. In FIG. 11, the flow channel is separate from the separation medium, whereas in FIG. 12, the flow channel is in fluid communication with the separation medium.
[0138]
FIG. 13 is an exemplary side view of a sample vessel 1101. The sample vessel 1101 comprises a container 11011 and a penetratable membrane 11012 at the end of the container 11011. The container 11011 retains a biological sample 11013.
[0139]
During use, at least a portion of the flow channel may traverse the penetratable membrane to bring the container in fluid communication with the separation medium. The traversing may be effected by a relative movement between the flow channel and the penetratable membrane. For example, the flow channel may move towards the penetratable membrane. Alternatively or additionally, the penetratable membrane may move towards the flow channel.
[0140]
FIGs. 14A-B are schematic diagrams for a process for bringing the container 11011 in fluid communication with the separation medium 1103 through the flow channel 1102.
[0141]
In FIG. 14A, the flow channel 1102, together with the separation medium 1103, and the penetratable membrane 11012 of the sample vessel 1101 are moved toward each other. In one embodiment, the flow channel 1102, together with the separation medium 1103, remains stationary and the penetratable membrane 11012 of the sample vessel 1101 is moved toward the flow channel 1102. In another embodiment, the penetratable membrane 11012 of the sample vessel 1101 remains stationary and the flow channel 1102, together with the separation medium 1103, is moved toward the penetratable membrane 11012. In still another embodiment, both the flow channel 1102, together with the separation medium 1103, and the penetratable membrane 11012 of the sample vessel 1101 are moved and their relative movement brings them toward each other.
[0142]
In FIG. 14B, the flow channel 1102 pierces through the penetratable membrane 11012 to bring the container 11011 in fluid communication with the separation medium 1103 through the flow channel 1102.
[0143]
If the flow channel is not in fluid communication with the separation medium in the first place, an additional step may be needed to bring the flow channel in fluid communication with the separation medium. In some cases, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium.
[0144]
FIG. 15A-D are schematic diagrams for an alternative process for bringing the container 11011 in fluid communication with the separation medium 1103 through the flow channel 1102, whereas the flow channel 1102 is not in fluid communication with the separation medium 1103 in the first place.
[0145]
In FIG. 15A, the flow channel 1102, and the penetratable membrane 11012 of the sample vessel 1101 are moved toward each other. In one embodiment, the flow channel 1102 remains stationary and the penetratable membrane 11012 of the sample vessel 1101 is moved toward the flow channel 1102. In another embodiment, the penetratable membrane 11012 of the sample vessel 1101 remains stationary and the flow channel 1102 is moved toward the penetratable membrane 11012. In still another embodiment, both the flow channel 1102, and the penetratable membrane 11012 of the sample vessel 1101 are moved and their relative movement brings them toward each other.
[0146]
In FIG. 15B, the flow channel 1102 pierces through the penetratable membrane 11012 to bring the container 11011 in fluid communication with the flow channel 1102.
[0147]
In FIG. 15C, the separation medium 1103 is separated from the flow channel 1102 by an additional penetratable membrane 11031. The flow channel 1102, together with the sample vessel 1101, and the additional penetratable membrane 11031 are moved toward each other. In one embodiment, flow channel 1102, together with the sample vessel 1101, remains stationary and the additional penetratable membrane 11031 is moved toward the flow channel 1102. In another embodiment, the additional penetratable membrane 11031 remains stationary and the flow channel 1102, together with the sample vessel 1101, is moved toward the penetratable membrane 11012. In still another embodiment, both the flow channel 1102, together with the sample vessel 1101, and additional penetratable membrane 11031 are moved and their relative movement brings them toward each other.
[0148]
In FIG. 15D, the flow channel 1102 pierces through the additional penetratable membrane 11031. The additional penetratable membrane 11031 and the separation medium 1103 are disposed such that the piercing of the additional penetratable membrane 11031 by the flow channel 1102 brings the container 11011 in fluid communication with the separation medium 1103 through the flow channel 1102.
[0149]
The system may further comprise one or more computer processors. The one or more computer processors may be individually or collectively programmed to conduct methods as described elsewhere herein. For example, the one or more computer processors may be individually or collectively programmed to pierce the penetratable membrane to bring the container in fluid communication with the separation medium through the flow channel, as described above.
[0150]
The one or more computer processors may be individually or collectively programmed to apply positive or negative pressure to subject the biological samples to flow from the container through the flow channel to the separation medium. For example, the positive pressure may be applied by squeezing the container.
[0151]
FIG. 16 is a schematic diagram for a process of subjecting the biological sample 11013 to flow from the container 11011 through the flow channel 1102 to the separation medium 1103. In FIG. 16, a positive pressure 1104 is placed upon the container 11011. Under the positive pressure 1104, the biological sample 11013 flows through the flow channel 1102 into the separation medium 1103. Alternatively, a negative 1104a may be drawn from the separation medium 1103. Under the negative pressure 1104a, the biological sample 11013 flows through the flow channel 1102 into the separation medium 1103.
[0152]
In an embodiment, the entire biological sample 11013 is transferred into the separation medium 1103 by flowing through the flow channel 1102. In another embodiment, substantially the entire biological sample 11013 is transferred into the separation medium 1103 by flowing through the flow channel 1102, but there may be residual biological sample remaining in the container 11011. In still another embodiment, a portion of the biological sample 11013 is transferred into the separation medium 1103 by flowing through the flow channel 1102.
[0153]
The one or more computer processors may be individually or collectively programmed to further apply the flow-inducing field across the separation medium. The conditions for the application of the flow-inducing field may be sufficient to direct the biological sample through the separation medium to separate the biological sample or portion thereof. For example, the separation may be on the basis of mass or charge.
[0154]
FIGs. 17A-B are schematic diagrams for a process of applying a flow-inducing field 1105 across the separation medium 1103 to direct the biological sample 11013 through the separation medium 1103. In FIG. 17A, after the biological sample 11013 is transferred to the separation medium 1103, a flow-inducing field 1105 is applied across the separation medium 1103. Under the influence of the flow-inducing field 1105, the biological sample 11013 moves across the separation medium 1103. In FIG. 17B, after a time period under the flowing-inducing field 1105, components 11013a and 11013b of the biological sample 11013 are separated on the separation medium 1103.
[0155]
Although in FIG. 17A-B, the flow channel 1102 and the sample vessel 1101 are depicted as remained in fluid communication with the separation medium 1103 during the action of the flow-inducing field 1105, the embodiment of the present disclosure is not limited as such. In some other embodiments, any or both of the flow channel 1102 and the sample vessel 1101 may be removed from the separation medium 1103 as long as it does not affect the action of the flow-inducing field 1105, or does not allow opening through which contamination to the separation medium 1103 from external environment may occur. In some embodiments, the connection between the separation medium 1103 and the fluid channel 1102 is resealable upon removal of the fluid channel 1102 from the separation medium 1103. In some embodiments, the additional penetratable membrane 11031 is resealable upon removal of the fluid channel 1102 from the separation medium 1103 such that it does not allow opening through which contamination to the separation medium 1103 from external environment may occur.
[0156]
Although in FIG. 17A-B, the flow-inducing field 1105 is shown as applied in a horizontal orientation from left to right across the separation medium 1103, the embodiment of the present disclosure is not limited as such. In some other embodiments, the flowing-inducing field 1105 can assume any orientation, direction, angle, vector, strength, intensity, density, etc., as appropriate. Moreover, all these parameters for the flow-inducing field 1105 may be changed or even reversed during its action, as long as the effect of the flowing-inducing field 1105 over time is such that components of the biological sample 11012 are separated at the end of its action.
[0157]
Although in FIG. 17B, only two components 11012a and 11012b of the biological sample 11012 are shown to have been separated from each other, the embodiment of the present disclosure is not limited as such. In some other embodiments, more than two components of the biological sample 11012 may be separated among one another due to the action of the flow-inducing field 1105. In some other embodiments, any one or more components of the biological sample 11012 may be separated from another or more than one other components of the biological sample 11012 due to the action of the flow-inducing field 1105. In some other embodiments, any subset comprising one or more components of the biological sample 11012 may be separated from any other subset (s) comprising one or more other components of the biological sample 11012 due to the action of the flow-inducing field 1105. In some other embodiments, any multiple subsets comprising one or more components of the biological sample 11012 mutually exclusive among the subsets may be separated from one another subsets due to the action of the flow-inducing field 1105.
[0158]
The one or more computer processor may be individually or collectively programmed to bring the flow channel in fluid communication with the separation medium. In some cases, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium. As described above, the additional penetratable membrane, other than being a separate entity from the aforesaid penetratable membrane, may be subject to any description or limitation on the aforesaid penetratable membrane. For example, the additional penetratable membrane may seal the separation medium in the absence of an object inserted through the penetratable membrane. In some cases, the additional penetratable membrane may comprise a slit. In some cases, the additional penetratable membrane may be resealable. The additional penetratable membrane may be formed of a polymeric material.
[0159]
The system may further comprise a holder for receiving and securably holding the sample vessel. FIG. 18 is a side view of a holder 1106 with the sample vessel 1101 held therein. Although the holder 1106 is depicted as a round-angle rectangle with at least one protrusion, it may assume any shape or dimension as long as such a shape or dimension does not hinder the holder 1106 from performing the function as described elsewhere herein. Moreover, although the sample vessel 1101 is depicted as embedded in the holder 1106 with only the end of penetratable membrane exposed, the embodiment of the present disclosure is not limited as such. In some other embodiments, the sample vessel 1101 may have other portion (s) of it exposed from the holder 1106. In some other embodiments, the sample vessel 1101 may have the other end which does not have the penetratable membrane exposed from the holder 1106. In some other embodiments, the sample vessel 1101 may have its side wall exposed from the holder 1106. In some other embodiments, the sample vessel 1101 may be entirely embedded in the holder 1106.
[0160]
At least a portion of the flow channel may be secured in a housing. In some embodiments, one end of the flow channel may be exposed. In some alternative embodiments, both ends of the flow channel may be embedded in the housing.
[0161]
During use, the holder may be brought in contact with the housing. As an alternative, the holder may not be brought in contact with the housing, but in some cases may be moved in proximity to the housing. The holder may be brought in contact with the housing in a first position. Alternatively, the holder may be brought in proximity to the housing in the first positoin. In the first position, the penetratable membrane may not be pierced. Any approaches that allow the holder and the housing to be brought in contact with or in proximity to each other can be used. In some embodiments, the holder may be configured to accommodate the insertion of housing which brought about the contact. In some embodiments, the housing may be configured to accommodate the insertion of holder which brought about the contact. In some embodiments, both the housing and the holder may be configured to allow mating of the both which brought about the contact. In some embodiments, the contact is a tangible contact. In some embodiments, the contact is an intangible contact. In some embodiments, the housing and the holder may be brought into contact by any suitable mechanical elements, such as springs or hinges. The term “intangible contact” as defined herein indicates contact by an intangible field, for example, an electric field, a magnetic field, an electro-magnetic field, a gravity field, etc.
[0162]
During use, the penetratable membrane may be pierced. The penetratable membrane may be pierced by directing the housing or the holder to a second position. In some embodiments, once in the second position, the housing and the holder are stabilized relative to each other. The term “stabilized or stabilization” as reference to elements herein indicates that the elements in question are held in an arrangement which will not be upset by small disturbs, but can be dismantled without undue effort. Non-limiting examples of such a fastening element include a snap fit, a quick release skewer, a pressure sensitive adhesive, and the like.
[0163]
FIG. 19A-C are schematic diagrams depicting the relative movement between the holder 1106 and a housing 1107 for the flow channel 1102 which brings about the piercing of the penetratable membrane by the flow channel 1102.
[0164]
FIG. 19A shows the holder 1106 for receiving and securably holding the sample vessel 1101, as well as the housing 1107 for securing at least a portion of the flow channel 1102. Although the housing 1107 is depicted as a round-angle rectangle, it may assume any shape or dimension as long as such a shape or dimension does not hinder the housing 1107 from performing the function as described elsewhere herein. Moreover, although the flow channel 1102 is depicted as embedded in the housing 1107 with only one exposed, the embodiment of the present disclosure is not limited as such. In some other embodiments, the flow channel 1102 may have other portion (s) of it exposed from the housing 1107. In some other embodiments, the flow channel 1102 may have the other end exposed from the housing 1107. In some other embodiments, the flow channel 1102 may have its side wall exposed from the housing 1107. In some other embodiments, the flow channel 1102 may be entirely embedded in the housing 1107.
[0165]
In FIG. 19B, the holder 1106 is brought into contact with the housing 1107 in a first position. Although the holder 1106 is depicted as contacting the housing 1107 via at least one protrusion 11061, the embodiment of the present disclosure is not limited as such. In some embodiments, the holder 1106 and the housing 1107 may take any configurations as exemplified by the top panel of FIG. 20. In some embodiments, the holder 1106 and the housting 1107 may be in proximity to each other in the first position.
[0166]
In some other embodiments, it may be the housing 1107 that has protrusions via which the housing 1107 is contacted with the holder 1106 (FIG. 20A) . In some embodiments, the housing 1107 has protrusions whereas the holder 1106 has concaves into which the protrusions may fit such that to bring the housing 1107 and the holder 1106 in contact (FIG. 20B) . In some other embodiments, both the holder 1106 and the housing 1107 have protrusions via which they may contact each other (FIG. 20C) . In some other embodiments, the housing 1107 and the holder 1106 may contact each other via springs (FIG. 20D) or hinges (FIG. 20E) . The contact between the housing 1107 and the holder 1106 may be a tangible contact as described above, as well as any other tangible contact that does not hinder the housing 1107 and the holder 1106 from performing the function as described elsewhere herein. It is within the skill and expertise of a person of ordinary skill in the art to conceive of and design physical contacts on the basis of the examples illustrated here.
[0167]
Alternatively, the contact between the housing 1107 and the holder 1106 may be an intangible contact (FIG. 20F) . The term “intangible contact” as defined herein indicates contact by an intangible field, for example, an electric field, a magnetic field, an electro-magnetic field, a gravity field, etc., rather than visible elements or approaches, as exemplified in the above paragraph. The intangible field may be disposed such that although the housing 1107 and the holder 1106 do not visibly contact each other, the field will influence the action of them as if they are visibly contacted with each other.
[0168]
In FIG. 19C, the housing 1107 and/or the holder 1106 may be directed to a second position. In the second position, the penetratable membrane 11012 is pierced by the flow channel 1102. In the second position, the flow channel 1102 pierces through the penetratable membrane 11012 to bring the container 11011 in fluid communication with the flow channel 1102.
[0169]
Although in FIG. 19C, the upper edge of the housing 1107 is depicted as touching the penetratable 11012 in the second position, the embodiment of the present disclosure is not limited as such. In some other embodiments, in the second position, the housing 1107 is separated from the penetratable 11012 with a gap, as long as the dimension of the gap still allows the penetratable 11012 to be pierced by the flow channel 1102. In some embodiments, in the second position, the housing 1107 and the holder 1106 are fastened by a fastening element such that they are stabilized relative to each other. The term “stabilized or stabilization” as reference to elements herein indicates that the elements in question are held in an arrangement which will not be upset by small disturbs, but can be dismantled without undue effort. Non-limiting examples of such a fastening element include a snap fit, a quick release skewer, a pressure sensitive adhesive, and the like.
[0170]
FIG. 20A-F illustrate several alternative embodiments of the configuration between the housing and the holder that allows they are in contact in the first positions (the upper panel) and directing them to the second positions (the lower panel) allows the flow channel to pierce the penetratable membrane. For sake of conciseness and clarity, the housing, the holder, the components of the sample vessel, as well as the flow channel are not numbered, but a person of ordinary skill in the art will readily understand that similarly shaped objects indicate the same objects across this figure and are also comparable to other similarly shaped objects in other figures. For example, the long and slim rectangular shaped object in the middle of each panel indicates the flow channel.
[0171]
FIG. 20A illustrates a configuration in which the housing has at least one protrusion 11071 which is in contact with the holder in the first position. The housing and/or the holder is directed to the second position while the holder in contact with the protrusions 11071 such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel.
[0172]
FIG. 20B illustrates a configuration in which the housing has at least one protrusions 11072 and the holder has at least one concaves 11062 into which the protrusions of the housing are fitted in the first position. The housing and/or the holder is directed to the second position while the protrusions 11072 move deeper into the concaves 11062 such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel. Alternatively, the protrusions may be disposed on the holder and the concaves may be disposed on the housing. Under such a configuration, the housing and/or the holder is directed to the second position while the protrusions move deeper into the concaves such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel.
[0173]
FIG. 20C illustrates a configuration in which the housing has at least one protrusions 11073 and the holder also has at least one protrusions 11063 into which the protrusions of the housing and the holder are contacted in the first position. The housing and/or the holder is directed to the second position while the protrusions 11073 move relatively to the protrusions 11063 such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel.
[0174]
FIG. 20D illustrates a configuration in which the holder has at least one springs 11064 which are in contact with the housing in the first position. The housing and/or the holder is directed to the second position while the springs are compressed against the housing such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel. Alternatively, the springs may be disposed on the housing instead of the holder and under such a configuration, the housing and/or the holder is directed to the second position while the springs are compressed against the holder such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel.
[0175]
FIG. 20E illustrates a configuration in which the housing has at least one hinges 11064 which are in contact with the holder in the first position. The housing and/or the holder is directed to the second position while the arms of the hinges rotate to sharper angles such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel. Alternatively, the hinges may be disposed on the holder instead of the housing and under such a configuration, the housing and/or the holder is directed to the second position while the arms of the hinges rotate to sharper angles such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel.
[0176]
FIG. 20F illustrates a configuration in which the housing and the holder contact with each other in an intangible contact. Both the housing and the holder are under the influence of one or more fields such that the housing are effected to move towards the general direction as indicated by the axis 11076, and the holder re effected to move towards the general direction as indicated by the axis 11066. The term “general direction” as described herein indicates within an angle of 5, 4, 3, 2, or 1 degree around a reference axis. It is apparent that the holder and/or the housing may not need to move relatively toward exact opposite directions to bring about the second position of the housing and/or the holder such that the flow channel pierces the penetratable membrane to bring the container in fluid communication with the flow channel.
[0177]
It should be understood that when the house and/or the hold is described as “in a/the first position” or “in a/the second position” , it refers to relative positions between the house and the holder. A similar way of understanding also applies to circumstances such as “the holder is brought in contact with the housing” or “directing the housing or the holder to a second position” . In any of these circumstances, even though an action may be described as exerted on either the housing or the holder, it should be understood that due to the relativity of motion, the same action may be exerted on the other object or both objects in the opposite direction (s) and bring about the same effect. Such alternative embodiments are also encompassed in the present disclosure.
[0178]
It should also be understood that although in FIGs. 19 and 20, the holder and the housing are depicted in a vertical configuration and the holder is depicted to be above the housing, the embodiment of the present disclosure is not limited as such. The holder and the housing may be disposed in any configuration with the two in any relative position. In some embodiments, the holder and the housing may be in a vertical configuration and the housing is above the holder. In some embodiments, the holder and the housing may be in a horizontal configuration. In some embodiments, the holder and the housing may be in an inclining configuration. In some other embodiments, the holder and the housing may be in other configurations which allow the holder and the housing to perform the aforesaid relative movement towards each other to bring about the piercing of the penetratable membrane.
[0179]
Despite that in the descriptions of FIGs. 18-20, the protrusions, concaves, springs, and the hinges are often referred to in pairs, it should be understood that they are for illustrative purpose only and the embodiment of the present disclosure is not limited as such. The housing and/or the holder may comprise one or more protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise one protrusion, concave, spring, hinge, or other similar or equivalent element. In some embodiments, the housing and/or the holder may comprise a total of two protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of three protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of four protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of five protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of six protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of seven protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of eight protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of nine protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of ten protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of more than ten protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof.
[0180]
The protrusions, concaves, springs, hinges, and other similar or equivalent elements as described herein may be integral to the housing and/or holder, or may be detachably or fixedly connected to the housing and/or holder.
[0181]
In any of the embodiments as described above, a fastening element may be employed to stabilize the housing and the holder in the second position for a time period sufficient for transferring any biological sample retained in the container to the fluid channel. Alternatively, no fastening element may be employed, but the housing and the holder are manually or mechanically stabilized in the second position for a time period sufficient for transferring any biological sample retained in the container to the fluid channel.
[0182]
Although neither the FIG. 19 nor FIG. 20 depicts the separation medium, the separation medium may be in fluid communication with the separation medium in any of the embodiments as described above. For example, FIG. 21A-C are schematic diagrams depicting the relative movement between the holder 1106 and a housing 1107 for the flow channel 1102 which brings about the piercing of the penetratable membrane by the flow channel 1102, wherein the flow channel 1102 is in fluid communication with a separation medium 1103. In FIG. 21A, the holder 1106 and the housing 1107 are not in contact. In FIG. 21B, the holder 1106 is brought in contact with the housing 1107 in a first position, where the penetratable membrane 11012 is not pierced. In FIG. 21C, the housing or the holder is directed to a second position where the penetratable membrane is pierced which brings the sample vessel 1101 in fluid communication with the separation medium via the flow channel 1102.
[0183]
It should be understood that the any embodiment described in the aforesaid discussion regarding FIGs. 19-20 may also be implemented with the flow channel 1102 in fluid communication with the separation medium 1103. For example, the embodiment as depicted in FIG. 210B with the holder having at least one concaves and the housing having two protrusions can also be implemented with the flow channel 1102 in fluid communication with the separation medium 1103.
[0184]
Alternatively, the flow channel may not be in fluid communication with the separation medium when the housing or the holder is in the second position. In such circumstances, the flow channel may be brought in fluid communication with the separation medium. The flow channel may be brought in fluid communication with the separation medium by directing the housing to a third position. Therefore, after the housing is directed to a second position as described herein, the housing may be directed to a third position which brings the flow channel in fluid communication with the separation medium. In some embodiments, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium. In some embodiments, the housing may be directed to the third position which allows the flow channel to pierce an additional penetrable membrane separating the flow channel from the separation medium, thereby bringing the sample vessel in fluid communication with the separation medium via the flow channel.
[0185]
As described above, the additional penetratable membrane may seal the separation medium in the absence of an object inserted through the penetratable membrane.
[0186]
In some embodiments, the additional penetratable membrane may comprise a slit. In some embodiments, the additional penetratable membrane may be resealable. The additional penetratable membrane may be formed of a polymeric material. Any description about the penetratable membrane of the sample vessel may also be applicable to the additional penetratable membrane.
[0187]
In some embodiments, the separation medium may be enclosed an additional housing. The additional housing may have an opening which is sealed by the additional penetratable membrane. The additional penetratable membrane may seal the opening on the additional housing, thereby sealing the separation medium in the absence of an object inserted through the penetratable membrane.
[0188]
When the separation medium is enclosed in the additional housing, any embodiment as depicted in FIGs. 19-20 or discussed above in reference to FIGs. 19-20 may be implemented with the additional housing substituting the holder, the additional penetratable membrane substituting the penetratable membrane, and the separation medium substituting the container, so as to bring the housing to the third position which allows the flow channel to pierce an additional penetrable membrane separating the flow channel from the separation medium, thereby bringing the sample vessel in fluid communication with the separation medium via the flow channel.
[0189]
FIG. 22A-C illustrates an exemplary process in which the housing 1107 is brought to a third position to allow the flow channel 1102 to pierce the additional penetrable membrane 11031. As illustrated in FIG. 22A, the additional housing 1108 has an opening sealed by the additional penetrable membrane 11031. The additional housing 1108 comprises at least one protrusion 11081. In FIG. 22B, the housing 1107 is brought to contact with the additional housing 1108 via the protrusion 11081. In FIG. 22C, the housing 1107 is brought to a third position which allows the flow channel to pierce the additional penetrable membrane 11031, thereby bringing the sample vessel 1106 in fluid communication with the separation medium 1103 via the flow channel 1102.
[0190]
Alternatively, the housing 1107 is not brought into contact with the additional housing 1108 prior to being brought to the third position. For example, the housing 1107 may be brought in proximity to the additional housing 1108 rior to being brought to the third position.
[0191]
One or more of the first, second and third positions may be vertical positions. Alternatively, one or more of the first, second and third positions may be any other suitable positions, including horizontal positions, or any position that is at angle to the vertical plane. For example, one or more of the first, second and third positions may be at an angle of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, or 90 degrees, or degrees of any numerical therebetween relative to the vertical plane.
[0192]
In some cases, the housing may further comprise a first covering element. The first covering element may cover an end of the flow channel when said housing is in said first position. The first covering element may allow the end of the flow channel to emerge from the first covering element when the housing or the holder is in the second position. As a result, the end of the flow channel may traverse the penetratable membrane.
[0193]
In some embodiments, the housing 1107 may further comprise a first covering element 11077 which may cover an end of the flow channel 1102 when the housing 1107 is in the first position as described elsewhere herein. FIGs. 23A-F illustrate exemplary configurations in which a first covering element covers and seals an end of the flow channel and exemplary processes for using these configurations in accordance with the present disclosure. FIG. 23A depicts an embodiment of the present disclosure utilizing the first covering element 11077 which covers an end of the flow channel 1102. As depicted in FIG. 23B, when the housing 1107 is in the first position, the first covering element 11077 covers the end of the flow channel 1102 such that it does not emerge from the housing 1107. When the housing and/or the holder is directed to the second position, as depicted in FIG. 23C, the covering element 11077 allows the flow channel 1102 to emerge from the first covering element 11077 such that it traverses the penetratable membrane 11012 to bring the sample vessel 1101 in fluid communication with the flow channel 1102.
[0194]
In some embodiments, the first covering element 11077 may have its shaped altered upon contact with the holder 1106. The compression of the first covering element 11077 substantially along the direction of the flow channel 1102 allows the flow channel 1102 to emerge from the first covering element 11077 such that it can traverses the penetratable membrane 11012.
[0195]
In some embodiments, the first covering element 11077 may have its shaped altered with manual or mechanical control. For example, one or more computer processors as described elsewhere herein may instruct an actuator to compress the first covering element 11077. Alternatively, a user may manually compress the first covering element 11077. The compression of the first covering element 11077 substantially along the direction of the flow channel 1102 allows the flow channel 1102 to emerge from the first covering element 11077 such that it can traverses the penetratable membrane 11012.
[0196]
Alternatively, the first covering element 11077 may be separated from the housing 1107 by a gap as depicted in FIG. 23D. As depicted in FIG. 23E, when the housing 1107 is in the first position, the first covering element 11077 covers the end of the flow channel 1102 such that it does not emerge from the housing 1107. When the housing and/or the holder is directed to the second position, as depicted in FIG. 23F, the first covering element 11077 is not compressed, but translates along the axis of the flow channel 1102 towards the housing 1107, which allows the flow channel 1102 to emerge from the first covering element 11077 such that it traverses the penetratable membrane 11012 to bring the sample vessel 1101 in fluid communication with the flow channel 1102. Although FIG. 23F depicts the first covering element 11077 touches the housing 1107 when the housing and/or the holder is at the second position, it does not have to be so. It is conceivable that when the housing and/or the holder is at the second position, a smaller gap may still remain between the the first covering element 11077 and the housing 1107.
[0197]
In some cases, the housing may further comprise a second covering element. The second covering element may cover another end of the flow channel when (a) the housing is in the first position or (b) the housing or the holder is in the second position. The second covering element may allow the other end of the flow channel to emerge from the second covering element when the housing is in the third position. As a result, the other end of the flow channel pierces an additional penetratable membrane. The additional penetratable membrane may separate the flow channel from the separation medium.
[0198]
In some embodiments, the housing 1107 may further comprise a second covering element 11078 which may cover another end of the flow channel 1102 when (a) the housing 1107 is in the first position or (b) the housing 1107 and/or the holder 1106 is in the second position. FIGs. 24A-F illustrate exemplary configurations in which a second covering element covers and seals another end of the flow channel and exemplary processes for using these configurations in accordance with the present disclosure. FIG. 24A depicts an embodiment of the present disclosure utilizing the second covering element 11078 which covers the other end of the flow channel 1102. As depicted in FIG. 24B, when the housing 1107 and the holder 1106 are in the second position, the second covering element 11078 covers the end of the flow channel 1102 such that it does not emerge from the housing 1107. When the housing is directed to the third position, as depicted in FIG. 24C, the second covering element 11078 allows the flow channel 1102 to emerge from the second covering element 11078 such that it traverses the additional penetratable membrane 11031 to bring the separation medium 1103 in fluid communication with the flow channel 1102. In some embodiments, the flow channel 1102 has already been in fluid communication with the sample vessel 1101 when the flow channel 1102 emerges from the second covering element 11078 and traverses the additional penetratable membrane 11031, such that the traversing of the additional penetratable by the flow channel 1102 brings the sample vessel 1101 in fluid communication with the separation medium via the flow channel 1102.
[0199]
In some embodiments, the second covering element 11078 may have its shaped altered upon contact with the second housing 1108. The compression of the first covering element 11077 substantially along the direction of the flow channel 1102 allows the flow channel 1102 to emerge from the second covering element 11078 such that it can traverses the additional penetratable membrane 11031.
[0200]
In some embodiments, the second covering element 11078 may have its shaped altered with manual or mechanical control. For example, one or more computer processors as described elsewhere herein may instruct an actuator to compress the second covering element 11078. Alternatively, a user may manually compress the second covering element 11078. The compression of the second covering element 11078 substantially along the direction of the flow channel 1102 allows the flow channel 1102 to emerge from the second covering element 11078 such that it can traverses the additional penetratable membrane 11031.
[0201]
Alternatively, the second covering element 11078 may be separated from the housing 1107 by a gap as depicted in FIG. 24D. As depicted in FIG. 24E, when the housing 1107 and the holder 1106 are in the second position, the second covering element 11078 covers the end of the flow channel 1102 such that it does not emerge from the housing 1107. When the housing and/or the holder is directed to the third position, as depicted in FIG. 24F, the second covering element 11078 is not compressed, but translates along the axis of the flow channel 1102 towards the housing 1107, which allows the flow channel 1102 to emerge from the second covering element 11078 such that it traverses the additional penetratable membrane 11031 to bring the separation medium 1103 in fluid communication with the flow channel 1102. In some embodiments, the flow channel 1102 has already been in fluid communication with the sample vessel 1101 when the flow channel 1102 emerges from the second covering element 11078 and traverses the additional penetratable membrane 11031, such that the traversing of the additional penetratable by the flow channel 1102 brings the sample vessel 1101 in fluid communication with the separation medium via the flow channel 1102. Although FIG. 24F depicts the first covering element 11077 touches the housing 1107 when the housing and/or the holder is at the second position, it does not have to be so. It is conceivable that when the housing and/or the holder is at the second position, a smaller gap may still remain between the first covering element 11077 and the housing 1107.
[0202]
The first covering element 11077 and/or the second covering element 11078 may be integral to the housing. Alternatively, the first covering element 11077 and/or the second covering element 11078 may be fixedly or detachably connected to the housing. Alternatively, the first covering element 11077 and/or the second covering element 11078 may be separated from the housing by a gap.
[0203]
The aforesaid shape alteration may be any alteration that allows the flow channel to emerge from the first or the second covering element. Non-limiting shape alteration may include compression, stretching, distortion, creeping, disintegration, deformation, fracture, rupture, etc. Elimination or reduction of a gap between the first or the second covering element and the housing may qualify as the “shape alteration” as mentioned elsewhere herein.
[0204]
It should be understood that although the two covering elements are called the first and the second covering element, it does not necessarily indicates that both have to be present on the housing 1107. In some embodiments, the housing comprises the first covering element. In some embodiments, the housing comprises the second covering element. In some embodiment, the housing comprises both the first covering element and the second covering element. In some embodiments, the housing comprises no covering element.
[0205]
In some cases, the housing may comprise a securing element. The securing element may prevent relative movement between the flow channel and the securing element. The securing element may not cover ends of the flow channel. In some embodiments, the securing may be integral to the housing. In some embodiment, the housing itself may prevent relative movement between the flow channel and the securing element. FIG. 25 is a side illustration of a housing 1107 with a first covering element 11077, a second covering element 11078, and a securing element 11079. The securing element 11079 secures the flow channel 1102 such that when the first covering element 11077 and/or the second covering element 11078 are effected to allow the flow channel 1102 to move relative to at least a portion of them, but not move relative to the securing element 11079, such that either or both ends of the flow channel 1102 emerge from the first covering element 11077 and/or the second covering element 11078 as a results of these relative movements.
[0206]
The securing element may be separated from either or both of the first covering element and the second covering element by a gap. The gap may allow either or both of the first covering element and the second covering element to move relative to the flow channel, whereas the securing element does not move relative to the flow channel.
[0207]
In any of the embodiments of the system in accordance with the present disclosure as described herein, the system may further comprise a cannula. The cannula may comprise a hollow tube that can serve as at least a part of the flow channel as described elsewhere herein. In some embodiments, the cannula may comprise at least a portion of the flow channel extending therethrough. In some embodiments, the cannula may accommodate the whole flow channel therewithin.
[0208]
The cannula may pierce the penetratable membrane. The cannula may comprise a piercing element for piercing the penetratable membrane and/or the additional penetratable membrane as described elsewhere herein. The piercing of the penetratable membrane by the cannula may bring the flow channel in fluid communication with the container. Alternatively or additionally, the piercing of the additional penetratable membrane by the cannula may bring the flow channel in fluid communication with the separation medium. The piercing element may be a tip, or any other sharp element that can pierce the penetratable membrane and/or the additional penetratable membrane. In some embodiments, the cannula may include a tip at an end thereof. The tip may pierce the penetratable membrane. The piercing of the penetratable membrane by the tip may bring the flow channel in fluid communication with the container. Alternatively or additionally, the piercing of the penetratable membrane by the tip may bring the flow channel in fluid communication with the separation medium.
[0209]
The piercing of the penetratable membrane and/or the additional penetratable membrane may be performed manually or automatically, such as with a motor or an actuator. In some embodiments, the system may further comprise an actuator. The actuator may pierce the penetratable membrane. The actuator may pierce the additional penetratable membrane. The piercing of the penetratable membrane by the actuator may bring the flow channel in fluid communication with the container. Alternatively or additionally, the piercing of the additional penetratable membrane by the actuator may bring the flow channel in fluid communication with the separation medium.
[0210]
The system may further comprise a compartment. The compartment may have the separation medium. For example, where the separation medium is an electrophoresis gel, the compartment may accommodate the electrophoresis gel. The compartment may comprise or may be a buffer chamber containing buffer for the electrophoresis.
[0211]
The system may further comprise a unique identifier on the compartment. The unique identifier may be a barcode. The barcode may be a one-dimensional barcode or a two-dimensional barcode. The unique identifier may involve contactless technique. The contactless technique allows information to be extracted from the compartment by placing the compartment close to a contactless. The contactless detector may use RFID (radio frequency identification) technique to extract information from the compartment. For example, a user may use a smartphone app that utilizes the RFID technique to communicate with the compartment for identification. In some embodiments, the unique identifier may be a radio-frequency identification tag.
[0212]
The system may further comprise a detector. The detector may detect the biological sample or portion thereof. The detector may detect the biological sample or portion thereof in the separation medium. For example, the system may have a detector that may be capable of detecting signals from one or more lanes of the electrophoresis in real-time. The signal may be an optical signal. The detector may be capable of detecting optical signals from the gels in multiple lanes simultaneously. The detector may capture an image of the top surfaces of the gels in the multiple lanes. The image may be a still image or may include video-rate images. Other details of the detedctor may be found elsewhere herein.
[0213]
Method for Sample Analysis
[0214]
In another aspect, the present disclosure provides a method for sample analysis. The method may comprise (a) receiving a sample vessel comprising (i) a container and (ii) a penetratable membrane at an end of the container. Alternatively, the method may comprise receiving a plurality of sample vessels.
[0215]
The method may involve a sample vessel. In some embodiments, the method for sample analysis as described herein may involve a plurality of sample vessels. In some embodiments where there is a plurality of sample vessels, the sample vessels may be arranged in a rack or an array. In some embodiments, the sample vessels may be held in a microplate before being used in the method. In some embodiments, the microplate may be separable to strips or single wells to allow flexible loading of the sample vessels.
[0216]
In some embodiments, the plurality of sample vessels are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more than 16 sample vessels.
[0217]
The sample vessel may comprise a container and a penetratable membrane at an end of the container. In some embodiments, where the method involves a plurality of sample vessels, a given sample vessel of the plurality of sample vessels may comprise the container and the penetratable membrane.
[0218]
The container as described herein may be a vial, test tube, Eppendorf tube, PCR tube, flask, bottle, syringe and/or other container elements, into which a sample can be placed. In some embodiments of the present disclosure, the container is a PCR tube. Other details of containers may be found elsewhere herein.
[0219]
The penetratable membrane as described herein may be any membrane intended for separation purpose. The penetratable membrane can be soft or rigid. The penetratable membrane can be a synthetic membrane. In some embodiments, the penetratable membrane may be a polymeric membrane, a ceramic membrane, a liquid membrane, a metallic membrane, a wooden membrane, or the like. In some embodiments of the present disclosure, the penetratable membrane may be a polymeric membrane. The polymeric membrane may be formed of a polymeric material. Non-limiting examples of polymeric material that may be used to form the polymeric membrane include cellulose acetate (CA) , nitrocellulose (CN) , cellulose esters (CE) , polysulfone (PS) , polyether sulfone (PES) , polyacrilonitrile (PAN) , polyamide, polyimide, polyethylene (PE) , polypropylene (PP) , polytetrafluoroethylene (PTFE) , polyvinylidene fluoride (PVDF) , polyvinylchloride (PVC) , and mixtures and copolymers thereof.
[0220]
The penetratable membrane may be of any form or shape, as long as it is capable of sealing the container in the absence of an object inserted through the penetratable membrane. Non-limiting examples of the forms that the penetratable membrane can assume include caps, tops, screw caps, friction fits, lids, plugs, hinged caps and the like.
[0221]
The penetratable membrane may seal the container in the absence of an object inserted through the penetratable membrane. In some embodiments, the penetratable membrane may be an integral part of the container in a configuration that seals the container. In some embodiments, the penetratable membrane may be deployed in an open or closed configuration relative to the container, where when the penetratable membrane is deployed in the closed configuration, it seals the container. In some embodiments, the penetratable membrane may be a different entity from the container. In the latter case, the penetratable membrane may seal the container by any applicable element, for example, by sealing tape, by pressure sensitive adhesive, by glass glue, or the like.
[0222]
In some embodiments, the penetratable membrane may be resealable after, for example, insertion of an object through the penetratable membrane. The resealing of the penetratable membrane may be effected by any applicable element, for example, by sealing tape, by pressure sensitive adhesive, by glass glue, or the like.
[0223]
In some embodiments, the container may retain a sample, in some cases a biological sample. The sample may be any sample analysis of which is of interest to the user of the system or method of the present disclosure. Other details of samples may be found elsewhere herein.
[0224]
In some cases, the penetratable membrane may comprise a slit. In some cases, the penetratable membrane may be intact before being penetrated.
[0225]
The method may further comprise (b) piercing the penetratable membrane to bring the biological sample in fluid communication with a flow channel. At least a portion of said flow channel may traverse the penetratable membrane. The flow channel may be in fluid communication with a separation medium. The biological sample or a portion thereof may be subject to separation upon application of a flow-inducing field across the separation medium. For example, the separation may be on the basis of mass or charge.
[0226]
The method may further involve a separation medium. The separation medium may be any separation medium capable of separating biological samples. Non-limiting examples of a separation medium that can be used in the present disclosure include gel, chromatographic column, paper, ceramic, capillary column, and the like.
[0227]
In one aspect, the separation medium may be a polymeric separation medium. Non-limiting examples of a polymeric separation medium include agarose, acrylamide, starch, resin, silica, and the like. In some embodiments of the present disclosure, the polymeric separation medium may be an agarose gel.
[0228]
The biological sample or a portion thereof may be subject to separation upon application of a flow-inducing field across the separation medium. The flow-inducing field may be any field that drives relative movement among different components of the biological sample or a portion thereof. Non-limiting examples of a flow-inducing field include an electrical field, a magnetic field, an electromagnetic field, a gravity field, pressure field, and the like.
[0229]
Under the influence of the flow-inducing field, different components of the biological sample or a portion thereof may be separated due to difference in their physical or chemical properties. The different components may be separated due to difference in any one or more of the properties selected from the group consisting of mass, charge, geometry, affinity to an agent, hydrophobicity, hydrophilicity, electro-osmotic properties, or any combination thereof.
[0230]
In some aspects, the flow-inducing field may be an electric field. The electric field may be generated by electrodes. In an aspect, the electric field may be generated by at least two electrodes. In an aspect, the system may further comprise two electrodes on ends of the separation medium, wherein the at least two electrodes may provide the electric field. In some aspects, the system may comprise an apparatus for gel electrophoresis. The present disclosure provides other details of electrophoretic methods suitable for methods and systems provided herein.
[0231]
In some embodiments of the present disclosure, the separation may be on the basis of mass or charge. In some embodiments, the separation may be based on a ratio of mass to charge. Alternatively, the flow-inducing field may be a pressure field. For example, the pressure field may be an osmotic pressure field, such as in forward osmosis or reverse osmosis. In some embodiments, the forward osmosis mechanism is employed to allow the separation medium to induce the flow. In some embodiments, the reverse osmosis mechanism is employed to allow the separation medium to induce the flow.
[0232]
In some embodiments, the flow-inducing field may employ both the electric field and the pressure field, such as in electro-osmosis. In such cases, the system comprises an apparatus capable of conducting an electro-osmotic separation, such as a capillary electrophoretic apparatus.
[0233]
The system may further comprise a flow channel. The flow channel may be in fluid communication with the separation medium. Alternatively, the flow channel may not be in fluid communication with the separation medium before use, and the flow channel may only be brought into fluid communication with the separation medium during use. In some cases, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium.
[0234]
The additional penetratable membrane, other than being a separate entity from the aforesaid penetratable membrane, may be subject to any description or limitation on the aforesaid penetratable membrane. For example, the additional penetratable membrane may seal the separation medium in the absence of an object inserted through the penetratable membrane. In some cases, the additional penetratable membrane may comprise a slit. In some cases, the additional penetratable membrane may be resealable. The additional penetratable membrane may be formed of a polymeric material.
[0235]
FIG. 11 and FIG. 12 show two schematic diagrams of a sample vessel 1101, a flow channel 1102, and a separation medium 1103, as described above. FIG. 13 is an exemplary side view of a sample vessel 1101 as described above.
[0236]
During use, at least a portion of the flow channel may traverse the penetratable membrane to bring the container in fluid communication with the separation medium. The traversing may be effected by a relative movement between the flow channel and the penetratable membrane. For example, the flow channel may move towards the penetratable membrane. Alternatively or additionally, the penetratable membrane may move towards the flow channel.
[0237]
FIGs. 14A-B are schematic diagrams for a process for bringing the container 11011 in fluid communication with the separation medium 1103 through the flow channel 1102, as described above.
[0238]
If the flow channel is not in fluid communication with the separation medium in the first place, an additional step may be needed to bring the flow channel in fluid communication with the separation medium. In some cases, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium.
[0239]
FIG. 15A-D are schematic diagrams for an alternative process for bringing the container 11011 in fluid communication with the separation medium 1103 through the flow channel 1102, whereas the flow channel 1102 is not in fluid communication with the separation medium 1103 in the first place, as described above.
[0240]
The method may further comprise (c) subjecting the biological sample to flow from the container through the flow channel to the separation medium. The biological sample may be subjected to flow from the container through the flow channel to the separation medium upon application of positive pressure or negative pressure. The positive pressure may be applied by squeezing the container.
[0241]
The method may further involve use of one or more computer processors. The one or more computer processors may be individually or collectively programmed to conduct methods as described elsewhere herein. For example, the one or more computer processors may be individually or collectively programmed to pierce the penetratable membrane to bring the container in fluid communication with the separation medium through the flow channel, as described above.
[0242]
The one or more computer processors may be individually or collectively programmed to apply positive or negative pressure to subject the biological samples to flow from the container through the flow channel to the separation medium. For example, the positive pressure may be applied by squeezing the container.
[0243]
The one or more computer processors may be individually or collectively programmed to further apply the flow-inducing field across the separation medium. The conditions for the application of the flow-inducing field may be sufficient to direct the biological sample through the separation medium to separate the biological sample or portion thereof. For example, the separation may be on the basis of mass or charge.
[0244]
FIG. 16 is a schematic diagram for a process of subjecting the biological sample 11013 to flow from the container 11011 through the flow channel 1102 to the separation medium 1103, as described above.
[0245]
The method may further comprise (d) applying the flow-inducing field across the separation medium. The conditions for the application of the flow-inducing field may be sufficient to direct the biological sample through the separation medium to separate the biological sample or portion thereof. For example, the separation may be on the basis of mass or charge.
[0246]
FIGs. 17A-B are schematic diagrams for a process of applying a flow-inducing field 1105 across the separation medium 1103 to direct the biological sample 11013 through the separation medium 1103, as described above.
[0247]
In step (c) of the method, the flow channel may be brought in fluid communication with the separation medium. In some cases, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium. As described above, the additional penetratable membrane, other than being a separate entity from the aforesaid penetratable membrane, may be subject to any description or limitation on the aforesaid penetratable membrane. For example, the additional penetratable membrane may seal the separation medium in the absence of an object inserted through the penetratable membrane. In some cases, the additional penetratable membrane may comprise a slit. In some cases, the additional penetratable membrane may be resealable. The additional penetratable membrane may be formed of a polymeric material.
[0248]
In the step (a) of the method, the sample vessel may be received in a holder that receives and securably holds the sample vessel. At least a portion of the flow channel may be secured in a housing. Moreover, in step (b) of the method, the holder may be brought in contact with the housing. Moreover, in step (b) of the method, the holder may be brought in contact with the housing in a first position. In the first position, the penetratable membrane may not be pierced. Moreover, in step (b) of the method, the penetratable membrane may be pierced. The penetratable membrane may be pierced by directing the housing or the holder to a second position. In the step (b) of the method, the flow channel may be brought in fluid communication with the separation medium. The flow channel may be brought in fluid communication with the separation medium by directing the housing to a third position. One or more of the first, second and third positions may be vertical positions.
[0249]
The system may further comprise a holder for receiving and securably holding the sample vessel. FIG. 18 is a side view of a holder 1106 with the sample vessel 1101 held therein, as described above.
[0250]
At least a portion of the flow channel may be secured in a housing. In some embodiments, one end of the flow channel may be exposed. In some alternative embodiments, both ends of the flow channel may be embedded in the housing.
[0251]
During use, the holder may be brought in contact with the housing. As an alternative, the holder may not be brought in contact with the housing, but in some cases may be moved in proximity to the housing. The holder may be brought in contact with the housing in a first position. Alternatively, the holder may be brought in proximity to the housing in the first positoin. In the first position, the penetratable membrane may not be pierced. Any approaches that allow the holder and the housing to be brought in contact with or in proximity to each other can be used. In some embodiments, the holder may be configured to accommodate the insertion of housing which brought about the contact. In some embodiments, the housing may be configured to accommodate the insertion of holder which brought about the contact. In some embodiments, both the housing and the holder may be configured to allow mating of the both which brought about the contact. In some embodiments, the contact is a tangible contact. In some embodiments, the contact is an intangible contact. In some embodiments, the housing and the holder may be brought into contact by any suitable mechanical elements, such as springs or hinges. The term “intangible contact” as defined herein indicates contact by an intangible field, for example, an electric field, a magnetic field, an electro-magnetic field, a gravity field, etc.
[0252]
During use, the penetratable membrane may be pierced. The penetratable membrane may be pierced by directing the housing or the holder to a second position. In some embodiments, once in the second position, the housing and the holder are stabilized relative to each other. The term “stabilized or stabilization” as reference to elements herein indicates that the elements in question are held in an arrangement which will not be upset by small disturbs, but can be dismantled without undue effort. Non-limiting examples of such a fastening element include a snap fit, a quick release skewer, a pressure sensitive adhesive, and the like.
[0253]
FIG. 19A-C are schematic diagrams depicting the relative movement between the holder 1106 and a housing 1107 for the flow channel 1102 which brings about the piercing of the penetratable membrane by the flow channel 1102, as described above.
[0254]
FIG. 20A-F illustrate several alternative embodiments of the configuration between the housing and the holder that allows they are in contact in the first positions (the upper panel) and directing them to the second positions (the lower panel) allows the flow channel to pierce the penetratable membrane, as described above.
[0255]
It should be understood that when the house and/or the hold is described as “in a/the first position” or “in a/the second position” , it refers to relative positions between the house and the holder. A similar way of understanding also applies to circumstances such as “the holder is brought in contact with the housing” or “directing the housing or the holder to a second position” . In any of these circumstances, even though an action may be described as exerted on either the housing or the holder, it should be understood that due to the relativity of motion, the same action may be exerted on the other object or both objects in the opposite direction (s) and bring about the same effect. Such alternative embodiments are also encompassed in the present disclosure.
[0256]
It should also be understood that although in FIGs. 19 and 20, the holder and the housing are depicted in a vertical configuration and the holder is depicted to be above the housing, the embodiment of the present disclosure is not limited as such. The holder and the housing may be disposed in any configuration with the two in any relative position. In some embodiments, the holder and the housing may be in a vertical configuration and the housing is above the holder. In some embodiments, the holder and the housing may be in a horizontal configuration. In some embodiments, the holder and the housing may be in an inclining configuration. In some other embodiments, the holder and the housing may be in other configurations which allow the holder and the housing to perform the aforesaid relative movement towards each other to bring about the piercing of the penetratable membrane.
[0257]
Despite that in the descriptions of FIGs. 18-20, the protrusions, concaves, springs, and the hinges are often referred to in pairs, it should be understood that they are for illustrative purpose only and the embodiment of the present disclosure is not limited as such. The housing and/or the holder may comprise one or more protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise one protrusion, concave, spring, hinge, or other similar or equivalent element. In some embodiments, the housing and/or the holder may comprise a total of two protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of three protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of four protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of five protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of six protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of seven protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of eight protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of nine protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of ten protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof. In some embodiments, the housing and/or the holder may comprise a total of more than ten protrusions, concaves, springs, hinges, other similar or equivalent elements, or combinations thereof.
[0258]
The protrusions, concaves, springs, hinges, and other similar or equivalent elements as described herein may be integral to the housing and/or holder, or may be detachably or fixedly connected to the housing and/or holder.
[0259]
In any of the embodiments as described above, a fastening element may be employed to stabilize the housing and the holder in the second position for a time period sufficient for transferring any biological sample retained in the container to the fluid channel. Alternatively, no fastening element may be employed, but the housing and the holder are manually or mechanically stabilized in the second position for a time period sufficient for transferring any biological sample retained in the container to the fluid channel.
[0260]
Although neither the FIG. 19 nor FIG. 20 depicts the separation medium, the separation medium may be in fluid communication with the separation medium in any of the embodiments as described above. For example, FIG. 21A-C are schematic diagrams depicting the relative movement between the holder 1106 and a housing 1107 for the flow channel 1102 which brings about the piercing of the penetratable membrane by the flow channel 1102, wherein the flow channel 1102 is in fluid communication with a separation medium 1103, as described above.
[0261]
It should be understood that the any embodiment described in the aforesaid discussion regarding FIGs. 19-20 may also be implemented with the flow channel 1102 in fluid communication with the separation medium 1103. For example, the embodiment as depicted in FIG. 210B with the holder having at least one concaves and the housing having two protrusions can also be implemented with the flow channel 1102 in fluid communication with the separation medium 1103.
[0262]
Alternatively, the flow channel may not be in fluid communication with the separation medium when the housing or the holder is in the second position. In such circumstances, the flow channel may be brought in fluid communication with the separation medium. The flow channel may be brought in fluid communication with the separation medium by directing the housing to a third position. Therefore, after the housing is directed to a second position as described herein, the housing may be directed to a third position which brings the flow channel in fluid communication with the separation medium. In some embodiments, the flow channel may be brought in fluid communication with the separation medium upon the flow channel piercing an additional penetrable membrane separating the flow channel from the separation medium. In some embodiments, the housing may be directed to the third position which allows the flow channel to pierce an additional penetrable membrane separating the flow channel from the separation medium, thereby bringing the sample vessel in fluid communication with the separation medium via the flow channel.
[0263]
As described above, the additional penetratable membrane may seal the separation medium in the absence of an object inserted through the penetratable membrane.
[0264]
In some embodiments, the additional penetratable membrane may comprise a slit. In some embodiments, the additional penetratable membrane may be resealable. The additional penetratable membrane may be formed of a polymeric material. Any description about the penetratable membrane of the sample vessel may also be applicable to the additional penetratable membrane.
[0265]
In some embodiments, the separation medium may be enclosed an additional housing. The additional housing may have an opening which is sealed by the additional penetratable membrane. The additional penetratable membrane may seal the opening on the additional housing, thereby sealing the separation medium in the absence of an object inserted through the penetratable membrane.
[0266]
When the separation medium is enclosed in the additional housing, any embodiment as depicted in FIGs. 19-20 or discussed above in reference to FIGs. 19-20 may be implemented with the additional housing substituting the holder, the additional penetratable membrane substituting the penetratable membrane, and the separation medium substituting the container, so as to bring the housing to the third position which allows the flow channel to pierce an additional penetrable membrane separating the flow channel from the separation medium, thereby bringing the sample vessel in fluid communication with the separation medium via the flow channel.
[0267]
FIG. 22A-C illustrates an exemplary process in which the housing 1107 is brought to a third position to allow the flow channel 1102 to pierce the additional penetrable membrane 11031, as described above.
[0268]
One or more of the first, second and third positions may be vertical positions. Alternatively, one or more of the first, second and third positions may be any other suitable positions, including horizontal positions, or any position that is at angle to the vertical plane. For example, one or more of the first, second and third positions may be at an angle of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, or 90 degrees, or degrees of any numerical therebetween relative to the vertical plane.
[0269]
In some cases, the housing may further comprise a first covering element. The first covering element may cover an end of the flow channel when said housing is in said first position. The first covering element may allow the end of the flow channel to emerge from the first covering element when the housing or the holder is in the second position. As a result, the end of the flow channel may traverse the penetratable membrane.
[0270]
FIGs. 23A-F illustrate exemplary configurations in which a first covering element covers and seals an end of the flow channel and exemplary processes for using these configurations in accordance with the present disclosure, as described above.
[0271]
In some cases, the housing may further comprise a second covering element. The second covering element may cover another end of the flow channel when (a) the housing is in the first position or (b) the housing or the holder is in the second position. The second covering element may allow the other end of the flow channel to emerge from the second covering element when the housing is in the third position. As a result, the other end of the flow channel pierces an additional penetratable membrane. The additional penetratable membrane may separate the flow channel from the separation medium.
[0272]
FIGs. 24A-F illustrate exemplary configurations in which a second covering element covers and seals another end of the flow channel and exemplary processes for using these configurations in accordance with the present disclosure, as described above.
[0273]
In some cases, the housing may comprise a securing element. The securing element may prevent relative movement between the flow channel and the securing element. The securing element may not cover ends of the flow channel. The securing element may not cover ends of the flow channel. In some embodiments, the securing may be integral to the housing. In some embodiment, the housing itself may prevent relative movement between the flow channel and the securing element. FIG. 25 is a side illustration of a housing 1107 with a first covering element 11077, a second covering element 11078, and a securing element 11079, as described above.
[0274]
The securing element may be separated from either or both of the first covering element and the second covering element by a gap. The gap may allow either or both of the first covering element and the second covering element to move relative to the flow channel, whereas the securing element does not move relative to the flow channel.
[0275]
In the step (b) of the method, the system may further comprise a cannula. The cannula may comprise a hollow tube that can serve as at least a part of the flow channel as described elsewhere herein. In some embodiments, the cannula may comprise at least a portion of the flow channel extending therethrough. In some embodiments, the cannula may accommodate the whole flow channel therewithin.
[0276]
The cannula may pierce the penetratable membrane. The cannula may comprise a piercing element for piercing the penetratable membrane and/or the additional penetratable membrane as described elsewhere herein. The piercing of the penetratable membrane by the cannula may bring the flow channel in fluid communication with the container. Alternatively or additionally, the piercing of the additional penetratable membrane by the cannula may bring the flow channel in fluid communication with the separation medium. The piercing element may be a tip, or any other sharp element that can pierce the penetratable membrane and/or the additional penetratable membrane. In some embodiments, the cannula may include a tip at an end thereof. The tip may pierce the penetratable membrane. The piercing of the penetratable membrane by the tip may bring the flow channel in fluid communication with the container. Alternatively or additionally, the piercing of the penetratable membrane by the tip may bring the flow channel in fluid communication with the separation medium.
[0277]
The piercing of the penetratable membrane and/or the additional penetratable membrane may be performed manually or automatically, such as with a motor or an actuator. In some embodiments, the system may further comprise an actuator. The actuator may pierce the penetratable membrane. The actuator may pierce the additional penetratable membrane. The piercing of the penetratable membrane by the actuator may bring the flow channel in fluid communication with the container. Alternatively or additionally, the piercing of the additional penetratable membrane by the actuator may bring the flow channel in fluid communication with the separation medium.
[0278]
The method may further comprise using a compartment having the separation medium. For example, where the separation medium is an electrophoresis gel, the compartment may accommodate the electrophoresis gel. The compartment may comprise or may be a buffer chamber containing buffer for the electrophoresis.
[0279]
The method may further comprise using a unique identifier on the compartment. The unique identifier may be a barcode. The unique identifier may be a radio-frequency identification tag. The unique identifier may involve contactless technique. The contactless technique allows information to be extracted from the compartment by placing the compartment close to a contactless. The contactless detector may use RFID (radio frequency identification) technique to extract information from the compartment. For example, a user may use a smartphone app that utilizes the RFID technique to communicate with the compartment for identification. In some embodiments, the unique identifier may be a radio-frequency identification tag.
[0280]
The method may further comprise using a detector to detect the biological sample or portion thereof. The detector may detect the biological sample or portion thereof in the separation medium. For example, the system may have a detector that may be capable of detecting signals from one or more lanes of the electrophoresis in real-time. The signal may be an optical signal. The detector may be capable of detecting optical signals from the gels in multiple lanes simultaneously. The detector may capture an image of the top surfaces of the gels in the multiple lanes. The image may be a still image or may include video-rate images. Other details of the detector may be found elsewhere herein.
[0281]
Non-transitory computer-readable medium
[0282]
In a further aspect, the present disclosure provides a non-transitory computer-readable medium. The non-transitory computer-readable medium may comprise machine executable code that, upon execution by one or more computer processors, implements the method of the present disclosure for sample analysis.
[0283]
Computer control system
[0284]
The present disclosure provides computer control systems that are programmed to implement methods of the disclosure. FIG. 26 shows a computer system 2601 that is programmed or otherwise configured to collect, process, and/or analyze a biological sample as described herein. The computer system 2601 can regulate various aspects of the system and/or the method of the present disclosure, such as, for example, collection, processing, and/or analysis of a biological sample. The computer system 2601 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.
[0285]
The computer system 2601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 2605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 2601 also includes memory or memory location 2610 (e.g., random-access memory, read-only memory, flash memory) , electronic storage unit 2615 (e.g., hard disk) , communication interface 2620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 2625, such as cache, other memory, data storage and/or electronic display adapters. The memory 2610, storage unit 2615, interface 2620 and peripheral devices 2625 are in communication with the CPU 2605 through a communication bus (solid lines) , such as a motherboard. The storage unit 2615 can be a data storage unit (or data repository) for storing data. The computer system 2601 can be operatively coupled to a computer network ( “network” ) 2630 with the aid of the communication interface 2620. The network 2630 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 2630 in some cases is a telecommunication and/or data network. The network 2630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 2630, in some cases with the aid of the computer system 2601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 2601 to behave as a client or a server.
[0286]
The CPU 2605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 2610. The instructions can be directed to the CPU 2605, which can subsequently program or otherwise configure the CPU 2605 to implement methods of the present disclosure. Examples of operations performed by the CPU 2605 can include fetch, decode, execute, and writeback.
[0287]
The CPU 2605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 2601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC) .
[0288]
The storage unit 2615 can store files, such as drivers, libraries and saved programs. The storage unit 2615 can store user data, e.g., user preferences and user programs. The computer system 2601 in some cases can include one or more additional data storage units that are external to the computer system 2601, such as located on a remote server that is in communication with the computer system 2601 through an intranet or the Internet.
[0289]
The computer system 2601 can communicate with one or more remote computer systems through the network 2630. For instance, the computer system 2601 can communicate with a remote computer system of a user (e.g., a subject, or a healthcare professional) . Examples of remote computer systems include personal computers (e.g., portable PC) , slate or tablet PC’s (e.g., iPad, Galaxy Tab) , telephones, Smart phones (e.g., iPhone, Android-enabled device, ) , or personal digital assistants. The user can access the computer system 2601 via the network 2630.
[0290]
Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 2601, such as, for example, on the memory 2610 or electronic storage unit 2615. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 2605. In some cases, the code can be retrieved from the storage unit 2615 and stored on the memory 2610 for ready access by the processor 2605. In some situations, the electronic storage unit 2615 can be precluded, and machine-executable instructions are stored on memory 2610.
[0291]
The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
[0292]
Aspects of the systems and methods provided herein, such as the computer system 2601, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.
[0293]
Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer (s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
[0294]
The computer system 2601 can include or be in communication with an electronic display 2635 that comprises a user interface (UI) 2640 for providing, for example, instructions and options as to the collection, processing, and/or analysis of a biological sample. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.
[0295]
Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 2605. The algorithm can, for example, implement the methods of the present disclosure for sample analysis.
[0296]
Container
[0297]
Any suitable reaction vessel may be used in the system or method of the present disclosure. In some embodiments, a container comprises a body that can include an interior surface, an exterior surface, an open end, and an opposing closed end. In some embodiments, a container can comprise a cap. The cap can be configured to contact the body at its open end, such that when contact is made the open end of the container is closed. In some cases, the cap is permanently associated with the container such that it remains attached to the container in open and closed configurations. In some cases, the cap is removable, such that when the container is open, the cap is separated from the container. In some embodiments, a container can be sealed, in some cases hermetically sealed. The container can be fluid-tight.
[0298]
A container can be of varied size, shape, weight, and configuration. In some examples, a container can be round or oval tubular shaped. In some embodiments, a container can be rectangular, square, diamond, circular, elliptical, or triangular shaped. A container can be regularly shaped or irregularly shaped. In some embodiments, the closed end of a container can have a tapered, rounded, or flat surface. For example, a flat cap, rounded, cap, or tapered cap can be provided. Non-limiting examples of types of a container include a tube, a well, a capillary tube, a cartridge, a cuvette, a centrifuge tube, or a pipette tip.
[0299]
Any dimensions can be provided for a container. The container can be configured to contain at least about 0.2 microliters (mL) or 0.5 mL of sample. The container can be configured to contain at least about 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 90 mL, 95 mL, 100 mL, 110 mL, 120 mL, 120 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL. The container can be configured to contain at most about 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 90 mL, 95 mL, 100 mL, 110 mL, 120 mL, 120 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL. The container can be configured to contain about 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 30 mL, 35 mL, 40 mL, 45 mL, 50 mL, 55 mL, 60 mL, 65 mL, 70 mL, 75 mL, 80 mL, 90 mL, 95 mL, 100 mL, 110 mL, 120 mL, 120 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL. The container can have a volume configured to contain no more than a volume falling into a range between two of the values described herein. The container can have a volume from about 20 mL to about 200 mL. The container can have a volume from about 50 mL to about 200 mL. The container can have a volume from about 100 mL to about 200 mL.
[0300]
The container can have a height of at least about 0.25 centimeters (cm) , 0.5 cm, 0.75 cm, 1 cm, 1.25 cm, 1.5 cm, 1.75 cm, 2 cm, 2.25 cm, 2.5 cm, 2.75 cm, 3 cm, 3.25 cm, 3.5 cm, 3.75 cm, 4 cm, 4.25 cm, 4.5 cm, 4.75 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. The container can have a height of at most about 0.25 centimeters (cm) , 0.5 cm, 0.75 cm, 1 cm, 1.25 cm, 1.5 cm, 1.75 cm, 2 cm, 2.25 cm, 2.5 cm, 2.75 cm, 3 cm, 3.25 cm, 3.5 cm, 3.75 cm, 4 cm, 4.25 cm, 4.5 cm, 4.75 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. The container can have a height of about 0.25 centimeters (cm) , 0.5 cm, 0.75 cm, 1 cm, 1.25 cm, 1.5 cm, 1.75 cm, 2 cm, 2.25 cm, 2.5 cm, 2.75 cm, 3 cm, 3.25 cm, 3.5 cm, 3.75 cm, 4 cm, 4.25 cm, 4.5 cm, 4.75 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm. The container can have a height greater than any of the values described herein. The container can have a height falling into a range between any two of the values described herein.
[0301]
The container can have a cross-sectional area of at least about 0.25 square centimeters (cm 2) , 0.5 cm 2, 0.75 cm 2, 1 cm 2, 1.25 cm 2, 1.5 cm 2, 1.75 cm 2, 2 cm 2, 2.25 cm 2, 2.5 cm 2, 2.75 cm 2, 3 cm 2, 3.25 cm 2, 3.5 cm 2, 3.75 cm 2, 4 cm 2, 4.25 cm 2, 4.5 cm 2, 4.75 cm 2, or 5 cm 2. The container can have a cross-sectional area of at most about 0.25 square centimeters (cm 2) , 0.5 cm 2, 0.75 cm 2, 1 cm 2, 1.25 cm 2, 1.5 cm 2, 1.75 cm 2, 2 cm 2, 2.25 cm 2, 2.5 cm 2, 2.75 cm 2, 3 cm 2, 3.25 cm 2, 3.5 cm 2, 3.75 cm 2, 4 cm 2, 4.25 cm 2, 4.5 cm 2, 4.75 cm 2, or 5 cm 2. The container can have a cross-sectional area of about 0.25 square centimeters (cm 2) , 0.5 cm 2, 0.75 cm 2, 1 cm 2, 1.25 cm 2, 1.5 cm 2, 1.75 cm 2, 2 cm 2, 2.25 cm 2, 2.5 cm 2, 2.75 cm 2, 3 cm 2, 3.25 cm 2, 3.5 cm 2, 3.75 cm 2, 4 cm 2, 4.25 cm 2, 4.5 cm 2, 4.75 cm 2, or 5 cm 2. The container can have a cross-sectional area less than any of the values described herein. The container can have a cross-sectional area falling into a range between any two of the values described herein.
[0302]
Containers can be constructed of any suitable material with non-limiting examples of such materials that include glasses, metals, plastics, and combinations thereof. Containers can be made from optically transparent or translucent materials that can permit an optical signal from within the container to leave the container. The containers can be made from a material that may or may not filter an optical signal exiting the container. In some instances, the containers can be formed from a clear material that can permit a detector to view the interior of the containers. In some instances, the interior of the containers can be imaged. Alternatively, an amount of optical signal exiting the container can be detected and measured.
[0303]
Samples
[0304]
The systems and methods of the present disclosure involve analysis of one or more samples. A sample can comprise a target nucleic acid. A sample can comprise an agent that detects amplified target nucleic acid (e.g., a detectable nucleic acid binding agent) . A sample can comprise reagents for conducting nucleic acid amplification. Depending on the nature of the target nucleic acid that is to be amplified, reagents can comprise reverse transcriptase for conducting reverse-transcriptase coupled PCT, dNTPs, or Mg 2+ ions. The sample may comprise products of nucleic acid amplification. The sample may comprise amplified nucleic acids.
[0305]
The sample can be a biological sample. The biological sample can be taken from a subject. For example, the sample can be taken from a living subject directly. In some embodiments, the biological sample can include breath, blood, urine, feces, saliva, cerebrospinal fluid and sweat. Any suitable biological sample that comprises nucleic acid can be obtained from a subject. A biological sample can be solid matter (e.g., biological tissue) or can be a fluid (e.g., a biological fluid) . In general, a biological fluid can include any fluid associated with living organisms. Non-limiting examples of a biological sample include blood (or components of blood –e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, cavity fluids, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cord blood, emphatic fluids, and/or other excretions or body tissues.
[0306]
A subject can be a living subject or a dead subject. The subject can be a human or an animal. In some examples, the subject can be mammal. Examples of subjects can include, but are not limited to simians, avians, canines, felines, equines, bovines, ovines, porcines, delphines, rodents (e.g., mice, rats) , or insects.
[0307]
A biological sample can be obtained from a subject by various approaches. Non-limiting examples of such approaches to obtain a biological sample directly from a subject include accessing the circulatory system (e.g., intravenously or intra-arterially via a syringe or other needle) , collecting a secreted biological sample (e.g., feces, urine, sputum, saliva, etc. ) , surgically (e.g., biopsy) , swabbing (e.g., buccal swab, oropharyngeal swab) , pipetting, and breathing. Moreover, a biological sample can be obtained from any anatomical part of a subject where a desired biological sample is located.
[0308]
A biological sample obtained directly from a subject can generally refer to a biological sample that has not been further processed after being obtained from the subject, with the exception of any approach used to collect the biological sample from the subject for further processing. For example, blood is obtained directly from a subject by accessing the subject’s circulatory system, removing the blood from the subject (e.g., via a needle) , and entering the removed blood into a receptacle. The receptacle can comprise reagents (e.g., anti-coagulants) such that the blood sample is useful for further analysis. In another example, a swab can be used to access epithelial cells on an oropharyngeal surface of the subject. After obtaining the biological sample from the subject, the swab containing the biological sample can be contacted with a fluid (e.g., a buffer) to collect the biological fluid from the swab. Alternatively, pre-processing can occur on the biological sample prior to being provided to the device.
[0309]
In some embodiments, a biological sample has not been purified when provided in a reaction vessel. In some embodiments, the nucleic acid of a biological sample has not been extracted when the biological sample is provided to a reaction vessel. For example, the RNA or DNA in a biological sample may not be extracted from the biological sample when providing the biological sample to a reaction vessel. Moreover, in some embodiments, a target nucleic acid (e.g., a target RNA or target DNA) present in a biological sample may not be concentrated prior to providing the biological sample to a reaction vessel. Alternatively, dilution or concentration of the sample can occur prior to being provided to a device.
[0310]
The sample can have a target nucleic acid to be amplified. The target nucleic acid can be amplified to generate an amplified product. A target nucleic acid can be a target RNA or a target DNA. In cases where the target nucleic acid is a target RNA, the target RNA can be any type of RNA. In some embodiments, the target RNA is viral RNA. In some embodiments, the viral RNA can be pathogenic to the subject. Non-limiting examples of pathogenic viral RNA include human immunodeficiency virus I (HIV I) , human immunodeficiency virus II (HIV II) , orthomyxoviruses, influenza viruses (e.g., H1N1, H3N2, H5N1, H7N9) , hepevirus, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, Epstein-Barr virus, mononucleosis, cytomegalovirus, SARS, West Nile Fever, Ebola virus, polio, and measles.
[0311]
In cases where the target nucleic acid is a target DNA, the target DNA can be any type of DNA. In some embodiments, the target DNA is viral DNA. In some embodiments, the viral DNA can be pathogenic to the subject. Non-limiting examples of DNA viruses include herpes simplex virus, smallpox, and chickenpox. In some cases, a target DNA can be a parasite DNA, such as malaria parasite or plasmodium. In some cases, a target DNA can be a bacterial DNA. The bacterial DNA can be from a bacterium pathogenic to the subject such as, for example, Mycobacterium tuberculosis –a bacterium known to cause tuberculosis.
[0312]
The sample can also include an agent that detects amplified target nucleic acid. The agent can be a reporter agent that can yield a detectable signal whose presence or absence is indicative of the presence of an amplified product. The intensity of the detectable signal can be proportional to the amount of amplified product. For example, the detectable signal can be directly linearly proportional, exponentially proportional, reversely proportional, or have any other type of proportional relationship to the amount of amplified product. In some cases, where amplified product is generated of a different type of nucleic acid than the target nucleic acid initially amplified, the intensity of the detectable signal can be proportional to the amount of target nucleic acid initially amplified. For example, in the case of amplifying a target RNA via parallel reverse transcription and amplification of the DNA obtained from reverse transcription, reagents necessary for both reactions can also comprise a reporter agent can yield a detectable signal that is indicative of the presence of the amplified DNA product and/or the target RNA amplified. The intensity of the detectable signal can be proportional to the amount of the amplified DNA product and/or the original target RNA amplified. The use of a reporter agent also enables real-time amplification methods, including real-time PCR for DNA amplification.
[0313]
Reporter agents can be linked with nucleic acids, including amplified products, by covalent or non-covalent linkages. Non-limiting examples of non-covalent linkages include ionic interactions, Van der Waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof. In some embodiments, reporter agents can bind to initial reactants and changes in reporter agent levels can be used to detect amplified product. In some embodiments, reporter agents can only be detectable (or non-detectable) as nucleic acid amplification progresses. In some embodiments, an optically-active dye (e.g., a fluorescent dye) can be used as can be used as a reporter agent. An agent for detecting amplified target nucleic acid can be a nucleic acid binding dye. The dye can be a DNA-intercalating dye. Non-limiting examples of dyes include Eva green, SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, phenanthridines and acridines, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine, SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue) , SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green) , SYTO-81, -80, -82, -83, -84, -85 (orange) , SYTO-64, -17, -59, -61, -62, -60, -63 (red) , fluorescein, fluorescein isothiocyanate (FITC) , tetramethyl rhodamine isothiocyanate (TRITC) , rhodamine, tetramethyl rhodamine, R-phycoerythrin, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC) , Sybr Green I, Sybr Green II, Sybr Gold, CellTracker Green, Eva Green, 7-AAD, ethidium homodimer I, ethidium homodimer II, ethidium homodimer III, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, cascade blue, dichlorotriazinylamine fluorescein, dansyl chloride, fluorescent lanthanide complexes such as those including europium and terbium, carboxy tetrachloro fluorescein, 5 and/or 6-carboxy fluorescein (FAM) , 5- (or 6-) iodoacetamidofluorescein, 5- { [2 (and 3) -5- (Acetylmercapto) -succinyl] amino} fluorescein (SAMSA-fluorescein) , lissamine rhodamine B sulfonyl chloride, 5 and/or 6 carboxy rhodamine (ROX) , 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA) , BODIPY fluorophores, 8-methoxypyrene-1, 3, 6-trisulfonic acid trisodium salt, 3, 6-Disulfonate-4-amino-naphthalimide, phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 755, and 800 dyes, or other fluorophores.
[0314]
In some instances, a reporter agent can be a sequence-specific oligonucleotide probe that can be optically active when hybridized with an amplified product. Due to sequence-specific binding of the probe to the amplified product, use of oligonucleotide probes can increase specificity and sensitivity of detection. A probe can be linked to any of the optically-active reporter agents (e.g., dyes) described herein and can also include a quencher capable of blocking the optical activity of an associated dye. Non-limiting examples of probes that can be useful used as reporter agents include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.
[0315]
A reporter agent can be an RNA oligonucleotide probe that can include an optically-active dye (e.g., fluorescent dye) and a quencher positioned adjacently on the probe. The close proximity of the dye with the quencher can block the optical activity of the dye. The probe can bind to a target sequence to be amplified. Upon the breakdown of the probe with the exonuclease activity of a DNA polymerase during amplification, the quencher and dye are separated, and the free dye regains its optical activity that can subsequently be detected.
[0316]
A reporter agent may be a molecular beacon. A molecular beacon can include, for example, a quencher linked at one end of an oligonucleotide in a hairpin conformation. At the other end of the oligonucleotide is an optically active dye, such as, for example, a fluorescent dye. In the hairpin configuration, the optically-active dye and quencher are brought in close enough proximity such that the quencher is capable of blocking the optical activity of the dye. Upon hybridizing with amplified product, however, the oligonucleotide assumes a linear conformation and hybridizes with a target sequence on the amplified product. Linearization of the oligonucleotide results in separation of the optically-active dye and quencher, such that the optical activity is restored and can be detected. The sequence specificity of the molecular beacon for a target sequence on the amplified product can improve specificity and sensitivity of detection.
[0317]
In some embodiments, a reporter agent can be a radioactive species. Non-limiting examples of radioactive species include 14C, 123I, 124I, 125I, 131I, Tc99m, 35S, or 3H.
[0318]
In some embodiments, a reporter agent can be an enzyme that is capable of generating a detectable signal. Detectable signal can be produced by activity of the enzyme with its substrate or a particular substrate in the case the enzyme has multiple substrates. Non-limiting examples of enzymes that can be used as reporter agents include alkaline phosphatase, horseradish peroxidase, I 2-galactosidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, and luciferase.
[0319]
The sample can be provided with reagents necessary for nucleic acid amplification within the device. In some instances, a reagent can comprise one or more of the following: (i) a reverse transcriptase, (ii) a DNA polymerase, and (iii) a primer set for the target nucleic acid (e.g., RNA) . Some examples of reagents can include a commercially available pre-mixture (e.g., Qiagen One-Step RT-PCR or One-Step RT-qPCR kit) comprising reverse transcriptases (e.g., Sensiscript and Omniscript transcriptases) , a DNA Polymerase (e.g., HotStarTaq DNA Polymerase) , and dNTPs.
[0320]
Nucleic acid amplification
[0321]
The practice of any of the subject methods typically may involve conducting a nucleic acid amplification assay. In any of the various aspects, a nucleic acid amplification reaction can involve any of a variety of methods for nucleic acid amplification. In general, “amplification” refers to any process by which the copy number of a target sequence is increased. A variety of suitable nucleic acid amplification reactions are available. Non-limiting examples of nuclei acid amplification reactions include polymerase chain reaction, real-time polymerase chain reaction, isothermal amplification, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, solid phase amplification, nucleic acid sequence-based amplification (NASBA) , linear amplification, and digital PCR reaction.
[0322]
The polymerase chain reaction (PCR) uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase copy numbers of the target sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from RNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA (see e.g. U.S. Pat. Nos. 5,322,770 and 5,310,652, each of which is entirely incorporated herein by reference) .
[0323]
In some embodiments, the nucleic acid amplification reaction is a PCR reaction. Conditions favorable to the amplification of target sequences by PCR can be determined by methods known in the art, can be optimized at a variety of steps in the process, and depend on characteristics of elements in the reaction, such as target type, target concentration, sequence length to be amplified, sequence of the target and/or one or more primers, primer length, primer concentration, polymerase used, reaction volume, ratio of one or more elements to one or more other elements, and others, some or all of which can be altered. In general, PCR involves the steps of denaturation of the target to be amplified (if double stranded) , hybridization of one or more primers to the target, and extension of the primers by a DNA polymerase, with the steps repeated (or “cycled” ) in order to amplify the target sequence. Steps in this process can be optimized for various outcomes, such as to enhance yield, decrease the formation of spurious products, and/or increase or decrease specificity of primer annealing. Methods of optimization are known in the art and include adjustments to the type or amount of elements in the amplification reaction and/or to the conditions of a given step in the process, such as temperature at a particular step, duration of a particular step, and/or number of cycles. In some embodiments, an amplification reaction comprises at least 5, 10, 15, 20, 25, 30, 35, 50, or more cycles. In some embodiments, an amplification reaction comprises no more than 5, 10, 15, 20, 25, 35, 50, or more cycles. Cycles can contain any number of steps, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more steps, and cycled steps may be preceded and/or followed by one or more steps not included in those steps that are cycled (e.g. an initial melting step or a final incubation step) . Steps can comprise any temperature or gradient of temperatures, suitable for achieving the purpose of the given step, including but not limited to, primer annealing, primer extension, and strand denaturation. Steps can be of any duration, including but not limited to about, less than about, or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 180, 240, 300, 360, 420, 480, 540, 600, or more seconds, including indefinitely until manually interrupted. Cycles of any number comprising different steps can be combined in any order. In some embodiments, different cycles comprising different steps are combined such that the total number of cycles in the combination is about, less that about, or more than about 5, 10, 15, 20, 25, 30, 35, 50, or more cycles.
[0324]
In some embodiments of any of the various aspects, the nucleic acid amplification reaction comprises 3’ -end extension of one or more primers, e.g. about, more than about, or less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primers. In some embodiments, primer extension in the nucleic acid amplification reaction involves only one pair of primers. In other embodiments, primer extension in the nucleic acid amplification reaction involves multiple pairs of primers, such as 2, 3, 4, 5, or more primer pairs. In some embodiments, a pair of primers consists of a first primer and a second primer, wherein the first primer comprises a sequence that is hybridizable to at least a portion of one or more target polynucleotides, and further wherein the second primer comprises a sequence that is hybridizable to at least a portion of the complement of a first primer extension product. When the target polynucleotide is double-stranded, the sequence of the second primer that is hybridizable to at least a portion of the complement of a first primer extension product may also be hybridizable to at least a portion of the complementary strand of the target polynucleotide. When an amplification reaction contains a plurality of primer pairs, the plurality of primer pairs may be distinct (as in two different primers for each pair) , overlapping (such as one forward primer paired with two or more different reverse primers) , or combinations of distinct pairs and overlapping pairs. An amplification primer can be of any suitable length, such as about, less than about, or more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or more nucleotides, any portion or all of which may be complementary to the corresponding target sequence (e.g. about, less than about, or more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides) . Typically, when a primer comprises a complementary portion and a non-complementary portion, the portion that is complementary to a target sequence is located at the 3’ -end of the primer. Primer pairs can be designed to amplify a target sequence of any desired length. As used herein, “amplicon” refers to the target sequence that is amplified from the target polynucleotide in the nucleic acid amplification reaction, in single-or double-stranded form. When an amplicon is amplified by a pair of primers, the amplicon is generally flanked by the pair of primers, such that one primer hybridizes at the 5’ end of the target sequence and the other primer hybridizes to the complement of the 3’ end of the target sequence. In some embodiments, the amplicon is about, or less than about 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 25, or fewer nucleotides in length. In some embodiments, the amplicon is about, or more than about, 50, 100, 200, 300, 400, 500, 750, 1000, or more nucleotides in length. In some embodiments, amplicon length is between any two of these endpoints, such as 25-1000, 30-500, 50-400, 50-250, 50-150, or 100-200 nucleotides in length. Primers may be selected based on conformance to any of a variety of design considerations, which may be used alone or in combination with any other design consideration disclosed herein or known in the art. Additional non-limiting examples of optional design considerations for primers include: avoiding runs of the same nucleotide (e.g. 3, 4, 5, or more of the same nucleotide in a row) ; proximity to the probe without overlapping probe hybridization site (e.g. about or less than about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 30, 40, 50, 75, 100, or more nucleotides between the 3’ end of a primer and the 5’ end of a probe along the same strand) ; G-C content within about 20%-80%, melting temperature (T m) within a selected range (e.g. about 55-65℃, 58-62℃, or 58-60℃) ; having no more than two G and/or C bases within the last five nucleotides at the 3’ end; primers in a pair having similar T m (e.g. the same T m, or T m’s within about 1-2℃ of each other) ; minimal secondary structure (e.g. about or fewer than about 5, 4, 3, 2, or 1 Watson-Crick paired bases when optimally folded, such as by analysis with mFold (see e.g. Zuker et al., Nucl. Acid Res, 2003, 31: 3406-3415) ) ; minimal hybridization between primers in a reaction as homodimers or heterodimers (e.g. about or fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 Watson-Crick paired bases when optimally aligned) ; and minimal hybridization between a primer and corresponding probe (e.g. about or fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 Watson-Crick paired bases when optimally aligned) . In some embodiments, primers specifically amplify amplicons that are about or at least about 25, 50, 75, 100, 125, 150, or 175 nucleotides in length, and have about or at least about 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, or more sequence identity with a sequence in Table 1 (or a complement thereof) when optimally aligned. Methods and algorithms for determining optimal sequence alignment are known in the art, any of which may be used to determine percent sequence identity. One example of an algorithm for determining sequence identity between two sequences includes the Basic Local Alignment Search Tool (BLAST) , as maintained by the National Center for Biotechnology Information at blast. ncbi. nlm. nih. gov.
[0325]
Primer extension in a nucleic acid amplification reaction can be carried out by any suitable polymerase known in the art, such as a DNA polymerase, many of which are commercially available. DNA polymerases can comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity, or DNA-dependent and RNA-dependent DNA polymerase activity. DNA polymerases can be thermostable or non-thermostable. Examples of DNA polymerases include, but are not limited to, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Klenow fragment, and variants, modified products and derivatives thereof. In some embodiments, enzymes produced using bacteria are highly purified, such that a no-template control amplification reaction does not produce an amplification signal above background levels after about, or more than about 25, 30, 35, 40, 45, or more cycles of a PCR reaction.
[0326]
In some embodiments of any of the various aspects, nucleic acid amplification products are detected during and/or at the completion of the amplification process. Amplification product detection can be conducted in real time in an amplification assay. In some embodiments, the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems. Non-limiting examples of DNA-binding dyes include green, SYBR blue, DAPI, propidium iodine, Hoechst, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like.
[0327]
In some embodiments, sequence specific oligonucleotide probes are employed in the nucleic acid amplification reaction to facilitate the detection and/or quantification of the amplified products. Probe-based quantitative amplification relies on the sequence-specific detection of a desired amplified product, such as by specific hybridization between a probe and a target sequence within an amplification product. Examples of target-specific probes include, without limitation, probes and molecular beacons. Generic methods for performing probe-based quantitative amplification are known in the art (see e.g. U.S. Pat. No. 5,210,015, which is entirely incorporated herein by reference) . Hybridization can be performed under various stringencies. Suitable hybridization conditions are generally such that the recognition interaction between the probe and target polynucleotide is both sufficiently specific and sufficiently stable as to provide preferential hybridization between an oligonucleotide probe and/or primer and the intended target sequence. Conditions that increase the stringency of a hybridization reaction are known in the art, and include optimization of annealing temperature and/or salt concentration. An oligonucleotide probe can be of any suitable length, such as about, less than about, or more than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or more nucleotides, any portion or all of which may be complementary to the corresponding target sequence (e.g. about, less than about, or more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides) . In some embodiments, a plurality of probes are used in a single nucleic acid amplification reaction, such as about or less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more probes. In some embodiments, a single nucleic acid amplification reaction contains only two probes, such as one that specifically hybridizes to a sequence from one or more Gram-positive bacteria (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) and a second that specifically hybridizes to a sequence from one or more Gram-negative bacteria (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) . In some embodiments, a single nucleic acid amplification reaction contains only one probe, such as a probe that specifically hybridizes to a sequence that is identical among a plurality of different bacterial species (e.g. about or more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more species) and/or identical among bacteria from a plurality of different genera (e.g. about or more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more genera) . Probes may be selected based on conformance to any of a variety of design considerations, which may be used alone or in combination with any other design consideration disclosed herein or known in the art. Additional non-limiting examples of optional design considerations for probes include: avoiding runs of the same nucleotide (e.g. 3, 4, 5, or more of the same nucleotide in a row) ; proximity to an amplification primer hybridization site without overlapping (e.g. about or less than about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 30, 40, 50, 75, 100, or more nucleotides between the 3’ end of a primer and the 5’ end of a probe along the same strand) ; G-C content within about 20%-80%, melting temperature (T m) within a selected range (e.g. about 8-10℃ higher than a corresponding primer T m) ; and having more C’s than G’s ; no G on the 5’ end. In some embodiments, a probe specifically hybridizes to amplicons that are about or at least about 25, 50, 75, 100, 125, 150, or 175 nucleotides in length, and have about or at least about 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, or more sequence identity with a sequence in Table 1 when optimally aligned. Methods and algorithms for determining optimal sequence alignment are known in the art, any of which may be used to determine percent sequence identity. One example of an algorithm for determining sequence identity between two sequences includes the Basic Local Alignment Search Tool (BLAST) , as maintained by the National Center for Biotechnology Information at blast. ncbi. nlm. nih. gov.
[0328]
For a convenient detection of the probe-target complexes formed during the hybridization assay, the nucleotide probes can be conjugated to a detectable label. Suitable detectable labels can include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical, or chemical approaches. A wide variety of appropriate detectable labels are known in the art, which include fluorescent labels, chemiluminescent labels, radioactive isotope labels, enzymatic labels, and ligands. The detection methods used to detect or quantify the hybridization intensity will typically depend upon the label selected above. For example, radiolabels may be detected using photographic film or a phosphoimager. Fluorescent markers may be detected and quantified using a photodetector to detect emitted light. In some embodiments, each of a plurality of probes in a single reaction is conjugated to a different detectable label (e.g. fluorescent dyes with different emission spectra) , such that signal corresponding to amplification of different targets can be differentiated. Enzymatic labels are typically detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and finally colorimetric labels are detected by simply visualizing the colored label.
[0329]
In some embodiments, hybridization of a bound probe is detected using a TaqMan assay (PE Biosystems, Foster City, Calif. ; See e.g., U.S. Pat. Nos. 5,962,233 and 5, 538, 848, each of which is herein incorporated by reference) . The assay is performed during a PCR reaction. The TaqMan assay exploits the 5’ -3’ exonuclease activity of DNA polymerases such as AMPLITAQ DNA polymerase. A sequence-specific probe is included in the PCR reaction. A typical TaqMan probe is an oligonucleotide with a 5’ -reporter dye (e.g., a fluorescent dye) and a 3’ -quencher dye. During PCR, if the probe is bound to its target, the 5’ -3’ nucleolytic activity of the AMPLITAQ polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter. A variety of reporter-quencher pairs are known in the art. Some pairs interact through fluorescence resonance energy transfer (FRET) . Molecules commonly used in FRET as reporters or quenchers include, but are not limited to, fluorescein dyes (e.g., FAM, JOE, and HEX) , rhodamine dyes (e.g., R6G, TAMRA, ROX) , cyanine dyes (e.g., Cy3, Cy3.5, Cy5, Cy5.5, and Cy7) , DABCYL, and EDANS. Whether a fluorescent dye acts as a reporter or a quencher is defined by its excitation and emission spectra, and by the fluorescent dye with which it is paired. For example, FAM is most efficiently excited by light with a wavelength of 488 nm, and emits light with a spectrum of 500 to 650 nm, and an emission maximum of 525 nm. FAM is a suitable reporter label for use with, e.g., TAMRA as a quencher, which has its excitation maximum at 514 nm. Examples of non-fluorescent or dark quenchers that dissipate energy absorbed from a fluorescent dye include the Black Hole Quenchers TM marketed by Biosearch Technologies, Inc, (Novato, Calif., USA) . The Black Hole Quenchers TM are structures comprising at least three radicals selected from substituted or unsubstituted aryl or heteroaryl compounds, or combinations thereof, wherein at least two of the residues are linked via an exocyclic diazo bond (see, e.g., International Publication No. WO2001086001) . Other dark quenchers include Iowa Black quenchers (e.g., Iowa Black FQ TM and Iowa Black RQ TM) , Dark Quenchers (Epoch Biosciences, Inc, Bothell, Wash. ) , and Zen TM quenchers (Integrated DNA Technologies, Inc. ; Coralville, IA) . Additional non-limiting examples of quenchers are also provided in U.S. Pat. No. 6,465,175, which is entirely incorporated herein by reference.
[0330]
In some embodiments, hybridization of a bound probe is detected using a molecular beacon oligonucleotide probe, such as described in U.S. Pat. No. 5,925,517, PCT Application No. WO1995013399, and U.S. Pat. No. 6,150,097, each of which is entirely incorporated herein by reference. In a typical molecular beacon, a central target-recognition sequence is flanked by arms that hybridize to one another when the probe is not hybridized to a target strand, forming a hairpin structure, in which the target-recognition sequence is in the single-stranded loop of the hairpin structure, and the arm sequences form a double-stranded stem hybrid. When the probe hybridizes to a target, a relatively rigid helix is formed, causing the stem hybrid to unwind and forcing the arms apart. A FRET pair, such as the fluorophore EDANS and the quencher DABCYL (or other pairs described herein or known in the art) , may be attached to the arms by alkyl spacers. When the molecular beacon is not hybridized to a target strand, the fluorophore's emission is quenched. When the Molecular Beacon is hybridized to a target strand, the FRET pair is separated, and the fluorophore's emission is not quenched. Emitted fluorescence signals the presence of target strands. Signal can be detected during a nucleic acid amplification reaction, such as with a fluorimeter at the end of each cycle in a PCR reaction. Signal intensity increases with an increasing amount of target sequence.
[0331]
As disclosed in Whitcombe et al., Detection Of PCR Products Using Self-probing Amplicons and Fluorescence, Nature Biotechnology 17: 804-807 (August 1999) , detection of PCR products may be accomplished with self-probing amplicons. A Scorpion Primer carries a 5’ extension comprising a probe element, a pair of self-complimentary stem sequences, and a fluorophore/quencher pair. The extension is “protected” from being copied by the inclusion of a blocking hexethylene glycol (HEG) monomer. After a round of PCR extension from a primer, a newly synthesized target region is now attached to the same strand as the probe. Following a second round of denaturation and annealing, the probe and target hybridize, the probe subsequently fluorescing. Accordingly, a “probe” as described herein, may be present as a portion of a primer.
[0332]
In some embodiments of any of the various aspects, primers and probes are selected to maximize sensitivity of target polynucleotide detection. In some embodiments, sensitivity is measured in terms of cycle threshold (C T) value. In the initial cycles of PCR, there is little change in fluorescence signal. This defines the baseline for an amplification plot (aplot of fluorescence intensity over cycle number) . An increase in fluorescence above the baseline indicates the detection of accumulated PCR product. A fixed fluorescence threshold can be set above the baseline. The parameter C T is defined as the fractional cycle number at which the fluorescence passes the fixed threshold, typically an intensity that is statistically significant above the baseline or background and in the log-linear phase of amplification. Software for calculating the threshold level of fluorescence in a given reaction or set of reactions is typically included in real-time PCR analysis software packages. One common method for setting the threshold is determining the baseline (background) average signal and setting a threshold 10-fold higher than the baseline average signal. Alternatively, a threshold value may be set at about 10 times the standard deviation of baseline emission. A plot of the log of initial target copy number for a set of standards versus C T is typically a straight line. Quantification of the amount of target in unknown samples is accomplished by measuring C T and using the standard curve to determine starting copy number. In some embodiments, detection has a linear range of detection over about or more than about 3, 4, 5, 6, 7, 8, or more logs. In some embodiments, amplification of about or less than about 10pg, 5pg, 4pg, 3pg, 2pg, 1pg, 0.5pg, 0.1pg, or range between any of these (e.g. 0.5-4pg, 1pg-5pg, 1pg-3pg, etc) of genomic DNA from any one of the bacterial species detectable by a probe in the amplification reaction has a C T of less than 30. In some embodiments, amplification of about or less than about 15000, 10000, 5000, 2500, 1500, 1000, 500, 200, 100, 50, or fewer starting copies of a target sequence detectable by a probe in the amplification reaction has a C T of less than 30. In some embodiments, amplification of about 1pg of genomic DNA from any one of the bacterial species detectable by a probe in the amplification reaction has a C T of about or less than about 30, 29, 28, 27, 26, 25, or lower. In some embodiments, the C T for a negative control sample is at least 2 cycles (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cycles) higher than the C T for a sample containing about 100pg, 10pg, 5pg, 4pg, 3pg, 2pg, 1pg, 0.5pg, 0.1pg, or range between any of these (e.g. 0.5-4pg, 1pg-5pg, 1pg-3pg, 5pg-10pg, etc) of genomic DNA from any one of the bacterial species detectable by a probe in the amplification reaction. Typically, a negative control is an amplification reaction that has all reaction reagents, but no template is added (e.g. add water instead of template, or polynucleotides known to lack a target amplicon, such as human genomic DNA in the case of a bacteria-specific amplicon) . In some embodiments, bacterial contamination is detected in nucleic acid from about or less than about 25mL, 20mL, 15mL, 10mL, 5mL, 4mL, 3mL, 2mL, 1mL, 0.1mL or less of a platelet concentrate. In some embodiments, amplification is performed on a platelet sample or portion thereof without first incubating the sample to promote bacterial growth, such as at a temperature above about 30℃, 35℃, or 37℃. In some embodiments, detection yields a detectable signal indicative of bacterial contamination of a platelet sample having a bacterial load of about or less than about 50, 25, 10, 5, 4, 3, 2, 1, 0.1 or fewer colony forming units per mL (CFU/mL) in a platelet sample. In some embodiments, detection yields a detectable signal indicative of bacterial contamination of a platelet sample when nucleic acids derived from fewer than 50000, 40000, 30000, 25000, 20000, 15000, 10000, 7500, 5000, 2500, 1250, 1000, 750, 500, 250, 100, 50, 25, 10, 5, or fewer CFU are present in the detection reaction. In some embodiments, a detectable signal is obtained for a reaction containing nucleic acids derived from 5 to 50000 CFU, 500 to 25000 CFU, 1000 to 10000 CFU, or 25 to 2500 CFU. In some embodiments, detection is completed prior to transfusion of a donated platelet sample, and if a positive signal is detected (indicating bacterial contamination) , the donated platelets are not transfused into a recipient. In some embodiments, a positive signal for a sample having contamination at or above any of the disclosed detection thresholds is detected within about 48, 24, 12, 6, 4, 2, or fewer hours from obtaining the sample from a subject (e.g. from withdrawing blood from a subject) .
[0333]
In some embodiments, primer pairs are immobilized on a solid support. Examples of solid supports include, but are not limited to, inorganic materials such as silica based substrates (e.g. glass, quartz, fused silica, silicon, or the like) , other semiconductor materials, and organic materials such as polymer materials (e.g. polymethylmethacrylate, polyethylene, polypropylene, polystyrene, cellulose, agarose, or any of a variety of organic substrate materials conventionally used as supports for reactive media) . In addition to the variety of materials useful as solid supports, solid support structures may be in any of a variety of physical configurations, including but not limited to microparticles, beads, nanoparticles, nanocrystals, fibers, microfibers, nanofibers, nanowires, nanotubes, mats, planar sheets, planar wafers or slides, multiwell plates, optical slides including additional structures, capillaries, microfluidic channels, and the like. In some embodiments, amplification on a solid support comprises bridge amplification. General methods of bridge amplification are known in the art. See for example WO/1998/044151 and WO/2000/018957, each of which is entirely incorporated herein by reference.
[0334]
Power Supply
[0335]
The electrophoresis apparatus can comprise a power supply. The power supply can be external to the apparatus housing or integrated within the apparatus housing. The power supply can comprise adaptors for connection to external power sources. External power sources can include, but are not limited to: residential, commercial, or industrial building power, 12 V DC sources, off-grid sources, renewable energy sources, solar panels, batteries or other energy storage devices, motor vehicles, and motor vehicle batteries.
[0336]
The power supply may include one or more batteries of the electrophoresis apparatus. Any description of any power supply may apply to the batteries or vice versa. Any description of a battery or batteries may apply to a battery pack and vice versa. A battery pack may include one or more batteries. Multiple batteries may be connected to one another in series, in parallel, or any combination thereof.
[0337]
The power supply can be configured to run on a low voltage input signal. The low voltage input can provide a voltage of at most about 64 V, 50 V, 48 V, 36 V, 30 V, 29 V, 28 V, 27 V, 26 V, 25 V, 24 V, 23 V, 22 V, 21 V, 20 V, 19 V, 18 V, 17 V, 16 V, 15 V, 14 V, 13 V, 12 V, 11 V, 10 V, 9 V, 8 V, 7 V, 6 V, 5 V, 4 V, 3 V, 2 V, or 1 V DC. In some embodiments, the low voltage input may be greater than one or more values of described herein, or may fall in a range between any two of the values described herein. In some cases, the low voltage input can provide at most about 14.5 V DC. In some cases, the low voltage input can provide at most about 12.5 V DC. The power supply may optionally provide a voltage of no more than about 24 V or 12 V to the electrophoresis apparatus.
[0338]
The power supply can provide an output voltage to the electrodes, resulting in a field strength or voltage gradient needed for electrophoresis. The output voltage can be about 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 15 V, 20 V, 25 V, 30 V, 35 V, 40 V, 45 V, 50 V, 60 V, 70 V, 80 V, 90 V, 100 V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1 kV, 2 kV, 3 kV, 4 kV, 5 kV, 6 kV, 7 kV, 8 kV, 9 kV, 10 kV, 15 kV, 20 kV, 25 kV, 30 kV, 35 kV, 40 kV, 45 kV, 50 kV, 60 kV, 70 kV, 80 kV, 90 kV, or 100 kV. The output voltage can be at least about 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 15 V, 20 V, 25 V, 30 V, 35 V, 40 V, 45 V, 50 V, 60 V, 70 V, 80 V, 90 V, 100 V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1 kV, 2 kV, 3 kV, 4 kV, 5 kV, 6 kV, 7 kV, 8 kV, 9 kV, 10 kV, 15 kV, 20 kV, 25 kV, 30 kV, 35 kV, 40 kV, 45 kV, 50 kV, 60 kV, 70 kV, 80 kV, 90 kV, or 100 kV. The output voltage can be at most about 1 V, 2 V, 3 V, 4 V, 5 V, 6 V, 7 V, 8 V, 9 V, 10 V, 15 V, 20 V, 25 V, 30 V, 35 V, 40 V, 45 V, 50 V, 60 V, 70 V, 80 V, 90 V, 100 V, 200 V, 300 V, 400 V, 500 V, 600 V, 700 V, 800 V, 900 V, 1 kV, 2 kV, 3 kV, 4 kV, 5 kV, 6 kV, 7 kV, 8 kV, 9 kV, 10 kV, 15 kV, 20 kV, 25 kV, 30 kV, 35 kV, 40 kV, 45 kV, 50 kV, 60 kV, 70 kV, 80 kV, 90 kV, or 100 kV. The field strength can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 48 V/cm. The field strength can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 48 V/cm. The field strength can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 48 V/cm. In some cases, the field strength can be between about 1 and about 2 V/cm. In some cases, the field strength can be between about 4 and about 10 V/cm. In some cases, the field strength can be between about 5 and about 8 V/cm. In some cases, the field strength can be between about 5 and about 10 V/cm. In some cases, the field strength can be between about 10 and about 20 V/cm.
[0339]
The field strength can be adjustable. The field strength can vary during the course of an electrophoresis. Different electrophoresis techniques can be implemented within the electrophoresis apparatus, including but not limited to, gel electrophoresis, pulsed field gel electrophoresis, isoelectric focusing, mobility shift electrophoresis, affinity electrophoresis, and isotachophoresis.
[0340]
The power supply can be compatible with a car power adaptor or cigarette lighter adaptor. The sockets and plugs used for connection with a car power adaptor can be those defined in the ANSI/SAE J563 specification. In some cases, the adaptor socket and plug can be size A, with the receptacle having an inside diameter of about 20.93 mm to about 21.01 mm and the plug body having a diameter of about 20.73 mm to about 20.88 mm. In some cases, the adaptor socket and plug can be size B, with the receptacle having an inside diameter of about 21.41 mm to about 21.51 mm and the plug body having a diameter of about 21.13 mm to about 21.33 mm. The plug can include a light to indicate a connection has been made. The power supply can be capable of drawing power from a cigarette lighter adaptor or other charging port of a motor vehicle. The power supply can draw power while the motor vehicle is running or not running. The power supply can draw power while the motor vehicle is in motion or is not in motion.
[0341]
The power supply can comprise various circuit elements, such as control elements, drivers, transformers, rectifiers, and sampling elements. The power supply can be capable of providing high voltage. The power supply can comprise pulse width modulation circuits or chips. The power supply can comprise drivers, such as MOS drivers. The power supply can comprise oscillators, such as Royer circuits. The power supply can comprise transformers, such as step-up voltage transformers. The power supply can comprise rectifiers, such as full-bridge rectifier circuits. The power supply can comprise sampling circuits, such as voltage sampling circuits. The power supply can comprise controls or regulators, such as a closed-loop voltage regulator circuit. The power supply can produce a constant or about constant high voltage output. The electrophoresis apparatus can comprise a voltage display circuit 710, which can display voltage information from a high voltage power supply circuit 720, as shown in FIG. 7. Apparatuses can also comprise light control circuits 730. Circuits can be used in the control of an integrated apparatus or system 740, comprising a high voltage source 750, a display 760, and a light source 770 in one integrated system.
[0342]
For example, the high voltage power supply circuit can comprise the circuit shown in FIG. 8. An input voltage signal (e.g., a low voltage input such as a 12 V input) 810 can be fed to a pulse width modulation (PWM) chip 820 and a MOS driver circuit with Royer circuit 830. Input voltage signals can comprise a low voltage signal, such as any low voltage signal described elsewhere in this disclosure. The PWM chip can also drive the MOS driver circuit with Royer circuit. Output from the MOS driver circuit with Royer circuit can be fed into a step-up voltage transformer or converter 840. The stepped-up voltage signal can be rectified with a rectifier circuit 860 (e.g., a full-bridge rectification circuit) to produce a high voltage DC output 870. Signal from the rectifier circuit can also be sampled with a voltage sampling circuit 850 and used in a closed-loop voltage regulator circuit to produce a constant voltage output.
[0343]
Various components of the electrophoresis apparatus can run from the low voltage input, such as 24 V or 12 V or other low voltage values described elsewhere herein. In some cases, the high voltage source for the electrodes can run from the low voltage input. In some cases, the high voltage source for the electrodes and the voltage control and display circuits can run from the low voltage input. In some cases, the integrated apparatus or system, comprising a high voltage source, a display, and a light source in one integrated system can run from the low voltage input. In some cases, the high voltage source for the electrodes, the control and display circuits, the light source, and the communication equipment can run from the low voltage input. In some cases, the high voltage source for the electrodes, the control and display circuits, the light source, the communication equipment, and the detector can run from the low voltage input.
[0344]
An electrophoresis apparatus may be capable of performing electrophoresis when running on a low voltage, such as a voltage less than about 24 V or 12 V or other low voltage values described elsewhere herein. The electrophoresis apparatus may be capable of powering one or more electrodes to cause the sample to migrate through the gel. Sample may be capable of migrating through the gel as the electrophoresis apparatus receives a low voltage. In some instances, a low voltage, such as a voltage less than about 24 V or 12 V or any other low voltage values described elsewhere herein, may be used to perform the combination of electrophoresis and detecting. The electrophoresis apparatus may be capable of powering one or more electrodes to cause the sample to migrate through the gel while powering a detector that detects the progress of the sample migration in real-time. Sample may be capable of migrating through the gel, and the detector may be capable of detecting the sample progress in real-time, as the electrophoresis apparatus receives a low voltage. Optionally, the low voltage may also power a light source of the electrophoresis. The electrodes, the electrodes and detector, the electrodes and detector and light source, the detector and light source, the electrodes and light source, or any combination thereof may be powered by using a low voltage value. Thus, real-time electrophoresis may occur, powered by a low voltage.
[0345]
The low voltage may be powered from a low voltage external power source. For example, the electrophoresis application may be powered by a motor vehicle, an off-grid power source, an energy storage device, or any other external power source as described elsewhere herein.
[0346]
The overall power consumption of the electrophoresis apparatus can be low. In some cases, the overall power consumption of the electrophoresis apparatus is less than or equal to about 12 W. In some instances, a low degree of power may be used for electrophoresis, or the combination of electrophoresis and detection. For instance, about 100 W may be used to perform the electrophoresis and detecting. In some instances, a low power may be less than or equal to about 250 W, 200 W, 150 W, 130 W, 120 W, 110 W, 100 W, 90 W, 85 W, 84 W, 83 W, 80 W, 75 W, 70 W, 65 W, 60 W, 55 W, 50 W, 45 W, 40 W, 35 W, 30 W, 25 W, 20 W, 15 W, 14 W, 13 W, 12 W, 11 W, 10 W, 9 W, 8 W, 7 W, 6W, 5 W, 4 W, 3 W, 2 W, 1 W, 500 mW, 100 mW, 50 mW, 10 mW, 5 mW, or 1 mW. In some cases, a low power may be from about The amount of power used to operate the device may be less than or equal to any of the values described herein. Alternatively, the amount of power used to operate the device may be greater than equal to any of the values described herein. The amount of power used to operate the device may fall into a range between any two of the values described herein. The amount of power used to operate the electrophoresis apparatus (e.g., electrodes) and detector may have a total less than any of the values described herein. The amount of power used to operate the electrophoresis apparatus and detector may have a total greater than any of the values described herein. The amount of power used to operate the electrophoresis apparatus and detector may fall into a range between any two of the values described herein.
[0347]
An electrophoresis apparatus may receive power via a single power cable. In some cases, a single power cable provides power for electrophoresis operation In some cases, a single power cable provides power for electrophoresis operation and a light source. In some cases, a single power cable provides power for electrophoresis operation and a detector. In some cases, a single power cable provides power for electrophoresis operation, and a light source, and a detector. In some cases, a single power cable provides power for all the elements enclosed in the electrophoresis apparatus housing. An electrophoresis apparatus may receive power via two power cables. In some cases, a two power cables provide power for electrophoresis operation In some cases, two power cables provide power for electrophoresis operation and a light source. In some cases, two power cables provide power for electrophoresis operation and a detector. In some cases two power cables provide power for electrophoresis operation, and a light source, and a detector. In some cases, two power cables provide power for all the elements enclosed in the electrophoresis apparatus housing. In some cases, power for a light source is provided via a separate power cable. The electrophoresis apparatus can connect with power sources such as wall outlets, battery packs, or motor vehicle power systems, either through an adaptor and a power cable, or directly through a power cable.
[0348]
An electrophoresis apparatus may be powered by a vehicle in accordance with an embodiment of the invention. A device may be electrically connected to a charging port of a vehicle. Electrical energy may flow from the charging port to the device. The vehicle may be a self-propelled vehicle having one or more propulsion unit.
[0349]
The device may be a portable device capable of conducing electrophoresis. The device may be useful for real-time electrophoresis. The device may be capable of operating using low voltage of power. The device may be capable of operating using less than 12 V of power, or any other voltage of power described elsewhere herein. The device may be capable of fitting within a vehicle. The device may be capable of fitting onto a seat of a vehicle. The device may be capable of resting on a lap of an individual sitting within a vehicle.
[0350]
The vehicle may be a passenger vehicle. The vehicle may be sedan, hatchback, station wagon, truck, SUV, mini-van, van, jeep, tank, unmanned vehicle, or any other type of automotive vehicle capable of self-propulsion. In some instances, the vehicle may be an airplane, helicopter, train, monorail, subway, boat, ship, or any other type of vehicle. The vehicle may be propelled with aid of an internal combustion engine. The vehicle may be propelled with aid of an electric motor. The vehicle may have a vehicle battery that may power one or more component of the vehicle. The vehicle may be capable of fitting about two, three, four, five, six or more people therein. The vehicle may include one or more propulsion units, such as wheels that may permit the vehicle to move in an environment.
[0351]
The vehicle may have a charging port thereon. The charging port may be in an interior of the vehicle. The charging port may be may be a cigarette lighter receptacle for an automobile. The charging port may be a DC power source. The charging port may be a 12 V receptacle. The charging port may include a socket configured to receive a charging connector. The charging port may be a 12 V auxiliary power outlet of the vehicle. In some instances, the charging port may be a 5 V outlet. The charging port may be a USB standard 5 V outlet. The charging port may provide any low voltage value, such as those described elsewhere herein. The charging port may be provided in accordance with ANSI/SAE J563 specifications, as described elsewhere herein.
[0352]
The charging port may provide power that may originate from a battery of a vehicle. A device electrically connected to a charging port may be powered by a battery of a vehicle. The battery of a vehicle may be a car battery or any type of automotive battery. The vehicle battery may be a starting, lighting, ignition (SLI) battery. The vehicle battery may be a lead-acid battery. Optionally, the vehicle battery may include six galvanic cells that may deliver a total of about 12 V or less. In some instances, a vehicle may have multiple automotive batteries that may deliver a total of about 24 V or less. In some instances, a vehicle may have one or more automotive batteries that may deliver a total of about 48 V or less.
[0353]
When a power connector of the electrophoresis device is connected to the charging port, power may flow from the charging port of the vehicle to the device. The power may flow when the vehicle is operational. The vehicle may or may not be in motion while the vehicle is in operation. The vehicle may be operational when it is powered on and/or the engine is running. The vehicle may be in operation when the vehicle’s ignition is not completely turned off. The vehicle may be in operation when one or more wheels of the vehicle are turning. The vehicle may be in operation when the vehicle is in parking mode with the ignition on. The vehicle may be in operation if the vehicle headlights or radio may be turned on. Power may or may not flow to the device when the vehicle is not in operation.
[0354]
The power may be used to directly operate the device. The power may be used to charge an energy storage unit. The energy storage unit may be used to operate the device. In some instances, one or more set of protocols may be used to govern whether the power flowing to the device is used to directly operate the device or charge an energy storage device that may be used to power the device. In some instances, both actions may simultaneously occur.
[0355]
The device may be connected to a charging port via a power connector. The power connector may include a plug that may fit into the charging port. The device may come equipped with a power connector that may be configured to directly connect to the charging port. The power connector may include one or more prongs, pins, indentations, or conductive surfaces.
[0356]
The charging port may be capable of providing low voltage power to the device to permit operation of the electrophoresis device. The charging port may be on-board the vehicle. The charging port may be any off-grid charging port. The charging port may be powered by a vehicle battery. The charging port may be any other type of charging port electrically connected to any type of external power source as described elsewhere herein.
[0357]
Alternatively, the electrophoresis device may be connected to a charging port via a power connector and an adaptor. The power connector may not directly fit into the charging port, or may not be configured to regulate the power coming from the charging port for operation of the vehicle. The adaptor may provide one or more of these functions. The adaptor may be provided between the power connector of the device and the charging port.
[0358]
The adaptor may be configured to physically fit into the charging port. The adaptor may be configured to mechanically and/or electrically connect to the charging port. The power connector may not be capable of directly mechanically and/or electrically connecting to the charging port. In some instances, the adaptor may or may not provide some power regulation or conversion when providing power to the power connector. For example, the adaptor may convert DC to AC. In another example, the adaptor may modify or regulate voltage and/or current from the charging port to the power connector.
[0359]
Any description herein of connecting the device to the charging port may or may not include the use of one or more adaptors.
[0360]
An electrophoresis device may be deployed with aid of one or more vehicles. The device may be a portable device that can be carried within a vehicle. The vehicle may provide power to the device at a low voltage power, such as 12 V or other voltage values described elsewhere herein. The power provided to the device may be used to charge an energy storage unit of the device and/or directly power one or more other component of the device. The power may be provided to the device via a charging port while the vehicle is turned on. The power may be provided to the device while the vehicle is stationary or while the vehicle is in motion. The device may thus advantageously be deployed to multiple locations. These may include remote locations that may otherwise not have the power sources capable of powering the device. These may include remote locations where rolling blackouts may occur so reliable access to power may not be provided.
[0361]
The electrophoresis device may receive a sample at a location. The device may conduct electrophoresis at the location or while the device is in transit to another location. The device may receive the sample while the device is outside the vehicle, or may receive the sample while the device is within the vehicle. The device may receive the sample while the vehicle is stationary or in motion.
[0362]
The device may be connected to a charging port of the vehicle while it is in operation. Alternatively, the device may be disconnected from a charging port of the vehicle while it is in operation. The device may have an energy storage unit that may store energy while the device is connected to the vehicle. When the device is disconnected from the vehicle, the energy storage unit may be used to power the device. This may permit the device to be charged while in transit to a location. The device may then be taken out of the vehicle and used to conduct electrophoresis at the location using the stored energy. If the device depletes the charge of the energy storage unit, the device may be re-connected to the vehicle to power the device and/or charge the energy storage unit. Thus, as long as a vehicle is available, a ready power source may be provided for the device. This may advantageously couple transport of a device to a remote location with powering the device at any location to which it has been transported.
[0363]
Any description herein of a vehicle may also apply to any other type of power source, such as those described elsewhere herein.
[0364]
One or more different locations may be provided. The locations may or may not be remote from one another. Infrastructure such as roads (or paved roads) may or may not exist between the various locations.
[0365]
In some instances, samples may be provided from subjects that are in the proximity of the device. For example, samples from subjects at or near a first location may be provided. In other instances, samples may be provided from subjects that are at other locations. The remote samples may be sent from the other locations to a facility. In some instances, this may delay results getting back to the subjects or individuals at the other locations.
[0366]
A vehicle may be sent to another location (e.g., a second location) . The vehicle may have a device for conducting electrophoresis. The device may optionally be electrically connected to the vehicle while the vehicle is in operation. The device may be powered and/or charged by the vehicle when the vehicle is in operation. The device may be powered and/or charged by the vehicle while the vehicle is in motion (e.g., from a first location to a second location) . Permitting a device to be charged while the vehicle is in transit may permit the device to be at a substantially charged state when the device arrives at the destination. In some instances, the device may be used at the destination to perform electrophoresis at the location. The device may be powered by the vehicle at the location. For example, a car or other type of vehicle may be turned on and used to power a device while the device is running the electrophoresis at the location. Alternatively, the device may operate at the location using an energy storage device that has already been charged. The energy storage device may have been charged while the device was in transit. Charging the device while the device is in transit may advantageously provide greater flexibility that may allow the vehicle or apparatus to be transported from one location to another. The locations need not have grid power sources, or the use of the device need not rely on grid power sources. Furthermore, the device may be charged to a ready-to-use state while in transit which may save time when the device arrives at a destination.
[0367]
In some instances, one or more subjects may provide a sample at a destination. The electrophoresis may occur at the destination. Point of care (POC) testing may permit the results to be provided at the destination. In some instances, real-time electrophoresis or detection may occur, which may permit results to be provided in real-time or instantaneously to subjects at the location. This may permit the nucleic acid amplification device to be brought to otherwise remote locations and allow testing that may provide much faster results than other situations. This may be advantageous for disease prognosis and/or treatment. This may also aid in the detection and prevention of spreading infectious diseases.
[0368]
In some instances, the testing may occur at the destination location. In some instances, the samples may be collected and/or loaded into the device at the destination location. The device may be used to perform electrophoresis on the sample at the destination location. The results may be delivered at the destination location. In other implementations, a vehicle may receive the device and depart the destination location. The vehicle may be on its way to another location, such as a lab or facility. The device may be capable of performing electrophoresis in the vehicle while the vehicle is in operation. The device may be capable of performing electrophoresis while the vehicle is in transit. The device may be powered by the vehicle to perform the electrophoresis. In some instances, after the samples have been loaded into the device at a location, the vehicle may make its way to another location. The amplification may occur and/or be completed while the vehicle is in transit. This may save time in getting the device to another location where it may be needed. The results may be detected with aid of an on-board detector. The results may be relayed to a user of the device in real-time. The results may be relayed back to the location from which the samples were collected. In some instances, the results may be relayed to a facility which may perform additional analysis.
[0369]
Powering the device using the vehicle, and permitting nucleic acid amplification and detection while the device is in the vehicle en route may provide greater flexibility and time saving measures. The vehicle transit time may be used, rather than being ‘down time. ’ This may aid in maximizing or improving the use of the device when the device is deployed to different locations.
[0370]
Electrophoresis Gel and Lanes
[0371]
Electrophoresis involves the migration of species in a sample through a matrix or medium, such as a gel, in the presence of an electric field. The physical properties of the matrix and of the sample species can affect the rate of migration, allowing separation of different species within a sample. Relevant physical properties of sample species include size, electrical charge, and conformation. Electrophoresis can be conducted within an apparatus, which can provide a matrix (e.g., a gel) , buffer solution, and electrodes for generating an electric field.
[0372]
The electrophoresis apparatus can comprise an electrophoresis matrix (e.g., a gel) . The matrix can be divided into lanes separated by solid barriers. For example, FIG. 1 and FIG. 2A show a schematic of an electrophoresis matrix 110, from three-quarters and top-down views, respectively. The electrophoresis matrix can comprise gel lanes 120. Physical barriers 125 may provide physical separation between samples running in neighboring gel lanes or channels. In some cases, the physical barrires 125 may be precluded. The electrophoresis matrix can comprise troughs or chambers 130 for holding buffer or other fluids. The electrophoresis matrix can comprise electrodes 140. FIG. 3 shows a side-view schematic of a gel lane, with electrodes 350 positioned on the ends, and with buffer 360 positioned in buffer chambers between the electrodes and the gel 370.
[0373]
Gel or matrix lanes, defined by solid barriers, can be open on top rather than enclosed. For example, the gel lanes may optionally not be covered. The gel lanes may have sides enclosed by the solid barriers while the top is open and does not contact a solid barrier. That is, gel lanes can be partially or totally physically separated side-to-side while sharing a common overhead space, whereby a fluid (e.g., buffer, air) can be in contact or communication with some or all of the lanes. For instance, a top surface of a gel in a first lane may be in fluid communication with a top surface of a gel of a second lane. A shared overhead space may span all the lanes in the electrophoresis matrix. Alternatively, the shared overhead space may span a subset of the lanes in the electrophoresis matrix. In some embodiments, a top surface of the gel in the lanes may be visually discernible from over the electrophoresis matrix. Gel or matrix lanes can comprise capillary channels, capillary tubes, capillary gels, or microfluidic channels. Barriers can extend completely or partially into the depth of the gel or matrix. Barriers can extend at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%through the thickness of the gel or matrix. Barriers can extend the entire length or part of the length of the gel or matrix lane. Barriers can extend at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%of the length of the gel or matrix lane. Barriers can prevent, reduce, or inhibit diffusion or other transport of material (e.g., sample material) between adjacent lanes. Barriers can reduce or inhibit transport of material between adjacent lanes by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%compared to a gel without barriers.
[0374]
Barriers can comprise one or more connectors between gel lanes. For example, FIG. 2B and FIG. 2C show connectors 260 allowing transport of material between gel lanes. Such transport can be facilitated by additional electrodes oriented to provide an electric field in the direction of the connectors. Connectors can be used, for example, to move sample or portions of sample from one lane to another. Connectors can contain electrophoresis gel or other transport medium. Connectors can connect a pair of lanes or any number of lanes; for example, connectors in adjacent barriers can allow transport of material across two, three, four, five, or more lanes. One, two, three, four, five, or more connectors can connect adjacent lanes. Connectors can be perpendicular to lanes; that is, the angle between a connector and a lane can be about 90°. The angle between a connector and a lane can be at least about 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, or 170°. The angle between a connector and a lane can be at most about 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, or 170°. The angle between a connector and a lane can be about 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, or 170°. In some cases, different gel lanes can comprise different types or compositions of gel from each other. Some compositions of gel can have different densities or porosities, which can provide better resolution for specific types or sizes of sample material. For example, a sample comprising DNA molecules from 20 bases to 2000 bases in size can be loaded into a first gel lane; then after some separation (e.g., by an electric field generated by a first pair of electrodes) , smaller DNA molecules (e.g., 20 bases to 200 bases) can be shifted (e.g., by an electric field generated by a second pair of electrodes, oriented perpendicularly to the first pair of electrodes) into a second gel lane with higher concentration gel for further higher resolution separation, while separation of larger DNA molecules (e.g., 200 bases to 2000 bases) in the sample continues in the first gel lane. Electrodes and gel composition are discussed further in this disclosure.
[0375]
An electrophoresis apparatus may include a frame. The frame can be used for casting, shaping, or holding an electrophoresis matrix or gel. The frame may include a bottom surface and one or more side surfaces. The frame may or may not include a top surface. In some instances, the frame may include a bottom surface and four side surfaces extending up from the perimeter of the bottom surface. The bottom surface may have any shape, and the side surfaces may extend upward from the bottom surface along the perimeter of the bottom surface shape. An upper surface of the bottom of the frame may contact a bottom surface of gel or electrophoresis matrix. The top surface of the electrophoresis gel or matrix can be considered the surface of the gel opposite the surface of the gel that contacts the bottom surface.
[0376]
The electrophoresis matrix can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 lanes. The electrophoresis matrix can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 lanes. The electrophoresis matrix may comprise less than any of the number of lanes described, or a number of lanes falling within a range between any two of the values described.
[0377]
Matrix or gel lanes can comprise different geometrical configurations. Matrix or gel lanes can be parallel with respect to each other. Matrix or gel lanes can be non-parallel with respect to each other. Matrix or gel lanes can have a common width or can vary in width. Matrix or gel lanes can have a common length or can vary in length. Matrix or gel lanes can extend for about 100%, 90%, 80%, 70%, 60%, or 50%of the length of the frame. Matrix or gel lanes can extend for at least about 100%, 90%, 80%, 70%, 60%, or 50%of the length of the frame. Matrix or gel lanes can extend for at most about 100%, 90%, 80%, 70%, 60%, or 50%of the length of the frame. Matrix or gel lanes can have a width of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mm. Matrix or gel lanes can have a width of at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mm. Matrix or gel lanes can have a width of at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 mm. Matrix or gel lanes can have a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cm. Matrix or gel lanes can have a length of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cm. Matrix or gel lanes can have a length of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cm.
[0378]
The matrix or gel lanes may be parallel to one or more sides of the frame. The solid barriers may be parallel to one or more sides of the frame. In some instances, the matrix or gel lanes may be perpendicular to one or more sides of the frame. The solid barriers may be perpendicular to one or more sides of the frame. The solid barriers may or may not have a height that is less than or equal to a height of one or more sides of the frame. Alternatively, the solid barriers may have a height that is greater than or equal to the height of one or more sides of the frame.
[0379]
The matrix or gel can comprise sample loading areas and separation areas. Sample loading areas can comprise wells for holding sample fluids. Sample loading areas (e.g., wells) can receive samples simultaneously, for example from multi-pipette or auto pipette. The spacing and/or sizing of the sample loading areas can be designed to match to the dimensions of a particular loading device, such as a multi-pipette head. Sample loading areas can correspond to, line up with, or be located inside gel lanes, so that material from a particular sample loading area migrates through a particular lane during operation and remains separated from material from other samples.
[0380]
Various types of sample material can be separated in the matrix or gel, including but not limited to, nucleic acids (e.g., DNA, RNA, PNA) , nucleic acid fragments, direct PCR products, proteins (e.g., enzymes, antibodies, structural proteins, storage proteins, transport proteins, motor proteins, hormonal proteins, receptor proteins) , protein fragments, peptides, and particles. Different lanes within a gel can be used to separate material of the same type simultaneously. Different lanes within a gel can be used to separate material of different types simultaneously. Different lanes within a gel can be used to separate different size ranges of a material. Enhanced resolution within each of a subset of size ranges of sample material can be provided by the use of different gel compositions tailored for each size range. In some cases, molecules that differ in size or molecular weight by at least 1, 2, 3, 4, or 5 orders of magnitude can be separated and resolved within one gel. For example, nucleic acid molecules between 10 bases and 10 kb can be separated and resolved within one gel. In another example, a sample can comprise three size ranges of sample molecules, and a portion of the sample can be loaded into three gel lanes; the first size range is well-resolved in the first lane, the second size range is well-resolved in the second lane, and the third size range is well-resolved in the third lane. In some instances, molecules may be separated in different lanes at different rates. Molecules may traverse the lanes in different rates. The rates may differ from one another by greater than or equal to about 10%, 20%, 30%, 40%, 50%, 70%, 100%, 150%, 200%, 300%, 400%, 500%, 700%, or 1000%.
[0381]
Different types of gel can be used, including but not limited to agarose, polyacrylamide, and starch. Gel can be used to separate proteins, nucleic acids, and particles. Polyacrylamide gel can be used to separate nucleic acids, including small fragments of nucleic acids (e.g., about 5-500 bp) . Agarose gel can be used to separate proteins, including proteins above about 200 kDa. Agarose gel can be used to separate nucleic acids, including nucleic acids from size about 50 bp up to and including nucleic acids several Mb in size.
[0382]
Different gel compositions can be used. The porosity of the gel can be affected by the composition of the gel. Different porosity gels can provide improved resolution for particular size ranges of samples. Agarose gel can comprise at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, or 3.5%agarose. Agarose gel can comprise at most about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, or 3.5%agarose. Agarose gel can comprise about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, or 3.5%agarose. In some cases, agarose gel can comprise between about 0.7%and about 2%agarose. In some cases, agarose gel can comprise between about 0.7%and about 3%agarose. Polyacrylamide gel can comprise at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%polyacrylamide. Polyacrylamide gel can comprise at most about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%polyacrylamide. Polyacrylamide gel can comprise about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%polyacrylamide. In some cases, polyacrylamide gel can comprise between about 6%and about 15%polyacrylamide. For example, between different lanes, the gel composition can vary between lanes by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, or 700%. Between lanes, the gel composition can have porosities differing by one, two, three, four, five or more orders of magnitude. Physical barriers can be used to separate between gel or matrix lanes of different porosities or materials.
[0383]
A different percentage of polymers or a different mix of polymers can produce a gel especially suited for resolution of a particular size range. For example, 0.7%agarose gel can provide good resolution for nucleic acid fragments between about 5 and 10 kb. For example, 2%agarose gel can provide good resolution for nucleic acid fragments between about 0.2 and 1.0 kb. Gel lanes within an apparatus can be loaded with gels of different or the same type. For example, some gel lanes can be loaded with agarose gel and some lanes can be loaded with polyacrylamide gel. Gel lanes can within an apparatus can be loaded with gels of the same or of different densities or porosities. For example, some gel lanes can be loaded with a 6%polyacrylamide gel while other gel lanes are loaded with a 12%polyacrylamide gel.
[0384]
Gels can comprise or be used in conjunction with buffers, reagents, detergents, dyes, and other components. Gels can comprise or be used in conjunction with denaturants for nucleic acids, such as urea, DMSO, glyoxal, or methylmercury hydroxide. Gels can comprise or be used in conjunction with denaturants for proteins, such as sodium dodecyl sulfate (SDS) , beta-mercaptoethanol or dithiothreitol. Gels can comprise buffers, such as loading buffer, Tris, Bis-Tris, imidazole, EDTA, Tris/Acetate/EDTA (TAE) , Tris/Borate/EDTA (TBE) , or lithium borate (LB) . The buffers used at each electrode can be the same or different. Gels can comprise or be used in conjunction with dyes, including but not limited to, xylene cyanol, Cresol Red, Orange G, bromophenol blue, intercalating dyes (e.g., ethidium bromide, SYBR Green, EvaGreen) , and protein stains (e.g., silver stain, Coomassie Brilliant Blue) .
[0385]
Buffer chambers can be shared among all lanes, or a subset of the lanes. Alternatively, each lane can have its own buffer chamber. The buffer chamber configuration can be the same at both ends of the gel or can differ; for example, lanes can share buffer chambers at the top of the gel, while at the bottom of the gel lanes have individual buffer chambers.
[0386]
Gel can be cast with the aid of various tools, such as a gel casting frame or a comb for forming wells. For example, FIG 4 and FIG. 5 show a lane electrophoresis apparatus 480 with the aid of a gel casting frame 490 and a comb 400. The comb can be used to form wells in the gel for loading sample before the gel solidifies. Electrophoresis gel, for example in liquid form, can be added to a lane electrophoresis apparatus with the aid of a pump or other fluid handling equipment. For example, FIG. 6 shows the use of a pump, such as an auto pipette 610 with associated pipette tips 620, may be used to add gel to a lane electrophoresis apparatus 480 or gel casting frame 490. In some cases, the buffer chamber can be filled with buffer prior to adding liquid gel to the lanes, in order to avoid contact between the liquid gel and the electrode. A pump, such as an auto pipette, can also be used for loading samples into wells. Alternatively, samples may be loaded into wells using systems or methods in accordance with the present disclosure. Gel can be cast using a gel casting system. For example, a gel casting system may comprise a pump, a pipe, tube, or other conduit, a gel injector (e.g., auto pipette) , and a heating zone. Gel can be pre-made or partially pre-made prior to loading; for example, gel or gel precursor can be loaded onto the apparatus but still require some further activation prior to use, such as cross-linking.
[0387]
The electrophoresis apparatus can comprise electrodes. In some cases, electrodes can be integrated into the electrophoresis apparatus or gel tray, such as in FIG. 1 and FIG. 2. In some cases, electrodes 1040 can be separated from the electrophoresis apparatus 910 or its gel tray, for example as shown in FIG. 10. Electrodes can be shared among all lanes, or a subset of the lanes. Alternatively, each lane can have its own electrode or electrode tip. The electrode configuration can be the same at both ends of the gel or can differ; for example, lanes can share electrodes at the top of the gel, while at the bottom of the gel lanes have individual electrodes.
[0388]
The electrodes may be positioned to directly contact the gel when the gel is provided within the frame. The electrodes may pass through the trough or chamber for holding buffer or other solutions. The electrodes may or may not contact sides of the frame. The length of the electrodes may be oriented substantially perpendicular to the lengths of the gel lanes. The length of the electrodes may span some or all of the gel lanes. The electrodes may or may not contact the gel. The electrodes may or may not be embedded in the gel. The electrodes may or may not be elevated over a bottom surface of the frame. In some cases, the electrode at the sample loading end of the matrix or gel can be the anode and the electrode at the other end of the matrix or gel can be the cathode. In some cases, the electrode at the sample loading end of the matrix or gel can be the cathode and the electrode at the other end of the matrix or gel can be the anode. In some cases, an electrode can comprise multiple electrode tips, with each tip aligned to a specific lane. The polarity or orientation of electrodes or electrode tips can be the same for all lanes. Alternatively, the polarity or orientation of electrodes or electrode tips can be different for different lanes within the matrix; that is, some lanes can have an anode at the first end and cathodes at the second end while other lanes are oriented in the opposite direction.
[0389]
The electrophoresis apparatus can comprise multiple sets of electrodes. In some cases, the electrophoresis apparatus can comprise a first set of electrodes and a second set of electrodes oriented orthogonally to the first set of electrodes. The second set of electrodes can be used to transport sample material through connectors between lanes, as described further in this disclosure. For example, FIG. 2B and FIG. 2C show connectors 260, a first vertical set of electrodes 140, and a second horizontal set of electrodes 250. Additional sets of electrodes can be oriented orthogonally to the first set of electrodes. The angle between the orientation of a first set of electrodes and a second set of electrodes can be about 0°, 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, 170°, or 180°. The angle between the orientation of a first set of electrodes and a second set of electrodes can be at least about 0°, 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, 170°, or 180°. The angle between the orientation of a first set of electrodes and a second set of electrodes can be at most about 0°, 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, 140°, 150°, 160°, 170°, or 180°. The voltage supplied to multiple sets of electrodes, or the field strength generated by multiple sets of electrodes, can be the same or can be different between electrode sets.
[0390]
Optionally, an electrophoresis device may have a detector that may be capable of detecting signals from one or more lanes in real-time. In some instances, the detector may be configured to detect optical signals from the one or more lanes. The detectors may be capable of visually discerning the top surface of the gels in the one or more lanes. Leaving the lanes uncovered may permit the detector to detect optical signals from the top surfaces of the gels in the lane, in real-time. The detector may be capable of detecting optical signals from the gels in multiple lanes simultaneously. The detector may capture an image of the top surfaces of the gels in the multiple lanes. The image may be a still image or may include video-rate images. Optionally, no intermediary materials or covers may be provided between the top surface of the gel and the detector. A direct line of sight may be presented between the top surface of the gel and the detector. In some alternative embodiments, an optically transmissive material may be provided between the gel surface and the detector.
[0391]
The electrophoresis apparatus can comprise a drawer or drawer-like structure with a moveable gel tray. The drawer can be movable manually or automatically, such as with a motor or actuator. For instance, an actuator may receive a command signal from a controller to cause the drawer to move. The drawer or tray can be accessible via a panel, such as a sliding panel. The sliding panel can be movable manually or automatically, such as with a motor or actuator. For instance, an actuator may receive a command signal from a controller to cause the sliding panel to move.
[0392]
The drawer may move between an open position and a closed position. In some cases, in the open position the matrix or gel is accessible for sample loading. In some cases, in the closed position the matrix or gel is inaccessible for sample loading. In some cases, in the open position the matrix or gel is exposed to elements of the ambient environment. In some cases, in the closed position the matrix or gel is isolated from elements of the ambient environment. Elements of the ambient environment can include but are not limited to light, gas (e.g., air) , fluids, liquids, particulates, organisms, viruses, reagents, samples or sample material (e.g., proteins, nucleic acids) , light, and temperature.
[0393]
The motion of the drawer or tray can be entirely or substantially horizontal. The motion of the drawer or tray can be entirely or substantially vertical. The motion of the drawer or tray can include horizontal and vertical elements. The drawer or tray can comprise a lock, stop, or other mechanism to prevent accidental opening. The drawer or tray can be capable of being completely removed from the electrophoresis apparatus. The gel tray can comprise elements including a buffer trough or chamber, a gel region, sample wells, and electrodes. The gel tray may include a frame, which can be used to contain or form gel. The gel tray may comprise a lower gel tray panel, an upper gel tray panel, a buffer stopper or weir, a gel region upper panel, and electrodes. The drawer can be used to access the gel, for example to remove a used gel or add a new gel. For example, a gel tray may be slid open from an electrophoresis apparatus with coupled detector, such that the gel is visible and exposed to the environment, the sample wells are accessible for sample loading, and the gel is physically accessible and can be removed and replaced with a different gel or matrix. The gel or matrix can be removed by hand or with an implement, such as a lever, tweezers, or tongs. The drawer or tray can be capable of being completely removed from the apparatus. If the drawer or tray is completely removed from the apparatus, the gel or matrix can be turned upside down and the gel can be removed with the aid of gravity.
[0394]
Detector
[0395]
The electrophoresis apparatus can be used in conjunction with a detector. In some cases, the detector can be integrated within the electrophoresis apparatus housing. For instance, electrophoresis apparatus housing may enclose the detector. The electrophoresis apparatus housing may enclose the detector, along with one or more gel lanes. The electrophoresis apparatus housing may enclose the detector and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose the detector, a light source, and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, a light source, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose the detector, communications equipment, and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, communications equipment, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose the detector, communications equipment, a light source, and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, communications equipment, a light source, the matrix or gel, and a power supply. In some cases, the detector can be enclosed within its own housing. The detector housing can enclose the detector and a light source. The detector housing can enclose the detector and communications equipment. The detector housing can enclose the detector, a light source, and communications equipment. The detector can be located in a sliding panel, such as a top sliding panel providing access to an observation window. In some cases, a sliding panel enclosing the detector can be positioned in an open state for external viewing of the electrophoresis matrix (e.g., via observation window) or positioned in a closed state for imaging of the electrophoresis matrix with the detector. The sliding panel can be movable manually or automatically, such as with a motor or actuator. For instance, an actuator may receive a command signal from a controller to cause the sliding panel to move.
[0396]
Alternatively, the detector can be contained in its own separate housing. For example, the detector may be in its own housing but coupled to or capable of being coupled to the electrophoresis apparatus. The detector housing can be coupled to the electrophoresis housing. The detector housing can be attached in a removable manner to the electrophoresis housing. The coupling between the detector housing and the electrophoresis housing can prevent all or most ambient light from reaching the gel or the detector. The detector housing can comprise a shade, sleeve, or other fixture to shield the interior of the detector housing and/or electrophoresis housing from environmental light.
[0397]
The detector can comprise an image sensor or image sensors. The image sensor can be capable of optical detection. The image sensor can comprise a charge-coupled device (CCD) sensor, including a cooled CCD. The image sensor can comprise an active-pixel sensor (APS) , such as a CMOS or NMOS sensor. The detector can comprise a laser sensor. The detector can comprise a photodiode, such as an avalanche photodiode. The detector can comprise a photomultiplier tube (PMT) . The sensors can comprise a single sensor or multiple sensors, of the same type or of different types.
[0398]
The detector can image subjects, such as a matrix or a region of a matrix. The detector can image at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%of the matrix in one field of view. The detector can image subjects before, during, or after operation of the electrophoresis apparatus. The detector can image in real-time while the electrophoresis apparatus is operating, for example during the electrophoretic separation of sample material with a matrix.
[0399]
The detector can record single still images or can record video. The detector can sample at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 90, 120, 150, 180, 210, 240, 270, or 300 times per minute. The detector can sample at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 90, 120, 150, 180, 210, 240, 270, or 300 times per minute. The detector can sample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 90, 120, 150, 180, 210, 240, 270, or 300 times per minute. The detector can sample at a rate of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 Hz. The detector can sample at a rate of at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 Hz. The detector can sample at a rate of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 Hz.
[0400]
The detector can have particular resolution or sensitivity. The detector can comprise at least about 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 pixels on a side. The detector can comprise at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 megapixels. The detector can have a resolution of at least about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06 mm, 0.05 mm, 0.04 mm, 0.03 mm, 0.02 mm, 0.01 mm, 0.009 mm, 0.008 mm, 0.007 mm, 0.006 mm, 0.005 mm, 0.004 mm, 0.003 mm, 0.002 mm, 0.001 mm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, or 100 nm.The detector can have a resolution of at most about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06 mm, 0.05 mm, 0.04 mm, 0.03 mm, 0.02 mm, 0.01 mm, 0.009 mm, 0.008 mm, 0.007 mm, 0.006 mm, 0.005 mm, 0.004 mm, 0.003 mm, 0.002 mm, 0.001 mm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, or 100 nm. The detector can have a resolution of about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 0.09 mm, 0.08 mm, 0.07 mm, 0.06 mm, 0.05 mm, 0.04 mm, 0.03 mm, 0.02 mm, 0.01 mm, 0.009 mm, 0.008 mm, 0.007 mm, 0.006 mm, 0.005 mm, 0.004 mm, 0.003 mm, 0.002 mm, 0.001 mm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, or 100 nm.
[0401]
The detector can comprise a light source. The light source can be integrated into the detector housing or the electrophoresis apparatus housing. The light source can comprise a lamp, such as an incandescent, halogen, fluorescent, gas-discharge, arc, or LED lamp. The light source can comprise a laser. The light source can produce a specific wavelength or range or wavelengths, such as UV. The light source can comprise filters for controlling the output wavelength or wavelengths. The light source can comprise multiple light sources, of the same or of different types, which can be used separately or in combination. The light source can be enclosed by its own housing or by the electrophoresis apparatus housing.
[0402]
The detector can comprise various optical elements, including but not limited to filters, lenses, collimators, mirrors, reflectors, beam splitters, and diffusers. A matrix or gel can be exposed to light, for example light from a light source. Light can pass through one or more optical elements between the light source and the matrix, or light can travel directly from the light source to the matrix. The detector can receive light from the matrix. Light can pass through one or more optical elements between the matrix and the detector, or light can travel directly from the matrix to the detector. Light can be collimated. Light may be evenly distributed over multiple gel lanes. The multiple gel lanes may be illuminated with light of substantially equal intensity. Light may be directed in a manner to be substantially perpendicular to the gel upper surface. Light can be multi-directional. Light can comprise one wavelength, a narrow band of wavelengths, a broad band of wavelengths, or a full spectrum. Light can comprise visible light wavelengths. Light can comprise ultraviolet wavelengths. Light can comprise infrared wavelengths. Light can be focused by a lens. Images produced by the detector can comprise bright field images. Images produced by the detector can comprise dark field images. Images produced by the detector can comprise fluorescent images.
[0403]
The detector can comprise a filter or filters, including but not limited to wavelength filters (e.g., color filters, UV filters, IR filters) , dichroic filters, and polarizing filters. The filters can comprise multiple filters, of the same or of different types, which can be used separately or in combination.
[0404]
The detector can comprise a lens or lenses. The lens can be a macro or “close-up” lens. The lens can be a zoom lens. The lens can be an infrared lens. The lens can be an ultraviolet lens. The lens can be a wide angle lens, including but not limited to wide angle lenses, ultra wide angle lenses, and fisheye lenses. The lenses can comprise multiple lenses, of the same or different types, which can be used separately or in combination.
[0405]
The detector can comprise an element for removing image distortion or aberration, such as barrel or fisheye distortion, pincushion distortion, mustache distortion, monochromatic aberrations (e.g., piston, tilt, defocus, spherical aberration, coma, astigmatism, field curvature, image distortion) , or chromatic aberrations (e.g., axial, longitudinal, lateral, transverse) . Such an element can comprise computer systems programmed to implement instructions for partially or fully correcting image distortion. For example, Brown’s distortion model or the Brown-Conrady model can be used to correct for radial distortion and tangential distortion.
[0406]
The detector can comprise communication equipment, such as wired (e.g., USB) or wireless (e.g., Wi-Fi, Bluetooth) communication equipment. The communication equipment can comprise equipment for radio. The communication equipment can comprise equipment for free-space optical (FSO) communication, such as visible or infrared (IR) communication. The communication equipment can comprise equipment for wired communication, including but not limited to universal serial bus (USB) , fiber-optics, peripheral component interconnect (PCI) , PCI Express (PCIe) , or Thunderbolt. The communication equipment can comprise equipment for Wi-Fi, such as IEEE 802.11 a, b, g, or n Wi-Fi. The communication equipment can comprise equipment for cellular data service, such as GSM, CDMA, GPRS, 3G, (e.g., W-CDMA, EDGE, CDMA2000) , or 4G (e.g., Long Term Evolution (LTE) , Mobile WiMAX) . The communication equipment can comprise equipment for mobile satellite communications. The communication equipment can comprise equipment for Bluetooth communication. The communication equipment can comprise multiple types of communication equipment, such as USB and Wi-Fi, or Bluetooth and Wi-Fi.
[0407]
The communication equipment can transmit information from the detector, such as images recorded by the detector (e.g., images of a gel) . The communication equipment can transmit information, such as images, in real time. The communication equipment can communicate with the electrophoresis apparatus and/or control systems thereof. The communication equipment can communicate with remote computer systems, such as desktop computers, laptop computers, tablet computers, smartphone devices, or servers. The communication equipment can communicate with display devices, such as handheld display devices. The communication equipment can transmit information, such as images, to a user at a separate location or facility.
[0408]
Remote Monitoring
[0409]
The electrophoresis apparatus can comprise equipment for the remote monitoring and control of operations and results. Information can be transmitted between the electrophoresis apparatus and a remote device over a network. Alternatively, information can be transmitted between the electrophoresis apparatus and a remote device directly, via wired or wireless communications. In some instances, the networks may be local area networks (LAN) or wide area networks (WAN) , such as the Internet. The networks may optionally be telecommunications networks, such as GSM, 3G, or 4G networks. Additional networks and communications are further discussed elsewhere in this disclosure.
[0410]
The apparatus can comprise communication equipment, such as wired (e.g., USB) or wireless (e.g., Wi-Fi, Bluetooth) communication equipment. The communication equipment can comprise equipment for radio. The communication equipment can comprise equipment for free-space optical (FSO) communication, such as visible or infrared (IR) communication. The communication equipment can comprise equipment for wired communication, including but not limited to universal serial bus (USB) , fiber-optics, peripheral component interconnect (PCI) , PCI Express (PCIe) , or Thunderbolt. The communication equipment can comprise equipment for Wi- Fi, such as IEEE 802.11 a, b, g, or n Wi-Fi. The communication equipment can comprise equipment for cellular data service, such as GSM, CDMA, GPRS, 3G, (e.g., W-CDMA, EDGE, CDMA2000) , or 4G (e.g., Long Term Evolution (LTE) , Mobile WiMAX) . The communication equipment can comprise equipment for mobile satellite communications. The communication equipment can comprise equipment for Bluetooth communication.
[0411]
The communication equipment can transmit information from the apparatus, such as operational data (e.g., voltage, field strength, running time) or images recorded by the detector (e.g., images of a gel) . The communication equipment can receive information, such as commands to start operation, stop operation, or change operational conditions. Communication equipment can transmit information from the apparatus and/or detector, such as imaging data (e.g., images or videos) or operational data (e.g., voltage, field strength, run time) . Communication equipment can transmit information in real time, or on a delay. Communication equipment can be used to provide instructions to the apparatus and/or detector, such as instructions to begin electrophoresis, stop electrophoresis, alter the applied voltage or electric field strength, take an image or video, transmit an image or video, begin taking images or videos at a specified time or rate, alter the imaging rate, or stop taking images or videos. Instructions can be given to be carried out immediately or at a future time. In some instances, instructions may be provided by a user via a user interface. The user may be remote user. Alternatively, instructions may be generated with aid of one or more processors. An automated or semi-automated system may be provided that may generate instructions off-board the electrophoresis apparatus. The communication equipment can transmit and receive information in real time. The communication equipment can communicate with remote computer systems, such as desktop computers, laptop computers, tablet computers, smartphone devices, or servers. The communication equipment can communicate with display devices, such as handheld display devices. The communication equipment can communicate with various devices, including but not limited to a laptop computer, a desktop computer, or a mobile device.
[0412]
The communication equipment can transmit and receive information to/from a user at a separate location or facility. The separate location or facility can be located in a different room. The separate location or facility can be located in a different city. The separate location or facility can be located in a different state. The separate location or facility can be located in a different country. The separate location or facility can be located on a different continent. The separate location or facility can be located in a different time zone. The separate location or facility can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, or 20,000 km away.
[0413]
Physical Shape
[0414]
The electrophoresis apparatus housing can comprise different shapes and form factors. Imaging or detecting equipment 1720 can be located in a separate housing from the electrophoresis apparatus, and can be configured to mount onto the electrophoresis apparatus for imaging. The apparatus can comprise a control interface. The control interface may permit a user to interact directly with the device. For example, the user may turn the device on or off, or control one or more aspects of the electrophoresis and/or detection. The apparatus can comprise a matrix. The matrix may be part of a lower base or body of the electrophoresis apparatus. The apparatus can comprise power cables or connectors. The power cables or connectors may provide power from an external power source. Optionally, the external power source may be a low voltage power source, such as a 24 V or 12 V power source, or any other voltage value mentioned elsewhere herein. The apparatus or detector housing can comprise a shield, sleeve, or other structure to block outside light. The apparatus or detector can comprise communications equipment, cables, or connectors. The connectors may optionally connect to a lower base of the electrophoresis apparatus or an external device. Data collected by the detector may be transmitted wirelessly directly to the base or to an external device. Alternatively, data collected by the detector may be transferred via a wireless connection to the base. The base may optionally transmit data from the detector to an external device via a wired or wireless connection.
[0415]
The electrophoresis apparatus housing can comprise a sliding gel trays previously described. The sliding gel tray may move laterally relative to a base of the electrophoresis device. Detection apparatus may be permitted to remain attached while the gel tray moves laterally. Thus, the matrix may be accessed by a user without having to remove the detection apparatus.
[0416]
The mounting of the imaging or detecting equipment can utilize a variety of methods or motions. For instance, the detecting equipment may move laterally and/or vertically relative to the electrophoresis apparatus base. This motion may be provided to attach and/or detach the detecting equipment from the electrophoresis apparatus base.
[0417]
Mounting can be aided by connectors, tabs, or other adaptors on the apparatus housing. For example, tabs may protrude from electrophoresis apparatus housing and may mate with portions of detection equipment that may be configured to attach to the electrophoresis apparatus. The detection equipment apparatus may include portions that may slide underneath the tabs and be held in place by the tabs. The detection equipment may fit into a perimeter from which the tabs extend. Optionally, a shoulder may be provided upon which the detection equipment may rest.
[0418]
The imaging or detector equipment housing can comprise different shapes and form factors. In some instances, the detector equipment may have a curved surface (e.g., concave or convex) or a straight surface. The detector equipment may have a greater height than width, or vice versa.
[0419]
The form factor of the electrophoresis housing can be small. In some instances, the electrophoresis housing may be substantially flat, or may have a smaller height than width. A control interface, gel, and/or connector may be provided. The electrophoresis housing may have any of the dimensions described elsewhere herein for an electrophoresis device.
[0420]
The electrophoresis apparatus housing can comprise one or more sliding panels , allowing access to various components such as an observation window or a gel matrix tray. A sliding panel can be movable manually or automatically, such as with a motor or actuator. For instance, an actuator may receive a command signal from a controller to cause a sliding panel to move.
[0421]
The electrophoresis apparatus housing can comprise one or more controls, such as a main switch, a light tuning control, a light power switch, or a voltage control) . In some cases, a control can be visible only when the apparatus power is on.
[0422]
Some or all of the components necessary for conducting electrophoresis can be integrated within the electrophoresis apparatus housing. For example, the gel tray and power supply can both be integrated into the same housing. A light source for imaging or detection can be integrated within the apparatus housing. Communications equipment, such as wireless (e.g., Wi-Fi, Bluetooth) or wired (e.g., USB) communication equipment, can be integrated within the housing. The electrophoresis apparatus housing may enclose the detector and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose the detector, a light source, and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, a light source, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose the detector, communications equipment, and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, communications equipment, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose the detector, communications equipment, a light source, and the matrix or gel. The electrophoresis apparatus housing may enclose the detector, communications equipment, a light source, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose the matrix or gel. The electrophoresis apparatus housing may enclose the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose a light source, and the matrix or gel. The electrophoresis apparatus housing may enclose a light source, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose communications equipment, and the matrix or gel. The electrophoresis apparatus housing may enclose communications equipment, the matrix or gel, and a power supply. The electrophoresis apparatus housing may enclose communications equipment, a light source, and the matrix or gel. The electrophoresis apparatus housing may enclose communications equipment, a light source, the matrix or gel, and a power supply.
[0423]
In some cases, the electrophoresis apparatus housing can have a maximum height of less than about 50 cm, 45 cm, 40 cm, 30 cm, 25 cm, or 20 cm, 15 cm, 10 cm, or 5 cm. In some cases, the electrophoresis apparatus housing can have a height of less than about 30 cm. In some cases, the electrophoresis apparatus housing can have a maximum dimension (e.g., height, length, width, diagonal, or diameter) of less than about 30 cm, 25 cm, 20 cm, 15 cm, or 10 cm. In some cases, the electrophoresis apparatus housing can have a lateral dimension (e.g., width, length) of less than about 30 cm, 25 cm, 20 cm, 15 cm, or 10 cm. In some cases, the electrophoresis apparatus housing can have a lateral dimension (e.g., width, length) of less than about 10 cm. In some cases, the electrophoresis apparatus housing can have a height to width ratio of less than about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5. In some cases the electrophoresis apparatus can have a height to width ratio of less than about 1.
[0424]
In some cases, the electrophoresis apparatus can have a maximum mass of less than about 10 kg, 9 kg, 8 kg, 7 kg, 6 kg, 5 kg, 4 kg, 3 kg, 2 kg, 1 kg, 0.9 kg, 0.8 kg, 0.7 kg, 0.6 kg, or 0.5 kg. In some cases, the electrophoresis apparatus and detector can have a combined maximum mass of less than about 20 kg, 19 kg, 18 kg, 17 kg, 16 kg, 15 kg, 14 kg, 13 kg, 12 kg, 11 kg, 10 kg, 9 kg, 8 kg, 7 kg, 6 kg, 5 kg, 4 kg, 3 kg, 2 kg, 1 kg, 0.9 kg, 0.8 kg, 0.7 kg, 0.6 kg, or 0.5 kg.
[0425]
In some cases, the electrophoresis apparatus can have a small footprint, that is the horizontal surface area or the area of a surface covered when the apparatus is placed on that surface. In some cases, the electrophoresis apparatus can have a footprint of less than or equal to about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, or 50 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 1000 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 200 and 1000 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 250 and 1000 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 300 and 1000 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 900 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 800 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 700 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 600 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 500 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 400 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 300 cm 2. In some cases, the electrophoresis apparatus can have a footprint between about 100 and 250 cm 2.
[0426]
In some cases, the electrophoresis apparatus can have a small total volume. The total volume of the electrophoresis apparatus can be less than or equal to about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 liters. The total volume of the electrophoresis apparatus can be between about 1 liter and about 10 liters. The total volume of the electrophoresis apparatus can be between about 1 liter and about 5 liters. The total volume of the electrophoresis apparatus can be between about 1 liter and about 3.5 liters.
[0427]
In some cases, the electrophoresis apparatus can be portable. The electrophoresis apparatus can be sized to be capable of being carried by a human. The electrophoresis apparatus can be sized to be capable of being carried in one hand. The electrophoresis apparatus can be handheld.
[0428]
EXAMPLES
[0429]
Example 1
[0430]
FIG. 27 illustrates an example of the system of sample analysis in accordance with the present disclosure. The system comprises an electrophoretic gel 2703, a cannula housing 2707, tubes 2701, and a tube holder 2706. The various elements of the system are further illustrated individually in FIGs. 28A-C. The electrophoretic gel 2703 comprises a slot 27031 for loading samples to be subject to electrophoresis (FIG. 28A) .
[0431]
The tube holder 2706 comprises a sample loading clap 27061 into which a tube rack 27062 can be inserted (FIG. 28B) . The sample loading clap 27061 can be snap fit with the tube rack 27061. The sample loading clap 27061 is equipped with a set of springs 27066 (see FIG. 29E, lower panel) which are compressed when the sample loading clap 27061 is snap fit with the tube rack. The sample loading clap 27061 further comprises tabs 27065 for releasing the snap fit. When the snap fit is released, the compressed springs 27066 can eject the tube rack 27062 from the sample loading clap 27061. The tube rack 27062 is configured to accommodate one or more tubes 2701.
[0432]
The cannula housing 2707 comprises an embedded securing element 27079 (see lower panel of both FIG. 29B) for securing cannulae 2702 and a covering element 27078 for covering an end of the cannulae 2702 (FIG. 28C) . The cannulae 2702 have flow channels extended therethrough and tips exposed on the other end not covered by the covering element 27078.
[0433]
The aforesaid system is used for loading samples contained in the tubes 2701 to electrophoresis in the electrophoretic gel 2703, as illustrated in FIG. 29A-G.
[0434]
First, the tubes 2701 are fit into the tube holder 2706, and the tube holder is inverted (FIG. 29A) . The inverted tube holder 2706 (with the tubes 2701 fit therein) is then aligned with the exposed tips of the cannulae secured in the cannula housing 2707 (FIG. 29B, upper left panel) . The inverted tube holder 2701 is then pushed against the cannula housing 2707 (FIG. 29B, upper right panel) such that the cannulae 2702 piercing the tubes 2701 (FIG. 29B, lower panel) to bring the tubes in fluid communication with the flow channels within the cannulae 2702.
[0435]
Subsequently, the combination 2706/2707 of the inverted tube holder (with the tubes 2701 fit therein) and the cannula housing is then aligned with the slot 27031 in the electrophoretic gel 2703 (FIG. 29C, upper left panel) . The combination 2706/2707 is then pushed against the electrophoretic gel 2703 (FIG. 29C, upper right panel) such that the cannulae 2702 piercing the covering element 27078 (FIG. 29C, lower panel) to bring the tubes 2701 in fluid communication with the slot 27031 via the flow channels within the cannulae 2702.
[0436]
FIG. 29D illustrates the process by which the tubes 2701 are brought in fluid communication with the slot 27031 via the flow channels within the cannulae 2702 as described above. As shown in FIG. 29D, upper panel, there is a gap between the covering element 27078 and the securing element 27079. When the combination 2706/2707 (with the tubes 2701 fit therein) is then pushed against the electrophoretic gel 2703, the gap is closed by the movement of the covering element 27078 effected by contact with the electrophoretic gel 2703, which movement results in the cannulae 2702 piercing the covering element 27078 to bring the tubes 2701 in fluid communication with the slot 27031 in the electrophoretic gel 2703.
[0437]
Subsequently, the tabs 27065 are pressed down (FIG. 29E, upper panel) such that the snap fit between the sample loading clap 27061 and the tube rack 27062 is released, resulting in the compressed spring 27066 in the sample loading clap 27061 ejecting the tube rack 27062 from the sample loading clap 27061 (FIG. 29E, lower panel) .
[0438]
Following the ejection of the tube rack 27062, the tubes 2701 are squeezed by squeezing the sample loading clap 27061 as illustrated in FIG. 29F. The squeezing of the tubes 2701 allows samples contained in the tubes 2701 to be transported via the flow channel 2702 to the slot 27031 in the electrophoretic gel 2703 for analysis (FIG. 29F, lower panel) .
[0439]
After the transport of the samples into the electrophoretic gel 2703, the tube holder 2706 may be removed from the system for future reuse (FIG. 29G, left panel) . The electrophoretic gel 2703, the cannula housing 2707, and the tubes 2701 may be disposable following sample analysis (FIG. 29G, right panel) .
[0440]
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

[Claim 1]
A system for sample analysis, comprising: a sample vessel comprising (i) a container that retains a biological sample and (ii) a penetratable membrane at an end of said container that seals said container in the absence of an object inserted through said penetratable membrane; a separation medium that subjects said biological sample or a portion thereof to separation upon application of a flow-inducing field across said separation medium, which separation is on the basis of mass or charge; a flow channel in fluid communication with said separation medium, wherein during use, at least a portion of said flow channel traverses said penetratable membrane to bring said container in fluid communication with said separation medium; and a controller comprising one or more computer processors that are individually or collectively programmed to (i) pierce said penetratable membrane to bring said container in fluid communication with said separation medium through said flow channel, (ii) subject said biological sample to flow from said container through said flow channel to said separation medium, and (iii) apply said flow-inducing field across said separation medium under conditions that are sufficient to direct said biological sample through said separation medium to separate said biological sample or portion thereof on the basis of mass or charge.
[Claim 2]
The system of claim 1, wherein said one or more computer processors are programmed to apply positive or negative pressure to subject said biological sample to flow from said container through said flow channel to said separation medium.
[Claim 3]
The system of claim 1, further comprising a holder for receiving and securably holding said sample vessel.
[Claim 4]
The system of claim 3, wherein at least a portion of said flow channel is secured in a housing, and wherein during use, said holder is brought in contact with said housing.
[Claim 5]
The system of claim 4, wherein during use, (1) said holder is brought in contact with or in proximity to said housing in a first position in which said penetratable membrane is not pierced, and (2) said penetratable membrane is pierced by directing said housing or said holder to a second position.
[Claim 6]
The system of claim 5, wherein said flow channel is brought in fluid communication with said separation medium by directing said housing to a third position.
[Claim 7]
The system of claim 5, wherein said housing further comprises a first covering element that covers an end of said flow channel when said housing is in said first position, wherein said first covering element allows said end of said flow channel to emerge from said first covering element when said housing or said holder is in said second position such that said end of said flow channel traverses said penetratable membrane.
[Claim 8]
The system of claim 7, wherein said housing further comprises a second covering element that covers another end of said flow channel when (a) said housing is in said first position or (b) said housing or said holder is in said second position, wherein said second covering element allows said another end of said flow channel to emerge from said second covering element when said housing is in a third position such that said another end of said flow channel pierces an additional penetratable membrane separating said flow channel from said separation medium.
[Claim 9]
The system of claim 1, further comprising a cannula that pierces said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[Claim 10]
The system of claim 9, wherein said cannula comprises at least a portion of said flow channel extending therethrough.
[Claim 11]
The system of claim 9, wherein said cannula includes a tip at an end thereof, which tip pierces said penetratable membrane to bring said flow channel in fluid communication with said container.
[Claim 12]
The system of claim 1, wherein said separation medium is a polymeric separation medium.
[Claim 13]
The system of claim 12, wherein said separation medium comprises an agarose gel.
[Claim 14]
The system of claim 1, wherein said flow-inducing field is an electric field.
[Claim 15]
The system of claim 14, further comprising at least two electrodes on ends of said separation medium, wherein said at least two electrodes provide said electric field.
[Claim 16]
The system of claim 1, wherein said flow-inducing field is a pressure field.
[Claim 17]
The system of claim 1, further comprising an actuator that pierces said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[Claim 18]
The system of claim 1, further comprising a plurality of sample vessels, wherein a given sample vessel of said plurality of sample vessels comprises said container and said penetratable membrane.
[Claim 19]
The system of claim 1, further comprising a compartment having said separation medium.
[Claim 20]
The system of claim 19, further comprising a unique identifier on said compartment.
[Claim 21]
The system of claim 20, wherein said unique identifier is a barcode.
[Claim 22]
The system of claim 20, wherein said unique identifier is a radio-frequency identification tag.
[Claim 23]
The system of claim 1, wherein said penetratable membrane comprises a slit.
[Claim 24]
The system of claim 1, wherein said penetratable membrane is resealable.
[Claim 25]
The system of claim 1, wherein said penetratable membrane is formed of a polymeric material.
[Claim 26]
The system of claim 1, further comprising a detector that detects said biological sample or portion thereof.
[Claim 27]
The system of claim 26, wherein said detector detects said biological sample or portion thereof in said separation medium.
[Claim 28]
The system of claim 1, wherein said one or more computer processors are individually or collectively programmed to bring said flow channel in fluid communication with said separation medium.
[Claim 29]
The system of claim 28, wherein said flow channel is brought in fluid communication with said separation medium upon said flow channel piercing an additional penetratable membrane separating said flow channel from said separation medium.
[Claim 30]
The system of claim 4, wherein said housing comprises a securing element that prevents relative movement between said flow channel and said securing element.
[Claim 31]
The system of claim 30, wherein said securing element does not cover ends of said flow channel.
[Claim 32]
A method for sample analysis, comprising: (a) receiving a sample vessel comprising (i) a container that retains a biological sample and (ii) a penetratable membrane at an end of said container that seals said container in the absence of an object inserted through said penetratable membrane; (b) piercing said penetratable membrane to bring said biological sample in fluid communication with a flow channel, wherein at least a portion of said flow channel traverses said penetratable membrane, wherein said flow channel is in fluid communication with a separation medium that subjects said biological sample or a portion thereof to separation upon application of a flow-inducing field across said separation medium, which separation is on the basis of mass or charge; (c) subjecting said biological sample to flow from said container through said flow channel to said separation medium; and (d) applying said flow-inducing field across said separation medium under conditions that are sufficient to direct said biological sample through said separation medium to separate said biological sample or portion thereof on the basis of mass or charge.
[Claim 33]
The method of claim 32, wherein in (c) , said biological sample is subjected to flow from said container through said flow channel to said separation medium upon application of positive pressure or negative pressure.
[Claim 34]
The method of claim 33, wherein said positive pressure is applied by squeezing the container.
[Claim 35]
The method of claim 32, wherein in (a) , said sample vessel is received in a holder that receives and securably holds said sample vessel.
[Claim 36]
The method of claim 35, wherein at least a portion of said flow channel is secured in a housing, and wherein (b) comprises bringing said holder in contact with said housing.
[Claim 37]
The method of claim 36, wherein (b) comprises (1) bringing said holder in contact with or in proximity to said housing at a first position in which said penetratable membrane is not pierced, and (2) piercing said penetratable membrane by directing said housing or said holder to a second position.
[Claim 38]
The method of claim 37, further comprising bringing said flow channel in fluid communication with said separation medium by directing said housing to a third position.
[Claim 39]
The method of claim 37, wherein said housing further comprises a first covering element that covers an end of said flow channel when said housing is in said first position, wherein said first covering element allows said end of said flow channel to emerge from said first covering element when said housing or said holder is in said second position such that said end of said flow channel traverses said penetratable membrane.
[Claim 40]
The method of claim 39, wherein said housing further comprises a second covering element that covers another end of said flow channel when (i) said housing is in said first position or (ii) said housing or said holder is in said second position, wherein said second covering element allows said another end of said flow channel to emerge from said second covering element when said housing is in a third position such that said another end of said flow channel pierces an additional penetratable membrane separating said flow channel from said separation medium.
[Claim 41]
The method of claim 32, wherein (b) comprises using a cannula to pierce said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[Claim 42]
The method of claim 41, wherein said cannula comprises at least a portion of said flow channel extending therethrough.
[Claim 43]
The method of claim 41, wherein said cannula includes a tip at an end thereof, which tip pierces said penetratable membrane to bring said flow channel in fluid communication with said container.
[Claim 44]
The method of claim 32, wherein said separation medium is a polymeric separation medium.
[Claim 45]
The method of claim 44, wherein said separation comprises an agarose gel.
[Claim 46]
The method of claim 32, wherein said flow-inducing field is an electric field.
[Claim 47]
The method of claim 46, further comprising at least two electrodes on ends of said separation medium, wherein said at least two electrodes provide said electric field.
[Claim 48]
The method of claim 32, wherein said flow-inducing field is a pressure field.
[Claim 49]
The method of claim 32, further comprising using an actuator to pierce said penetratable membrane, thereby bringing said flow channel in fluid communication with said container.
[Claim 50]
The method of claim 32, further comprising a plurality of sample vessels, wherein a given sample vessel of said plurality of sample vessels comprises said container and said penetratable membrane.
[Claim 51]
The method of claim 32, further comprising a compartment having said separation medium.
[Claim 52]
The method of claim 51, further comprising a unique identifier on said compartment.
[Claim 53]
The method of claim 52, wherein said unique identifier is a barcode.
[Claim 54]
The method of claim 52, wherein said unique identifier is a radio-frequency identification tag.
[Claim 55]
The method of claim 32, wherein said penetratable membrane comprises a slit.
[Claim 56]
The method of claim 32, wherein said penetratable membrane is resealable.
[Claim 57]
The method of claim 32, wherein said penetratable membrane is formed of a polymeric material.
[Claim 58]
The method of claim 32, further comprising using a detector to detect said biological sample or portion thereof.
[Claim 59]
The method of claim 58, wherein said detector detects said biological sample or portion thereof in said separation medium.
[Claim 60]
The method of claim 32, wherein (c) further comprises bringing said flow channel in fluid communication with said separation medium.
[Claim 61]
The method of claim 60, wherein said flow channel is brought in fluid communication with said separation medium upon said flow channel piercing an additional penetratable membrane separating said flow channel from said separation medium.
[Claim 62]
The method of claim 36, wherein said housing comprises a securing element that prevents relative movement between said flow channel and said securing element.
[Claim 63]
The method of claim 63, wherein said securing element does not cover ends of said flow channel.
[Claim 64]
A non-transitory computer-readable medium comprising machine-executable code that, upon execution by one or more computer processors, implements a method for sample analysis, the method comprising: (a) receiving a sample vessel comprising (i) a container that retains a biological sample and (ii) a penetratable membrane at an end of said container that seals said container in the absence of an object inserted through said penetratable membrane; (b) piercing said penetratable membrane to bring said biological sample in fluid communication with a flow channel, wherein at least a portion of said flow channel traverses said penetratable membrane, wherein said flow channel is in fluid communication with a separation medium that subjects said biological sample or a portion thereof to separation upon application of a flow-inducing field across said separation medium, which separation is on the basis of mass or charge; (c) subjecting said biological sample to flow from said container through said flow channel to said separation medium; and (d) applying said flow-inducing field across said separation medium under conditions that are sufficient to direct said biological sample through said separation medium to separate said biological sample or portion thereof on the basis of mass or charge.

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