Photoresponsive redox detection and discrimination

• US 4,849,330 A
• Filed: 05/03/1985
• Issued: 07/18/1989
• 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:
  US06/730,231
• Time in Patent Office: 1,537 Days
• Field of Search: 436/805, 436/806, 436/84, 436/501, 436/807, 436/827, 436/904, 204/1 T, 204/403, 204/412, 324/71.5, 435/4, 435/7, 435/25, 435/26, 435/28, 357/25
• 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 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 creating a potential at said element with respect to said counterelectrode;

first and second container means for retaining electrically responsive media in communication with a first and second one of said two sites, respectively;

means adapted for providing impedance between each of said sites and said counterelectrode; 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 a portion of said element is coated with a lipid bilamellar layer for contact with said media.

6. A device according to claim 5, wherein said lipid bilamellar layer is coated on said irradiation receiving surface and has a specific binding pair member bound to said layer.

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

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

9. A device according to claim 1, wherein said means adapted for providing impedance comprises a plurality of resistive portions of said counterelectrode, each resistive portion being proximate to one of said sites.

10. A device according to claim 1, wherein said means adapted for providing impedance comprises said first and second container means adapted to retain said media, said media being resistive.

11. A device according to claim 1, wherein said means for determining the photoresponse comprises means for varying the intensity of said irradiation means to maintain a constant photoresponse and measuring the level of said varying.

12. A device according to claim 1, wherein said means for determining the photoresponse comprises means for varying the intensity of said irradiation means with an alternating signal and measuring the resulting AC current in said photoresponsive element.

13. A device according to claim 1, wherein said means for determining the photoresponse comprises means for varying said potential to maintain a constant photoresponse and measuring the varying of said potential.

14. A device according to claim 1, wherein said means for determining the photoresponse comprises means to maintain said potential and the intensity of said irradiation means fixed and measuring a DC component of the photoresponse.

15. 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 monolithic doped silicon semiconductor wafer having an irradiation receiving surface;

irradiation means for sequentially irradiating at least two sites on said surface with an alternating signal;

a counterelectrode;

means for creating a potential at said wafer with respect to said counterelectrode;

first and second container means for retaining a resistive electrically responsive media in which said substance is to be determined in communication with a first and second one of said two sites, respectively; and

means for determining the electrical photoresponse during irradiation of a site by varying said potential to maintain a constant photoresponse and measuring the level of said varying.

16. A device according to claim 15 wherein said means for determining comprises:

means for rectifying an alternating electrical photoresponse signal to produce a rectified signal; and

means for providing to said counterelectrode a feedback signal proportional to said rectified signal.

17. A device according to claim 16 wherein said determining means further comprises:

means for filtering said alternating electrical photoresponse signal; and

means for filtering said rectified signal.

18. 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;

maintaining a separate electrical communication having impedance between each of a plurality of sites on a photoresponsive surface of said element and said counterelectrode through said electrically responsive medium;

irradiating said surface at said sites at different times in a predetermined sequence for defined periods of time at each of said sites, each site being associated with a different one of said volumes; 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.

19. A method according to claim 18, 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.

20. A method according to claim 18, wherein said circuit includes means for varying the light intensity in relation to said photoresponse.

21. A method according to claim 18, 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.

22. A method according to claim 18, wherein said substance is a result of an enzymatic reaction.

23. A method according to claim 22, wherein said enzyme is an oxidoreductase.

24. A method according to claim 23, wherein said oxidoreductase is a peroxidase.

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

26. A method according to claim 22, wherein said substance is reduced or oxidized indirectly through one or more intermediate compounds by the product of said enzyme.

27. A method according to claim 18, wherein said substance is an inorganic ion.

28. A method according to claim 18, wherein said photoresponsive element is a monolithic doped semiconductor.

29. A method according to claim 18, wherein said volumes are portions of at least one stream.

30. A method according to claim 18, wherein each of said volumes is physically separated from the other volumes.

31. A method according to claim 18, wherein said substance is methylene blue, Meldola blue, or phenazine methosulfate.

32. A method according to claim 18, 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.

33. 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 electrode and counterelectrode and maintaining said electrodes in a predetermined potential relationship;

(d) means for providing separate resistive couplings between said counterelectrode and a plurality of sites on said surface;

(e) 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

(f) 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 said 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.

34. A method according to claim 33, wherein said photoresponsive semiconductor is a doped monolithic silicon wafer.

35. A method according to claim 33, wherein said specific binding means is a second ligand and receptor.

36. A method according to claim 35, wherein said second ligand and receptor are a saccharide and a lectin.

37. A method according to claim 35, wherein said second ligand and receptor are a ligand and a natural receptor or antibody.

38. A method according to claim 33, wherein said label is an enzyme.

39. A method for determining the presence of an enzyme substrate in a medium, wherein the product of said substrate results in the production of 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;

providing separate resistive couplings between said counterelectrode and each of said sites;

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.

40. A method according to claim 39, wherein said enzyme is horseradish peroxidase.

41. A method according to claim 39, wherein said enzyme is glucose oxidase.

42. 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;

providing separate resistive couplings between said counterelectrode and each of said sites;

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.

43. A method according to claim 42, wherein said microorganism is bound by a specific receptor in juxtaposition to said site.

44. 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;

providing separate resistive couplings between said counterelectrode and each of said sites;

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.