Devices and methods for detecting analytes using electrosensor having capture reagent

• US 6,670,115 B1
• Filed: 10/27/2000
• Issued: 12/30/2003
• Est. Priority Date: 11/24/1999
• Status: Expired due to Fees

• Current Assignee: Biotronics Technology Incorporated
• Original Assignee: Biotronics Technology Incorporated
• Inventors: Zhang, Honghua
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Padmanabhan, Kartic
• Application Number:
  US09/699,140
• Time in Patent Office: 1,159 Days
• Field of Search: 422/56, 422/57, 422/58, 422/61, 422/68.1, 204/193, 204/400, 204/403, 204/412, 204/280, 435/4, 435/5, 435/7.1, 435/7.71, 435/7.72, 435/7.93, 435/7.94, 435/970, 435/975, 435/287.1, 436/501, 436/514, 436/518, 436/524, 436/538
• US Class Current: 435/5
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The present invention relates to devices comprising electrosensors containing capture reagents, their preparation thereof, and their use for detecting, preferably, quantitative measurement, of analyte in a liquid sample. In particular, the invention relates to an enzyme electrosensor, e.g., electroimmunosensor, device for electrochemical detection and preferably, real-time measurement, which is suitable for use at point-of-care settings by unskilled personnel.

Claims

1. A device for detecting an analyte in a liquid sample, which device comprises:

a) a solid support;

b) an electrosensor immobilized on said solid support, said electrosensor comprises a working electrode and another electrode used as auxiliary and/or reference electrode;

c) a capture reagent immobilized on said working electrode, said capture reagent binds to an analyte, if said analyte is present in a liquid sample; and

d) conductive leads for connecting said electrodes to a readout device for electrochemical measurement,

wherein the device further comprising a sample application area that is separate, but in fluid communication with the electrosensor via a wicking member having an open mesh structure that does not comprise an immobilized capture reagent, and wherein the wicking member having an open mesh structure provides for two dimensional transport of fluids from the application area to the electrosensor and faster flow rate, lower background noise and higher signal to noise ratio compared to a wicking member not having an open mesh structure.

2. The device of claim 1, wherein the solid support comprises a material selected from the group consisting of polyester, polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), paper, nylon, fiberglass, polyethylene, nitrocellulose, and a combination thereof.

3. The device of claim 1, wherein the electrosensor comprises a working electrode, an auxiliary electrode and a reference electrode.

4. The device of claim 3, wherein the working electrode and/or auxiliary electrode comprise(s) a screen-printed carbon conductor and the reference electrode comprises a screen-printed silver or silver/silver chloride conductor.

5. The device of claim 1, wherein the capture reagent is selected from the group consisting of a cell, a cellular organelle, an inorganic molecule, an organic molecule and a mixture thereof.

6. The device of claim 5, wherein the organic molecule is selected from the group consisting of an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin, a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and a complex thereof.

7. The device of claim 6, wherein the protein is an antibody, avidin/strepavidin, protein A or protein G.

8. The device of claim 1, wherein the capture reagent is immobilized on the working electrode in a buffered organic solution containing a ketone or an aliphatic alcohol.

9. The device of claim 8, wherein the aliphatic alcohol is isopropyl alcohol.

10. The device of claim 1, wherein the capture reagent specifically binds to the analyte.

11. The device of claim 1, wherein the analyte is selected from the group consisting of a cell, a cellular organelle, an inorganic molecule, an organic molecule and a mixture thereof.

12. The device of claim 1, wherein the analyte is alpha-fetoprotein, prostate-specific antigen, cardiac troponins, c-reactive protein (CRP), or human chorionic gonadotropin, or a marker for HBV, HAV, HCV or HIV infection.

13. The device of claim 1, further comprising an analyte bound to the capture reagent, said analyte contains a label that generates electrode current.

14. The device of claim 13, wherein the label is an enzyme.

15. The device of claim 14, wherein the enzyme is horseradish peroxidase and the device further comprises an enzymatic substrate that is hydrogen peroxide and an electron transfer mediator that is ferrocene, or a derivative thereof, benzoquinone, ascorbic acid or 3,3′

,5,5′

tetramethylbenzidine.

16. The device of claim 1, further comprising a cover casing having a liquid sample application aperture and a detection aperture.

17. The device of claim 1, wherein the sample application area contains movably bound labeled analyte, said labeled analyte is dissolved or suspended into the sample liquid and carried to the capture reagent immobilized on the electrosensor by the sample fluid, and said label on said analyte generates electrocurrent.

18. The device of claim 1, wherein the sample application area contains movably bound labeled detection reagent, said labeled detection reagent is dissolved or suspended into the sample liquid, binds to the analyte, if there is any, carried to the capture reagent immobilized on the electrosensor by the sample fluid to form a sandwich comprising the immobilized capture reagent-analyte-labeled detection reagent, and said label on said detection reagent generates current.

19. The device of claim 18, wherein the movably bound labeled detection reagent specifically binds to the analyte, if there is any, in the sample fluid.

20. The device of claim 18, wherein the movably bound labeled detection reagent is an antibody.

21. The device of claim 18, wherein the label is an enzyme.

22. The device of claim 21, wherein the enzyme is horseradish peroxidase and the device further comprises an enzymatic substrate that is hydrogen peroxide and an electron transfer mediator that is ferrocene, or a derivative thereof, benzoquinone, ascorbic acid or 3,3′

,5,5′

tetramethylbenzidine.

23. The device of claim 1, wherein the wicking member comprises a mesh having mesh opening in the range from about 0.45 μ

m to about 100 μ

m.

24. The device of claim 1, further comprising a filter in the application area, said filter removes insoluble or insuspendable material(s) from the sample fluid.

25. The device of claim 24, wherein the filter removes insoluble or insuspendable material(s) from a sample blood.

26. The device of claim 1, further comprising an absorptive sink in fluid communication with the electrosensor, said sink having sufficient porosity and capacity to absorb excess liquid or allow excess liquid to be washed out of the device.

27. The device of claim 26, wherein the absorptive sink is a pad of absorbent material.

28. The device of claim 1, which comprises an absorptive sink, an electrosensor and an application area that are linearly arranged in order.

29. The device of claim 1, further comprising an enzyme substrate and an electron transfer mediator localized on or in proximity to the electrosensor, said substrate and mediator is controllably released.

31. A kit for detecting an analyte in a liquid sample, which kit comprises:

a) the device of claim 1; and

b) an effective amount of an electron transfer mediator and a substrate.

34. The device of claim 1, wherein the wicking member comprises a nylon net.

35. The device of claim 34, wherein the nylon net has a mesh opening from 11 μ

m to 100 μ

m.

36. The device of claim 34, wherein the nylon net has a mesh opening from 80 μ

m to 100 μ

m.

30. A device for detecting an analyte in a liquid sample, which device comprises:

a) a base sensor strip having a working electrode, a reference electrode, and an auxiliary electrode coated on a plastic substrate, whereon a capture reagent is immobilized on the working electrode, said sensor strip having conductive leads for attaching the electrodes to a readout device for electrochemical measurement;

b) a cover casing having a liquid sample application aperture and a detection aperture;

c) an application zone for receiving a fluid containing an analyte from the application aperture, said application zone, in dry unused form, containing a labeled detection reagent specifically binds to said analyte, if said analyte is present said fluid, wherein said labeled reagent is released into mobile form when in contact with the liquid sample;

d) a detection zone in fluid communication with the electrodes in the presence of a liquid sample received from the application aperture;

e) a wicking member having an open mesh structure that does not comprise an immobilized capture reagent and that carries the liquid sample from the application zone to the detection zone by capillary action, wherein the wicking member having an open mesh structure provides for two dimensional transport of fluids from the application area to the electrosensor and faster flow rate, lower background noise and higher signal to noise ratio compared to a wicking member not having an open mesh structure and wherein said analyte is sandwiched between the detection reagent and the capture reagent immobilized on the electrode surface; and

f) an absorbent sink placed in partial contact with the wicking member at the end of the flow path to absorb any excess fluid from the detection zone.

32. A device for detecting an analyte in a liquid sample, which device comprises a sample application area that is in fluid communication with an electrosensor via a wicking member, wherein the wicking member has an open mesh structure that does not comprises an immobilized capture reagent that binds to an analyte, and wherein the wicking member having an open mesh structure provides for two dimensional transport of fluids from the application area to the electrosensor and faster flow rate, lower background noise and higher signal to noise ratio compared to a wicking member not having an open mesh structure.

33. The device of claim 32, wherein the wicking member comprises a nylon mesh having mesh opening in the range from about 0.45 μ

m to about 100 μ

m.