AU2016200514B2 - Electrochemical test strip - Google Patents

Abstract

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 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. 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 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.

Description

P/OO/OOl Regulation 3.2 2016200514 29 Jan 2016

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: -2- 2016200514 29 Jan 2016 ELECTROCHEMICAL TEST STRIP Statement of Related Applications

This application is a divisional of Australian application number 2013204842, which in turn is a divisional of Australian patent application number 2008279274, the entire disclosure of each 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 determination 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 Patent Nos. 5437999, 5650062, 5700695, 6484046, and 6942770, and Application Nos. US20040225230 and US20060169599.

In known electrochemical sensors for determination of glucose the reactions depicted in Fig. 1A and IB may be used. Glucose present in the sample is oxidized in reaction 11 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. -3 - 2016200514 29 Jan 2016

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 arrangement (solid line). The mediator is freely diffusible between the two electrodes in both cases. The x-axis (time) starts when the measurement potential is supplied. An initial current spike 21 is observed in both traces that results from the charging of the double layer on, and consuming 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 flux 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 diffusing 15 to the working electrode. This is balanced by a flux 16 of oxidized mediator going the other way

Determination of the analyte concentration in solution can be made at various points along the current traces. When the electrodes are in a closely spaced facing arrangement 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 analyte concentration and the estimate of the current can be improved by averaging data in this region over a time period. When the electrodes are side-by-side the analyte concentration can be determined from the data at the peak value 21 or in the decrease 22, 24.

To use data from 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. -4 - 2016200514 29 Jan 2016

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 arrives rapidly and stabilizes rapidly at the working electrode 13 during the course of data collection. The transition between the working 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. - 5 - 2016200514 29 Jan 2016

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 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 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 2016200514 28 Feb 2017 -6- (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 and wherein the overall transfer of electrons from the counter electrode to the working electrode is accomplished by diffusion of reduced mediator from the counter electrode to the conductive region of the second substrate and then diffusion of reduced mediator from the conductive region of the second substrate to the working electrode.

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. 2016200514 28 Feb 2017 -6a-

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. -7- 2016200514 29 Jan 2016

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.

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. 2016200514 29 Jan 2016 - 8 -

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 predefined 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.

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. -9- 2016200514 29 Jan 2016

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, 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, the time necessary for diffusion to start affecting 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 (bending) 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-- 10- 2016200514 29 Jan 2016 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 time. In addition, the accuracy of the 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 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. IB. 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 -11 - 2016200514 29 Jan 2016 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 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 - 12- 2016200514 29 Jan 2016 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 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. 2016200514 29 Jan 2016 - 13 -

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 (2)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. 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 and counter electrodes to a contact element in a connector portion of the test strip, wherein portions of the second substrate and the spacer layer are removed to expose the contact elements in the connection portion of the test strip and wherein the overall transfer of electrons from the counter electrode to the working electrode is accomplished by diffusion of reduced mediator from the counter electrode to the conductive region of the second substrate and then diffusion of reduced mediator from the conductive region of the second substrate to the working electrode.
2. 2. An electrochemical test strip according to claim 1, 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.