CA2694085A1 - Electrochemical test strip - Google Patents

Abstract

An electrochemical test strip is formed from a first insulating substrate layer, a second substrate layer, and an inter-vening 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 dertermination 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 sub-strates (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

ELECTROCHEMICAL TEST STRIP
Statemezit of Related Applications This application elairras priority fr~~n US Provisional Application No.
60/951,264, filed July 23, 2007, which application is ineor~3orated herein by reference in its entiretv in all jurisdictions permitving such incorporation.

Backgrauiid of the h-ivei-ition This ap-plieation relates to a d.esign for small voltime electrochemical test strips fiuital~le for ariperQ~rietric deterininatian of analytes in a liquid test sample.
Disposable, single-use eleetroc;hernieal test strips are commonly employed in the deterniination of analytes, particularly by diabetes in the determination of blood ~lu.ec~se le~rels. Advancements in design. of these strips have irequently focused on the ability to use smaller samples, since smaller blood sa:rnples can be obtaii-ied with less pain. Examples of such test stri-ps can be seen, for example for US Patents Nos.
5437999, 5650062, 55,7100695, 6484046, and 6942770 and ApplieatiQtis Nos.
US20040225230, and L; S200601695~9A1.

In lnow:i electroehemieal sensors for deternlination of aluease the reactions depicted in Fig. 1.4. and lB may be used. Glucose present in the sample is oxidized i-n reaction 11 bv an enzyme, such as glucose oxidase to fonrf, ucOr,ie acid ior gluconolactone) and reduced enzy-rne. The reduced enzyme is regenerated to its active oxidized I-orm by interaction 12 with oxidized mediator, l'ar example ferricyanide. When there is an appropriate potential difference between the working and counter electrodes, the resulting redtieed mediator is converted 13 to oxidized mediator at the dvorking electrode, with concurrent oxidation of reduced niediator at the counter electrode 14.
ln addition the reduced mediator may dififuse 155 from the c0witer electrode and whezi it reaches the working electrode it can bc, converted 17 to oxidized mediator and difffite 16 to the counter electrode complete the cycle.

Fig. 2 shows two exem~lary current traces in. sariple cells according to the prior art, in which the working.and counter electrodes are disposed in. a closely spaced facing (sand,wich) arrangement (dotted line) a.nd a more operrly spaced side-by-side arraiigement (solid line). The z:ied7.ator is f-eeiy diffusible between the two electrodes in both cases. The x-axis (time) starts wlZeii the measurement potential is supplied. t^U-1 initial current spike 21 is observed in both traces that results from the charging of the double laver on, and consuming the mediator 130 close to, the partiaix 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 flt:x of mediator a-rriving at the workir-g eiectrade, resuitincy from the depletion of mediator in the vie.inity of the electrode. In the solid trace this persists to longer times and lower currents 23 beeaLise of continued depIetior~~ of the mediator. ih the dotted (ine a limiting current 24 is reached, wh:ch is caused by a stable flux ufreduced mediator being C'enerated at the nearby counter electrode 14 and diffiisinb 15 to the working electrode. This is balanced by a flux 16 of oxidized mediator going the other way Determination of the analvte concentration in solution can be made at various -joints along the current traces. NV anen the electrodes are in a closely spaced facing arrangement this includes at the peak value 2:, the plateau level 24, or during the decrease 22 in bet-ween; the plateau current 24 has a simple linear relationship witii analyte concentration and the estimate of the current ear~ be irraproved by aver aping data:n this region over a time period. When the e` eetrodes are side-bv-s zde the analyte concentration can be deten-iiined from the data at t:ae peak value '?' 1 or in the decrease 22, 24.

To use data ftom the decrease 22, '24 it is possible to recadeulate the einrent data as the inverse square of current. The results of such a caleuiation on the data from boti-i traces of figure 2 are shown in. Fig. 3, with the :minima 31 corresponding to the Peaks 21, the initial straight slo-pes ' :) 2? corresponding to the curved decline in current 22, and the continuation of the curving decline 23 bei.~iz a continuation 33 of the initial straight slope 32 for the side-by-side geometry. The plateau for the sandwich geometry 24 also mai-lifests as a plate-au 34.

The results with the sandwich izeometry of Figs. 21 and 3 require a sample cell with electrodes that are sufficiently close that flux of reduced mediator 15 beinc, generated 1.4 at the counter electrode arrives rapidly and stabilizes rapidiy at the working Z--eleetrode I3 during the cntirse of data collection. The transition between the working eiectrode being una.j'fe;eted. h;~ the counter electrode ar~~. being in a steady state with the r'l~x f-om the counter e3eet ode produces the eurvebetween 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. ~;~~~r~.Ã~r~ly assigned I_;~ Patent Publication No. 2005/0258036, which is incorporated herein by reaere.nee discloses an approach to this problem, adapted for use in the context of fiacirg eieetrodes. The elee~.~rodes are in close proximiev, which allows the system to reach the stable plateau quickly. This fa-,%ours small sample chambers and hence small sample sizes.

However, this approach is not well-suited to eleetrodes, disposed on the same substrate, i.e., to side-'by-side eleedrodes. I'he results with the side-by-side geometry of Figs. 2 and 3 require a sar:-~ple cell that is sufficiently large that flu..~ of reduced mediator being generated at the eoupter electrode 14 does not arrive at the working electrode during lhe course of measurerneiit. This favours large sariiple chambers and hence large sample sizes. Decreased sample sizes can be achieved simply by placing the electrodes closer togeti-ier, but placing the electrodes closer together means flux 15 from the counter electrode arrives at the workang electrode ar-,d generates an additional signal 17, causing the region 33 to bend, reducing the aeeura.c~ of the estimate of concentration ftom the slope. However, the side-by side geometry nieans that a steadv state will not be set up as rapidly as iii the sandwich geometry and so the bending will last a 1Qp-g tinie before reaeMng a stable plateaa where more reliable data will be available to estimate the analyte concentration.

The present iriventi r, 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 oi 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, aa-id an intervening insulating spaeer laver. An opening in the insulating spacer laver defines a tes'L cell which is in eontact with the imier surface of'the first sub-strate on one side and the i~-ner 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 thick-ness of the spacer lavez=.

Working a-nd eountor elect-rodes appropriate for the analyte to be detected are disposed on tl-ie first insulating substrate in a location witl~an the test cell. The working and counter electrodes are associated with eonduefive leads that allow connection of the electrodes to a meter for determination of analyte. At least tl-ie inner surface o: the second substrate is eonduetavee at least in a region facing the working and coLinter electrodes. No fu.zictional eoTuieCtion. of t-his condiietive surface of the second substrate to the meter is required.

In use, a potential difference is kpplied betveen the working and couiiter electrodes.
Because of the presence of the conductive surface on the secoiitl substrate, the relevant diffusion length is not dependent on the distance between working and counter electrodes, but is i:istead dependent on the distance between the first and second substrates (i.e., on tl:e tftickness of the spacer layer). This means that shorter rreasuremeTit times can be achieved without having to reduce the spacing of the working and counter electrodes.

Brief Description of the Dravyings Figs. 1A a.nd. B show reactions from a glucose detector.
Fig. 2 shows a schematic of two exemplary current traces as a R;.,~-~etion of time in accordance with the pnor art.
Fig. 3 shows a schematic of two exemplary current traces presented as the inverse sqtiare of current as a function of time in accordance with the prior art.
Figs, 4A and 4.13 show a cross section of a test strip in ae,cordan= with the prior art, and ffie relevant diffusion paths in such as test strip.
Figs. 5A ancl. 5B show a cross section of a test stdip in accordance with the invention, and the relevant diffusion paths in such as test strip.
Fig 6 shows the reactions releva-nt to detern)iriation of aralyte eoneentratiori in a test strp in accordance with tlie invention.
Fig. 7 shows a cross section througb- the test cell of an embodiment of a test strip aceordin_g to the iiiveiition.
Fig. 8 shows a top view of a test strip in accordance with the iiivei-ition.

Detailed Description oi'the Invention This application relates to electrochemical test strips. ID. the detailed deseni.ption that follows, the invention will be discussed primarily in the context detenninatior~ of blood Oueose levels. This use of oTie pri:mary detection system, however, should Tiot be taken as limiting the scope of the invention, as the invention can be used inthe detection of anv analyte that can be detected using an eleet-roehemical. test stnp.
~efinitions In the speci:ication and claims of this application, the following clefinitions are relevant. Numerical values in tl-ie speeif eatior. and claims o-f this application should be u-nderstood to include numerical values 'w`nieh are the same when reduced to the same number oi' significant figures anc' numerical values which differ from tl-ie stated value by less than the experimental error of conventional measurement technique of pe deseribed in the present application to deterrnine the value.
the ty The terrn "analyte" as used in the specification and claims of this application means a cor~~onert of a sample to be measured. The analyte may be one tli.at is direetlv oxidized or reduced in the electrochemical test strip, or one tl-iat is oxidized or reduced througli the use of an enzyme and/or a redox mediator. Non-limiting exam-ples of specific analytes include glucose, hema~labin, cholesterol and vitai~.in. 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,n.ediators include ferricvanicle, p-benzoquinon.e; phenazine metLosulfate, methylere blue, ferrocene derivatives, osmium mediators, for example as described in US Patents Nos.

5,589,3216; 5,710,011, 5,846,102 and 6,262,264, whieb. are incorporated herein by reference, rutlaeziiiLm mediators, such as rutheniw. arlfi-ies, and rutl-ieniuM. complexes as deseribed in TiS Patents Nos. 5,410,059.

The term, "determination of an analyte" refers to qualitative, semi-quantitative and quantitative processes for evaluatin~ a sample. Ln a qualitative e~%altiation, a result indicates whether or not analyte was detected in the sample. In a semi-quantitative evaltiation, the result indicates whether or not analyte is prese.-It above some pre-defined threshold. In a quantitative evaluation, the result is a numerieal indication of the arr:ount of analyte -oresent.

The terni "electrochemical test strip" refers to a strip having at least two electrodes, ai-id any necessary reagents ior determination of an analyte in a sam-ole placed betvveen the electrodes. In preferred erabodiments, the eleetroehem ieal test strip is disposable after a single use, and has connectors for attaelunent to a separate and reusable meter that contains the electronics for apply~~.g potenti.al, anal.yR,ri~.g sign.als 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 st.rps, coneentric or nested.
rirr.gs, zj:ested spirals or ar-y other suitable, spatial arrangement.

'I`he term "sandwich geometry electrodes" refers to a pair of eleetrodes disposed in a closely spaced fac:ing arrangement with space for the sample in be' 'weene P:-ior A-rt: StriD.s Fig. 4A shows a cross section t~irougli the test cell of a test strip according to the prior art. The st-H.p has a first ansu'ating substrate 41 on which are disposed working electrode 42 and counter eleetrode 43. Insulating spacer layer 44 separates the first insulati.n; 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 tlaird.

In the am perometric deteririiriation of an analyte a potential difference is applied between electrodes 41- aiid 43. For exanple, in the determination of glucose as deseribed above, the working electrode 42 is suitably fixed at a potential of 4-300 mV
relative to the counter eleetrode. The sittialion when flux from the counter electrode 15 arrives at the worlcing electrode and is oxidized 17 has been. discussed in terms of the increase in the electrochemical current, where the region 33 is ca-LFsed to bend, red-acing the accuracy of the estimate of concentration from the slope.
Diffusion of mediator 15, 16 occurs between the electrodes along the 'lizies sha~wn. in Fig. 4B. ~~lus, the ti-tne necess-ary for diffusion to star', affecting the slope of the region 32, 33, is dependent upon the spacing beriveen electrodes 42 a.n.d 43, However, the side-by side geometi-y means that a steady state will not be set up as rapidly as in the sandwich geome~.~-y because there is a large portion of the volume 4%where mediator will have to diff-Lise a long distance before reachiTig 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 ~~(ux. between the electrodes will aifeet the testing time, sample volume and accuracy of the systemõ To avoid the inaccuracy of deviations (bending) in the re9%oTi 33 the sample must be suaieiently large the-, all data is collected before a:ly fliLx ftom the counter electrode affieets data collection at the working electrode. For smaller sample sizes the reduced time before tMs occurs limits the arnouizt of linear data 32 that can be collected and he-nce 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 ef ieieni at doing, this. The bending will therefore last a long time before reaching a stable plateau, giving an extended test tirn.e. 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 'Lhe repeatabilityr of separation of the adjacent edges of the two electrodes inihe disposable test strip. This is likely to introduce significant errors.

Test strips of the I-avention 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 ii-isulatin.g substrate 41 on which are disposed. working electrode 42 and counter electrode 43. Insulating spacer layer 44 separates the first insulating substrate 41. f~orn the second insulating substrate 45.
However, insulating substrate 45 has a conductive coating 50 on its iruier surface. An opening in the spaeea layer 44 defines two dimensions of the test cell 46, while the thie~ess of the spacer layer 44 defines the third. The eoi.aduetive eoating5Ã3 is exposed in the test cell 46 and faces the wQrkir-g and counter electrodes 42, 43.

Ln the am-neroinetxie determination of an analyte using the test strip depicted. in Fig.
5A, a potential differenee is app;ied between electrodes 4L and 43. For example, in the deterrniration of glucose as described above, the working electrode 42 is suitably tixed. at a potential of +300 mV relative to the counter electrode. Diffusion of mediator 15, 16 of course occurs between the electrodes as s~own in Fig. I B.
However, because of the presence of the conductive coating 50, this diffusion is not a limiting factor in the tim<, required to established a steady state euiTent.
Rather; the relevar~t limiting diffusion is that extending -from the eleetTodes 42, 43 to the facing surface fthe conductive coating 50 as shown by the lines in Fig. 5B. Thus, tl-ie time necessary for di:fuslon is dependent upori the thickness of the spacer layer 44.

This change in the relevant portioiis of the diffusion occurs because the conductive coating 40 short circuits the test cell. The reievant reactions are s.~owm in Fig 6. where reduced mediator is ox:dized 601 at the working electrode 602 and the electron transfer out of solution is balaizeed at the woricin~ electrode 603 by reduction of the oxidized rriediator 604. Rather than diffusing directly 6et,,veen the electrodes as irs 15, 16 much of the mediator flux is transformed at the conductive layer 605: the flux o-f reduced mediator from the counter electrode 606 can give up. electrons 607, into conductive laver 605 and diffibse 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 eonduetiv e? a:yer onto oxidized mediator where they are in abundance. An abu-ndant supply of oxidized mediator is ~ound in the flux of oxidized med; atQr 610 produced by tlae working electrode reaction 601. The overall transfer of reduced mediator from the coiinter electrode 603 to the workin9 electrode 602 is completed by dif'~.:si+~r of reduced mediator 611 from the conductive 1a-ver 605 to the workin~ electrode 602.
The reactions 607, 609 shoNvn in Fig 6;nvolve simultaneous electron transfer into and out of the conductive layer 605. This is possible, in fact it is a requirement, because the eonduetive'ayer 605 acts as an electrode and the cheinieal potential of a solution in contact with an electrode wi1': always t~y to reach equilibri~~m with the electrode potential. Since the layer 605 is conductive, it can only be at a single unifor.~i poteTitial throughout, so it is the chemical potentials of the various parts of the sc3lution i.*~ contact with the conductive later 605 that are brought into equilibrium through electron transfer. This electron transfer is effectively instantaneous in the conductive laver 6051 so it provides a very rapid path for electron t.ar,sfer f~or~~ regiens of the test cell that would otherwise take much time to reach a steady state.
Tn addition, the proximity of the caDduetive layer to the electrodes and their parallel orientation render much of the diffusion effectively r:e-0-imensional.

The siinple addition of acanduetive layer over side-by-side electredes therefore rapidl~Y reduces the time it takes to generate a steady-state tlux bet~vee~
tA~ie electrodes.
This allows much smaller sample sizes, since flux between the electrodes is no longer undesired. It allows an i-nproved accuracy because the fiux is now arri'ving principally at the electrode surfaces rather than along their acljacent edges and so is dependent on the far more controllable electrode area than. the electrode separation. I:t also produces a near one-dimensional diffusion at a rapidly reached steadv state and so the ability to probe the system by electrochemical teeh-niques and apply usef~l corrections such as those described in US Patent p-Lfblication No. US20050109637A1, and US patent N'o.
5284125 are at their most effective.

Fig. 7 shows a cross section through the test cell of ar: embodimenL of a test strip according to tl-ie inventioii. Ir~ Fig. 7, the second insulating laver 35 and the conductive e.oatir~~ 40 are replaced with aconduetive laver 70.

f ig. 8 shows a top view of a test str pof the t~rpe shs~~-r~ in Fig. 8. Test cell 3 6 is opened to the exterrior to permit introduction of sarnple via channel. 61 formed in the spacer layer. A vent 82 is fom-ied through the conductive layer 70 (or alternatively through the insulating substrate 31 te facilitate flow into the test cell.
~%o~ l~in~ and counter electrodes 32, 33 are cQrriected to conductive leads 84, 85, respectively. At the end opposite the channel 8 1, a portion ef the conductive la-,rer 60 and the spacer layer are cut away to expose a part of the insulating substrate 31 and af l.eads 84 and 85 to allow connection of these leads with a meter. In the embodiment show-n in Fig.
8, part 83, 83` of the conductive laver 60 is leftin place at the edges of the test strip.
This not anlv provides better dimensional stability but electrical contact with t[lis layer can be used to de;.ect insertion of a test strip into a test meter, for example as described in US Patents Nos. 4,999,582, 5,282,950 aiid 6,61 8,81 9, 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 ori part 83 and another part on part 83'.

The crnbsdimerit disclosed in Fia. 8 can also facilitate detection of sample mtreduction into the cell. For exam-ole, by moriitorin- for a change in resistance between t,he conductive layer 80 and. either of the working or counter electrodes 32, 33; the 'mtreduction of sample into the test cell can be monitored, aiad used as a signal to activate applieation. 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 insuiating outer suz-faee can also be used providin~
that a portion of the insulation is removed or a lead is forrsed to allow contact with the conductive surface. It should be understood, however, that no e;ectrieal sigal needs to be applied to or measured from this conductive coating or layer in order to achieve the benefits of reduced test'nig time.

Claims (11)

1. An electrochemical test strip for detection of an analyte comprising a first insulating substrate layer, a second substrate layer, and 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 and the inner surface of the second substrate on the other side;
(b) 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 disposed on the first insulating substrate in a location within the test cell, said working and counter electrodes being associated with conductive leads that allow connection of the electrodes to a meter for determination of analyte wherein a region of the second substrate facing the working and counter electrodes is conductive.

2. The test strip of claim 1, wherein the working electrode comprises a enzyme and a redox mediator.

3. The test strip of claim 2, wherein the enzyme is glucose oxidase.

4. The test strip of claim 1, wherein the entirety of the second substrate layer is conductive.

5. The test strip of claim 4, wherein the working electrode comprises a enzyme and a redox mediator.

6. The test strip of claim 5, wherein the enzyme is glucose oxidase.

7. The test strip of claim 1, wherein the second substrate layer comprises a second insulating substrate and a conductive coating.

8. The test strip of claim 7, wherein the working electrode comprises a enzyme and a redox mediator.

9. The test strip of claim 8, wherein the enzyme is glucose oxidase.

10. A method for detecting an analyte in a sample comprising the steps of:
introducing the sample into the test cell of any one of claims 1 to 9;
applying a potential between the working and counter electrodes to generate a current indicative of analyte in the sample; and measuring the current to obtain a determination of analyte in the sample.

11. The method of claim 10, wherein the entirety of the second substrate layer is conductive, further comprising the step of monitoring the resistance between the conductive second substrate layer and the working and/or counter electrodes as a measure of filling of the test cell with sample.