Determination of glycoprotein and glycosylated hemoglobin in blood

• US 6,054,039 A
• Filed: 08/18/1997
• Issued: 04/25/2000
• Est. Priority Date: 08/18/1997
• Status: Expired due to Fees

• Current Assignee: Paul Shieh
• Original Assignee: Paul Shieh
• Inventors: Shieh, Paul
• Primary Examiner(s): Tung, T.
• Assistant Examiner(s): Noguerda, Alex
• Application Number:
  US08/914,283
• Time in Patent Office: 981 Days
• Field of Search: 204/403, 205/792, 205/777.5, 205/778, 435/817
• US Class Current: 205/778
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

A method of determining the concentration of glycoproteins and glycosylated hemoglobin in whole blood and whole blood components by means of an amperometric biosensor and an amperometric biosensor for this determination are provided. In one embodiment, whole blood is introduced into a version of an amperometric sensor having a component that removes erythrocytes. Redox mediators are used to obtain a current flow based on the oxidation of fructosamine derivatives that can be correlated with the concentration of glycosylated proteins in the fraction of the blood from which erythrocytes have been excluded. To obtain the concentration of glycosylated hemoglobin, whole blood is introduced into a version of the sensor which includes a component that produces lysis of the erythrocytes yielding a current flow proportional to the total quality of glycosylated proteins including glycosylated hemoglobin. The glycosyltaed hemoglobin concentration is obtained by subtracting the glycoprotein concentration in the absence of erythrocytes from the glycoprotein concentration of the lysed whole blood. The sensor generally comprises a sensing electrode having a first redox mediator dispersed in an electrically conductive medium such as an electrically conductive graphite formulation; a reference electrode such as a standard silver-silver chloride electrode; a reagent strip containing a pH buffer and a second redox mediator system in a gel medium; and a whole blood treatment component consisting of either a membrane or other means to filter erythrocytes from whole blood or a means to lyse erythrocytes. In a preferred form, that has high sensitivity, the sensing electrode and the reference electrode may be formed as coatings on separate non-conductive strips such as polyester film with these strips arranged so that they form "the bread" of a sandwich in which the electrode coatings are face-to-face and the reagent strip and the filtration or lysing component form the "filling" of the sandwich. The filtration or cell lysing component covers an opening in the reference electrode through which samples are introduced, and is superimposed on the reagent strip.

Claims

1. An amperometric sensor for assaying the concentration of a fructosamine moiety contained in a biological fluid in the presence of interfering oxidizable substances also contained in the fluid, comprising:

a sensing electrode, the sensing electrode comprising;

a non-conductive support member strip,

with the non-conductive support member strip coated with an electrically conductive layer, with the electrically conductive layer containing;

a first redox mediator; and

a reference electrode comprising;

a non-conductive support member strip, with the support member strip 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 water absorbent carrier, the water absorbent carrier impregnated with a mixture comprising;

a second redox mediator that can be reduced by a fructosamine derivative,

at least one surfactant,

at least one stabilizer,

a buffering agent to maintain a pH from about 8 to about 12; and

a whole blood treatment component selected from the group consisting of;

an erythrocyte filtration component and an erythrocyte lysing component;

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 treatment component superimposed on and in physical contact with the reagent strip; and

with the reference electrode superimposed on the whole blood treatment component so that the electrically conductive formulation coating the reference electrode is in physical contact with the whole blood treatment component; and

with the whole blood treatment component completely covering the opening in the reference electrode;

so that the sensing electrode and the reagent strip and the whole blood treatment component and the reference electrode form a sandwich.

2. The sensor of claim 1 in which the first redox mediator contained in the electrically conductive layer of the sensing electrode is selected from the group consisting of dimethyl ferrocene, 7,7,8,8-tetracyanoquinodimethane, tetrathiafulvalene, nicklocene, N-methylacridinium, tetrathiatetracene, N-methylphenazinium, and mixtures thereof.

3. The sensor of claim 2 in which the second redox mediator impregnated in the reagent strip is selected from the group consisting of nitroblue tetrazolium, methylene blue, thionin and mixtures thereof.

4. The sensor of claim 3 in which the erythrocyte filtration component comprises a component selected from the group of components consisting of membranes and fibrous matrices having a pore size sufficient to retain erythrocytes while allowing passage of a liquid fraction of whole blood.

5. The sensor of claim 4 in which the erythrocyte lysing component comprises a filtration component further comprising a coating selected from the group consisting of octanoyl-N-methylglucamide surfactant, Triton X surfactant, lauryl sulfate salts, alpha olefin sulfonates, sulfosuccinate salts, sorbitol, lauric diethanolamide, lauryl dimethylamine oxide, lauryl amidopropyldimethylamine oxide, betaine, amidobetaine glycinate, sulfobetaine, stearyl dimethylbenzylammonium chloride, di(C₁₂ -C₁₈) dimethyl ammonium chloride and mixtures thereof.

6. The sensor of claim 5 in which the erythrocyte filtration component and the erythrocyte lysing component are comprised of materials selected from the group consisting of polysulfones, polyamides, celluose derivatives, polyesters, polyvinylalcohol polymers, glass fibers and mixtures thereof.

7. The sensor of claim 6 in which the erythrocyte filtration component comprises a polysulfone membrane and the erythrocyte lysing component comprises a polysulfone membrane further comprising a coating of octanoyl-N-methylglucamide surfactant.

8. The sensor of claim 7 in which the electrically conductive layer of the sensing electrode comprises a formulation containing an electrically conductive material selected from the group consisting of electrically conductive carbon or electrically conductive graphite, and the first redox mediator contained in the electrically conductive layer of the sensing electrode is dimethyl ferrocene and the second redox mediator impregnated in the reagent strip is nitroblue tetrazolium and the reagent strip further comprises, stabilizers, surfactants and a buffer to maintain a pH of about 10 to a pH of about 11.

9. The sensor of claim 8 in which the non-conductive support member strip of the sensing electrode comprises a non-conductive polymeric film and the non-conductive support member strip of the reference electrode comprises a non-conductive polymeric film.

11. A method for the amperometric determination of the concentration of fructosamine derivatives contained in a sample of whole blood or fractions thereof, in the presence of interfering oxidizable substances also contained in the sample, comprising the steps of:

providing an amperometric biosensor for fructosamine derivatives having high sensitivity at low applied potentials, the biosensor comprising;

a sensing electrode, with the sensing electrode comprising;

a non-conductive support strip, with the non-conductive support strip coated with an electrically conductive layer, with the electrically conductive layer containing a first redox mediator, and

a reference electrode, with the reference electrode comprising;

a non-conductive support strip, with the non-conductive support strip 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, with the reagent strip comprising;

a water absorbent carrier, with the water absorbent carrier containing a second redox mediator comprising at least one compound that can be reduced by a fructosamine derivative, and a buffering agent to maintain a pH from about 8 to about 12; and

a whole blood treatment component with the whole blood treatment component comprising;

a component selected from the group consisting of an erythrocyte filtration component and an erythrocyte lysing component,

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 treatment component superimposed on and in physical contact with the reagent strip; and

with the reference electrode superimposed on the whole blood treatment component so that the electrically conductive formulation coating the reference electrode is in physical contact with the whole blood treatment component and with the whole blood treatment component completely covering the opening in the reference electrode;

so that the sensing electrode and the reagent strip and the whole blood treatment component and the reference electrode form a sandwich;

contacting the whole blood treatment component of the biosensor with an aliquot of a whole blood sample and,

maintaining a potential of 500 mV or less across the sensing electrode and the reference electrode and,

measuring the current passing between the sensing electrode and the reference electrode and,

comparing the current measured to a calibration curve of the concentration of a fructosamine versus current for the biosensor, whereby the concentration of the fructosamine is substantially precisely determined in the presence of the interfering substances.

10. The sensor of claim 11 in which the sensing electrode further comprises a conductive protrusion and the reference electrode further comprises a conductive protrusion, with the protrusions having a gap separating them, with the protrusions providing points of contact for electrical connection.

12. The method of claim 11 in which the sensing electrode comprises a non-conductive polymeric film coated with an electrically conductive layer comprising an electrically conductive carbon or graphite formulation containing the first redox mediator, with the first redox mediator selected from the group consisting of dimethyl ferrocene, 7,7,8,8-tetracyanoquinodimethane, tetrathiafulvalene, nicklocene, N-methylacridinium, tetrathiatetracene, N-methylphenazinium, and mixtures thereof.

13. The method of claim 12 in which the second redox mediator is selected from the group consisting of nitroblue tetrazolium, methylene blue, thionin and mixtures thereof.

14. The method of claim 13 in which the erythrocyte filtration component comprises a component selected from the group of components consisting of membranes and fibrous matrices having a pore size sufficient to retain erythrocytes while allowing passage of a liquid fraction of the whole blood.

15. The method of claim 14 in which the erythrocyte lysing component comprises a filtration component further comprising a coating selected from the group consisting of octanoyl-N-methylglucamide, Triton X surfactant, lauryl sulfate salts, alpha olefin sulfonates, sulfosuccinate salts, sorbitol, lauric diethanolamide, lauryl dimethylamine oxide, lauryl amidopropyldimethylamine oxide, betaine, amidobetaine glycinate, sulfobetaine, stearyl dimethylbenzylammonium chloride, di(C₁₂ -C₁₈) dimethyl ammonium chloride and mixtures thereof.

16. The method of claim 15 in which the erythrocyte filtration component and the erythrocyte lysing component are comprised of materials selected from the group consisting of polysulfones, polyamides, celluose derivatives, polyesters, polyvinylalcohol polymers, glass fibers and mixtures thereof.

17. The method of claim 16 in which the erythrocyte filtration component comprises a polysulfone membrane and the erythrocyte lysing component comprises a polysulfone membrane further comprising a coating of octanoyl-N-methylglucamide surfactant.

18. The method of claim 17 in which the first redox mediator contained in the electrically conductive layer of the sensing electrode comprises dimethyl ferrocene and the second redox mediator contained in the reagent strip comprises nitroblue tetrazolium and the reagent strip further comprises, stabilizers, surfactants and sodium carbonate buffer.

19. The method of claim 11 in which the potential maintained across the sensing electrode and the reference electrode does not exceed 200 mV.

20. A method for the amperometric determination of the concentration of glycosylated hemoglobin contained in a sample of whole blood in the presence of interfering oxidizable substances also contained in the whole blood sample comprising the steps of:

providing a first and a second amperometric biosensor for fructosamine derivatives, with the first biosensor having a whole blood treatment component comprising an erythrocyte filtration component and the second biosensor having a whole blood treatment component comprising an erythrocyte lysing component, with each biosensor further comprising;

a sensing electrode, with the sensing electrode comprising;

a non-conductive support strip, with the non-conductive support strip coated with an electrically conductive layer, with the electrically conductive layer containing a first redox mediator;

a reference electrode, with the reference electrode comprising;

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

a reagent strip, with the reagent strip comprising;

a water absorbent carrier, with the water absorbent carrier containing a second redox mediator comprising at least one compound that can be reduced by a fructosamine derivative, and a buffering agent to maintain a pH from about 8 to about 12;

with the electrically conductive 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 treatment component superimposed on and in physical contact with the reagent strip; and

with the reference electrode superimposed on the whole blood treatment component so that the electrically conductive formulation coating the reference electrode is in physical contact with the whole blood treatment component and with the whole blood treatment component completely covering the opening in the reference electrode;

so that the sensing electrode and the reagent strip and the whole blood treatment component and the reference electrode form a sandwich; and

introducing an aliquot of a whole blood sample into each biosensor; and

maintaining a potential of 500 mv or less across the sensing electrode and the reference electrode of each biosensor and,

measuring the current passing between the sensing electrode and the reference electrode of each biosensor, and

comparing the current measured for each biosensor to a calibration curve of the concentration of a fructosamine derivative versus current corresponding to each biosensor to determine the corresponding concentration of the fructosamine derivative measured by each biosensor, and subtracting the concentration of fructosamine derivative determined with the first biosensor having the erythrocyte filtration component from the concentration of fructosamine derivative determined with the second biosensor having the erythrocyte lysing component to obtain the concentration of glycosylated hemoglobin in the whole blood.

21. The method of claim 20 in which the sensing electrode of each biosensor further comprises a non-conductive polymeric film coated with an electrically conductive layer comprising an electrically conductive carbon or graphite formulation containing the first redox mediator, with the first redox mediator selected from the group consisting of dimethyl ferrocene, 7,7,8,8-tetracyanoquinodimethane, tetrathiafulvalene, nicklocene, N-methylacridinium, tetrathiatetracene, N-methylphenazinium, and mixtures thereof.

22. The method of claim 21 in which the second redox mediator of each biosensor is selected from the group consisting of nitroblue tetrazolium, methylene blue, thionin and mixtures thereof.

23. The method of claim 22 in which the erythrocyte filtration component comprises a component selected from the group of components consisting of membranes and fibrous matrices having a pore size sufficient to retain erythrocytes while allowing passage of the liquid fraction of the whole blood.

24. The method of claim 23 in which the erythrocyte lysing component comprises a filtration component further comprising a coating selected from the group consisting of octanoyl-N-methylglucamide, Triton X surfactant, lauryl sulfate salts, alpha olefin sulfonates, sulfosuccinate salts, sorbitol, lauric diethanolamide, lauryl dimethylamine oxide, lauryl amidopropyldimethylamine oxide, betaine, amidobetaine glycinate, sulfobetaine, stearyl dimethylbenzylammonium chloride, di(C₁₂ -C₁₈) dimethyl ammonium chloride and mixtures thereof.

25. The method of claim 24 in which the erythrocyte filtration component and the erythrocyte lysing component are comprised of materials selected from the group consisting of polysulfones, polyamides, celluose derivatives, polyesters, polyvinylalcohol polymers, glass fibers and mixtures thereof.

26. The method of claim 25 in which the erythrocyte filtration component comprises a polysulfone membrane and the erythrocyte lysing component comprises a polysulfone membrane further comprising a coating of octanoyl-N-methylglucamide.

27. The method of claim 26 in which the electrically conductive layer of the sensing electrode of each biosensor comprises electrically conductive graphite or carbon and the first redox mediator contained in the electrically conductive layer of each biosensor comprises dimethyl ferrocene and the second redox mediator contained in the reagent strip of each biosensor comprises nitroblue tetrazolium and each strip further comprises, stabilizers, surfactants and sodium carbonate buffer.

28. The method of claim 20 in which the potential maintained across the sensing electrode and the reference electrode does not exceed 200 mV.