WO2002014535A2 - Electrochemical strip test for small volumes - Google Patents

Electrochemical strip test for small volumes Download PDF

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Publication number
WO2002014535A2
WO2002014535A2 PCT/GB2001/003615 GB0103615W WO0214535A2 WO 2002014535 A2 WO2002014535 A2 WO 2002014535A2 GB 0103615 W GB0103615 W GB 0103615W WO 0214535 A2 WO0214535 A2 WO 0214535A2
Authority
WO
WIPO (PCT)
Prior art keywords
sample
reagents
glucose
volume
cell
Prior art date
Application number
PCT/GB2001/003615
Other languages
French (fr)
Other versions
WO2002014535A3 (en
Inventor
Bernadette Yon-Hin
James Mccann
Original Assignee
Cambridge Sensors Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cambridge Sensors Limited filed Critical Cambridge Sensors Limited
Priority to AU2001277625A priority Critical patent/AU2001277625A1/en
Publication of WO2002014535A2 publication Critical patent/WO2002014535A2/en
Publication of WO2002014535A3 publication Critical patent/WO2002014535A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels

Definitions

  • This invention relates to an electrochemical test cell for the measurement of reactive species.
  • a sensor can be used for the measurement of enzymes, enzyme substrates, cofactors or mediators.
  • One particular application is the measurement of glucose for use in diabetes.
  • Diabetes mellitus is the most common endocrine disease and affects between 3-8% of the population.
  • Various studies, such as the UK Prospective Diabetes Study (UKPDS, Lancet, 12th September 1998) have pointed out the importance of maintaining a normal glucose level in the blood or plasma, in order to delay the onset of diabetic complications.
  • the present invention is based on the realisation that, by using appropriate chemistry etc, an adequate signal can be achieved if a cavity is provided that is smaller than has previously been used, i.e. which holds 500 nL or less of blood.
  • the invention allows the detection of glucose in whole blood, using a simple to use strip test, characterised in that the volume of sample required is very small, e.g. less than 200 nL and down to 50 nL.
  • a device of the invention can be made from known materials. However, it has been found that its size can be reduced, because sufficient current density can be achieved that there is adequate sensitivity for the analyte, typically 90-100 nA mM glucose.
  • the small volume of blood that can be utilised in the invention means that the sample can be taken from a part of the body having a low vasculature. Thus, for example, the sample can be taken from the arm.
  • the drawings show a cell or sample area 1 , a cover 2, a spacer layer 3, a reference/counter electrode 4, a working electrode 5, a cell-defining layer 6, a counter electrode 7, a reference electrode 8, and a strip/sample electrode 9.
  • Electrochemical strips for the detection of glucose in whole blood typically comprise an working, reference and counter electrode, or combination thereof, for the detection and quantitation of the activity of reagents or reaction products on the strip.
  • these reagents may include enzymes, enzyme products or mediators accepting electrons from redox enzymes on the strip.
  • the electrodes are preferably printed or deposited onto an elongate substrate, configured for ease of use by the diabetic and for insertion into a reader.
  • a substrate may also include other tests of use for the diabetic such as, but not limited to, glycosylated haemoglobin, glucose and potassium.
  • Some of the reagents may be deposited directly onto the electrodes themselves or onto other structures incorporated in the device, including wetting meshes such as is known in the art.
  • the mesh is preferably as described in WO-A- 00/28068.
  • the use of a mesh in the cavity e.g. having an open area of about 30%, can reduce the sample volume.
  • Appropriate reagents may be placed on the electrochemical strip test by methods including, but not limited to, screen printing and dosing the enzymes or enzyme-mediator couples.
  • such reagents may be, for example: glucose oxidase and ferrocene/ferricyanide glucose oxidase and oxygen to yield hydrogen peroxide glucose dehydrogenase and ferricyanide glucose dehydrogenase and NAD and Meldola's Blue
  • the effect of interferents in the test may be reduced by including enzymes or chemicals to reduce ascorbate interference, e.g. ascorbate oxidase.
  • Haematocrit interference may be reduced by including saponin, digitonin, or other lysing agents.
  • PVP, PVA and other polymers may be included, to increase linearity.
  • Surfactants may be added to the device, to aid filling.
  • the assay may also incorporate features such as filling via the side of the elongate substrate, to assist the diabetic in using the device. This is described in WO-A-00/28086 and also WO-A-98/55856 (the contents of both of which are incorporated herein by reference).
  • the output from the device may also be quantitated on small electronic instrument. Analysis of the output from the device may be via coulometry, amperometry, analysis or cyclic voltamogram or other electrochemical analytical methods known in the art.
  • the arrangement of the device may include provision for the sequential exposure of reagents to the sample, to allow the reactions to proceed in a sequential or time-delayed manner, to optimise the readout.
  • the device comprises two electrodes placed approximately 100 ⁇ m or more apart: a counter/reference electrode preferably made of Ag/AgCI and a second conductor of either Au, Pt, Pt/C or C.
  • a well is constructed by laminating a plastics layer around the cell or printing a layer around the cell, as shown in Figure 1.
  • a top cover may then be added to complete the cell.
  • the electrodes are approximately 100 ⁇ m wide and 1000 ⁇ m long.
  • the reagents are dosed onto the device. Strips made with a Au, Pt or Pt/C conductor may contain glucose oxidase. Hydrogen peroxide is detected electrochemically on the strip.
  • Strips made of a carbon conductor may contain a redox enzyme (glucose oxidase or glucose dehydrogenase) and a mediator for the enzyme (potassium ferricyanide or phenoxazines including Meldola's Blue).
  • a device comprises 3 conducting electrodes, namely a working electrode (carbon, Pt, Au or Pt/C), a reference electrode made of Ag/AgCI and a counter electrode (C, Pt, Au or Pt/C), as shown in Figure 2.
  • the reagents, i.e. enzyme, cofactor and mediator are deposited on the working electrode as a single or multiple layers. Alternatively, the reagents are dried over the whole of the cell.
  • a channel is created over the reagents by laminating a thin plastic layer or printing a layer of adhesive around the cell.
  • a clear cover with an air exit hole is placed over the device to complete the test strip.
  • the cell volume can be further reduced by sandwiching a mesh layer between the substrate containing the conducting electrodes and reagents and the cover.
  • a device comprises 3 conducting electrodes, namely a working electrode (carbon, Pt, Au or Pt/C), a reference/counter electrode made of Ag/AgCI and a strip/sample detection electrode (C, Pt, Au or Pt/C), as shown in Figure 3.
  • the reagents i.e. enzyme, cofactor and mediator, are deposited on the working electrode as a single or multiple layers. Alternatively, the reagents are dried over the whole of the cell. A channel is created over the reagents by laminating a thin plastic layer or printing a layer of adhesive around the cell. A clear cover with an air exit hole is placed over the device to complete the test strip.
  • the cell volume can be further reduced by sandwiching a mesh layer between the substrate containing the conducting electrodes and reagents and the cover.

Abstract

A device for the electrochemical detection of an analyte in a sample, comprises electrodes and appropriate reagents on a substrate defining a volume within which the sample can be detected, wherein said volume is 500 nL or less.

Description

ELECTROCHEMICAL STRIP TEST FOR SMALL VOLUMES Field of the Invention
This invention relates to an electrochemical test cell for the measurement of reactive species. Such a sensor can be used for the measurement of enzymes, enzyme substrates, cofactors or mediators. One particular application is the measurement of glucose for use in diabetes. Background of the Invention
Diabetes mellitus is the most common endocrine disease and affects between 3-8% of the population. Various studies, such as the UK Prospective Diabetes Study (UKPDS, Lancet, 12th September 1998) have pointed out the importance of maintaining a normal glucose level in the blood or plasma, in order to delay the onset of diabetic complications.
Many diabetics monitor their disease by a regime of tests at home. A simple means of monitoring their disease is therefore desirable. Electrochemical strip tests for glucose, electrolytes, lipids and enzymes have all been widely discussed in the patent and other literature. For example, US-A-4224125 discloses an enzyme electrode system in which an enzyme such as glucose oxidase is used to sense glucose. US-A-5820551 discloses such a strip electrode fabricated by screen printing. Summary of the Invention
The present invention is based on the realisation that, by using appropriate chemistry etc, an adequate signal can be achieved if a cavity is provided that is smaller than has previously been used, i.e. which holds 500 nL or less of blood. In particular, the invention allows the detection of glucose in whole blood, using a simple to use strip test, characterised in that the volume of sample required is very small, e.g. less than 200 nL and down to 50 nL.
In most respects, a device of the invention can be made from known materials. However, it has been found that its size can be reduced, because sufficient current density can be achieved that there is adequate sensitivity for the analyte, typically 90-100 nA mM glucose. The small volume of blood that can be utilised in the invention means that the sample can be taken from a part of the body having a low vasculature. Thus, for example, the sample can be taken from the arm. Brief Description of the Drawings Figures 1 to 3 of the drawings are each associated plan and side views of different illustrative embodiments of the invention. The drawings show a cell or sample area 1 , a cover 2, a spacer layer 3, a reference/counter electrode 4, a working electrode 5, a cell-defining layer 6, a counter electrode 7, a reference electrode 8, and a strip/sample electrode 9. Description of the Invention
Electrochemical strips for the detection of glucose in whole blood typically comprise an working, reference and counter electrode, or combination thereof, for the detection and quantitation of the activity of reagents or reaction products on the strip. Examples of these reagents may include enzymes, enzyme products or mediators accepting electrons from redox enzymes on the strip.
The electrodes are preferably printed or deposited onto an elongate substrate, configured for ease of use by the diabetic and for insertion into a reader. Such a substrate may also include other tests of use for the diabetic such as, but not limited to, glycosylated haemoglobin, glucose and potassium. Some of the reagents may be deposited directly onto the electrodes themselves or onto other structures incorporated in the device, including wetting meshes such as is known in the art. The mesh is preferably as described in WO-A- 00/28068. The use of a mesh in the cavity, e.g. having an open area of about 30%, can reduce the sample volume. Appropriate reagents may be placed on the electrochemical strip test by methods including, but not limited to, screen printing and dosing the enzymes or enzyme-mediator couples. Especially for the detection of glucose, such reagents may be, for example: glucose oxidase and ferrocene/ferricyanide glucose oxidase and oxygen to yield hydrogen peroxide glucose dehydrogenase and ferricyanide glucose dehydrogenase and NAD and Meldola's Blue The effect of interferents in the test may be reduced by including enzymes or chemicals to reduce ascorbate interference, e.g. ascorbate oxidase. Haematocrit interference may be reduced by including saponin, digitonin, or other lysing agents. PVP, PVA and other polymers may be included, to increase linearity.
Surfactants may be added to the device, to aid filling.
The assay may also incorporate features such as filling via the side of the elongate substrate, to assist the diabetic in using the device. This is described in WO-A-00/28086 and also WO-A-98/55856 (the contents of both of which are incorporated herein by reference).
The output from the device may also be quantitated on small electronic instrument. Analysis of the output from the device may be via coulometry, amperometry, analysis or cyclic voltamogram or other electrochemical analytical methods known in the art. The arrangement of the device may include provision for the sequential exposure of reagents to the sample, to allow the reactions to proceed in a sequential or time-delayed manner, to optimise the readout.
In a typical configuration that embodies the invention, the device comprises two electrodes placed approximately 100 μm or more apart: a counter/reference electrode preferably made of Ag/AgCI and a second conductor of either Au, Pt, Pt/C or C. A well is constructed by laminating a plastics layer around the cell or printing a layer around the cell, as shown in Figure 1. A top cover may then be added to complete the cell. The electrodes are approximately 100 μm wide and 1000 μm long. Before laminating the top cover onto the system, the reagents are dosed onto the device. Strips made with a Au, Pt or Pt/C conductor may contain glucose oxidase. Hydrogen peroxide is detected electrochemically on the strip. Strips made of a carbon conductor may contain a redox enzyme (glucose oxidase or glucose dehydrogenase) and a mediator for the enzyme (potassium ferricyanide or phenoxazines including Meldola's Blue). In a second embodiment of the invention, a device comprises 3 conducting electrodes, namely a working electrode (carbon, Pt, Au or Pt/C), a reference electrode made of Ag/AgCI and a counter electrode (C, Pt, Au or Pt/C), as shown in Figure 2. The reagents, i.e. enzyme, cofactor and mediator, are deposited on the working electrode as a single or multiple layers. Alternatively, the reagents are dried over the whole of the cell. A channel is created over the reagents by laminating a thin plastic layer or printing a layer of adhesive around the cell. A clear cover with an air exit hole is placed over the device to complete the test strip. The cell volume can be further reduced by sandwiching a mesh layer between the substrate containing the conducting electrodes and reagents and the cover.
In a third embodiment of the invention, a device comprises 3 conducting electrodes, namely a working electrode (carbon, Pt, Au or Pt/C), a reference/counter electrode made of Ag/AgCI and a strip/sample detection electrode (C, Pt, Au or Pt/C), as shown in Figure 3. The reagents, i.e. enzyme, cofactor and mediator, are deposited on the working electrode as a single or multiple layers. Alternatively, the reagents are dried over the whole of the cell. A channel is created over the reagents by laminating a thin plastic layer or printing a layer of adhesive around the cell. A clear cover with an air exit hole is placed over the device to complete the test strip. Again, the cell volume can be further reduced by sandwiching a mesh layer between the substrate containing the conducting electrodes and reagents and the cover.

Claims

1. A device for the electrochemical detection of an analyte in a sample, which comprises electrodes and appropriate reagents on a substrate defining a volume within which the sample can be detected, wherein said volume is 500 nL or less.
2. A device according to claim 1 , wherein the volume is 50 to 200 nL.
3. A device according to claim 1 or claim 2, wherein the analyte is glucose.
4. A device according to claim 3, wherein the reagents comprise glucose oxidase.
5. A device according to claim 3, wherein the reagents comprise glucose dehydrogenase.
6. A device according to claim 4, wherein the reagents comprise ferrocene/ferricyanide.
7. A device according to claim 5, wherein the reagents comprise ferricyanide.
8. A device according to claim 5, wherein the reagents comprise NAD and Meldola Blue.
9. A method for the electrochemical detection of an analyte in a sample, which comprises introducing the sample into the cavity of a device according to any preceding claim, and detecting the current.
10. A method according to claim 9, wherein the sample is whole blood.
PCT/GB2001/003615 2000-08-11 2001-08-13 Electrochemical strip test for small volumes WO2002014535A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001277625A AU2001277625A1 (en) 2000-08-11 2001-08-13 Electrochemical strip test for small volumes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0019694A GB0019694D0 (en) 2000-08-11 2000-08-11 Electrochemical strip test for small volumes
GB0019694.9 2000-08-11

Publications (2)

Publication Number Publication Date
WO2002014535A2 true WO2002014535A2 (en) 2002-02-21
WO2002014535A3 WO2002014535A3 (en) 2002-06-13

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GB (1) GB0019694D0 (en)
WO (1) WO2002014535A2 (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1447665A2 (en) * 2003-02-11 2004-08-18 Bayer Healthcare, LLC Method for reducing effect of hematocrit on measurement of an analyte in whole blood, and test kit and test article useful in the method
EP1486778A2 (en) * 2003-06-09 2004-12-15 I-Sens, Inc. Electrochemical biosensor
WO2005029070A1 (en) * 2003-09-20 2005-03-31 Cambridge Sensors Limited Biosensors having reduced haemocrit effect
WO2009094297A3 (en) * 2008-01-22 2009-09-17 Home Diagnostics, Inc. Improved reagent compositions for use in electrochemical detection
US7727467B2 (en) 2003-06-20 2010-06-01 Roche Diagnostics Operations, Inc. Reagent stripe for test strip
US7972861B2 (en) 2004-05-14 2011-07-05 Bayer Healthcare Llc Methods for performing hematocrit adjustment in glucose assays and devices for same
US8071030B2 (en) 2003-06-20 2011-12-06 Roche Diagnostics Operations, Inc. Test strip with flared sample receiving chamber
US8287703B2 (en) 1999-10-04 2012-10-16 Roche Diagnostics Operations, Inc. Biosensor and method of making
US8298828B2 (en) 2003-06-20 2012-10-30 Roche Diagnostics Operations, Inc. System and method for determining the concentration of an analyte in a sample fluid
US8663442B2 (en) 2003-06-20 2014-03-04 Roche Diagnostics Operations, Inc. System and method for analyte measurement using dose sufficiency electrodes
AU2012268873B2 (en) * 2008-01-22 2014-07-03 Trividia Health, Inc. Improved reagent compositions for use in electrochemical detection

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US7569126B2 (en) 2004-06-18 2009-08-04 Roche Diagnostics Operations, Inc. System and method for quality assurance of a biosensor test strip

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Cited By (30)

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US8551308B2 (en) 1999-10-04 2013-10-08 Roche Diagnostics Operations, Inc. Biosensor and method of making
US8287703B2 (en) 1999-10-04 2012-10-16 Roche Diagnostics Operations, Inc. Biosensor and method of making
CN1558235B (en) * 2003-02-11 2010-05-26 拜尔健康护理有限责任公司 Method for determining the existence and/or content of an analyte in a blood sample, and detecting element
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EP1447665A3 (en) * 2003-02-11 2005-12-28 Bayer HealthCare LLC Method for reducing effect of hematocrit on measurement of an analyte in whole blood, and test kit and test article useful in the method
US7323315B2 (en) 2003-02-11 2008-01-29 Bayer Healthcare Llc Method for reducing effect of hematocrit on measurement of an analyte in whole blood
EP1447665A2 (en) * 2003-02-11 2004-08-18 Bayer Healthcare, LLC Method for reducing effect of hematocrit on measurement of an analyte in whole blood, and test kit and test article useful in the method
EP1486778A2 (en) * 2003-06-09 2004-12-15 I-Sens, Inc. Electrochemical biosensor
EP1486778A3 (en) * 2003-06-09 2005-01-05 I-Sens, Inc. Electrochemical biosensor
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US8298828B2 (en) 2003-06-20 2012-10-30 Roche Diagnostics Operations, Inc. System and method for determining the concentration of an analyte in a sample fluid
US7749437B2 (en) 2003-06-20 2010-07-06 Roche Diagnostics Operations, Inc. Method and reagent for producing narrow, homogenous reagent stripes
US8663442B2 (en) 2003-06-20 2014-03-04 Roche Diagnostics Operations, Inc. System and method for analyte measurement using dose sufficiency electrodes
US7879618B2 (en) 2003-06-20 2011-02-01 Roche Diagnostics Operations, Inc. Method and reagent for producing narrow, homogenous reagent strips
US8586373B2 (en) 2003-06-20 2013-11-19 Roche Diagnostics Operations, Inc. System and method for determining the concentration of an analyte in a sample fluid
US7727467B2 (en) 2003-06-20 2010-06-01 Roche Diagnostics Operations, Inc. Reagent stripe for test strip
US8071030B2 (en) 2003-06-20 2011-12-06 Roche Diagnostics Operations, Inc. Test strip with flared sample receiving chamber
US8119414B2 (en) 2003-06-20 2012-02-21 Roche Diagnostics Operations, Inc. Test strip with slot vent opening
US8142721B2 (en) 2003-06-20 2012-03-27 Roche Diagnostics Operations, Inc. Test strip with slot vent opening
US8211379B2 (en) 2003-06-20 2012-07-03 Roche Diagnostics Operations, Inc. Test strip with slot vent opening
US8222044B2 (en) 2003-06-20 2012-07-17 Roche Diagnostics Operations, Inc. Test strip with flared sample receiving chamber
WO2005029070A1 (en) * 2003-09-20 2005-03-31 Cambridge Sensors Limited Biosensors having reduced haemocrit effect
US7972861B2 (en) 2004-05-14 2011-07-05 Bayer Healthcare Llc Methods for performing hematocrit adjustment in glucose assays and devices for same
WO2009094297A3 (en) * 2008-01-22 2009-09-17 Home Diagnostics, Inc. Improved reagent compositions for use in electrochemical detection
US8062490B2 (en) 2008-01-22 2011-11-22 Nipro Diagnostics, Inc. Reagent compositions for use in electrochemical detection
US7678250B2 (en) 2008-01-22 2010-03-16 Home Diagnostics, Inc. Reagent compositions for use in electrochemical detection
AU2009206598B2 (en) * 2008-01-22 2014-04-03 Trividia Health, Inc. Improved reagent compositions for use in electrochemical detection
AU2012268873B2 (en) * 2008-01-22 2014-07-03 Trividia Health, Inc. Improved reagent compositions for use in electrochemical detection

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Publication number Publication date
GB0019694D0 (en) 2000-09-27
WO2002014535A3 (en) 2002-06-13
AU2001277625A1 (en) 2002-02-25

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