Highly sensitive amperometric bi-mediator-based glucose biosensor

• US 6,033,866 A
• Filed: 12/08/1997
• Issued: 03/07/2000
• Est. Priority Date: 12/08/1997
• Status: Expired due to Fees

• Current Assignee: Biomedix, Inc.
• Original Assignee: Biomedix, Inc.
• Inventors: Shieh, Paul, Goldberg, Esfir, Guo, Dingli
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US08/986,974
• Time in Patent Office: 820 Days
• Field of Search: 435/14, 435/4, 435/25, 435/28, 435/817, 435/289.1, 435/283.1, 205/263, 205/571
• US Class Current: 435/14
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

A highly sensitive sensor for the amperometric assay of glucose in aqueous media having a face-to-face sandwich configuration is provided which comprises a sensing electrode strip containing a first redox mediator in an electrically conductive coating and a reference electrode strip in simultaneous contact with a reagent strip containing a second redox mediator which is sandwiched between the active electrode surfaces. An opening is provided in the reference electrode for the introduction of samples. In one embodiment, a whole blood separation membrane is interposed between the reference electrode and the reagent strip to filter red blood cells and other particles from whole blood to enable direct assay of glucose without sample preparation. The sensing electrode comprises a non-conductive support member having an electrically conductive layer containing the first redox mediator. The reference electrode is typically a Ag/AgCl electrode formed by coating an ink containing Ag/AgCl dispersed in a resin on a non-conductive base. The reagent strip is a porous or fibrous carrier, typically a paper, impregnated with a mixture containing the second redox mediator, glucose oxidase, horseradish peroxidase, at least one surfactant and at least one stabilizer comprising an aqueous thickening agent. In one version of the sensor, the sensing electrode comprises a support member of polyester film coated with an electrically conductive graphite composition containing dimethylferrocene as the first redox mediator and the second redox mediator comprises potassium ferrocyanide.

Claims

1. An amperometric sensor for the determination of glucose in aqueous media comprising:

a sensing electrode, the sensing electrode comprising;

a non-conductive support member, with the non-conductive support member comprising;

a non-conductive polymeric film;

the non-conductive polymeric film coated with an electrically conductive layer, with the electrically conductive layer containing;

a first redox mediator; and

a reference electrode comprising;

a non-conductive polymeric film, the polymeric film coated with an electrically conductive formation comprising Ag/AgCl dispersed in a resin formulation, and with the reference electrode having an opening; and

a reagent strip, the reagent strip comprising;

a carrier strip that is a porous or fibrous water absorbent matrix, the carrier strip impregnated with a mixture comprising;

glucose oxidase,

horseradish peroxidase,

a second redox mediator that can be oxidized by hydrogen peroxide under catalysis by horseradish peroxidase,

at least one surfactant,

at least one stabilizer,

a buffering agent to maintain a pH from about 4 to about 8;

with the electrically conducting layer of the sensing electrode and the electrically conductive formulation of the reference electrode facing each other; and

with the reagent strip superimposed on and in physical contact with the electrically conducting layer of the sensing electrode, and with the reference electrode superimposed on the reagent strip so that the electrically conductive formulation coating the reference electrode is superimposed on the reagent strip and in physical contact with the reagent strip;

so that the sensing electrode and the reagent strip and the reference electrode form a sandwich.

2. The sensor of claim 1 in which the non-conductive support member of the sensing electrode comprises a polymeric film strip and the electrically conductive layer of the sensing electrode comprises an electrically conductive carbon or graphite formulation containing the first redox mediator.

3. The sensor of claim 2 in which the reference electrode comprises a Ag/AgCl reference electrode strip comprising a non-conductive polymeric film support member coated with a layer of an electrically electrode formulation comprising Ag and AgCl dispersed in a resin formulation.

4. The sensor of claim 3 in which the first redox mediator contained in the electrically electrode layer of the sensing electrode is selected from the group consisting of dimethylferrocene, ferricinium, ferrocene monocarboxylic acid, 7,7,8,8-tetracyanoquinodimethane, tetrathiafulvalene, nickelocene, N-methylacridinium, tetrathiatetracene, N-methylphenazinium, hydroquinone and mixtures thereof.

5. The sensor of claim 4 in which the second redox mediator contained in the reagent strip is selected from the group consisting of 3,3'"'"'5,5'"'"'-tetramethylbenzidine, 3-methyl-2-benzothiazolinone hydrazone hydrochloride and 3-dimethylaminobenzoic acid, o-dianisidine, o-toluidine, sulfonated 2,4-dichloro-phenol and 4-amino phenazone benzidine, 3-methyl-2-benzothiozolinone hydrazone and 3-(dimethylamino) benzoic acid, 3-methyl-2-benzothiozolinone hydrazone and 2-methoxy-4-allyl phenol, 4-aminoantipyrene-dimethylaniline and 4-aminoantipyrene-4-methoxynaphthol, (Fe(CN)₆^-4), HCN, I^-, Co(NH₃)₆⁺², Sn⁺², S^-2, Tl⁺² and mixtures thereof.

6. The sensor of claim 5 in which the surfactant is selected from the group consisting of cholic acid, Triton X-100, polyethylene glycol, sodium lauryl sulfate, sodium lauryl sarcosinate, hydroxypropyl methylcellulose, tetrapropylene diphenyloxide disulphonate sodium salt, capryloamphocarboxypropionate, polyoxyethylene-2-cetyl ether, 2,4,7,9-tetramethyl-5-decyne-4,7-diol ethoxylate, octanoyl-N-methyl-D-glucamide, decanoyl-N-methyl-D-glucamide and mixtures thereof.

7. The sensor of claim 6 in which the stabilizer is selected from the group consisting of gelatin, bovine serum albumin, glutamate, L-arginine, Gantrez, mannitol, gum arabic, low viscosity polypep, methocel and mixtures thereof.

8. The sensor of claim 7 in which the buffer is selected from the group consisting of citrate, succinate, tris-(hydroxymethyl)aminomethane, phosphate, 2(N-morpholino) ethanesulfonic acid and mixtures thereof.

9. The sensor of claim 8 in which the polymeric film comprising the non-constructive support member of the sensing electrode and the non-conductive support member of the reference electrode comprises polyester and the first redox mediator is 1,1'"'"'dimethylferrocene and the second redox mediator is selected from the group consisting of 3,3'"'"',5,5'"'"'tetramethylbenzidine, potassium ferrocyanide and mixtures thereof.

10. The sensor of claim 9 in which the surfactant is selected from the group consisting of 2,4,7,9-tetramethyl-5-decyne-4,7-diol ethoxylate, octanoyl-N-methyl-D-glucamide and mixtures thereof.

11. The sensor of claim 1 further comprising a whole blood separation membrane, the whole blood separation membrane having an effective pore size of about 1 micron to about 10 microns, with the whole blood separation membrane interposed between the reagent strip and the reference electrode and with the whole blood separation membrane superimposed on and in physical contact with the reagent strip and with the reference electrode superimposed on the whole blood separation membrane so that the electrically conductive formulation coating the reference electrode is in physical contact with the whole blood separation membrane;

and with the whole blood separation membrane completely covering the opening in the reference electrode.

12. The sensor of claim 11 in which the whole blood separation membrane is selected from the group consisting of PrimeCare (Code S) polysulfone membrane;

Gelman Cytosep 1660 membrane;

PrimeCare (Code S) polysulfone membrane coated with octanoyl-N-methyl-D-glucamide;

polycarbonate membrane having a coating selected from the group consisting of polyvinylpyrrolidone, polyvinylalcohol, polyacrylic acid, alginic acid, gelatin, ethylcellulose, polyethylene glycol monostearate; and

glass fiber membranes having a coating selected from the group consisting of polyvinylpyrrolidone, polyvinylalcohol, polyacrylic acid, alginic acid, gelatin, ethylcellulose, polyethylene glycol monostearate.

13. A method for assaying a sample of whole blood for the concentration of glucose contained in the whole blood and fractions thereof, comprising the steps of:

a) providing an amperometric biosensor for glucose comprising;

a sensing electrode, the sensing electrode comprising;

a non-conductive support member, with the non-conductive support member comprising;

a non-conductive polymeric film;

the non-conductive polymeric film coated with an electrically electrode layer, with the electrically conductive layer containing;

a first redox mediator; and

a reference electrode comprising;

a non-conductive polymeric film, the polymeric film coated with an electrically conductive formulation comprising Ag/AgCl dispersed in a resin formulation, and with the reference electrode having an opening; and

a reagent strip, the reagent strip comprising;

a carrier strip that is a porous or fibrous water absorbent matrix, the carrier strip impregnated with a mixture comprising;

glucose oxidase,

horseradish peroxidase,

a second redox mediator that can be oxidized by hydrogen peroxide under catalysis by horseradish peroxidase,

at least one surfactant,

at least one stabilizer,

a buffering agent to maintain a pH from about 4 to about 8; and

a whole blood separation membrane, the whole blood separation membrane comprising a porous matrix;

with the porous matrix having an effective pore size of about 1 micron to about 10 microns;

with the electrically conducting layer of the sensing electrode and the electrically conductive formulation of the reference electrode facing each other; and

with the reagent strip superimposed on and in physical contact with the electrically conducting layer of the sensing electrode, and with the whole blood separation membrane superimposed on and in physical contact with the reagent strip; and

with the reference electrode superimposed on the whole blood separation membrane;

so that the electrically conductive formulation coating the reference electrode is in physical contact with the whole blood separation membrane, and with the whole blood separation membrane completely covering the opening in the reference electrode;

so that the sensing electrode and the reagent strip and the whole blood separation membrane and the reference electrode form a sandwich;

b) introducing a whole blood sample into the opening of the reference electrode;

c) maintaining a potential of about -80 mV to about -125 mV across the sensing electrode and the reference electrode;

d) measuring the current passing between the sensing electrode and the reference electrode;

e) comparing the current measured to a calibration curve of the concentration of glucose versus current at the potential used in step c) to obtain the concentration of glucose in the whole blood sample.

14. The method of claim 13 in which the electrically conductive layer of the sensing electrode of step a) comprises an electrically conductive carbon or graphite formulation and the first redox mediator is selected from the group consisting of dimethylferrocene, ferricinium, ferrocene monocarboxylic acid, 7,7,8,8-tetracyanoquinodimethane, tetrathiafulvalene, nickelocene, N-methylacridinium, tetrathiatetracene, N-methylphenazinium, hydroquinone and mixtures thereof.

15. The method of claim 14 in which the second redox mediator contained in the reagent strip of step a) is selected from the group consisting of 3,3'"'"'5,5'"'"'-tetramethylbenzidine, 3-methyl-2-benzothiazolinone hydrazone hydrochloride and 3-dimethylaminobenzoic acid, o-dianisidine, o-toluidine, sulfonated 2,4-dichloro-phenol and 4-amino phenazone benzidine, 3-methyl-2-benzothiozolinone hydrazone and 3-(dimethylamino) benzoic acid, 3-methyl-2-benzothiozolinone hydrazone and 2-methoxy-4-allyl phenol, 4-aminoantipyrene-dimethylaniline and 4-aminoantipyrene-4-methoxynaphthol, (Fe(CN)₆^-4), HCN, I^-, Co(NH₃)₆⁺², Sn⁺², S^-2, Tl⁺² and mixtures thereof.

16. The method of claim 15 in which the porous matrix comprising the whole blood separation membrane of step a) is selected from the group consisting of PrimeCare (Code S) polysulfone membrane;

Gelman Cytosep 1660 membrane;

PrimeCare (Code S) polysulfone membrane coated with octanoyl-N-methyl-D-glucamide;

polycarbonate membrane having a coating selected from the group consisting of polyvinylpyrrolidone, polyvinylalcohol, polyacrylic acid, alginic acid, gelatin, ethylcellulose, polyethylene glycol monostearate; and

glass fiber membranes having a coating selected from the group consisting of polyvinylpyrrolidone, polyvinylalcohol, polyacrylic acid, alginic acid, gelatin, ethylcellulose, polyethylene glycol monostearate.

17. The method of claim 16 in which the at least one surfactant impregnated in the reagent strip of step a) is selected from the group consisting of cholic acid, Triton X-100, polyethylene glycol, sodium lauryl sulfate, sodium lauryl sarcosinate, hydroxypropyl methylcellulose, tetrapropylene diphenyloxide disulphonate sodium salt, capryloamphocarboxypropionate, polyoxyethylene-2-cetyl ether, 2,4,7,9-tetramethyl-5-decyne-4,7-diol ethoxylate, octanoyl-N-methyl-D-glucamide, decanoyl-N-methyl-D-glucamide and mixtures thereof.

18. The method of claim 17 in which the stabilizer in the reagent strip of step a) is selected from the group consisting of gelatin, bovine serum albumin, glutamate, L-arginine, Gantrez, mannitol, gum arabic, low viscosity polypep, methocel and mixtures thereof.

19. The method of claim 18 in which the buffering agent in the reagent strip of step a) is selected from the group consisting of citrate, succinate, tris-(hydroxymethyl) aminomethane, phosphate, 2(N-morpholino) ethanesulfonic acid and mixtures thereof.

20. A method of enhancing the sensitivity of an amperometric strip sensor for the assay of an analyte comprising the steps of:

a) forming a sensing electrode, the sensing electrode comprising;

a non-conductive support member, with the non-conductive support member comprising;

a non-conductive polymeric film;

the non-conductive polymeric film coated with an electrically conductive layer, with the electrically conductive layer containing;

a first redox mediator; and

b) forming a reference electrode comprising;

a non-conductive polymeric film, the polymeric film coated with an electrically conductive formulation comprising Ag/AgCl dispersed in a resin formulation,

c) providing an opening in either electrode for introduction of a sample; and

d) forming a reagent strip, the reagent strip comprising;

a carrier strip that is a porous or fibrous water absorbent matrix, the carrier strip impregnated with a mixture comprising;

reagents that produce a flow of electrons when contacted by the analyte,

at least one surfactant,

at least one stabilizer,

e) arranging the sensing electrode and the reference electrode and the reagent strip so that the electrically conductive layer of the sensing electrode and the electrically conductive formulation of the reference electrode face each other with the reagent strip superimposed on and in physical contact with the electrically conducting layer of the sensing electrode, and with the reference electrode superimposed on the reagent strip so that the electrically conductive formulation coating the reference electrode is in physical contact with the reagent strip;

so that the sensing electrode and the reagent strip and the reference electrode form a sandwich.

21. The method of claim 20 further comprising formation of a filtration or treatment layer and interposing the filtration or treatment layer between the reagent strip and the opening in either electrode so that he filtration or treatment layer covers the opening.

22. The method of claim 21 in which the filtration or treatment layer completely covers the reagent strip so that the reagent strip does not make physical contact with the electrode having the opening.