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1. WO2020141350 - PH-MODULATED IMAGING OF TARGETS CLOSE TO A SOLID SURFACE

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[ EN ]

CLAIMS

What is claimed is:

1. A method for imaging a biospecimen, comprising

(a) labeling the biospecimen with a pH-sensitive label to form a labeled biospecimen,

(b) submerging the labeled biospecimen and a surface of an electrode in a buffered solution having a pH value, wherein

the buffered solution comprises a pH modulating agent;

the surface optionally comprises microstructures;

the labeled biospecimen is coupled to the surface or to the microstructures; and the microstructures, when present, define a volume between the biospecimen and the surface, through which the pH modulating agent diffuses,

(c) applying a potential or a current to the electrode, whereupon the pH modulating agent causes a change in the pH value in a zone adjacent to the surface of the electrode, thereby causing the pH-sensitive label within the zone to produce an optical signal, and

(d) detecting the optical signal thereby imaging the labeled biospecimen.

2. The method of claim 1, wherein the pH modulating agent is a quinone derivative, an aminophenol derivative, an aniline derivative, a benzidine derivative, a hydrazine derivative, phenol -Ru(2, 2' -bipyridine)32+, or a combination thereof.

3. The method of claim 2, wherein the pH modulating agent is a quinone derivative of any of formula (I)-(XII)

wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of: H; CnH2n+1; Cl; F; I, Br, OM, NO2, OH, OCnH2n, OCnH2nOH, O(CnH2nO)yOH, O(CnH2nO)yOCnH2n+1, O(CnH2nO)y COOH; O(CnH2nO)yCOOM; COOH; COOM; COOCnH2n+1; CONHCnH2n+1; CON(CnH2n+1)2; SO3H; SO3M; NH2; NHCnH2n+1; N(CnH2n+1)2; NHCnH2nOH; NHCnH2nNH2; N(CnH2nOH)2; N(CnH2nNH)2; NHCOCnH2n+1; NCnH2nCOCnH2n+1;

NCnH2nCOCnH2nOH; NCnH2nCOCnH2nNH2; NCnH2nCOCnH2nSH; SH; SCnH2n+1; SCnH2nOH; S(CnH2nO)yOH; S(CnH2nO)yOCnH2n+1; S(CnH2nO)yCOOH; S(CnH2nO)yCOOM; OCnH2nSH; O(CnH2nO)ySH; O(CnH2nO)ySCnH2n+1; CnH2n; CnH2nOCnH2n; CnH2nSCnH2n; CnH2nNHCnH2n; CnH2nN(CnH2n+1)CnH2n; CnH2n+1; CnH2nOH; CnH2n+1OCnH2n; CnH2nOCnH2nOH;

CnH2nO(CnH2nO)yCOOH; CnH2nO(CnH2nO)yCOOM; CnH2nCOOH; CnH2nCOOM;

CnH2nCOOCnH2n+1; CnH2nCONHCnH2n+1; CnH2nCONH(CnH2n+1)2; CnH2nS03H; CnH2nS03M; CnH2nNH2; CnH2nNHCnH2n+1; CnH2nN(CnH2n+1)2; CnH2nNHCnH2nOH; CnH2nNHCnH2nNH2; CnH2nN(CnH2nOH)2; CnH2nN ( CnH2hNH2)2 ; CnH2nNHCOCnH2n+1; CnH2nNCnH2nCOCnH2nOH; CnH2nNCnH2nCOCnH2nNH2; CnH2nN CnH2nCO CnH2nSH; CnH2nSH; CnH2n+1S CnH2n;

CnH2nSCnH2nOH; CnH2nS( CnH2nO)yOH; CnH2nS(CnH2nO)yOCnH2n+1; CnH2nS( CnH2nO)yCOOH; CnH2nS( CnH2nO)yCOOM; sugars; peptides; and amino acids,

wherein

M is any metal cation or NH4+,

n is an integer from 1 to 109, and

y is an integer from 1 to 109.

4. The method of claim 1, wherein the pH sensitive label is a fluorescent dye, a fluorescent protein, an enzyme, or a combination thereof.

5. The method of claim 4, wherein the pH sensitive label is a fluorescent dye selected from the group consisting of pHrodo, Protonex, Oregon Green, LysoSensor Green, pHAb, fluorescein, FAM, rhodamine B derivatives, and SNARF, or a fluorescent protein, or an enzyme selected from the group of HRP, glucose oxidase, and alkaline phosphatase.

6. The method of claim 1, wherein the optical signal is colorimetric signal, a

chemiluminescent signal, or a fluorescent signal.

7. The method of claim 1, wherein the potential in (c) is defined by a waveform capable of being modulated based on open-loop and/or closed-loop control scheme to change the size of the pH modulated zone.

8. The method of claim 1, wherein the coupling between the biospecimen to the

micro structures in (b) is at a higher degree than the coupling of the biospecimen to the surface without the microstructure, or wherein the concentration of the pH modulating agent in the volume between the biospecimen and the surface with the microstructures is higher than the concentration of the pH modulating agent in the volume between the biospecimen and the surface without the microstructure

9. The method of claim 1, further comprising conducting imaging the biospecimen in an array of controlling and/or imaging modules, each module comprising an independent cycle of performing (a)-(d) , wherein the array of controlling and/or imaging modules is a complementary metal-oxide semiconductor (CMOS) array, an electrode array, or a thin-film transistor (TFT) array.

10. The method of claim 1, wherein biospecimen is a fixed tissue, a single cell, a cell selected from the group consisting of a fixed cell and a live cell, a vesicle, a protein, a peptide, DNA, RNA, or a combination thereof.

11. A method for imaging a biospecimen, comprising

(a) labeling the biospecimen with a pH-sensitive label to form a labeled biospecimen,

(b) submerging the labeled biospecimen and a surface of an electrode in a buffered solution having a pH value, wherein

the surface comprises a coating;

the coating comprises a pH modulating agent;

the coating optionally comprises microstructures;

the buffered solution optionally comprises the pH modulating agent; the labeled biospecimen is coupled to the coating or to the microstructures; and the microstructures, when present, define a volume between the biospecimen and the surface, through which the pH modulating agent in the buffered solution, when present, diffuses,

(c) applying a potential or a current to the electrode, whereupon the pH modulating agent causes a change in the pH value in a zone adjacent to the coating, thereby causing the pH-sensitive label within the zone to produce an optical signal, and

(d) detecting the optical signal thereby imaging the labeled biospecimen.

12. The method of claim 11, wherein the pH modulating agent is a quinone derivative, an aminophenol derivative, an aniline derivative, a benzidine derivative, a hydrazine derivative, phenol -Ru(2, 2' -bipyridine)32+, or a combination thereof.

13. The method of claim 12, wherein the pH modulating agent is a quinone derivative of any of formula (I)-(XII)

wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from the group consisting of: H; CnH2n+1; C1; F; I, Br, OM, NO2, OH, OCnH2n, OCnH2nOH, O(CnH2nO)yOH, O(CnH2nO)yOCnH2n+l, O(CnH2nO)yCOOH; O(CnH2nO)yCOOM; COOH; COOM; COOCnH2n+1; CONHCnH2n+1; CON(CnH2n+1)2; SO3H; SO3M; NH2; NHCnH2n+1; N(CnH2n+1)2; NHCnH2nOH; NHCnH2nNH2; N(CnH2nOH)2; N(CnH2nNH)2; NHCOCnH2n+1; NCnH2nCOCnH2n+1;

NCnH2nCOCnH2nOH; NCnH2nCOCnH2nNH2; NCnH2nCOCnH2nSH; SH; SCnH2n+1; SCnH2nOH; S(CnH2nO)yOH; S(CnH2nO)yOCnH2n+1; S(CnH2nO)yCOOH; S(CnH2nO)yCOOM; OCnH2nSH; O(CnH2nO)ySH; O(CnH2nO)y SCnH2n+1; CnH2n; CnH2nOCnH2n; CnH2nSCnH2n; CnH2nNHCnH2n; CnH2nN(CnH2n+1)CnH2n; CnH2n+1; CnH2nOH; CnH2n+1OCnH2n; CnH2nOCnH2nOH;

CnH2nO(CnH2nO)yCOOH; CnH2nO(CnH2nO)yCOOM; CnH2nCOOH; CnH2nCOOM;

CnH2nCOOCnH2n+1; CnH2nCONHCnH2n+1; CnH2nCONH(CnH2n+1)2; CnH2nS03H; CnH2nS03M; CnH2nNH2; CnH2nNHCnH2n+1; CnH2nN(CnH2n+1)2; CnH2nNHCnH2nOH; CnH2nNHCnH2nNH2; CnH2nN(CnH2nOH)2; CnH2nN (CnH2hNH2)2 ; CnH2nNHCOCnH2n+1; CnH2nNCnH2nCOCnH2nOH; CnH2nNCnH2nCOCnH2nNH2; CnH2nNCnH2nCOCnH2nSH; CnH2nSH; CnH2n+1SCnH2n;

CnH2nSCnH2nOH; CnH2nS(CnH2nO)yOH; CnH2nS(CnH2nO)yOCnH2n+1; CnH2nS(CnH2nO)yCOOH; CnH2nS(CnH2nO)yCOOM; sugars; peptides; and amino acids,

wherein

M is any metal cation or NH4+,

n is an integer from 1 to 109, and

y is an integer from 1 to 109.

14. The method of claim 11, wherein the pH sensitive label is a fluorescent dye, a fluorescent protein, an enzyme, or a combination thereof.

15. The method of claim 14, wherein the pH sensitive label is a fluorescent dye selected from the group consisting of pHrodo, Protonex, Oregon Green, LysoSensor Green, pHAb, fluorescein, FAM, rhodamine B derivatives, and SNARF, or a fluorescent protein, or an enzyme selected from the group of HRP, glucose oxidase, and alkaline phosphatase.

16. The method of claim 11, wherein the coating comprises a polymer and the pH

modulating agent is integrated in the polymer as a part of a backbone of the polymer or as a side chain of the polymer.

17. The method of claim 11, wherein the potential in (c) is defined by a waveform capable of being modulated based on open-loop and/or closed-loop control scheme to change the size of the pH modulated zone.

18. The method of claim 11, wherein the coupling of the biospecimen to the coating with the micro structure in (b) is at a higher degree than the coupling of the biospecimen to the coating without the microstructure, or wherein the concentration of the pH modulating agent in the volume between the biospecimen and the surface with the microstructures is higher than the concentration of the pH modulating agent in the volume between the biospecimen and the surface without the microstructure.

19. The method of claim 11, further comprising conducting imaging the biospecimen in an array of controlling and/or imaging modules, each module comprising an independent cycle of performing (a)-(d) , wherein the array of controlling and/or imaging modules is a complementary metal-oxide semiconductor (CMOS) array, an electrode array, or a thin-film transistor (TFT) array.

20. The method of claim 11, wherein biospecimen is a fixed tissue, a single cell, a cell selected from the group consisting of a fixed cell and a live cell, a vesicle, a protein, a peptide, DNA, RNA, or a combination thereof.