AU2013204842B2 - Electrochemical test strip - Google Patents

Abstract

An electrochemical test strip is formed from a first insulating substrate layer, a second substrate layer, and an intervening insulating spacer layer. An opening in the insulating spacer layer defines a test cell which is in contact with the inner surface of the first 5 substrate on one side and the inner surface of the second substrate on the other side. The size of the test cell is determined by the area of substrate exposed and the thickness of the spacer layer. Working and counter electrodes appropriate for the analyte to be detected are disposed on the first insulating substrate in a location within the test cell. The working and counter electrodes are associated with conductive leads that allow 10 connection of the electrodes to a meter for determination of analyte. The second substrate is conductive at least in a region facing the working and counter electrodes. No functional connection of this conductive surface of the second substrate to the meter is required. When a potential difference is applied between the working and counter electrodes, because of the presence of the conductive surface on the second 15 substrate, the relevant diffusion length is not dependent on the distance between working and counter electrodes, but is instead dependent on the distance between the first and second substrates (i.e., on the thickness of the spacer layer). This means that shorter measurement times can be achieved without having to reduce the spacing of the working and counter electrodes. WO 2009/015077 PCT/US2008/070630 44--- 46 -44 4) 43 Fig 5B

Description

P/00/001 Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention title: ELECTROCHEMICAL TEST STRIP The following statement is a full description of this invention, including the best method of performing it known to us: - la ELECTROCHEMICAL TEST STRIP Statement of Related Applications This application is a divisional of Australian patent application no. 2008279274, the entire disclosure of which is incorporated herein by reference. Background of the Invention This application relates to a design for small volume electrochemical test strips suitable for amperometric detennination of analytes in a liquid test sample. Disposable, single-use electrochemical test strips are commonly employed in the determination of analytes., particularly by diabetes in the determination of blood glucose levels. Advancements in design of these strips have frequently focused on the ability to use smaller samples, since smaller blood samples can be obtained with less pain. Examples of such test strips can be seen, for example for US Patents Nos. 5437999, 5650062, 5700695, 6484046, and 6942770 and Applications Nos. US20040225230, and U S20060169599AL In known electrochemical sensors for deteml ination of glucose the reactions depicted in Fig. 1A and 1B may be used. Glucose present in the sample is oxidized in reaction II by an enzyme, such as glucose oxidase to form gluconic acid (or gluconolactone) and reduced enzyme. The reduced enzyme is regenerated to its active oxidized form by interaction 12 with oxidized mediator, for example ferricyanide. When there is an appropriate potential difference between the working and counter electrodes, the resulting reduced mediator is converted 13 to oxidized mediator at the working electrode, with concurrent oxidation of reduced mediator at the counter electrode 14. In addition the reduced mediator may diffuse 15 from the counter electrode and when it reaches the working electrode it can be converted 17 to oxidized mediator and diffuse 16 to the counter electrode complete the cycle. Fig. 2 shows two exemplary current traces in sample cells according to the prior art, in which the working and counter electrodes are disposed in a closely spaced facing (sandwich) arrangement (dotted line) and a more openly spaced side-by-side WO 2009/015077 PCT/US20081070630 2 arrangement (solid line). The mediator IS freely diffusib e between the two electrodes in both cases. The x-axis (time) starts when the measurement potential is supplied. An initial current spike 2 1 is observed in both traces that results from the charging of the double layer on. and consurming the mediator 13 close to, the portion of the electrode surface covered at the time of the measurement potential is first applied. Thereafter, there is a decline in current 22 because of the smaller f lux of mediator arriving at the working electrode, resulting from the depletion of mediator in the vicinity of the electrode. In the solid trace this persists to longer times and lower currents 23 because of continued depletion of the mediator. In the dotted line a limiting current 24 is reached, which is caused by a stable flux of reduced mediator being generated at the nearby counter electrode 14 and diffLising 15 to the working electrode. This is balanced by a flux 16 of oxidized mediator going the other way Determination of the analytc concentration in solution can be made at various points along the current traces. When the electrodes are in a closely spaced facing arrangeinent this includes at the peak value 21, the plateau level 24, or during the decrease 22 in between; the plateau current 24 has a simple linear relationship with aialyte concentration and the estimate of the current can be improved by averaging data in this rgio Over a time period. When the electrodes are side-by-side the analyze concentration can be determined from the data at the peak value 21 or in the decrease 22, 24. To use data front the decrease 22, 24 it is possible to recalculate the current data as the inverse square of current. The results of such a calculation on the data from both traces of figure 2 are shown in Fig. 3, with the minima 31 corresponding to the peaks 21, the initial straight slopes 32 corresponding to the curved decline in current 22, and the continuation of the curving decline 23 being a continuation 33 of the initial straight slope 32 for the side-by-side geometry. The plateau for the sandwich geometry 24 also manifests as a plateau 34. The results with the sandwich geometry of Figs. 2 and 3 require a sample cell with electrodes that are sufficiently close that flux of reduced mediator 15 being generated 14 at the counter electrode arrves rapidly and stabilizes rapidly at the working electrode 13 during the course of data collection. The transition between the working -3 electrode being unaffected by the counter electrode and being in a steady state with the flux from the counter electrode produces the curve between the straight parts 32 and 34 in Fig. 3. To minimize the time required for the test, it is desirable to decrease the time required for diffusion to occur, and for a steady state current to be established. Commonly assigned US Patent Publication No. 2005/0258036, which is incorporated herein by reference discloses an approach to this problem, adapted for use in the context of facing electrodes. The electrodes are in close proximity, which allows the system to reach the stable plateau quickly. This favours small sample chambers and hence small sample sizes. However, this approach is not well-suited to electrodes disposed on the same substrate, i.e., to side-by-side electrodes. The results with the side-by-side geometry of Figs. 2 and 3 require a sample cell that is sufficiently large that flux of reduced mediator being generated at the counter electrode 14 does not arrive at the working electrode during the course of measurement. This favours large sample chambers and hence large sample sizes. Decreased sample sizes can be achieved simply by placing the electrodes closer together, but placing the electrodes closer together means flux 15 from the counter electrode arrives at the working electrode and generates an additional signal 17, causing the region 33 to bend, reducing the accuracy of the estimate of concentration from the slope. However, the side-by side geometry means that a steady state will not be set up as rapidly as in the sandwich geometry and so the bending will last a long time before reaching a stable plateau where more reliable data will be available to estimate the analyte concentration. The present invention provides a simple approach to decreasing the time required to arrive at a steady state current for an electrochemical test strip with side-by-side electrodes and increasing the accuracy of the estimated concentration of the analyte that can be achieved without increasing the complexity of the manufacturing process. Summary of the Invention The present invention provides an electrochemical test strip comprising a first insulating substrate layer, a second substrate layer, and an intervening insulating spacer layer. An opening in the insulating spacer layer defines a test cell which is in contact with the inner surface of the -4 first substrate on one side and the inner surface of the second substrate on the other side. The size of the test cell is determined by the area of substrate exposed and the thickness of the spacer layer. Working and counter electrodes appropriate for the analyte to be detected are disposed on the first insulating substrate in a location within the test cell. The working and counter electrodes are associated with conductive leads that allow connection of the electrodes to a meter for determination of analyte. At least the inner surface of the second substrate is conductive, at least in a region facing the working and counter electrodes. No functional connection of this conductive surface of the second substrate to the meter is required. In use, a potential difference is applied between the working and counter electrodes. Because of the presence of the conductive surface on the second substrate, the relevant diffusion length is not dependent on the distance between working and counter electrodes, but is instead dependent on the distance between the first and second substrates (i.e., on the thickness of the spacer layer). This means that shorter measurement times can be achieved without having to reduce the spacing of the working and counter electrodes. In a preferred embodiment, the present invention provides a combination of a test meter and an electrochemical test strip for detection of an analyte in a sample, (i) said electrochemical test strip comprising: a first insulating substrate; a second substrate; an intervening insulating spacer layer; wherein (a) an opening in the insulating spacer layer defines a test cell which is in contact with the inner surface of the first substrate on one side of the test cell and the inner surface of the second substrate on the second side of the test cell; and (b) the volume of the test cell is defined by the area of the first and second substrates exposed through the opening and the thickness of the spacer layer; working and counter electrodes appropriate for detection of the analyte disposed on the first insulating substrate in a location within the test cell, -5 first and second conductive leads connected to the working and counter electrodes, respectively, and extending from the working and counter electrodes to a contact element in a connector portion of the test strip; and a conductive element that is on or is a part of the second substrate disposed facing the working and counter electrode cross the test cell, and (ii) a test meter comprises a electronic circuitry for determination of analyte in a sample applied to the test strip when connected to the meter, wherein the meter applies a potential difference between the working and counter electrodes, and analyzes signal from the working and counter electrode to determine analyte in the sample, but does not utilize a signal measured at the conductive element to determine analyte in the sample. In another preferred embodiment, the present invention provides an electrochemical test strip for detection of an analyte in a sample, said electrochemical test strip comprising: an insulating first substrate; a conductive second substrate; an intervening insulating spacer layer; wherein (a) an opening in the insulating spacer layer defines a test cell which is in contact with the inner surface of the first substrate on one side of the test cell and the inner surface of the second substrate on the second side of the test cell; and (b) the volume of the test cell is defined by the area of the first and second substrates exposed through the opening and the thickness of the spacer layer; working and counter electrodes appropriate for detection of the analyte disposed on the first insulating substrate in a location within the test cell, and first and second conductive leads connected to the working and counter electrodes respectively and extending from the working an counter electrodes to a contact element in a connector portion of the test strip, wherein portions of the second substrate and the space layer are removed to expose the contact elements in the connection portion of the test strip. In this embodiment, it is preferred that no signal is measured at the conductive second substrate in the detection of analyte in a sample applied to the strip.

-6 Brief Description of the Drawings Figs. 1 A and B show reactions from a glucose detector. Fig. 2 shows a schematic of two exemplary current traces as a function of time in accordance with the prior art. Fig. 3 shows a schematic of two exemplary current traces presented as the inverse square of current as a function of time in accordance with the prior art. Figs. 4A and 4B show a cross section of a test strip in accordance with the prior art, and the relevant diffusion paths in such as test strip. Figs. 5A and 5B show a cross section of a test strip in accordance with the invention, and the relevant diffusion paths in such as test strip. Fig 6 shows the reactions relevant to determination of analyte concentration in a test strip in accordance with the invention. Fig. 7 shows a cross section through the test cell of an embodiment of a test strip according to the invention. Fig. 8 shows a top view of a test strip in accordance with the invention. Detailed Description of the Invention This application relates to electrochemical test strips. In the detailed description that follows, the invention will be discussed primarily in the context determination of blood glucose levels. This use of one primary detection system, however, should not be taken as limiting the scope of the invention, as the invention can be used in the detection of any analyte that can be detected using an electrochemical test strip. Definitions In the specification and claims of this application, the following definitions are relevant. Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

- 6a The term "analyte" as used in the specification and claims of this application means a component of a sample to be measured. The analyte may be one that is directly oxidized or reduced in the electrochemical test strip, or one that is oxidized or reduced through the use of an enzyme and/or a redox mediator. Non-limiting examples of specific analytes include glucose, hemoglobin, cholesterol and vitamin C. The term "redox mediator" as used in the specification and claims of this application means a chemical species, other than the analyte, that is oxidized and/or reduced in the course of a multi step process transferring electrons to or from the analyte to an electrode of the electrochemical cell. Non-limiting examples of mediators include ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, ferrocene derivatives, osmium mediators, for example as described in US Patents Nos. 5,589,326; 5,710,011, 5,846,702 and 6,262,264, which are incorporated herein by reference, ruthenium mediators, such as ruthenium amines, and ruthenium complexes as described in US Patents Nos. 5,410,059. The term "determination of an analyte" refers to qualitative, semi-quantitative and quantitative processes for evaluating a sample. In a qualitative evaluation, a result indicates whether or not analyte was detected in the sample. In a semi-quantitative evaluation, the result indicates whether or not analyte is present above some pre-defined threshold. In a quantitative evaluation, the result is a numerical indication of the amount of analyte present. The term "electrochemical test strip" refers to a strip having at least two electrodes, and any necessary reagents for determination of an analyte in a sample placed between the electrodes. In preferred embodiments, the electrochemical test strip is disposable after a single use, and has connectors for attachment to a separate and reusable meter that contains the electronics for applying potential, analyzing signals and displaying a result. The term "side-by-side electrodes" refers to a pair of electrodes disposed on a common substrate surface. The electrodes may be parallel strips, concentric or nested rings, nested spirals or any other suitable spatial arrangement.

- 6b The term "sandwich geometry electrodes" refers to a pair of electrodes disposed in a closely spaced facing arrangement with space for the sample in between. As herein, except where the context requires otherwise, the term 'comprises' and its variants are not intended to exclude the presence of other integers, components or steps. In this specification, references to prior art are not intended to acknowledge or suggest that such prior art is part of the common general knowledge in Australia or that a person skilled in the relevant art could be reasonably expected to have ascertained, understood and regarded it as relevant. Prior Art Strips Fig. 4A shows a cross section through the test cell of a test strip according to the prior art. The strip has a first insulating substrate 41 on which are disposed working electrode 42 and counter electrode 43. Insulating spacer layer 44 separates the first insulating substrate 41 from the second insulating substrate 45. An opening in the spacer layer 44 defines two dimensions of the test cell 46, while the thickness of the spacer layer 44 defines the third. In the amperometric determination of an analyte a potential difference is applied between electrodes 42 and 43. For example, in the determination of glucose as described above, the working electrode 42 is suitably fixed at a potential of +300 mV relative to the counter electrode. The situation when flux from the counter electrode 15 arrives at the working electrode and is oxidized 17 has been discussed in terms of the increase in the electrochemical current, where the region 33 is caused to bend, WO 2009/015077 PCTUS2008/070630 7 reducing the accuracy of the estimate of concentration from the slope. Diffusion of mediator 15, 16 occurs between the electrodes along the lines shown in Fig. 4B. Thus, tie time necessary for diffusion to start affetin the slope of the region 32, 33, is dependent upon the spacing between electrodes 42 and 43. However, the side-by side geometry means that a steady state will not be set up as rapidly as in the sandwich. geometry because there is a large portion of the volume 47 where mediator will have to diffuse a long distance before reaching equilibrium with the two electrodes. Only once this steady state is reached will the data reach a simple plateau like 24. 34 where more reliable data will be available to estimate the analyte concentration. The large portion of the volume 47 that is remote from the main flux between the electrodes will affect the testing time, sample volume and accuracy of the system. To avoid the inaccuracy of deviations bendingn) in the region 33 the sample must be sufficiently large that all data is collected before any flux from the counter electrode affects data collection at the working electrode. For smaller sample sizes the reduced time before this occurs limits the amount of linear data 32 that can be collected and hence the accuracy of the concentration estimate that can be made from it. Steady state data will only be available after the entire region 47 has been brought into steady state and the side-by-side geometry is not efficient at doing this. The bending will therefore last a long time before reaching a stable plateau, giving an extended test timei In addition, the accuracy of tie concentration estimate will be sensitive to the distance between the electrodes and for the side-by-side geometry this will be defined by the repeatability of separation of the adjacent edges of the two electrodes in the disposable test strip. This is likely to introduce significant errors. Test strips of the Invention Fig. 5A shows a cross section through the test cell of an embodiment of a test strip according to the invention. The strip has a first insulating substrate 41 on which are disposed working electrode 42 and counter electrode 43. Insulating spacer layer 44 separates the first insulating substrate 41 from the second insulating substrate 45. However, insulating substrate 45 has a conductive coating 50 on its inner surface. An opening in the spacer layer 44 defines two dimensions of the test cell 46, while the -8 thickness of the spacer layer 44 defines the third. The conductive coating 50 is exposed in the test cell 46 and faces the working and counter electrodes 42, 43. In the amperometric determination of an analyte using the test strip depicted in Fig. 5A, a potential difference is applied between electrodes 42 and 43. For example, in the determination of glucose as described above, the working electrode 42 is suitably fixed at a potential of +300 mV relative to the counter electrode. Diffusion of mediator 15, 16 of course occurs between the electrodes as shown in Fig. 1B. However, because of the presence of the conductive coating 50, this diffusion is not a limiting factor in the time required to established a steady state current. Rather, the relevant limiting diffusion is that extending from the electrodes 42, 43 to the facing surface of the conductive coating 50 as shown by the lines in Fig. 5B. Thus, the time necessary for diffusion is dependent upon the thickness of the spacer layer 44. This change in the relevant portions of the diffusion occurs because the conductive coating 50 short circuits the test cell. The relevant reactions are shown in Fig 6. where reduced mediator is oxidized 601 at the working electrode 602 and the electron transfer out of solution is balanced at the working electrode 603 by reduction of the oxidized mediator 604. Rather than diffusing directly between the electrodes as in 15, 16 much of the mediator flux is transformed at the conductive layer 605: the flux of reduced mediator from the counter electrode 606 can give up electrons 607 into conductive layer 605 and diffuse back to the counter electrode 608 in the oxidized form without ever reaching the working electrode. The electrons transferred 607 into the conductive layer 605 must be balanced by electrons transferred 609 out of the conductive layer onto oxidized mediator where they are in abundance. An abundant supply of oxidized mediator is found in the flux of oxidized mediator 610 produced by the working electrode reaction 601. The overall transfer of reduced mediator from the counter electrode 603 to the working electrode 602 is completed by diffusion of reduced mediator 611 from the conductive layer 605 to the working electrode 602. The reactions 607, 609 shown in Fig 6 involve simultaneous electron transfer into and out of the conductive layer 605. This is possible, in fact it is a requirement, because the conductive layer 605 acts as an electrode and the chemical potential of a solution in contact with an electrode will always try to reach equilibrium with the electrode -9 potential. Since the layer 605 is conductive, it can only be at a single uniform potential throughout, so it is the chemical potentials of the various parts of the solution in contact with the conductive later 605 that are brought into equilibrium through electron transfer. This electron transfer is effectively instantaneous in the conductive layer 605 so it provides a very rapid path for electron transfer from regions of the test cell that would otherwise take much time to reach a steady state. In addition, the proximity of the conductive layer to the electrodes and their parallel orientation render much of the diffusion effectively one-dimensional. The simple addition of a conductive layer over side-by-side electrodes therefore rapidly reduces the time it takes to generate a steady-state flux between the electrodes. This allows much smaller sample sizes, since flux between the electrodes is no longer undesired. It allows an improved accuracy because the flux is now arriving principally at the electrode surfaces rather than along their adjacent edges and so is dependent on the far more controllable electrode area than the electrode separation. It also produces a near one-dimensional diffusion at a rapidly reached steady state and so the ability to probe the system by electrochemical techniques and apply useful corrections such as those described in US Patent Publication No. US20050109637A1, and US patent No. 6284125 are at their most effective. Fig. 7 shows a cross section through the test cell of an embodiment of a test strip according to the invention. In Fig. 7, the second insulating layer 35 and the conductive coating 40 are replaced with a conductive layer 70. Fig. 8 shows a top view of a test strip of the type shown in Fig. 8. Test cell 36 is opened to the exterior to permit introduction of sample via channel 81 formed in the spacer layer. A vent 82 is formed through the conductive layer 70 (or alternatively through the insulating substrate 31 to facilitate flow into the test cell. Working and counter electrodes 32, 33 are connected to conductive leads 84, 85, respectively. At the end opposite the channel 81, a portion of the conductive layer 70 and the spacer layer are cut away to expose a part of the insulating substrate 31 and of leads 84 and 85 to allow connection of these leads with a meter. In the embodiment shown in Fig. 8, part 83, 83' of the conductive layer 70 is left in place at the edges of the test strip. This not only provides better dimensional stability but electrical contact with this - 10 layer can be used to detect insertion of a test strip into a test meter, for example as described in US Patents Nos. 4,999,582, 5,282,950 and 6,618,819, which are incorporated herein by reference. The electrical contact could be between two points on either one of the parts 83, and 83' or it could be between one point on part 83 and another part on part 83'. The embodiment disclosed in Fig. 8 can also facilitate detection of sample introduction into the cell. For example, by monitoring for a change in resistance between the conductive layer 70 and either of the working or counter electrodes 32, 33, the introduction of sample into the test cell can be monitored, and used as a signal to activate application of a measurement potential. The use of sample detection in this way is known in the art, for example from US patents Nos 5,108,564 and 5,266,179, which are incorporated herein by reference. While the embodiment of Fig. 8 is convenient for use in insertion and sample detection, embodiments with an insulating outer surface can also be used providing that a portion of the insulation is removed or a lead is formed to allow contact with the conductive surface. It should be understood, however, that no electrical signal needs to be applied to or measured from this conductive coating or layer in order to achieve the benefits of reduced testing time.

Claims (12)

1. A combination of a test meter and an electrochemical test strip for detection of an analyte in a sample, (i) said electrochemical test strip comprising: a first insulating substrate; a second substrate; an intervening insulating spacer layer; wherein (a) an opening in the insulating spacer layer defines a test cell which is in contact with the inner surface of the first substrate on one side of the test cell and the inner surface of the second substrate on the second side of the test cell; and (b) the volume of the test cell is defined by the area of the first and second substrates exposed through the opening and the thickness of the spacer layer; working and counter electrodes appropriate for detection of the analyte disposed on the first insulating substrate in a location within the test cell, first and second conductive leads connected to the working and counter electrodes, respectively, and extending from the working and counter electrodes to a contact element in a connector portion of the test strip; and a conductive element that is on or is a part of the second substrate disposed facing the working and counter electrode cross the test cell, and (ii) a test meter comprises a electronic circuitry for determination of analyte in a sample applied to the test strip when connected to the meter, wherein the meter applies a potential difference between the working and counter electrodes, and analyzes signal from the working and counter electrode to determine analyte in the sample, but does not utilize a signal measured at the conductive element to determine analyte in the sample.

2. A combination according to claim 1, wherein the test strip further comprises an enzyme and a redox mediator disposed within the test cell.

3. A combination according to claim 2, wherein the analyte is glucose and the enzyme is - 12 glucose oxidase or glucose reductase.

4. A combination according to any one of claims 1 to 3, wherein the conductive element cover the entire exposed area of the second substrate.

5. A combination according to any one of claims I to 4, wherein the entirety of the second substrate is a conductive layer.

6. A combination according to any one of claims 1 to 4 wherein the second substrate comprises a second insulating substrate and a conductive layer.

7. A combination according to any one of claims 1 to 6, wherein the meter measures a change in conductance to between the conductive element and the working electrode or the counter electrode to assess sample addition to the test cell.

8. A combination according to any one of claims 1 to 7, wherein the meter measures the electrical connection between two points on the second substrate in electrical contact with the conductive element to assess insertion of the test strip into the meter.

9. An electrochemical test strip for detection of an analyte in a sample, said electrochemical test strip comprising: an insulating first substrate; a conductive second substrate; an intervening insulating spacer layer; wherein (a) an opening in the insulating spacer layer defines a test cell which is in contact with the inner surface of the first substrate on one side of the test cell and the inner surface of the second substrate on the second side of the test cell; and (b) the volume of the test cell is defined by the area of the first and second substrates exposed through the opening and the thickness of the spacer layer; working and counter electrodes appropriate for detection of the analyte disposed on the first insulating substrate in a location within the test cell, and - 13 first and second conductive leads connected to the working and counter electrodes respectively and extending from the working an counter electrodes to a contact element in a connector portion of the test strip, wherein portions of the second substrate and the space layer are removed to expose the contact elements in the connection portion of the test strip, and wherein no signal is measured at the conductive second substrate in the detection of analyte in a sample applied to the strip.

10. An electrochemical test strip claim 9, wherein lateral edges of the second substrate and the spacer layer are left in place along the length of the test strip when the portions of the second substrate and the space layer are removed.

11. A combination according to claim 1 substantially as hereinbefore described with reference to any one of the drawings or examples.

12. An electrochemical test strip according to claim 9, substantially as hereinbefore described with reference to any one of the drawings or examples.