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1. WO2016176366 - DEVICE AND METHODS OF USING DEVICE FOR DETECTION OF HYPERAMMONEMIA

Примечание: Текст, основанный на автоматизированных процессах оптического распознавания знаков. Для юридических целей просьба использовать вариант в формате PDF

DEVICE AND METHODS OF USING DEVICE FOR DETECTION OF

HYPERAMMONEMIA

GOVERNMENT SUPPORT

This disclosure was made jointly by the NIH and with government support under

HHSN268201200360P awarded by the NIH. The United States government has certain rights in the disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is an international application designating the United States of America and filed under 35 U.S.C. §120, which claims priority to U.S. Provisional Serial Number 62/153,409, filed on April 27, 2015, which is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates generally to devices that quantify and identify the presence or absence of ammonia or ammonimum ion in a sample of bodily fluid, water, or other environmental sample. In some embodiments, the disclosure relates to diagnosing a subject with an hyperammonemia by detecting the presence, absence, or quantity of ammonia or ammonium ion in a sample of bodily fluid. In some embodiments, the device is a biosensor only requiring a sample of whole bodily fluid for detection and/or quantification of ammonia or ammonium ion.

BACKGROUND OF THE DISCLOSURE

Elevated ammoma levels, oftentimes called hyperammonemia, is a potentially fatal symptom associated with a variety of diseases such as cirrhosis of the liver and urea cycle disorders found in neonatal infants. Left untreated, hyperammonemia can lead to cognitive developmental issues, seizures, other neurological problems, and death. The current testing methods include fluorometry and tandem mass spectroscopy performed by central laboratories, which could take multiple days to produce a reliable diagnosis. These methods involve large, cumbersome, and expensive machinery, which prevents testing of ammonia levels at the bedside or home once the disorder has been identified. Therefore, a system for a point of care testing device may be desired, as this may allow administration of treatment to occur more rapidly, in turn improving the neurological development of infants as well as making cirrhosis more manageable. Devices able to test for hyperammonemia may also be modified

inexpensively to detect amino acid levels for applications in diagnosing and treating aminoacidopathies and other diseases.

SUMMARY OF DISCLOSURE

The present disclosure encompasses the recognition that hyperammonemia can be identified and/or characterized by identifying the levels or quantities of ammonia or ammonium ion in any sample, including a bodily fluid and, in some embodiments, including human and non-human whole blood samples. In some embodiments, the present disclosure relates to identifying the quantity, presence, or absence of ammonia or ammonium ion in bodily fluids by contacting a bodily fluid to a device disclosed herein. In some embodiments, the methods disclosed herein do not comprise contacting the bodily fluid with any reagent or external stimuli prior to identifying or quantifying whether or how much one or more ammonia or ammonium ion are present in the bodily fluid.

The present disclosure relates to a biosensor comprising: at least a first and second vessel; a fluid exchange opening positioned between the first and the second vessel; a membrane positioned across or over the fluid exchange opening; and a catalyst in solid phase within the second vessel or within a conduit in fluid communication with the second vessel; wherein the membrane comprises an ionomer.

The present disclosure also relates to a biosensor comprising: at least a first and second vessel; a fluid exchange opening positioned between the first and the second vessel; a membrane positioned across or over the fluid exchange opening; and a phenolic reagent stored in solid phase within the second vessel or within a conduit in fluid communication with the second vessel; wherein the membrane comprises an ionomer.

The present disclosure also relates to a biosensor comprising: at least a first and second vessel configured for receving a volume of a sample from a point exterior to the biosensor; a fluid exchange opening positioned between the first and the second vessel; a membrane positioned across or over the fluid exchange opening; and a catalyst in liquid phase or solid phase a hypohalite in liquid phase; an alkali buffer in liquid phase; a phenolic reagent in liquid phase or solid phase; and a fluid circuit comprising, in fluid communication: the first and second vessel; a reagent conduit; and a detection vessel positioned distal from the first and second vessel; wherein the catalyst is sodium nitroprusside, and, if the catalyst is in solid phase, the biosensor comprises from about 5.8 to about 7.3 micrograms of sodium nitroprusside or a salt thereof in solid phase; and, if the catalyst is in liquid phase, the sodium nitroprusside or a salt thereof is at a concentration greater than about 7 μΜ.

The present disclosure also relates to a system comprising the biosensor disclosed herein in operable connection to at least one computer storage memory. In some embodiments, the system comprises at least a first and second vessel configured for receving a volume of a sample from a point exterior to the biosensor; a fluid exchange opening positioned between the first and the second vessel; a membrane positioned across or over the fluid exchange opening; and a catalyst in liquid phase or solid phase a hypohalite in liquid phase; an alkali buffer in liquid phase; a phenolic reagent in liquid phase or solid phase; and a fluid circuit comprising, in fluid communication: the first and second vessel; a reagent conduit; and a detection vessel positioned distal from the first and second vessel; wherein the catalyst is sodium nitroprusside, and, if the catalyst is in solid phase, the biosensor comprises from about 5.8 to about 7.3 micrograms of sodium nitroprusside or a salt thereof in solid phase; and, if the catalyst is in liquid phase, the sodium nitroprusside or a salt thereof is at a concentration greater than about 7 μΜ; in operable connection to at least one computer storage memory. In some embodiments, the system comprises a computer processor in operable connection with the at least one: light emitting diode (LED), amplification circuit, battery, or stepper motor. In some embodiments, the system comprises a digital display in operable connection to at least one electrically conductive support by an electrical circuit capable of carrying an a electrical signal corresponding to a measurement of a wavelength, current, and/or voltage differential from a diode, spectrophotometer, voltmeter and/or amperoeter to the digital display, wherein the digital display is a configured to display concentration value of ammonia, ammonium ion and/or an amino acid in a sample when the at least one electrically conductive support is in contact with the sample for a time period sufficient for the at least one catalyst to catalyze the indophenol reaction. In some embodiments, the system compriss a solid support, such as a test strip or a chip or cartridge comprising a fluid circuit comprising: a reaction vessel in fluid

communication with a reagent conduit and a detection vessel. Each reagent disclosed herein may be added to the sample at the reaction vessel, mixing taskes place in the reagent conduit and detection occurs at the detection vessel. The chipr, cartridge or test strip may be inserted into a handheld device or tabletop device comprising diode, spectrophotometer, voltmeter and/or

amperometer and a digital display such that, when the test strip, cartridge or chip is contacted to the device, the first and second electrodes become operably connected to a closed electrical circuit comprising the voltmeter and/or amperometer and the digital display, and, upon contact with a sample, hypohalite, an alkali buffer, catalyst and at least one indophenol reagent or indophenol related compound catalyze an indophenol reaction resulting in a current on the first electrode corresponding to a concentration value of ammonia in the sample, such concentration value readable on the display of the device. In some embodiments, the reaction vessel is in fluid communication with a conduit configured to receive a sample from a point exterior to the test strip, cartridge, chip or device. In some embodiments, the reaction vessel is bifurcated laterally or vertically by a membrane comprising or consisting of an ionomer, such that the reaction vessel is split into a first and second vessel.

The present disclosure also relates to a kit comprising a biosensor or test strip comprising: at least a first and second vessel; a fluid exchange opening positioned between the first and the second vessel; at least one conduit in fluid communication with the at least first vessel, the at least one conduit configured to receive a fluid from a point external to the biosensor; and a membrane positioned at the fluid exchange opening; a catalyst in liquid phase or solid phase a hypohalite in liquid phase; an alkali buffer in liquid phase; a phenolic reagent in liquid phase or solid phase; and a fluid circuit comprising, in fluid communication: the first and second vessel; a reagent conduit; and a detection vessel positioned distal from the first and second vessel; wherein the catalyst is sodium nitroprusside, and, if the catalyst is in solid phase, the biosensor comprises from about 5.8 to about 7.3 micrograms of sodium nitroprusside or a salt thereof in solid phase; and, if the catalyst is in liquid phase, the sodium nitroprusside or a salt thereof is at a concentration greater than about 7 μΜ; and wherein the membrane comprises an ionomer.

The present disclosure also relates to a kit comprising a solid support that comprises: at least a first and second vessel; a fluid exchange opening positioned between the first and the second vessel; at least one conduit in fluid communication with the at least first vessel, the at least one conduit configured to receive a fluid from a point external to the biosensor; and a membrane positioned at the fluid exchange opening; a catalyst in liquid phase or solid phase; a hypohalite in liquid phase; an alkali buffer in liquid phase; a phenolic reagent in liquid phase or solid phase; and a fluid circuit comprising, in fluid communication: the first and second vessel; a reagent conduit; and a detection vessel positioned distal from the first and second vessel; wherein the catalyst is sodium nitroprusside, and, if the catalyst is in solid phase, the biosensor comprises from about 5.8 to about 7.3 micrograms of sodium nitroprusside or a salt thereof in solid phase; and, if the catalyst is in liquid phase, the sodium nitroprusside or a salt thereof is at a concentration greater than about 7 μΜ; and wherein the membrane comprises an ionomer.

The present disclosure also relates to a method of determining or identifying a concentration of an ammonia or ammonium ion in a sample comprising: contacting a sample to any biosensor disclosed herein, or any system disclosed herein; or any test strip disclosed herein; and determining a quantity of ammonia or ammonium ion in the sample.

The present disclosure also relates to a method of quantifying a concentration of ammonia or ammonium ion in a comprising contacting a sample of bodily fluid to any biosensor disclosed herein, or any system disclosed herein; or any test strip disclosed herein.

The present disclosure also relates to a method of diagnosing a metabolic disease in a subject comprising: (a) contacting a sample of bodily fluid to the to any biosensor disclosed herein, or any system disclosed herein; or any test strip disclosed herein; (b) quantifying one or more concentration values of ammonia in the sample; (c) comparing the one or more concentration values of ammonia in the sample to a threshold value of ammonia concentration identified as being in a healthy range; and (d) identifying the subject as having a metabolic disease if the one or more concentration values of ammonia in the sample exceed or fall below the threshold value.

The present disclosure also relates to a method of determining patient responsiveness to a therapy comprising: (a) contacting a sample of bodily fluid to any biosensor disclosed herein, or any system disclosed herein; or any test strip disclosed herein; (b) quantifying one or more ammonia or ammonium ion concentration values; (c) comparing the one or more concentration values to one or more threshold values associated with a metabolic disease.

The present disclosure also relates to a test strip comprising a solid support comprising: at least a first and second vessel configured for receving a sample from a point exterior to the biosensor; a fluid exchange opening positioned between the first and the second vessel; a membrane positioned across or over the fluid exchange opening; and a catalyst in liquid phase or solid phase; a hypohalite in liquid phase; an alkali buffer in liquid phase; a phenolic reagent in liquid phase or solid phase; and a fluid circuit comprising, in fluid communication: the first and second vessel; a reagent conduit; and a detection vessel positioned distal from the first and second vessel; wherein the catalyst is sodium nitroprusside, and, if the catalyst is in solid phase, the biosensor comprises from about 5.8 to about 7.3 micrograms of sodium nitroprusside or a salt thereof in solid phase; and, if the catalyst is in liquid phase, the sodium nitroprusside or a salt thereof is at a concentration greater than about 7 μΜ; and wherein the membrane comprises an ionomer.

The present disclosure also relates to a method of manufacturing any biosensor disclosed herein, or any system disclosed herein; or any test strip disclosed herein comprising affixing the membrane between the first and/or second vessel.

The present disclosure also relates to a method of detecting the presence, absence, or quantity of amino acid in a sample comprising: (a) contacting a sample of bodily fluid to any biosensor disclosed herein, or any system disclosed herein; or any test strip disclosed herein; or any catridge disclosed herein; (b) quantifying one or more ammonia or ammonium ion concentration values; (c) correlating the one or more ammonia or ammonium ion concentration values to one or more quantities of an amino acid.

The present disclosure also relates to a method of treating a metabolic disease comprising: (a) contacting a sample of bodily fluid to the to any biosensor disclosed herein, or any system disclosed herein; or any test strip disclosed herein; (b) quantifying one or more concentration values of ammonia in the sample; (c) comparing the one or more concentration values of ammonia in the sample to a threshold value of ammonia concentration identified as being in a healthy range; and (d) identifying the subject as having a metabolic disease if the one or more concentration values of ammonia in the sample exceed or fall below the threshold value; (e) administering a therapeutically effective amount of a therapeutic agent to treat metabolic disease.

The disclosure relates to a method of detecting or quantifying ammonia in a blood sample from a subject comprising: wiping a portion of the subject from which the blood will be drawn with saline solution; inserting a sharp device into the portion of the body from which blood will be drawn at a an angle and depth in the skin sufficient to draw at least a droplet of blood from the point of insertion; contacting at least a droplet of blood from the surface of the body to a sensor surface. In some embodiments, the method of detecting or quantifying ammonia in a blood sample from a subject is free of wiping the portion of the subject from which the blood will be drawn with a pad, swab or solution comprising iodine or alcohol. In some embodiments, wiping a portion of the body from which the blood will be drawn with a swab, wipe, or solution comprising saline but free of iodine, detergent or alcohol.

BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of embodiments of the present invention will be apparent from the following detailed description of the exemplary embodiments. The following detailed description should be considered in conjunction with the accompanying figures in which:

FIG. 1 depicts a photograph of 3D printed modular pieces snapped together around Nafion to form the bisected well.

FIG. 2 depicts a CAD sketch of the top piece of a disposable cartridge, with dimensions in mm. Channels 1 through 5 are labeled.

FIG. 3 : CAD sketch of the bottom piece of the chip with channel 6 labeled.

FIG. 4: depicts the representations of a concentration profile at i) t=0 seconds (s) ; ii) t=13 s , and iii) t=24 s after a whole blood sample in 40 microliters is loaded into well number 6.

FIGs. 5A and 5B depict a disposable cartridge with reagents contained in blister packs and a reader. FIG. 5A depicts a diagram of the cartridge, comprising three blister packs. FIG. 5B depicts a sample cartridge reader.

FIG. 6 is an exemplary view of a system having the ability to detect ammonia or ammonium ion levels in a given sample applied to a first and second vessel separated by a membrane positioned at an fluid exchange opening.

FIG. 7 is an exemplary view of a system comprising multiple vessels within which more than one indophenol reaction mat be performed in parallel.

FIG. 8 shows exemplary reaction otherwise known as Berthelot's Reaction or an indophenol reaction.

FIG. 9 shows an exemplary embodiment of a microfluidic testing device.

FIG. 10 shows an exemplary flowchart for a method of quantitative point of care

hyperammonemia sensing using embodiments of the disclosure.

FIG. 11 shows an exemplary embodiment of a blood test strip for use with an electronic testing device.

FIG. 12 shows an exemplary embodiment of a device comprising an electronic circuit comprising an electrode exposed to a vessel configured for performanceof the indophenol reaction; an analog to digital converter, a microchip in electronic communication with a display.

FIG. 13 shows the chemical composition of Nafion.

FIG. 14 depicts the detection of 500 micromolar ammonia solutions using various concentrations of sodium nitroprusside and the indophenol reaction. Appropriate amounts of the other reagents were used in this optimization. A concentration range of 1330 - 1996 micromolar sodium nitroprusside is optimal for detection of ammonia.

FIG. 15 depicts the response of solutions used for the indophenol reaction to 500

micromolar ammonium chloride over the course of 245 days at room temperature. The response to the ammonia chloride was stable for the entire experiment.

FIG. 16 depicts a calibration curve for detecting blood ammonia concentrations using a cartridge of the disclosure. Concentrations of blood ammonia were generated by either spiking whole blood samples or allowing them to hydrolyze at room temperature. The calibration curve has an R2 of 0.999 and a measured sensitivity of 0.288 a.u./μΜ of ammonia. The calibration curve covers concentrations of blood ammonia ranging from 20 to 500 μΜ of ammonia and thus cover the entire physiologically normal to highly elevated range. This calibration curve was utilized to determine the limit of detection and limit of quantification, which were 8 μΜ and 27 μΜ

respectively.

FIG. 17 depicts a calibration curve for comparing the detection of blood ammonia concentrations using the Siemens EXL, a clinical chemical analyzer, versus a cartridge of the disclosure.

FIG. 18 depicts a calibration curve produced through the use of dried 2-phenylphenol and sodium nitroprusside reconstituted using sodium hypochlorite, sodium hydroxide and ammonium solution. The produced curve was linear from concentrations of 0-500 micromolar ammonium.

FIG. 19 depicts the measured ammonia concentration from a fingerstick blood draw after various fingertip washing methods.

FIGS. 20A and 20B depict a radar graph showing the detection of amno acids.

FIG. 21 A and 21B show the review of conentrations of reagents used in the experiments.

DETAILED DESCRIPTION OF THE DISCLOSURE

Various terms relating to the methods and other aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1%) from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, the terms "attach," "attachment," "adhere," "adhered," "adherent," or like terms generally refer to immobilizing or fixing, for example, a group, a compound or enzyme, to a surface, such as by physical absorption, chemical bonding, and like processes, or combinations thereof.

As used herein, the terms "biopsy" means a cell sample, collection of cells, or bodily fluid removed from a subject or patient for analysis. In some embodiments, the biopsy is a bone marrow biopsy, punch biopsy, endoscopic biopsy, needle biopsy, shave biopsy, incisional biopsy, excisional biopsy, or surgical resection. In any of the methods disclosed herein, the method may comprise a step of isolating a section of tissue by any biopsy technique described above and grinding the tissue or extacting blood from the tissue to create fluid sample.

As used herein, the terms "bodily fluid" means any fluid from a isolated from a subject including, but not necessarily limited to, blood sample, serum sample, a whole blood sample, urine sample, mucus sample, saliva sample, and sweat sample. The sample may be obtained from a subject by any means such as intravenous puncture, biopsy, swab, capillary draw, lancet, needle aspiration, collection by simple capture of excreted fluid.

As used herein, the word "exemplary" means "serving as an example,

instance or illustration." The embodiments described herein are not limiting, but rather are exemplary only. It should be understood that the described embodiment are not necessarily to be construed as preferred or advantageous over other embodiments. Moreover, the terms

"embodiments of the invention", "embodiments" or "invention" do not require that all

embodiments of the invention include the discussed feature, advantage or mode of operation. In addition, those skilled in the art may appreciate the wide variations in sizing scales that may be incorporated into the disclosed or related designs for use with samples many orders of magnitude larger or smaller than those disclosed.

As used herein, the term "aminoacidopathy" is meant to refer to those diseases and disorders characterized by dysfunction of a metabolic catalysis of amino acids thate results in over production or under production of amino acids in the body of a subject. . Examples of aminoaciopathies ar elisted in the definition of a metabolic disease, terms that are used interchangeably in this application.

As used herein the terms "electronic medium" mean any physical storage employing electronic technology for access, including a hard disk, ROM, EEPROM, RAM, flash memory, nonvolatile memory, or any substantially and functionally equivalent medium. In some

embodiments, the software storage may be co-located with the processor implementing an embodiment of the disclosure, or at least a portion of the software storage may be remotely located but accessible when needed.

As used herein, "sequence identity" is determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). To use the term "homologus to" is synonymous with a measured "sequence identity." In some embodiments, if an embodiment comprises a nucleic acid sequence or amino acid sequence with a percent sequence identity the term refers to a disclosed nucleic acid sequence or amino acid sequence possessing a homology to a disclosed sequence over its entire length.

The term "subject" is used throughout the specification to describe an animal from which a sample of bodily fluid is taken. In some embodiment, the animal is a human. For diagnosis of those conditions which are specific for a specific subject, such as a human being, the term "patient" may be interchangeably used. In some instances in the description of the present disclosure, the term "patient" will refer to human patients suffering from a particular disease or disorder. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop a metabolic disease, such as hyperammonemia. In some embodiments, the subject may be diagnosed as having at least one aminoacidopathy. In some embodiments, the subject is suspected of having or has been diagnosed with hyperammonemia. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop hyperammonemia. In some embodiments, the subject may be a mammal which functions as a source of the isolated sample of

bodily fluid. In some embodiments, the subject may be a non-human animal from which a sample of bodily fluid is isolated or provided. The term "mammal" encompasses both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. Any methods disclosed hereing may comprise testing a blood sample from a mammal, human, non-human, or any other non-human animal disclosed herein.

As used herein, "conservative" amino acid substitutions may be defined as set out in Tables A, B, or C below. Metabolic enzymes include those amino acid sequences wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides disclosed herein. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution, whjich may be a part of any amino acid disclosed herein, is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.

Table A— Conservative Substitutions I

Side Chain Characteristics Amino Acid

Aliphatic

Non-polar G A P I L V F

Polar - uncharged C S T M N Q

Polar - charged D E K R

Aromatic H F W Y

Other N Q D E

Alternately, conservative amino acids can be grouped as described in Lehninger,

(Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71-77) as set forth in Table B.

Table B— Conservative Substitutions II

Side Chain Characteristic Amino Acid

Non-polar (hydrophobic)

Aliphatic: A L I V P

Aromatic: F W Y

Sulfur-containing: M

Borderline: G Y

Uncharged-polar

Hydroxyl: S T Y

Amides: N Q

Sulfhydryl: C

Borderline: G Y

Positively Charged (Basic): K R H

Negatively Charged (Acidic): D E

Alternately, exemplary conservative substitutions are set out in Table C.

Table C— Conservative Substitutions III

Original Residue Exemplary Substitution

Ala (A) Val Leu He Met

Arg (R) Lys His

Asn (N) Gin

Asp (D) Glu

Cys (C) Ser Thr

Gin (Q) Asn

Glu (E) Asp

Gly (G) Ala Val Leu Pro

His (H) Lys Arg

He (I) Leu Val Met Ala Phe

Leu (L) He Val Met Ala Phe

Lys (K) Arg His

Met (M) Leu He Val Ala

Phe (F) Trp Tyr He

Pro (P) Gly Ala Val Leu He

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr Phe He

Tyr (Y) Trp Phe Thr Ser

Val (V) He Leu Met Ala

It should be understood that the polypeptides comprising polypeptide sequences associated with the extracellular matrix described herein are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues.

As used herein, the term "prognosing" means determining the probable course and/or outcome of a disease.

As used herein, the terms "indophenol related compound" mean a small chemical compound that is a reaction product of an indophenol reaction. In some embodiment, it comprises at least one carbon atom in a 4, 5, 6-membered ring and emits a visible wavelength of light upon excitation of the small chemical compound by light emitted by from light source. In some embodiments, the small chemical compound is a product of the indophenol reaction and emits a wavelength of light visible to the human eye upon excitation of the chemical compound by light emitted from a light source. In some embodiments, the small chemical compound emits a wavelength from about 400 nm to about 700 nm when it is excited by light from a light source. In some embodiments, the small chemical compound emits a wavelength of about 635 nm when it is excited by light from a light source. In some embodiments, the biosensor, device, and/or system comprises a light source and at least one diode and/or spectrophotometer, or other device capable of measuring the light emitted by the indophenol or the indophenol related compound when said indophenol or indophenol related compound is exposed to light.

The term "vessel" as used herein is any chamber, indentation, container, receptacle, or space. In some embodiments, a vessel is a well capable of holding no more than about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μΐ^ of total volume. In some emboidments, the reaction vessel comprises the first and second vessels separated by a membrane and each of the first or second vessels is no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 \iL of total volume. In some embodiments, the total volume of the first and second vessels combinaed is no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 \iL of total volume. The biosensor, chip, cartridge, test strip or solid support disclosed herein can include multiple vessels in fulid communication with each other. In some embodiments, the biosensor, chip, cartridge, test strip or solid support comprises a reaction vessel which is configured to receive a sample or portion of a sample. In some embodiments, the biosensor, chip, cartridge, test strip or solid support comprises a

a detection vessel, which is configured to be near to substantially near a diode or some other discloded device capable of stimulating the contents of the detection vessel and enabling detection of the amount of ammonia in the vessel. In some embodiments, the biosensor, chip, cartridge, test strip or solid support comprises a reagent conduit, which may be branched or unbranched, linear, curved, or not linear, that connects the reaction vessel to the detection vessel. In some

embodiments, at least a portion of the reagent conduit comprises at least one, two or more components of the indophenol reaction in solid phase, such as a powder. In some embodiments, at least a portion of the reagent conduit comprises a nonlinear portion which has two or more parallel paths of fluid flow before fluid passing through the reagent conduit reaches the detection vessel. The parallel portions of fluid path enable mixing of all of the reagents and completion of a sufficient number of indophenol reaction prior to detection of the ammonia in a sample. In some

embodiments, the biosensor, chip, cartridge, test strip or solid support comprises a conduit, which may be branched or unbranched, linear, curved, or not linear, that connects the reaction vessel to one, two, three, four, five or more reagent storage vessels. The reagent storage vessels may be from about 5 microliters to about 100 microliters in volume and store any of the disclosed regaents in liquid phase. In some embodiments, the regaent storage vessel is from about 5 microliters to about 50 microliters in volume. In some embodiments, the regaent storage vessel is from about 5 microliters to about 40 microliters in volume. In some embodiments, the regaent storage vessel is from about 5 microliters to about 30 microliters in volume. In some embodiments, the regaent storage vessel is from about 5 microliters to about 20 microliters in volume. In some embodiments, the regaent storage vessel is from about 5 microliters to about 10 microliters in volume.

The term "membrane" means any monomer or polymer in a solid phase. In some

embodiments, the membrane comprises an ionomer. In some embodiments, the membrane is incapable of gas chromatography. In some embodiments, the membrane comprises an ionomer and is formed ni the shape of a sheet which is capable of extension over or between one or more openings.

The terms "point of care" disclosed herein refer to a device, biosensor, system, test strip, or cartridge, either individually or configured to function with one or more additional components, capable of analyzing the presence, absence, or quantity of a reaction product, such as ammonia, and/or a sample component, such as an amino acid, within a time period no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 minutes. In some embodiments, the terms refer to a device, biosensor, system, chip, test strip, or cartridge, either individually or configured to function with one or more additional components, capable of analyzing the presence, absence, or quantity of ammonia and/or an amino acid within a time period no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or 40 minutes, or capable analyzing the presence, absence, or quantity of ammonia and/or an amino acid at or substantially near the point from which the sample was taken. For instance, in some embodiments, the sample may be taken from a subject suspected of or previously diagnosed with hyperammonemia or a hyperammonemia-related disorder. Without sending and analyzing the ammonia content of a sample to a different location from the source of the sample, in some embodiments, the point of care device or biosensor or system is a point of care device which is capable of detecting the presence, absence, or quantity of ammonia or ammonium ion in a sample.

The term "fluid exchange opening" means any space or void through which a fluid may pass from one vessel to an adjacent vessel or another vessel in fluid commuinication with the one vessel.

The terms "individually comprise" in respect to a claimed element or elements mean that only one claimed element comprises each of the listed elements and not in combination with any other element named.

The terms "a compound comprising a phenol substituent" means any molecule comprising a phenyl group attached to a 4, 5, 6, or more-membered atomic ring comprising at least one carbon atom.

The term "ionomer" as used herein refers to any polymer comprising an ion. In some embodiments, the ionomer is a perflurinated ionomer. In some embodiments, the ionomer comprises Formula I or a salt thereof.

Formula I:


X2

Where F2-S02, or F2-CF2-C02CH3

X2=CF3, or, if X! is F2, X2 is null

Where Y=CF2-S02F, CF2-CF-S02F, or CF3-C02CH3

In some embodiments, the ionomer comprises one or a combination of:

CF2=CF-0-CF2-CF-0-CF2-CF2-S02F

CF3,

CF2=CF-0-CF2-CF-0-CF2-CF2-CF2-S02F

CF3,

CF2=CF-0-CF2-CF-0-CF2-CF3-C02CH3

CF3,

CF2=CF-0-CF2-CF2-CF2-C02CH3,

CF2=CF-0-CF2-CF2-S02F,

[-(CF2-CF2)n-CF-CF2-]m

0-CF-CF2-0-CF2-SO-3M+

CF3,

or a salt thereof, wherein n and m are any positive integer. In some embodiments, n and/or m are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, n and/or m are independently variable and any positive integer from about 1 to about 1000. In some embodiments, n and/or m are independently variable and any positive integer from about 1 to about 500.

The term "bodily fluid" means any sample taken from an animal including a human, or non-human animal.

As used herein, the term "functional fragment" means any portion of a disclosed

polypeptide that is of a sufficient length to retain at least partial biological function that is similar to or substantially similar to the function of the wild-type polypeptide upon which the fragment is based. In some embodiments, a functional fragment of a polypeptide associated with the function of a metabolic enzyme is a polypeptide that comprises at least 70%, 75%, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity of any polypeptides disclosed herein and has sufficient length to retain at least partial binding affinity to one or a plurality of substrates that bind to the polypeptide. In some embodiments, the fragment is a fragment of any polypeptide disclosed herein and has a length of at least about 10, about 20, about 30, about 40, about 50 , about 60, about 70, about 80, about 90, or about 100 contiguous amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed herein and has a length of at least about 50 amino acids. In some

embodiments, the fragment is a fragment of any polypeptide disclosed herein and has a length of at least about 100 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed herein and has a length of at least about 150 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed herein and has a length of at least about 200 amino acids. In some embodiments, the fragment is a fragment of any polypeptide disclosed herein and has a length of at least about 250 amino acids.

As used herein, the terms "polypeptide sequence associated with the metabolic enzyme" means any polypeptide or fragment thereof, modified or unmodified by any macromolecule (such as a sugar molecule or macromolecule), that is a metabolic enzyme as diclosed herein or a functional fragment thereof. In some embodiments the polypeptide sequence is is synthetic or recombinantly produced in any multicellular or unicellular organism. In some embodiments, a polypeptide sequence associated with the extracellular matrix is any polypeptide which sequence comprises any of the polypeptides disclosed in Table 2. In some embodiments, a polypeptide sequence associated with the metabolic enzyme is any polypeptide sequence comprising any of the polypeptides disclosed in Table 2 or a sequence that shares 85,90,95, 96, 97, 98, or 99% sequence identity with the polypeptides disclosed in Table 2 or a functional fragment thereof. In some embodiments, a polypeptide sequence associated with the metabolic enzyme consists of any of the polypeptides

disclosed in Table 2 or a sequence that shares 85, 90, 95, 96, 97, 98, or 99% sequence identity with the polypeptides disclosed in Table 2.

The term "salt" refers to acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. Examples of these acids and bases are well known to those of ordinary skill in the art. Such acid addition salts will normally be

pharmaceutically acceptable although salts of non-pharmaceutically acceptable acids may be of utility in the preparation and purification of the compound in question. Salts include those formed from hydrochloric, hydrobromic, sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic, succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.

In some embodiments, the device, system, membrane, or vessel, may comprise any of the disclosed reagents or formula disclosed herein or any salt. Salts may be formed by reacting the free base, or a salt, enantiomer or racemate thereof, with one or more equivalents of the appropriate acid. In some embodiments, salts of the present invention refer to salts of the disclosed reagents or formula disclosed herein having at least one basic group or at least one basic radical. In some embodiments, salts of the present invention refer to salts of the disclosed reagents or formula disclosed herein having a free amino group, a free guanidino group, a pyrazinyl radical, or a pyridyl radical that forms acid addition salts. In some embodiments, salts of the present invention refer to salts of the disclosed reagents or formula disclosed herein that are acid addition salts of the subject compounds with (for example) inorganic acids, such as hydrochloric acid, sulfuric acid or a phosphoric acid, or with suitable organic carboxylic or sulfonic acids, for example aliphatic mono-or di-carboxylic acids, such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid or oxalic acid, or amino acids such as arginine or lysine, aromatic carboxylic acids, such as benzoic acid, 2-phenoxy-benzoic acid, 2-acetoxybenzoic acid, salicylic acid, 4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such as nicotinic acid or isonicotinic acid, aliphatic sulfonic acids, such as methane-, ethane- or 2-hydroxyethane-sulfonic acid, or aromatic sulfonic acids, for example benzene-, p-toluene- or naphthalene-2-sulfonic acid. When several basic groups are present mono-or poly-acid addition salts may be formed. The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, for example, water, dioxane, ethanol, tetrahydrofuran or diethyl ether, or a mixture of solvents, which may be removed in vacuo

or by freeze drying. The reaction may also be a metathetical process or it may be carried out on an ion exchange resin. Salts according to the present invention may be found in their anhydrous form or as in hydrated crystalline form (i.e., complexed or crystallized with one or more molecules of water).

As used herein, the term "antibody" refers to any immunoglobulin, whether natural or wholly or partially synthetically produced. In some embodiments, an antibody is a complex comprised of 4 full-length polypeptide chains, each of which includes a variable region and a constant region, e.g., substantially of the structure of an antibody produced in nature by a B cell. In some embodiments, an antibody is a single chain. In some embodiments, an antibody is cameloid. In some embodiments, an antibody is an antibody fragment. In some embodiments, an antibody is chimeric. In some embodiments, an antibody is bi-specific. In some embodiments, an antibody is multi-specific. In some embodiments, an antibody is monoclonal. In some embodiments, an antibody is polyclonal. In some embodiments, an antibody is conjugated (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins). In some embodiments, an antibody is a human antibody. In some embodiments, an antibody is a mouse antibody. In some embodiments, an antibody is a rabbit antibody. In some embodiments, an antibody is a rat antibody. In some embodiments, an antibody is a donkey antibody. In some embodiments, the biosensor or system described herein comprises an antibody or plurality of antibodies.

Characteristic: As is used herein, the term "characteristic" refers to any detectable feature of a sample of bodily fluid that allows it to be distinguished from a comparable sample of bodily fluid. In some embodiments, a characteristic is an amount or identity of ammonia or ammonium ion in bodily fluid, in an environmental sample, or water sample. In some embodiments, a characteristic is an amount, sequence of, or modification of a amino acid. In some embodiments a characteristic is an amount of a carbohydrate. In some embodiments, a characteristic is an amount of a small molecule.

Comparable: As is used herein, the term "comparable" is used to refer to two entities that are sufficiently similar to permit comparison, but differing in at least one feature.

Metabolic Enzyme: As is used herein, the term "metabolic enzyme" means an enzyme responsible for catalysis of at least one step in the metabolic pathway of one or more amino acids. In some embodiments, the metabolic enzyme is phenylalanine dehydrogenase, glutamate

dehydrogenase, respective functional fragments or a combination thereof or a fusion protein thereof.

As used herein the terms "metabolic disease" is any one of a group of disorders caused by a defect in an enzymatic step in the metabolic pathway of one or more amino acids or in a protein mediator necessary for transport of certain amino acids into or out of cells. In some embodiments, the metabolic disease is chosen from: Argininemia (ARG, arginase deficiency) Argininosuccinate acidemia (ASA, argininosuccinase) Citrullinemia type I (CIT-I, argininosuccinate synthetase) Citrullinemia type II (CIT-II, citrin deficiency) Defects of biopterin cofactor biosynthesis (BIOPT-BS) Defects of biopterin cofactor regeneration (BIOPT-RG) Homocystinuria (HCY, cystathionine beta synthase) Hyperphenylalaninemia (H-PHE) Hypermethioninemia (MET) Maple syrup urine disease (MSUD, branched-chain ketoacid dehydrogenase) Phenylketonuria (PKU, phenylalanine hydroxylase) Tyrosinemia type I (TYR-1, fumarylacetoacetate hydrolase), Tyrosinemia type II (TYR-II, tyrosine aminotransferase), and Tyrosinemia type III (TYR-III, hydroxyphenylpyruvate dioxygenase) where the parenthetical phrases after each disease state represent an abbreviation for the disease accompanies by the enzyme that is generally defective in the subject suffering from the disease state. In some embodiments, the metabolic disease is a urea cycle disorder or

hyperammonemia.

Polypeptide: The term "polypeptide", as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term "polypeptide" is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying or significantly reducing activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, 75%, 80%, or 85%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term "polypeptide" as used herein.

As used herein, the term "therapeutically effective amount" refers the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician. The therapeutic effect is dependent upon the disorder being treated or the biological effect desired. As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and/or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects. The amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject's response to treatment. In some embodiments, the therapeutically effective amount comprises the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician to treat or prevent metabolic disease, such as hyperammonemia.

As used herein, the term "threshold value" is the concentration of ammonia or ammonium ion or amino acid in a sample of bodily fluid that indicates whether the amount of ammonia or ammonium ion or amino acid in the sample is considered abnormally high or low resulting in a diagnosis or suspected diagnosis of a particular disorder, such as a metabolic disease. For instance, in the case of a blood sample, known threshold values for certain aminoacidopathies are indicated in Table 1 below:

Table 1 : Aminoacidopathies and their associated amino acid markers detectable in a sample


PKU, H-PHE Phenylalanine > 130 umol/L BIOPT-BS and BIOPT-RG Phe/Tyr > 2.0

TYR-I, TYR-II, and TYR-III Tyrosine > 250 umol/L

In some embodiments, information about a threshold value or reference sample of bodily fluid is obtained prior to or simultaneously with information about an experimental sample of bodily fluid. In some embodiments, information about a reference cell or cell type is historical. In some embodiments, information about a threshold value or reference sample of bodily fluid is stored for example in a computer-readable storage medium. In some embodiments, comparison of a particular concentration value with a threshold value or reference sample of bodily fluid differentiates the concentration values of ammonia in an experimental sample of bodily fluid with the threshold values thereby allowing a comparison that results in diagnosing a subject with one or more metabolic diseases or a change in severity of one or more metabolic diseases.

Reference electrode: As will be understood from context, a reference electrode or control electrode is an electrically conductive support such as an electrode placed in a circuit with an at least one electrically conductive support comprising hydrogel and/or immobilized enzymes disclosed herein, to permit a relevant comparison of voltage difference between the reference or control electrode and the at least one electrically conductive support comprising hydrogel and/or immobilized enzymes disclosed herein.

Sample: As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs;

washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises bodily fluid. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some

embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term "sample" refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a "processed sample" may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc. in some embodiments, the methods disclosed herein do not comprise a processed sample. In some embodiments, the methods disclosed herein comprise taking a sample from water or other environmental surface, processing the sampel to include a known volume, and exposing the sample to the biosensor, system, or test strip disclosed herein.

As used herein "whole blood" means blood that is taken directly from the subject and unprocessed by filtration or additives prior to manipulation, in some embodiments, whole blood may comprise anti-coagulants. In some embodiments, whole blood is free of anti-coagulants.

In some embodiments, the system, test strip, device, biosensor, chip and/or cartridge comprises a concentration of any one or combination of the reagents disclosed on pages 78-84 of this disclosure.

Table 2

Enzyme Gene Sequence Accession

Numbers

Phenylalanine ATGGAAATCTTCGAGGAAATCAAACGGCGGGGACACGAGCAA AEW06037.1

Dehydrogenase ATTCTGTTCAATTATGATCGGGCTTCCG YP 005257709.1

GTTTGAAAGCAATTATCGCCATTCACAATACTACGTTGGGGC AEH47572.1

CGGCGTTGGGCGGGTGCCGAATGTTACC YP 004587653.1

YP 004581770.1

GTATCAAACGGAAGAGGCGGCCCTCGAGGATGCGCTGCGGTT

AEH07849.1

GTCGGAAGGGATGACCTATAAAGCGGCC

ACF96938.1

GCCGCCGGGCTCGATTTCGGCGGGGGCAAAACGGTGATTATC YP 007466124.1 GGGGAT C CGATGAAAGAC AAGT C CGAGG EZP75760.1

CCCTGTTTCGTGCGCTCGGGCGTTTTATCGAGACCTTGAAAG AGT95551 .1

GCCGTTACCTTACGGGAGAAGACGTAGG EWG09095.1

AACCAACGAAGAAGATTTTGTCTGGGCTCGTCGGGAAACCCG YP 008456272.1 TTATGTTGTCGGATTGCCGCCGGCTTAT EME23486.1

GGCGGGTCCGGCGATACGGGTGACAATACCGCGCGCGGCGTC EJS99791 .1

ATTCAAGCGATGCGCGCCGCGTTGATGC EIT85807.1

AAA22646.1

ACCGGTACGGTTCGCCGGATCTCCAGGGCCGGCGGATTGCCG

EDL64419.1

T C C AAGGG C TGGG C AAAGT AGG C T AT C A

EAR66050.1

TGTGGCGCGACGGGCCATCGAGGCCGGCGCTCGAGTGATTGC BAA08816.1

GGCCGATAT CAAT C CGCATGTAGT CGGC

CGAGTGGCGTCCGCTTGGGGGATTGAAGCCACCGATCCGTGG GCTGTGGTGGAAACCCCCTGCGATATTT

TCGCCCCCTGTGCGTTGGGTAACGTCATTACGGAACGGACCG TGTCCGCCCTCCAATGTCAGGTGGTGGC

CGGTTCGGCCAACAATCAGCTGGCGGATGATCGACTGGCCGA TGATTTAGCTGCCCGCGGCATTCTCTAT

GCGCCGGATTTTATTGCGAATGCCGGCGGATTGATTCAGGTG GCGGATGAAATTCGGGGATATCATGAAG

AACGGGT CCGT CAT C AAAT AGACGGGATTT ATGACGT C CTGC TCGAGATTTTTCGGAAGGCGGACGCCTC

CGGCCGATCAACCGTGGCGGTTGCGGTAGACGAGGCGCGTCG

CCGTTTGGACACCATTCAGGCCATCCAC

CGCCTGTACGGATCATAG

Phenylalanine CTGCAGGTCAACGGATCATATTCTACACATATATAATGCACTCCAATTGA AAA34179.2

Ammonia-Lyase CATAATACATAACGTGACAT ADR78835.1

ATGATACATTTATTAATATTAATTGTCACATTTACACTTCACATATTAAA AAA99500.1 ATACTCTCGTATGAATGCAA AAC18871 .1

AAC 18870.1

TTTGAAACATATTTTAAATTAATTGATTGATATATATTGAACAAAACCTA AAA33805.1 ACAAAAATGCACCCTCTTGG AIC66437.1

TTCACAAAGAAACTTTCTTCTATTTCTCACTTATTTCTGCTAGTGTCTTT AGY49231 .1

CCTATTCAAAGCCATCATTT AEW43005.1

CCATCAACCTTCACAATACCATGTTTAAAAAGTCATTAAAAATCAATTTT AFP24940.1

TTAAATAGAAAAAAACAAGA AER58180.1

AGATGGAAATCACTTGGTTGGTACTATATATTTAGTTGTTAAGTTTGACT ADD12041 .1

CAT AC CGTGT ATTGAC CAAT AEE81750.1

AT AAAT AAAAT C T T AT T T C AAAT AAAT T C AAAAGT T C AAT AAAT AT AT AT AAP59440.1 TCGTTCATAACTTATAATAA AAP59439.1

AAP59438.1

AATTGATTATACATAGTCCTCCCCCATTCACTTTTACTGATCAATTATTT ACG80829.1 C T AAAAT AT AT TATTACTTT ACG80828.1

TACTTGTTATTTTTAATAAATTAAGAAAATATAATACTCCCTTCGTTTTT ACG56648.1

AAAAAAATACCTAGTTTGAC ACG56647.1

TTGAAACGGAGTTTAATAAAAGAAAGAAGACTTGTTAATCTTGTGATTCT

AAATTAAAGTTATGTCAAAT

GTACCAAAATGTCCTTTAATCTTGTGGTCTTAAACATGTCACATGAAAAA

TTAAAGTGTTTCCAAAAAAA

GAAAGGGGTCAATGTCATTCTTTTTTAAACAGACTAAAAAAGAAATAAAC

TCATTCTTTTTGAAACGGAG

AGAGTAATTTTTTCCACGTTTTACTCATTAATATTAAATATTATTCTCTA

GAT C AT C C T AT AAG AT C T AA

TAGTGGACATCAATTAATACCTATGTCACTTATTATTATTTTAATAATTG

TAT CAAGT C AAAT AAT AAC A

AGTAAAAATGGAGTACCTACTATTAATCTTCAACAACCACAATTTACTAG

TTTTTTCCTAGCAACCCCCT

CTCACATATTTCACCATTTACTGGTTTTTTCCTAGCAACCCCCTCTCACA

TATTTTGTTTACCAACCATC

ATTTGTTCCTCTATATATACTCACCACATGATAGATACATATATATACCA

CAACCAAAACAAAAGGTTTT

ATAAGTTCACAACATTTTTTATATACATACAAATAAACTCTAACCATTTT

CTCTTCACTAAAATTTCTTC

ATTACAAATCTAACAATTTACTTGATCCAATGGCACCATCAATTGCACAA

AATGGACATATTAATGGAGA

AGTAGCTATGGATTTGTGCAAGAAATCAATCAATGATCCATTGAATTGGG

AAATGGCTGCTGATTCTTTA

AGAGGCAGCCATTTGGATGAAGTGAAAAAGATGGTGGATGAATTTAGAAA

GCCAATTGTGAAACTTGGGG

GTGAAACTTTGTCAGTTGCACAAGTTGCATCCATTGCAAATGTTGATGAC

AAAAGTAATGGGGTTAAAGT

GGAACTTTCTGAAAGTGCAAGGGCTGGTGTGAAAGCTAGTAGTGATTGGG

TTATGGATAGTATGAGTAAA

GGTACAGATAGTTATGGTGTTACTGCTGGATTTGGAGCAACATCTCATAG

AAGAACAAAAAATGGTGGTG

CTCTTCAAAAAGAACTTATTAGGTAAACAAACTATTTTTTTTCGTTATAT

ATACTAACAATGTAAAGAAT

TTAATATTTTTTTGTTATATATACTAACAATGTAAAAAATTTAATATTTT TTTGTTATATATACTAACAA

TGTAAAGAATTTAATATTTTTTTGTTATACATAGCTTATCGACTACTTAA GTGCTCCATTGATAAAGATT

TTTTTTTGTTTTTACGCGAAGGGGATTCGGATGAATTCAGTTAAAATGTG ATCTTAATGAATTATGATAT

TTTTTTGTAGGTTCTTGAATGCTGGAGTTTTTGGTAATGGAATAGAATCA TTTCACACATTGCCACATTC

AGCAACAAGGGCAGCTATGCTTGTTAGGATCAACACTCTGCTTCAAGGCT ACTCTGGCATTAGATTTGAG

ATCTTGGAAGCAATCACTAAGTTGATCAATAGCAACATCACCCCGTGTTT GCCTCTCCGTGGCACGATCA

CTGCCTCGGGTGATCTCGTCCCTTTGTCCTATATTGCTGGTTTGCTCACT GG C AG AC C T AAT T C C AAGG C

TGTTGGACCCAATGGTGAGAAACTTAATGCTGAGGAAGCTTTCTGCGTGG CTGGTATTAGTGGTGGATTT

TTCGAGTTGCAGCCTAAGGAAGGACTTGCACTTGTGAATGGCACAGCAGT TGGTTCTGCTATGGCATCAA

TAGTCCTGTTTGAGTCCAATATCTTTGCTGTTATGTCTGAAGTTTTATCA GCGATTTTTACTGAAGTGAT

GAACGGAAAGCCCGAATTCACTGACTATTTGACACACAAGTTGAAGCATC ACCCTGGTCAGATTGAGGCT

GCTGCTATTATGGAACACATTTTGGATGGAAGCTCTTATGTGAAGGTAGC TCAGAAGCTCCATGAAATGG

ATCCTCTTCAAAAACCAAAGCAAGATCGTTATGCTCTCCGAACATCTCCA CAATGGCTTGGACCTCAGAT

TGAAGTCATTCGTGCTGCAACTAAGATGATCGAGAGGGAGATTAACTCAG TGAACGAC AAT C C ATTGAT C

GATGTTTCAAGAAACAAGGCCTTACATGGTGGCAACTTCCAAGGAACCCC TATTGGTGTCTCCATGGATA

ATACAAGATTGGCCCTTGCATCAATTGGTAAATTGATGTTTGCCCAATTC TCAGAGCTTGTCAACGACTA

TTACAACAACGGGTTGCCATCTAATCTGACAGCAGGAAGGAATCCAAGCT TGGACTATGGTTTCAAGGGC

GCTGAAATCGCGATGGCTTCTTACTGCTCGGAACTTCAATTCTTGGCAAA TCCAGTGACTAACCATGTCT

AAAGTGCTGAGCAACACAACCAAGATGTGAATTCCTTGGGCTTAATTTCA G C C AGG AAAAC AG C T AAGG C

TGTTGATATCTTGAAGATAATGTCATCAACCTATCTCGTGGCTCTTTGCC AAGCTATTGACTTACGACAT

TTGGAGGAAAACTTGAAGAGTGTTGTCAAGAACACAGTTAGCCAAGTAGC TAAGAGAACTTTGACAATGG

GTGCTAATGGTGAACTTCATCCAGCAAGATTCAGCGAAAAAGAATTGCTT CGAGT CGTGGAT AGAGAAT A

CTTGTTTGCCTATGCTGATGATCCCTGCAGCTCCAACTACCCTTTGATGC AGAAGCTGAGACAAGTCCTT

GTTGATCAAGCAATGAAGAATGGTGAAAGTGAGAAGAATGTCAACAGCTC AAT C T T C C AAAAG AT TGG AG

CTTTCGAGGACGAATTAATCGCTGTGTTGCCTAAAGAAGTTGAGAGTGTA AGAGCTGTTTTTGAAAGTGG

CAACCCTTTAATTCGTAACAGGATCACAGAATGCAGATCATATCCATTGT

ACAGGTTGGTGAGAGAAGAA

CTTGGAACAGAATTGTTGACGGGTGAAAAAGTTCGATCACCTGGTGAGGA

GATTGATAAAGTGTTTACAG

CAATATGTAATGGACAGATTATTGATCCATTGTTGGAGTGTCTGAAGAGC

TGGAATGGTGCTCCTCTTCC

AATCTGCTAAATGTGTTATTCTTTCAAGTTCTTTTTTTGTACCTTTTAGT

GAATTACTAGAATTATAATG

ATGTTATGAACTTATATTAAAAAAAAATATTTTTGACTATAAAATTTAGT

TTTGTTATTGAAATTAAAGG

CTCAATCTGTGTTCTTTCCTTCTGTTATCTGAATATTATAAGAATTCAAG

TAATCTTTTAGCTTTGTGAA

CATGATGACATGCTTTCTT

Histidine Ammonia- ATGATCACGCTTACCCCCGGCCACCTGACCCTCCCGCAACTGCGCCAGAT BAG44062.1

Lyase CGCGCGCGAGCCCGTGCAGC YP_00522592

TGACGCTGGATCCGGCCAGCTTCGCGAAGATCGACGCGGGCGCGAAGGCC 3.1

GTGTCCGACATCGCCGCGAA CDF52938.1

ABR76232.1

GGGCGAGCCGGCGTACGGCATCAACACGGGCTTCGGTCGTCTGGCCAGCA AAL 19728.1 CGCATATCCCGCACGATCAG AEW60321 .1

CTCGAATTGCTGCAGAAGAACCTCGTGCTGTCGCATGCAGTCGGTGTCGG AEW51583.1

CGAGCCGATGGCGCGTTCGT ABQ54772.1

CGGTGCGTCTGCTGATCGCGCTGAAGCTGTCGAGCCTCGGCCGCGGCCAT AAX64695.1

TCGGGCATTCGCCGCGAAGT AAU27462.1

GATGGACGCGCTGATCAAGCTGTTCAACGCCGACGTGCTGCCGCTGATTC WP 0210000

CGGTGAAGGGCTCGGTCGGC 87.1

GCATCGGGCGACCTCGCGCCGCTCGCGCACATGTCGGCCGTGCTGCTCGG YP 00518568

CGTCGGCGAAGTGTTCATTC 2.1

YP 0012501 1

GCGGCGAGCGCGCGAGCGCGGTGGACGGGTTGCGCGTCGCGGGCCTCGCG

8.1

CCGCTGACGCTGCAGGCGAA

EFC47317.1

GGAAGGCCTCGCGCTGCTGAACGGTACGCAGGCGTCGACGGCGCTCGCGC AAH89809.1

TCGACAACCTGTTCGCGATC BAH62483.1

GAAGACCTGTACCGCACGGCGCTCGTCGCCGGCGCGCTGTCGGTCGATGC XP 00268006

GGCGGCCGGCTCGGTGAAGC 1 .1

CGTTCGACGCGCGCATCCACGAACTGCGCGGCCATCGCGGCCAGATCGAT AA07341 1 .1

GCGGCGGCCGCGTATCGCGA CAI79696.1

GCTGCTCGAAGGCTCGGCGATCAACCTCTCGCATCGCGACTGCGGCAAGG CAI79696.1

TGCAGGATCCGTACAGCCTG

CGCTGCCAGCCGCAGGTGATGGGCGCGTGCCTGGACCAGATGCGTCATGC

GGCCGACGTGCTGCTCGTCG

AGGCGAACGCGGTATCGGACAACCCGCTGATCTTCCCGGATACCGGCGAA

GTGCTGTCGGGCGGCAATTT

CCATGCGGAGCCCGTCGCGTTCGCGGCCGACAACCTCGCGCTCGCGGCTG

CGGAAATCGGCGCGCTGGCC

GAGCGCCGCATCGCGCTGCTGATCGACGCGACGCTGTCGGGCCTGCCGCC

GTTCCTCGTGAAGGATGGCG

GCGTGAACTCGGGCTTCATGATTGCGCACGTGACGGCAGCTGCGCTCGCA

TCGGAGAACAAGACGCTCGC

GCATCCGGCGTCGGTCGATTCGCTGCCGACCTCGGCGAACCAGGAAGACC

ACGTGTCGATGGCGACGTTC

GCGGCACGCAAGCTGGCCGACATCGCCGACAACACGAAGCACATCCTCGC

GATCGAACTGCTCGCGGCCG

CGCAGGGCGTCGATCTGCGCGAGAACGAGACGAGCCCGAAGCTCGCGGAA

GTGATGAAGACGATTCGCAG

CAAGGTCGCGCATTACGAGCTCGACCACTACTTTGCGCCGGACATCGCCG

TGATCGCGAAGCTCGTCGTC

GAGCGCGCGTTCGCGAAGCACTGCCCGTTCGCCTTCGCATCGGAGCAGTA A

Tyrosine Ammonia- GTGACGCAGGTCGTGGAACGTCAGGCTGATCGGCTCAGCAGCAGGGAGTA YP 00703999

Lyase CCTGGCCCGGGTCGTGCGCA 9.1

GCGCCGGGTGGGACGCCGGTCTCACCTCGTGCACCGACGAGGAGATCGTC Q8GMG0.1 CGGATGGGCGCGAGCGCGCG WP 0151032

37.1

CACCATCGAGGAGTACCTGAAGTCCGACAAGCCCATCTACGGCCTGACGC

CCH33126.1 AGGGCTTCGGTCCGCTGGTG AGZ04575.1

CTGTTCGACGCCGACTCGGAGCTGGAGCAGGGCGGCTCGCTGATCTCGCA GAK34477.1

CCTGGGCACCGGCCAGGGCG AIG26365.1

CGCCACTGGCCCCGGAGGTGTCGCGGCTGATCCTCTGGCTGCGCATCCAG WP 0308142

AACATGCGCAAGGGGTACTC 63.1

GGCGGTCTCGCCGGTGTTCTGGCAGAAGCTCGCCGACCTGTGGAACAAGG WP 0305926

GGTTCACCCCGGCGATCCCC 22.1

CGGCACGGCACGGTCAGCGCGAGCGGCGACCTGCAACCGCTGGCGCACGC WP 0305838

CGCGCTCGCCTTCACCGGTG 02.1

WP 0302258

TCGGCGAGGCGTGGACCCGGGACGCCGACGGCCGGTGGTCCACCGTGCCG

85.1

GCCGTGGACGCGCTCGCCGC

WP 0301070

GCTGGGGGCGGAGCCGTTCGACTGGCCGGTGCGCGAGGCGCTGGCGTTCG 56.1

TCAACGGGACCGGCGCGAGC WP 0102616

CTCGCGGTGGCTGTGCTCAACCACCGGTCCGCCCTGCGGCTGGTCCGCGC 15.1

CTGCGCCGTGCTCTCCGCGC WP 0090658

GGCTGGCGACCCTGCTGGGGGCCAATCCCGAGCACTACGACGTGGGGCAC 1 1 .1

GGTGTCGCGCGCGGCCAGGT WP_0290439

CGGTCAGCTGACCGCGGCGGAGTGGATCCGGCAGGGGCTGCCCCGGGGCA 04.1

TGGTGCGCGACGGCAGCCGC WP 0290276

07.1

CCGCTCCAGGAGCCGTACAGCCTGCGGTGCGCGCCGCAGGTGCTCGGCGC

WP 0290256

GGTGCTCGACCAGCTCGACG

70.1

GCGCGGGCGACGTGCTGGCGCGGGAGGTCGACGGCTGCCAGGACAACCCG WP 0290239

ATCACCTACGAGGGCGAGCT 88.1

GCTGCACGGCGGCAACTTCCACGCCATGCCGGTGGGTTTCGCCTCCGACC WP 0290202

AGATCGGGTTGGCCATGCAC 80.1

ATGGCCGCCTACCTGGCCGAGCGCCAGCTGGGTCTGCTGGTCAGCCCGGT WP 0286735

GACCAACGGCGACCTGCCGC 81 .1

CCATGCTCACCCCGCGCGCCGGGCGCGGTGCCGGGCTGGCCGGGGTGCAG

ATCAGCGCGACCTCGTTCGT

CTCGCGGATCCGGCAGCTGGTGTTCCCCGCCTCGCTGACCACCCTGCCGA

CCAACGGCTGGAACCAGGAC

CACGTGCCGATGGCGCTCAACGGGGCGAACTCGGTGTTCGAGGCGTTGGA

GCTCGGCTGGCTGACGGTCG

GGTCGCTGGCGGTGGGCGTCGCGCAGCTCGCGGCCATGACCGGCCACGCC

GCGGAGGGCGTCTGGGCGGA

GCTGGCCGGGATCTGCCCGCCGCTGGACGCCGACCGCCCGCTGGGCGCCG

AGGTGCGCGCCGCGCGCGAC

CTGCTGTCCGCGCACGCGGACCAACTGCTCGTCGACGAGGCAGACGGGAA GGATTTCGGATGA

Glutamate ATGTCAGCAAAGCAAGTCTCGAAAGATGAAGAAAAAGAAGCTCTTAACTT P39633.3 Dehydrogenase ATTTCTGTCTACCCAAACAA KEG08275.1

TCATTAAGGAAGCCCTTCGGAAGCTGGGTTATCCGGGAGATATGTATGAA NP_00123385 CTCATGAAAGAGCCGCAGAG 0.1

NP_00126803

AATGCTCACTGTCCGCATTCCGGTCAAAATGGACAATGGGAGCGTCAAAG

9.1

TGTTCACAGGCTACCGGTCA

AEW04907.1

CAGCACAATGATGCTGTCGGTCCGACAAAGGGGGGCGTTCGCTTCCATCC YP_00716125 AGAAGTTAATGAAGAGGAAG 5.1

TAAAGGCATTATCCATTTGGATGACGCTCAAATGCGGGATTGCCAATCTT YP 00525657 CCTTACGGCGGCGGGAAGGG 9.1

CGGTATTATTTGTGATCCGCGGACAATGTCATTTGGAGAACTGGAAAGGC YP 00493265 TGAGCAGGGGGTATGTCCGT 2.1

GCCATCAGCCAGATCGTCGGTCCGACAAAGGATATTCCAGCTCCCGATGT YP 00444244 GTACACCAATTCGCAGATTA 4.1

YP 00441234

TGGCGTGGATGATGGATGAGTACAGCCGGCTGCGGGAATTCGATTCTCCG

8.1

GGCTTTATTACAGGTAAACC YP 00441098

GCTTGTTTTGGGAGGATCGCAAGGACGGGAAACAGCGACGGCACAGGGCG 6.1

TCACGATTTGTATTGAAGAG YP 00437273

GCGGTGAAGAAAAAAGGGATCAAGCTGCAAAACGCGCGCATCATCATACA 1 .1

GGGCTTTGGAAACGCGGGTA YP 00436766

GCTTCCTGGCCAAATTCATGCACGATGCGGGCGCGAAGGTGATCGGGATT 7.1

TCTGATGCCAATGGCGGGCT YP 00436636

CTACAACCCAGACGGCCTTGATATCCCTTATTTGCTCGATAAACGGGACA 6.1

GCTTTGGTATGGTCACCAAT YP 00434396

8.1

TTATTTACTGACGTCATCACAAATGAGGAGCTGCTTGAAAAGGATTGCGA YP 00434335 TATTTTAGTGCCTGCCGCGA

6.1

TCTCCAATCAAATCACAGCCAAAAACGCACATAACATTCAGGCGTCAATC YP 00426176 GTCGTTGAAGCGGCGAACGG 6.1

CCCGACAACCATTGATGCCACTAAGATCCTGAATGAAAGAGGCGTGCTGC YP 00427038 TTGTGCCGGATATCCTAGCG 2.1

AGTGCCGGCGGCGTCACGGTTTCTTATTTTGAATGGGTGCAAAACAACCA YP 00409996 AGGATATTATTGGTCGGAAG 1 .1

AAGAGGTTGCAGAAAAACTGAGAAGCGTCATGGTCAGCTCGTTCGAAACA YP 00396781 ATTTATCAAACAGCGGCAAC 1 .1

ACATAAAGTGGATATGCGTTTGGCGGCTTACATGACGGGCATCAGAAAAT

CGGCAGAAGCATCGCGTTTC

CGCGGATGGGTCTAA

Glutamate ATGTCCATCAAAGACGCTGTAAAACTGATTGAAGAAAGCGAAGCCCGCTT CBX2231 1 .1 Ammonia-Lyase TGTCGATTTGCGCTTTACCG

ATACCAAAGGCAAGCAGCACCACTTTACCGTGCCTGCGCGCATCGTGTTG GAAGACCCCGAAGAGTGGTT

CGAAAACGGACAGGCGTTTGACGGTTCGTCCATCGGCGGCTGGAAAGGCA TTCAGGCTTCCGATATGCAG

CTTCGCCCCGATCCCGCCACGGCGTTTATCGATCCTTTTTATGATGATGT

TACCGTCGTCATTACCTGCG

ACGTTATCGATCCCGCCGACGGTCAGGGTTACGACCGCGACCCGCGCTCC

ATCGCACGCCGCGCCGAAGC

CTATTTGAAATCTTCCGGTATCGGCGACACGGCATACTTCGGTCCCGAAC

CCGAGTTTTTCGTCTTCGAC

GGCGTAGAATTTGAAACCGATATGCACAAAACCCGTTACGAAATCACGTC

CGAAAGCGGCGCATGGGCCA

GCGGCCTGCATATGGACGGTCAAAACACCGGCCACCGCCCTGCCGTCAAA

GGCGGTTACGCGCCCGTCGC

GCCGATTGACTGCGGTCAGGATTTGCGTTCCGCGATGGTAAACATTTTGG

AAGGACTCGGCATCGAAGTC

GAAGTGCACCACAGCGAAGTCGGTACCGGCAGCCAAATGGAAATCGGCAC

GCGCTTCGCCACCTTGGTCA

AACGCGCCGACCAAACCCAAGACATGAAATATGTGATTCAAAATGTCGCC

CACAACTTCGGCAAAACCGC

CACCTTCATGCCCAAACCCATTATGGGCGACAACGGCAGCGGTATGCACG

TTCACCAATCCATCTGGAAA

GACGGTCAAAACCTGTTCGCAGGCGACGGCTATGCCGGCTTGAGCGACAC

CGCGCTCTACTACATCGGCG

GCATCATCAAACACGCCAAAGCCCTGAACGCGATTACCAATCCGTCCACC

AACTCCTACAAACGCCTTGT

GCCGCACTTTGAAGCGCCGACCAAACTGGCATATTCCGCCAAAAACCGTT

CCGCTTCCATCCGTATTCCG

TCTGTGAACAGCAGCAAGGCGCGCCGCATCGAAGCGCGTTTCCCCGACCC

GACCGCCAACCCGTACTTGG

CGTTCGCTGCCCTGCTGATGGCGGGTTTGGACGGCATTCAAAACAAAATC

CATCCGGGCGATCCTGCCGA

TAAAAATCTCTACGACCTGCCGCCGGAAGAAGACGCGCTCGTCCCGACCG

TTTGCGCTTCTTTAGAAGAA

GCCCTCGCCGCGCTCAAAGCCGACCACGAATTCCTCTTACGCGGCGGCGT

GTTCAGCAAAGACTGGATCG

ACAGCTACATCGCCTTTAAAGAGGAAGATGTCCGCCGCATCCGTATGGCG

CCGCATCCGCTGGAATTTGA

AATGTATTACAGCCTGTAA

Threonine AGGAGGTGTTTTAATAATGAAAGGTTTTGCAATGCTCAGTATCGGTAAAG NP 622353.1

Dehydrogenase TCGGTTGGATTGAAAAAGAA EPX86072.1

AAGCCTACTCCCGGCCCTTTTGACGCTATTGTAAGACCTCTAGCTGTGGC AFT82159.1 CCCTTGCACTTCGGACGTTC YP 00679615

8.1

ATACCGTTTTTGAAGGTGCTATTGGCGAAAGACATAACATGATACTCGGT

EJZ15419.1 CACGAAGCTGTAGGTGAAGT YP 00172763

AGTTGAAGTAGGTAGTGAGGTAAAAGATTTTAAACCTGGTGATCGCGTTG 0.1

TGGTACCAGCTATTACCCCT AC A82186.1

GATTGGCGAACCTCTGAAGTGCAAAGAGGATATCACCAACACTCTGGTGG AGZ44086.1

AATGCTGGCAGGCTGGAAAT AEB44998.1

TTTCGAATATAAAAGATGGTGTTTTTGGTGAATTTTTTCATGTGAACGAT YP 00873713

GCTGATATGAATTTAGCACA 9.1

TCTGCCTAAGGAAATTCCATTGGAAGCTGCAGTTATGATTCCCGATATGA EPX87740.1 TGACTACTGGCTTTCACGGA YP 00440559

8.1

GCCGAACTGGCAGATATAGAATTAGGTGCGACGGTAGCGGTTTTGGGTAT

BAN60779.1

TGGCCCAGTAGGTCTTATGG EPE39095.1

CAGTCGCTGGTGCCAAATTGCGGGGTGCTGGAAGGATTATCGCAGTAGGC EPC57128.1 AGTAGACCAGTTTGTGTAGA EME23086.1

TGCTGCAAAATACTATGGAGCTACTGATATTGTAAACTATAAAGATGGTC ACI75705.1

ACI75704.1 CTATCGACAGTCAGATTATG

ACI75703.1

GATTTAACGGAAGGCAAAGGTGTTGATGCTGCCATCATCGCTGGAGGAAA

ACI75702.1 TGTTGACATCATGGCTACAG

CAGTTAAGATTGTTAAACCTGGTGGCACCATCGCTAATGTAAATTACTTT

GGCGAAGGAGATGTTTTGCC

TGTTCCTCGTCTTGAATGGGGTTGCGGCATGGCTCATAAAACTATAAAAG

GCGGGCTATGCCCCGGTGGA

CGTCTAAGAATGGAAAGACTGATTGACCTTGTTGTTTATAAGCGTGTCGA

TCCTTCTAAGCTCGTCACTC

ACGTTTTCCGGGGATTTGACAATATTGAAAAAGCCTTTATGTTGATGAAA

GAC AAAC C AAAAGAC CT AAT

CAAACCTGTTGTAATATTAGCATAA

Threonine Ammonia- ATGGCTGACTCGCAACCCCTGTCCGGTACCCCGGAAGGTGCCGAATATTT EGP22802.1

Lyase AAGAGCGGTGCTGCGCGCGC AIL15845.1

CGGTCTACGAAGCGGCGCAGGTCACGCCGCTACAGAAAATGGAAAAACTG KFJ1441 1 .1

TCGTCGCGTCTCGATAACGT B22317

ESE06785.1

GATTCTGGTGAAGCGCGAAGATCGCCAGCCAGTTCATAGCTTTAAGTTGC

ESD87895.1 GCGGCGCATACGCCATGATG

ESD77040.1

GCGGGCCTGACGGAAGAACAAAAAGCACACGGCGTGATTACCGCTTCTGC ESD56952.1

AGGTAACCACGCGCAGGGCG ESD26867.1

TCGCGTTTTCTTCCGCACGGTTAGGCGTGAAGGCGCTGATCGTCATGCCA ESDI 8649.1

ACCGCCACCGCCGATATCAA ESC98561 .1

AGTTGATGCGGTGCGCGGCTTTGGCGGCGAAGTGCTGCTTCACGGCGCAA ESA95751 .1

ATTTCGATGAAGCGAAAGCG ESA86931 .1

AAAGCGATCGAACTGTCACAGCAGCAGGGTTTCACCTGGGTACCGCCGTT ESA78951 .1 CGAT CAT C CGATGGTGAT CG ESA72735.1

ESA67809.1

CCGGGCAAGGCACGCTGGCGCTGGAACTGCTCCAGCAGGACGCCCATCTC ERL21545.1 GACCGCGTATTTGTACCGGT ERK40933.1

CGGCGGCGGCGGTCTGGCAGCGGGTGTGGCGGTGCTGATCAAACAACTGA ERJ97484.1

TGCCGCAAATCAAAGTAATC ERH28800.1

GCCGTGGAAGCGGAAGATTCCGCCTGCCTGAAAGCGGCGCTGGATGCGGG

TCATCCCGTTGATCTGCCCC

GCGTGGGGCTGTTTGCTGAAGGCGTCGCGGTAAAACGCATCGGCGATGAA

ACCTTCCGTTTGTGCCAGGA

GTATCTTGACGACATCATCACCGTCGATAGCGATGCCATCTGTGCGGCGA

TGAAAGATCTGTTCGAAGAT

GTGCGCGCGGTGGCGGAACCTTCCGGCGCGCTGGCGCTGGCGGGGATGAA

AAAATACATCGCCCAGCACA

ACATTCGCGGTGAACGGCTGGCGCATATTCTTTCCGGTGCTAACGTGAAC

TTTCACGGTCTGCGCTACGT

CTCGGAACGCTGCGAACTGGGCGAACAGCGTGAAGCGTTGTTGGCGGTGA

CCATTCCGGAAGAAAAAGGC

AGCTTCCTCAAATTCTGCCAACTGCTTGGCGGGCGTTCGGTCACCGAGTT

CAACTACCGTTTTGCCGATG

CCAAAAACGCCTGCATCTTTGTCGGCGTGCGCTTAAGCCGTGGCCTCGAA

GAGCGCAAAGAAATTTTGCA

GATGCTCAACGACGGTGGCTACAGCGTGGTTGATCTCTCCGACGACGAAA TGGCGAAGCTGCATGTGCGC

TATATGGTTGGCGGGCGTCCATCGCATCCGTTGCAGGAACGCCTATACAG CTTCGAATTCCCGGAATCAC

CGGGCGCGCTGCTGCGCTTCCTCAACACGCTGGGTACGCACTGGAACATC TCGCTGTTCCATTATCGCAG

CCACGGTACCGACTACGGGCGCGTACTGGCGGCGTTCGAGCTTGGCGATC ATGAACCGGATTTTGAAACC

CGGTTGAATGAACTGGGCTACGATTGCCACGACGAAACCAATAACCCGGC

GTTCAGGTTCTTTTTGGCGG

GTTAG

Serine ATGAGCGGTACCATCCTCATCACCGGCGCCACGTCCGGCTTCGGACAGGC ADY67207.1

Dehydrogenase CACGGCGCGGCGTTTCGTCA YP_00444429

AGGAAGGCTGGAAGGTCATCGGCACAGGTCGGCGGGCGGAACGGCTGGAG 8.1

GCGCTGGCGCAAGAACTCGG EAZ63492.1

XP_00138751

CTCCGCCTTTCACGGCGCTGCCTTCGATGTTACCGACGAAGATGCCACTA

5.1

GAAAGGCACTTGCGGCTTTG

BAB07807.1

CCGGAAGGTTTCCGGGACATCGATATTCTCGTCAACAATGCGGGGCTTGC EMS96834.1 GCTCGGCACCGCACCTGCAC EKJ96295.1

CGCAGGTGCCGCTGAAAGACTGGCAGACCATGGTGAACACCAACATCACC EHJ96027.1 GGTCTTTTGAACATCACCCA EHH03760.1

CCATCTTTTGCCCACGTTGATCGACCGCAAGGGCATTGTCATCAACCTTT WP_0287070 CCTCGGTAGCTGCGCACTGG 25.1

CCCTATGCGGGCGGCAATGTCTATGCCGGAACGAAAGCCTTCCTGCGGCA NP_356536.1 ATTCTCGCTCGGTCTGCGCT AEQ50417.1

AAK89321 .1

CCGACCTGCATGGCAAGGGCGTGCGCGTCACCTCGATCGAACCGGGCATG

YP_00489816 TGCGAAACGGAATTCACGCT

7.1

TGTTCGCACCGGCGGCAATCAGGATGCCTCGGACAATCTTTACAAGGGCG YP_064393.1 TCAATCCGATCACGGCCGAG WP_0035224

GATATCGCCAATACGATCCATTGGGTCGCCTCGCAGCCCAAACATATCAA 80.1

CATCAACAGCCTCGAACTCA EGP55658.1

TGCCGGTCAACCAGTCCTTTGCCGGTTTCCAAGTGCATCGGGAAAGTTGA EGL63994.1

KFC62486.1

WP_0313543

48.1

Serine Ammonia- ATGATGACCAAAAACGAAATCCAAAAGTGGGTAAAGGAATTCCCGCTGCT KFL14920.1 Lyase TGAAACGATCATGGCGGCCG AIF56070.1

AAGAGGTATTTTGGCGCAATCCAAAATATCACGCGTTTGCGCAAGCTATT KFI03369.1 CGAACGATTCCTTTACGCGA KFH36969.1

KFH35774.1

ACGCGATGTCAAGGAGGCCGAAGAGCGATTGCGCCGCTTTGCCCCCTACA

KFF561 12.1 TCGCGAAAGTGTTTCCCGAG

WP_0314091

ACGCGAACGGCCCACGGTATCATCGAATCCCCTTTAGTGCGGATTCCGAA 41 .1

CATGAAACAGCGTTTGGAAA KFC30598.1

AGATGTTTCAGACCAACATCGAGGGGGATCTGTTGCTAAAATGCGACAGC KEZ84476.1 CATCTTCCCATCTCCGGATC KEY95863.1

GATCAAGGCGAGAGGGGGAATCTACGAGGTTCTGAAACATGCGGAAGAAC KER46054.1 TCGCTCTGGCAAACCATATG WP_0300249

ATCACCATGGGGGATGACTATGCGGTCATGGCCAGCGAAGAATTCCGGCA 49.1

GTTCTTTTCCCGCTATTCGC KEK24273.1

TTGTCGTTGGTTCGACGGGAAATTTAGGCTTGAGTATCGGCATCATCGGG KEK22892.1 GCGCAGCTTGGGTTCCGCGT KEK18491 .1

TACCGTTCATATGTCAGCCGATGCGAAACAATGGAAAAAAGACTTGTTGC KEK12402.1 GAAGCAAAGGGGTTGCGGTC WP_0297612

12.1

ATCGAACATCTCACCGACTACAACAAGGTGGTGGAAGAGGCGCGAAGACA

WP_0297581 GT C CGC CGAGGAT C CAACGT

74.1

CGTATTTTATCGATGATGAGAACTCGATCCATCTGTTTTTAGGCTATGCG WP_0297140 GTGGCGGCGTTTCGGCTGAA 78.1

AAAGCAATTAGAGGACATGAACATCACGGTTGATGAAAACCACCCGCTCT WP_0295983 TTGTATATCTTCCTTGCGGC 16.1

GTCGGCGGCGGTCCGGGCGGGGTGACGTTTGGGCTGAAGCTCGTGTACGG CGATCATGTCCATTGCTTTT

TCGCTGAGCCGACGCATTCGCCTTGCATGTTGCTCGGCCTGATGACGGGA CAGCACGACCGCGTGTCGGT

GCAAGATTTTGGCCTCGACAATAAGACCGAAGCGGACGGGCTAGCGGTGG GGCGGCCGTCAAGGTTGGTG

GGGAACATGCTTGAGAACGTCATCAGCGGCGTCTATACGGTGGACGATGC GACGCTTTACCGCTTGCTCG

CGGCGATGGTGGAAACGGAGGAAATCTATTTAGAGCCGTCCGCCTTGGCG GGGGTGGCGGGGCCTGTTCG

GCTGTTTCGTGATTTGGCGGGGCAAACGTACGTAGAGGCAAACGGTTTGA AAGAAAAGATGAAAAACGCC

GTCCATATTGGCTGGGCGACAGGCGGAAGCATGGTGCTAAAGGATGTGAT GG AGG CCTATTAT CGGG AAG

GCGTGCGCATCGAAACGATGACAGGGAACGGTTTTTCTGAAGGACGATAA

Leucine ATGCTGATGTTCGAAGAAATCCAGGCGCGCGGCCACGAGAGCGTCACGCT YP_00416978

Dehydrogenase GCTGCACCACGCCCCCAGCG 5.1

GCCTGCGCGCCGTGCTCGCCGTGCACTCCACCGTGCTCGGCCCTGCCATT AD V66120.1 GCCGGCTGCCGCCTGATGCC ADY26991 .1

AEW05136.1

CTGCACCGAGGAACGCGCCGTGCGCGACGCCCTCGCCCTCAGCGAGTCCG YP_00525680 TCACGCTCAAGGCCGCCCTC

8.1

GCGGGCCTGAACTACGGCGGGGGCGCGTGCGTCATGCTCCCCCCGGAAGG YP_00425660 CGGCGACATCGACGGGCACG 8.1

CCCGCGAGGCGCTGTTCCGCGCGCTCGGCCGGCAGATCCGTTACCGCGGT YP_00434624 GGCCGCGTCATCCTCACCGA 5.1

GGACGTCGGCGTGACCGGCCGCGACATCGCCTTCGCCGCGCAGGAAACCG AEA45407.1 ACAGCACCATGGGCATGCAC YP_00410199

ACCGACACGCCCACCGTCACCGCGTACGGCGTGTACCGCGGCATCAAGGC 2.1

CGCCGCGCGCGCCTACCTCG YP_00410199

1 .1

GCGGCGAGAGCATGCGCGGCGTGCGCGTCGCGCTGCTCGGCGCGGGCGCA

YP_00382593 GTCGGGCGCACCCTCGCGCA

2.1

GCACCTGCACCGCGAGGGCGCGCGCCTCACCGTCGCAGACCTGATGTCTG ADU51265.1 AGCGCGCGCAGGCCCTCGCG ADU51264.1

GACGACCTCGGCGAGCGCGTCACCGTCGTGAGCGCCGCTGACATCTTCGA ADL08309.1 CGTGCCGTGCGACGTATTCG AFY88585.1

CGCCGTGCGCGTTCGGGCACAGCATCAAAAGCGCCGACGTGCCCCGCTTG YP_00405400 CAGTGCCGGGTGATCGCCGG 7.1

CAGCGAACACCACCCGCTCAGCCACAACGGCGAGACGCTCGTGCGCGAAG YP_00709245 CGGGC AT C AC AT AC AT C C CG 4.1

YP 00382521

GACTTCGCCATCAACAGCGCCGGCCTGATGAGCGCCGCGCAGAACCTCAG 6.1

CATCGAAACGGCGGCGGAAC ADR21899.1

GCGTGTACGAGAGCGTCGCGCAGATCTGCGCGACCGCGCAGAAGTACGAG ADL07593.1 AAGCCGCCGCACGTCGTCGC

CCGTAAACTCGCGCTGCGCCGCATCGAACTGATCGGCTCCATCAGCGGCC AGTACGCCGGCCAGTAA

Aspartate TCATGTGCCAACACGTATGTTATCACTTAAAATTTTTAGTAAAGTGACTG ADP76847.1 Dehydrogenase CTGAATATGCTGCCAAAACA YP_00400360

CTTGTTTTTGGATTTAATTCACACACAGTGTTTTTTGTTATAGATTTAAA 9.1

CTCTCCAAAATCTCCTTTAA ADE37476.1

AEH60264.1

CATGGACTTCATGGATATTGTGTTCAACTTCAGGATCTGCAATTATCTTT

AEH50568.1 ACATCCGCATCTATTCCAGA

YP_00461548

GGCTAGACTTAATGCCGCAGCAACGTTAATATTCACTGGAAATTTTTTAA 3.1

CAGCTTCTGAGGATTTCCCT YP_00465966

TTAAACACGACCTCCTTTTTTTTGGTCTTAACACCTAACGAAGTAGGTGA 4.1

TTTTCTCGTTATAAGTTTTA YP_00354312

TTTCTTTTATCTTACCTAAGGATGCGGCTTTTACACCATCTAAACCAATT 1 .1

ATTGCACCGGAAGGTATGTA YP_00389589

TATATTAGCTCCTGATTCTCTAGATTCCTTTATCAATCTTCTTCTAACTT 1 .1

TCTCATCTAATAGTGCACCC ADN37453.1

ADV47603.1

ACACTCATAATCAAAACATCTATACCTCTACTAATTATATTGGGCACAAT YP_00416310 TTCTTTTACTGCCTCTTGAG

1 .1

AAGCAGATTCAATTATCAAATCAACTCCATTGAACATTTCTTCTACCTTT ADY50896.1 TTTACGGCAGTGCCATTTGT ABX33598.1

TAAATTTGCTAGCTTCTTAGCTTTTCTAAAATTTCTGTCATAAAAATATT YP_00427271 TTAATTTTATTTTTTTGATA 8.1

TCTTGTTTTAAGACAAGGTTAACTATTGTATTTGCAATTGCACCACATCC ADN60949.1 TATAATCCCACATCTCAT ACL18032.1

ACL16745.1 ABX00971 .1 ADD08173.1

Aspartate Ammonia- ATGTCCTCGCCTGCATCATCGCGCATCGAAAAAGACCTGCTTGGTGTTCT ELS44542.1 Lyase CGAAGTACCTGCCAACGCGT EXL32019.1

ATTACGGCATCCAGACCCTGCGAGCGGTGAACAACTTTCACCTCTCCGGC EPF69098.1 GTGCCGCTTTCGCACTACCC EDZ32290.1

ACC77466.1

GAAACTGGTAGTCGCGCTGGCCATGGTCAAGCAGGCGGCAGCGGATGCAA

ETO09916.1 ACCATCAGCTCGGACACCTC

ETN58394.1

AATGACGCCAAGCATGCGGCGATCAGCGAGGCCTGTGCCCGCCTGATCCG AGZ94384.1 CGGCGACTTCCACGATCAGT EGU12843.1

TCGTGGTCGACATGATCCAGGGCGGCGCTGGCACGTCGACCAACATGAAT AGQ54567.1 GCCAACGAAGTCATCGCCAA BAN21048.1

CATCGCTCTGGAAACCATGGGTTTCGAGAAAGGCGCATACAAACACCTGC ELU36465.1 AC C C C AAC AACGATGT C AAC ELU36464.1

ATGGCGCAGTCGACCAACGACGCCTACCCCACGGCGATCCGCTTGGGTCT EDS31003.1 GCTGCTGGGTCACGACGCTC BAM20634.1

AC048312.1

TGCTCGCCAGCCTTTCCAGCCTGATTCAGGCCTTCGCCGCCAAGGGCGAA

XP_00182883 GAATTCAACCATGTGCTGAA

3.2

GATGGGCCGCACCCAGTTGCAGGACGCCGTTCCAATGACCCTGGGTCAGG EAU92840.2 AATTCCGCGCCTTCGCCACC XP_00184988

ACCCTGACAGAAGACCTGAACCGCCTGCGCAGCCTGGCGCCAGAGCTGTT 0.1

GACCGAAGTGAACCTCGGCG XP 00165898

GAACCGCCATCGGCACCGGCATCAACGCCGACCCTGGCTATCAGAAGCTG 8.1

GCAGTCGATCGTCTGGCACT

CATCAGCGGCCAGCCTCTGGTGCCAGCAGCCGACCTGATCGAAGCGACCT

CCGACATGGGCGCCTTCGTG

TTGTTCTCGGGCATGCTCAAGCGTACTGCGGTCAAGCTGTCGAAAATCTG

CAACGACCTGCGCCTGCTGT

CCAGCGGCCCACGCACCGGCATCAACGAAATCAACCTGCCGGCACGTCAG

CCAGGCAGCTCGATCATGCC

CGGCAAGGTCAACCCGGTGATCCCGGAAGCGGTCAATCAGGTTGCCTTCG

AAATCATCGGCAACGACCTG

TCGCTGACCATGGCAGCCGAAGGAGGACAATTGCAGCTCAACGTGATGGA

GCCGCTGATCGCCTACAAGA

TCTTCGACTCGATCCGCCTGCTGCAGCGCGCCATGGACATGCTGCGCGAG

CACTGCATCGTCGGCATCAC

AGCCAACGAACAGCGCTGCCGCGAGCTGGTCGAGCATTCGATCGGTCTGG

TCACCGCCCTGAACCCTTAC

ATCGGTTACGAGAACTCCACCCGTATCGCCCGCATCGCGCTGGAAACCGG

CCGCGGCGTGCTGGAACTGG

TGCGTGAGGAAGGTCTGCTCGACGACGCCATGCTCGACGACATCCTGCGC

CCGGAAAACATGATCGCTCC

GCGTCTGGCCCCCTTGAAGGCCTGA

Valine TCAGCGACCGCGGGCCTCGGCCATCCGCTGCTCGGCGATCCGGTCGGCCG YP 00793265

Dehydrogenase CCGCGGCGGGCGGAATGCCG 2.1

TCCGCCTTCGCACGTGCGAATATTTCCAGCGTGGTGTCGAAGATCTTCGT AGK78767.1 CGCCTTCGCCTTGCACCGGT NP 628270.1

YP 00197323

CGAAGTCGAACCCGTGCAGCTCGTCGGCGACCTGGATCACGCCGCCGGCG

4.1

TTGACCACATAGTCGGGTGC

AIJ14557.1

GTAGAGGACCGACCGGTCGGCCAGGTCCTTCTCGACACCCGGGTGGGCCA YP 00752320

GCTGGTTGTTGGCCGCGCCG 9.1

CACACCACCTTCGCCGTGAGCACCGGAACGGTCGCGTCGTTGAGCGCGCC WP 0156594

GCCGAGCGCGCAGGGCGCGT 26.1

AGATGTCGAGACCCTCGGTGCGGATCAGCGTCTCGGTGTCCGCCACCACG CCK29082.1

GTGACCTCGGGGTGCAGATC CAR62534.1

GGTGATCCGGCGCACCGACTCCTCGCGTACGTCGGTGATCACGACCTCGG AGT93561 .1 CCCCGTCGGAGAGCAGGTGC AEK45617.1

ADI08852.1

TCGACGAGGTGGTGGCCCACCTTGCCGACCCCGGCGACGCCGACCTTGCG

YP_00845428

GCCGCGCAGCGTCGGGTCGC

2.1

CCCACAGGTGCTGGGCCGAGGCCCGCATGCCCTGGAAGACACCGAACGCG ESQ05180.1

GTGAGGACGGAGGAGT CGC C ESP98677.1

GGCGCCGCCGTTCTCGGGGGAGCGGCCGGTGGTCCAGCGGCACTCCCTGG YP 00553507

CGACGACGTCCATGTCGGCG 4.1

ACGTAGGTGCCGACATCGCAGGCGGTGACGTACCGGCCGCCGAGCGAGGC YP 00496398

GACGAACCGGCCGTAGGCCA 3.1

GGAGGAGTTCCTCCGTCTTGATCTTCTCCGGGTCGCCGATGATGACGGCC EOD63988.1 TTGCCGCCACCGTGGTCGAG EME98953.1

EME52779.1

TCCGGCCAGGGCGTTCTTGTACGACATCCCGCGCGACAGGTTCAGCGCGT

CGGCGACGGCCTCGGCCTCG

GTCGCGTACGGGTAGAAGCGGGTGCCGCCGAGGCCGGGGCCCAGGGCGGT

GGAGTGGAGGGCGATGACGG

CCTTGAGGCCGGTGGCACGGTCCTGGCAGATCACGACTTGCTCGTGACCC

CCCTGATCCGAGTGGAACAG

GGTGTGCAGGACGCCGTTAGTCACATCGGTCAC

Glycine CTAGTTGTAAAAGTCGAGGGAGGCGCAACTGCACATGAGGTGACGATCTC KEG12217.1

Dehydrogenase CGTAAACCCCGTCAATGCGA ADH66904.1

CCCACAGTCGGCCAGTACTTTTCAACGTACGAGTAAGGATAAGGGAATGC YP 00367941

CGCCAAACGCCGATCATATG 0.1

YP 00350749

GTTTGTCCCATTTATCATCGGTGACACATCTTGCCGTGTGTGGTGCATTC

1 .1

TTCAAAACATTGTTATCCAC

ADN74845.1

TGGTTGTTCACCTTTTTCAATGGCGGCAATCTCACCTCGAATGGAAATTA ADD28471 .1

GTGCATCTGCCAAGCGATCC YP 00444520

AACTCCCGCTTGGGTTCTGATTCGGTGGGTTCAATCATTAAAGTCCCGGG 3.1

TACAGGAAACGCCAGTGTTG YP 00391 191

GCGAGTGAATTCCGTAGTCCATCAACCGTTTGGCCACGTCCTCCGCCTCA 9.1

ATATGAGCTGTCTTCTTGAA ADQ81869.1

CCGTCGAAGATCAACGATAAACTCATGAGCGCAGTAGTTTTCTCCACCCA AEH88507.1 GGAAAAGAATCGTATAATGG YP 00713821

9.1

TTCTCTAGGCGCTTCTTCAAGTAGTTTGCATTCAAAACGGCGTACTCTGT

YP 00461260

ACAAGTTTTGAGCCCGTGTG

1 .1

ATCCAAGCATTAACATCAACATGTACGATATCGGAAGAATTGATGCTGAT YP 00417031

CCGTACGCTGATTGTGAGAC 8.1

TTGGCCGAATGGCTGTGAACCGCCAACTTTTTGGTTGAAAACAGAATTTG YP 00416355

GCAAAAAGGGGGCCAGATGT 9.1

TGACGGACAGCTATAGGGCCCATTCCGGGGCCGCCACCACCATGGGGAAT YP 00404537

TGAAAACGTCTTGTGGAGAT 5.1

TAATGTGGCACACGTCGCCACCGATATATCCAGGGCCTGTATAGCCAACC YP 00478719

ATGGCGTTAAGATTTGCCCC 0.1

YP 00714236

ATCAATGTAGCATTGTCCACCGTAGTAGTGCGCCATTGATGTAATGGATA

1 .1

AAATATCCTTGTCAAACAAG

YP 00706789

CCATACGTACTTGGATATGTTATCATGATACACGACAACTCCTTTGCGTG 6.1

TTTTTGGCAAGATTTCTCCA YP 00710078

GGTCATTGATATCAACCCTGCCGTTAGACAAGCATTTCACCAAGACAATA 8.1

TTCATTCCTGCCAATGTTGC YP 00477304

CGAAGCTGGATTCGTACCATGCGCACTCTCTGGAATCAAACAGACGTTGC 3.1

GGTGTCCTTCCTTCATTGAT

AGATGGTACGCACGAATAACACGAAGCCCAGCGTATTCACCTTGGGCGCC

ACTATTAGGCTGAAGCGATA

CCGCATCCAGACCGGTAATTTCCCTTAACTTTTGCTCAAGATCTAGACAC

AACGCACTGTACCCTCGCAC

TTGGTCCACTGGGGCAAGGGGATGCACATTGGTGAATTCTGGCCAAGAGA

GTGGTAACATAGCAGCGGCA

GGGTTAAGCTTCATGGTGCAAGATCCCAACGGGACGCAACCATGCGTAAG

GCCGTAATCCTTTCGTTGTA

GACGATGAATATAGCGCATCAGTTCACTTTCACTCTTGTACTTTTGAAAC

GTTGAGTGTTTCAGGAAATC

AGACTTCCGCACCAGATCCAACGGTAGTACCGATTTCTGATCGGCTATTT

TGGAAAGGGCTGCGACGACG

GGAAGCTTCAACCCTGCAGCCTCCAAAAGTGACACAATGTGTCCATCCGT

TGTTGCCTCATCCACAGAAA

TGGAGACAGTCCCATTACTGTAATCAACAAAAACATTAATACCCTTCTCA

ACACATCGTGTCTTGTAATC

CTCCGCTGTAATGCCTTTTAGGTTCACAGTAACAGTGTCGAAAAATGCAC

TGTTTACCACAGAGTGTCCT

ACTGATTCCATACCAACAGCGAGCACTTTCGCCTTGCCGTGTATCTCATT

GGCAATCTCATTTAGACCAT

CTGGACCATGGTAGGCGGCATAAAACCCACTCACGTTGGCCAATAACGCT

TGTGCAGTACAGATATTTGA

TGTGGCGCGCTCACGCTTAATATGTTGTTCACGTGTCTGCAGCGCCATGC

GTATGGATGGCTCTCCGGCA

GAATCCTTACTGACGCCGATCACACGTCCCGGCATCAACCTCTTAAACTG

CTCCTTGACAGCAAAGAACG

CGGCGTGAGGACCTCCATATCCTAGTGGAACACCAAAACGCTGGGAGGAT

CCCACAACCACATCTGCATT

CATTTCACCAGGTGGCTTGACAAGAACACAAGCCATCAAGTCGGTCCCAC

AGCAACTAATGACACCGTGC

TTCTTTGCATTCTCGAACAGTGGTGAGAAGTCATGAAGCATGCCCATCGC

ATCTGGTGTTTGTACAAGGA

TACCAAACAAGGAACTGTCAGTCCAGTCAATCAGATTCGTGTCGCCCACG

ACGACGTTTATCTTGAGCGG

TTCGGCTCTTGTCTTAACCATCTCAATGCAGGATGGAAAAACAGTTTTTG

ATACGAAGAACGTATTCCGC

TTTCGTTGACCATGCTGAAAAGCAAGATGCATCGCCTCGGATGATGCTGT

CGCTTGGTCAAGAAGAGATG

CATTTGCCACATCCATCTTTGTCAAATCCATAACCATGGTTTGGAAATTC

AAAAGGGACTCCAGACGTCC

TTGTGCAATCTCAGCTTGGTATGGTGTGTAGGGTGTGTACCATCCAGGAT

TTTCAATGACGTTGCGAAGT

ATGACAGGAGGAGTAATGGACTCGTAGTACCCCTGACCAATCATGCTTTT

TAGTACCTTGTTTCGCGCAC

CAAGAGAGCGCACGAGTGCGAGAGCATCCATCTCACTCATAGCCGCCACC

TCCGTCAAGGGTGGGCGTAC

AATATCCCCTGGAATAGCAGCCGTCATCAAATCAGAGAGACTCTCTTTTC

CAACCGTTCGAAGCATCGAC

ATTGTCTCAGCCGTTGTTGGACCAATATGGCGGTTAATATAGCTGTCCGT

GGCAGTCCATCGAACAAATG

TCACGCATGGCAAAGAGCCACGAAACAAACGACGGTACAT

Alanine ATGATCATTGGCCTGCCGAAAGAGATCAAAGTTAAGGAAAACCGCGTGGC YP 00417139

Dehydrogenase ACTCACGCCCGGGGGCGTCG 5.1

CCAGCCTCGTGCGCCGCGGCCACACCGTCATCGTGGAACGCAGCGCCGGC ADV67730.1 GTGGGCAGCGGCATCCAGGA ADY25885.1

ADV48359.1

CACCGAGTACGAGCAGGCCGGCGCGCAGCTCGGCAGCGCCGCCGAGGCGT

AFZ35471 .1

GGGCCGCGCAGATGGTCGTG

AFZ05172.1

AAGGTCAAGGAGCCCATCAAGAGCGAATACGGGTACCTCCGCCCGGACCT AEW05285.1 GCTGCTGTTCACGTACCTGC AEW04533.1

ACCTCGCTGCGGACCAGCCCCTCACGGACGCCCTGCTGAGCGCCGGCACG AEM70054.1

ACCGCCGTTGCGTACGAGAC YP 00525695

GGTGCAGCTCGACGACCGCAGCCTGCCGCTGCTCACGCCCATGAGTGAGG 7.1

TCGCGGGCCGCCTGAGCGTG YP 00525620

CAGGCCGGCGCGTACCACCTGCAAAAGCCCATCGGCGGGCGCGGCGTGCT 5.1

GCTCGGCGGCGTGCCGGGCG YP 00445049

TGCAGGCGGGCCACGTCGTCGTGATTGGCGGCGGCGTCGTCGGCACGAAC 2.1

YP 00436810 GCCGCGAAAATGGCCATGGG

3.1

CCTCGGCGCGAAGGTCACGGTGCTGGACGTGAACCACGGGCGCCTCTCGT YP 00434043 ACCTCGACGACGTGTTCTTC 2.1

GGGAAGCTCACCACCATGATGAGCAACGAGGCGAACATCCGCTCCATCCT YP 00426160 GCCCGAAGCGGACCTCGTGA 9.1

TCGGCGGCGTGCTGATCCCCGGGGCGAAGGCGCCGCACCTTGTCACGCGC YP 00425550 GACATGCTGGCGACCATGCA 2.1

GGAAGGCAGCGTCATCGTCGACGTGGCGGTGGACCAGGGCGGATGCGTGG YP 00416385 AGACCATTCACGCGACGACG 7.1

CACGACGATCCCACGTACATCGTGGACGGCGTGATCCACTACGGCGTGGC YP 00478747

6.1

GAACATGCCGGGCGCGGTGC YP 00713243

CGCGCACCAGCACGTTCGCGCTCACGAACCAGACCATTGGGTACGTGCTG

7.1

CAGCTCGCGGACAAGGGCGT YP 0071 1358

GGAGGCACTCAGCGCCAGCAAGCCGCTGCTGCGTGGCCTGAACACCATCG 8.1

GCGGGAAGCTGACGTACGCG

GGCGTCGCGGAAGCGTTCGGCCTGACGTACACCGCGCCTGAAGTGGCGCT GGCGTAA

Proline ATGGAGCCCACTATGAGCCAATTCGAACAGCTGTACCGCCAGGTGGCCCT ADY26965.1

Dehydrogenase CAGTGTCGCCGGCAACCCGG ADI14996.1

TCGTGGAAAAAGTCTTGAGCAAGCAGGGCTGGGCGCTGGCGCAGCGTTTT YP_00443747 GTATCGGGCGAGACGGCGCA 0.1

YP_00436897

GGACGCCATCAAGGCCATCAAGCGGCTGGAAGCCCAGGGCATCTCCGGCA

4.1

ACCTCGACCTGCTGGGCGAG

YP_00434574

TTCGTGAACACCCCGGAACCCGCCAATGCCAACACCGAGATGATTCTGGC 4.1

GACCATTGACCAGGTGCACG YP_00434068

CGGCGGGCCTCACGCCCTACAACAGCGTGAAAATGTCGGCGCTGGGCCAA 4.1

GGGCAGACCGCGCCGGACGG YP_00425658

CCAGGACCTCGGCTACGTCAACACCCGCCGCGTCGTGGAGCGGGCCAAGC 2.1

GCTACGGCGGCTTCGTCAAT YP_00417068

CTGGACATGGAAGACCACACCCGCGTGGACTCGACTCTGCAGATTTTCCG 0.1

CCGCCTGGTCAAGGAGTTCG YP_00370553

9.1

GCCACCAGCATGTGGGAACGGTGTTGCAGGCCTACCTGCACCGCTCGGAA

AEA44906.1 GACGACCGCCGCAGCCTGGA

AEB12864.1

CGACCTGCGCCCCAACCTCCGCATGGTGAAGGGCGCCTACCTGGAGCCCG ADV67015.1 CCTCCGTCGCCCTGCAGAGC AEE14339.1

AAAACCGACATTGACGCCGCCTACCGCCGCCTGGTCTACGAGCACCTCAA AEA34625.1 GGCCGGCAACTACTGCAACG EFH87253.1

TGGCCACCCACGACCACCACATCATCTACGACGTGATGCACTTTGCGCTG NP_868270.1 GCCCACGGCATCCCTAAGGA ADQ16526.1

CCAGTTCGAATTCCAGCTGCTGTACGGCATCCGCGAGGACCTGCAGCGCG AEL26370.1 AATTGGCCGAGGCCGGCTAC AFK04422.1

AEM71761 .1

ACGGTGCGCTCGTACATTCCTTTCGGCAAGGACTGGTACGGCTACTACTC GCGCCGCATCGCCGAGCGCC

CGCAGAACGTGATGTTCGTGCTGCGCGGCCTGCTGTAA

Lysine ATGAAAAACATTGTGGTTATCGGCGCGGGCAATATCGGTTCGGCAATCGC BAH80102.1

Dehydrogenase CTGGATGCTGGCCGCATCAG AAV93559.1

GCGATTATCGCATCACGGTTGCCGATCGTTCAGCCGATCAGCTGGCCAAT YP_165503.1 GTGCCGGCGCATGAACGGGT AIA01810.1

AIA03878.1

CGACATCGTCGACATTACCGACCGTCCCGCTCTGGAAGCACTGCTAAAAG

AIA03381 .1 GCAAATTCGCCGTGCTCTCC

AIA00889.1

GCCGCCCCCACCGAATTCCACCTGACGGCGGGTATTGCCGAAGCGGCCGT EXU 92064.1 TGCCGTCGGCACGCATTATC EOT00338.1

TCGATCTCACCGAAGACGTGGAATCCACCCGCAAGGTCAAGGCGCTGGCG NP_882461 .1 GAAACGGCCGAAACCGCGCT EIJ80893.1

CATTCCGCAATGCGGCCTCGCCCCCGGCTTCATCTCCATCGTCGCTGCCG AIA06975.1 ATCTCGCCGTCAAGTTCGAC AIA05885.1

AAGCTGGACAGCGTGCGCATGCGCGTCGGCGCTCTGCCGCAATATCCGTC EXU88317.1 CAATGCGCTCAACTACAACC EOT04629.1

AIA07859.1

TCACCTGGAGTACCGACGGGCTGATCAACGAATATATCGAGCCCTGCGAA

AIA04644.1 GGATTCGTCGAAGGCCGCCT

AIA04440.1

CACCGCCGTTCCGGCCCTTGAGGAGCGCGAGGAGTTCTCGCTCGATGGCA AIA03522.1 TCACCTACGAGGCGTTCAAC AIA02686.1

ACCTCGGGCGGTCTCGGTACGCTTTGCGCGACGCTGGAAGGCAAGGTGCG GACCATGAACTACCGCACTA

TCCGTTATCCCGGCCATGTGGCGATCATGAAGGCGCTTTTGAACGACCTC AACCTGCGCAACCGCCGCGA

TGTGCTGAAGGACCTGTTCGAAAACGCCCTGCCCGGCACCATGCAGGATG TGGTCATCGTCTTCGTCACC

GTCTGCGGCACCCGCAACGGCCGCTTCCTGCAGGAAACCTATGCCAACAA GGTCTATGCCGGCCCGGTTT

CCGGCCGGATGATGAGCGCCATCCAGATCACTACCGCCGCCGGCATCTGC ACGGTTCTCGACCTGCTCGC

GGAAGGCGCCCTGCCGCAGAAGGGCTTCGTTCGACAGGAGGAAGTGGCGC TGCCGAAGTTCCTCGAAAAC

CGGTTTGGCCGGTATTATGGCTCGCATGAGCCGCTGGCGCGGGTTGGGTG A

The disclosure relates to an ammonia or ammonium ion biosensor for measuring a total concentration of ammonia in blood. The ammonia biosensor may comprise, in some embodiments, a measuring electrode which include as components, a mediator and an enzyme, which selectively act on the plurality of specific amino acids each serving as a substrate, and a counter electrode. In the amino-acid biosensor, the enzyme has a substrate affinity to each of the plurality of specific amino acids. The enzyme is operable to catalyze a reaction in each of the plurality of specific amino acids as a substrate so as to form a reaction product. The mediator is operable, during amino-acid concentration measurement, to carry electrons between the reaction product and the measuring electrode. Further, the amino-acid biosensor is designed to apply a voltage between the measuring electrode and the counter electrode at a measurement point in such a manner that, in an analytical curve representing a relationship between an applied voltage and a current value in a specific concentration for each of the plurality of specific amino acids, the applied voltage is a voltage allowing the variety of the current values for the amino acids in the same concentration and at the same applied voltage.

In some embodiments, the measuring electrode (at least a first electrode) further comprises a a hydrogel that comprises a coenzyme or reduction agent as a component. In some embodiments, the enzyme consists of a dehydrogenase. Further, the reaction product consists of a reduced coenzyme derived by reduction of the coenzyme, and the mediator is operable, during the amino-acid concentration measurement, to carry electrons from the reduced coenzyme to the measuring electrode.

The disclosure also relates to a system comprising the biosensors disclosed herein and a computer processor and a display. In some embodiments, the disclosure relates to a computer-implemented method of quantifying ammonia concentration in a sample, for example, a light detector such as a CMOS camera as disclosed in PCT/US2015/026546, which is included herein by reference in its entirety.

In some embodiments, a biosensor or system disclosed herein is used in conjunction with one or a combination of the following:

1. a power source in electrical connection with the electrodes and capable of supplying an electrical potential difference between the electrodes sufficient to cause diffusion limited electro-oxidation of the reduced form of the mediator at the surface of the working electrode; and

2. at least one meter, (such as a spectrophoteomter, voltmeter and/or amperometer) in electrical connection with the electrodes and capable of measuring the diffusion limited current produced by of the reduced form of the mediator with the above-stated electrical potential difference is applied.

The meter will normally be adapted to apply an algorithm to the current measurement, whereby an ammonia or ammonium ion concentration is provided and visually displayed.

Improvements in such power source, meter, and biosensor system are the subject of commonly assigned U.S. Pat. No. 4,963,814, issued Oct. 16, 1990; U.S. Pat. No. 4,999,632, issued Mar. 12, 1991; U.S. Pat. No. 4,999,582, issued Mar. 12, 1991; U.S. Pat. No. 5,243,516, issued Sep. 7, 1993; U.S. Pat. No. 5,352,351, issued Oct. 4, 1994; U.S. Pat. No. 5,366,609, issued Nov. 22, 1994; White et al., U.S. Pat. No. 5,405,511, issued Apr. 11, 1995; and White et al., U.S. Pat. No. 5,438,271, issued Aug. 1, 1995, the disclosures of which are hereby expressly incorporated by reference.

Ammonia or ammonium ion concetrations from a plurality of samples may be analyzed in parallel. For example, human and non-human body fluids such as whole blood, plasma, sera, lymph, bile, urine, semen, cerebrospinal fluid, spinal fluid, lacrimal fluid and stool specimens as well as other biological fluids readily apparent to one skilled in the art may be measured by palcing multiple test strips in a device disclosed herein at one time. Fluid preparations of tissues from humans and non-human animals can also be assayed, along with foods, water samples, fermentation products and environmental substances, which potentially contain environmental contaminants. In some embodiments, human serum is assayed with the disclosed biosensor. In some embodiments, the biosensor comprises or is configured to assay whole blood. In some embodiments, the term sample specifically excludes samples from a subject and refers to foods, water samples,

fermentation products and environmental substances, which potentially contain environmental contaminants.

After reaction is complete, a power source (e.g., a battery) applies a potential difference between electrodes. When the potential difference is applied, the amount of oxidized form of the mediator at the auxiliary electrode and the potential difference must be sufficient to cause diffusion-limited electro-oxidation of the reduced form of theat least one mediator at the surface of the working electrode. In some embodiments, the working electrode comprises a hydrogel disclosed herein. A current measuring meter (not shown) measures the diffusion-limited current generated by the oxidation of the reduced form of the mediator at the surface of the working electrode. The measured current may be accurately correlated to the concentration of ammonia or ammonium ion and/or one or more amino acids in sample when the following requirements are satisfied:

1. The rate of the indophenol reaction based upon the concentration of indophenol reagents is governed by the rate of diffusion of the ammonia from the sample in a first vessel to the second vessel comprising a surface of the working electrode.

To manufacture biosensor a roll of metallized film is fed through guide rolls into an ablation/washing and drying station. A laser system capable of ablating bottom plate element 14 is known to those of ordinary skill in the art. Non-limiting examples of which include excimer lasers, with the pattern of ablation controlled by mirrors, lenses, and masks. A non-limiting example of

such a system is the LPX-300 or LPX-200 both commercially available from LPKF Laser

Electronic GmbH, of Garb sen, Germany.

In the laser ablator, the metallic layer of the metallized film is ablated in a pre-determined pattern, to form a ribbon of isolated electrode sets. The metallized film is further ablated, after the isolated electrode sets are formed to create recesses positioned adjacent the electrochemical area. The ribbon is then passed through more guide rolls, with a tension loop and through an optional inspection camera. The camera is used for quality control in order to check for defects.

Reagent is compounded and may be applied in a liquid form to the center of one or more vessels or electrochemical area at a dispensing and drying station. Reagent application techniques are well known to one of ordinary skill in the art as described in U.S. Pat. No. 5,762,770, the disclosure of which is expressly incorporated herein by reference. It is appreciated that reagent may be applied to the biosensor, chip, test strip, or solid support disclosed herein in a liquid or other form and dried or semi-dried onto the center of the electrochemical area in accordance with this disclosure. If using liquid reagents, the liquid reagent may be stored in a sealed or unsealed storage vessel such that volume of reagents (anywhere from about 1 microliter to about 100 microliters)may be attached or part of the surface of the biosensor, chip, test strip, or solid support. If sealed, the seal prevent mixing of any of the indophenol reagent prior to introduction of the sample to the biosensor, chip, test strip, or solid support. If sealed, the biosensor, chip, test strip, or solid support may comprises a means to break or puncture or otherwise disrupt the seal so that fluid contents of the storage vessel can use a fluid path to he first or second vessel (a part of the reaction vessel). In some embodiments, the sealed storage vessels are blisterpacks. In some embodiments, the sealed storage vessels are blisterpacks with a portion of the seal adjacent to or substantially proximate to a conduit through which liquid phase reagents may flow to the reaction vessel upon breaking the seal. In some embodiments, reagenets in solid phase may be placed in a regeant storage vessel or in the reagent conduit itself, whereby by liquid phase indophenol reagents may be mixed with solid phase indophenol reagents upon rupture, breakage or disruption of any seals holding back fluid flow in the fluid circuit.

In some embodiments, the biosensor, system, chip, test strip, or solid support disclosed herein comprises the following indophenol reagents in solid or liquid form: a hyophalite, an alkali buffer, a phenolic reagent, and a catalyst. In some embodiments, the biosensor, chip, test strip, or solid support may comprise two alkali buffers sodium acetate and sodium hydroxide in liquid phase

(at concentrations from about 1 μΜ to about 1 M each) in individual reagent storage vessels. In some embodiments, the biosensor, chip, test strip, or solid support may comprise the hypohalite in liquid phase within a reagent storage vessel.

In some embodiments, the indophenol reagents, such as the phenolic reagent and the catalyst are stroed in solid phase. The disclosure relates to the placement of solid phase indophenol reagents in vessels or conduits with in the fluid circuit so that the biosensor, chip, test strip, or solid support may stored for long periods of time. If some solid phase reagents are used, the biosensor, chip, test strip, or solid support may be sotered for no fewer than about 50, 75, 100, 125, 150, 175, 200, 225, or about 245 days at room temperature or below. In some embodiments, room temperature is from about 60 to about 80 degrees Farenheit. In some embodiments, room temperature is about 70 degrees Farenheit. In some embodiments, storage of the reagents in solid phase allows for longterm storage of the biosensor, chip, test strip, or solid support for no fewer than about 50, 75, 100, 125, 150, 175, 200, 225, or about 245 days at or below 0 degrees Celsius.

In addition, a roll or top plate element material is fed into an assembly station along with a roll of spacer material. Liners on either side of the spacer material are removed in that station and the top plate element or surface scaffold is applied to one side of the spacer material to form a top plate element/spacer subassembly. The top plate element/spacer subassembly is slit into the appropriate width for a row of biosensors. Next, a new release liner is added to the side of the spacer material opposite the cover and the subassembly is wound into a roll.

The ribbon of the reagent-coated bottom plate element is unwound and fed into a sensor assembly station along with the top plate element/spacer subassembly. The liner is removed from the spacer and the subassembly is placed on bottom plate elementto cover reagent. Next, the assembled material is cut to form individual biosensors, which are sorted and packed into vials, each closed with a stopper, to give packaged sensor test strips.

Although ablating recesses is described herein, it is appreciated that the method of forming recesses in bottom plate element is also not limited. For example, the recesses may be formed by etching (e.g., using photoligographic methods) or otherwise removing a portion of the surface of top plate element. The nearest electrode edge is approximately about 10 μιη to about 500 μιη from the recess, or about 100 μιη to about 400 μιη from the recess, or from about 200 μιη to about 300 μιη from the recess. Biosensors that are formed with recesses in accordance with this disclosure yield a reagent profile with generally uniform thickness of chemistry. A generally uniform thickness of chemistry allows for more accurate sample analysis.

The processes and products described above include a disposable biosensor, especially for use individually as a diagnostic device or in combination with other components such as a pump system or spectrophotometer configured to diagnose hyperammonemia, abnormal function, or abnormally high or low amounts of ammonia in a sample, in some embodiments, the system disclosed herein comprises a unit configured to receive the biosensor, chip, test strip, or solid support dsiclsoed herein such that insertion of the biosensor, chip, test strip, or solid support into the unit aligns the microfluidic pathways to channels on the unit, whereby a sample may be introduced to the biosensor, chip, test strip, or solid support while nested in the unit and readings from detection equipment, such as a spectrophotometer, may be positioned at near or substantially near a detection vessel such that measurements within the detection vessel may be taken and sent digitally to a display that provides an operator with a reading fof how much ammonia is in the sample. In such embodiments, an operator may engage a microneedles or other means of breaking seals of reagent storage vessels so that liquid phase and solid phase indophenol reganets may mix with a sample in the fluid circuit for a sufficient time to complete an indophenol reaction. After the reaction is complete and a reading is sent to a display for the operator, the biosensor, chip, test strip, or solid support may be removed from the detection unit and disposed of. The unit may be re-used as necessary to accept a plurality of biosensor, chip, test strip, or solid supports disclosed herein.

Variations on the Indophenol Reaction

The disclosure relates to contacting a sample with one or a plurality of reagents in independently variable phases of dried, powdered or aqueous phases. The reaction has four major components: a compound comprising a phenyl group (or phenolic reagent), a hypohalite, a catalyst and an alkali buffer. When these reagents are exposed to ammonia, an indophenol compound is produced that, when exposed to a light source at a particular wavelength, absorbs and/or emits a particular wavenlength of light. In some embodiments, any of the methods disclosed herein make comprise a step of detecting the presence, absence, or quantity of ammonia or ammonium ion by measuring the absorbance of the contents of indophenol reaction products at the first vessel or the second vessel, or the detection vessel.

Family of Phenols or Phenolic reagents

Different compounds comprising a phenyl group can be used as long as the compound comprises a 4, 5, or 6-membered ring with at least one carbon atom and a unsubstituted 'para-position.'

Phenol Thymol Salicylate 2-phenylphenol


Napthol Guaiacol m-Cresol Chlorophenol

Sodum dichloroisocyanurate


Family of Catalysts/Coupling Agents

Sodium Nitroprusside Chromium Iron Manganese


Alkali Conditions

Any buffer capable of creating an alkali microenvironment for the reaction to take place with ammonia from a sample may be used. In some embodiments, a vessel comprising an alkali buffer with pH from about 8.5 to about 13 can be used in the biosensor, test strip, or system disclosed herein. Any compound that can create these alkali conditions can be used including sodium and potassium hydroxide, or sodium or potassium acetate. In some embodiments, the alkali buffer is in a powdered form, lyophilized, or aqueous solution in a vessel located within the biosensor or kit disclosed herein.

Electrode

In some embodiments, the biosensor, system or test strip disclosed herein comprise one or more electrodes. In some embodiments, the one or more electrodes transmit current variation generated by the reaction between the indophenol reagents and ammonia or ammonium ion from a sample and/or transmit current variation generated by a battery source to the light source or other equipment necessary to provide a readout of the levels of ammonia in a sample, for instance, in the case of a spectrophotometer to measure absorbance of a reactant vessel in the biosensor. In some embodiments, the electrodes comprise metal. In some embodiments, the electrodes comprise a

carbon scaffold upon which a metal is deposited. In some embodiments, the electrodes comprise a carbon scaffold of carbon nanotubes.

Electrode structures which are suitable for the present disclosure and methods for the production of such structures have already been suggested in biosensor technology for other purposes. In this regard, reference is made to U.S. Pat. No. 6,645,359 and its content is incorporated herein by reference in its entirety. Electrodes or Electrically conductive tracks are created or isolated on first surface. Tracks represent the electrodes of biosensor. As used herein, the phrase "electrode set" is a set of at least two electrodes, for example 2 to 200, or 3 to 20, electrodes. These electrodes may, for example, be a working (or measuring) electrode and an auxiliary electrode. In some embodiments, tracks cooperate to form an interdigitated electrode array positioned within the periphery of recesses and leads that extend from array and between recesses toward end.

Tracks are constructed from electrically conductive materials. Non-limiting examples of electrically-conductive materials include aluminum, carbon (such as graphite), cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as an amalgam), nickel, niobium, osmium, palladium, platinum, rhenium, rhodium, selenium, silicon (such as highly doped polycrystalline silicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium, mixtures thereof, and alloys, oxides, or metallic compounds of these elements.

Preferably, tracks include gold, platinum, palladium, iridium, or alloys of these metals, since such noble metals and their alloys are unreactive in biological systems. In some embodiments, the track is a working electrode made of silver and/or silver chloride, and track is an auxiliary electrode that is also made of silver and/or silver chloride and is substantially the same size as the working electrode.

Tracks are isolated from the rest of the electrically conductive surface by laser ablation. Techniques for forming electrodes on a surface using laser ablation are known. Techniques for forming electrodes on a surface using laser ablation are known. See, for example, U.S. patent application Ser. No. 09/411,940, filed Oct. 4, 1999, and entitled "LASER DEFINED FEATURES FOR PATTERNED LAMINATES AND ELECTRODE", the disclosure of which is expressly incorporated herein by reference. Tracks are preferably created by removing the electrically conductive material from an area extending around the electrodes. Therefore, tracks are isolated from the rest of the electrically-conductive material on a surface by a gap having a width of about 5 μπι to about 500 μπι, preferably the gap has a width of about 100 μπι to about 200 μπι.

Alternatively, it is appreciated that tracks may be created by laser ablation alone on bottom substrate. Further, tracks may be laminated, screen-printed, or formed by photolithography.

Multi-electrode arrangements are also possible in accordance with this disclosure. For example, it is contemplated that a biosensor may be formed that includes an additional electrically conductive track. In a three-electrode arrangement, the first track is a working electrode, the second is a counter electrode, and the third electrode is a reference electrode. It is also appreciated that an alternative three-electrode arrangement is possible where tracks are working electrodes and a third electrode is provided as an auxiliary or reference electrode. It is appreciated that the number of tracks, as well as the spacing between tracks in array may vary in accordance with this disclosure and that a number of arrays may be formed as will be appreciated by one of skill in the art. in some embodiments, the electrodes are embedded on or attached to a solid support, such as a test strip comprising a plastic and/or paper material.

Micro-electrode arrays are structures generally having two electrodes of very small dimensions, typically with each electrode having a common element and electrode elements or micro-electrodes. If "interdigitated" the arrays are arranged in an alternating, finger-like fashion (See, e.g., U.S. Pat. No. 5,670,031). These are a sub-class of micro-electrodes in general.

Interdigitated arrays of micro-electrodes, or ID As, can exhibit desired performance characteristics; for example, due to their small dimensions, ID As can exhibit excellent signal to noise ratios.

Interdigitated arrays have been disposed on non-flexible substrates such as silicon or glass substrates, using integrated circuit photolithography methods. ID As have been used on non-flexible substrates because ID As have been considered to offer superior performance properties when used at very small dimensions, e.g., with feature dimensions in the 1-3 micrometer range. At such small dimensions, the surface structure of a substrate (e.g., the flatness or roughness) becomes significant in the performance of the IDA. Because non-flexible substrates, especially silicon, can be processed to an exceptionally smooth, flat, surface, these have been used with ID As. In some embodiments, the at least one electrode is a component of any IDA disclosed herein.

Membrane

In some embodiments, the membrane positioned at a fluid exchange opening comprises an ionomer. In some embodiments, the membrane comprises one or a combination of the following polymers:






wherein each of the variables p, q, r, s, t, u, v, w, x, y, and z are independently variable and are 0 or any positive integers; and wherein R is independently selected from an amine, hydroxy, hydroxyl, carbonyl, H, =0, -OH, -COOH, -N, -CH3, -CH2-X, halo, aryl, arylalkoxy, arylalkyl, alkynyl,

alkenyl, alkylene, alkyl, akyl-halo, arylamido, alkylheterocycle, alkylamino, alkylguanidino, alkanol, alkylcarboxy, cycloalkyl, heteroaryl, heteroarylalkyl, heteroarylalkoxy, or heterocyclyl; or any salt thereof. In some embodiments, the variables p, q, r, s, t, u, v, w, x, y, and z are independently variable and are 0 or about 100, 1000, 10000, or 100000 or more whereby, optionally, a manufacturer may orm long micrometer width sheets of polymer that can sover a width of a vessel from about 1 to about 1000 micrliters in diameter.

In some embodiments, the R group is acidic or an electronegative substiuent. In some embodiments, the variables p, q, r, s, t, u, v, w, x, y, z are independently variable and are 0 or positive integers from about 1 to about 200. In some embodiments, the variables p, q, r, s, t, u, v, w, x, y, z are independently variable and are 0 or positive integers from about 10 to about 100. In some embodiments, the variables p, q, r, s, t, u, v, w, x, y, z are independently variable and are 0 or positive integers from about 10 to about 100 across many species within a matrix of material comprising many species of polymer. A- represents the anionic or acidic groups that can include sulfonate, carboxylate, or other similar functional group. M+ represents the counter ion and may include H+, Li+, Na+, or similar cation. Letters (p-z) accompanied by parenthesis or brackets represent repeat units that can range from 0 to any integer value. Any polymer containing any combination of Carbon (C), Fluorine (F), Sulfur (S), Oxygen (O), Hydrogen (H), Nitrogen (N), Phosphorous (P), or any similar element, which may be used to create an ionic exchange membrane may also be utilized.

Ion exchange membranes can be constructed from polymers including perfluorinated ionomers (1&2), polyphosphazene based ionomers (3), polystyrene based ionomers (4), polystyrene based block-co-polymer ionomers (5), and poly(arlyene ether sulfone) based ionomers (6).

Total acid content for ionic exchange membranes may range from about 0.57 to about 3.5 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 4.0 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 3.0 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.9 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.8 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.7 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.6 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.5 meq/g. In some embodiments, the total acid content for ionic

exchange is from about 0.57 to about 2.4 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.3 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.2 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.1 meq/g. In some embodiments, the total acid content for ionic exchange is from about 0.57 to about 2.0 meq/g.

Membranes constructed from these ionomers may range in thickness from about .025 to about .69 mm in thickness. In some embodiments the membrane is from about .001 to about 069 mm in thickness. In some embodiments the membrane is from about .001 to about 068 mm in thickness. In some embodiments the membrane is from about .001 to about 067 mm in thickness.

In some embodiments the membrane is from about .001 to about 066 mm in thickness. In some embodiments the membrane is from about .001 to about 065 mm in thickness. In some embodiments the membrane is from about .001 to about 064 mm in thickness. In some embodiments the membrane is from about .001 to about 063 mm in thickness. In some embodiments the membrane is from about .001 to about 062 mm in thickness. In some embodiments the membrane is from about .001 to about 061 mm in thickness. In some embodiments the membrane is from about .001 to about 060 mm in thickness. In some embodiments the membrane is from about .001 to about 059 mm in thickness. In some embodiments the membrane is from about .001 to about 058 mm in thickness. In some embodiments the membrane is from about .001 to about 050 mm in thickness. In some embodiments the membrane is from about .001 to about 040 mm in thickness. In some embodiments the membrane is from about .001 to about 030 mm in thickness. In some embodiments the membrane is from about .001 to about 020 mm in thickness. In some embodiments the membrane is from about .001 to about 010 mm in thickness. In some embodiments the membrane is from about .025 to about 065 mm in thickness. In some embodiments the membrane is from about .025 to about 064 mm in thickness. In some embodiments the membrane is from about .025 to about 063 mm in thickness. In some embodiments the membrane is from about .025 to about 062 mm in thickness. In some embodiments the membrane is from about .025 to about 061 mm in thickness. In some embodiments the membrane is from about .025 to about 060 mm in thickness. In some embodiments the membrane is from about .025 to about 059 mm in thickness. In some embodiments the membrane is from about .025 to about 058 mm in thickness. In some

embodiments the membrane is from about .025 to about 050 mm in thickness. In some embodiments the membrane is from about .025 to about 040 mm in thickness. In some embodiments the membrane is from about .025 to about 030 mm in thickness. In some embodiments the membrane is from about .025 to about 020 mm in thickness. In some embodiments the membrane is from about .025 to about 010 mm in thickness.

Higher total acid content and smaller membrane thickness leads to faster diffusion times. Membranes may be formed through extrusion casting, drop casting, hot pressing, or similar method.

Cartridges and Disposable Devices

The biosensor, device, system, and or test strip may be or comprise a cartridge. In some embodiments, the cartridge is disposable after one use or can be used more than once per ammonia or ammonium ion detection event. In some embodiments, the cartridge comprises a plurality of microfluidic conduits in fluid communication with a storage portion, a mixing portion and a readout portion of the cartridge. The storage portion comprises a plurality of compartments that store one or a combination of indophenol reagents either crystalized, dried, lyophilized or in solution. In some embodiments, the compartments may be partitioned from an adjacent conduit by plastic wall or other inert material. The mixing portion of the cartridge comprises a trunk-shaped conduit where one or more reagents being stored mix after they are released from the storage portion of the device. The reagents may mix with a sample and/or each other at different points in the microfluidic channels adajacent to the storage portion of the device. In some embodiments of the device the readout portion of the microfluidic conduits is adjacent to the mixing portion of the device. In some embodiments of the device, the cartridge comprises only a storage portion and a readout portion, wherein the readout portion comprises a microfluidic conduit configured to align to an instrucment that measures the amount of ammonia or ammonium in a sample but also allows mixing of samples prior to any detection or quantification step takes place through the instrument. In some

embodiments, the cartridge does not comprise an instrument for detection of the amount of ammonia or ammonium ion in an sample (spectrophotometer), but is configured to align the readout portion of the cartridge to a instrument capable of determining the amount of ammonia or ammonium ion in a sample. In some embodiments, the cartridge comprises an instrument for detection of the amount of ammonia or ammonium ion in an sample, such as a photodiode. In some embodiments, the cartridge comprises readout portion comprising microfluidic conduits for

detection or quantification adjacent to the mixing portion of the device. In some embodiments, the cartridge comprises an instrument for detection of the amount of ammonia or ammonium ion in an sample, such as a photodiode, such instrument comprising a light source aligned to or with the readout portion of the device such that light from the light source may penetrate the readout portion and such instrument may detct the presence, absence or absorbance of wavelength of light in the readout portion.

In some embodiments, the cartridge comprises a microfluidic circuit comprising a storage portion in fluid communication with a mixing portion which is also in fluid communication with a readout portion. Fluid in such an embodiment is designed to flow from the storage portion to the mixing portion, and from the mixing portion to the readout portion of the cartridge. In some embodiments the storage portion comprises one compartment or vessel for each indophenol reagent. In some embodiments, the storage portion comprises a first compartment comprising a hypohalite (such as hypochlorite), a second compartment comprising an basic buffer (such as NaOH), and a third compartment comprising at least one indophenol reagent or indophenol related compound (such as 2-phenylphenol). In some embodiments, the storage portion comprises a fourth

compartment comprising a catalyst or coupling reagent (such as Sodium Nitroprusside). In some embodiments, the storage portion comprises a fifth compartment comprising an alkali buffer (such as sodium acetate or calcium acetate or zinc acetate). In some embodments, the cartridge comprises a fluid exchange opening between a microfluidic conduit the compartment comprising a an alkali buffer (such as sodium acetate or calcium acetate or zinc acetate). In some embodiments, a membrane disclosed herein is positioned over at least a portion of the fluid exchange opening such that when a sample comes in contact with the alkali buffer, ammonia can be transported from the first vessel across the membrane into the second vessel or adjacent microfluidic conduit.

In some embodiments, the storage portion comprises a compartment optionally comprising an electrode. In some embodiments the compartment optionally comprising an electrode is adjacent to a compartment comprising the alkali buffer in solid or liquid phase, such compartment having an opening through which a sample may be deposited into the cartridge from a point exterior to the cartridge. In some embodiments, the cartridge comprises a sixth compartment comprising an opening and optionally comprising an electrode, such compartment having an opening through which a sample may be deposited into the cartridge from a point exterior to the cartridge. In some embodiments, the cartridge comprises a sixth compartment comprising an opening and optionally

comprising an electrode, such compartment having an opening through which a sample may be deposited into the cartridge from a point exterior to the cartridge; wherein the cartridge further comprises a a compartment comprising an alkali buffer that is positioned at or substantially near the compartment comprising the opening, such that, upon inserting a sample into the compartment with an opening, the alkali buffer is transported to the compartment comprising the opening and mixes with the sample.

In some embodiments, a compartment has a volume of no more than about 100 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 100 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 90 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 80 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 70 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 60 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 50 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 40 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 30 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 20 microliters of fluid. In some embodiments, one or more compartments in the cartridge has a volume of no more than about 10 microliters of fluid.

FIG. 1 decpits a photograph of 3D printed modular pieces snapped together around Nafion to form a bisected well. A bisected well containing blood in one section and a concentrated alkali solution in the other would provide a means for cation exchange of the whole blood to occur, yielding a strong recovery of the ammonium. A computer-aided design of the well that is both reusable and modular was 3D printed. As seen in FIG. 1, two modular pieces were 3D printed from acrylonitrile-butadiene-styrene thermoplastic. The pieces will snap together with the membrane in the middle, forming a Nafion bisected well. This design was chosen to provide a uniform platform for all future experiments involving this sensing mechanism. Silicone gasketing material, at a 1/64" thickness, was glued to the inner face of each well-half to ensure a water tight seal. The wells were then back-filled with polydimethylsiloxane to improve their mechanical properties.

FIGs. 2 through 4 depict an embodiment of the disclosure that is a cartridge. One half of the cartridge is depicted in FIG. 2 while the opposite facing half of the cartridge is depicted in FIG. 3. The two halves of the cartridge may be secured together by one or a plurality of micrscrews, dowels or fastners. The two halves of the cartridge may be made out of one or a plurality of inert materials such as a plastic and/or glass.

The cartridge half disclosed in FIG. 2 comprises a first, second, third, fourth and fifth storage compartment. FIG. 2 depicts a first, second, third, fourth and fifth compartment (labeled 1, 2, 3, 4, and 5 respectively) that define a volume immediately adjacent to, but partitioned from, a microfluic conduit on a bottom half of the cartridge. The partition is delineated by the small solid dash bisecting the space between the compartment and the microfluidic conduit (labeled 10, 11, 12, 13, and 14 for each of the compartments 1, 2, 3, 4, and 5 respectively. In this embodiment, the first compartment comprises hypohalite, the second compartment comprises an basic buffer, the third compartment comprises a catalyst, the fourth compartment comprises a indophenol reagent (such as a phenolic compound), and the fifth compartment comprises an alkali buffer, which, if in aqueous solution, may be at a concentration from about 500 mM to about 1 M sodium acetate. The storage portion of the microfluidic circuit comprises the storage points 1, 2, 3, 4, 5, and 6. Any membrane disclosed herein may be placed at or near position 14 such that , upon introduction of a sample such as whole blood in the compartment 6 of FIG. 3, mixing of the reagents can occur. Fluid from compartment 5 is mixed with the sample and ammonia ions in solution may be transferred from 5 and 6 into the mixing portion of the device 20 across the membrance. The reagents in compartments 1 through 4 are also released such that after a period of about 4-5 seconds, all reagents have entered the mixing portion of the device 20. The upper branched portion of the mixing portion 20 mixes the indophenol reagents contained in the compartments 1, 2, 3 and 4 while the ammonia from the sample and te buffer in 5 and 6 mix in the lower trunk of the cartridge. Once in use, FIG. 4 depicts the anticipated flow of fluid from each compartment to the mixing portion of the cartridge. Lighter shades of grey show the bolus of reagents from each compartment as they travel at 0 seconds (i); at 13 second (ii); and 24 seconds (iii) through the microfluid circuit. After mixing is complete in the mixing portion of the device, all reagents mix in the portion of the mixing portion closest proximae to the readout portion 25 of the circuit. At the readout portion of the device, the cartridge may have an opening though which light may travel and expose the fluid to a certain measurable wavelength of light. An instrument such as a photodiode may be present near or adjacent to the readout portion of the device so that measurements of absorbance may be taken.

FIGs. 5A and 5B depict an embodiment of the disclosure that is another type of cartridge. As shown in FIG. 5 A, the cartridge will comprise 2-3 blister packs of reagents, with B l, B2, and B3 indicating blister pack placement. The chanber labeled F is the Nafion membrane and chamber that will take in the blood sample. The chamber labeled O is the overflow chamber for excess sample. The light grey regions are channels by which the liquid reagents can move, with the circle W being the read-out well. The dark grey region indicates placement of dried reagents, for example, sodium nitroprusside and 2-phenlyphenol, which are more stable as dried powered then in liquid or suspended form.

The cartridge in FIG. 5A comprises 2-3 blister packs. The blister pack for sodium

hypochlorite and sodium hydroxide could be combined into one blister pack. The blister packs can have a volume ranging from 5-50 microliters. The region on the chip to the left of the dotted line would sit outside the measurement device once inserted while the region to the right would sit inside the measurement device. A blood sample of a volume of 5-100 microliters would be inserted into the a container on the device by either pipette, capillary action or mechanical action. The Nafion membrane would subdivide the container. One side of the subdivision would contain the freshly added blood sample. On the other side of the membrane would be an empty subdivision that would be filled with the sodium acetate solution from Blister 1. The blister would be depressed using a passive device or a motor such as a stepper motor. Depressing the blister pack would inject sodium acetate solution into the empty subdivision. This would initiate the process of extracting ammonia from the whole blood sample into the sodium acetate solution. From this point, Blister 21 Blister 3 would be depressed injecting the sodium hypochlorite and sodium hydroxide into the Nafion subdivision. This would mix these two reagents with the now ammonium containing sodium acetate solution and push the aggregate mixture into the dark, long channel containing the dried 2-phenylphenol and sodium nitroprusside. This action would reconstitute and dissolve these two reagents so that the final solution contains the sodium acetate, ammonium, sodium hypochlorite, sodium hydroxide, sodium nitroprusside and 2-phenylphenol. This solution would then be pushed by further depression of the blisters through the 'mixing region' of the fluidic chip until it reaches the measurement well of the device. The colorimetric reaction would then proceed and the resultant color measured by a device. For example, the cartridge could be inserted into slot A of the chip reader shown in FIG. 5B.

In some embodiments, the cartridge comprises at least one electrode that detects the presence or absence of ammonia or ammonium ion in a sample in a vessel configured to receive a sample from a point external to the cartridge. Once the electrode is activated by the presence of a sample, the storage portion of the cartridge open and release their contents such that a solution from each compartment is released into the mixing portion of the microfluidic conduits. The microfluidic conduits are of a length sufficient to mix all of the reagents from each compartment such that, by the time total fluid volume of reactants reach the readout portion of the cartridge, an indophenol reaction has taken place and an indophenol reaction product (such as indophenol or an indophenol related compound) have formed in the microfluidic conduits.

Table 3

Exam les of Indo henol Rea ent Concentration Ran es


Hydrogel

The biosensor comprises a hydrogel in some embodiments. The hydrogel may be a cross- linked polymeric material that swells in water but does not dissolve. It is envisioned that the hydrogel may be capable of absorbing at least about 1 to about 10 times, and in one embodiment at least about 100 times, its own weight of a liquid. The hydrogel chosen for use in the biosensor should depend directly on the method of functionalization. It is envisioned that the hydrogel may be biocompatible. In some embodiments, the hydrogel comprises sodium alginate. In some

embodiments, the hydrogel comprises from about 0.1% to about 5% alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.1% to about 4% alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.1% to about 3% alginate

weight/volume. In some embodiments, the hydrogel comprises from about 0.1% to about 2% alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.1% to about

1%) alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.1% to

about 1% alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.2% to about 1%) alginate weight/volume. In some embodiments, the hydrogel comprises sodium alginate. In some embodiments, the hydrogel comprises from about 0.3% to about 1% alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.4% to about 1% alginate weight/volume In some embodiments, the hydrogel comprises from about 0.5% to about 1%) alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.6% to about 1%) alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.7% to about 1%) alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.8%) to about 1%) alginate weight/volume. In some embodiments, the hydrogel comprises from about 0.9%) to about 1%> alginate weight/volume. In some embodiments, the hydrogel comprises from about 1.0% to about 3.0% alginate weight/volume. In some embodiments, the hydrogel comprises from about 1.0% to about 2.0% alginate weight/volume. In some embodiments, the hydrogel comprises from about 1.0% to about 1.5% alginate weight/volume. In some embodiments, the hydrogel comprises about 1%, about 2%, or about 3% alginate weight/volume. In some embodiments, the hydrogel comprises sodium alginate. The aliginate may be any individual polymer of alginate used in bulk form or repitive pattern of monomers, G blocks, M blocks, and/or GM blocks. In some embodiments the alignate comprises the formula:


where m and n are any positive integer. In some embodiments m and n are indepedently variable and any positive integer from about 1 to about 1000. In some embodiments, the hydrogel may be polymerized from acrylic monomers. The acrylic monomer may be one or a combination of the following: acrylamido-glycolic acid, acrylamido-methyl-propa-ne-sulfonic acid, acrylamido-ethylphosphate, diethyl-aminoethyl-acrylamide-, trimethyl-amino-propyl-methacrylamide, N-octyl aery 1 amide, N-phenyl-acrylamide and tert-butyl-acrylamide. In embodiments in which the device contains a cross-linking agent, exemplary cross-linking agents may be N,N'-methylene-bis- acrylamide, Ν,Ν'-methylene-bismethacrylamide, di ally ltatardi amide and poly(ethylene glycol)dimethacrylate. Examples of suitable hydrogels may also include silicon wafers, borosilicate glass substrates, 2-hydroxyethyl methacrylate (HEMA), N-Isopropylacrylamide (NIPAAm), and polyethylene glycol (PEG).

The hydrogel may include any number of molecules. For example, the hydrogel may include a polymerized monomer or hydrogel a cross linking agent and optionally a chemical or UV-light activated inducer agent. Examples of such monomers or dimers include vinyl acetates, vinyl pyrrolidones, vinyl ethers, olefins, styrenes, vinyl chlorides, ethylenes, acrylates, methacrylates, nitriles, acrylamides, maleates, epoxies, epoxides, lactones, ethylene oxides, ethylene glycols, ethyloxazolines, amino acids, saccharides, proteins, anhydrides, amides, carbonates, phenylene oxides, acetals, sulfones, phenylene sulfides, esters, fluoropolymers, imides, amide-imides, etherimides, ionomers, aryletherketones, amines, phenols, acids, benzenes, cinnamates, azoles, silanes, chlorides, and epoxides, Ν,Ν'-methylenebisaciylamide, methylenebismethacrylamide ethyleneglycol-dimethacrylate, Ν,Ν'-methylenebisaciylamide, polyethyleneglycoldiacrylate (PEGDA), polyethyleneglycoldimethacrylate (PEGDMA), polyethyleneglycoldiacrylate (PEGDA), polyethyleneglycoldimethacrylate (PEGDMA), poly(vinyliden fluoride) (PVdF) based polymer, a polyacrylonitrile (PAN) based polymer, a polymethylmethacrylate (PMMA) based polymer, a polyvinyl chloride (PVC) based polymer, and a mixture of the poly(vinyliden fluoride) (PVdF) based polymer, polyacrylonitrile (PAN) based polymer, polymethylmethacrylate (PMMA) based polymer, and polyvinyl chloride (PVC) based polymer, and mixtures of any two or more thereof. IN some embodiments, the hydrogel does not comprise 3,4-dihydroxybenzoic acid (3, 4-DHB) or an analog thereof.

Cross linking agents and optionally the chemical or UV-light activated inducer agent may include Ν,Ν'-methylenebisaciylamide, methylenebismethacrylamide ethyleneglycol-dimethacrylate and agent Ν,Ν'-methylenebisaciylamide. Irgacure 2959 (Ciba); 2,2-dimethoxy-2-phenylacetophenone, 2-methoxy-2-phenylacetone, benzyl-dimethyl-ketal, ammonium sulfate, benzophenone, ethyl benzoin ether, isopropyl benzoin ether, .alpha. -methyl benzoin ether, benzoin phenyl ether, 2,2-diethoxy acetophenone, 1,1-dichloro acetophenone, 2-hydroxy-2-methyl-l-phenylpropane 1-on, 1 -hydroxy cyclohexyl phenyl ketone, antraquinone, 2-ethyl antraquinone, 2-chloroantraquinone, tioxantone, isopropyltioxantone, chloro tioxantone, 2,2-chlorobenzophenone, benzyl benzoate, and benzoyl benzoate, TEMED, and ammonium persulfate (APS). In some

embodiments, hydrogel comprises a protein, peptide, glycoprotein, proteoglycans,

glycosaminoglycans, and/or carbohydrate that is secreted by cells into the extracellular

environment. In some embodiments, the secreted protein, peptide, glycoprotein, proteoglycans, glycosamainoglycans, and/or carbohydrate, or structures composed thereof.

In some embodiments, the disclosure relates to a coated biosensor device comprising at least one coating, wherein the biosensor comprises a metabolic enzyme covalently bound or immobilized to the coating, wherein the metabolic enzyme shares at least 70% sequence identify to SEQ ID NO: 1 or SEQ ID NO:2 or shares at least 70% sequence identify to functional fragments of SEQ ID NO: 1 or SEQ ID NO:2. In some embodiments, the disclosure relates to a coated biosensor device comprising at least one coating, wherein the biosensor comprises a metabolic enzyme covalently bound or immobilized within the coating, wherein the coating comprises a composition comprising a hydrogel matrix, said matrix comprising any one or combination of: alginate, trehalose, at least one electron mediator, and at lest one reduction agent. In some embodiments, the disclosure relates to a coated biosensor device comprising at least one coating, wherein the biosensor comprises a metabolic enzyme covalently bound or immobilized to the coating, wherein the coating comprises a composition comprising a hydrogel matrix, said matrix comprising any one or combination of: poly(ethylene glycol) dimethyacrylate with a molecular weight of about 1000 (PEGDMA-1000), 2-hydroxy-2 methyl propiophenone (HMPP) and at least one acrylate, wherein the acrylate is selected from the group consisting of methacrylic acid (MAA) and methyl methacrylate (MMA), wherein the ratio of PEGDM A: Acrylate is from about 10:90 mol % to about 70:30 mol %, and said HMPP is at a concentration of from about 0.2% to about 0.6%, total weight.

In some embodiments, the hydrogel solution prior to curing comprises trehalose or an analog thereof at a concentration from about 1 nM to about 999 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 10 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 9 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 8 mM. In some emboidiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 7 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 6 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 5 mM. In some emboidiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 4 mM. In some emboidiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 3 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 2 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 1 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 10 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 100 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 200 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 300 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 400 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 500 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 600 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 700 μΜ to about 1 mM. In some embodiments, the hydrogel solution prior to curing comprises trehalose at a concentration from about 800 μΜ to about 1 mM. In some embodiments, the hydrogel solution (prior to contacting with the electrode) comprises trehalose at a concentration from about 900 μΜ to about 1 mM.

Mediators

In some embodiments, the biosensor is free of an electorn mediator, in some embodiments, the biosensor is free of at least one or a combination of electron mediators selected from: thionine, o-phenylenediamine, methylene blue, and toluidine blue.

In some embodiments, the reaction surface comprises an electron mediator. The mediator facilitates transport of electrons to the electrode. In some embodiments, the mediator is attached to the electrode. In some embodiments, the mediator is embedded in the hydrogel. In some embodiments, the hydrogel comprises one or a combination of mediators chosen from: mediator 2-Acrylamido-2-methylpropanel, sulfonic acid IV, ethacrylic acid, 2-Sulfoethyl methacrylate, and 2-Propene-l-sulfonic acid. U.S. Pat. No. 4,254,222 (1981; Owen) and U.S. Pat. No. 4,351,899

(1982; Owen) disclose an assay for .beta.-hydroxybutyrate where 3-hydroxybutyrate is oxidized to acetoacetate by .beta.-hydroxybutyrate dehydrogenase (HBDH) in the presence of nicotinamide adenine dinucleatide (NAD.sup.+). The reduced NADH produced from this reaction, in turn, reacts with a tetrazolium dye to form a colored formazan compound. The degree and intensity of the color transition correlates to the concentration of .beta.-hydroxybutyrate in the sample solutions. U.S. Pat. No. 5,510,245 (1996; Magers) and U.S. Pat. No. 5,326,697 (1994; Magers) disclose an improved calorimetric method that utilizes a reductive pathway based on lipoamide dehydrogenase (LADH) and a thiol-sensitive indicator dye such as Ellman's reagent. It was found the NADH, produced from the .beta.-hydroxybutyrate dehydrogenase enzyme reaction, can interact with lipoamide dehydrogenase (LADH) and D,L-lipoamide to form athiol compound (6,8-dimercaptooctamide). The 6,8-dimercaptooctamide then interacts with a thiol-responsive indicator dye such as Ellman's reagent. Upon reaction, the thiol-sensitive indicator dye undergoes a detectable color transition that can be used to measure the level of 3-hydrobutyrate in the blood sample. The colorimetric methods for 3-hydrobutyrate suffer the disadvantages of poor stability, interference from co-existing species such as ascorbate, glutathione etc. in the blood, and insufficient sensitivity and accuracy. NAD- and NADP-dependent enzymes are of great interest insofar as many have substrates of clinical value, such as glucose, D-3-hydroxybutyrate, lactate, ethanol, and cholesterol. Amperometric electrodes for detection of these substrates and other analytes can be designed by incorporating this class of enzymes and establishing electrical communication with the electrode via the mediated oxidation of the reduced co factors NADH and NAD PH. NAD- and NADP-dependent enzymes are generally intracellular oxidoreductases. The oxidoreductases are further classified according to the identity of the donor group of a substrate upon which they act. The category of oxidoreductases is also broken down according to the type of acceptor utilized by the enzyme. The enzymes of relevance have NAD+ or NADP+ as acceptors. These enzymes generally possess sulphydryl groups within their active sites and hence can be irreversibly inhibited by thiol -reactive reagents such as iodoacetate. An irreversible inhibitor forms a stable compound, often through the formation of a covalent bond with a particular amino acid residue that is essential for enzymatic activity. U.S. Pat. No. 6,541,216 (2003; Wilsey et al.) discloses a biosensor and method to test blood ketone bodies using an amperometric meter. The test strip has a reagent that is reactive with β-hydroxybutyrate in sample solution to generate an electrical output signal, which is related to the concentration of .beta.-hydroxybutyrate in the sample solution. The reagent in this method includes ferricyanide salt as mediator, .beta.-hydroxybutyrate dehydrogenase as the first enzyme operative to catalyze the oxidation of .beta.-hydroxybutyrate, NAD+ as a cofactor corresponding to the first enzyme, and diaphorase as the second enzyme operative to catalyze the oxidation of a reduction form of the cofactor (NADH). The oxidation form of the mediator will accept the electron from the second enzyme and generates an electrical signal at the electrode surface, which is related to the concentration level of .beta.-hydroxybutyrate. U.S. Pat. No. 6,736,957 (2004; Forrow et al.) and a research paper (N.J. Forrow et.al, Biosensors & Bioelectronics, 2005, 20, 1617-1625) disclose an amperometric biosensor for .beta.-hydroxybutyrate based on the discovery of NAD+ and NADP-mediator compounds that do not bind irreversibly to thiol groups in the active sites of intracellular dehydrogenase enzymes. These mediator compounds such as 1,10-phenanthroline quinone (1,10-PQ), which is used as an electron mediator in their electrochemical measurement system, can increase the stability and reliability response in amperometric electrodes constructed from NAD- and NADP-dependent enzyme. The dry reagents include 1, 10-phenanthroline quinone (1,10-PQ), .beta.-hydroxybutyrate dehydrogenase andNAD+ as the cofactor. This sensor shows reliable and sensitive response to the concentration levels of .beta.-hydroxybutyrate in blood samples. Meldola's Blue (MB) was also studied as a mediator in the system, but it was found that MB did not work well in their electrochemical test system due to the inhibition of .beta.-hydroxybutyrate dehydrogenase enzyme activity by MB and poor long term stability of the test strips.

The dehydrogenase enzymes such as, for example, glucose dehydrogenase, D-3-hydroxybutyrate dehydrogenase (FIBDH), and lactate dehydrogenase et.al are known to be common dehydrogenases for construction of biosensors. As disclosed by Forrow et al., there are certain mediators that are considered efficient mediators for NADH but are irreversible enzyme inhibitors such as Meldola's blue, 4-methyl-l,2-benzoquinone (4-MBQ), 1-methoxy phenazine methosulphate (1-Meo-PMS) and 2,6-dichloroindophenol (DCIP), which cause losing the activity of enzymes, insensitive response and poor stability in sensors containing dehydrogenase enzymes. In some embodiments, the biosensor, system, or test strip comprise any one or more of the mediators disclosed herein. In some embodiments, the mediator is chosen fromone or a combination of:

ortho-quinones, para-quinones and quinoneimines in their basic structural elements. The representative examples of the quinoid structure type include, but are not limited to, benzo-.alpha.-phenazoxonium chloride, Meldola's Blue (MB), 3,4-methyl-l,2-benzoquinone, 1-methoxy

phenazine methosulphate, 1,10-phenanthroline quinone (1,10-PQ). in some embodiments, the at least one mediator is selected from one or a mixture of the following compounds:


cr

ΐ¾Β 58 biOB

M tbyfcac Ste

Cof actors/Reduction Agent

NAD+

FAD+

Ascorbic Acid

Flavin mononucleotide

Flavin adenine dinucleotide

Coenzyme F420

Glutathione

Heme

Pyrroloquinoline quinone

Enzymes

Any one or more metabolic enzymes may be chosen to used with the present disclosure. Metabolic enzymes that can be used individually or in combination with the biosensor, system or test strip disclosed herein include: any bacterial clone of phenylalanine dehydrogenase, histidine ammonia lyase, mistidine oxidase, pheylalanine lyase, glutamate dehydrogenase. In some embodiments the enzyme is chosen from any one or combination of enzymes disclosed below or their respective functional fragments that are at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) homoglous to the full-length enzyme or nucleic acid encoding such enzyme.

Organism Enzyme GenBank Accession No Seq ID No

Thermoactinomyces phenylalanine dehydrogenase D00631.1 2 intermedius

Solanum lycopersicum phenylalanine ammonia-lyase XM_004246602 7

Thermoactinomyces phenylalanine dehydrogenase DD421709.1 8 intermedius

Caenorhabditis remanei phenylalanine dehydrogenase XM_003102740 9

Arabidopsis thaliana glutamate dehydrogenase NM_121822.3 10

Spirochaeta africana Hisitidine ammonia lyase NC_017098.1

SEQ ID NO:2

MRDVFEMMDRYGHEQVIFCRHPQTGLKAIIALHNTTAGPALGGCRMIPYASTDEALEDVL

RL SKGMT YKC SL AD VDFGGGKM VIIGDPKKDK SPELFRVIGRF VGGLNGRF YTGTDMGTN

PEDFVHAARESKSFAGLPKSYGGKGDTSIPTALGVFHGMRATARFLWGTDQLKGRVVAIQ

GVGKVGERLLQLLVEVGAYCKIADIDSVRCEQLKEKYGDKVQLVDVNRIHKESCDIFSPCA

KGGVV DDTIDEFRCLAIVGSANNQLVEDRHGALLQKRSICYAPDYLVNAGGLIQVADEL

EGFHEERVLAKTEAIYDMVLDIFHRAK ENITTCEAADRIVMERLKKLTDIRRILLEDPRNS

ARR

SEQ ID NO: 7

MASSIVQNGHWGEAMDLCKKSINV1WPLNWEMAAESLRGSHLDEVKKMVDEFRKPIVK

LGGETLTVAQVASIANVDNKSNGVKVELSESARAGVKASSDWVMDSMGKGTDSYGVTTG

FGATSHRRTKNGGALQKELIRFLNAGVFGNGTESSHTLPHSATRAAMLVRINTLLQGYSGI

RFEILEAITKLINSNITPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVGPNGEKLNAEEAF

RVAGVTSGFFELQPKEGLALVNGTAVGSGMASMVLFESNILAVMSEVLSAIFAEVMNGKP

EFTD YLTHKLKHHPGQIEA AAF EHILDGS S YVK AAQKLHEMDPLQKPKQDRYALRT SPQ

WLGPQffiVIRAATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNTRLALASIG

KLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGLKGAEIAMASYCSELQFLANPVTNHV

QSAEQHNQDWSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRSAVKNTVS

Q VAKRTLTMGANGELHP ARFCEKELLRVVDREYVF AYADDPC S ST YPLMQKLRQVLVDH

AMKNGESEKNVNSSIFQKIVAFEDELKAVLPKEVESARAVVESGNPAIPNRITECRSYPLYR

LVRQELGSELLTGEKVRSPGEEIDKVFTAMCNGQIIDPLLECLKSWNGAPLPIC

SEQ ID NO: 8

atgcgcgacg tgtttgaaat gatggaccgc tatggccacg agcaggtcat tttttgccgt

61 catccgcaaa ccggtctcaa agcgatcatc gccttgcata atacaaccgc ggggccggct

121 ttgggtggat gccgcatgat cccgtatgct tcgacggacg aagccttgga ggatgttttg

181 cggttgtcca aaggcatgac ctataaatgc agtctggcgg atgtggactt tggcggggga

241 aaaatggtta tcatcggcga tccgaaaaaa gataaatcgc cggagttgtt tcgcgtgatc 301 ggccgttttg tgggcgggtt aaacggccgt ttctataccg gaaccgacat gggaaccaat

361 ccggaagatt ttgtccatgc cgccagggaa tcgaaatctt ttgccggatt gccgaaatcg

421 tacggcggaa agggggacac atccattccc accgcgctcg gggtgtttca cggaatgcgg

481 gccaccgccc ggtttttatg ggggacggat cagctgaaag ggcgtgtggt tgccatccaa

541 ggagtcggca aggtgggaga gcgcttgttg cagcttttgg tcgaagtggg ggcttactgc

601 aaaattgccg acatcgattc ggtgcgatgc gaacagctga aagaaaagta tggcgacaag

661 gtccaattgg tggatgtgaa ccggattcac aaggagagtt gcgatatttt ctcgccttgc

721 gccaaaggcg gcgtggtcaa tgatgacacc attgacgagt tccgttgcct ggccattgtc

781 ggatccgcca acaaccaact ggtggaagac cggcatgggg cactgcttca aaaacggagc

841 atttgttatg cacccgatta tctggtgaat gccggcgggc tgattcaagt ggctgatgaa

901 ctggaaggct tccatgaaga gagagtgctc gccaaaaccg aagcgattta tgacatggtc

961 ctggatattt ttcaccgggc gaaaaatgag aatattacca cttgtgaggc agcggaccgg

1021 atcgtgatgg agcgtttgaa aaagttaacc gatattcgcc ggatcttgtt ggaggatccc

1081 cgcaacagcg caaggaggta a

SEQ ID N0:9

MDFKAKLLAEMAKKRKAVSGLEVKEGGAKFVRGADLESKRTQEYEAKQEELAIKKRKAD

DEILQESTSRAKIVPEVPEAEFDEKTPMPEIHARLRQRGQPILLFGESELSVRKRLHQLEIEQP

EL EGWE EMQTAMKFIGKEMDKAVVEGTADSATRHDIALPQGYEEDNWKSIEHASTLL

GVGDEMKRDCDIILSICRYILARWARDL DRPLDVKKTAQGMHEAAHHKQTTMHLKSLM

TSMEKYNVN DIRHHLAKICRLLVIERNYLEANNAYMEMAIGNAPWPVGVTRSGIHQRPG

SAKAYVSNIAHVL DETQRKYIQAFKRLMTKLQEYFPTDPSKSVEFVKKSV

SEQ ID NCv lO

MNALAAT R FKLAARLLGLDSKLEKSLLIPFREIKVECTIPKDDGTLASFVGFRVQHDNA

RGPMKGGIRYFffE VDPDE VN AL AQLMT WKT A VAKIP YGGAKGGIGCDP SKL SI SELERLTR

VFTQKIHDLIGIHTDVPAPDMGTGPQTMAWILDEYSKFHGYSPAVVTGKPIDLGGSLGRDA

ATGRGVMFGTEALL EHGKTISGQRFVIQGFGNVGSWAAKLISEKGGKIVAVSDITGAIKN

KDGIDIPALLKHTKEHRGVKGFDGADPIDPNSILVEDCDILVPAALGGVINRENA EIKAKFI

IEAA HPTDPDADEILSKKGVVILPDIYANSGGVTVSYFEWVQNIQGFMWEEEKVNDELKT

YMTRSFKDLKEMCKTHSCDLRMGAFTLGV RVAQATILRGWGA

SEQ ID NO: !

MNTVTNQWKAVDIFTQIRDHEQVVFC DKNTGLKAIIAIHDTTLGPALGGCRMYPYATVE

DALFDVLRLSKGMTYKCLAADVDFGGGKAVIIGDPHKDKTPELFRAFGQFVESLNGRFYT

GTDMGTTPDDFVHAMKETNCIVGVPEEYGGSGDSSVPTALGVIYGIQAT KVIWGSDELH

GKTYAIQGLGKVGRKVAERLLKEGADLYVCDIHPTAIEAIVSYAKKLGANVKVVQGTEIY

RTDADIFVPCAFGNVVNDNTIHVLKVKAIVGSANNQLLDVRHGQLLKEKGILYAPDYIVNA

GGLIQVADELYGL KERVLQKTKAIYSTLLHIYSRAEADHITTIEAA RFCEERLQQRSRRN

DFF THRKQPK WDIRR

Solid Support

There are many forms of ammonia- or ammonium ion-measuring devices; one common type is represented by hand-held electronic meters which receive blood samples via enzyme-based test strips. In using these systems, the patient may for example lances a finger or alternate body site to obtain a blood sample, the strip is inserted into a test strip opening in the meter housing, the sample is applied to the test strip and the electronics in the meter convert a current generated by the enzymatic reaction in the test strip to a amino acid concentration value.

Solid supports of the disclosure may be solid state but are a flexible substrate. According to the disclosure, the interdigitated array or at least one electrode is disposed proximal to, e.g., on, a flexible substrate. To act as a flexible substrate, a material must be flexible and also insulating, and is typically relatively thin. The substrate should be capable of adhering components of an IDA, or additional components of a sensor, to its surface. Such thin, insulative, flexible substrates are known in the art of flexible circuits and flex circuit photolithography. "Flexible substrates" according to the present disclosure can be contrasted to non-flexible substrates used in integrated circuit (IC) photolithography but not in flexible circuit photolithography. Examples of non-flexible substrates used in IC photolithography include silicon, aluminum oxide, and other ceramics. These non-flexible substrates are chosen to be processable to a very flat surface. Typical flexible substrates for use in the disclosure are constructed of thin plastic materials, e.g., polyester, especially high temperature polyester materials; polyethylene naphthalate (PEN); and polyimide, or mixtures of two or more of these. Polyimides are available commercially, for example under the trade name Kapton®, from I.E. duPont de Nemours and Company of Wilmington, Del. (duPont). Polyethylene naphthalate is commercially available as Kaladex®, also from duPont. A particularly preferred flexible substrate is 7 mil thick Kaladex®film.

Interdigitated arrays of the disclosure can be used in applications generally known to incorporate electrodes, especially applications known to involve interdigitated arrays of electrodes. Various applications are known in the arts of electronics and electrochemistry, including applications relating to process and flow monitoring or control, and chemical analytical methods. The arrays may be particularly useful as a component of an electrochemical sensor, where there is added value, benefit, or cost efficiency, to the use of a flexible substrate, or where there is value, benefit, or cost efficiency in having an interdigitated array of dimensions relatively larger than the dimensions of interdigitated arrays conventionally disposed on non-flexible substrates.

An interdigitated array of the disclosure can, for example, be included in an electrochemical sensor (sometimes referred to as a "biosensor" or simply "sensor") used in electrochemical detection methods. Electrochemical detection methods operate on principles of electricity and chemistry, or electrochemistry, e.g., on principles of relating the magnitude of a current flowing through a substance, the resistance of a substance, or a voltage across the substance given a known current, to the presence of a chemical species within the substance. Some of these methods can be referred to as potentiometric, chronoamperometric, or impedance, depending on how they are practiced, e.g., whether potential difference or electric current is controlled or measured. The methods and sensors, including sensors of the disclosure, can measure current flowing through a substance due directly or indirectly to the presence of a particular chemical compound (e.g., an analyte or an electroactive compound), such as a compound within blood, serum, interstitial fluid, or another bodily fluid, e.g., to identify levels of amino acids, blood urea, nitrogen, cholesterol, lactate, and the like. Adaptations of some electrochemical methods and electrochemical sensors, and features of their construction, electronics, and electrochemical operations, are described, for example, in U.S. Pat. Nos. 5,698,083, 5,670,031, 5, 128,015, and 4,999,582, each of which is incorporated herein by reference.

In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of anticoagulant. In some embodiments, the volume of the anticoagulant disclosed herein in a volume of about 10 microliters. In some embodiments, the volume of the anticoagulant disclosed herein in a volume of about 20 microliters. In some embodiments, the volume of the anticoagulant disclosed herein in a volume of about 30 microliters. In some embodiments, the volume of the anticoagulant disclosed herein in a volume of about 40 microliters. In some embodiments, the volume of the anticoagulant disclosed herein in a volume of about 50 microliters. In some embodiments, the volume of the anticoagulant disclosed herein in a volume of about 100 microliters.

In some embodiments, the methods disclosed herein comprise a step of mixing a sample comprising blood with an anticoagulant such as heparin, Acenocoumarol, phenprocoumon,

Atromentin, Brodifacoum, Phenindione, Coumadin or the like. In some embodiments the biosensor, cartridge, device, or test strip comprise a mechanical shaker mechanism configured to shake one or more volumes within the at least one vessel, microfluidic conduit, or mixing portion of the biosensor, cartridge, device, or test strip.

Methods

The disclosure relates to a method of diagnosing or prognosing a clinical outcome of a subject with hyperammonemia or a hyperammonia related disorder, comprising contacting a sensor, system, or test strip disclosed herein with a sample of bodily fluid, and quantifying a level of ammonia or ammonium ion in the sample; and comparing the level of amino acid in the sample to a threshold value of what is considered normal level of amino acid level in the bodily fluid. In some embodiments, the method relates to to a method of diagnosing or prognosing a clinical outcome of a subject suspected of having or having been previously diagnosed with hyerpammonemia or a hyperammonemia-related disorder and/or at least one aminoacidopathy.

In some embodiments, the method relates to to a method of diagnosing or prognosing a clinical outcome of a subject suspected of having or having been previously diagnosed with at least one hyerpammonemia or a hyperammonemia-related disorder. The ranges of what ammonia or ammonium ion levels are considered normal for each age type are below in Table 4. If, after performing the quantification steps provided herein, the amount of ammonia or ammonium ion in the sample solution exceeds or falls below the ranges provided, diet regimen, exercise regimen, and/or medical treatment may be initiated or changed such that ammonia or ammonium ion levels are monitored until the subject's levels have stabilized or fall within what is considered a healthy range.

Table 4

Ammonia Ran es

The disclosure relates to a method of detecting the presence or absence or quantity of ammonia or ammonium related disorder in bodily fluids. The disclosure also relates to a method of quantifying the concentration of ammonia or ammonium ion in bodily fluids of a subject.

Quantification can occur at the point-of-care due to the quick enzymatic reaction readout caused by the generation of a detectable current within a circuit after exposure of a sample from a subject to one or a plurality of vessels comprising any one or combination of indophenol reagents disclosed herein. In some embodiments, the device or system described herein may be utilized to detect if a person has abnormally high or low levels of ammonia in the blood, after which an electronic message or display may then be provided to the user of the device or system or activated on a display by one or more processors or microchips that remotely or directly access one or more storage memories comprising one or rmore concentration values of ammonia or ammonium ion in sample of the subject. In some embodiments, multiple concentration values may be obtained either simultaneously or in series, compared or analyzed by the one or more processors operably connected to the device or system disclosed herein. In some embodiments, multiple concentration values of a subject over a time period may be compared or analyzed by the one or more processors operably connected to the device or system disclosed herein, after which a message comprising the concentration value and/or threshold values are displayed. In some embodiments, the message optionally includes a signal indicating that the subject should seek medical treatment or alter diet to control ammonia or ammonium ion levels in the subject.

The disclosure also relates to a method of diagnosing a subject with a liver dysfunction comprising:

(a) contacting a sample of bodily fluid from a subject to the to the biosensor, system or test strip disclosed herein;

(b) quantifying one or more concentration values of ammonia in the sample;

(c) comparing the one or more concentration values of ammonia in the sample to a threshold value of ammonia concentration identified as being in a healthy range; and

(d) identifying the subject as having a metabolic disease if the one or more concentration values of ammonia in the sample exceed or fall below the threshold value. In some embodiments, if the sample is blood or whole blood, the method comprises contacting the sample with an anticoagulant before or simultaneously with step (a).

The disclosure also relates to a method of diagnosing a subject with hyperammonemia comprising:

(a) contacting a sample of bodily fluid from a subject to the to the biosensor, system or test strip disclosed herein;

(b) quantifying one or more concentration values of ammonia in the sample;

(c) comparing the one or more concentration values of ammonia in the sample to a threshold value of ammonia concentration identified as being in a healthy range; and

(d) identifying the subject as having a metabolic disease if the one or more concentration values of ammonia in the sample exceed or fall below the threshold value. In some embodiments, if the sample is blood or whole blood, the method comprises contacting the sample with an anticoagulant before or simultaneously with step (a).

The disclosure also relates to a method of quantifying the amount of amino acid in sample comprising:

(a) contacting a sample of bodily fluid from a subject to the to the biosensor, system or test strip disclosed herein;

(b) quantifying one or more concentration values of ammonia in the sample;

(c) comparing the one or more concentration values of ammonia in the sample to a threshold value of ammonia concentration identified correlating to amino acid quantity; and

(d) identifying the amino acid levels if the one or more concentration values of ammonia in the sample exceed or fall below the threshold value.

Any amnio acid may be detected using wherein the biosensor, system or test strip disclosed herein comprises an enzyme disclosed herein or a functional fragment that has 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to any enzyme disclosed herein. One of ordinary skill in the art would know, for instance, that to detect the presence, absence, or quantity of amino acids listed on Table 5, one or more recombinant or synthetic enzymes disclosed herein or a functional fragment thereof that has 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to any sequence (either nucleic acid or encoded amino acid) disclosed herein.

In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 52 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 54 to about 70 mmol/liter. In some

embodiments, the phenolic reagent or indophenol reagent is used in a range from about 56 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 58 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 60 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 62 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 64 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range

from about 66 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 68 to about 70 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 68 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 66 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 64 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 62 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 60 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 58 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 56 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 54 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in a range from about 50 to about 52 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is used in concentration about 59 mmol/liter. In some embodiments, the phenolic reagent or indophenol reagent is 2-phenylphenol.

In some embodiments, the basic buffer is used in a range from about 50 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 120 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 140 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 160 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 180 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 200 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 220 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 240 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 260 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 280 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 300 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 320 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 340 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 360 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 380 to about 500 mmo [/liter. In some embodiments, the basic buffer is used in a range from about 400 to about 500 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 420 to about 500 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 440 to about 500 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 460 to about 500 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 480 to about 500 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 480 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 460 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 440 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 420 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 400 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 380 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 360 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 340 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 320 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 300 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 280 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 260 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 240 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 220 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 200 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 180 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 160 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 140 mmo [/liter. In some embodiments, the basi c buffer is used in a range from about 100 to about 120 mmo [/liter. In some embodiments, the basi c buffer is used in a concentration about 50 mmol/liter.

In some embodiments, the basic buffer is sodium hydroxide. In some embodiments, the basic buffer is used in a concentration about 100 mmol/liter. In some embodiments, the basic buffer is sodium hydroxide. In some embodiments, the basic buffer is used in a concentration about 200 mmol/liter. In some embodiments, the basic buffer is sodium hydroxide. In some embodiments, the basic buffer is used in a concentration about 300 mmol/liter. In some embodiments, the basic buffer is sodium hydroxide. In some embodiments, the basic buffer is used in a concentration about 400 mmol/liter. In some embodiments, the basic buffer is sodium hydroxide. In some embodiments, the basic buffer is used in a concentration about 500 mmol/liter. In some embodiments, the basic buffer is sodium hydroxide.

In some embodiment, the catalyst is sodium nitroprusside or any nitroprusside salt. In some embodiments, the sodium nitroprusside or nitroprusside salt is from about 7.1 micromolar solution to about 9 mM solution in liquid phase. In some embodiments, the catalyst is used in a

concentration of about 8 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 9 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 10 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 25micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 50 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 75 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 100 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 200 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 300 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about400 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 500 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 600 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 700 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 900 micromoles/liter. In some embodiments, the catalyst is used in a concentration of about 1 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 2 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 3 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 4 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 5 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 6 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 7 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 8 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 9 millimoles/liter. In some embodiments, the catalyst is used in a concentration of about 10 millimoles/liter. In some embodiments, the catalyst is sodium

nitroprusside.

In some embodiments, the hypohalite is used in a range from about 50 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 52 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 54 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 56 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 58 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 58 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 60 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 62 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 64 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 66 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 68 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 70 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 72 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 74 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 76 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 78 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 80 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 82 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 82 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 84 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 86 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 90 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 92 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 94 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 96 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 98 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 100 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 102 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 104 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 106 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 108 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 110 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 112 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 114 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 116 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 118 to about 120 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 118 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 116 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 114 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 112 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 110 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 108 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 106 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 104 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 102 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 100 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 98 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 96 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 94 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 92 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 90 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 88 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 86 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 84 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 82 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 80 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 78 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 76 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 74 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 72 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 70 mmo /liter. In some embodiments, the hypohal ite is used in a range from about 50 to about 68 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 66 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 64 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 62 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 60 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 58 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 56 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 54 mmol/liter. In some embodiments, the hypohalite is used in a range from about 50 to about 52 mmol/liter. In some embodiments, the hypohalite is used in a concentration about 100 mmol/liter. In some embodiments, the hypohalite is sodium hypochlorite. In some embodiments, the hypohalite reagent range is + or - 50% from the stated value disclosed herein.

In some embodiments, the alkali buffer is used in a range from about 0.5 to about 1.0 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.6 to about 1.0 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.7 to about 1.0 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.8 to about 1.0 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.9 to about 1.0 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.9 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.8 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.7 mol/liter. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.6 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 1.0 mol/liter. In some embodiments, the alkali buffer is one or a combination of: calcium acetate, calcium chloride, zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valent salt thereof. In some embodiments, the alkali buffer is sodium/calcium acetate. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.6 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 1.0 mol/liter. In some embodiments, the alkali buffer is one or a combination of: calcium acetate, calcium chloride, zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valent salt thereof. In some embodiments, the alkali buffer is sodium/calcium acetate. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.6 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 1.0 mol/liter. In some embodiments, the alkali buffer is one or a combination of: calcium acetate, calcium chloride, zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valent salt thereof. In some embodiments, the alkali buffer is sodium/calcium acetate. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.6 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 1.0 mol/liter. In some embodiments, the alkali buffer is one or a combination of: calcium acetate, calcium chloride, zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valent salt thereof. In some embodiments, the alkali buffer is sodium/calcium acetate. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.6 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 1.0 mol/liter. In some embodiments, the alkali buffer is one or a combination of: calcium acetate, calcium chloride, zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valent salt thereof. In some embodiments, the alkali buffer is sodium/calcium acetate. In some embodiments, the alkali buffer is used in a range from about 0.5 to about 0.6 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 0.5 mol/liter. In some embodiments, the alkali buffer is one or a combination of: calcium acetate, calcium chloride, zinc acetate, zinc chloride, or any equivalent mono, di, or tri, -valent salt thereof. In some embodiments, the alkali buffer is sodium/calcium acetate. In some embodiments, the alkali buffer is used in a concentration about 0.6 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 0.7 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 0.8 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 0.9 mol/liter. In some embodiments, the alkali buffer is used in a concentration about 0.75 mol/liter.


In some embodiments, the ratio of volume of indophenol reagent to final volume of mixed solution should be 1/1.2. As an example, in some embodiments, 5 microliters of whole blood would result in a final reaction solution volume of 6 microliters, 10 microliters of blood would result in a final reaction volume of 12 microliters, and a 20 microliter smaple of blood would result in a final reaction volume of 24 microliters.

The disclosure relates to a method of diagnosing liver dysfunction or hyperammonemia in a subject comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) detecting the presence, absence, or quantity of ammonia;

(c) correlating the quantity of ammonia to the levels of amino acid in the sample;

(d) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 100 micromoles/liter of sample.

The disclosure relates to a method of diagnosing liver dysfunction or hyperammonemia in a subject comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) detecting the presence, absence, or quantity of ammonia;

(c) correlating the quantity of ammonia to the levels of amino acid in the sample;

(d) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 90 micromoles/liter of sample.

The disclosure relates to a method of diagnosing liver dysfunction or hyperammonemia in a subject comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) detecting the presence, absence, or quantity of ammonia;

(c) correlating the quantity of ammonia to the levels of amino acid in the sample;

(d) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 80 micromoles/liter of sample.

The disclosure relates to a method of diagnosing liver dysfunction or hyperammonemia in a subject comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) detecting the presence, absence, or quantity of ammonia;

(c) correlating the quantity of ammonia to the levels of amino acid in the sample;

(d) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 70 micromoles/liter of sample.

A method of treating a subject with liver dysfunction or hyperammonemia comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 70 micromoles/liter of sample; and

(c) treating the subject by administering therapeutically effective amounts of steroids, arginine supplements, sodium benzoate, phenyl acetate, and/or a glucose solution.

A method of treating a subject with liver dysfunction or hyperammonemia comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 80 micromoles/liter of sample; and

(c) treating the subject by administering steroids, arginine supplements, sodium benzoate, phenyl acetate, and/or a glucose solution.

A method of treating a subject with liver dysfunction or hyperammonemia comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 90 micromoles/liter of sample; and

(c) treating the subject by administering steroids, arginine supplements, sodium benzoate, phenyl acetate, and/or a glucose solution.

A method of treating a subject with liver dysfunction or hyperammonemia comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) diagnosing the subject as having liver dysfunction or hyperammonemia if the ammonia levels are quantified as above about 100 micromoles/liter of sample; and

(c) treating the subject by administering steroids, arginine supplements, sodium benzoate, phenyl acetate, and/or a glucose solution.

In any of the above methods, the method comprises detecting the ammonia or ammonium ion levels in whole blood, water, or a sample taken from a microenvironment such as a test solution reconstituted from a swab taken from a microenvironment.

The disclosure relates to a method of diagnosing a metabolic disorder in a subject comprising:

(a) contacting a sample of the subject to a system, cartridge, test strip, biosensor or device disclosed herein;

(b) detecting the presence, absence, or quantity of ammonia;

(c) correlating the quantity of ammonia to the levels of amino acid in the sample;

(d) diagnosing the subject as having a metabolic disorder if the amino acid levels are quantified as above those levels set forth in Table 1.

In some embodiments, any methods disclosed herein comprises taking multiple steps of detecting the presence, absence, or quantity of ammonia in a sample by performing 1, 2, 3, or more tests simultaneously or in series.

In some embodiments, the step of detecting the presence, absence, or quantity of ammonia comprises detecting the wavelength emitted or absorbed by a indophenol reaction product. In any of the above methods, the step of detecting the presence, absence, or quantity of ammonia comprises detecting the wavelength emitted or absorbed by a indophonel reaction product by looking at the visible light in one or more vessels. In some embodiments, the step of detecting the presence, absence, or quantity of ammonia comprises detecting the wavelength absorbed by a indophenol reaction product wherein the wavelength from about 500nm to about 700nm.

In some embodiments, any of the above methods, the step of detecting the presence, absence, or quantity of ammonia comprises detecting the wavelength emitted or absorbed by a indophonel reaction product. In some embodiments, any of the above methods, the step of detecting the presence, absence, or quantity of ammonia comprises the step of using a fingerstick to extract blood from a subject wherein the method does not comprise a step of swiping the finger with a swab, wipe, or pad of alchol, detergent, or iodine. In some embodiments, the methods comprise pre-wiping the subject with a wipe, swab, or pad step of saline solution wiping at the portion of the blood draw prior to extracting blood from th e patient an contacting it to the test strip, device, chiop or solide support described herein. I would say you can use a ratio of 1.2 for blood to final solution volume.

In some embodiments, any of the above methods do not comprise a step of converting liquid to a gas or any step involving gas chromatography.

In some embodiments, any of the above biosensor cartridges, devices, or methods comprise mixing a volume of any of the reagents disclosed herein in a volume of from about 10 microliters to about 150 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise comprise mixing a volume of any of the reagents disclosed herein in a volume of from about 10 microliters to about 100 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of from about 10 microliters to about 150 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 10 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 9 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 8 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 7 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 6 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 20 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 30 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 40 microliters. In some embodiments, any of the above biosensor cartridges, devices, or methods comprise a volume of any of the reagents disclosed herein in a volume of about 50 microliters.

In some embodiments, the disclosure relates to a computer-implemented method of quantifying ammonia or ammonium ions and/or amino acid concentration in a sample.

In some embodiments, the disclosure relates to a system comprising a processor that performs a computer-implemented method of quantifying amino acid concentration in a sample of a subject. In some embodiments, the system comprises a processor optinally located at a remote location and accessible by internet connection, operably connected to a computer storage memory that stores subejct's concentration values over time. In some embodiments, the subject or the subject's healthcare provider may accesses the internet to communicate with a server linked to the computer storage memory. Subject data reports may be generated and obtained by the subject after initiating a retrieve command through the processor. In some embodiments, the system comprises a computer program-product that performs a function convert current signals generated by a biosensor disclosed herein to concentration of a particular amino acid and/or ammonia in a sample. In some embodiments, the disclosure relates to a system including at least one processor and a computer readable memory, said computer readable memory having stored thereon program code for quantifying amino acid concentration in a sample of bodily fluid comprising: means for storing data associated with a subject; means for, responsive to receiving a level of current response from a biosensor or its computer storage memory, presenting a concentration value to a user as part of a user interface. In some embodiments, the user is the subject or healthcare provider of the subject. In some embodiments, the disclosure relates to a system that comprises at least one processor, a program storage, such as memory, for storing program code executable on the processor, and one or more input/output devices and/or interfaces, such as data communication and/or peripheral devices and/or interfaces. In some embodiments, the user device and computer system or systems are communicably connected by a data communication network, such as a Local Area Network (LAN), the Internet, or the like, which may also be connected to a number of other client and/or server computer systems. The user device and client and/or server computer systems may further include appropriate operating system software.

The present disclosure relates generally to definition and/or use of concentration values that characterize a subject's modification of behavior. In some embodiments, the concentration values corresponding to the concentration of amino acids in a sample of bodily fluid may characterize the degree to which a subject is advised to modify a diet or seek medical treatment.

In some embodiments, the present disclosure provides biosensors or test strips for use in diagnostic assays. In some embodiments the biosensor and/or test strips are provided as part of a diagnostic or detection kit. In certain embodiments, kits for use in accordance with the present disclosure may include one or more reference samples; instructions (e.g., for processing samples, for performing tests, for interpreting results, etc.); media; and/or other reagents necessary for performing tests.

The disclosure provides a test strip comprising: a solid support, a at least a first vessel in fluid communication with at least one conduit, wherein the test strip comprises a hydrogel disclosed herein. In some embodiments, the solid support is a slide optionally coated with a polymer. In some embodiments, the solid support is coated with a polymer. In some embodiments, the polymer is polyacrylamide. In some embodiments, the solid support is a material chosen from: polysterene (TCPS), glass, quarts, quartz glass, poly(ethylene terephthalate) (PET), polyethylene, polyvinyl difluoride (PVDF), polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE),

polymethylmethacrylate (PMMA), polycarbonate, polyolefin, ethylene vinyl acetate,

polypropylene, polysulfone, polytetrafluoroethylene, silicones, poly(meth)acrylic acid, polyamides, polyvinyl chloride, polyvinylphenol, and copolymers and mixtures thereof. In some embodiments, the test strip is a paper product. In some embodiments, the at least one electrode is attached to the solid support.

According to some embodiments, the disclosure provides a software component or other non-transitory computer program product that is encoded on a computer-readable storage medium, and which optionally includes instructions (such as a programmed script or the like) that, when executed, cause operations related to the calculation of amino acid concentration values. In some embodiments, the computer program product is encoded on a computer-readable storage medium that, when executed: quantifies one or more ammonia or ammonium ion concentration values; normalizes the one or more ammonia or ammonium ion concentration values over a control set of data; creates an amino acid profile or signature of a subject; and displays the profile or signature to a user of the computer program product. In some embodiments, the computer program product is encoded on a computer-readable storage medium that, when executed: calculates one or more ammonia or ammonium ion concentration values, normalizes the one or more ammonia or ammonium ion concentration values, and creates an amino acid signature, wherein the computer program product optionally displays the amino acid signature and/or one or more ammonia or

ammonium ion concentration values on a display operated by a user. In some embodiments, the disclosure relates to a non-transitory computer program product encoded on a computer-readable storage medium comprising instructions for: quantifying one or more ammonia or ammonium ion concentration values; and displaying the one or more ammonia or ammonium ion concentration values to a user of the computer program product.

In some embodiments, the step of calculating one or more ammonia or ammonium ion concentration values comprises quantifying an average and standard deviation of counts on replicate trials of contacting the device or test strip with one or more samples of bodily fluids.

In some embodiments, the one or more hydrogel coated electrodes are attached to a solid phase support. In some embodiments, a solid phase support comprises any solid or semi-solid surface. In some embodiments, a solid phase comprises any traditional laboratory material for growing or maintaining cells in culture including petri dishes, beakers, flasks, test tubes, microtitre plates, and/or culture slides. In some embodiments, a solid phase comprises a glass slide, a plastic slide, a paper test strip, or combination thereof.

In some embodiments, the one or more hydrogel coated electrodes are attached to discrete addressable sites on a solid phase support. In some embodiments, a solid phase comprises polyamides, polyesters, polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g. polyvinylchloride), polycarbonate, polytetrafluoroethylene (PTFE), nitrocellulose, cotton, polyglycolic acid (PGA), cellulose, dextran, gelatin, glass, fluoropolymers, fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, silicon substrates (such as fused silica, polysilicon, or single silicon crystals) or combinations thereof.

In some embodiments, the disclosure relates to a catalogue of medical records relating to a subject comprising test results from the one or plurality of methods described herein. Such catalogue, in some embodiments, being stoed on a computer readable medium being accessible remotely through a wireless internet connection.

As described above, certain embodiments of the present disclosure may be used to distinguish between samples of bodily fluid obtained from a subject who does or is suspected of having an hyperammonemia and a subject who does not have a metabolic disease. This system is potentially useful, for example, when testing whole blood samples of a subject to determine whether disease is present. Diagnosing a patient using one or more ammonia or ammonium ion

concentration values would include, for example, comparing one or more ammonia or ammonium

ion concentration values of a sample from a subject with the measured reference values or threshold values of a subject.

The disclosure also relates to methods of treating or preventing a metabolic disease comprising:

(a) contacting a sample of bodily fluid to the to the biosensor , system, test strip, chip or solid support disclosed herein;

(b) quantifying one or more concentration values of ammonia in the sample;

(c) comparing the one or more concentration values of ammonia in the sample to a threshold value of ammonia concentration identified as being in a healthy range; and

(d) identifying the subject as having a metabolic disease if the one or more concentration values of ammonia in the sample exceed or fall below the threshold value; and

(e) administering a therapeutically effective amount of a therapeutic agent to treat metabolic disease.

In some embodiments, the metabolic isease is hyperammonemia. In some embodiments, the therapeutic agent is glycerol phenylbutyrate or a salt thereof. In some embodiments, the therapeutic agent is Ravicti®.

Kits

In some embodiments, kits in accordance with the present disclosure may be used to quantify amino acid concentration is samples of bodily fluid.

The disclosure further provides for a kit comprising one or a plurality of containers that comprise one or a plurality of the polypeptides or fragments disclosed herein. In some

embodiments, the kit comprises a test strip and/or a biosensor comprising a test strip , or any animal-based derivative of serum that enhances the culture or proliferation of cells. In some embodiments, the kit comprises: a biosensor disclosed herein, any test strip disclosed herein, and a computer program product disclosed herein optionally comprising instructions to perform any one or more steps of any method disclosed herein. In some embodiments, the kit does not comprise cell media. In some embodiments, the kit comprises a solid support comprising a membrane disclosed herein and/or embedded with at least one electrode disclosed herein optionally comprising any one or combination of a hypohalite, an aqueous basic solution, and at least one compound comprising a phenyl group in one or a a pluarality of containers. In some embodiments, the kit comprises a device to affix a hydrogel to a solid support.

The kit may contain two or more containers, packs, or dispensers together with instructions for preparation of an array. In some embodiments, the kit comprises at least one container comprising the biosensor or system described herein and a second container comprising a solution for maintenance, use, and/or storage of the biosensor such as storage buffer . In some

embodiments, the kit comprises a composition comprising any molecule disclosed herein in solution or lyophilized or dried and accompanied by a rehydration mixture. In some embodiments, the molecules and rehydration mixture may be in one or more additional containers. In some embodiments, the kit comprises a composition comprising any one or combination of

The compositions included in the kit may be supplied in containers of any sort such that the shelf-life of the different components are preserved, and are not adsorbed or altered by the materials of the container. For example, suitable containers include simple bottles that may be fabricated from glass, organic polymers, such as polycarbonate, polystyrene, polypropylene, polyethylene, ceramic, metal or any other material typically employed to hold reagents or food; envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, and syringes. The containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components of the compositions to mix. Removable membranes may be glass, plastic, rubber, or other inert material.

The kit may contain a biosensor described herein and/or a test strip comprising

ahypohalite, an aqueous basic solution, and at least one compound comprising a phenyl group. The kit may also contain a sold support such as a test strip comprising any membrane disclosed herein.

Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrates, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, zip disc, videotape, audio tape, or other readable memory storage device. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.

The disclosure also provides a kit comprising: a biosensor comprising: a solid support and a plurality of electrodes, wherein at least one electrode comprises a hydrogel disclosed herein, in some embodiments, the hydrogel comprises an immobilized metabolic enzyme or a functional

fragment thereof; and optionally comprising at least one vessel comprising a hyohalite, an aqueous basic buffer, in liquid or solid phase, and at least one compound comprising a phenyl group. In some embodiments, the kit further comprises at least one of the following: a sample, and a set of instructions, optionally accessible remotely through an electronic medium.

Generally referring to FIGs. 6-13, a system, method, and apparatus for point of care hyperammonemia sensors may be described. In the exemplary embodiments described by the figures, samples may be tested for ammonia levels, amino acid levels, or other compound levels by being in concert with certain reagents to utilize an indophenol reaction. Color change in the reaction may be measured and correspond to certain concentrations of specific compounds and molecules by manual comparison to an extensive color-matching sheet or automated electronic analysis with the use of calibration curves.

FIG. 6 shows one exemplary embodiment of a system demonstrating the ability to detect ammonia levels in various samples. A well 100 may be made of plastic, wood, metal, composite materials, or a combination thereof. Additionally, well 100 may be comprised of synthetic compounds or polymers, such as silicone. Well 100 may further be divided into two or more sections, and may be separated by a membrane filter 105 interposed in or near the center of well 100. Membrane filter 105 may be made of a cation exchange filter such as Nafion, or similar perfluorinated ionomers to allow for only the passage of small positively charged and neutral molecules between sections. Therefore, membrane filter 105 may be selected to allow for the passage of various molecules or biological components based on charge, size, or similar characteristics. Other membrane filters may consequently be used for desired functionality, such as acrylamide, poly( ethylene glycol) diacrylate, poly(2-hydroxylethyl methacrylate), poly(vinyl alcohol), or other similar polymeric hydrogels. The selection of membrane filter 105 for a hyperammonemia sensor may depend on the membranes ability to allow for the passage of molecules such as ammonia, and the ability to limit the passage of proteins, amino acids, and other molecules or compounds.

Still referring to FIG. 6, reagent section 101 may contain reagents such as phenol, 2-phenylphenol, sodium salicylate, other phenolic reagents or polymers, or a combination thereof. Further, reagent section 101 may also contain bleach, hypochlorite, chloramine T, a similar anion, or a combination thereof, catalysts such as nitroprusside, and a basic buffer such sodium hydroxide or potassium hydroxide to maintain alkali conditions. Sample section 102 may contain serum, blood, plasma, or other liquid desired to be tested. Membrane filter 105 may only allow the passage of ammonia from section 102 to section 101. A chemical reaction, described in FIG. 8, may take place upon reception of ammonia or similar molecule into section 101, turning the reagents a blue color, as shown in section 103. Section 104 may describe the tested sample after the reaction takes place. Color sheets may be available for a qualitative comparison between colors representing specific ammonium concentrations.

In order for the cation exchange membrane, such as Nafion, to be useable for this application, a certain washing procedure and method may be disclosed. The membrane may be washed in a hydrogen peroxide aqueous solution, which may be at boiling temperatures.

Additionally, the membrane may be washed in deionized water, ethylenediaminetetraacetic acid or other chelating agents, sulfuric acid, and other similar aqueous materials. The membrane may be exposed to extreme temperatures and pressures to further ensure washing.

FIG. 7 shows an embodiment of a device fitted with multiple wells. Wells 200 may be depressions or fossa in a mounting plate 203. Mounting plate 203 may be comprised of plastic, wood, metals, composite materials, or a combination thereof. Additionally, mounting plate 203 may be comprised of synthetic compounds or polymers, such as silicone. As shown, mounting plate 203 carries three wells 200, yet those skilled in the art may appreciate the ability for a mounting plate 203 to carry substantially more or fewer wells as desired. Membrane filter 205 may be made of Nafion or similar membranes, and may be disposed of in any angle, such as a vertical placement as shown in FIG. 7, a horizontal placement, or a different angle as desired. Reagent section 201 may be filled with phenol, 2-phenylphenol, other phenolic reagents, or a combination thereof; bleach, hypochlorite, chloramine T, a similar anion, or a combination thereof; sodium hydroxide, potassium hydroxide, or a similar basic buffer to maintain alkali conditions; and one or more catalysts, such as nitroprusside. Sample section 202 may be filled with serum, blood, plasma, or similar material desired to be tested. The various wells 200 may be interconnected to facilitate the fluid flow between respective sections in order to test samples multiple times to further accuracy, or to test samples with multiple different membrane filters or reagents. Generally, if sample section 202 contains sufficient levels of ammonia, the ammonia may diffuse through membrane filter 205 and into reagent section 202, which may allow the reaction to be described in FIG. 8 to take place.

FIG. 8 shows exemplary reactions that may take place in a point of care hyperammonemia sensor, sometimes known as an indophenol reaction or Berthelot's Reaction. Reactions 300, 330, and 360 may take place upon diffusion of ammonia from one section of a well to another through a membrane filter, as described in FIGs. 6-7. Anion 302 may be hypochlorite, as shown, bleach, calcium hypochlorite, sodium hypochlorite, or other similar anions. Anion 302 may then react with ammonia 301, and produce chloramine 303, or similar ammonia derivative. Chloramine 303 may then react with further reagents, such as phenol 331. A phenol-cholarmine intermediate 333 may further react with additional phenol 331 molecules, producing indophenol 363 which may appear visibly blue in color. Phenol 331 may also be replaced with 2-phenylphenol for further efficacy, with other phenolic reagents such as sodium salicylate, with phenol polymers, or with a

combination thereof. The color change in the reagent section of the well or depression may demonstrate the presence of ammonia in the sample section.

FIG. 9 shows a further exemplary embodiment of a testing device, comprising of a microfluidic. The microfluidic 400 may be suitable for home use in a similar fashion to blood glucose meters to provide ongoing, rapid, reliable testing for hyperammonemia, various

aminoacidopathies, and other similar applications. The device 401 may be manufactured of plastic, wood, metal, composites, or a combination thereof, or a synthetic polymer or compound, such as silicone. A user may use a lancet to excrete a small amount of blood from the tip of a finger or other location on the body, and apply a small amount of blood, serum, plasma, or similar component at opening of a conduit channel 402. The sample may be transported through conduit channel 402 by capillary action and reach sample section 403. Sample section 403 may be separated from reagent section 404 by a cation exchange membrane 405, such as Nafion, whereby allowing ammonia to diffuse through membrane 405 into reagent section 404. Prior to the application of a blood sample, a squeezable reservoir 406 containing either dry or liquid bleach, hypochlorite, chloramine T, or similar anion may be manually or electronically stimulated, allowing for the flow of bleach into interposed reagent section 404. The bleach may be separate from reagents in reagent section 404 to ensure accurate and timely chemical reactions. Reagent section 404 may contain liquid or dry components of reagents disclosed in FIGs. 6-8, such as phenol, 2-phenylphenol, other phenolic reagents, or a combination thereof; sodium hydroxide, potassium hydroxide, or a similar basic buffer to maintain alkali conditions; and may also contain one or more catalysts, such as nitroprusside. Upon the presence of a certain level of ammonium in the sample, the reagent section 404 may turn into a blue color, which may be compared to a separate or included color schematic for the user to identify.

Still referring to FIG. 9, the microfluidic 400 may be used multiple times or manufactured to be a single-use device. Additionally, changes may be implemented to the design and range of chemicals used to determine amino acid levels in samples. Those skilled in the art may also appreciate the ability for a device or similar device to conform to various biological or non-biological samples, such as saliva, urine, waste water, or perhaps various chemicals to be used in a laboratory or medical setting.

In addition to the qualitative methods of determining presence or levels of ammonia in applicable samples, a quantitative apparatus, system, and method may be disclosed.

FIG. 10 shows an exemplary flowchart of a sequence of events that may take place to accurately and quantitatively identify the amount of ammonia in a sample, and is closely related to the exemplary apparatus and system disclosed in FIGs. 11-12. Additionally, those skilled in the art may appreciate that quantitative analysis in this, or a similar fashion, be added to any of the apparatuses or systems disclosed in FIGs. 5-8.

FIG. 10 therefore shows an exemplary flowchart of steps for a quantitative point of care hyperammonemia sensor. It may be appreciated that these steps may be interchangeable

chronologically, may be altered significantly, or eliminated while receiving similar results. Block 501 may refer to a test strip of any size, similar to sizing of the testing strips of blood glucose meters. The insertion mechanism block 501 test strip may be manual or automated. Upon insertion into a device, block 502 may further disclose the initiation of a series of events that may take place under program control. A bleach reservoir may be opened, manually or automatically, into a reagent section within the device. The reagent section, sample section, or both may contain reagents necessary for an indophenol reaction, or reagents used for diagnosing aminoacidopathies or similar diseases and conditions. Block 503 may further disclose the application of a blood sample by way of lancet excretion. The blood sample may be substituted for other biological samples, which may then be transported through a conduit channel to a sample section separated by a reagent section by a cation exchange membrane, such as Nafion. Block 503 may further initiate a microchip under program control which may serve as a timing device, allowing for consistent timing between various steps. This microchip may direct a photodiode or photoresistor to remain inactive for a desired duration to allow for an adequate period of time for certain reactions to take place.

Still referring to FIG. 10, Block 504 may further disclose the diffusion of ammonia or similar compound from sample section to reagent section to initiate any reaction. After a determined period of time, the reagent section may turn blue in the presence of ammonia. The degree of coloration may be dependent on the amount of ammonia in the sample section, which will allow for accurate quantitative analysis. Block 505 may further disclose the initiation of a photodiode or photoresistor near the reagent section to measure the degree of coloration. The photodiode or photoresistor may change the current of the system based on the coloration, whereby block 506 may disclose the step of converting photodiode or photoresistor signal from an analog to a digital signal. Block 507 may further disclose the reception of a digital signal to a microchip under program control. Upon reception, a microchip of block 507 may utilize a predetermined calibration curve in order to correlate a signal to an accurate ammonia concentration value, as further disclosed in block 508. Block 509 may further disclose a transmission of data from the microchip to a display device, which may be either physically or wirelessly connected to microchip, for user accessibility. This method may include the use of fewer or significantly more microchips and controllers under additional program control. Further microchips may be useful for various tests, display mechanisms, data analysis, and both visual and auditory aesthetics. Microchips may also facilitate

communication between an exemplary device and an at-home computer, cell phone, TV, or other common display and communication devices.

FIG. 11 shows an exemplary embodiment of a blood test strip for use with an electronic device further disclosed in FIG. 12. The testing strip may be large or small in nature, for use in either laboratory settings or personal home use. Conduit channel 601 may be the reception point of a sample to be tested. A blood droplet, excreted by lancet, may be placed on distal edge of conduit channel 601, where capillary action may transport sample into sample section 602. Sample section 602 may be U-shaped to increase surface area with a cation exchange membrane 604, such as Nafion. On the opposing side of membrane 604, a reagent section 603 may be filled with reagents commonly used with an indophenol or Berthelot's reaction. Bleach, or a similar anion, may be located in a separated reservoir either on the testing strip or within the electronic device in order to ensure the reactivity of certain reagents.

FIG. 12 shows an exemplary embodiment of a testing device under program control and a display device for the presentation of quantitative analysis. A blood test strip 701, such as a strip disclosed in FIG. 11, may be inserted into a port or aperture located on testing device 700, and a blood droplet 702 may be dispensed onto a conduit located distally on blood test strip 701. Upon insertion, an injection mechanism 770 may either automatically or manually add bleach or a similar anion to a reagent section 703. Bleach, chloramine T, or similar dry or liquid anion may be stored in reservoir 775, and may be refillable as desired. A photodiode or photoresistor 771 may remain inactive for a predetermined period of time until a fill sensor within microchip 773 directs the photodiode or photoresister to generate a signal corresponding to the coloration of reagent section 703. Photodiode or photoresistor 771 may then alter the current or voltage of the system with or without the means of an instrumentational amplifier and emit a signal sent to an analog-to-digital converter 772. Upon conversion to a digital signal, this may be sent to microchip 773 for analysis and further program control. Microchip 773 may compute signal and equate to a concentration of ammonium, or specific amino acids, within sample section 702 by means of pre-programmed calibration curves. Microchip 773 may then send data and information to display device 774 for user readability. Display device 774 may be wholly integrated into testing device 700, or may be connected to testing device 700 physically or wirelessly. Additionally, an alternate embodiment of testing device 700 may incorporate multiple microchips for further program control, and may be connected wirelessly or physically to an external display device, such as a computer, cell phone, TV, LCD screen, printer, or similar display and communication devices. Testing device 700 may also be in communication with devices at hospitals or laboratories for ease of information transfer to a user's doctor or medical facility.

FIG. 12 shows the chemical composition of Nafion. Other similar cation exchange membranes or perfluorinated ionomer membranes may also be used interchangeably.

Any and all journal articles, patent applications, issued patents, or other cited references disclosed herein are incorporated by reference in their respective entireties.

PCT Application Serial No. PCT/US2013/065548.

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EXAMPLES

Example 1

The presented work demonstrates how the systematic investigation of previously known technologies yielded the fabrication of an effective blood ammonia sensor. The indophenol reaction, in tandem with a polyelectrolyte membrane, was explored as a means to quantify ammonia concentrations in whole blood.

The ammonia-indophenol standard curve was produced using a range of ammonium chloride concentrations in lx phosphate buffered saline (PBS) of 0 to 750 μΜ. The following concentrations were utilized in the indophenol reaction: 59mM 2-phenylphenol in ethanol, 7μΜ sodium nitroprusside in water, 500mM sodium hydroxide in water, and 0.2-0.25% aqueous hypochlorite. These concentrations were mixed in a 1 : 1 : 1 :0.5 ratio with an equal volume of the ammonium solution of interest and allowed to react at room temperature for 10 minutes. The absorbance of the resulting solution was measured at a wavelength of 635nm.

Example 2

The reagents utilized in the indophenol reaction were investigated for long term stability. Aqueous solutions of hypochlorite, sodium nitroprusside, sodium hydroxide and a solution of 2-phenylphenol in ethanol were stored in separate 50mL falcon tubes, with limited exposure to light. At intervals of 3, 5, 7, 15, 21, 28, 35, 50, 75 and 100 days the hypochlorite, sodium nitroprusside, sodium hydroxide and 2-phenylphenol were utilized to develop a standard curve using ammonia concentrations ranging from 0-750μΜ. Significant deviations from the original standard curve indicated the degradation of the stored reagents. It should be noted that fresh ammonia samples were utilized at each test interval.

Response to Amino Acids

Primary amines can also undergo the indophenol reaction. Total amino acid concentrations in blood can be as high as 2.5mM, therefore the selectivity of 2-phenylphenol was determined in the indophenol reaction. ImM solutions of each of the 21 amino acids was prepared in IX PBS. The same protocol utilized with the indophenol reagents for the ammonia standard curve was utilized with each amino acid solution. 10 minutes after the indophenol reagents and amino acid solution was mixed, its absorbance at 635nm was measured using a plate reader. The response was directly compared to the response seen from a ImM solution of ammonium chloride and expressed as a percentage of the ammonium response.

Sensor Design

While the following tests and experiements can be used with any of the cartridges disclosed herein, the specific data shown below came from the blister-pack and dried reagent cartridge as disclosed in FIG. 5A.

Hypochlorite Concentrations Effect on Indophenol Response to Blood Ammonia

To reduce interference from reducing species in blood, higher concentrations of

hypochlorite than conventionally utilized were employed in the indophenol reaction with ammonia extracted from whole sheep's blood. 1, 2, 3, 5, and 10X concentrations of hypochlorite were utilized and the resulting absorbance at 635nm was recorded.

Proper Reagent Concentrations

In order to correctly state the concentrations, it is easier to provide them in terms of final concentration after mixed together and with ammonium. Ideally, any starting concentration should work as long as the final concentration is the same as what is listed below. For convenience, I have also listed the concentrations of reagents used to produce these final concentrations. Table 5 below assumes an ammonia solution of a volume of 20 microliters was added. A general 20% swing of concentrations would be valid for each reagent other than sodium nitroprusside, but the listed values are optimal.

Table 5


Optimized Sodium Nitroprusside

As shown in FIG. 14, concentrations of 7-8754 micromolar sodium nitroprusside solution was utilized to detect 500 micromolar ammonia solutions using the indophenol reaction.

Appropriate amounts of the other reagents were used in this optimization. It is evident from the above figure that a concentration of 1330-1996 micromolar were optimal for detection of ammonia.

Stability

As shown in FIG. 15, One major advantage of using the indophenol reaction for determining ammonia concentrations is that it does not require any biological components such as enzymes, which are high in cost and prone to stability issues. The shelf life of the solutions used for the indophenol reaction was evaluated over the course of 245 days. The response to the range of ammonia chloride concentrations was stable for the entire course of the current study. As seen in FIG. 15, the response to 500 μΜ ammonium chloride did not change significantly over the course of 245 days. It should be noted that this was not performed using the laboratory prototype test cassette, which significantly lowers the day-to-day variability of the measurements as seen in the following sections.

Calibration Curve and Sensitivity

A calibration curve was generated using the laboratory prototype test cassettes as seen in FIG. 16. Concentrations of blood ammonia were generated by either spiking whole blood samples or allowing them hydrolyze at room temperature. Seven measurement points produced the curve, which fall within the FDA guideline for pre-validation of a bioanalytical method. The calibration curve has an excellent R2 of 0.998. Additionally the measured sensitivity was 0.288 a.u./μΜ of ammonia. This sensitivity will allow for high-resolution measurements. It should be noted that the calibration curve covers concentrations of blood ammonia ranging from 20-500 μΜ and thus covers the entire physiologically normal to highly elevated range. This calibration curve was utilized to determine the limit of detection and limit of quantification, which were 8 μΜ and 27 μΜ

respectively.

The investigated system for evaluating blood ammonia levels demonstrated a high degree of correlation between blood ammonia and sensor response. In the range of 2S-1 SO )1M, the most clinically critical concentrations, the relative standard deviation was just 12.23%. The

method could discern levels of SO and 100 )1M with a p=0.000 1, indicating the system is extremely reliable in differentiating these concentrations.

The sensor has a 30 minute response time, and the interference from other small molecules and proteins was greatly reduced. The components used are stable at room temperature for up SO days and inexpensive. This presented method could lead the way for PoC devices

for whole blood ammonia detection. Ultimately, such a device would assist in the rapid diagnosis of urea cycle disorders, which are not included in newborn screening programs. Due to their exclusion from newborn screening, UCDs are often misdiagnosed and treated after severe

hyperammonemic episodes. Additionally, measured blood ammonia levels can often result in false positives due to the difficulty in sample processing, which can involve several preparative steps and logistical hurdles. The future development of a point-of-care device would provide an attending physician with the option for rapid diagnosis, especially in the intensive care unit setting. This is particularly imperative as high ammonia levels can be indicative of many disorders outside of UCDs, such as organic acidemias, fatty acid oxidation disorders and other rare inborn errors of metabolism. At home testing is another option for such a point-of-care device in the management ofUCDs. Dietary adjustments could be made more readily, however, due to parental expectations and use, this option should be examined carefully and approached strategically to ensure the patient's best interest is taken into account.

Variability

The Clinical Laboratory Standards Institute guideline for measuring variability was utilized to determine the test cassette variability. Low, medium and high concentrations of ammonia were measured twice a day for 5 days, as shown on Table 6 below.. The variability displayed in the above table is the standard deviation of each measurement divided by the average measurement. For each concentration, the variability was less than 15%, which falls within the FDA guidelines for required variability limits for a bioanalytical method. This translates to 2.5 μΜ resolution at low

concentrations, -10 μΜ resolution at medium concentrations and 25 μΜ resolution at high concentrations.

Table 6

Ammonia Concentration (μΜ) 25 125 500

Variability 10% 11.5% 5%

Correlation with Clinical Test

The cartridge's ability to accurately measure blood ammonia was directly correlated with measurements taken by the Siemens EXL, a clinical chemical analyzer currently used in hospital laboratories around the nation. Measurements taken were converted into blood ammonia values by use of the calibration curve seen in FIG. 17. Blood ammonia samples were split into 40 μΐ^ and 200 μΐ. aliquots. The 40 μΐ^ aliquot was utilized for the sample cartridge and the 200 [iL aliquot for the Siemens EXL. The aliquot for the Archimedes meter was used immediately within the device, which produced a measurement in 25 minutes. The aliquot for the Siemens EXL was centrifuged in a 4oC centrifuge for 10 minutes to yield the plasma fraction. This plasma fraction was then inserted into the Siemens EXL and a result was obtained 10 minutes later. The two measured values were then plotted against each other as seen in FIG. 17 blood ammonia measurements were utilized to generate the correlational curve. As seen, the cartridge blood ammonia meter produced results that correlated tightly with the Siemens EXL with an r2 of 0.997.

Calibration Curve with Dried Reagents

To demonstrate the viability of drying reagents used in the indophenol reaction, two components of reaction, namely the sodium nitroprusside and the 2-phenylphenol were drop casted and air-dried prior to use. The two reagents would be reconstituted using the other two reagents used in the colorimetric indophenol reaction, specifically sodium hypochlorite and sodium hydroxide. Subsequently to reconstitution the ammonium sample would be added and the developed color measured. As seen in FIG. 18, this approach produced a linear curve for concentration of ammonium ranging from 0-500 micromolar. It should be noted that the resulting 'reacting' solution contained the appropriate concentrations of each reagent.

Example 3: Measuring Ammonia in Whole Blood with a Fluidic Device

Materials

2-phenylphenol, sodium nitroprusside, sodium hydroxide, sodium hypochlorite, sodium acetate, and ammonium chloride were purchased from Sigma- Aldrich. Nafion 111 was purchased from Ion-Power.

Methods

Sensor Design and Construction

A bisected well containing whole human blood in one section and a solution of sodium acetate in the other provide a means for cation exchange of the whole blood to occur, yielding a high recovery of the ammonium. Modular well-halves were 3D printed from acrylonitrilebutadiene-styrene thermoplastic. The pieces snap together with the 1 cm2 Nafion 111 membrane in the middle, forming a Nafion bisected well. This design was chosen to provide a uniform platform for all future experiments involving this sensing method. A silicone gasket, at a 1/64 inch thickness, was glued to the inner face of each well-half to ensure a water tight seal.

Extracting Ammonia

Sodium acetate was utilized to extract ammonia through ion-exchange. Concentrations of 0.1, 0.5 and 1M sodium acetate were prepared. Using fresh Milli-Q water (18.5 ΜΩ), to ensure no ammonia contaminants were present. Bisected wells were prepared, with 100 μΐ of 500 μΜ of ammonium chloride in one section and 45 μΐ of the sodium acetate solution in the other. Ion-exchange took place for 20 minutes before 35 μΐ of the now ammonia -enriched sodium acetate was tested.

Reducing Interference from Blood-Borne Small Molecules

Reducing agents in blood such as uric acid can negatively interfere with the indophenol reaction. Increasing concentrations of the hypochlorite were utilized in a modified version of the indophenol reaction to eliminate this negative interference. 500 μΜ solutions of ammonium

chloride were prepared in PBS and in whole human blood. The ammonia was extracted from these samples using previously described ion-exchange protocol. The concentration ofhypochlorite was varied between 0.2S-2.S% to examine its effectiveness in reducing interference.

Sensor Response to Ammonia in Whole Blood

The 3D printed wells were constructed with 1 cm2 pieces of Nafion membrane. Whole human blood was spiked using ammonia chloride to generate concentrations of ammonia of 25, 50, 75, 100, 150, 200, 250, 300, 400 and 500 μΜ In one section of the well, 100 μΐ of the ammonia containing blood sample was added. In the opposing section 1 M sodium acetate was added. Ion-exchange of ammonia was allowed to occur for 20 minutes. To a 384 well plate, 3S )1L of ammonia extracted sample, 10 )1L of 2-phenylphenol, 10 )1L of NaOH, 5 μΐ of 0.75 % hypochlorite and an appropriate amount of sodium nitroprusside were added. The absorbance of the resulting indophenol reaction was measured at 635 nm after 10 minutes using a microplate reader.

Preparation of Sample

• Prepare stock solutions of 59mM 2-phenylphenol in ethanol, 7 μΜ sodium nitroprusside in water, 500mM sodium hydroxide in water and 0.75% sodium hypochlorite in water.

• Prepare a stock solution of 1M sodium acetate in water.

• Molds are produced from a 3D printer for the 2 individual pieces of the device.

• Fill each mold with the PDMS elastomer and heat at 60°C for one hour.

• Remove each side of the device from the mold with a spatula.

• Remove the 25 μπι thick Nafion 111 from plastic backing and cut it into a 1.5X1.5 cm square.

• Glue the square of Nafion over the well in the channel 6 using PDMS.

• Glue the top piece of the device to the bottom piece using PDMS, ensuring channel 6 lines up with channel 5.

• Heat the device to 60°C for one hour.

Ammonia Exchange

• Insert a needle through the bottom of the device into well 6, and fill with 40 μΐ of blood.

• Fill channels 1-4 with 5 μΐ of 2-phenylphenol, sodium nitroprusside, sodium hydroxide and sodium hypchlorite respectively .

• Fill channel 5 with 20 μΐ of sodium acetate solution.

• Wait 20 minutes for ion exchange to occur.

Collection of Data

• Apply a flow rate of 1 mm/sec to channels 1 through 5 for 24 seconds.

• Wait ten minutes for the indophenol reaction to proceed.

• Insert the device into the custom photo-spectrometer to acquire absorbance data.

• Compare the measured absorbance to a standard curve to determine unknown ammonia concentration.

FIG. 20 depicts the results of amino acid detection usng the indophenoal reaction. 1 mM concentrations of each of the 21 amino were tested using the indophenol reaction. The absorbance measured at 635 nm for each amino acid after the indophenol reaction was calculated as percentage of the response from indophenol reaction with 1 mM ammonia chloride. The radar graph displays the percent response as compared to 1 mM ammonia chloride. The highest response was threonine, which produced an absorbance value at just 5% of ammonia's response.

FIG. 21 A and 21B show the review of conentrations of reagents used in the experiments. The ion-exchange of ammonia through the use of aN afion membrane is the mechanism of recovery of the analyte. Sodium salt solutions of different concentrations were tested for their effectiveness in exchanging with the ammonia from a PBS solution. It was expected that higher concentrations of sodium salts would yield larger recoveries of ammonia. Bisected wells were prepared with Nafion membranes. A 500 μΜ solution of ammonium chloride in PBS was placed on the 'analyte' side of the bisected well and solutions of sodium acetate in the opposing bisection. As seen in Figure 21 A, a concentration of 1 M gave the largest recovery in 20 minutes. This recovery was about 40% from whole blood and about 70% from a IX PBS solution. The indophenol reaction is also sensitive to the presence of blood-borne reducing agents such as uric and ascorbic acid. These molecules, amongst others, can react with intermediates of the indophenol reaction as well as the hypochlorite. This effect was evident when initially testing serial dilutions of ammonia in whole blood. The response plateaued at higher concentrations of ammonia due to side reactions with indophenol reagents. To circumvent this issue, larger concentrations of hypochlorite were introduced to the reactionwhen detecting 500 μΜ ammonia in both whole blood and PBS. As seen in Figure21B, with a 0.25% hypochlorite solution, the response to ammonia in whole blood was less than 20% of the response to ammonia in PBS. Increasing the hypochlorite concentration resulted in larger responses to whole blood, with a 50% response as compared to PBS, at an optimum concentration of 0.75%). At larger concentrations the response to both whole blood and PBS began to degrade.

Example 4: Fingerstick Ammonia Measurements

Fingerstick ammonia measurements are known to produce false positive values. The prevailing theory assumed that hemolysis from shearing forces from the fingerstick contaminated the blood sample with ammonia from tissue. To demonstrate that hemolysis is not the cause for contamination venous draws and fingerstick ammonia were acquired within the same hour. As seen in Table 7 below, fingerstick ammonia values, while varied, did not correlate with the degree of hemolysis observed. This preliminary data, in conjunction with previously reported data, demonstrates that hemolysis is not a predictor of a false positive read in ammonia concentration. Another source of false elevations from fingerstick ammonia likely comes from dried sweat on the fingertip. Sweat ammonia values can be as high as 1000-3000 micromolar, which can easily cause a false positive. This theory was tested by exposing saline solution to a fingertip and then measuring the corresponding ammonia concentration in the saline solution, the results of which can be seen in FIG. 19. For an "unwashed" finger, the measured ammonia concentration was 74 micromolar. Alcohol and iodine swabs, which are the most commonly used swabs for fingertip pre-treatment before a fingerstick, lowered the measured concentration to 37 and 26 micromolar respectively. Handwashing and saline wipes lowered the measured ammonia concentration to 14 and 11 micromolar respectively. This demonstrates the poor cleaning of the fingertip can significantly contribute to false elevations in fingerstick blood ammonia measurements. It also demonstrates that certainly cleansing swabs, primarily those containing saline solution, are more effective than traditional swabs from removing ammonia from the fingertip.

Table 7

Measured Blood Hemolysis

NH4+ Draw Appearance

Type

9 Venous None

6 Venous None

51 Fingerstick None

34 Fingerstick Pink

25 Fingerstick Light Pink

Fingerstick Light Pink

The foregoing description and accompanying figures illustrate the principles, preferred embodiments and modes of operation of the disclosure. However, the disclosure should not be construed as being limited to the particular embodiments or applications discussed above. Additional variations, modifications, and applications of the embodiments discussed above will be appreciated by those skilled in the art. Additional variations and modifications may include, but are not limited to, the detection of a variety of different amino acids, such as phenylalnine, histidine, tyrosine, glutamate, threonine, serine, leucine, isoleucine, aspartate, valine, glycine, alanine, tryptophan, proline, lysine, arginine, or others. Detection of these amino acids may involve placing dehydrogenase enzymes or other ammonia lyase enzymes in the sample section of the well, along with the blood, serum, or plasma. Possible applications for the detection of the presence of ammonia or ammonium ion is to diagnose phenylketonuria or other aminoacidopathies.

Therefore, the above-described embodiments should be regarded as illustrative rather than restrictive. Accordingly, it should be appreciated that variations to those embodiments can be made by those skilled in the art without departing from the scope of the disclosure as defined by the following claims.