Method and apparatus for determining the amount of an analyte in a fluid

Abstract

A method for determining the amount of an analyte in a fluid using an electrochemical aptamer-based sensor is provided. The method involves applying a step change in potential to a working electrode of the sensor and determining the distribution of the aptamer's redox reporter from the measured current. The amount of analyte around the sensor is then determined from the distribution.

Description

[Technical Field]

[0001] The present invention relates generally to electrochemical sensors useful for determining the amount of an analyte in biological fluids such as blood and interstitial fluid. More specifically, the present invention provides electrochemical sensors operated by an improved chronoamperometric method that provide more reliable analyte determination over a range of conditions. [Background technology]

[0002] Several classes of electrochemical sensors are selective and capable of real-time continuous detection of target analytes as well as single-point measurements, including exogenous agents (e.g., pharmaceutical compounds and toxins) and also endogenous agents (e.g., metabolites, proteins, hormones, etc.).

[0003] These sensors may include electrodes with associated binding elements that undergo a conformational change upon analyte binding. This conformational change alters the accessibility of the redox reporter to the electrode surface, thereby resulting in an analyte-induced change in electron exchange between the redox reporter and the electrode. In some situations, analyte binding brings the redox reporter closer to the electrode surface, thereby increasing the rate of electron exchange and, in turn, increasing the current through the electrode. In other situations, binding displaces the reporter distal to the electrode surface, resulting in the opposite effect. Nevertheless, analyte binding results in a detectable change in the electrode current.

[0004] Electrochemical sensors can be implemented in many forms, including a microneedle-based patch applied to the skin so that the electrodes contact interstitial fluid. The tips of the microneedles function as sensor electrodes, and a binding element is associated with the tips. This configuration provides a minimally invasive platform for real-time, continuous in vivo target analyte detection that is sensitive and selective enough to function in the complex matrix of interstitial fluid.

[0005] While prior art sensors are undoubtedly useful, they present a number of problems.

[0006] For example, the response of a whole blood glucose sensor can vary according to the viscosity of the blood, which in turn can vary according to temperature, hematocrit, and lipid content. In most prior art devices, such undesirable variations are addressed by using correction factors when calculating the amount of analyte. A separate temperature sensor may also be provided, and the sensor is calibrated taking into account pooled population data.

[0007] Signal drift is a significant issue in continuous electrochemical sensors. Prior artisans have proposed methods to quantify bound analytes using electrochemical square-wave voltammetry, which uses measurements and multiple square-wave frequencies to account for signal drift over time. However, such methods require careful selection of specific frequency pairs where the signal drift at the two frequencies empirically matches. Uncertainty remains as to whether the selected frequencies are applicable in all situations, for example, at different temperatures or for different devices subject to manufacturing variations.

[0008] Further problems arise from variations in sensor output resulting from electrode area, layer thickness, and the diffusion coefficient and density of redox species adjacent to the electrode surface. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to overcome, or at least ameliorate, these shortcomings in the prior art by providing an easy to perform and extremely rapid method whereby quantification of the amount of analyte bound proximally or distally to an electrode may be determined in a manner that is more immune to variations in the sensing environment and manufacturing variations between sensors, as well as changes in the device over time both before and during use.

[0010] One aspect of the present invention is to provide improvements in the operation of electrochemical sensors to improve or overcome any one or more of undesirable output fluctuations, inaccurate outputs, unclear outputs, and irreproducible outputs. A further aspect of the present invention is to eliminate the need for auxiliary means within the electrochemical sensor to address any one or more of undesirable output fluctuations, inaccurate outputs, unclear outputs, and irreproducible outputs. A further aspect of the present invention is to provide a useful alternative to prior art methods of interrogating electrochemical sensors.

[0011] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. No suggestion or representation is made that any or all of these matters formed part of the prior art or were common general knowledge in the art relevant to the present invention by virtue of existing prior to the priority date of each provisional claim of this application. [Means for solving the problem]

[0012] In a first aspect, not necessarily its broadest aspect, the present invention provides a method for determining the amount of an analyte in a fluid, the method comprising: applying a first potential change to an electrochemical sensor working electrode, the electrode having associated therewith (i) a binding element and (ii) an associated redox-active species spatially confined within a layer adjacent to the electrode surface; measuring, at a plurality of time points, the current resulting from application of at least the first potential change; determining the distribution of redox active species by referring to the measured current value; and c) using the determined distribution of the redox active species to determine the amount of analyte bound to the binding member.

[0013] In an embodiment of the first aspect, the change in potential is substantially instantaneous.

[0014] In an embodiment of the first aspect, the change in potential is effected as a step change.

[0015] In one embodiment of the first aspect, the first potential change is configured to change the redox state of the redox active species from one redox state to another redox state, resulting in a transfer of electrons between the redox active species and the electrode surface.

[0016] In one embodiment of the first aspect, the first potential change is configured such that the current flowing through the electrode surface as a result of the redox-active species changing the redox state is controlled by mass transport of the redox-active species through a layer adjacent to the electrode surface.

[0017] In one embodiment of the first aspect, the potential resulting from the first potential change is maintained for at least about 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, or 100 ms.

[0018] In an embodiment of the first aspect, the method includes applying an initial potential before applying the first potential change.

[0019] In one embodiment of the first aspect, the initial potential is selected such that the magnitude of the first potential change is at least about 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, or 1.0 V.

[0020] In one embodiment of the first aspect, the initial potential is maintained for at least about 0.05 seconds, 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.4 seconds, 0.5 seconds, 0.6 seconds, 0.7 seconds, 0.8 seconds, 0.9 seconds, 1.0 seconds, 1.1 seconds, 1.2 seconds, 1.3 seconds, 1.4 seconds, 1.5 seconds, 1.6 seconds, 1.7 seconds, 1.8 seconds, 1.9 seconds, or 2.0 seconds.

[0021] In an embodiment of the first aspect, the method includes measuring at least a first current value after application of a first potential change.

[0022] In one embodiment of the first aspect, the first current value is measured substantially immediately after application of the first potential change.

[0023] In an embodiment of the first aspect, the first of the first current values ​​is measured less than about 0.1 ms, 0.2 ms, 0.3 ms, 0.4 ms, 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, or 1.0 ms after application of the first potential change.

[0024] In one embodiment of the first aspect, the method includes measuring at least first and second current values ​​in time series after application of a first potential change.

[0025] In one embodiment of the first aspect, the method includes measuring at least first, second, and third current values ​​in time sequence after application of a first potential change.

[0026] In one embodiment of the first aspect, the method includes measuring at least first, second, third, and fourth current values ​​in time sequence after application of a first potential change.

[0027] In one embodiment of the first aspect, the measurements of the first current value, the second current value (if measured), the third current value (if measured), and the fourth current value (if measured) are taken within a period during which the potential resulting from the first potential change is maintained.

[0028] In one embodiment of the first aspect, the first current value, the second current value (if implemented), the third current value (if measured), and the fourth current value (if measured) are average current values, and the average current value is determined by reference to a plurality of current values ​​determined up to, over, or after a certain point in time.

[0029] In one embodiment of the first aspect, the average current value is determined by reference to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 current values.

[0030] In an embodiment of the first aspect, the method includes generating at least one current ratio by referencing a first current value and a second current value.

[0031] In one embodiment of the first aspect, the first current value is measured substantially immediately after application of the first potential change.

[0032] In an embodiment of the first aspect, the first current value is measured within about 0.1 ms, 0.2 ms, 0.3 ms, 0.5 ms, 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, 1.0 ms, 2.0 ms, 3.0 ms, 4.0 ms, or 5.0 ms after application of the first potential change.

[0033] In an embodiment of the first aspect, the second current value is measured after the first current value.

[0034] In an embodiment of the first aspect, the second current value is measured within about 1.0 ms, 2.0 ms, 3.0 ms, 4.0 ms, 5.0 ms, 6.0 ms, 7 ms, 8 ms, 9 ms, 10 ms, 11 ms, 12 ms, 13 ms, 14 ms, 15 ms, 16 ms, 17 ms, 18 ms, 19 ms, 20 ms, 21 ms, 22 ms, 23 ms, 24 ms, or 25 ms after the first current value is measured or after the first potential change is applied.

[0035] In an embodiment of the first aspect, the method includes applying a second potential change to the electrochemical sensor working electrode after the first potential change.

[0036] In an embodiment of the first aspect, the sign of the second potential change is the same as the sign of the first potential change.

[0037] In an embodiment of the first aspect, the sign of the second potential change is opposite to the sign of the first potential change.

[0038] In one embodiment of the first aspect, the second potential change is used to estimate the capacitive charging current of the sensor device and the current due to redox species not confined in the layer adjacent to the electrode, and therefore the background current draw.

[0039] In one embodiment of the first aspect, the background current draw is subtracted from the first current value, the second current value (if measured), the third current value (if measured), or the fourth current value (if measured). Optionally, the background current draw may be multiplied by a factor before subtracting it from the first, second, third, or fourth current value where they are measured.

[0040] In an embodiment of the first aspect, the first and / or second potential change is a substantially instantaneous change.

[0041] In one embodiment of the first aspect, the first and / or second potential change is a substantially step change.

[0042] In one embodiment of the first aspect, the method comprises: applying a first potential change and at least a second potential change; combining a selected current resulting from the at least second potential change with a selected current from the first potential change to derive a current; and determining an analyte concentration from the derived current.

[0043] In one embodiment of the first aspect, the at least second potential change varies from the potential applied at the end of the first potential change.

[0044] In one embodiment of the first aspect, the selected currents are combined by subtracting a function of the magnitude of the current resulting from the at least second potential change from the magnitude of the current resulting from the first potential change, and the time at which the current is sampled after the potential change is substantially the same for both the first potential change and the at least second potential change.

[0045] In one embodiment of the first aspect, the magnitude of the current resulting from at least the second potential change is multiplied by a coefficient and subtracted from the current resulting from the first potential change.

[0046] In one embodiment of the first aspect, the method includes combining currents resulting from the first change in potential and the at least second change in potential by using currents measured simultaneously after the change in potential and the magnitude of the change in potential used to generate the currents, and extrapolating the measured currents to a value corresponding to zero change in potential.

[0047] In an embodiment of the first aspect, the extrapolation is a linear extrapolation.

[0048] In an embodiment of the first aspect, the extrapolation is a non-linear extrapolation.

[0049] In a second aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a working electrode having associated therewith (i) a binding element and (ii) an associated redox-active species spatially confined within a layer adjacent to the electrode surface; a microprocessor-based controller; and The microprocessor-based controller provides an electrochemical sensor device or system configured to carry out the method of any embodiment of the first aspect.

[0050] In one embodiment of the second aspect, the microprocessor-based controller is in electrical communication with the working electrode or in wired or wireless network communication with another microprocessor-based controller that is in electrical communication with the working electrode.

[0051] In an embodiment of the second aspect, a microprocessor-based controller is configured to access and execute program instructions to perform the method of any embodiment of the first aspect.

[0052] In an embodiment of the second aspect, the electrochemical sensor device or system comprises a variable power supply electrically connected to the working electrode, and the program instructions direct the power supply to apply a potential to the working electrode according to the method of any embodiment of the first aspect.

[0053] In an embodiment of the second aspect, the electrochemical sensor device or system comprises a current measurement circuit configured to measure a current through the working electrode.

[0054] In one embodiment of the second aspect, the electrochemical sensor device or system comprises an electronic memory operatively associated with the microprocessor-based controller and the current measurement circuit, the electronic memory configured to store one or more currents measured by the current measurement circuit.

[0055] In one embodiment of the second aspect, the microprocessor-based controller is configured to process the one or more currents measured by the current measurement circuit to provide an amount of the analyte.

[0056] In a third aspect, the present invention provides a computer readable medium comprising program instructions configured to carry out the method of any embodiment of the first aspect. [Brief explanation of the drawings]

[0057] Unless otherwise indicated herein, drawing features labeled with the same numerals when used across different drawings are to be construed as the same features, or at least functionally similar features.

[0058] With the exception of graphs, the drawings are not made to any particular scale or dimensions, and are not intended to be entirely accurate representations of various embodiments.

[0059] [Figure 1] Figure 1 shows a graph of f values ​​versus vancomycin concentration (in micromolar). The points are calculated f values ​​corrected for the f value in the absence of vancomycin. The curve shown is obtained by fitting a Langmuir binding isotherm to the data. This graph represents the response of a gold electrode coated with the vancomycin aptamer and tested at 32 °C. [Figure 2] This is a graph of f values ​​versus vancomycin concentration (in micromolar). The points are calculated f values ​​corrected for the f value in the absence of vancomycin. The curve shown is obtained by fitting a Langmuir binding isotherm to the data. The graph represents the response of a gold electrode coated with the vancomycin aptamer and tested at 41 °C. [Figure 3] 1 is a graphical representation of the potentials applied during the course of the experiment detailed in Example 1. [Figure 4] 1 is a graph of f values ​​versus time (min). The graph shows the f values ​​and current values ​​obtained at t1 (0.5 ms) for different electrodes interrogated under different temperature conditions. The f values ​​were corrected for background response. [Figure 5] 1 is a graph of current (microamps) versus time. The graph shows the current over time for a sensor where five different potential step sizes are used. [Figure 6] 1 is a graph of current (microamps) versus potential step size (volts). The graph shows an example of sensor current at a particular time plotted against the size of the potential step used. [Figure 7] 1 is a graph of current (microamps) versus time (seconds). The graph shows current over time for one potential step size for various vancomycin concentrations. [Figure 8] 1 is a graph of current (microamps) versus time (seconds). The graph shows the current extrapolated to zero potential step size over time for various vancomycin concentrations. [Figure 9] 1 is a graph of f values ​​versus vancomycin concentration (in micromolar). The graph shows f values ​​calculated using the current from one of the potential step sizes and from the current extrapolated to zero potential step size for various vancomycin concentrations. The f values ​​were corrected for background response. [Figure 10] 1 is a graph of current (microamps) versus time (seconds). The graph shows the current over time for the first potential change for various vancomycin concentrations. [Figure 11] 11 is a graph of current (microamps) versus time (seconds) for various vancomycin concentrations, showing the current over time for a second potential change beginning at the end potential of the first potential change used to generate the current shown in FIG. [Figure 12] 1 is a graph of current (microamps) versus time (seconds). The graph shows the current over time, where the current from the second potential change was multiplied by 2.5 and subtracted from the current from the first potential change. [Figure 13] 12 is a graph of f values ​​versus vancomycin concentration (in micromolar). The graph shows f values ​​calculated using the currents from FIG. 10 and using the currents from FIG. 12 for various vancomycin concentrations. The f values ​​were corrected for background response. DETAILED DESCRIPTION OF THE INVENTION

[0060] After considering this description, it will be apparent to one skilled in the art how the present invention may be implemented in various alternative embodiments and applications. However, while various embodiments of the present invention are described herein, it is understood that these embodiments are presented for purposes of illustration only, and not limitation. Accordingly, this description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention. Furthermore, statements of advantages or other aspects apply to particular exemplary embodiments and not necessarily to all embodiments, or indeed to any embodiments encompassed by the claims.

[0061] Throughout the description and claims of this specification, the term "comprise" and variations of that term, such as "comprising" and "comprises," are not intended to exclude other additives, ingredients, integers, or steps.

[0062] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may.

[0063] When the terms "determine," "determining," and "determined" are used, these terms are not intended to be construed as necessarily implying any perfectly precise determination, although that meaning is not excluded. These terms may be construed to include estimation, approximation, or even indication.

[0064] In a first aspect, the present invention provides a method for determining the amount of an analyte in a fluid, the method comprising: applying a first potential change to an electrochemical sensor working electrode, the electrode having associated therewith (i) a binding element and (ii) an associated redox-active species spatially confined within a layer adjacent to the electrode surface; measuring, at a plurality of time points, the current resulting from application of at least the first potential change; determining the distribution of redox active species by referring to the measured current value; and c) using the determined distribution of the redox active species to determine the amount of analyte bound to the binding member.

[0065] In some embodiments of the method, the potential applied to the working electrode of the device is changed from a potential sufficient to convert and maintain redox species in one redox state to another potential sufficient to substantially immediately change the redox state of any redox-active species sufficiently close to the electrode to allow electron transfer across the electrode / solution interface. Expressed another way, the potential is changed to a potential such that the current flowing through the electrode surface as a result of the redox-active species changing redox state is primarily or substantially controlled by mass transfer of the redox-active species toward the electrode. As a result of the change in potential, a net current flows through the electrode surface, and typically the current varies in magnitude over time.

[0066] While not wishing to be bound by theory in any way, it is proposed that, despite the redox-active reporter being tethered to the working electrode surface (e.g., by an aptamer) and therefore capable of limited movement only within the layer adjacent to the electrode, the redox-active species nevertheless diffuses substantially freely within the layer. Therefore, the movement of the redox-active species can be modeled with some accuracy (though not necessarily complete accuracy) based on Fick's first and second laws of diffusion. Thus, even though tethered, the redox-active species can nevertheless move from regions of high concentration to regions of low concentration, with the magnitude of the flux being proportional to the difference in concentration, in the manner expected for a freely diffusible (i.e., untethered) species. Furthermore, the concentration gradient of the redox-active species in the layer changes over time, as expected for a freely diffusible species.

[0067] Fick's first and second laws were applied to a system in which the redox-active species were spatially confined to a layer adjacent to the electrode surface but were free to diffuse within that layer. It was therefore possible to model the diffusion of the confined redox species and, therefore, the change in current over time (called a "current transient") due to the movement of the redox-active species relative to the electrode surface, taking into account that electron transfer increases as the redox-active species moves closer to the surface.

[0068] The resulting modeled current transients resulting from the migration of redox-active species revealed a dependence on (i) the surface area of ​​the electrode, (ii) the thickness of the layer adjacent to the electrode, (iii) the diffusion coefficient of the redox species in the layer adjacent to the electrode, (iv) the overall concentration of the redox species in the layer adjacent to the electrode, and (v) the distribution of the redox species within the layer adjacent to the electrode at the time the potential step was applied. These evidences have been found to have practical importance with respect to methods for interrogating electrochemical sensor devices, determining the distribution of redox-active species, and determining the amount of analyte bound to the binding element.

[0069] Further investigations were performed to demonstrate the practical relevance of single current measurements and current ratios determined during the period immediately following the application of a potential change. Surprisingly, at longer times after the potential change, the ratio of the two measured currents was found to depend strongly on the thickness of the layer adjacent to the electrode and the diffusion coefficient of the redox species, but only weakly on the initial distribution of the redox species. Meanwhile, the ratio of currents at shorter times after the potential step was more strongly dependent on the initial distribution of the redox species than the currents at longer times, and also on the thickness of the layer adjacent to the electrode and the diffusion coefficient of the redox species. Furthermore, both of these current ratios were modeled to be insensitive to the overall concentration of the redox species and the electrode area.

[0070] These modeled behaviors suggested that the later current ratios can be useful in combination with the thickness of the layer adjacent to the electrode to obtain a combined measure of the redox species diffusion coefficient, which can be applied to the earlier current ratios to obtain an estimated measure of the initial distribution of the redox species, and that this estimate is largely insensitive to changes in the overall concentration of the redox species in the layer adjacent to the electrode, the thickness of the layer adjacent to the electrode, and the diffusion coefficient of the redox species in the layer adjacent to the electrode and the area of ​​the electrode.

[0071] The equation for the current over time derived from the model is shown as equation (1) below.

number

[0072] Thus, f can be used to determine the distribution of redox-active species when the initial potential is applied and at the moment before the change in potential is applied. The distribution of redox-active species can be used to determine the amount of analyte bound to the binding element of the sensor.

[0073] Note from equation (1) that by taking the ratio of the currents at two different times, the electrode area and redox species concentration terms cancel out. Furthermore, at sufficiently long times, the exponential term for n>0 becomes sufficiently small relative to the exponential term for n=0, so that equation (1) can be approximated by equation (2) as follows:

number

[0074] Therefore, by taking the ratio of the currents at two different times to which equation (2) applies, only the exponential terms do not cancel, resulting in D / l 2 An estimate of can be obtained.

[0075] According to this method, the current at at least three different times during the current transient can be determined: i(t1), i(t2), and i(t3). Optionally, the current at a fourth time, i(t4), can be determined. Time t1 is selected to be a short time after the potential step change is applied, t3 and optionally t4 are selected to be longer times after the potential step is applied, and t2 is selected to be between t1 and t3 or optionally between t1 and t4.

[0076] At least two current ratios can then be determined. In preferred embodiments of the invention, the ratios i(t1) / i(t3) and i(t2) / i(t3) are determined. In other embodiments of the invention, the ratios i(t1) / i(t4) and / or i(t2) / i(t4) are determined. In some embodiments of the invention, current ratios at additional times can be calculated and used to improve the method by providing additional estimates of the derived parameters. Any of the ratios i(t1) / i(t4) and / or i(t2) / i(t4) can be used to calculate further estimates of the f-value to aid in the verification or accuracy of those calculated using the i(t1) / i(t3) and i(t2) / i(t3) ratios.

[0077] In a further embodiment of this method, a first potential change at the electrode is followed by a second potential change at the electrode. In this embodiment, the electrode is held at the potential resulting from the first potential change for a time sufficient to electrochemically oxidize or reduce substantially all of the redox-active species present in the confined layer. The electrode potential is then changed in the direction of the initial potential. For example, if the electrode potential is initially held at a value at which the redox-active species is reduced, the second potential change is in the direction of a stronger reduction potential. If the electrode potential is initially held at a value at which the redox-active species is oxidized, the second potential change is in the direction of a stronger oxidation potential. The potential applied after the second potential change is selected so that the faradaic current resulting from the sensed redox species is substantially absent from the current flowing after this potential is applied. The current resulting from the second potential change can be used to obtain an estimate of the non-faradaic current flowing at the electrode due to, for example, capacitive double layer charging, as well as any faradaic current from redox species not confined in the layer adjacent to the electrode, and can be subtracted from the current used to calculate the current ratio disclosed above to improve the accuracy of the results. The current from the second potential change can optionally be multiplied by a factor before being subtracted from the current used to calculate the current ratio disclosed above. In an alternative to this embodiment, the second potential change at the electrode is in a direction opposite to that of the initial potential. The potential applied after the second step is selected so that the faradaic current resulting from the sensed redox species is substantially absent from the current that flows after this potential is applied. To achieve this according to this alternative, the potential step should be selected so as not to substantially change the redox state of the sensed redox species. In this alternative, the current that flows after the second potential step is applied will be opposite in sign to the current that flows after the first potential step is applied. The current from the second potential change can optionally be multiplied by a factor before being added to the current used to calculate the current ratio disclosed above to improve the accuracy of the results. This alternative may be desirable, for example, in situations where moving to a more oxidizing potential can result in additional faradaic current that is not associated with the sensed redox species.

[0078] In a further embodiment of the present invention, multiple potential steps of different magnitudes are applied to the sensor. According to this embodiment, at least two potential steps are applied, with the starting or ending potentials of the potential steps being different for different potential steps. For illustrative purposes only, if the starting potential of the first step is −0.4 V and the ending potential is −0.15 V, the second potential step can start at −0.45 V and end at −0.15 V, and if a third potential step is used, it can start at −0.5 V and end at −0.15 V. Alternatively, the first step can start at −0.4 V and end at −0.15 V, the second potential step can start at −0.4 V and end at −0.1 V, and if a third potential step is used, it can start at −0.4 V and end at −0.05 V. The difference in starting or ending potentials between the multiple potential steps is selected so that the current contributed by the Faradaic electron transfer across the electrode interface contributed by the sensed redox species does not change substantially for different potential steps, while the capacitive charging current and possible external Faradaic current vary proportionally with the potential step size. The first current value from the first potential step at a selected time t1 is combined with the first current value at time t1 from the second potential step and any subsequent potential steps to extrapolate the first current value to a theoretical value at zero potential step size. Any suitable extrapolation function can be used, and the technique used is appropriate for the type of current-to-potential step size dependence. For example, if the measured current varies linearly with potential step size, a linear least-squares regression can be applied to the current values ​​at selected times for various potential steps against the potential step size, and the regression intercept can be used as the extrapolated current at zero potential step size. The extrapolated current at zero potential step size is used as an estimate of the sensed redox species faradaic current at this point, and is substantially free of interference from currents whose magnitude varies with potential step size, such as electron transfer rate controlled capacitive charging currents and faradaic currents.If more than two different potential step sizes are used in this embodiment, the degree of correlation in the extrapolated plot can be used as a measure of the accurate functioning of the device. The extrapolation process is repeated for the current values ​​at the second and third times, and the measured fourth time, and the extrapolated values ​​are used to calculate the f value, as given elsewhere in this disclosure.

[0079] An advantage of some embodiments of the present invention is that a method is provided that allows for estimation of the distribution of redox species without specific knowledge of the electrode area, the thickness of the tethered redox layer, the total amount of redox species, or the number of electrons transferred per mole of redox species. To the best of Applicant's knowledge, no chronoamperometric method has been previously disclosed that can accomplish this. Furthermore, no method has been previously known that derives the distribution using a simple ratio of currents at different times.

[0080] Another advantage of some embodiments of the present invention is that they provide a chronoamperometric method that is significantly faster than prior art interrogation methods (such as square-wave voltammetry), which can take seconds to minutes to execute. The methods described herein, in some embodiments, can be executed in milliseconds to tens of milliseconds. For example, the electrode is optionally held at an initial potential for a short period (on the order of milliseconds to seconds), and then the potential is stepped and held at a second potential for a period of time, typically up to tens of milliseconds. Due to the speed of execution and the fact that the redox species are bound to the electrode, the potential stepping can be repeated multiple times over a short period of time to obtain multiple estimates of the desired parameter, which can be averaged or otherwise combined to reduce random fluctuations in the results.

[0081] In one embodiment of the present invention, multiple potential steps of different magnitudes are applied to the sensor. According to this embodiment, at least two potential steps may be applied, with the starting or ending potentials of the potential steps being different. A first current value from the first potential step may be combined with a first current value from the second potential step and any subsequent potential steps and extrapolated to an extrapolated value at zero potential step size. This process may be repeated for the second, third, and fourth measured current values.

[0082] The difference in the starting or ending potential between the multiple potential steps may be selected so that the current contributed by oxidizing or reducing the sensed redox species does not change substantially for different potential steps, but capacitive charging currents and other possible extraneous currents change with the potential step size.

[0083] If the capacitive charging current and other possible external currents are proportional to the potential step size, a linear extrapolation method, such as linear least squares, can be used to regress the measured current against the potential step size, and the intercept of this regression is used as the extrapolated value.

[0084] If the capacitive charging current and / or other possible extraneous currents vary nonlinearly with the potential step size, a nonlinear extrapolation method, as known in the art, may be used to regress the measured current against the potential step size, with the intercept of this regression being used as the extrapolated value. Extrapolating the measured current versus potential step size to zero potential step size may eliminate or substantially reduce the contribution of the capacitive charging current and other extraneous currents that vary with the potential step size to the obtained extrapolated current value. This, in turn, may improve the ability of the extrapolated current to distinguish between different target analyte concentrations compared to situations where a non-extrapolated current is used as a measure of the target analyte concentration, since a greater proportion of the extrapolated current is attributable to the presence of the target analyte.

[0085] The present invention may be embodied in the form of an apparatus or system configured to facilitate the performance of the methods described herein.

[0086] The apparatus of the present invention may be embodied in the form of a wearable device that is substantially self-contained, allowing measurements to be taken while a subject is performing normal activities and / or over an extended period of time. The wearable device may be a collar, bracelet, strap, adhesive, or patch. The wearable device may include transdermal microneedles, one of which serves as the working electrode of the sensor by contacting the subject's interstitial fluid and detecting an analyte therein.

[0087] The wearable device may further include a housing structure that encloses one or more other components, such as a processor-based microcontroller configured to electrically communicate with at least one electrode and generally including a power source, a data processing unit, an electronic memory, and a wireless transmitter / receiver.

[0088] When embodied as a system, the components may be distributed in different physical locations but still operate in an integrated manner. For example, software instructions may be stored and executed by a smartphone or other remote process in data communication with a microprocessor-based controller within the wearable device.

[0089] As will be appreciated, the methods described herein may be deployed in part or in whole through one or more microprocessors executing computer software, program code, and / or instructions on a processor. A microprocessor may be any type of computing or processing device capable of executing program instructions, code, binary instructions, etc.

[0090] Any microprocessor may access a storage medium (such as electronic memory) via an interface that may store methods, codes, and instructions as described herein and elsewhere. The storage medium is associated with the processor for storing methods, programs, codes, program instructions, or other types of instructions that may be executed.

[0091] The computer software, program code, and / or instructions may be stored on and / or accessed by computer-readable media, which may include computer components, devices, and recording media that hold digital data used in computing for some interval of time, semiconductor storage devices known as random access memory (RAM), and mass storage devices for more permanent storage, typically non-volatile memory such as read-only memory (ROM).

[0092] The methods and systems described herein may transform physical and / or intangible items from one state to another. The methods and systems described herein may also transform data representing physical and / or intangible items from one state to another.

[0093] Software products may be created using structured programming languages ​​such as C, object-oriented programming languages ​​such as C++, or any other high-level or low-level programming languages ​​(including assembly languages, hardware description languages, and database programming languages ​​and techniques) that can be stored, compiled, or interpreted for execution on a microprocessor, as well as heterogeneous combinations of processors, processor architectures, or combinations of different hardware and software, or any other machine capable of executing program instructions.

[0094] Thus, in one aspect, any method may be implemented in computer-executable code that performs its steps when executed on one or more microprocessors. In another aspect, the method may be embodied in a system that performs its steps, may be distributed in some manner across devices, or all of the functionality may be integrated into a dedicated stand-alone device or other hardware. In another aspect, the means for performing the steps associated with the processes described above may include any of the hardware and / or software described above. All such permutations and combinations are intended to fall within the scope of the present disclosure.

[0095] The present invention may be embodied in a set of program instructions executable on one or more microprocessors. Such an instruction set may include any one or more of the following instruction types:

[0096] Data processing and memory operations may include instructions to set a register to a fixed constant value, or to copy data from a memory location to a register, or vice versa, to store the contents of a register, the result of a calculation, or to retrieve stored data and perform a calculation on it at a later time, or to read or write data from a hardware device.

[0097] Arithmetic and logical operations may include instructions to add, subtract, multiply, or divide the values ​​of two registers, place the result in a register, and possibly set one or more condition codes in a status register, to perform bitwise operations, e.g., AND and OR corresponding bits in a pair of registers, negate each bit in a register, or compare two values ​​in registers (e.g., to determine whether one is less than the other, or whether they are equal).

[0098] Control flow operations can include instructions that branch to another location in the program and execute instructions there, conditionally branch to another location if a condition is true, indirectly branch to another location, or call another block of code while saving the location of the next instruction as a point to return to.

[0099] Coprocessor instructions may include instructions that load / store data to / from the coprocessor, or exchange with CPU registers, or instructions that perform coprocessor operations.

[0100] The processor of the computer of this system may include "complex" instructions in its instruction set. A single "complex" instruction does something that would take many instructions on another computer. Such instructions are typified by instructions that take multiple steps, instructions that control multiple functional units, or instructions that appear on a scale larger than the bulk of the simple instructions implemented by a given processor. Some examples of "complex" instructions include storing many registers on the stack at once, moving large blocks of memory, complex integer and floating-point arithmetic (such as sine, cosine, and square root), SIMD instructions, a single instruction that performs operations on many values ​​in parallel, performing atomic test-and-set instructions or other read-modify-write atomic instructions, and instructions that perform ALU operations with operands from memory rather than registers.

[0101] An instruction may be defined according to its parts. According to more traditional architectures, an instruction includes an opcode that specifies an operation to perform, such as adding the contents of memory to a register, and zero or more operand specifiers, which may specify registers, memory locations, or literal data. An operand specifier may have an addressing mode that determines its meaning, or may be in a fixed field. In very long instruction word (VLIW) architectures, which include many microcode architectures, multiple simultaneous opcodes and operands are specified in a single instruction.

[0102] Some types of instruction sets have no opcode field (such as the Transport Trigger Architecture (TTA) or Force Virtual Machine), but only operands. Other unusual "zero operand" instruction sets, such as some stack machines including NOSCs, lack an operand specifier field.

[0103] Conditional instructions often have a predicate field, i.e., several bits that encode a particular condition that causes an operation to be performed rather than not performed. For example, a conditional branch instruction may be executed; if the condition is true, the branch is taken, resulting in execution proceeding to a different part of the program that is not executed; if the condition is false, the branch is not taken, resulting in execution continuing sequentially. Some instruction sets also have conditional moves, resulting in the move being performed and data being stored to a target location if the condition is true; if the condition is false, the move is not executed and the target location is not modified. Similarly, the IBM z / Architecture has conditional stores. Some instruction sets include a predicate field for each instruction, called the branch predicate.

[0104] The instructions that make up a program are rarely specified using their internal numeric form (machine code); they may be specified using assembly language, or more typically, generated from a programming language by a compiler.

[0105] The present invention will now be more fully described by reference to the following non-limiting examples.

[0106] Example 1: Interrogation of an aptamer-based biosensor by applying a potential change to the working electrode

[0107] This example utilized an electrochemical biosensor with an aptamer capable of selectively binding to vancomycin. The aptamer was labeled with methylene blue as a redox-active reporter.

[0108] The labeled aptamer was attached to the surface of a gold electrode via a thiol bond with a methylene blue label at the distal end of the aptamer. The gold electrode served as the working electrode in the biosensor device. Upon selective binding of vancomycin, the aptamer changed conformation, thereby bringing the methylene blue label closer to the electrode surface.

[0109] The biosensor device further included a silver / silver chloride reference electrode and a platinum counter electrode.

[0110] The working and counter electrodes were immersed in solutions containing various concentrations of vancomycin. Using a potentiostat (PalmSens BV, Houten, Netherlands), the working electrode was held at an initial potential of -0.4 V relative to the silver chloride reference electrode for 1 second. The potential of the gold electrode was then changed stepwise to -0.2 V and maintained at that potential (again using the potentiostat) for 50 milliseconds. The current through the working electrode was recorded over 50 milliseconds at 20-microsecond intervals. To reduce possible random fluctuations in the current measurements, a 20-point running average of the current was calculated for each time point.

[0111] The mean current was determined for time points 0.5 ms (i(t1)), 6.7 ms (i(t2)), and 17 ms (i(t3)), which were obtained with reference to the change in potential. Referring to Figure 3, the initial potential, the change in potential, and time points t1, t2, and t3 are shown graphically.

[0112] The current ratios were determined as follows: i(t1) / i(t3) and i(t2) / i(t3) were calculated. The f value was determined using equation (3), which was derived using equations (1) and (2). In equation (3), terms up to n=10 were used to approximate an infinite series of exponential functions.

number

number

[0113] Note that the ratio i(t2) / i(t3) is present in the calculation of f, although it is not explicitly shown. To explain further, the variable r2 is actually:

number

[0114] Three separate measurements of f were made and averaged to obtain the f values ​​graphed in FIGS.

[0115] The calculated f values ​​were found to be in good agreement with the expected Langmuir binding isotherm over the range of concentrations shown.

[0116] Example 2: Demonstration of low variability in aptamer-based biosensors across a range of conditions and electrodes

[0117] The experimental arrangement described in Example 1 was used in this second example to demonstrate the ability of the interrogation method to correct for inter-electrode variations, temperature variations, and drift in electrode response over time.

[0118] Four different electrodes (E1, E2, E3, and E4) were used: two electrodes (E1, E2) were operated at 32°C, and the remaining electrodes (E3, E4) were operated at 41°C.

[0119] These electrodes were tested in a phosphate buffered saline solution that did not contain vancomycin, the target of the aptamer in the sensing layer.

[0120] Referring to Figure 4, the ability of the method to account for inter-electrode variations, temperature variations, and drift in electrode response over time is illustrated.

[0121] The sensors were continuously queried for 25 hours, and the current was plotted for each electrode at t1 (0.5 ms) at approximately 1 hour intervals in Figure 4. This i(t1) data represents the actual variation in sensor response caused by electrode-to-electrode variations during manufacturing, test temperature, and degradation of the sensing layer on the electrode over time. The data shown is the average of three repeated current transients.

[0122] Figure 4 also shows data from the same test, but analyzed using the present method to obtain f values ​​according to equation (3), background corrected by subtracting the average of the zero-time responses between the electrodes.

[0123] These results demonstrate the ability of the present invention to significantly reduce inter-electrode variations, temperature-induced variations, and signal output drift over time in the sensor response.

[0124] Example 3: Demonstration of the AMULTIPULSE method for improving discrimination between different target analyte concentrations in aptamer-based biosensors

[0125] The experimental configuration described in Example 1 was used in this third example to demonstrate the ability of the multipulse method to improve the ability to distinguish between different target analyte concentrations. The data shown in Figures 5-9 are for a single sensing electrode tested in 0-25 micromolar solutions of vancomycin in phosphate-buffered saline at 37°C. Five different interrogation pulses were used. Each pulse terminated at a potential of -0.15 V. The first pulse began at a potential of -0.4 V, the second pulse began at a potential of -0.425 V, the third pulse began at a potential of -0.45 V, the fourth pulse began at a potential of -0.475 V, and the fifth pulse began at a potential of -0.5 V. Each potential is shown relative to a silver / silver chloride reference electrode. The current shown is the average of five repeated pulses.

[0126] Figure 5 shows the currents recorded over time for five different pulses, labeled P1 to P5 in the legend, in the absence of vancomycin.

[0127] Using the data shown in Figure 5, Figure 6 shows an exemplary plot of current at various times (shown in seconds in the legend) plotted against the size of the potential step used for the pulse in volts (V). The dotted line is the linear least-squares regression line. This figure shows the excellent linearity of these plots with respect to time, confirming that linear least-squares regression is an appropriate technique for extrapolating current to zero potential step size in this case.

[0128] Figure 7 shows current versus time plots using potential steps of -0.4 V to -0.15 V for various concentrations of vancomycin present in the test solution from 0 to 25 micromolar, with the legend indicating the concentration of vancomycin present in micromolar.

[0129] FIG. 8 shows the current extrapolated to zero potential step size using five different pulse sizes for the same vancomycin solution used in FIG.

[0130] The improved separation between the current plots for different vancomycin concentrations in Figure 8 compared to Figure 7 demonstrates the ability of the multipulse method to improve concentration discrimination in the sensor response.

[0131] Figure 9 shows the f values ​​calculated using the currents shown in Figure 7 (circular points labeled P1 in the legend) and the f values ​​calculated using the currents shown in Figure 8 (diamond points labeled Extrap. in the legend), corrected for the zero vancomycin concentration value, demonstrating the improved dynamic range and therefore sensitivity of the results when using the extrapolated current data.

[0132] Example 4: Demonstration of a double-pulse method to improve discrimination between different target analyte concentrations in an aptamer-based biosensor

[0133] The experimental configuration described in Example 1 was used in this example to demonstrate the ability of the double-pulse method to improve the ability to discriminate between different target analyte concentrations. The data shown in Figures 10-13 are for a single sensing electrode tested in 0-25 micromolar solutions of vancomycin in phosphate-buffered saline at 37°C. Two different query potential steps were used. The first potential step started at a potential of -0.45 V and ended at a potential of -0.2 V. The second potential step started at a potential of -0.2 V and ended at -0.1 V. Each potential is shown relative to a silver / silver chloride reference electrode.

[0134] Figure 10 shows the current versus time plot using potential steps of -0.45 V to -0.2 V for various concentrations of vancomycin present in the test solution, from 0 to 25 micromolar; the legend indicates the concentration of vancomycin present in micromolar. The current shown is the average of five repetitive potential pulses. Here we show how the current varies with vancomycin concentration.

[0135] Figure 11 shows current versus time plots using potential steps of -0.2 V to -0.1 V for various concentrations of vancomycin present in the test solution, from 0 to 25 micromolar; the legend indicates the concentration of vancomycin present in micromolar. The current shown is the average of five repeated potential pulses, demonstrating that the current is essentially invariant with vancomycin concentration.

[0136] Figure 12 shows current versus time plots for various vancomycin concentrations where the currents shown in Figure 11 were multiplied by 2.5 and subtracted from the currents in Figure 10. The factor of 2.5 was used to compensate for the potential step size used to generate the currents shown in Figure 10, which was 0.25 V, while a potential step size of 0.1 V was used to generate the currents in Figure 11, where the magnitude of the capacitive charging current is expected to be proportional to the potential step size.

[0137] Figure 13 shows f values ​​calculated using the currents shown in Figure 10 (circular points labeled P1 in the legend) and the currents shown in Figure 11 (diamond points labeled P1-2.5P2 in the legend), demonstrating the improved dynamic range, and therefore improved sensitivity, of the results when the second potential step current is subtracted from the first potential step current. The f values ​​have been corrected for background values.

[0138] Those skilled in the art will appreciate that the invention described herein is susceptible to further variations and modifications other than those specifically described, and it is to be understood that the invention includes all such variations and modifications that are within the spirit and scope of the invention.

[0139] The present invention is described primarily by referring to aptamer-based biosensors in which a redox reporter is covalently linked to an aptamer. The aptamer may be a DNA or RNA aptamer, or some analogs such as XNA (heterogeneous nucleic acid) or PNA (peptide nucleic acid). Given the benefit of this disclosure, those skilled in the art will be able to implement the present invention in a variety of alternative formats.

[0140] For example, the binding element may be an antibody, an antibody fragment (such as a Fab fragment), a natural or synthetic polymer containing an analyte binding site, a peptide, a small molecule, an antigen, or any other element that can be attached to an electrode and specifically bind to an analyte of interest.

[0141] Furthermore, the redox-active reporter need not be covalently bound to the binding member. In some embodiments, the redox-active species is the analyte itself, which is bound to the binding member and thereby tethered and spatially restricted. In other embodiments, the redox-active reporter is directly bound to the analyte itself or is associated with a separate species (such as a redox-labeled antibody) that is bound to the analyte.

Claims

1. 1. A method for determining the amount of an analyte in a fluid, comprising: applying a first potential change to an electrochemical sensor working electrode, the electrode having associated therewith (i) a binding element and (ii) an associated redox-active species spatially confined within a layer adjacent to the electrode surface; measuring, at a plurality of time points, the current resulting from application of at least the first potential change; determining the distribution of the redox active species by referring to the measured current value; and using the determined distribution of redox active species to determine the amount of analyte bound to the binding member.

2. The method of claim 1 , wherein the first potential change is substantially instantaneous.

3. The method of claim 1 , wherein the first potential change is provided as a step change.

4. 2. The method of claim 1, wherein the first potential change is configured to change the redox state of the redox active species from one redox state to another redox state, resulting in a transfer of electrons between the redox active species and the electrode surface.

5. 2. The method of claim 1, wherein the first potential change is configured such that current flowing through the electrode surface as a result of the redox-active species changing its redox state is controlled by mass transport of the redox-active species through the layer adjacent to the electrode surface.

6. 10. The method of claim 1, wherein the potential resulting from the first potential change is maintained for at least about 10 ms, 20 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, or 100 ms.

7. The method of claim 1 , comprising applying an initial potential before applying the first potential change.

8. 8. The method of claim 7, wherein the initial potential is selected such that the magnitude of the first potential change is at least about 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, or 1.0 V.

9. 8. The method of claim 7, wherein the initial potential is maintained for at least about 0.05 seconds, 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.4 seconds, 0.5 seconds, 0.6 seconds, 0.7 seconds, 0.8 seconds, 0.9 seconds, 1.0 seconds, 1.1 seconds, 1.2 seconds, 1.3 seconds, 1.4 seconds, 1.5 seconds, 1.6 seconds, 1.7 seconds, 1.8 seconds, 1.9 seconds, or 2.0 seconds.

10. The method of claim 1 , further comprising measuring at least a first current value after applying the first potential change.

11. The method of claim 10 , wherein the first current value is measured substantially immediately after application of the first potential change.

12. 11. The method of claim 10, wherein the first current value is measured less than about 0.1 ms, 0.2 ms, 0.3 ms, 0.4 ms, 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, or 1.0 ms after application of the first potential change.

13. 11. The method of claim 10, comprising measuring at least first and second current values ​​in chronological order after application of the first potential change.

14. 11. The method of claim 10, comprising measuring at least first, second, and third current values ​​in chronological order after application of the first potential change.

15. 11. The method of claim 10, comprising measuring at least first, second, third, and fourth current values ​​in chronological order after application of the first potential change.

16. The method of claim 10 , wherein the measurement of the first current value is performed during a period in which the potential resulting from the first potential change is maintained.

17. 11. The method of claim 10, wherein the first current value is an average current value, the average current value being determined by reference to a plurality of current values ​​determined up to, over, or after a point in time.

18. 18. The method of claim 17, wherein the average current value is determined by reference to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 current values.

19. The method of claim 13 , comprising generating at least one current ratio by referencing the first current value and the second current value.

20. 20. The method of claim 19, wherein the first current value is measured substantially immediately after application of the first potential change.

21. 21. The method of claim 20, wherein the first current value is measured within about 0.1 ms, 0.2 ms, 0.3 ms, 0.4 ms, 0.5 ms, 0.6 ms, 0.7 ms, 0.8 ms, 0.9 ms, 1.0 ms, 2.0 ms, 3.0 ms, 4.0 ms, or 5.0 ms after application of the first potential change.

22. 20. The method of claim 19, wherein the second current value is measured after the first current value.

23. 20. The method of claim 19, wherein the second current value is measured within about 1.0 ms, 2.0 ms, 3.0 ms, 4.0 ms, 5.0 ms, 6.0 ms, 7 ms, 8 ms, 9 ms, 10 ms, 11 ms, 12 ms, 13 ms, 14 ms, 15 ms, 16 ms, 17 ms, 18 ms, 19 ms, 20 ms, 21 ms, 22 ms, 23 ms, 24 ms, or 25 ms after the first current value is measured or after the application of the first potential change.

24. 11. The method of claim 10, further comprising applying a second potential change to the electrochemical sensor working electrode after the first potential change.

25. 25. The method of claim 24, wherein the sign of the second potential change is the same as the sign of the first potential change.

26. 25. The method of claim 24, wherein the second potential change is used to estimate the capacitive charging current of the sensor device and the faradaic current from redox species not confined in a layer adjacent to the electrode, and therefore the background current draw.

27. 27. The method of claim 26, wherein the background current draw is subtracted from the first current value.

28. 25. The method of claim 24, wherein the first and / or second potential changes are substantially instantaneous.

29. 25. The method of claim 24, wherein the first and / or second potential changes are substantially step changes.

30. applying a first potential change and at least a second potential change; combining a selected current resulting from the at least second potential change with a selected current resulting from the first potential change to derive a current; and determining the analyte concentration from the derived current.

31. 31. The method of claim 30, wherein the at least a second potential change varies from a potential applied at the end of the first potential change.

32. 31. The method of claim 30, wherein the selected currents are combined by subtracting a function of the magnitude of the current resulting from the at least second potential change from the magnitude of the current resulting from the first potential change, and wherein the time at which the current is sampled after the potential change is substantially the same for both the first potential change and the at least second potential change.

33. 33. The method of claim 32, wherein the magnitude of the current resulting from the at least second potential change is multiplied by a coefficient and subtracted from the current resulting from the first potential change.

34. 31. The method of claim 30, comprising combining currents resulting from the first potential change and the at least second potential change by using currents measured simultaneously after potential changes and the magnitude of the potential changes used to generate the currents, and extrapolating the measured currents to a value corresponding to zero change in potential.

35. 35. The method of claim 34, wherein the extrapolation is a linear extrapolation.

36. 35. The method of claim 34, wherein the extrapolation is a non-linear extrapolation.

37. a working electrode having associated therewith (i) a binding element and (ii) an associated redox-active species spatially confined within a layer adjacent to the electrode surface; a microprocessor-based controller; and 10. An electrochemical sensor device or system, wherein the microprocessor-based controller is configured to perform the method of claim 1.

38. 38. The electrochemical sensor device or system of claim 37, wherein the microprocessor-based controller is in electrical communication with the working electrode or is in wired or wireless network communication with another microprocessor-based controller that is in electrical communication with the working electrode.

39. 38. The electrochemical sensor device or system of claim 37, wherein the microprocessor-based controller is configured to access and execute program instructions to carry out the method of claim 1.

40. 40. The electrochemical sensor device or system of claim 39, comprising a variable power supply electrically connected to the working electrode.

41. 38. An electrochemical sensor device or system according to claim 37, comprising a current measurement circuit configured to measure the current through the working electrode.

42. 42. The electrochemical sensor device or system of claim 41, comprising an electronic memory operatively associated with the microprocessor-based controller and the current measurement circuitry, the electronic memory configured to store one or more currents measured by the current measurement circuitry.

43. 43. The electrochemical sensor device or system of claim 42, wherein the microprocessor-based controller is configured to process the one or more currents measured by the current measurement circuit to provide a quantity of an analyte.

44. A computer readable medium comprising program instructions configured to carry out the method of any one of claims 1 to 36.

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