EP3167282A1

EP3167282A1 - Analyte concentration measurement

Info

Publication number: EP3167282A1
Application number: EP15747510.4A
Authority: EP
Inventors: Zuifang Liu
Original assignee: Cilag GmbH International
Current assignee: Cilag GmbH International
Priority date: 2014-07-08
Filing date: 2015-07-08
Publication date: 2017-05-17
Also published as: WO2016005743A1; RU2017103730A3; BR112017000305A2; CA2953452A1; JP2017519990A; RU2017103730A; JP6619367B2; AU2015287447A1; RU2680266C2; CN106687803A; GB201412156D0; KR20170028408A

Abstract

A method of determining analyte concentration uses a redox reaction in an electrochemical cell that has at least two electrodes, one of which is a working electrode, at least one electrode being exposed to at least one redox mediator. The method uses at least one cycle of pulses, each cycle having at least a first potential and a second potential. The method comprises applying a first potential to initiate an accumulation phase that forces accumulation of a mediator concentration gradient at or close to the working electrode with a concentration that decreases towards the bulk solution, applying a second potential to initiate a measurement phase and deplete the established mediator concentration gradient at the working electrode, measuring current associated with the second potential of each cycle, and using the measured current to determine analyte concentration. In this way, the effects of diffusion interfering factors (DIF), especially haematocrit (Hct) can be mitigated.

Description

Analyte Concentration Measurement

Field of the Invention

The present invention relates to a diffusion based analyte concentration measurement that mitigates the effects of diffusion interfering factors.

Background of the Invention

Electrochemical-based sensors, such as self-monitoring blood glucose (SMBG) strips, are used for measuring/determining analyte concentration in fluid samples, for example whole blood. However, their accuracy can suffer from diffusion interfering factors (DIF), which affect analyte mass transfer in the test fluids, e.g. blood haematocrit (Hct) because red blood cells block diffusion pathway of the analyte (e.g. glucose). There is a need to develop technologies for DIF mitigation to meet product accuracy requirements.

DIF mitigation can be categorized into active approaches and passive approaches. The former relies on using DIF sensitive signals to have DIF "measurements" which are then used for DIF correction. A problem with active approaches is that they require extra mechanisms, such as additional strip elements, more measurement steps, and additional device/meter components/parts. In contrast, passive approaches use DIF insensitive signals or signals with negligible DIF effect for the analyte measurement.

US8105478B2 describes a method for selecting pulse lengths for measuring the concentration of a redox-active substance as a mediator in a molecular-biological detection system, in which suitable potentials are applied to a working electrode, to cause at least one of an oxidation process and a reduction process, which takes place as a redox reaction. The method comprises pulsing the potential of the working electrode and alternately forming measuring phases and relaxation phases; selecting the measuring-phase pulse lengths so that, towards the end of the pulse, the capacitive current is relatively small in comparison with the Faraday current; and selecting the relaxation-phase pulse lengths so that, towards the end of the pulse, the concentration gradient is relaxed so that at the beginning of a following measuring phase, the change in concentration of the mediator, brought about by the consumption of the mediator by the measurement itself, is reversed to the greatest possible extent approaching the original level. Summary of the invention

According to the present invention, there is provided a method of reducing or mitigating the effects of DIF in a method of determining analyte concentration using a redox reaction in an electrochemical cell that has at least two electrodes, one of which is a working electrode, at least one electrode being exposed to at least one redox mediator, using at least one cycle of pulses, each cycle having a first potential and a second potential, the method comprising: applying a first potential to initiate an accumulation phase that forces accumulation of a concentration gradient of the mediator at or close to the working electrode with a concentration that decreases towards the bulk solution; applying a second potential to initiate a measurement phase and deplete the established concentration gradient of the mediator; and measuring current associated with the second potential of each cycle during depletion of the established concentration gradient. The measured current can be used to calculate analyte concentration.

By forcing establishment of a concentration gradient of mediator with a concentration that decreases towards the bulk solution at the working electrode in an accumulation phase of the cycle, for glucose testing current sensitivity to Hct is reduced, whilst current sensitivity to glucose is enhanced.

The concentration gradient may gradually and continuously decrease towards the bulk solution. Alternatively, the concentration gradient may fluctuate, whilst generally decreasing towards the bulk solution.

The at least one electrode may be coated with at least one redox mediator. Alternatively or additionally, the electrode may be in a solution that includes at least one redox mediator, and so is exposed to the at least one redox mediator. The accumulation phase can have a reduction at the working electrode and the measurement phase can have an oxidation at the working electrode. Alternatively, the accumulation phase may have an oxidation and the measurement phase can have a reduction at the working electrode depending on nature of redox reaction involving the analyte and mediator(s). As is known in the art, whether the accumulation phase should have reduction or oxidation depends on the mediator state (oxidised or reduced) before it undergoes the heterogeneous reaction at electrode surface. Also, it will be appreciated that more than one mediator may be used for a series of redox reactions.

In addition to the first and second potentials, further potentials may be applied to the electrode(s). Such further potentials could be applied before the first potential or after the second potential. For example, an initiation potential may be applied prior to applying the at least one cycle of pulses, wherein the initiation potential has an open circuit or potential for substantially no redox reaction at the electrodes. The initiation potential may be applied prior to applying each cycle of pulses. In all cases, the second potential has to immediately follow the first potential, so that the accumulation phase that forces accumulation of the concentration gradient immediately precedes the measurement phase.

The second potential is such that after depletion of the established concentration gradient of the mediator another concentration gradient of the mediator builds up (the opposite concentration gradient is established), but with a concentration that increases towards the bulk solution.

The magnitudes of the first potential and the second potential may be symmetrical relative to a potential which elicits substantially zero current flow (E₀₎. The magnitudes of the first potential and second potential may be asymmetrical to E₀.

Durations of the first potential and the second potential may be the same. Durations of the first potential and the second potential may be different. Durations of the first potential and the second potential may be less than 10 minutes, preferably less than 1 minute, and most preferably less than 5 seconds. Durations of the first potential and the second potential may be between 5 to 100% of the total time of each pulse cycle.

According to another aspect of the invention, there is provided a test meter for determining analyte concentration using a redox reaction in an electrochemical cell that has at least two electrodes, one of which is a working electrode, at least one electrode exposed to at least a redox mediator, using at least one cycle of pulses, each cycle having a first potential and a second potential, the meter being configured to: apply a first potential to initiate an accumulation phase that forces accumulation of a concentration gradient of the mediator at or close to the working electrode with a concentration that decreases towards the bulk solution; apply a second potential to initiate a measurement phase and deplete the established concentration gradient of the mediator; and measure current associated with the second potential of each cycle. Preferably, the test meter is configured to calculate analyte concentration using the measured current.

The accumulation phase can be a reduction and the measurement phase can be an oxidation. Alternatively, the accumulation phase can be an oxidation and the measurement phase can be a reduction depending on the nature of the redox reaction involving the analyte and mediator(s). Also, it will be appreciated that more than one mediator may be used for a series of redox reactions.

The second potential may be such that after depletion of the established concentration gradient of the mediator another concentration gradient of the mediator builds up, but with a concentration that increases towards the bulk solution.

The magnitudes of the first potential and the second potential may be symmetrical relative to a potential that causes substantially zero current flow (E₀). The magnitudes of the first potential and second potential may be asymmetrical to E₀. Durations of the first potential and the second potential may be the same. Durations of the first potential and the second potential may be different. Durations of the first potential and the second potential may be less than 10 minutes, preferably less than 1 minute, and most preferably less than 5 seconds. Durations of the first potential and the second potential may be between 5 to 100% of the total time for each pulse cycle.

Brief Description of the Drawings

Various aspects of the invention will now be described by way of example only, and with reference to the following drawings, of which:

Figure 1 shows switching redox reactions at two electrodes by controlling potential;

Figure 2 shows an evolution of M_red (reduced mediator) concentration gradient at the working electrode E1 with the redox reaction switch in Figure 1 ;

Figure 3 shows a test waveform and a control waveform for applying to the electrodes of an electrochemical cell; Figure 4 shows current sensitivity to glucose versus measurement time for various samples taken using the waveforms of Figure 3, where the numbers in the legends are Hct levels as a percentage, and

Figure 5 shows the percentage biases to the nominal Hct of current sensitivity to glucose at 0.3 seconds of four of the oxidation pulses of the waveforms of Figure 3, where the numbers in legends are the pulse numbers of the waveforms in Figure 3.

Detailed Description of the Drawings

This present invention mitigates the effects of DIF current signals by switching and controlling redox reactions at the working and counter electrodes of electrochemical- based sensors that use mediators. This is done by creating a higher concentration of mediator near the working electrode than in the bulk sample during an accumulation phase, so that a mediator concentration gradient is present at the start of each measurement phase. Ideally, the mediator concentration gradient extends by at least 10nm from the working electrode into the bulk sample. The mediator concentration gradient should not reach the counter electrode, and so ideally the maximum extent of the mediator gradient is less than the separation of the working and counter electrodes. In many practical implementations, it is preferred that by the end of the accumulation phase and the start of the measurement phase, the mediator concentration gradient does not extend to beyond half way between the working and counter electrodes.

Figure 1 shows one pulse cycle and the initiated corresponding redox reactions of an electrochemical test strip with two electrodes, a working electrode E1 and a counter electrode E2. The two electrodes are covered with a reagent layer which contains redox mediator (M) and enzyme (Enz). To test the strip, the working and counter electrodes E1 and E2 respectively are in contact with a whole blood sample and an electrical potential (voltage) is applied between the two electrodes. This results in redox reactions both in the blood (homogeneous redox reactions) and at surfaces of the two electrodes (heterogeneous redox reactions).

For the heterogeneous redox reactions, oxidation occurs at one electrode and reduction at the other simultaneously. In Figure 1 , the potential is applied as a series of square waves (pulses). The redox reactions at the two electrodes are switched over by altering the pulses from a reduction potential (E_red) to an oxidation potential (E_ox). This can be done by controlling the magnitude of the potential and, if necessary, polarity. E₀ is a potential for substantially zero redox reactions (neither reduction nor oxidation). E_red bias (difference) to E₀ and E_ox bias to E₀ can be the same (i.e. symmetrical to E₀ as illustrated in Figure 1 ) or different. As illustrated in Figure 1 , under E_red, oxidized mediator (M_ox) undertakes reduction at the working electrode E1 (reaction 3), whilst reduced mediator (M_red) undertakes oxidation at the counter electrode E2 (reaction 2). At the same time, glucose (Glue) reacts with M_ox involving the enzyme (Enz) to produce M_red in blood (reaction 1 ). As a result, M_red is "accumulated" at the working electrode E1 (both reactions 1 and 3 produce M_red) to a concentration C, higher than the starting M_red concentration C₀ and a M_red concentration gradient is established with decreasing M_red concentration from the working electrode E1 surface towards the bulk solution (see Figure 2A to 2B). The M_red concentration gradient can be expressed as: C_g = (Q - C₀)/ d,. A larger C_g means more M_red are retained closer to the electrode surface.

In addition to rates of reactions 1 and 3 (Figure 1 ), which are proportional to glucose concentration, C_g is dependent on Hct. The higher Hct, the slower M_red diffusion away from the electrode surface and hence, the larger C_g is established during the reduction pulse.

Upon changing from E_red to E_ox, the heterogeneous redox reactions at the two electrodes are switched over (see Figure 1 ). In case of using the oxidation pulse (under E_ox) for glucose measuring, glucose concentration is determined by measuring the rate of reaction 5, e.g. by measuring current. Rate of reaction 5 is proportional to M_red available at the surface of the working electrode E1 . Reaction 5 proceeds at a sufficiently high rate to cause M_red depletion to be faster than its supply (through diffusion) at the surface of the working electrode E1 . This means that M_red concentration drops from , through C₀, ultimately to C_zero (see the dotted concentration gradient line in Figure 2C) with time. After the M_red concentration at the surface of the working electrode E1 decreases below C₀, C_g is negative and the rate of reaction 5 decreases with time in a pattern described by the Cottrell equation (see below), and the glucose measurement is Hct dependent.

nFA o

i⁼ r-r ^C<

jnt Here, i is the rate of reaction 5 expressed as current (amps) ; n is the number of electrons transferred in the heterogeneous redox reaction; F is the Faraday constant (96485 Coulombs/mol) ; A is the electrode area (cm²); D is the diffusion coefficient (cm²/sec); t is the test time (sec) ; and C₀ is the reactant starting concentration (mol/cm³).

Over the time window that C_g is positive the rate of reaction 5 is dependent on M_red diffusion and the M_red concentration gradient established during the previous reduction pulse. Increasing Hct decreases M_red diffusion, but increases the M_red concentration gradient. Therefore, the Hct effect on the glucose measurement is compensated by manipulating the redox reaction switch. To benefit from this compensated measurement, the current has to be measured in the concentration gradient depletion phase only, i.e. while the concentration gradient is being depleted. After the depletion phase, when the mediator concentration drops below its start level (i.e. the level prior to the accumulation phase), the current becomes Hct dependent.

Establishment of the M_red concentration gradient during the accumulation phase can be achieved in various ways, including, but not limited to, controlling the potential magnitude and/or polarity of the pulses applied, controlling the effective surface ratio of the two electrodes, controlling the pulse time, controlling reagent layer components and quantity ratios, or any combination of these. In the following examples, the potential magnitude of the pulses applied is used to force accumulation of the mediator concentration gradient and subsequently deplete the established mediator concentration gradient at the working electrode. However, other techniques for controlling the accumulation and depletion of mediator concentration are possible.

Figure 3 shows a control waveform W69 and test waveform W70. The difference between the test waveform W70 and the control waveform W69 is that the test waveform W70 has a higher potential magnitude for the reduction pulses (pulses 2, 4, 6, 8) than the control waveform W69 to enhance the establishment of M_red concentration gradient at the working electrode E1 during these pulses (see Figure 1 ).

Both the oxidation and reduction pulses of the test waveform are over-potential. By this it is meant that the potential is of a magnitude that is sufficient for the redox reactions at the electrodes to be dominated by diffusion of the mediator and/or analyte towards the electrode. Magnitudes of the oxidation and reduction pulses are selected and controlled by taking into account the electrochemical properties of the mediator and the electrodes. Both the oxidation and reduction pulses can have positive, negative or zero potentials.

Each waveform of Figure 3 has 9 square pulses. At the working electrode E1 , pulses 2, 4, 6, 8 lead to reduction (negative current in Figure 4) and pulses 3, 5, 7, 9 lead to oxidation (positive current in Figure 4). Pulse 1 is an initiation pulse that is applied once before the start of the repeated cycles of pulses (i.e. it is not part of the repeated cycle of pulses). Pulse 1 is an under-potential pulse wherein the rate of redox reactions at the electrodes is dominated by the kinetics of the heterogeneous redox reactions to allow hydration/dissolution of the strip reagent layer (potential of this pulse can be E₀ or close to E₀ to keep redox reaction at both the working electrode E1 and the counter electrode E2 to a minimum). Pulse 1 can also be an open circuit.

The total test time of the 9 pulses in this experiment was designed for 6.25 seconds, but the test can be completed in 5 seconds (i.e. without pulses 8 and 9) or less. The waveforms were applied to the strip using a potentiostat, so that a positive potential lead to oxidation and a negative/zero potential leads to reduction at the working electrode E1 . The oxidation current (i.e. positive pulse current resulting from reaction 5 in Figure 1 ) is used for glucose measurement. Techniques for determining glucose concentration using current measured during a redox reaction are well known in the art and so will not be described in detail.

Laboratory test results of W69 and W70 current transients are shown in Figure 4. The top row and the bottom row of Figure 4 show plots of current sensitivity to glucose (i.e. current in Amp generated per mg/dL glucose) against test time at five targeted glucose concentrations (TG) for W69 and W70, respectively. For each target glucose concentration, the same blood sample was tested for W69 and W70. Each line in the graphs represents mean current sensitivity of 6 replications. Each graph shows five mean current sensitivities at five target Hct levels (from 20% to 60%).

Figure 4 shows that in each graph of W69, there are clear separations between the lines of positive pulses (measuring pulses). Higher Hct corresponds to lower current sensitivity. This indicates that W69 measuring currents are sensitive to Hct variation. In contrast, for the graphs of W70, the line separation disappears or is remarkably decreased, i.e. Hct as a DIF is effectively accounted for. W70 leads to significantly higher current sensitivity than W69, i.e. W70 has enhanced current sensitivity to glucose compared to W69. The enhancement in current sensitivity is twofold: 1 ) W70 lines in Figure 4 have higher magnitude than W69 lines; 2) W70 lines of each pulse have similar/close magnitude (they are on top of each other if overlaid) whilst W69 lines of each pulse decreases with time, i.e. W70 retains current sensitivity throughout the test time whilst W69 does not. This is because the established M_red concentration gradient during the reduction pulse leads to enhanced reaction 5 of the subsequent oxidation pulse.

Figure 5 also show the mitigation of Hct, where percentage bias is the difference in current sensitivity between the Hct of interest and the nominal Hct (defined here as 42%, and so this has zero bias). As an example, the current sensitivity at 0.3 seconds of the oxidation pulses 3, 5, 7 and 9 (see Figure 3) are used. For W69 results, the bias to nominal Hct had a range of about 20% to -20% with increased Hct. In contrast, for W70 results, the bias to nominal Hct was reduced with a range less than +/-10%.

The above described embodiment uses control of the pulse magnitude to force the establishment of the concentration gradient. However, DIF mitigation, and in particular Hct mitigation, can be achieved by other means. For example, this could be done by increasing the effective surface ratio of the counter electrode E2 to the working electrode E1 , or increasing the duration of the accumulation phase pulses. Indeed, any technique that forces the accumulation of a concentration gradient of the mediator at the working electrode E1 could be used.

The present invention provides a simple and effective technique for mitigating the effects of DIF providing an Hct insensitive measurement. The method could be applied to existing products without making any changes to the strips.

A skilled person will appreciate that variations of the disclosed arrangements are possible without departing from the invention. For example, although the invention has been described with reference to a test strip with only two electrodes, the invention could equally be applied to strips with three or more electrodes. Equally, whilst in the specific tests conducted only a single initiation pulse was used, an initiation pulse could be applied prior to every cycle of accumulation and measurement pulses. Accordingly the above description of the specific embodiment is made by way of example only and not for the purposes of limitation. It will be clear to the skilled person that minor modifications may be made without significant changes to the operation described.

Claims

1. A method of determining analyte concentration using a redox reaction in an electrochemical cell that has at least two electrodes, one of which is a working electrode, at least one electrode being exposed to at least one redox mediator, using at least one cycle of pulses, each cycle having at least a first potential and a second potential, the method comprising:

applying a first potential to initiate an accumulation phase that forces accumulation of a mediator concentration gradient at or close to the working electrode with a concentration that decreases towards the bulk solution;

applying a second potential to initiate a measurement phase and deplete the established mediator concentration gradient at the working electrode;

measuring current associated with the second potential of each cycle, and using the measured current to determine analyte concentration.

2. A method as claimed in claim 1 , wherein the accumulation phase is a reduction and the measurement phase is an oxidation.

A method as claimed in claim 1 , wherein the accumulation phase is an oxidation and the measurement phase is a reduction.

A method as claimed in any of the preceding claims, wherein the second potential is such that after depletion of the established concentration gradient of the mediator another concentration gradient of the mediator builds up, but with a concentration that increases towards the bulk solution.

5. The method of the preceding claims, wherein the first potential and the second potential have magnitudes that are symmetrical relative to a potential that causes substantially zero current flow.

6. A method as claimed in any of claims 1 to 5, wherein the first potential and second potential have magnitudes that are asymmetrical relative to a potential that causes substantially zero current flow.

A method as claimed in any of the preceding claims, wherein durations of the first potential and the second potential are the same or different.

A method as claimed in any of the preceding claims comprising applying an initiation pulse prior to applying the at least one cycle of pulses, wherein the initiation pulse is an open circuit or a potential that causes substantially no redox reaction at the electrodes.

9. A method as claimed in claim 8, wherein an initiation pulse is applied prior to applying each cycle of pulses.

10. A method as claimed in claim 8 or claim 9, wherein the initiation pulse has a duration between zero to 20 minutes, preferably zero to 5 minutes, and most preferably zero to 5 seconds.

. A method as claimed in claim 9 or claim 10, wherein the initiation pulse duration of between zero to 95 percent of the total time of each pulse cycle.

A method as claimed in any of the preceding claims, wherein durations of the first potential and the second potential are less than 10 minutes, preferably less than 1 minute, and most preferably less than 5 seconds.

13. A method as claimed in any of the preceding claims, wherein durations of the first potential and the second potential are between 5 to 100 percent of the total time of each pulse cycle.

14. A test meter for determining analyte concentration using a redox reaction in an electrochemical cell that has at least two electrodes, one of which is a working electrode, at least one electrode being exposed to at least a redox mediator, using at least one cycle of pulses, each cycle having a first potential and a second potential, the meter being configured to:

apply a first potential to initiate an accumulation phase and force accumulation of a mediator concentration gradient at or close to the working electrode with a concentration that decreases towards the bulk solution;

apply a second potential to initiate a measurement phase and deplete the established mediator concentration gradient at the working electrode;

measure current associated with the second potential of each cycle, and use the measured current to determine analyte concentration

15. A test meter as claimed in claim 14, wherein the accumulation phase is a reduction and the measurement phase is an oxidation.

16. A test meter as claimed in claim 14, wherein the accumulation phase is an oxidation and the measurement phase is a reduction.

17. A test meter as claimed in any of claims 14 to 16, wherein the second potential is such that after depletion of the accumulated mediator concentration gradient another concentration gradient of the mediator at the working electrode builds up, but with a concentration that increases towards the bulk solution.

18. A test meter as claimed in any of claims 14 to 17 configured to calculate analyte concentration using the recorded current.

19. A test meter as claimed in any of claims 14 to 18, wherein the first potential and the second potential have magnitudes that are symmetrical relative to a potential that causes substantially zero current flow.

20. A test meter as claimed in any of claims 14 to 19, wherein magnitudes of the first potential and second potential are asymmetrical relative to a potential that causes substantially zero current flow.

21 . A test meter as claimed in any of claims 14 to 20, wherein durations of the first potential and the second potential are the same or different.

22. A test meter as claimed in any of claims 14 to 21 adapted to apply an initiation pulse prior to applying the at least one cycle of pulses, wherein the initiation pulse is an open circuit or a potential that causes substantially no redox reaction at the electrodes.

23. A test meter as claimed in claim 22 adapted to apply an initiation pulse prior to applying each cycle of pulses, so that each cycle includes an initiation potential as well as first and second potentials.

24. A test meter as claimed in claim 22 or claim 23, wherein the initiation pulse has a duration between zero to 20 minutes, preferably zero to 5 minutes, and most preferably zero to 5 seconds.

25. A test meter as claimed in claim 24, wherein the initiation pulse has a duration of between zero to 95 percent of the total time of each pulse cycle.

26. A test meter as claimed in any of claims 14 to 25, wherein durations of the first potential and the second potential are less than 10 minutes, preferably less than 1 minute, and most preferably less than 5 seconds.

27. A test meter as claimed in any of claims 14 to 26, wherein the combined duration of the first potential and the second potential is between 5 to 100 percent of the total time for each pulse cycle.