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WO2014072820A2 - Dispositifs et procédés chrono-ampérométriques de quantification électrochimique de substances à analyser - Google Patents

Dispositifs et procédés chrono-ampérométriques de quantification électrochimique de substances à analyser Download PDF

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WO2014072820A2
WO2014072820A2 PCT/IB2013/002926 IB2013002926W WO2014072820A2 WO 2014072820 A2 WO2014072820 A2 WO 2014072820A2 IB 2013002926 W IB2013002926 W IB 2013002926W WO 2014072820 A2 WO2014072820 A2 WO 2014072820A2
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electrode
concentration
mbe
current
electrodes
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WO2014072820A3 (fr
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Thomas William Beck
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/1468—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/1468—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means
    • A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means using enzyme electrodes, e.g. with immobilised oxidase
    • 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
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/02—Details of sensors specially adapted for in-vivo measurements
    • A61B2562/0295—Strip shaped analyte sensors for apparatus classified in A61B5/145 or A61B5/157
    • 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
    • G01N27/416—Systems
    • G01N27/49—Systems involving the determination of the current at a single specific value, or small range of values, of applied voltage for producing selective measurement of one or more particular ionic species

Definitions

  • the invention disclosed herein generally relates to chronoamperometric methods and devices for measuring the concentration of an analyte.
  • the invention also relates to the use of electrodes having an edge effect to measure the concentration and the diffusion coefficient of an analyte simultaneously.
  • the invention further relates to the use of pulses and alternating pulse voltages to measure the concentration of an analyte.
  • Electrochemical systems of this sort are called amperometric systems. By applying the voltage between two electrodes they can oxidize (or reduce) an analyte of interest and use the resulting current to estimate its concentration.
  • Glucose cannot be directly oxidized electrochemically, so electrochemical blood glucose sensors use an enzyme to oxidize the glucose. In doing so the enzyme is itself reduced. This is then re-oxidized by a mediator, which is the oxidized species of a redox couple.
  • a mediator is ferricyanide.
  • ferricyanide In oxidizing the enzyme the e.g. ferricyanide is reduced to ferrocyanide. Sufficient ferricyanide is present that it is always in excess to the amount of ferrocyanide produced. The ferrocyanide is now oxidized at what is called the working electrode (W/E), which is at a positive voltage with respect to the other electrode, generating a current.
  • This other electrode is called the counter electrode (C/E) and it completes the circuit, typically by converting ferricyanide to ferrocyanide. This is called mediated electron transfer.
  • C/E counter electrode
  • the analyte of interest can sometimes be oxidized or reduced electrochemically directly, in which case it can be measured directly by direct electron transfer.
  • This system which uses a W/E and a C/E is called a two electrode system.
  • a three electrode system is used.
  • the potential of the W/E is set relative to a reference electrode (R E) and the circuit is completed via a C/E.
  • the R/E does not pass current.
  • a three electrode system of this sort can also be used in the following invention.
  • a two electrode system can be used where the C/E does not use the redox couple to provide the C/E reaction - for instance Ag/ AgCl - as a sensor for an electrochemically oxidizable/ reducible analyte.
  • Ferrocyanide generated at the C/E can interfere with the estimation of the ferrocyanide produced by the enzyme at the working electrode, so the counter electrode is typically kept at a sufficient distance from the working electrode to ensure that it does not arrive there during the test and interfere with the working electrode.
  • a strip typically contains two or more electrodes in contact with, or in the vicinity of, a mixture of an enzyme and an oxidized mediator (e.g. ferricyanide) and other substances, such as buffer.
  • an oxidized mediator e.g. ferricyanide
  • the electrodes and reagent mix are contained within a cavity and the blood wicks into this and dissolves the reagents.
  • the current is dependent on a variety of parameters other than the concentration of the analyte (C). These include time after the potential is applied (t), the area of the working electrode (A) and the diffusion coefficient (D), which is a measure of the mobility of the analyte in the solution.
  • t time after the potential is applied
  • A area of the working electrode
  • D diffusion coefficient
  • D can vary with factors such as temperature, viscosity of the solution, and haematocrit.
  • an average value of D was assumed and used to calculate C, which however can result in significant inaccuracy in the measurement of C. Thus, there exists a need in the art to accurately determine D.
  • D can be measured from the time dependent behavior of the current.
  • the Verio system marketed by LifeScan has closely opposed electrodes, set an exact distance apart. As t increases there is an augmentation in current due to the diffusion of ferrocyanide from the counter electrode to the working electrode, allowing an estimation of D.
  • adjacent electrodes can be used to interact in the same fashion, though practically speaking this is more difficult.
  • Chronoamperometry is the name of the electrochemical technique in which the potential of the working electrode is stepped and the resulting current from faradaic processes occurring at the electrode (caused by the potential step) is monitored as a function of time. Alternate to stepping the potential the electrodes can be switched in at the desired voltage from open circuit.
  • ferro/ferricyanide see below section Chemistry of Electrochemical Blood Glucose Sensors.
  • ferrocyanide is oxidized at the anode to ferricyanide, and ferricyanide is reduced at the cathode back to ferrocyanide.
  • This redox couple is often referred to as an example throughout the specification, but one skilled in the art will recognize that any suitable redox couple can be used.
  • a supporting electrolyte is added to the solution to minimize solution resistance. This ensures that the voltage applied between the electrodes is primarily applied at the electrode interfaces and not across the solution between the electrodes.
  • the continued ferrocyanide flux causes the length of the diffusion layer to increase (the diffusion layer is the layer in which the ferrocyanide concentration is depleted with respect to the bulk concentration), so the slope of the concentration profile at the electrode surface decreases (Figure IB) and so does the current.
  • n number of electrons involved in the redox process
  • A is the cross-sectional area of the electrode D is the diffusion coefficient.
  • Figure 2 A shows the variation of the diffusion layer as t increases for this shielded electrode (where ⁇ 3 lt2-ti are in the ratio of 100:25: 1).
  • Electrochemical blood glucose sensors are discussed as examples, but one skilled in the art will understand that any solution based analyte can be determined using the same methodology.
  • GDH Glucose dehydrogenase
  • the most commonly used acceptor is ferricyanide.
  • the enzyme reaction is a two electron reaction, which converts two ferricyanide ions to two ferrocyanide per molecule of D-glucose.
  • a complication is that in solution D-glucose converts to ⁇ : ⁇ -0- Glucopyranose in the ratio 36:64 and the GDH only acts on the ⁇ -D-Glucopyranose. Conversion of the a to ⁇ form in solution is relatively slow, so when the enzyme reaction has acted to completion it has produced 1.28 ferrocyanide ions for each molecule of glucose in the original solution. By measuring the concentration of this ferrocyanide the sensor estimates the original concentration of glucose.
  • the Cottrell current is proportional to the concentration of the analyte being oxidized, and various constants. All these constants can be measured, and hence the current at an electrode can be used to determine the concentration of an analyte.
  • the diffusion coefficient is sensitive to factors such as the temperature of the solution and its composition. An average value of D can be used, but this is only useful if the variation in D is small compared to the accuracy desired.
  • D varies significantly (approximately two times) for a haematocrit range (which is the volume percentage (%) of red blood cells in blood) of 20 to 70%, which can typically be encountered in samples of diabetic blood. Additionally D varies by approximately 3% per degree Celsius.
  • Ferrocyanide (produced by the enzyme reaction discussed above) is oxidized at the anode, and to complete the circuit ferricyanide is reduced to ferrocyanide at the cathode.
  • C total concentration of redox couple
  • Do and D R are the diffusion coefficients for the oxidized and reduced species respectively
  • T is the thickness of the cell (S.J.Konopka, Anal Chem 1970 42 (14) ppl 741 - 1746).
  • the ratio of l(t) to the Cottrell current i.e. the current if the electrodes were not interfering
  • the excess current over Cottrell due to interaction of the electrodes is 0.3% to 14%.
  • Oldham considers edge effects in planar unshielded electrodes in his paper Edge Effects in Semiinfinite Diffusion (J. Electroanal. Chem., 122 (1981) 1 -17). In that Oldham states essentially that: "Provided that the linear dimensions of the electrode are much greater than the diffusion layer thickness, the shape of the electrode is completely characterized by its area A and its perimeter length, P. In fact the current is the sum of an areal term, that depends on A but not on P, and a perimetric term, that depends on P but not on A.”
  • a method for determining a concentration of an analyte in a medium comprises contacting the medium with one or more unshielded working electrode (W/E) and a counter electrode (C/E); wherein the W/E and C/E are non-interfering with each other; applying a voltage between the W/E and C/E; measuring a non-steady state current between the W/E and the C/E under the applied voltage over time (t), wherein the current is indicative of oxidation of a first species or reduction of a second species in the medium; plotting the measured current over 1/t 0'5 to produce a fitted straight line with a slope and a intercept; determining values of the slope and the intercept; and solving the concentration and diffusion coefficient of the first or second species of the redox couple simultaneously based on the determined values of the slope and the intercept
  • a method for determining a profile of concentration change of an analyte in a medium comprises contacting the medium with at least two unshielded working electrodes (W/E) and a counter electrode (C/E), wherein the W/E and C/E are arranged to be non-interfering with each other; wherein the W/E comprise at least a band electrode (BE) and at least a microband electrode (MBE), wherein the BE is wider than the MBE; applying a voltage between the W/E and C/E; measuring a non-steady state current between each of the at least two W/E and the C/E under the applied voltage over time, wherein the current is indicative of oxidation of a first species or reduction of a second species in the medium; calculating a difference current over time by subtracting a fraction of the measured current between the BE and the C/E from the measured current between the MBE and the C/E; wherein the fraction is the ratio between an area of
  • a method for determining a profile of concentration change of an analyte in a medium comprises contacting the medium with a working electrode (W/E) and a counter electrode (C/E); wherein the W/E and C/E are non-interfering with each other; applying a train of alternating positive and negative voltage pulses between the W/E and the C/E; measuring a current between the W/E and the C/E at the end of each applied voltage pulse, wherein the current is indicative of oxidation or reduction of a first species in the medium; wherein a second species is present in excess to the first species in the medium; and wherein the first species and second species form a redox couple; calculating a difference current between the currents measured for each pair of successive positive and negative voltage pulses; estimating a concentration (C t ) of the analyte for each calculated difference current; and generating the profile of concentration change by tracking the estimated concentration (C t ) over time
  • a device for electrochemical quantification of an analyte in a sample medium comprises one or more unshielded working electrode (W/E), a counter electrode (C/E), an insulating support, and a sensing meter.
  • W/E unshielded working electrode
  • C/E counter electrode
  • insulating support an insulating support
  • sensing meter an insulating support
  • the insulating support defines a cavity cell configured to receive the sample medium; the W/E and C/E are partially embedded in the insulating support such that each electrode exposes a surface to the cavity cell; the W/E and C/E are arranged to be non-interfering with each other; the W/E and C/E are capable of reversibly engaging with the sensing meter; and the sensing meter is configured to determine a concentration and a diffusion coefficient of the analyte simultaneously by applying a voltage between the W/E and C/E and monitoring the resulting current.
  • the present invention offers in several embodiments the advantage of measuring C and D independently and measuring C and D several times in a 5 second test, allowing measurement of the final concentration C ⁇ .
  • the present invention also offers in several embodiments the advantage of using very small volume of cells and samples, down to ⁇ 100 nL and completing the test in very short time, for example, less than 5 seconds.
  • Other advantages of the present invention include, but are not limited to, the ability to measure background electrochemical interferents, the ability to generate more accurate measurements than prior-existing methods and devices, and the ability to fabricate the device at a lower cost
  • Figure 1 A depicts an exemplary voltage step applied between the working electrode and the counter electrode of the present invention.
  • Figure IB depicts exemplary concentration profiles following the application of a voltage step according to the present invention.
  • Figure 1 C depicts an exemplary current transient during the application of a voltage step according to the present invention.
  • Figure 2A depicts the change in diffusion layer (the Cottrell behavior) with time for a shielded electrode according to the present invention.
  • Figure 2B depicts the change in diffusion layer (Cottrell behavior plus the Edge Effects) with time for an unshielded electrode according to the present invention.
  • Figure 3A depicts a diagrammatic representation of an opposing electrode cell according to the present invention.
  • Figure 3B depicts concentration profiles with time for a thin layer cell with opposing electrodes according to the present invention.
  • Figure 3C depicts i (t) Faradaic for a thin layer cell (opposed electrodes) and a shielded planar electrode of the same dimensions according to the present invention.
  • Figure 3D depicts an exemplary interdigitated array according to the present invention.
  • Figure 4 A depicts the top view of a (Micro)Band Electrode according to the present invention. The counter electrode is not shown.
  • Figure 4B depicts the side view of a (Micro)Band Electrode according to the present invention.
  • Figure 5A depicts a i (t) vs t plot showing the Cottrell behavior of a shielded planar electrode versus the Oldham behavior of the same electrode but unshielded (with edge effects) according to the present invention.
  • Figure 5B depicts a i (t) vs 1/t 0'5 plot showing the Cottrell behavior of a shielded planar electrode versus the Oldham behavior of the same electrode but unshielded (with edge effects) according to the present invention.
  • Figure 6 depicts a i (t) vs 1/t 0'5 plot showing the Cottrell behavior of a shielded planar electrode with different areas according to the present invention.
  • Figure 7 depicts a i (t) vs 1/t 0'5 plot of band electrodes of different widths, same length, according to the present invention.
  • Figure 8 depicts diagrammatic representation of dual band electrode according to the present invention
  • Figure 8A depicts one possible variation of concentration with time according to the present invention.
  • Figure 8B depicts data from Figure 8A approximated as discrete changes according to the present invention
  • Figure 9 depicts multiple Microband Electrode without overlap of diffusion layers according to the present invention
  • Figure 10 depicts a i (t) vs 1/t 0'5 plot for Band Electrode, Microband Electrode and Multiple Microband Electrode according to the present invention
  • Figure 11 A depicts a possible low volume cell configuration according to the present invention.
  • Figure 11B depicts a possible low volume cell configuration according to the present invention.
  • Figure 11C depicts a complete strip from Figure 11A according to the present invention
  • Figure 12A depicts data plotted using t zero as the Time of Application of the voltage pulse gathered from a Band Electrode of a width of 185 ⁇ , according to the present invention
  • Figure 12B depicts data plotted using optimized t zero as 2.1 ms after the time of application of the voltage pulse gathered from a Band Electrode of a width of 185 ⁇ , according to the present invention. Data are plotted using the same data of Figure 12A
  • Figure 13A depicts data plotted using t zero as the time of application of the voltage pulse gathered from a Band Electrode of a width of 268 ⁇ according to the present invention.
  • Figure 14A depicts values of slope for successive pulses measured with different rest periods at O/C
  • Figure 14B depicts values of slope for successive pulses measured with different rest periods at 0V
  • Figure 15A depicts estimated concentration with time calculated using forward/ reverse pulses at +/- 0.3V. The pulses were applied as the solution entered the cavity.
  • Figure 15B depicts estimated concentration with time calculated using forward/ reverse pulses at +/- 0.3V. The pulses were applied 100 s after the solution entered the cavity.
  • C/E is an abbreviation of Counter Electrode.
  • W/E is an abbreviation of Working Electrode.
  • i (t) Faradaic represents the total current generated by the reduction or oxidation of some chemical substance at an electrode. Such a chemical substance is in solution.
  • the symbol '3 ⁇ 4 Edge or lEdge represents the current caused by diffusion from the region non-normal to the electrode surface i.e. from the edges.
  • the symbol '3 ⁇ 4 oidham represents i( t ) Cottrell+ lEdge (generally - but not exclusively - for a band electrode).
  • n represents the number of electrons involved in the redox process.
  • the symbol "A" represents the cross-sectional area of the electrode.
  • C* represents the concentration in the bulk solution.
  • C t represents concentration of C at time t.
  • Cd represents double layer capacitance, which term refers to the capacitance of the electrical double layer. "The whole array of charged species and orientated dipoles existing at the metal -solution interface is called the electrical double layer. At a given potential, the electrode-solution interface is characterized by a double layer capacitance, Cd, typically in the range of 10 to 40 ⁇ /cm 2 .” ⁇ Electrochemical Methods (Second Edition), Bard and Faulkner, page 12.)
  • D represents diffusion coefficient
  • D 0 represents diffusion coefficient for the oxidized species.
  • D R diffusion coefficient for the reduced species.
  • T represents the thickness of a cell cavity
  • i ss represents steady state cell current
  • the symbol '3 ⁇ 4" represents cell current at time t.
  • S represents the slope of a line on a graph (generally of i (t) vs 1/t 0'5 ).
  • I nt represents the intercept of a line on a graph (generally the y axis intercept of i (t) vs 1/t 0'5 ).
  • W represents the width of an electrode
  • L represents the length of an electrode
  • P represents the perimeter length of an electrode
  • haematocrit refers to the volume percentage (%) of red blood cells in blood.
  • chronoamperometry refers to an electrochemical technique in which the potential of the working electrode is stepped against a counter electrode (or switched in from open circuit) and the resulting current from faradaic processes occurring at the electrode is monitored as a function of time.
  • redox couple refers to a reduced species and its corresponding oxidized form.
  • BE is an abbreviation of Band Electrode, which refers to a strip shaped electrode with substantially straight and parallel edges and a length that is greater than the width.
  • BE width is understood to mean the average width over the length.
  • MBE Microband Electrode
  • VNBE is an abbreviation of Very Narrow Band Electrode.
  • MDA Microdisk Array
  • EAE Edge Effect Electrodes
  • IOE Interacting Opposed Electrodes
  • non-interacting electrodes refers to electrodes that are spaced sufficiently apart that there is insignificant diffusion of product from the C/E to the W/E within the timescale of a single determination of D and C. In other words, for non-interfering electrodes, there is insignificant overlap of the diffusion layers of any of the electrodes with each other.
  • the term "interference between electrodes” refers to the diffusion of the product of one electrode and the reactant of the other electrode between the two electrodes in such a way that their concentration profiles interact.
  • O/C is an abbreviation of open circuit.
  • t p represents the length of a voltage pulse.
  • the term "bulk concentration” refers to the concentration which is unaffected by the diffusion profiles of the C/E and the W/E.
  • the present invention provides chronoamperometric methods and devices that use a single band electrode (BE).
  • BE has an edge which is substantially a straight line. (Substantially as there are manufacturing irregularities at the level of a few microns - see below). This substantial straightness of the edge of a BE allows the diffusion to be solved in 2 dimensions as opposed to the disk and sphere shaped electrode as known in the art, which are solved in 3 dimensions.
  • the edge of a BE is substantially straight.
  • the realities of manufacturing mean that the edge is not entirely even.
  • the edge as used in the equation increases from the geometric length towards the real length.
  • Equally for short times the roughness of the electrode surface increases the area from the geometric towards the real area. This is handled by excluding data collected for the transient in the first 10 ms to 50 ms or so, or applying a correction factor.
  • Manufacturing methods that are expected to give a roughness of the edges of the BE are e.g. the use of flex substrates/ substrates with rough surfaces, printing methods lacking edge definition, "scalloping" of the edge due to the laser using pulses, metal layer being uneven e.g. made up of overlapping grains.
  • a band electrode where W is e.g. > lOx the irregularities of the edges and surface will allow a good range to collect data - approx lOOx in time (W > 50 ⁇ ).
  • Small microdisks (d ⁇ 50 ⁇ ) will have less time range to collect data in, especially given the limit on the approximation of t ⁇ 0.01d 2 /D.
  • edges of a BE can be formed by etching, cutting, lasers etc.
  • the ends can both be defined by an overlay, or one can be so defined and the other can be defined by a laser etc.
  • Conveniently in manufacturing the ends of the BE can be formed by an overlay with a hole punched in it. In this case the accuracy of length is determined by the accuracy of the punch, a very well controlled dimension.
  • US 7972487 B2 teaches the construction of a disposable sensor with band electrode in the cavity wall. There is no mathematical analysis of the transient and no teaching about using this to calculate C and D.
  • the width or diameter of the working electrode bands or disks should be of such a dimension as to allow for some degree of radial or spherical diffusion to the working electrode surfaces. Typically, this dimension should be in the range of 5 to 50 ⁇ , and most preferably 10 to 50 um.”
  • microelectrode as: electrodes can be considered a micro-electrode array, especially if the diffusion occurs predominantly (e.g., greater than 50%) according to a non-planar path, or if the size of the electrodes is less than 100 um, e.g., less than 50 ⁇ .
  • US8388821 teaches the use of microdisk electrodes to measure D by plotting i t / i ss vs. 1/t 0'5 then using that to calculate C from i ss . This requires the use of a plurality of microdisks, an overlay being used to define the whole perimeter of the each disk, using the long term approximation for microdisk electrodes and measuring i ss . Diameters of the microdisks are 5 to 50 ⁇ .
  • the present invention provides a method and a device for determining C and D of an analyte in a medium simultaneously.
  • the simultaneous estimation of C and D is carried out using a W/E with a significant edge effect.
  • W/E of carefully controlled dimensions are placed in sufficient distance from each other so as to be non-interfering.
  • electrodes of carefully controlled dimensions are spaced sufficiently apart to avoid interference (this is defined as non-interfering).
  • such electrodes can be a band electrode and a distal counter electrode. If the solution in contact with the electrode surface extends beyond the area normal to the electrode surface the diffusion region is said to have edges and the current i (t) can considered to be made up of two components: one due to planar diffusion i (t) cottreii and one due to edge diffusion i( t )Ed g e (see above Equation 5).
  • electrodes having an edge current in other configurations are used.
  • BE Band Electrode
  • MBE Microband Electrode
  • a method and a device for accurately determining both C and D of an analyte in a medium simultaneously are provided.
  • the method and device use one or more band electrodes as the W/E.
  • Band Electrode and Microband electrode are to a large extent used interchangeably as described in this application.
  • microelectrodes are differentiated from (macro)electrodes based on the edge effect representing a "significant" proportion of the overall current/ diffusion to the electrode, or on L DL being of “comparable” size to the minimum dimension of the electrode.
  • MBE and BE are used in this application when discussing two band electrodes of different sizes, in which case it is convenient to call the smaller of the two "micro.” In the case of confusion, both terms should be considered as being BE.
  • the extent of the edge effect is covered in the math, not in the name.
  • the Cottrell current is dependent on A and 1/t 0'5 whereas the Edge Effect current is dependent on L and is time independent. Also:
  • the Oldham current is proportional to concentration (for constant A, D and t):
  • Figure 7 is a plot of i( t ) oidham vs 1/t 0'5 .
  • the Cottreii part of the current is the same, but all the lines are shifted up the y axis by lEdge- That is:
  • Equation 14's accuracy has been determined to be:
  • U.S. patent US8388821 and European patent EP2080023 describe the use of microelectrode arrays where an overlay with openings is used to expose a plurality of microelectrodes to the solution.
  • the prior invention differs from the present invention in using a plurality of microelectrodes and in necessitating the use of an overlay to define the electrode areas, and also in "measuring" the final steady state current (which exists for microdisk electrode, but does not for a band electrode).
  • the mathematical approximation is less robust and the device is more complicated to make and is less robust in performance than the present invention.
  • inaccuracies in measuring W and L cause errors in calculating C and D.
  • Dimensions of L of 1 mm or greater can be manufactured to 1% reliability or better. But if the width of an electrode can be determined to +/- 5 ⁇ , this is 1% on a 500 ⁇ wide band (BE), and 10% on a 50 ⁇ wide band (MBE).
  • a 50 ⁇ track can be manufactured to a greater accuracy than +/- 10%> width.
  • the use of this value should be seen as illustrative of a general point - that is, that as the width of the BE decreases there will be some point at which its accuracy and reproducibility is harder to maintain.
  • the tracks are manufactured on a smooth/ rigid substrate, such as silicon, rather than the flexible plastic substrates normally used in the manufacture of disposable sensors, then a 10 ⁇ track can be manufactured to 1% accuracy in width.
  • the opposing interacting electrodes strip has 3 dimension estimates used in the calculation of C: the length of the cavity, the width of the cavity and the thickness of the cavity.
  • the BE also has 3 dimension estimates: the length of cavity and width of the BE twice (i.e. W 2 ).
  • a method and a device for accurately determining both C and D of an analyte in a medium simultaneously are provided.
  • the method and device use an electrode having a microdisk or alternatively other shape as the W/E.
  • i(short t) 4FCDr[0.7854 + - 5 r/(4(Dt) a5 )] Equation 29 where r is the radius of the disk electrode.
  • microelectrode array having a plurality of disk shaped microelectrodes has been considered as an electrochemical glucose sensor in patents EP2080023 & US8388821. The authors cite the use of the long term solution to microdisks with electrodes of 5 to 50 ⁇ diameter, equation 28.
  • microdisks require the use of an insulating layer around the whole perimeter of the electrodes. This must be continuous (i.e. have no pin holes) yet be thin enough that it does not interfere significantly with the diffusion condition that the electrode be flush with its surroundings. Whilst the condition of a shielded electrode transitioning to an unshielded electrode can be handled it represents a complication and requires knowledge of the insulating (shielding layer) thickness. Additionally r must be accurately known, to at least 1%. These requirements are not impossible, but are onerous for an electrode of radius ⁇ 50um.
  • a variation on microelectrode arrays of circular disks is an "anti- microelectrode array of circular holes.” That is, a planar electrode where circular areas are either covered with an insulator, or the metal (or other electrode material) is removed e.g. by a laser or by photo lith. This is covered by Equation 13 and can be analyzed in a similar fashion. Many of the construction problems are simplified i.e., there is no need for an insulator to define the electrodes which is one large electrode with holes. The holes need not be limited to circular shapes.
  • the present invention provides a method and a device that are capable of increasing the speed of the test without adversely affecting accuracy in measurement.
  • a method and a device that use two or more electrodes of different width are provided.
  • a narrow electrode allows a more rapid accurate determination of the value of the intercept. This value is independent of the width of the electrode, so the use of a narrower electrode, where W will be less accurately known, does not have any adverse effect. Such uncertainties however will affect the accuracy of the analysis of the slope, which is dependent on width. But the use of the wider electrode allows the area of the electrode to be accurately measured and therefore CD 0'5 , which is calculated from the slope and area, can also be determined accurately.
  • the use of two or more W/E of different widths allows accurate determination of C and D at shorter times than either electrode on its own.
  • the wider and narrower BE can be of the same length, in which case they will have the same intercept, or different length in which case they will have different intercepts.
  • a DBE consists of two band electrodes of different widths. In the various discussions (below) of the use of the DBE it has been assumed that they are of the same length, but the two band electrodes of different lengths can be used and the maths modified in a straightforward way to take account of this.
  • a consideration of Table 2 shows an advantage in using the combination of a BE and an MBE to determine C more accurately than by using either individually.
  • One configuration of a DBE is shown in Figure 8. In this case the BE, MBE and C/E are shown side by side, but they can also be in other configurations, such as on different sides of a cavity cell.
  • the BE and MBE in a DBE are usually of the same length but the lengths can be different.
  • the BE is used to determine the slope (CD 0'5 ) and the MBE is used to determine intercept (CD). It will be noted from this analysis that the width of the MBE does not need to be known to determine the intercept.
  • any electrode of accurately known area (and perimeter) can give the same data. Equally the method is not limited to using a single MBE to determine the CD behavior. Multiple MBEs or a backwards and forwards track (e.g. zigzag, or the U-shaped equivalent) can also be used. All that is required is to be able to accurately determine P, the perimeter length. The ease of determining the perimeter length of a band electrode is a major advantage. The perimeter length of a microdisk area can only be known, for instance, by knowing the diameters of the microdisks in a microdisk array.
  • the present invention provides a method for, and a device that is capable of, calibrating the width of the narrower MBE, thereby providing more accurate measurement of the concentration of an analyte in a media.
  • a DBE system is configured to compare the slopes of the i (t) vs. 1/t 0'5 curves generated using the BE and the MBE of the system, which comparison allows calibration of the MBE width value with respect to the BE width.
  • the BE can be used to calibrate the MBE area more accurately as:
  • W MBE , W BE , S MBE and S BE are the widths and slopes of the MBE and the BE.
  • this calibration allows the width of the MBE to be determined as accurately as that of the BE, in which case it can be used on its own in subsequent measurements to determine C and D, which can be carried out in a shorter time.
  • a similar analysis can be carried out based on the areas of the BE and MBE if they are of different length.
  • the present invention provides a method and a device for tracking the concentration change of an analyte in a media.
  • a fraction of the current due to the BE is subtracted from that due to the MBE.
  • this difference current can be used to track the rate of the concentration change and to determine the end point concentration which will result after a relatively long time compared to that of the overall test (5 seconds for a typical blood glucose test), which situation can arise for example when:
  • C is increasing with time due to diffusion from one compartment to another, such as in the case where glucose diffuses out of blood cells and becomes available to enzyme to turnover and generate ferrocyanide.
  • the difference current, i D iff is defined as:
  • i (t) DL and i (t) s are the double layer charging current and the oxidation of surface groupings/ absorbed species i.e. the non-faradaic currents.
  • Ratio For an invariant C, by varying Ratio numerically the value can be determined which gives the most constant value of ioi ff with time, and this gives the most accurate estimate of iEd g e-
  • This also calibrates the width of the MBE which is then known to the same accuracy as the width of the BE.
  • a similar analysis can be carried out based on the areas of the BE and MBE if they are of different length.
  • the widths of the two electrodes are accurately known before the measurements relating to glucose are taken. These can be determined from manufacturing to a high accuracy, or from measurement of the double layer capacitance, or from measurement of the resistance of the electrodes before solution is added. Alternately the widths may be calibrated electrochemically by a measurement immediately after the solution is applied when there is very little glucose related current (this effect is even clearer if the enzyme is dried on the opposite side of the cell to the W/E). In this case the currents measured at short t are background currents, such as are discussed in Equation 32, and additionally electrochemical active interferents, such as ascorbic acid
  • the present invention provides a method and a device for accurately measuring the concentration of an analyte in a medium while the concentration is changing during the course of measurement.
  • the change in concentration is approximated as a series of discrete concentration changes, and each concentration increment is considered separately.
  • the current for each increment is calculated and summed vs. time, and the sum represents the current vs. time that is expected for that rate of concentration/time profile. Accordingly, C and D are determined based on the expected shape of the profile, even though C is changing during the course of measurement.
  • Different i-t transients can be simulated mathematically or digitally for changes in C t during the time of the transient.
  • the shape of the transient as it deviates from the shapes expected for constant C can then be used to determine dC/dt.
  • This allows the estimation of the "constant C" transient and therefore an estimation of D and the values of C t throughout the measurement.
  • This requires a relatively simple spreadsheet model, or alternatively digital simulation can be performed. This will allow the generation of look up correction factors e.g. if the concentration is estimated to vary by x% during the measurement period apply a correction of y% to the concentration estimate.
  • Equation 36g C ⁇ f(t) Equation 36g
  • C ⁇ the final concentration as t tends to infinity
  • f(t) an analytical time dependent function which models the enzyme kinetics etc.
  • Equation 36e can be analytically solved.
  • digital simulation can be used. The time dependence can be determined from the time dependence of lEdge and comparison with the Cottrell behavior will allow D and C to be determined.
  • lEdge can be determined by using Equation 33. If the concentration is to be tracked in this fashion the ideal dimensions of the two BE will need to be determined. For instance, for a 5 second test, widths of 100 and 200 ⁇ could be used. In this case they can be manufactured with sufficient accuracy to allow the subtraction to leave only the edge current. Or the widths can be determined by resistance measurements. Or measuring the slopes at short times - e.g. 1 to 10 ms - when the edge current is a small percentage of the Cottrell current allows the relative slopes to be used to calibrate the widths. Or allowance for the increasing edge current allows the slopes over a larger time range to be used. [0221] Knowing that the time dependence of C t is the same for both Cottrell and Edge currents the value of ratio used in Equation 34 can be adjusted for optimal fit. Now knowing both C t and f(t), C ⁇ can be calculated.
  • the second working electrode can be one, such as a very narrow band electrode (VNBE) or a microdisk array (MDA), where the dimensions are sufficiently small that they predominantly give a pseudo steady state current during the period of measurement.
  • VNBE very narrow band electrode
  • MDA microdisk array
  • the second W/E will give a current that is largely proportional to concentration (that is, a situation where the current is constant if C does not change).
  • concentration that is, a situation where the current is constant if C does not change.
  • This information can then be used to convert the "distorted" transient of the first working electrode (e.g. a BE) to the simple case where D can be accurately calculated.
  • MMBE Widely Spaced Multi-Microband Electrode
  • a method and a device for accurately determining both C and D of an analyte in a medium simultaneously are provided.
  • the method and device use multiple microband electrodes (multiple M BE , MMBE) as the W/E.
  • the current configuration leaves space for a C/E on one side, or one on both sides of the MBE.
  • the C/E can be on the other side of the cavity opposing the MBEs.
  • a MBE WL Equation 37 [0228] If all the MBEs are connected as in the diagram, like fingers to the palm of a hand, then they will act as a band electrode of area:
  • Row 4 of Table 4 shows a comparison of the errors expected for an MMBE with the previous three cases. This is open to further improvement if the determination of area can be improved. It is only one electrode to be measured electrochemically (compared to two for the DBE), and it is spread uniformly across a larger area ensuring it sees the average of the chemistry that is taking place.
  • the device comprises one or more working electrode(s) (W/E) and one counter electrode (C/E), embedded within an insulating substrate.
  • the insulating substrate defines a cavity cell, in which the electrodes are exposed to an analyte.
  • the electrodes are connected to a sensing meter through a connector.
  • the cavity cell assumes an open configuration.
  • the insulating substrate defines the bottom and sides of the cavity cell.
  • a base layer of the insulating substrate defines a bottom surface of the cavity cell and a cavity-defining layer of the insulating substrate is arranged on top of the base layer.
  • the cavity-defining layer has a punch that defines the sides of the cavity cell.
  • the electrodes are mounted on the base layer and are exposed to the open space through an open face of the cavity.
  • the electrodes and/or cavity cell are exposed to a sample or sample fluid by filling the sample through the open face into the cavity cell.
  • the electrodes and/or cavity cell are exposed to a sample or sample fluid by placing the electrodes in direct contact with a sample surface through the open face of the cavity.
  • the cavity cell assumes a closed configuration.
  • the cavity cell is sealed within the body of the insulating substrate.
  • a base layer of the insulating substrate defines a bottom surface of the cavity cell
  • a top layer of the insulating substrate defines a top surface of the cavity cell
  • a cavity-defining layer placed in between the top and bottom layers has a punch that defines the sides of the cavity cell.
  • the electrodes are mounted on the base layer and are exposed to the space within the cavity.
  • the top or bottom layer of the cavity cell has a vent hole that allows filling of a sample or sample fluid into the cavity and becomes accessible to the electrodes.
  • the cell uses either one or two BE (or BE plus MBE) or a plurality of BE and one counter electrode.
  • the counter electrode (C/E) can be on the opposite side of the cavity as the working electrode (W/E) or on the same side as the working electrode (W/E). The same side offers a simplification in design and cost reduction.
  • the electrodes are spaced far enough apart (approx 0.1 to 0.2 mm) not to interfere with each other during the course of the test.
  • the BE measurement is preferably completed by the time L DL ⁇ T/3, so that interference from the opposing C/E (the arrival of ferrocyanide) does not affect the accuracy of the measurement.
  • t is the time for a liquid of dynamic viscosity ⁇ and surface tension ⁇ to penetrate a distance L into the capillary whose pore diameter is d.
  • a cell of a shorter path length fills in a similar time for much lower values of T.
  • Such small values of path length can be realized with a single BE, or another edge effect type electrode, as described in this invention. Based on Equation 47, values of T as little as 25 ⁇ can be used.
  • a further advantage of having a thinner cell is that the enzyme/ mediator can be put on the opposite side of the cavity to the W/E.
  • the W/E can now be used as soon as the liquid fills the cell (especially if it has a very small amount of ferricyanide dried on it) and the background interferences measured and subtracted, but due to the thinness of the cell the enzyme/ reduced mediator will rapidly diffuse to the W/E, allowing time related measurements of the ferrocyanide to track the enzyme reaction (plus diffusion of glucose from the blood cells) to end point.
  • an unshielded electrode is arranged coplanar with the surface of the insulating support where the electrode is mounted.
  • the unshielded electrode is not coplanar with the insulating support surface.
  • the electrode can assume any angle of between 0 to 180 degrees with the insulating support surface.
  • the angle of edge effect ( ⁇ ) can be 0 ⁇ ⁇ ⁇ 180°, and for these configurations, the second term of Equation 13 is modified as (cp/90)nFCDP/2.
  • ⁇ 90.
  • the electrode is placed at the bottom of a cone with 45 degree sides, in which case the perimetric term is expected to be half that in Equation 13, as the angle of edge effect ( ⁇ ) is defined as 45 degrees for the cone configuration.
  • the electrode is placed on the top of a Mesa like plateau (of height greater than the length of the diffusion layer), in which case the perimetric term is expected to be twice that of Equation 13, as the angle of edge effect ( ⁇ ) is defined as 180 degrees for the plateau configuration.
  • the present invention provides a method for, and a device that is capable of, calibrating the width of the electrode, thereby providing more accurate measurement of the concentration of an analyte in a media.
  • a DBE system is configured to compare the slopes of the i (t) vs. 1/t 0'5 curves generated using the BE and the MBE of the system, which comparison allows calibration of the MBE width value with respect to the BE width (see also the above section "Calibration of MBE by BE".
  • a DBE system is able to calibrate the width of the electrodes by measuring double layer capacitance once the cell is filled, as the double layer capacitance of an electrode in solution is proportional to its area. Hence, by measuring one electrode vs another, the relative area of the two electrodes can be determined. Electrodes can be measured in pairs to make up a circuit. Absolute area involves knowing the area of one electrode or knowing the capacitance per cm 2 for that type of electrode in that solution.
  • a DBE system is built with tracks connecting to both ends of the working electrode(s) and to measure the resistance. This can be done as an absolute measurement of width if the metal layer is of a reproducible thickness and hence conductivity. However, given a more typical +/- 20% process variation across a production roll of metal coated support, it is preferable to make a relative measurement of the resistances of the MBE and BE (or of the C/E and the MBE) on the actual electrode immediately it is fitted into the connector of the accompanying meter. The calibration is of the MBE with respect to the BE (or the C/E) which is wider and therefore of a more accurately known width. The measurement includes the resistance(s) of the tracks to the working electrodes, but this can be allowed for.
  • the resistance of the C/E can be used to calibrate the width of the BE or MBE.
  • a resistive track of accurately known width can be defined on the strip in a region remote from the cavity and not exposed to the sample to calibrate the width of the BE or MBE.
  • This approach can be used, for example, when one BE or MBE is used, and if the C/E is opposing the BE or MBE (i.e. the C/E is placed on the opposite side of the cavity where the BE or MBE is placed).
  • FIG. 11 A One such configuration (one BE only) is shown in Figure 11 A. Measuring R between tracks 1 and 4 gives the resistance of the BE plus the wider tracks to both ends, just outside the cavity. Measuring R between tracks 3 and 4 gives the resistance of the calibration strip, which is of a width of 1 mm along its length, allowing accurate definition of its width.
  • iR drop down the tracks is a problem if the voltage across the electrode interface drops below that required to maintain the reaction in the diffusion limited region.
  • a further advantage of measuring the resistance of the tracks is that the actual voltage across the working electrode/ counter electrode(s) interface(s) can be calculated. If this voltage needs to reach, for example, 200 mV for the reaction of the redox couple to become reversible (i.e. the concentration of e.g. ferrocyanide at the electrode surface to reach zero) then the time at which this is achieved can be set at t zero in the data analysis (see below). Knowing the ohms per square ( ⁇ / ⁇ ) from the measurement of the BE plus track resistance and the geometry of the track to the counter electrode allows this voltage to be calculated.
  • one of the connectors to one side of the BE and C/E is used to supply current (i.e. tracks 1 and 2) and the other to measure the voltage across the electrode interface(s) (i.e. tracks 3 and 4).
  • the voltage applied to the meter connectors is adjusted to be sufficient to ensure that the voltage across the electrode interface(s) was for example > +200 mV.
  • the concentration of C can be estimated through a Cottrell analysis, either by measuring D or using an estimated value of D. If the concentration profile is allowed to recover to its pre- pulse distribution then the application of a second pulse gives a second estimate of concentration. D can be re-measured for the second pulse, or again, an estimated value can be used.
  • the present invention provides a method and a device for accurately measuring a constant concentration of an analyte in a medium using voltage pulses.
  • the present invention provides a method and a device for tracking concentration change of an analyte in a medium using voltage pulses. Pulse Length Strategy Depending on Whether D and/or C Vary With Time
  • D is measured using a pulse of around 0.2 sec or greater.
  • the use of repeated pulses of this length allows repeated measurements of D.
  • D does not vary while C varies.
  • D is measured once and the varying C is tracked with a multiple pulse train of shorter pulses.
  • pulses of 5 ms or shorter are used to track C by the measurement of one value only of i (t) .
  • more pulses are applied in any given time, giving more estimates of C.
  • D is measured by applying a longer pulse to at the start or end of the pulse train, or at any time during it.
  • D and C of an analyte in a medium are both changing.
  • D is measured at the beginning and end of the pulse train with shorter pulses between.
  • the electrode is switched to a potential where the reaction in Equation 48 is reversed and O is converted to R. In this case the concentration profile of R recovers more quickly.
  • the solution was 10 mM ferrocyanide/ lOOmM ferricyanide in 0.1M phosphate buffer (pH 7.4).
  • the measurements were made with a band electrode of width 250 ⁇ so the recoveries are different to those observed for an electrode of sufficient width not to be seeing edge effects.
  • the concentration profile is best recovered by applying a voltage of 0V (this is discussed in more detail in below section - Consideration of R only Present in Bulk of solution, and R Plus O Present in the Bulk of the Solution).
  • This calibration factor is a dimensionless number whose value depends only on N, the ratio of the recovery period at O/C (or 0V) to the length of the pulse. That is, it is independent of t p , the length of the pulse, and D, the diffusion coefficient.
  • these ratios S n /Si can be applied to the case where the concentration of R increases between pulses, provided it is produced uniformly across the bulk of the solution, or a region encompassing the diffusion layer produced during the pulse. In this case, however, it is necessary to apply the calibration factor according to when the reactant was produced.
  • Equation 49 For the first pulse the situation is described by Equation 49.
  • Equation 53 0297 Equation 53 0297]
  • both members of a redox couple are present in the solution.
  • ferrocyanide in the presence of excess ferricyanide.
  • the reaction at the W/E is ferrocyanide being oxidized to ferricyanide
  • the opposite reaction takes place at the C/E.
  • the potential between the electrodes is set such that the reactions at both electrodes are diffusion limited. In this case the reaction is limited by the concentration of ferrocyanide, as it is at the lower concentration.
  • the ferrocyanide concentration at the W/E is zero and the concentration of the ferricyanide at the C/E is the bulk concentration of the ferricyanide minus the bulk concentration of the ferrocyanide.
  • the bulk concentration is the concentration unaffected by the diffusion profiles of the C/E and the W/E.
  • a glucose sensor initially contains only ferricyanide and the enzyme.
  • the enzyme turns over the glucose and in doing so mediates with the ferricyanide, generating ferrocyanide.
  • the ferrocyanide is then available to be oxidized at the W/E. Enough ferricyanide is provided that it is always in excess. Measuring the ferrocyanide concentration can be used to determine the glucose concentration.
  • i(t) 2nd pulse "0.293nF ⁇ ( ⁇ / ⁇ t p )° '5 Equation 59 [0306]
  • This derivation is given in Bard and Faulkner ⁇ Electrochemical Methods, Second Edition), at page 210. Analysis of this diffusion behavior has been extended to continuous multiple forward and reverse pulses of equal length by Kambara (Polarographic Diffusion Current Observed with Square Wave Voltage by Tomihito Kambara Bull. Chem. Soc. Jpn., 27, 523 - 534).
  • each positive pulse is immediately followed by a negative pulse, and each negative pulse is immediately followed by a positive pulse, all pulses being of the same length t p
  • Equation 7.3.29 in Bard and Faulkner ⁇ Electrochemical Methods, Second Edition), at page 295).
  • this gives an expression for the sample current at the end of the m 4 pulse is the current at time m*t p (i m*tp ) which is the Cottrell Equation modified by a summation term:
  • the values of K m are independent of the value of t p , so the limit is approached in shorter times as t p decreases in direct proportion to the shortening of t p .
  • the values of K m are also independent of D, the diffusion coefficient. As stated earlier, these successive values converge very slowly on the infinite sum limit, only approaching within 1% for the 7,000 pulse, or 3,500th pulse pair.
  • Such a system can be used as part of a three-electrode system, or a two-electrode system where the C/E does not use the redox couple to provide the C/E reaction (for instance Ag/ AgCl) as a sensor for an electrochemically oxidisable/ reducible analyte.
  • C/E does not use the redox couple to provide the C/E reaction (for instance Ag/ AgCl) as a sensor for an electrochemically oxidisable/ reducible analyte.
  • Fixed concentrations can be measured or changing concentrations can be tracked.
  • R is used for ferrocyanide
  • O is used for ferricyanide as examples for the convenience of discussion.
  • R is used for ferrocyanide
  • O is used for ferricyanide as examples for the convenience of discussion.
  • a skilled person in the art will understand that the same principle discussed herein can be applied for other redox couples.
  • the first term is within 5% of the infinite limit, and the second term is within 1 %.
  • a pulse train can be used to track changes in the concentration of ferrocyanide (R) with time without being concerned over changing K DIF(P) , the calibration factor, for each pulse.
  • Pulses as above can be used with shielded or unshielded electrodes (W/E and C/E) which are adjacent or opposite.
  • Equation 60, 61 , and 62 58, 59 and 60 will hold.
  • +/-V could be +V 1 /-V 2 where Vi need not be the same as V 2 and they can range between 0.2 volt and 0.5 volt. This voltage is sufficient to allow the oxidation/ reduction of R and O to be diffusion limited.
  • Equation 62 the value of K DIF ( P ) in Equation 62 would tend to 1.21.
  • the lengths of the first and second pulse in a pair of positive and negative pulses do not need to be equal, though the ratio between the pulse lengths of the first and second pulses should be constant among all the pairs.
  • the series would be different, but the result would be the same. That is, the difference between the currents measured at the end of the forward and reverse pulses settles to a limit, (i.e. the same Equation 62 but with a different K DIF(P) .
  • K DIF(P) 2.42.
  • the present invention provides a method and a device for tracking a concentration change of an analyte in a medium by applying alternating voltage pulses between the W/E and the C/E.
  • a forward/ reverse pulse is applied to a band electrode to estimate D.
  • such an analysis is performed with digital simulation.
  • such an analysis is performed through a mathematical analysis, giving an exact or approximate solution.
  • Such an analysis has been carried out for a microdisk electrode for a single forward/ reverse pulse pair (Haruko Ikeuchi , Mitsuhiro Kanakubo, Journal of Electro analytical Chemistry 493 (2000) 93-99, Determination of diffusion coefficients of the electrode reaction products by the double potential step chronoamperometry at small disk electrodes).
  • Timescales of less than 0.005 sec primarily probe the plasma layer, whereas longer pulses give time for the diffusion layer to extend into the region including blood cells. In this case measuring the current response with time gives information on the diffusion coefficient of both layers.
  • the present invention provides methods for manufacturing a device for electrochemical quantification of analytes in a media.
  • the system consists of one C/E and 1 or more W/E(s). These can be adjacent or opposite, in any variation.
  • the electrodes can be metal, carbon, or semiconductor.
  • Exemplary materials for the electrodes include but are not limited to platinum, gold, palladium, iridium or alloys of these, graphite, carbon pastes, and tin oxide.
  • the W/E and C/E can any one of a group of suitable materials, including the noble metals.
  • other materials such as gold can be used in areas in contact with the test solution only.
  • disposable strips have the same material to make contacts with the meter, to make the electrodes and to make up the conducting strip that connects the two.
  • the connector, the connector track and the band electrode can all be formed from one material, such as gold.
  • a layer of gold can be sputtered onto an insulating substrate and the electrode, connector and connector track can be formed by laser cutting.
  • the material in the connector and/ or track area can be made from a second, lower cost conducting material, such as aluminium, and the band electrode from gold.
  • a second, lower cost conducting material such as aluminium
  • the band electrode from gold.
  • the material is coated on a roll made at right angles to the connector track in Figure 4A a stripe of aluminium can be sputtered in the region of the track and connector and a stripe of gold in the region of the electrodes. Some degree of overlap of the gold and aluminium layers is necessary to provide electrical contact between the two layers.
  • the electrode material can be formed in various forms, including but not limited to as an evaporated/ sputtered/ electrolessly deposited/ electroplated film on a flexible e.g. polymeric substrate or a rigid substrate such as silicon.
  • an adhesive metal/ carbon film can be used, or a carbon layer can be printed (screen printing, offset printing etc), or a carbon/ metal film can be adhered to a substrate by an intermediate adhesive layer, or a carbon paste electrode.
  • the electrodes can be formed into adjacent band electrodes, such as shown in Figure 8, , through the use of lasers, photolithography, sand blasting, water knives, or kiss cutting etc.
  • adjacent band electrodes such as shown in Figure 8
  • Two thin lines of electrode free material can define the edges of the electrodes, but the region between these edges of the two separate electrodes can still contain metal/carbon etc., so long as it is electrically isolated from each electrode.
  • tops and bottoms of the band electrodes can be defined by the use of an adhesive overlay, or an overlay with a hole punched through it, or two separate strips of overlay.
  • This overlay can also form the body of a cavity, and a second overlay can be applied to the first, defining the top of a cavity.
  • opposing, though possibly laterally displaced, electrodes can be formed on the underside of the second overlay, facing inwards.
  • One end of the covered band is necessary to make contact with a remote meter, but the other end can also be cut by e.g. a laser within the cavity (making 3 sides of the band defined by e.g. laser cutting and one by overlay).
  • a ribbed substrate can be used to deposit the e.g. metal film, leading to separate electrodes.
  • a carbon paste material can be use to fill the troughs of such a ribbed substrate, defining a band electrode/ electrodes.
  • One such carbon paste is made from high purity graphite powder and a pasting liquid such as nujol.
  • a typical device will consist of a substrate with adjacent electrodes, separated distally (i.e. the electrodes are non-interfering), a second insulating layer which is adhered by adhesive (pressure sensitive, heat sensitive etc.). This, along with the lateral edges, defines the area of the electrodes, and exposes the connector to the meter ( Figure 4A). Typically the length of the cavity will be defined by a punch. Then a third layer is applied to the second to define the top of the cavity. This third layer can be omitted.
  • the cavity can contain a mixture of reagents, such as an enzyme, mediator, buffer, supporting electrolyte etc., either on the electrodes, between them or on the opposite side of the cavity.
  • This chemistry can be applied as a solution and dried in place. When test solution is applied to the lateral openings to the cavity it is drawn in and dissolves these reagents.
  • the substrates can be on for example either cards or rolls.
  • Electrodes As disclosed herein, standard lithographic techniques can be used to form the electrodes or they can be formed on a single metal coated strip. In the latter case the metal can be left in place between the BE and C/E provided it was not in electrical contact. Techniques such as scribes, kiss-cuts, lasers, sand blasting, water knives etc. can be used.
  • a ⁇ 10 ⁇ track can be manufactured to ⁇ 1% accuracy. If the part of the sensor corresponding to the electrodes/ cavity is manufactured with such a substrate then this is cut & placed on the part of the sensor corresponding to the tracks. Calibration tracks can be included on the smooth/ rigid substrate in the non cavity region.
  • a sensing meter that is configured to cooperatively function with the electrodes.
  • the sensing meter is capable of reversible engaging with the electrodes.
  • the sensing meter is configured to apply a voltage between the W/E and C/E and measuring a current under the applied voltage.
  • the sensing meter is configured to apply a train of voltage pulses.
  • the frequency, pulse duration, and direction of the applied pulse train can be manipulated.
  • the sensing meter is configured to apply a train of alternating positive and negative voltage pulses between the electrodes.
  • the sensing meter is configured to apply voltages and measure currents between multiple electrodes simultaneously. In some embodiments, the sensing meter is configured to perform various mathematical calculations, approximation and estimations based on the measured current. In some embodiments, the sensing meter comprises a processor or algorithm, and various functions of the sensing meter are programmed and controlled by the processor or algorithm. Those with ordinary skill in the art will recognize suitable algorithms that can be programed to realize the functions of the sensing meter.
  • a meter can recognize that a strip has been inserted and switches on, prompting the addition of a sample fluid such as blood.
  • the addition of sample fluid is detected by applying a voltage to the electrodes and measuring a resistance. Once the blood is detected the voltage may switch off until the test begins, or it may start the test.
  • the sensing meter is configured to measure the double layer capacitance to check that the electrodes are covered by the sample fluid and the filling is completed so as to avoid an underestimate of the analyte concentration.
  • the sensing meter is configured to work with disposable consumables such as strips that are produced in batches and have different calibration methods or parameters for each batch.
  • the sensing meter is configured to provide a calibration number and prompt an end user to check whether the calibration number agrees with a batch number of the disposable consumables.
  • the sensing meter is configured to calibrate the device using a control solution.
  • the sensing meter can be any known amperometric or chronoamperometric device known to a person skilled in the art. A skilled person in the art will recognize that other embodiments are also possible for the sensing meter.
  • EEE Edge Effect Electrodes
  • IOE Interacting Opposed Electrodes
  • the EEE offer many advantages. For example, because there is no need to control the thickness of the cavity cell of the device, the cavity cell can be made thicker to allow more rapid fill of the sample.
  • the manufacturing cost of the EEE device of the present invention is reduced compared to prior-existing devices.
  • the thickness of the cavity cell i.e., of the insulator overlay, does not need to be precisely controlled during manufacture; also, metal parts can be put on only one side of the cavity; further, more standard connectors to meter can be used.
  • D is determined more quickly for an EEE cell than for an IOE cell of the same dimensions.
  • the width of the BE (MBE) can be reduced allowing shorter time scales to be probed than for an EEE cell of equivalent thickness.
  • the EEE cells can use very narrow electrodes allowing the fabrication of narrow cells. Such narrow cells will fill quickly allowing thin cells of around 50 ⁇ to be used with acceptable filling times (Washburn Equation, Equation 47), resulting in a small cell volume.. This is useful where expensive reagents are used in the cell, or as part of a larger cell where blood is separated into plasma by lateral wicking through a membrane and only small volumes of plasma are available to the cell.
  • interdigitated electrodes need to be manufactured to a high degree of accuracy. Small dimensions are necessary to get a test done in a reasonable time. Absolute knowledge of both electrode width and separation are needed. Electrodes varying in width will have an effect on accuracy as the gap between the electrodes will consequently vary. This is not the case for a band electrode where the only the average W needs be know, or for an MBE in a DBE format.
  • microelectrode arrays of circular disks of small diameter can be used in a similar fashion. These are spaced sufficiently far apart to avoid overlap of diffusion layers during the course of the measurement. As stated in above section Microdisk Electrodes and Microelectrode Arrays (of Circular Disks), the analytical solution for microdisks is only accurate in the regions t ⁇ 0.04 r 2 /D and t > r 2 /D.
  • microelectrodes of 5 to 50 ⁇ are required.
  • U.S. Patent 8388821 The production of such microelectrodes presents significant difficulties.
  • a conducting film is defined into circular disks by the application of an overlayer which seals closely with the conducting material beneath but does not extend over the electrode surface. This gives problems, both in the reproducible definition of the size and shape, and in sealing the edges of the electrodes to prevent solution leaking below the overlay.
  • This insulation has a minimum thickness of microns, resulting in the disks being recessed from the solution, complicating the diffusion situation, starting as shielded electrodes until the diffusion layer reaches the thickness of the overlay, then acting as essentially unschielded microdisks..
  • each electrode and its perimeter are small, a plurality is required to raise the signal to a magnitude that facilitates the measurement. It is necessary to accurately know the average diameter of the electrodes to analyze the signal and the variation must be small.
  • the technique of using the perimeter to give the CD term and a larger electrode to give the CD 0'5 term requires knowing the cumulative perimeter lengths of the microelectrodes. For the BE this is easily determined as the length of the band, whereas for the array of microelectrode disks it requires knowing the diameters of all the electrodes, or the average diameter if this is a tightly controlled parameter.
  • an electrochemical cell made up of at least one (i.e. one or a plurality of) non-interacting working electrode in combination with a counter electrode, with electrode and cell dimensions/ geometry chosen to allow the use of edge current effects to determine D and C for an analyte in solution by the application of a voltage between the C/E ad the W/E and the measurement of the resulting current.
  • enzyme that is a glucose oxidizing or reducing enzyme.
  • first pulse to determine concentration of background interferents (such as ascorbic acid) before significant reduced, or oxidized, part of a redox couple is generated by enzyme related activity.
  • background interferents such as ascorbic acid
  • Electrodes Unmodified Verio strip, electrode width 3.5 mm, electrode length 1.2 mm, opposing W/E Au & C/E Pd, separation 95 ⁇ .
  • Figure 15A shows estimated concentration with time calculated from the pulse method described in the above paragraph. The data is from two separate strips superimposed. The concentration estimate of 11.0 mM agrees well with the expected value.
  • the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

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Abstract

L'invention concerne des procédés et dispositifs relatifs à l'utilisation d'une électrode en bande, afin de créer un courant à deux composantes dans un capteur électrochimique, à utiliser de préférence avec un glucomètre. Les dimensions de l'électrode en bande sont inférieures à celles de la solution déposée sur elle, un composant du courant résultera donc d'une diffusion planaire et l'autre d'une diffusion de bordure. En analysant les courants tracés, le coefficient de diffusion et la concentration de la substance cible à analyser peuvent être déterminés simultanément, ce qui offre une précision accrue dans le domaine de la mesure de concentration. Les procédés et dispositifs de l'invention concernent également l'utilisation de plusieurs électrodes en bande de différentes largeurs, afin d'augmenter la vitesse de mesure, sans affecter la précision. Les procédés et dispositifs de l'invention concernent en outre l'utilisation de tensions pulsées alternatives, afin de mesurer la concentration d'une substance à analyser, en tirant parti du temps de récupération plus court pour le profil de concentration. Les impulsions alternatives ont également tendance à perturber le profil de concentration dans une seule couche de diffusion fine, ce qui permet d'interroger différentes couches de l'échantillon.
PCT/IB2013/002926 2012-11-08 2013-11-08 Dispositifs et procédés chrono-ampérométriques de quantification électrochimique de substances à analyser Ceased WO2014072820A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115684296A (zh) * 2021-07-28 2023-02-03 五鼎生物技术股份有限公司 电化学检测系统、测量仪及电化学检测方法
CN116678928A (zh) * 2022-02-23 2023-09-01 浙江纳智汇生物科技有限公司 一种基于酶抑制法的电化学检测设备、检测系统及检测方法
WO2023225191A1 (fr) * 2022-05-20 2023-11-23 Lifescan Ip Holdings, Llc Biocapteur et procédé de fabrication associé

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US6110354A (en) * 1996-11-01 2000-08-29 University Of Washington Microband electrode arrays
US20070235346A1 (en) * 2006-04-11 2007-10-11 Popovich Natasha D System and methods for providing corrected analyte concentration measurements
ES2375288T3 (es) * 2006-10-05 2012-02-28 Lifescan Scotland Limited Procedimiento para determinar concentraciones de analito corregidas con hematocrito.
US8101062B2 (en) * 2007-07-26 2012-01-24 Nipro Diagnostics, Inc. System and methods for determination of analyte concentration using time resolved amperometry
GB0814238D0 (en) * 2008-08-04 2008-09-10 Oxford Biosensors Ltd Enhancement of electrochemical response

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115684296A (zh) * 2021-07-28 2023-02-03 五鼎生物技术股份有限公司 电化学检测系统、测量仪及电化学检测方法
CN116678928A (zh) * 2022-02-23 2023-09-01 浙江纳智汇生物科技有限公司 一种基于酶抑制法的电化学检测设备、检测系统及检测方法
WO2023225191A1 (fr) * 2022-05-20 2023-11-23 Lifescan Ip Holdings, Llc Biocapteur et procédé de fabrication associé

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