Photoresponsive redox detection and discrimination

• US 4,704,353 A
• Filed: 04/27/1984
• Issued: 11/03/1987
• Est. Priority Date: 04/27/1984
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Parce, John W., Humphries, Gillian M. K., McConnell, Harden M.
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US06/604,800
• Time in Patent Office: 1,285 Days
• Field of Search: 204/1 T, 204/403, 204/412, 324/71.5, 435/7, 435/25, 435/26, 435/28, 436/84, 436/501, 436/805, 436/806, 436/807, 436/827, 436/904
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: G01N 27/305 optically transparent or photoresponsive electrodes
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 435/966 involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/827 Lectins
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators

Abstract

Devices and methods are provided for making a plurality of determinations of the local (site-specific) redox state of a liquid system, by employing a photoresponsive element, which can be independently irradiated at different sites to provide independently detectable signals.

Claims

1. A photoresponsive device for making at different sites a plurality of determinations of a redox substance capable of modifying an electrical signal resulting from irradiation of a photoresponsive element, said device comprising:

a photoresponsive element having an irradiation receiving surface;

irradiation means for irradiating at least two sites on said surface to provide distinguishable signals;

a counterelectrode;

means for polarizing said element with respect to said counterelectrode;

first container means for retaining media in which said substance is to be determined in a photoresponsive modifying relationship to at least two sites;

second container means for maintaining a first electrolyte in electrical communication with said photoresponsive element;

third container means for maintaining a second electrolyte in electrical communication with said counterelectrode;

means for providing electrical communication between electrolyte in said second and third container means without mixing of said first and second electrolytes; and

means for measuring the electrical photoresponse during irradiation of a site.

2. A device according to claim 1, wherein said photoresponsive element is a monolithic doped silicon semiconductor wafer.

3. A device according to claim 1, including a clear sheet in close spaced apart parallel juxtaposition to said irradiation receiving surface.

4. A device according to claim 1, wherein said irradiation means is an LED array programmed to provide light pulses.

5. A device according to claim 1, wherein said container means is a plurality of channels, each of said channels associated with an irradiation site.

6. A device according to claim 1, wherein said container means is a plurality of compartments, each of said compartments associated with an irradiation site.

7. A device according to claim 1, wherein a portion of said element is coated with a lipid bilamellar layer for contact with said media.

8. A device according to claim 7, wherein said lipid bilamellar layer is coated on said irradiation receiving surface and has a specific binding pair member bound to said layer.

9. A method for substantially simultaneously determining a redox substance in a plurality of volumes of an electrically responsive medium, employing a photoresponsive element having a common lead through an electrical circuit to a counterelectrode, wherein said substance is capable of modulating the photoresponse of said element;

said method comprising;

directing said medium into photoresponsive modifying relationship with a photoresponsive surface of said element;

maintaining electrical communication between said element and said counterelectrode through an electrically responsive fluid or gel;

irradiating said surface at a plurality of sites in relation to individual volumes at different times in a predetermined sequence for defined periods of time at each of said sites; and

determining by means of said circuit the presence of said substance in each of said volumes by the effect of said substance on the photoresponse resulting from said irradiation.

10. A method according to claim 9, wherein said circuit includes means for polarizing said element in relation to said counterelectrode and means for measuring the current necessary to restore the level of polarization during irradiation of said surface.

11. A method according to claim 9, wherein said circuit includes means for varying the light intensity in relation to said photoresponse.

12. A method according to claim 9, wherein said circuit includes means for fixing the potential between said photoresponsive element and counterelectrode and for fixing the intensity of said irradiation and determining the DC component of the photoresponse.

13. A method according to claim 9, wherein said substance is an inorganic ion.

14. A method according to claim 9, wherein said photoresponsive element is a monolithic doped semiconductor.

15. A method according to claim 9, wherein said volumes are portions of at least one stream.

16. A method according to claim 9, wherein each of said volumes is physically separated from the other volumes.

17. A method according to claim 9, wherein said substance is methylene blue, Meldola blue, or phenazine methosulfate.

18. A method according to claim 9, wherein said circuit maintains said element and counterelectrode at a predetermined potential difference and measures the current necessary to restore the potential difference upon irradiation of said surface.

19. A method according to claim 9, wherein said substance is a result of an enzymatic reaction.

20. A method according to claim 19, wherein said substance is reduced or oxidized indirectly through one or more intermediate compounds by the product of said enzyme.

21. A method according to claim 19, wherein said enzyme is an oxidoreductase.

22. A method according to claim 21, wherein said oxidoreductase is a peroxidase.

23. A method according to claim 21, wherein said enzyme is a dehydrogenase.

24. A method for determining the presence of an analyte in a sample, which analyte is a member of a first specific binding pair consisting of ligand and receptor, said method employing:

(a) a photoresponsive electrode having an irradiation surface;

(b) a counterelectrode;

(c) an electrical circuit connecting said photoresponsive electrodes and counterelectrode and maintaining said electrodes in a predetermined potential relationship;

(d) a binding surface for binding a member of a specific binding pair, which surface is said irradiation surface or a facing surface in close spaced apart juxtaposition to said irradiation surface; and

(e) specific binding means for specifically binding one of said members of said first specific binding pair to said binding surface;

said method comprising;

in an aqueous assay medium, combining components with said specific binding means, said assay components including a sample, a labeled specific binding pair member, which labeled specific binding pair member is a member of said first pair or capable of binding to, either directly or indirectly, a member of said first pair, wherein said label is a member of a system capable of modulating the electrical photoresponse of said photoresponsive electrode by means of a change in redox potential, and any additional members of said system;

irradiating a plurality of sites on said irradiation surface in relation to individual volumes of said combined components at different times in accordance with a predetermined schedule; and

determining the electrical photoresponse as modulated by said system by means of said circuit as indicative of the presence of said analyte in said sample.

25. A method according to claim 24, wherein said photoresponsive semiconductor is a doped monolithic silicon wafer.

26. A method according to claim 24, wherein said label is an enzyme.

27. A method according to claim 24, wherein said specific binding means is a second ligand and receptor.

28. A method according to claim 27, wherein said second ligand and receptor are a saccharide and a lectin.

29. A method according to claim 27, wherein said second ligand and receptor are a ligand and a natural receptor or antibody.

30. A method for determining the presence of an enzyme substrate in a medium, wherein the product of said substrate results in the production of a product, capable of producing either directly or indirectly a detectable redox potential, said method employing:

(a) a photoresponsive semiconductor electrode having an irradiation surface;

(b) at least one counterelectrode;

(c) an electrical circuit connecting said photoresponsive electrode and counterelectrode and maintaining said electrodes in a predetermined potential relationship;

(d) a binding surface for binding said enzyme at a plurality of sites;

said method comprising;

contacting said enzyme at each of said sites with an aqueous medium containing said substrate and any additional components necessary for the enzymatic catalyzed reaction;

irradiating said sites on said irradiation surface at different times in accordance with a predetermined schedule; and

determining the electrical photoresponse as modified by said system by means of said circuit as a measure of the amount of said substrate present.

31. A method according to claim 30, wherein said enzyme is horseradish peroxidase.

32. A method according to claim 30, wherein said enzyme is glucose oxidase.

33. A method for determining the presence of one or more microorganisms in a medium, said method employing:

(a) a photoresponsive electrode having an irradiation surface having a plurality of sites;

(b) at least one counterelectrode;

(c) an electrical circuit connecting said photoresponsive electrode and counterelectrode and maintaining said electrodes in a predetermined potential relationship;

said method comprising;

contacting said medium at each of said sites under conditions where said microorganism remains viable and produces at least one metabolic product capable of modulating the photoresponsive of said photoresponsive electrode by means of a change in the detectable redox potential of said medium;

irradiating said sites on said irradiation surface at different times in accordance with a predetermined schedule; and

determining the electrical photoresponse as modified by said metabolic product by means of said circuit as an indication of the presence of a microorganism.

34. A method according to claim 33, wherein said microorganism is bound by a specific receptor in juxtaposition to said site.

35. A method for detecting the presence of an analyte in a medium, said method employing:

(a) a photoresponsive electrode having an irradiation surface having a plurality of sites;

(b) at least one counterelectrode;

(c) an electrical circuit connecting said photoresponsive electrode and counterelectrode and maintaining said electrodes in a predetermined potential relationship;

said method comprising;

contacting said medium at each of said sites, wherein said medium contains horseradish peroxidase, hydrogen peroxide and methylene blue, and the amount of horseradish peroxidase in said medium is related to the amount of analyte in said medium, said horseradish peroxidase catalyzing the oxidation of methylene blue;

irradiating said sites on said irradiation surface at different times in accordance with a predetermined schedule; and

determining the electrical photoresponse as a result of the reaction of the methylene blue at each of said sites.