Method and device for determining an amount of an analyte in a fluid

Abstract

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

Description

Technical Field

[0001] The present invention generally relates to electrochemical sensors that can be used to determine the amount of an analyte in biological fluids such as blood and interstitial fluid. More particularly, the present invention provides an electrochemical sensor that operates by an improved chronoamperometry method that provides more reliable analyte determination under a range of conditions. Background Art

[0002] Some classes of electrochemical sensors are selective and capable of real-time, continuous detection of a target analyte as well as single-point measurements, where the target analyte includes exogenous agents (e.g., pharmaceutical compounds and toxins) and endogenous agents (e.g., metabolites, proteins, hormones, etc.).

[0003] These sensors can include an electrode with an associated binding element that undergoes a conformational change upon analyte binding. The conformational change alters the accessibility of a redox reporter molecule to the electrode surface, thereby producing an analyte-induced change in the electron exchange between the redox reporter molecule and the electrode. In some cases, analyte binding brings the redox reporter molecule closer to the electrode surface, thereby increasing the rate of electron exchange and, in turn, increasing the current passing through the electrode. In other cases, binding moves the reporter molecule away from the electrode surface, resulting in the opposite effect. In any case, analyte binding results in a detectable change in the electrode current.

[0004] Electrochemical sensors can be implemented in a variety of forms, one of which is a microneedle-based patch that is applied to the skin such that the electrodes contact interstitial fluid. The tips of the microneedles serve as sensor electrodes, where the binding element is associated with the tip. This arrangement 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 the sensors of the prior art are undoubtedly useful, they suffer from a number of problems.

[0006] For example, the response of a whole blood glucose sensor can vary depending on the viscosity of the blood, which in turn can vary depending on temperature, hematocrit, and lipid content. In most devices of the prior art, this undesirable variation is addressed by using a correction factor in the calculation of the analyte amount. A separate temperature sensor can be provided that is calibrated based on a population data pool.

[0007] Signal drift is an important problem in continuous electrochemical sensors. Those skilled in the art have proposed methods for using electrochemical square wave voltammetry to quantify bound analytes, where the problem of signal drift over time is addressed using measured values and multiple square wave frequencies. However, such methods require careful selection of specific frequency pairs, where the signal drift at two frequencies matches empirically. There remains uncertainty as to whether the selected frequencies are applicable in all cases (e.g., at different temperatures or for different devices affected by manufacturing variations).

[0008] Variations in the sensor output caused by the electrode area, layer thickness, and diffusion coefficient and density of redox species adjacent to the electrode surface give rise to additional problems.

[0009] The present invention seeks to overcome or at least ameliorate these deficiencies in the prior art by providing an easily implemented and extremely rapid method, whereby the quantification of the amount of analyte bound near or far from the electrode can be determined in a manner that is more immune to variations in the sensing environment and manufacturing variations from sensor to sensor and changes over time of the device before and during use.

[0010] One aspect of the present invention is to provide an improvement in the operation of an electrochemical sensor so as to ameliorate or overcome any one or more of the undesired variations, inaccurate outputs, imprecise outputs, and non - reproducible outputs in the output. Another aspect of the present invention is to dispense with auxiliary means in the electrochemical sensor for addressing any one or more of the undesired variations, inaccurate outputs, imprecise outputs, and non - reproducible outputs in the output. Another aspect of the present invention is to provide a useful alternative to the prior art methods of interrogating electrochemical sensors.

[0011] The discussion of documents, acts, materials, devices, articles, etc. is included in this specification solely to provide a context for the present invention. There is no implication or representation that any or all of these matters form part of the prior art base because they existed before the priority date of each provisional claim of this application, or are common general knowledge in the field related to the present invention. Summary of the Invention

[0012] In a first aspect, but not necessarily the broadest aspect, the present invention provides a method for determining the amount of an analyte in a fluid, the method comprising:

[0013] Applying a first change in potential to a working electrode of an electrochemical sensor, the electrode having associated therewith (i) a binding element and (ii) an associated redox - active species spatially constrained within a layer adjacent to the electrode surface;

[0014] Measure current values generated by the application of at least a first change in potential at multiple time points;

[0015] Determine the distribution of redox-active species by reference to the measured current values; and

[0016] Use the determined distribution of redox-active species to determine the amount of analyte bound to the binding element.

[0017] In one embodiment of the first aspect, the change in potential is substantially instantaneous.

[0018] In one embodiment of the first aspect, the change in potential is implemented as a step change.

[0019] In one embodiment of the first aspect, the first change in potential is configured to change the redox state of the redox-active species from one redox state to another such that electrons are transferred between the redox-active species and the electrode surface.

[0020] In one embodiment of the first aspect, the first change in potential is configured such that the current flowing across the electrode surface due to the change in redox state of the redox-active species is controlled by mass transport of the redox-active species through a layer adjacent to the electrode surface.

[0021] In one embodiment of the first aspect, the potential generated by the first change in potential 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.

[0022] In one embodiment of the first aspect, the method includes applying an initial potential prior to the application of the first change in potential.

[0023] In one embodiment of the first aspect, the initial potential is selected such that the magnitude of the first change in potential 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.

[0024] In one embodiment of the first aspect, the initial potential is maintained for at least about 0.05 s, 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, 1.0 s, 1.1 s, 1.2 s, 1.3 s, 1.4 s, 1.5 s, 1.6 s, 1.7 s, 1.8 s, 1.9 s, or 2.0 s.

[0025] In one embodiment of the first aspect, the method includes measuring at least a first current value after the application of the first change in potential.

[0026] In one embodiment of the first aspect, the first current value is measured substantially immediately after the application of the first change in potential.

[0027] In one embodiment of the first aspect, the first of the first current values is measured within 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 the application of the first change in potential.

[0028] In one embodiment of the first aspect, the method includes measuring at least a first current value and a second current value in chronological order after the application of the first change in potential.

[0029] In one embodiment of the first aspect, the method includes measuring at least a first current value, a second current value, and a third current value in chronological order after the application of the first change in potential.

[0030] In one embodiment of the first aspect, the method includes measuring at least a first current value, a second current value, a third current value, and a fourth current value in chronological order after the application of the first change in potential.

[0031] 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) occur during a period in which the potential generated by the first change in potential is maintained.

[0032] In one embodiment of the first aspect, the first current value, the second current value (if measured), the third current value (if measured), and the fourth current value (if measured) are average current values that are determined by reference to a plurality of current values determined at approximate time points, across time points, or after time points.

[0033] 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.

[0034] In one embodiment of the first aspect, the method includes generating at least one current ratio by reference to the first current value and the second current value.

[0035] In one embodiment of the first aspect, the first current value is measured substantially immediately after the application of the first change in potential.

[0036] In one embodiment of the first aspect, a 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 the application of a first change in potential.

[0037] In one embodiment of the first aspect, a second current value is measured after the first current value.

[0038] In one embodiment of the first aspect, a 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 measurement of the first current value or after the application of the first change in potential.

[0039] In one embodiment of the first aspect, the method includes applying a second change in potential to the working electrode of the electrochemical sensor after the first change in potential.

[0040] In one embodiment of the first aspect, the sign of the second change in potential is the same as the sign of the first change in potential.

[0041] In one embodiment of the first aspect, the sign of the second change in potential is opposite to the sign of the first change in potential.

[0042] In one embodiment of the first aspect, the second change in potential is used to estimate the capacitive charging current of the sensor device and the current due to redox species not confined to the layer adjacent to the electrode, and thus to estimate the background current consumption.

[0043] In one embodiment of the first aspect, the background current consumption 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 consumption can be multiplied by a factor before subtracting it from the first current value, the second current value, the third current value, or the fourth current value (if they are measured).

[0044] In one embodiment of the first aspect, the first and / or second change in potential is a substantially instantaneous change.

[0045] In one embodiment of the first aspect, the first and / or second change in potential is a substantially stepwise change.

[0046] In an embodiment of the first aspect, the method comprises the steps of:

[0047] Applying a first change in potential and at least a second change in potential;

[0048] Combining a selected current resulting from at least the second change in potential with a selected current from the first change in potential to derive a current; and

[0049] Determining an analyte concentration from the derived current.

[0050] In an embodiment of the first aspect, at least the second change in potential changes from the potential applied at the end of the first change in potential.

[0051] In an embodiment of the first aspect, the selected currents are combined by subtracting a function of the magnitude of the current resulting from at least the second change in potential from the magnitude of the current resulting from the first change in potential, wherein for both the first change in potential and at least the second change in potential, the time at which the current is sampled after the change in potential is substantially the same.

[0052] In an embodiment of the first aspect, the magnitude of the current resulting from at least the second change in potential is multiplied by a factor and subtracted from the current resulting from the first change in potential.

[0053] In an embodiment of the first aspect, the method comprises the steps of: combining the currents resulting from the first change in potential and at least the second change in potential by using the current measured simultaneously after the change in potential and the magnitude of the change in potential used to generate the current, to extrapolate the measured current to a value corresponding to zero change in potential.

[0054] In an embodiment of the first aspect, the extrapolation is a linear extrapolation.

[0055] In an embodiment of the first aspect, the extrapolation is a non - linear extrapolation.

[0056] In a second aspect, the present invention provides an electrochemical sensor device or system, comprising:

[0057] A working electrode having (i) a binding element associated therewith and (ii) an associated redox - active species spatially confined within a layer adjacent to the electrode surface; and

[0058] A microprocessor - based controller,

[0059] wherein the microprocessor - based controller is configured to perform the method of any embodiment of the first aspect.

[0060] In an embodiment of the second aspect, a microprocessor-based controller is electrically connected to the working electrode, or is network-connected, either wired or wirelessly, to another microprocessor-based controller that is electrically connected to the working electrode.

[0061] In an embodiment of the second aspect, the microprocessor controller is configured to access and execute program instructions to perform the method of any embodiment of the first aspect.

[0062] In an embodiment of the second aspect, the electrochemical sensor device or system includes a variable power source electrically connected to the working electrode, wherein the program instructions direct the power source to apply a potential to the working electrode according to the method of any embodiment of the first aspect.

[0063] In an embodiment of the second aspect, the electrochemical sensor device or system includes a current measurement circuitry configured to measure the current passing through the working electrode.

[0064] In an embodiment of the second aspect, the electrochemical sensor device or system includes an electronic memory operably associated with the microprocessor-based controller and the current measurement circuitry, the electronic memory being configured to store one or more currents measured by the current measurement circuitry.

[0065] In an embodiment of the second aspect, the microprocessor-based controller is configured to process one or more currents measured by the current measurement circuitry to provide an analyte quantity.

[0066] In a third aspect, the present invention provides a computer-readable medium that includes program instructions configured to execute the method of any embodiment of the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 is a plot of the f value versus the concentration of vancomycin in micromoles. The points are the calculated f values, corrected for the f value when vancomycin is absent. The shown curve is given by fitting the Langmuir binding isotherm equation to the data. This plot represents the response of a gold electrode coated with a vancomycin aptamer and tested at 32 °C.

[0068] Figure 2 is a plot of the f value versus the concentration of vancomycin in micromoles. The points are the calculated f values, corrected for the f value when vancomycin is absent. The shown curve is given by fitting the Langmuir binding isotherm equation to the data. This plot represents the response of a gold electrode coated with a vancomycin aptamer and tested at 41 °C.

[0069] Figure 3 is a graphical representation of the potential applied during the experiment detailed in Example 1.

[0070] Figure 4 It is a coordinate graph showing the relationship between the f value and time (in minutes). This coordinate graph shows the f values and current values taken at t1 (0.5 ms) for different electrodes interrogated under different temperature conditions. The f values are corrected for the background response.

[0071] Figure 5 It is a coordinate graph showing the relationship between current (in microamperes) and time. This coordinate graph shows the variation of current with time for a sensor using five different potential step sizes.

[0072] Figure 6 It is a coordinate graph showing the relationship between current (in microamperes) and potential step size (in volts). This coordinate graph shows an example of the sensor current at a specific time plotted against the magnitude of the potential step used.

[0073] Figure 7 It is a coordinate graph showing the relationship between current (in microamperes) and time (in seconds). This coordinate graph shows the variation of current with time for one of the potential step sizes at various vancomycin concentrations.

[0074] Figure 8 It is a coordinate graph showing the relationship between current (in microamperes) and time (in seconds). This coordinate graph shows the variation of current with time for the extrapolation to zero potential step size at various vancomycin concentrations.

[0075] Figure 9 It is a coordinate graph showing the relationship between the f value and the concentration of vancomycin (in micromoles). This coordinate graph shows the f values calculated using the current caused by one of the potential step sizes and the extrapolated current to zero potential step size for various vancomycin concentrations. The f values are corrected for the background response.

[0076] Figure 10 It is a coordinate graph showing the relationship between current (in microamperes) and time (in seconds). This coordinate graph shows the variation of current with time for the first change in potential at various vancomycin concentrations.

[0077] Figure 11 It is a coordinate graph showing the relationship between current (in microamperes) and time (in seconds). This coordinate graph shows, for various vancomycin concentrations, the current variation with time starting from the end potential of the first change in potential used to generate the current shown in Figure 10 for the second change in potential.

[0078] Figure 12It is a coordinate graph showing the relationship between current (in microamperes) and time (in seconds). This coordinate graph shows the variation of current over time, where the current from the second change in potential has been multiplied by 2.5 and subtracted from the current from the first change in potential.

[0079] Figure 13 It is a coordinate graph showing the relationship between the f value and the concentration of vancomycin (in micromoles). This coordinate graph shows the f values calculated using the current from Figure 10 for various vancomycin concentrations and the current from Figure 12 The f values are corrected for the background response.

[0080] Unless otherwise specified herein, when used across different figures, the features of the figures labeled with the same reference numerals are considered to be the same features, or at least functionally similar features.

[0081] Except for the coordinate graphs, the figures are not drawn to any specific scale or size and are not presented as a completely accurate representation of the various embodiments. Detailed Description

[0082] After considering this description, it will be apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, although various embodiments of the present invention will be described herein, it should be understood that these embodiments are presented by way of example only and not by way of limitation. Thus, such a description of the various alternative embodiments should not be construed as limiting the scope or breadth of the present invention. In addition, statements of advantages or other aspects apply to specific exemplary embodiments and do not necessarily apply to all embodiments, or indeed any embodiment covered by the claims.

[0083] Throughout this specification and the claims, the word "comprise" and variations of the word such as "comprising" and "comprises" are not intended to exclude other additives, components, integers or steps.

[0084] References to "an embodiment" or "embodiments" throughout this specification mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in an embodiment" or "in embodiments" that appear throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment.

[0085] Where the terms "determine", "determining" and "determined" are used, these terms are not intended to necessarily be construed as meaning any perfectly accurate determination, although that meaning is not excluded. These terms may be construed to include estimation, approximation or even indication.

[0086] In a first aspect, the present invention provides a method for determining the amount of an analyte in a fluid, the method comprising:

[0087] Applying a first change in potential to a working electrode of an electrochemical sensor, 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;

[0088] Measuring current values generated at least by the application of the first change in potential at a plurality of time points;

[0089] Determining the distribution of the redox-active species by reference to the measured current values; and

[0090] Using the determined distribution of the redox-active species to determine the amount of analyte bound to the binding element.

[0091] 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 a 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 permit electron transfer across the electrode / solution interface. Stated alternatively, the potential is changed such that the current flowing across the electrode surface as a result of the redox-active species changing redox state is predominantly or substantially controlled by mass transport of the redox-active species towards the electrode. As a result of the change in potential, a net current flows across the electrode surface, typically with the magnitude of the current varying with time.

[0092] Without wishing to be bound by theory in any way, it is proposed that redox-active reporter molecules are bound to the working electrode surface (e.g. by an aptamer) and are thus able to move only within a layer adjacent to the electrode, however the redox-active species diffuse substantially freely within the layer. Thus, the movement of the redox-active species can be modelled with a certain degree of accuracy (although not necessarily perfectly accurately) according to Fick's first and second laws of diffusion. Thus, even when bound, the redox-active species can still move from regions of high concentration to regions of low concentration in the manner expected for freely diffusible (i.e. unbound) species, with the magnitude of the flux being proportional to the difference in concentration. In addition, the concentration gradient of the redox-active species in the layer changes over time in the manner expected for freely diffusible species.

[0093] Fick's first and second laws are applied to systems in which the redox-active species are spatially confined within a layer adjacent to the electrode surface, but are able to diffuse freely within that layer. Thus, assuming that electron transfer increases when the redox-active species is closer to the surface, it is possible to model the diffusion of the confined redox species, and therefore the change in current over time (called "current transient") due to the movement of the redox-active species relative to the electrode surface.

[0094] The resulting simulated current transients resulting from the movement of redox-active species reveal 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 total 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 upon application of a potential step. These revelations have been found to have practical implications with respect to methods for interrogating electrochemical sensor devices, determining the distribution of redox-active species, and thereby determining the amount of analyte bound to a binding element.

[0095] Further studies were performed to demonstrate the practical relevance of the single current measurements and the current ratio determined at the time immediately after the applied potential change. It was surprisingly found that the ratio of the two measured currents at longer times after the applied potential change depends 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, whereas the ratio of the current at shorter times after the applied potential step compared to the current at longer times depends more strongly on the initial distribution of the redox species and strongly on the thickness of the layer adjacent to the electrode and the diffusion coefficient of the redox species. Furthermore, both current ratios were modeled to be insensitive to the total concentration of the redox species and to the electrode area.

[0096] The behavior of these simulations suggests that later current ratios can be used to obtain a combined measure of the redox species diffusion coefficient and the thickness of the layer adjacent to the electrode, which can be applied to earlier current ratios to obtain an estimated measure of the initial distribution of the redox species, where the estimate is largely insensitive to changes in the total 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.

[0097] The equation for the current versus time derived from the model is shown in equation (1) below:

[0098]

[0099] in:

[0100] i(t) is the current at time t,

[0101] z is the number of moles of electrons transferred per mole of redox species oxidized or reduced at the electrode surface,

[0102] F is the Faraday constant,

[0103] A is the area of the electrode,

[0104] D is the diffusion coefficient of the redox species in the layer adjacent to the electrode,

[0105] C0 is the total concentration of the redox species in the layer adjacent to the electrode,

[0106] l is the thickness of the layer adjacent to the electrode containing the redox species, and

[0107] f is the fraction of the redox species near or at the electrode surface when a potential step is applied.

[0108] Thus, f can be used to determine the distribution of redox-active species at the instant when the initial potential is applied and before the change in the applied potential. The distribution of redox-active species can be used to determine the amount of analyte bound to the binding element of the sensor.

[0109] 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. Further, at long enough times, the exponential term for n > 0 will be small enough relative to the exponential term for n = 0 such that Equation (1) can be approximated by Equation (2) as follows:

[0110]

[0111] Thus, by taking the ratio of the currents at two different times when applying Equation (2), only the exponential term does not cancel out, and an estimate of D / l 2 can be obtained.

[0112] According to this method, the currents at at least three different times during the current transient can be determined: i(t1), i(t2), and i(t3). Optionally, the current i(t4) at a fourth time can be determined. Time t1 is chosen to be a short time after the change in the applied potential step, t3 and optionally t4 are chosen to be longer times after the applied potential step, and t2 is chosen to be between t1 and t3 or optionally between t1 and t4.

[0113] Then the ratio of at least two currents can be determined. In a preferred embodiment of the present invention, the ratios i(t1) / i(t3) and i(t2) / i(t3) are determined. In other embodiments of the present invention, the ratios i(t1) / i(t4) and / or i(t2) / i(t4) are determined. In some embodiments of the present invention, the current ratio at an additional time can be calculated and used to improve the method by providing an additional estimate of the derived parameter. The alternative ratios i(t1) / i(t4) and / or i(t2) / i(t4) can be used to calculate an additional estimate of the f value to assist in verifying the accuracy of the calculation using the i(t1) / i(t3) and i(t2) / i(t3) ratios.

[0114] In another embodiment of the method, after a first change in the electrode potential, a second change in the electrode potential is performed. In this embodiment, the electrode is held at the potential generated by the first change in potential and for a sufficient time to substantially electrochemically oxidize or reduce all redox-active species present in the binding layer. Then the electrode potential is changed in the direction of the initial potential. For example, if the electrode potential was initially held at the value when the redox-active species was reduced, the second potential change will be in the direction of a more reducing potential. If the electrode potential was initially held at the value when the redox-active species was oxidized, the second step will be in the direction of a more oxidizing potential. The potential applied after the second step is selected such that there is substantially no Faraday current due to sensing redox species in the current flowing after the application of this potential. The current generated by the second potential change can be used to obtain an estimate of the non-Faraday current flowing at the electrode (due to, for example, capacitive double-layer charging) and any Faraday current from redox species not confined in the layer adjacent to the electrode, and subtracted from the current used to calculate the current ratios disclosed above to improve the accuracy of the result. The current from the second potential change can optionally be multiplied by a factor and then subtracted from the current used to calculate the current ratios disclosed above. In an alternative of this embodiment, the second change in the electrode potential is in the direction opposite to the direction of the initial potential. The potential applied after the second step is selected such that there is substantially no Faraday current due to sensing redox species in the current flowing after the application of this potential. To achieve this according to this alternative, the potential step should be selected such that it substantially does not change the redox state of the sensing redox species. In this alternative, the current flowing after the application of the second potential step will be opposite in sign to the current flowing after the application of the first potential step. The current from the second potential change can optionally be multiplied by a factor before adding it to the current used to calculate the current ratios disclosed above to improve the accuracy of the result. This alternative may be desirable in cases where, for example, stepping to a more oxidizing potential may cause an additional Faraday current unrelated to the sensing redox species to flow.

[0115] In another embodiment of the present invention, multiple steps of potentials of different magnitudes are applied to the sensor. According to this embodiment, at least two potential steps are applied, where the starting potential or the ending potential of the potential step is 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 step potential can start at -0.45 V and end at -0.15 V, and the third step potential (if used) starts at -0.5 V and ends at -0.15 V. Alternatively, the first step can start at -0.4 V and end at -0.15 V, the second step potential starts at -0.4 V and ends at -0.1 V, and the third step potential (if used) starts at -0.4 V and ends at -0.05 V. The difference in the starting potential or the ending potential between the multiple potential steps is selected such that the current contributed by the Faradaic electron transfer across the electrode interface by the sensed redox species is substantially unchanged for different potential steps, but where the capacitive charging current and possibly the extraneous Faradaic current vary proportionally to the potential step magnitude. The first current value from the first potential step at a selected time t1 is combined with the first current values at time t1 from the second and any subsequent potential steps to extrapolate the first current value to a theoretical value at zero potential step magnitude. Any suitable extrapolation function can be used, where the technique used is appropriate for the type of dependence of the current on the potential step magnitude. For example, if the measured current varies linearly with the potential step magnitude, linear least squares regression can be applied to the relationship between the current values at the selected time for various potential steps and the magnitude of the potential step, and the regression intercept is used as the extrapolated current at zero potential step magnitude. The extrapolated current at zero potential step magnitude is used as an estimate of the Faradaic current of the sensed redox species at this time, which is substantially not disturbed by currents whose magnitudes vary with the potential step magnitude, such as the capacitive charging current and the Faradaic current controlled by the electron transfer rate. When more than two different potential step magnitudes are used in this embodiment, the degree of correlation in the extrapolation plot can be used as a measure of the correct operation of the device. The extrapolation process is repeated for the current values at a second time and a third time and at a fourth time, where the measured and extrapolated values are used to calculate the f value as given elsewhere in this disclosure.

[0116] Advantages of some embodiments of the present invention are that a method is provided which allows the estimation of the distribution of redox species without specific knowledge of the electrode area, the thickness of the bound redox layer, the total amount of redox species, or the number of electrons transferred per mole of redox species. To the applicant's knowledge, the chronoamperometric method that can achieve this has not been previously disclosed. Furthermore, the method of using a simple ratio of currents at different times to derive the distribution has been unknown heretofore.

[0117] Another advantage of some embodiments of the present invention is to provide an amperometric method that is significantly faster than prior art interrogation methods (such as square wave voltammetry methods), which may take from several seconds to several minutes to execute. In some embodiments, the presently described method can be executed in a few milliseconds to a few tens of milliseconds. For example, the electrode is optionally held at an initial potential for a short time (on the order of a few milliseconds to one second), and then the potential is changed in a stepwise manner and held at a second potential typically for up to a few tens of milliseconds. Due to the speed of execution and the fact that redox species are bound to the electrode, the step of the potential can be repeated multiple times in a short time to obtain multiple estimates of the desired parameter, and the multiple estimates can be averaged or otherwise combined to reduce random variations in the results.

[0118] In one embodiment of the present invention, multiple steps of different magnitudes of potential are applied to the sensor. According to this embodiment, at least two potential steps can be applied, where the starting potential or the ending potential of the potential step is different for different potential steps. The first current value from the first potential step can be combined with the first current values from the second and any subsequent potential steps to extrapolate to an extrapolated value at zero potential step size. The process can also be repeated for the second current value, the third current value, and the fourth current value in the case of measurement.

[0119] The difference between the starting potential or the ending potential between multiple potential steps can be selected such that the current contributed by the oxidation or reduction of the sensed redox species is substantially unchanged for different potential steps, but where the capacitive charging current and other possible stray currents vary with the potential step size.

[0120] If the capacitive charging current and other possible stray currents are proportional to the potential step size, a linear extrapolation method such as linear least squares can be used to regress the relationship between the measured current and the potential step size, where the intercept of the regression is used as the extrapolated value.

[0121] If the capacitive charging current and / or other possible stray currents vary non-linearly with the potential step size, a non-linear extrapolation method known in the art can be used to regress the relationship between the measured current and the potential step size, where the intercept of the regression is used as the extrapolated value. Extrapolating the relationship between the measured current and the potential step size to zero potential step size can remove or significantly reduce the contribution of the capacitive charging current and other stray currents that vary with the potential step size to the obtained extrapolated current value. This in turn can improve the ability of the extrapolated current to distinguish between different target analyte concentrations, since a greater proportion of the extrapolated current is due to the presence of the target analyte compared to the case where the non-extrapolated current is used as a measure of the target analyte concentration.

[0122] The present invention can be implemented in the form of an apparatus or system configured to facilitate the execution of the methods described herein.

[0123] The apparatus of the present invention can be implemented in the form of a substantially self - contained wearable device, allowing measurements to be performed while the subject undergoes normal activities and / or over an extended period of time. The wearable device can be a collar, bracelet, band, adhesive, or patch. The wearable device can include transdermal microneedles, where one transdermal microneedle serves as the working electrode of a sensor by contacting the interstitial fluid of the subject and detecting an analyte therein.

[0124] The wearable device can also include a housing structure enclosing one or more other components, such as a processor - based microcontroller. The controller is configured to be in electrical communication with at least one electrode and will generally include a power source, a data processing unit, an electronic memory, and a wireless transmitter / receiver.

[0125] When implemented as a system, the components can be distributed in different physical locations, although still operating in an integrated manner. For example, software instructions can be stored and executed by a smart phone or other remote processor in data communication with a microprocessor - based controller in the wearable device.

[0126] As will be appreciated, the methods described herein can be deployed, in whole or in part, by one or more microprocessors that execute computer software, program code, and / or instructions on the processor. The microprocessor can be any kind of computing or processing device capable of executing program instructions, code, binary instructions, etc.

[0127] Any microprocessor can access a storage medium, such as an electronic memory, through an interface. The storage medium can store the methods, code, and instructions described herein and elsewhere. The storage medium is associated with the processor for storing methods, programs, code, program instructions, or other types of instructions that can be executed.

[0128] Computer software, program code, and / or instructions can be stored and / or accessed on a computer - readable medium, which can include: computer components, devices, and recording media that retain digital data for computing for a certain time interval; semiconductor storage devices known as random access memory (RAM); mass storage devices, which are typically used for more permanent storage, such as non - volatile memory, such as read - only memory (ROM).

[0129] The methods described herein can transform physical and / or intangible items from one state to another. The methods and systems described herein can also transform data representing physical and / or intangible items from one state to another.

[0130] Software products can 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 language (including assembly language, hardware description language, and database programming languages and techniques), which can be stored, compiled, or interpreted to run 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.

[0131] Thus, in one aspect, any method can be implemented in computer - executable code that, when executed on one or more microprocessors, performs its steps. In another aspect, the method can be implemented in a system that performs its steps and can be distributed among devices in various ways, or all functions can be integrated into a dedicated stand - alone device or other hardware. In yet another aspect, the apparatus for performing the steps associated with the above - mentioned process can include any one of the above - mentioned hardware and / or software. All such arrangements and combinations are intended to fall within the scope of the present disclosure.

[0132] The present invention can be implemented in a set of program instructions executable on one or more microprocessors. Such a set of instructions can include any one or more of the following types of instructions:

[0133] Data processing and memory operations, which can include instructions for: setting a register to a fixed constant value; copying data from a memory location to a register and vice versa; storing the contents of a register, the result of a calculation; or retrieving stored data to perform a calculation on it later; or reading and writing data to / from a hardware device.

[0134] Arithmetic and logical operations, which can include instructions for: adding, subtracting, multiplying, or dividing the values of two registers, placing the result in a register, and possibly setting one or more condition codes in a status register; performing bit - by - bit operations, such as conjuncting and disjuncting corresponding bits in a pair of registers, inverting each bit in a register; or comparing two values in a register (e.g., to determine if one value is smaller, or if they are equal).

[0135] Control - flow operations, which can include instructions for: branching to another location in the program and executing instructions there; conditionally branching to another location if a certain condition holds; indirectly branching to another location; or calling another code block while saving the location of the next instruction as the point to return to.

[0136] Coprocessor instructions, which can include instructions for loading data from / storing data to a coprocessor, or exchanging data with CPU registers, or performing coprocessor operations.

[0137] The processor of a computer in the system of the present invention may include "complex" instructions in its instruction set. What a single "complex" instruction does may require many instructions on other computers. Such instructions typically are instructions that take multiple steps, control multiple functional units, or instructions that occur on a larger scale than the large number of simple instructions implemented by a given processor. Some examples of "complex" instructions include: saving many registers on the stack at once; moving large chunks of memory; complex integer and floating-point operations (sine, cosine, square root, etc.); 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 using operands from memory rather than registers.

[0138] Instructions can be defined according to their parts. According to more traditional architectures, an instruction includes an opcode that specifies the operation to be performed, such as adding the contents of memory to a register, and zero or more operand specifiers, which can specify registers, memory locations, or literal data. The operand specifiers can have addressing modes that determine their meaning, or can be in fixed fields. In very long instruction word (VLIW) architectures, which include many microcode architectures, multiple simultaneous opcodes and operands are specified in a single instruction.

[0139] Some types of instruction sets do not have an opcode field (such as, for example, the Transport Triggered Architecture (TTA) or the Forth virtual machine), only operands. Other less common "0-operand" instruction sets lack any operand specifier fields, such as some stack machines that include NOSC.

[0140] Conditional instructions typically have a predicate field - several bits that encode a particular condition, such that the operation is performed rather than not performed. For example, a conditional branch instruction is executed, and if the condition is true, the branch is taken, such that execution proceeds to a different part of the program, and if the condition is false, the branch is not taken, such that execution continues sequentially. Some instruction sets also have conditional move instructions, such that if the condition is true, the move is performed and the data is stored in the destination location, and if the condition is false, it is not performed and the destination location is not modified. Similarly, the IBM z / architecture has conditional store instructions. Some instruction sets include a predicate field in every instruction; this is called branch prediction.

[0141] The instructions that make up a program rarely specify themselves in their internal numeric form (machine code); they can be specified using assembly language, or more typically, can be generated by a compiler from a programming language.

[0142] The present invention will now be described more fully by reference to the following non-limiting examples.

[0143] Example 1: Querying an aptamer-based biosensor by applying a change in potential to the working electrode

[0144] This example utilizes an electrochemical biosensor with an aptamer capable of selectively binding to vancomycin. The aptamer is labeled with methylene blue as a redox-active reporter molecule.

[0145] The labeled aptamer binds to the surface of the gold electrode via a thiol bond, with the methylene blue label at the distal end of the aptamer. The gold electrode serves as the working electrode in the biosensor device. Upon selective binding of vancomycin, the aptamer changes conformation, bringing the methylene blue label closer to the electrode surface.

[0146] The biosensor device also includes a silver / silver chloride reference electrode and a platinum counter electrode.

[0147] The working electrode and the counter electrode are immersed in a solution containing different concentrations of vancomycin. The working electrode is held at an initial potential of -0.4 V relative to the silver|silver chloride reference electrode for 1 second using a potentiostat (PalmSens BV, Houten, Netherlands). The potential of the gold electrode is then changed stepwise to -0.2 V and maintained at this potential (again using the potentiostat) for 50 milliseconds. The current passing through the working electrode is recorded at 20 - microsecond intervals within the 50 milliseconds. The 20 - point moving average of the current is calculated for each time point to reduce possible random variations in the current measurement.

[0148] The average current at the time point of 0.5 milliseconds (i(t1)), the average current at the time point of 6.7 milliseconds (i(t2)), and the average current at the time point of 17 milliseconds (i(t3)) are determined, and the time points are obtained by reference to the change in the reference potential. Refer Figure 3 , which graphically shows the initial potential, the change in potential, and the time points t1, t2, and t3.

[0149] The current ratios are determined as follows: Calculate i(t1) / i(t3) and i(t2) / i(t3). The f value is determined using Equation (3), which is derived using Equations (1) and (2). In Equation (3), terms up to n = 10 are used to approximate the infinite series of the exponential.

[0150]

[0151] where r1 is i(t1) / i(t3), and q is given by:

[0152]

[0153] It should be noted that the ratio i(t2) / i(t3) is present in the calculation of f, although not explicitly shown. For further explanation, the variable r2 is in fact:

[0154]

[0155] Three separate determinations of f are made and averaged to give Figure 1 and Figure 2 the f values plotted in

[0156] It was found that the calculated f values fit well to the expected Langmuir binding isotherm over the concentration range shown.

[0157] Example 2: Demonstration of low variability of the aptamer-based biosensor across a range of conditions and electrodes

[0158] In this second example, the experimental setup described in Example 1 was used to demonstrate the ability of the interrogation method to correct for electrode-to-electrode variations, temperature variations, and drift of the electrode response over time.

[0159] Four different electrodes (E1, E2, E3, and E4) were used. Two electrodes (E1, E2) were operated at 32 °C, while the remaining electrodes (E3, E4) were operated at 41 °C.

[0160] These electrodes were tested in a phosphate buffered saline solution that did not contain vancomycin (the target of the aptamer in the sensing layer).

[0161] Reference Figure 4 shows the ability of the method to address electrode-to-electrode variations, temperature variations, and drift of the electrode response over time.

[0162] The sensors were interrogated continuously over 25 hours, and the current at t1 (0.5 ms) for each electrode was plotted at approximately one-hour intervals in Figure 4 . This i(t1) data represents the true variations in sensor response due to electrode-to-electrode variations during fabrication, test temperature, and degradation of the sensing layer on the electrode over time. The data shown is the mean of three repeated current transients.

[0163] Figure 4 Data from the same test is also shown in

[0164] but analyzed using the present method to give f values according to Equation (3), which have been background corrected by subtracting the mean of the zero-time responses across the electrodes.

[0165] Example 3: Demonstration of the ability of the multi-pulse method to improve the discrimination of different target analyte concentrations in an aptamer-based biosensor

[0166] In this third example, the experimental setup described in Example 1 is used to demonstrate the ability of the multi-pulse method to improve the ability to discriminate between different target analyte concentrations. Figures 5 to 9 The data shown are for a single sensing electrode tested in phosphate buffered saline at 37 °C in vancomycin solutions ranging from 0 to 25 μM. Five different interrogation pulses were used. Each pulse ended at a potential of -0.15 V. The first pulse started at a potential of -0.4 V, the second pulse started at a potential of -0.425 V, the third pulse started at a potential of -0.45 V, the fourth pulse started at a potential of -0.475 V, and the fifth pulse started at a potential of -0.5 V. Each potential was referenced to a silver|silver chloride reference electrode. The current shown is the mean of five repeated pulses.

[0167] Figure 5 Shows the current recorded over time for five different pulses (labeled P1 to P5 in the legend) when there is zero vancomycin present.

[0168] Using Figure 5 the data shown in Figure 6 shows an example graph of the current plotted against the magnitude of the potential step used for the pulse in volts (V) at different times (shown in the legend in seconds). The dashed line is the linear least squares regression line. This graph demonstrates the excellent linearity of these plots over time, confirming that linear least squares regression is a suitable technique for extrapolating the current to zero potential step magnitude in this case.

[0169] Figure 7 Shows a graph of current versus time when using a potential step from -0.4 V to -0.15 V for various concentrations of vancomycin ranging from 0 to 25 μM present in the test solution, where the legend gives the concentration of vancomycin present in μM.

[0170] Figure 8 Shows for Figure 7 the same vancomycin solutions used in

[0171] The current extrapolated to zero potential step magnitude using five different pulse magnitudes. Figure 7 Compared to Figure 8 the improvement in the separation between the current graphs for different vancomycin concentrations in

[0172] Figure 9 Shows using Figure 7The current calculation shown (the circled point labeled P1 in the legend) and the f value corrected for a zero vancomycin concentration value using Figure 8 The current calculation shown (the diamond point labeled Extrap. in the legend) demonstrate the improved dynamic range of the results and thus the improved sensitivity when using extrapolated current data.

[0173] Example 4: Demonstration of the ability of the double-pulse method to improve the discrimination of different target analyte concentrations in an aptamer-based biosensor

[0174] In this example, the experimental setup described in Example 1 was used to demonstrate the ability of the double-pulse method to improve the ability to discriminate between different target analyte concentrations. Figures 10 to 13 The data shown are for a single sensing electrode tested in phosphate buffered saline at 37 °C in vancomycin solutions ranging from 0 to 25 μM. Two different interrogation 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 started at a potential of -0.2 V and ended at -0.1 V. Each potential was referenced to a silver|silver chloride reference electrode.

[0175] Figure 10 A graph of current versus time is shown for various concentrations of vancomycin ranging from 0 to 25 μM present in the test solution when using a potential step from -0.45 V to -0.2 V, where the legend gives the concentration of vancomycin present in μM. The current shown is the mean of five repeated potential pulses. This shows how the current varies with vancomycin concentration.

[0176] Figure 11 A graph of current versus time is shown for various concentrations of vancomycin ranging from 0 to 25 μM present in the test solution when using a potential step from -0.2 V to -0.1 V, where the legend gives the concentration of vancomycin present in μM. The current shown is the mean of five repeated potential pulses. This shows how the current remains essentially constant with vancomycin concentration.

[0177] Figure 12 A graph of current versus time is shown for various vancomycin concentrations when Figure 11 the current shown in Figure 10 is multiplied by 2.5 and subtracted from the current in Figure 10 The factor of 2.5 is used to compensate for the potential step size of 0.25 V used to generate the current shown in Figure 11 while a 0.1 V potential step size was used to generate the current in

[0178] Figure 13 Show the use of Figure 10 for the current calculation shown in (the circled dot labeled P1 in the legend) and the use of Figure 11 for the current calculation shown in (the diamond dot labeled P1 - 2.5P2 in the legend) the f-values, demonstrating the improved dynamic range of the result and thus the improved sensitivity when using a current in which the second potential step current is subtracted from the first potential step current. The f-values have been corrected for background values.

[0179] Those skilled in the art will recognize that the invention described herein is susceptible to further variations and modifications in addition to those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within the spirit and scope of the invention.

[0180] The invention has been described primarily by reference to aptamer-based biosensors, in which a redox reporter molecule is covalently bound to the aptamer. The aptamer can be a DNA or RNA aptamer, or some analogue such as XNA (xeno nucleic acid) or PNA (peptide nucleic acid). Given the benefits of this disclosure, those skilled in the art are able to practice the invention in a range of alternative formats.

[0181] For example, the binding element can be an antibody, an antibody fragment (such as a Fab fragment), a natural or synthetic polymer comprising an analyte binding site, a peptide, a small molecule, an antigen, or any other element that can bind to an electrode and specifically bind to an analyte of interest.

[0182] Furthermore, the redox-active reporter molecule does not need to be covalently linked to the binding element. In some embodiments, the redox-active species is the analyte itself, which binds to the binding element and thus becomes trapped and spatially restricted. In other embodiments, the redox-active reporter molecule binds directly to the analyte itself, or is associated with a separate species (such as a redox-labeled antibody) that binds to the analyte.

Claims

1. A method for determining the amount of an analyte in a fluid, the method comprising: Applying a first change in potential to a working electrode of an electrochemical sensor, 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 current values generated at least by the application of the first change in potential at a plurality of time points; Determining the distribution of the redox-active species by reference to the measured current values; And Using the determined distribution of the redox-active species to determine the amount of analyte bound to the binding element.

2. The method according to claim 1, wherein, The change in potential is substantially instantaneous.

3. The method according to claim 1 or claim 2, wherein The change in potential is implemented as a step change.

4. The method according to any one of claims 1 to 3, wherein, The first change in potential is configured to change the redox state of the redox-active species from one redox state to another such that electrons are transferred between the redox-active species and the electrode surface.

5. The method according to any one of claims 1 to 4, wherein The first change in potential is configured such that the current flowing across the electrode surface due to the redox-active species changing redox state is controlled by mass transport of the redox-active species through the layer adjacent to the electrode surface.

6. The method according to any one of claims 1 to 5, wherein The potential generated by the first change in potential 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 according to any one of claims 1 to 6, comprising applying an initial potential prior to the application of the first change in potential.

8. The method according to claim 7, wherein The initial potential is selected such that the magnitude of the first change in potential 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. The method according to claim 7 or claim 8, wherein The initial potential is maintained for at least about 0.05 s, 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, 1.0 s, 1.1 s, 1.2 s, 1.3 s, 1.4 s, 1.5 s, 1.6 s, 1.7 s, 1.8 s, 1.9 s, or 2.0 s.

10. The method according to any one of claims 1 to 9, comprising measuring at least a first current value after the application of the first change in potential.

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

12. The method according to claim 10 or claim 11, wherein 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 the application of the first change in potential.

13. The method according to any one of claims 10 to 12, comprising measuring at least a first current value and a second current value in chronological order after the application of the first change in potential.

14. The method according to any one of claims 10 to 12, comprising chronologically measuring at least a first current value, a second current value, and a third current value after the application of the first change in potential.

15. The method according to any one of claims 10 to 12, comprising chronologically measuring at least a first current value, a second current value, a third current value, and a fourth current value after the application of the first change in potential.

16. The method according to any one of claims 10 to 15, wherein, The measurements of the first current value, the second current value (in the case of measurement), the third current value (in the case of measurement), and the fourth current value (in the case of measurement) occur during a period in which the potential generated by the first change in potential is maintained.

17. The method according to any one of claims 1 to 16, wherein The first current value, the second current value (in the case of execution), the third current value (in the case of measurement), and the fourth current value (in the case of measurement) are average current values, which are determined by referring to a plurality of current values determined before, across, or after a time point.

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

19. The method according to any one of claims 10 to 18, comprising generating at least one current ratio by referring to the first current value and the second current value.

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

21. The method according to claim 20, wherein, 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 the application of the first change in potential.

22. The method according to any one of claims 19 to 21, wherein, The second current value is measured after the first current value.

23. The method according to any one of claims 19 to 22, wherein The second current value is measured after the measurement of the first current value or 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 application of the first change in potential.

24. The method according to any one of claims 1 to 23, comprising applying a second change in potential to the working electrode of the electrochemical sensor after the first change in potential.

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

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

27. The method according to claim 26, wherein, Subtract the background current consumption from the first current value, the second current value (if measured), the third current value (if measured), or the fourth current value (if measured).

28. The method according to any one of claims 1 to 27, wherein The first and / or second change in potential is a substantially instantaneous change.

29. The method according to any one of claims 1 to 28, wherein, The first and / or second change in potential is a substantially stepwise change.

30. The method according to any one of claims 1 to 29, comprising the steps of: Applying a first change in potential and at least a second change in potential; Combining a selected current generated by the at least second change in potential with a selected current from the first change in potential to derive a current; And Determining an analyte concentration from the derived current.

31. The method according to claim 30, wherein, The at least second change in potential changes from the potential applied at the end of the first change in potential.

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

33. The method according to claim 32, wherein, Multiply the magnitude of the current generated by the at least second change in potential by a factor and subtract it from the current generated by the first change in potential.

34. The method according to any one of claims 30 to 33, comprising the following steps: The currents generated by the first change in potential and the at least second change in potential are combined by using the current measured simultaneously after the change in potential and the magnitude of the change in potential of the potential used to generate the current to extrapolate the measured current to a value corresponding to a zero change in potential.

35. The method according to claim 34, wherein The extrapolation is a linear extrapolation.

36. The method according to claim 34, wherein, The extrapolation is a non-linear extrapolation.

37. An electrochemical sensor device or system, comprising: A working electrode having associated therewith (i) a binding element and (ii) an associated redox-active species spatially constrained within a layer adjacent the electrode surface; And A microprocessor-based controller, wherein the microprocessor-based controller is configured to perform or assist in performing the method according to any one of claims 1 to 36.

38. The electrochemical sensor device or system according to claim 37, wherein, The microprocessor-based controller is electrically connected to the working electrode or is network-connected, either wired or wirelessly, to another microprocessor-based controller electrically connected to the working electrode.

39. The electrochemical sensor device or system according to claim 37 or claim 38, wherein, The microprocessor controller is configured to access and execute program instructions to perform the method according to any one of claims 1 to 36.

40. The electrochemical sensor device or system according to claim 39, comprising a variable power source electrically connected to the working electrode, wherein, The program instructions direct the power source to apply a potential to the working electrode according to the method according to any one of claims 1 to 36.

41. The electrochemical sensor device or system according to any one of claims 37 to 40, comprising a current measurement circuitry configured to measure the current passing through the working electrode.

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

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

44. A computer-readable medium comprising program instructions configured to perform or facilitate the method according to any one of claims 1 to 36.

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