Method and apparatus for determining amount of analyte in fluid using periodic waveform

Abstract

A method for interrogating an aptamer-based electrochemical sensor using square wave voltammetry is provided. A redox reporter molecule is attached to each of the aptamers. The current generated by the interrogation is measured and used to determine the initial distribution of redox reporter molecules over the surface of the aptamer coated working electrode of the sensor. The measured current is used to determine the position or distribution of redox-active species relative to the surface, and in turn to determine the amount of analyte around the electrode.

Description

Technical Field

[0001] The present invention generally relates to an electrochemical sensor that can be used to determine the amount of an analyte in a fluid, the fluid including biological fluids such as blood and interstitial fluid. More particularly, the present invention provides an electrochemical sensor that operates by an improved method that provides for a more reliable analyte determination and an extended sensor life. 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, the target analyte including exogenous agents (e.g., pharmaceutical compounds and toxins) and endogenous agents (e.g., metabolites, proteins, hormones, etc.).

[0003] Electrochemical sensors have shown great promise in the field of human and animal health. In this scenario, the sensor can be implemented in the form of a microneedle-based patch applied to the skin. The microneedles form the working electrode, which is inserted into the skin to contact the interstitial fluid. The tip of the microneedle serves as the sensor electrode, and an analyte recognition element (such as an aptamer) is associated with the tip. This arrangement provides a minimally invasive platform for real-time, continuous in vivo detection of a target analyte, which is sensitive and selective enough to function in the complex matrix of interstitial fluid.

[0004] Electrochemical sensors typically include a working electrode coated in an analyte recognition element that undergoes a conformational change when an analyte binds. A redox reporter molecule (such as methylene blue) can be covalently linked to the analyte recognition element. The conformational change in the analyte recognition element alters the accessibility of the redox reporter molecule to the electrode surface, resulting in an analyte-induced change in the level of electron transfer between the redox reporter molecule and the electrode. In some cases, the binding of the analyte brings the redox reporter molecule closer to the electrode surface, increasing the level of electron transfer and, in turn, increasing the current passing through the electrode. In other cases, the binding causes the reporter molecule to move away from the electrode surface, resulting in the opposite effect. In any case, the binding of the analyte results in a detectable change in the electrode current.

[0005] Electrochemical sensors are typically interrogated by applying a potential across the working electrode and a counter electrode and then measuring the current after the potential is removed or changed.

[0006] One method of interrogating a sensor is square wave voltammetry, where the potential is applied to the working electrode in the form of a square wave. A voltammogram (i.e., current versus voltage) is generated, and the peak current passing through the working electrode is determined. The analyte quantity is determined by an earlier generated calibration curve that defines the relationship between the analyte quantity and the peak current. Square wave voltammetry can be customized for specific applications to provide an increase ("signal on") or decrease ("signal off") in the peak current in the presence of the target analyte. Signal drift and signal gain reduction can be issues, which are addressed by making measurements at two square wave frequencies and then using the measurements to generate a kinetic differential measurement (KDM) value. The KDM value is calculated by subtracting the normalized peak currents measured at the signal on and signal off frequencies and then dividing by their average. Average KDM values are collected over a range of target concentrations to create a calibration curve that is fit using the Hill-Langmuir isotherm.

[0007] Another method for interrogating an electrochemical sensor is cyclic voltammetry. In this method, the voltage across the working electrode and the reference electrode is modulated at a fixed rate between two values (V1 and V2). When the voltage reaches V2, the scan is reversed and the voltage is modulated back to V1. The voltage is measured between the reference electrode and the working electrode while the current is measured between the working electrode and the counter electrode. The measurements obtained are plotted as a voltammogram. For square wave voltammetry, the peak current is used to determine the quantity of analyte around the working electrode.

[0008] Another prior art interrogation method is chronoamperometry, which has been shown to enable aptamer-based sensing with no drift and sub-second resolution. The difference in the electron transfer rate between the bound and unbound analyte can be measured as a difference in the current decay lifetime. Such a lifetime can be related to the concentration of the target in the sample. Since the lifetime of chronoamperometry is a function of the fractional occupancy of the bound and unbound receptors, it is less sensitive to progressive changes at the sensor interface compared to the total current magnitude that depends on the total number of aptamers. A variant called intermittent pulse amperometry can be used to achieve millisecond resolution measurements of analyte binding kinetics. The output of each periodic pulse produces a forward chronoamperogram and a reverse chronoamperogram, which can be subtracted to produce a differential current proportional to the concentration of the target analyte.

[0009] Another prior art interrogation method is electrochemical impedance spectroscopy (EIS). EIS is a technique that has been found useful for analyzing interfacial properties related to biorecognition events such as aptamer-target folding that occur due to the binding of the target analyte. Biosensing using EIS is based on changes in the electron transfer resistance using redox probe pairs such as [Fe(CN)6]. Dynamic impedance measurements are typically made at a single frequency, and a calibration curve is used to convert the impedance measurements to analyte quantity.

[0010] Existing art methods for quantifying analytes using square wave voltammetry or amperometry present several challenges. For example, the determination of KDM by square wave voltammetry is temperature-dependent. The method requires normalization in the absence of the target analyte, making it unsuitable for in vivo use in the human body to determine the amount of endogenous analytes such as hormones. Amperometry is strongly affected by capacitive current and any potential offsets in the reference electrode.

[0011] Sensor degradation is another problem that can occur when repeatedly interrogating the sensor, such as in the case of continuously monitoring a target analyte in real time.

[0012] There are also issues regarding sensor accuracy, reproducibility, and the time required to perform the measurement.

[0013] In cases where the sensor is configured as a portable device, including a wearable device, the power consumption of the device electronics may limit the operating time due to the battery capacity. Therefore, it is desirable to reduce the power consumption in such cases.

[0014] One aspect of the present invention is to provide improvements in operating an electrochemical sensor in order to ameliorate or overcome any one or more of the problems described herein. Another aspect of the present invention is to provide useful alternatives to existing art methods of interrogating an electrochemical sensor.

[0015] The discussion of documents, acts, materials, devices, articles, etc. included in this specification is included only to provide 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

[0016] 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:

[0017] Providing an electrochemical sensor working electrode having a plurality of analyte recognition elements associated therewith, each of the analyte recognition elements having a redox active substance associated therewith;

[0018] Applying a potential to the working electrode according to a periodic waveform; and

[0019] Measuring the current value generated by the application of the potential.

[0020] In one embodiment of the first aspect, each of the plurality of analyte recognition elements is associated with the surface of the working electrode, and the measured current value(s) is / are used to determine the position or distribution of the redox-active substance relative to the surface.

[0021] In one embodiment of the first aspect, the position or distribution is an initial position or initial distribution.

[0022] In one embodiment of the first aspect, wherein the position or distribution of the redox-active substance is used to determine the degree of movement of the redox-active substance towards the surface of the working electrode, and thereby determine the amount of analyte recognized by the plurality of analyte recognition elements.

[0023] In one embodiment of the first aspect, the measured current value(s) is / are used to determine the degree of movement of the redox-active substance towards the surface of the working electrode, and thereby determine the amount of analyte recognized by the plurality of analyte recognition elements.

[0024] In one embodiment of the first aspect, the measured current value(s) is / are used to generate one or more current-potential relationships.

[0025] In one embodiment of the first aspect, each of the one or more current-potential relationships is a differential current-potential relationship.

[0026] In one embodiment of the first aspect, each of the one or more current-potential relationships provides a peak current, and the peak current is used to calculate one or more f values, each f value indicating the fraction of the redox-active substance at or near the surface of the working electrode.

[0027] In one embodiment of the first aspect, the f value is calculated according to Equation (3).

[0028] In one embodiment of the first aspect, the periodic waveform has a substantially fixed frequency, and the periodic waveform is superimposed on the base scan potential.

[0029] In one embodiment of the first aspect, the base scan potential has a stepped form.

[0030] In one embodiment of the first aspect, the periodic waveform has a duty cycle of approximately 50%.

[0031] In one embodiment of the first aspect, the periodic waveform is stepped so as to provide a substantially instantaneous potential change.

[0032] In one embodiment of the first aspect, the periodic waveform is a substantially square waveform.

[0033] In one embodiment of the first aspect, the periodic waveform is applied for a plurality of cycles.

[0034] In one embodiment of the first aspect, a periodic waveform is applied for at least about 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000, or 100000 cycles.

[0035] In one embodiment of the first aspect, the periodic waveform has a substantially fixed frequency.

[0036] In one embodiment of the first aspect, a substantially square waveform is applied according to square wave voltammetry.

[0037] In one embodiment of the first aspect, the measured current value(s) is / are used to provide a time-current relationship.

[0038] In one embodiment of the first aspect, the time-current relationship defines the variation of current over time.

[0039] In one embodiment of the first aspect, the variation of current over time is a current transient.

[0040] In one embodiment of the first aspect, the current transient depends on the rate of electron transfer between the redox-active species and the surface of the working electrode.

[0041] In one embodiment of the first aspect, the rate of transfer depends on the accessibility of the redox-active species to the surface of the working electrode.

[0042] In one embodiment of the first aspect, the accessibility of the redox-active species to the surface of the electrode depends on the proximity of the reporter molecule to the surface of the working electrode.

[0043] In one embodiment of the first aspect, the time-current relationship is used to determine the distribution of the redox-active species related to the proximity of the redox-active species to the surface of the working electrode.

[0044] In one embodiment of the first aspect, the method includes measuring the current at 2, 3, or more different time points within the period of the periodic waveform.

[0045] In one embodiment of the first aspect, the method includes measuring the current at 2, 3, or more different frequencies of the periodic waveform.

[0046] In one embodiment of the first aspect, the method includes calculating one or more current ratios based on the measured current.

[0047] In one embodiment of the first aspect, the time-current relationship is processed to determine the analyte amount.

[0048] In an embodiment of the first aspect, the process includes determining the rate of current decay that occurs after the application of a potential.

[0049] In an embodiment of the first aspect, the time-current relationship is used as an input to a method for determining the amount of analyte by the chronoamperometry method.

[0050] In an embodiment of the first aspect, the periodic waveform has a first frequency, and the method includes:

[0051] Applying a potential using the periodic waveform at the first frequency and measuring the current generated by the potential at a later time point in the cycle wave period and at one or two earlier time points.

[0052] In an embodiment of the first aspect, the time-current relationship is a current transient or is used to determine the peak current.

[0053] In an embodiment of the first aspect, a waveform having only a single frequency is applied to the working electrode.

[0054] In an embodiment of the first aspect, each of the plurality of analyte recognition elements is an aptamer or a functional equivalent thereof that has binding specificity for the analyte.

[0055] In an embodiment of the first aspect, a redox-active substance is linked to the analyte recognition element.

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

[0057] A working electrode having associated therewith (i) an analyte recognition element and (ii) an associated redox-active substance 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, the 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, an electrochemical sensor device or system includes a variable power source electrically connected to a working electrode, wherein 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, program instructions direct the application of the potential in a periodic waveform or a substantially square waveform.

[0064] In an embodiment of the second aspect, an electrochemical sensor device or system includes a current measurement circuitry configured to measure the current passing through the working electrode.

[0065] In an embodiment of the second aspect, an electrochemical sensor device includes an electronic memory operably associated with a 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.

[0066] 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.

[0067] In a third aspect, the invention includes a computer-readable medium comprising program instructions configured to execute the method of any embodiment of the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 Illustrated is a preferred method of the invention, wherein a square wave potential at three frequencies is applied to the working electrode of an aptamer-based electrochemical sensor, and the current output is measured and used to calculate an f value according to Equation (3) herein.

[0069] Figure 2 is a plot showing Figure 1 an implementation of the method, which depicts the f value response using the peak currents at Fon|Fnr|Foff in place of i(t1), i(t2), and i(t3) in the context of a working electrode having a vancomycin-sensitive aptamer and a methylene blue redox reporter molecule.

[0070] Figure 3 is a plot showing Figure 1 an implementation of the method, which depicts the response of an aptamer-based vancomycin-sensitive electrode to different concentrations of vancomycin (2 μM, 10 μM, and 200 μM) over a 24-hour period, the method using square wave voltammetry to interrogate and Equation (3) to calculate the f value.

[0071] Figure 4 is Figure 1An alternative to the method, in which a square wave potential at a single frequency is applied to the working electrode of an aptamer-based electrochemical sensor, and the current output is measured and used to calculate the f value according to Equation (3) herein.

[0072] Except for the coordinate plots, 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

[0073] 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 limitation. Thus, such a description of the various alternative embodiments should not be construed as limiting the scope or breadth of the present invention. Additionally, 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.

[0074] Throughout this specification and the claims, the word "comprising" and variations of the word such as "comprises" and "comprising" are not intended to exclude other additives, components, integers or steps.

[0075] References throughout this specification 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 present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" throughout this specification are not necessarily all referring to the same embodiment, but may refer to the same embodiment.

[0076] In cases where the terms "determine", "determining", and "determined" are used, these terms are not necessarily intended to be construed as meaning any completely accurate determination, although that meaning is not excluded. These terms may be construed to include estimating, approximating, or even indicating.

[0077] The term "aptamer" is intended to include DNA and RNA aptamers. "Functional equivalents" of aptamers include related species such as xenonucleic acid (XNA) and peptide nucleic acid (PNA).

[0078] 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:

[0079] An electrochemical sensor working electrode is provided, the working electrode having a plurality of analyte recognition elements associated therewith, each of the analyte recognition elements having a redox active substance associated therewith;

[0080] A change in potential is applied to the working electrode according to a periodic waveform;

[0081] The value of the current generated by the application of the potential is measured; and

[0082] The measured current is used to provide a time-current relationship.

[0083] It has been found that a viable or advantageous method for interrogating an electrochemical sensor is provided, wherein, in one embodiment, the interrogation potential is applied to the working electrode as a periodic waveform (such as a square waveform). The current generated by the applied potential is considered to be a function of time and is used in a time-based methodology (such as that utilized in amperometry, for example) to determine the amount of the target analyte.

[0084] In some embodiments of the method, the potential applied to the working electrode of the device as a periodic waveform (such as a square waveform) is changed from a potential sufficient to convert and maintain a redox substance in one redox state to another potential sufficient to substantially immediately change the redox state of any redox active substance sufficiently close to the electrode to allow electron transfer across the electrode / solution interface. Stated in an alternative manner, the potential (applied as a periodic waveform such as a square waveform) is changed such that the current flowing across the electrode surface as a result of the redox active substance changing its redox state is primarily or substantially controlled by the mass transport of the redox active substance 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 over time.

[0085] Without wishing to be limited in any way by theory, it is proposed that redox active reporter molecules are bound to the working electrode surface (e.g., by aptamers) and are thus able to move only within a layer adjacent to the electrode, yet the redox active substances diffuse substantially freely within the layer. Thus, the movement of the redox active substances can be modeled with a certain degree of accuracy (although not necessarily completely accurately) according to Fick's first and second laws of diffusion. Thus, even when bound, the redox active substances can still move from a region of high concentration to a region of low concentration in the manner expected for freely diffusible (i.e., unbound) substances, with the magnitude of the flux being proportional to the difference in concentration. In addition, the concentration gradient of the redox active substances in the layer changes over time in the manner expected for freely diffusible substances.

[0086] Apply Fick's first and second laws to a system where a redox-active species is spatially confined within a layer adjacent to an electrode surface but is able to diffuse freely within that layer. Thus, assuming that electron transfer increases as the redox-active species gets closer to the surface, it is possible to model the diffusion of the confined redox species and, thus, the variation in current (termed a "current transient") resulting from the movement of the redox-active species relative to the electrode surface.

[0087] The resulting modelled current transient generated by the movement of the redox-active species reveals 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 within the layer adjacent to the electrode, (iv) the total concentration of the redox species within the layer adjacent to the electrode, and (v) the distribution of the redox species within the layer adjacent to the electrode at the time of application of a potential step. It has been found that these revelations have practical implications for methods of interrogating an electrochemical sensor device, determining the distribution of a redox-active species, and thus determining the amount of analyte recognized by an analyte recognition element.

[0088] Further studies were conducted demonstrating the practical relevance of individual current measurements and current ratios determined at times immediately following the application of a potential change. It was surprisingly found that at longer times following the application of a potential change, the ratio of the two measured currents strongly depends on the thickness of the layer adjacent to the electrode and the diffusion coefficient of the redox species, but only weakly depends on the initial distribution of the redox species, while the ratio of the current at a shorter time following the application of a potential step to the current at a longer time depends more strongly on the initial distribution of the redox species and strongly depends on the thickness of the layer adjacent to the electrode and the diffusion coefficient of the redox species. Additionally, both of these current ratios were modelled as being insensitive to the total concentration of the redox species and the electrode area.

[0089] These simulated behaviors suggest that the later current ratio can be used to obtain a combined measure of the diffusion coefficient of the redox species and the thickness of the layer adjacent to the electrode, which can be applied to the earlier current ratio to obtain an estimated measure of the initial distribution of the redox species, where the estimate is largely insensitive to variations in the total concentration of the redox species within the layer adjacent to the electrode, the thickness of the layer adjacent to the electrode, the diffusion coefficient of the redox species within the layer adjacent to the electrode, and the area of the electrode.

[0090] The equation for the current as a function of time derived from the model is shown in Equation (1) below:

[0091]

[0092] Where:

[0093] i(t) is the current at time t,

[0094] z is the number of moles of electrons transferred per mole of redox species oxidized or reduced at the electrode surface,

[0095] F is the Faraday constant,

[0096] A is the area of the electrode,

[0097] D is the diffusion coefficient of the redox species in the layer adjacent to the electrode,

[0098] C0 is the total concentration of the redox species in the layer adjacent to the electrode,

[0099] l is the thickness of the layer adjacent to the electrode containing the redox species, and

[0100] f is the fraction of the redox species near or at the electrode surface when a potential step is applied.

[0101] Thus, f can be used to determine the distribution of the redox active species at the instant when the initial potential is applied and before the change in the applied potential. The distribution of the redox active species can be used to determine the amount of the analyte recognized by the analyte recognition element of the sensor.

[0102] 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 with n > 0 will be small enough relative to the exponential term with n = 0 such that Equation (1) can be approximated by Equation (2) as follows:

[0103]

[0104] 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 thus D / l 2 can be estimated.

[0105] 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.

[0106] 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.

[0107] In another embodiment of the method, a second change in the electrode potential is performed in the case of a periodic waveform (such as a square waveform), which occurs after the first change in the electrode potential. In this embodiment, by applying a periodic waveform, 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 substances present in the binding layer. Then the electrode potential is changed in the direction of the initial potential. For example, if the electrode potential is initially held at the value when the redox-active substance is reduced, the second potential change will be in the direction of a stronger reducing potential. If the electrode potential is initially held at the value when the redox-active substance is oxidized, the second step will be in the direction of a stronger oxidizing potential. The current generated by 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) and any Faradaic current from redox substances not confined in the layer adjacent to the electrode, and the current generated by the second potential change can be subtracted from the current used to calculate the current ratios disclosed above to improve the accuracy of the result.

[0108] The potentials achieved by the first, second, and third changes in potential can occur in the case of a periodic waveform such as a square waveform and can be related to the minimum potential value of the waveform, the maximum potential value of the waveform, and the minimum potential value of the square waveform, respectively (each potential being added to the base scan potential).

[0109] The inputs required for the chronoamperometry method involving Equation (3) are the currents (i(t1), i(t2), and i(t3)) corresponding to three different times after the application of a step potential, and the values of t1, t2, and t3.

[0110] It has been found that the peak current generated by square wave voltammetry can provide a step potential and thus, according to Equation (3), generate an f value, which in turn can be used to determine the concentration of the target analyte. In square wave voltammetry, a square wave with a slowly rising or falling potential during the scan is used to slowly scan the potential from one potential to another. If the potential is scanned within an appropriate window, a peak in the current at the potential during the scan is generated. The baseline current on either side of the peak is subtracted from the current value at the peak to produce a so-called "peak current" value. Unexpectedly, the peak current measured in this way can be used to determine the analyte concentration (including, for example, calculating f according to Equation (3)) in the case of chronoamperometry, as when using square wave voltammetry, the flowing current is completely or almost completely controlled by the electron transfer rate; that is, the current is limited by the potential applied to the electrode rather than the mass transport of the redox species to the electrode. It can be considered that this method violates the assumptions of the equations used to derive f as outlined herein.

[0111] It may be useful to use square wave voltammetry to generate the input values for the f equation, as subtracting the baseline current to calculate the square wave voltammetry peak current allows for correction of the charging current contribution and the background Faraday current not related to the redox species of interest. The times typically used when generating i(t1), i(t2), and i(t3) using square wave voltammetry are also longer than those required when using prior art chronoamperometry methods, which potentially reduces the charging current contribution and makes potential applications and current measurement tasks easier and / or cheaper due to the use of slower speed electronic components.

[0112] The present invention can be implemented by at least the following two different methods for obtaining a peak current by square wave voltammetry for use in the calculation of f.

[0113] The first method requires performing square wave voltammetry at three different frequencies to obtain three peak currents, one peak current being obtained from each frequency scan. This method is shown in Figure 1 and is detailed in Example 1 below.

[0114] The second method is a modification of the first method, which requires generating a square wave voltammogram at only one frequency (typically the lowest frequency required to generate the input data for the f equation). Instead of repeating the voltammogram scan three times at different frequencies, the current is sampled at three different times at the lowest frequency of interest during the square wave period. This method is shown in Figure 4 and is detailed in Example 2 below.

[0115] Compared with the first method, the advantage of the second method is that it only requires a single frequency scan instead of three scans, which saves time and the battery life of battery-powered sensing devices.

[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 confined redox layer, the total amount of redox species, or the number of electrons transferred per mole of redox species. Additionally or alternatively, applying the potential in the case of a periodic waveform such as a square waveform contributes to a lower variation of the charging current. Further, it allows scanning a range of potentials in order to identify the optimal potential for a given redox-active species such as methylene blue and accurately subtracting that potential from the non-redox-active species current.

[0117] Another advantage of some embodiments of the present invention is that a method is provided that is significantly faster than prior art interrogation methods, which may take from a few seconds to several minutes to perform. In some embodiments, the currently described method can be performed in 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 tens of 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 the 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 the random variation in the result.

[0118] The present invention can be implemented in the form of an apparatus or system configured to facilitate the performance of the methods described herein.

[0119] 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 is undergoing 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 the sensor by contacting the subject's interstitial fluid and detecting the analyte therein.

[0120] 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.

[0121] 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 the microprocessor - based controller in the wearable device.

[0122] As will be appreciated, the methods described herein can be deployed, in part or in whole, by one or more microprocessors that execute computer software, program code, and / or instructions on the processors. The microprocessor can be any kind of computing or processing device capable of executing program instructions, code, binary instructions, and the like.

[0123] Any microprocessor can access a storage medium (such as an electronic memory) through an interface, and 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.

[0124] 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 a period of time for computing; 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).

[0125] 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.

[0126] Software products can be created using a structured programming language such as C, an object-oriented programming language 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 microprocessors and heterogeneous combinations of processors, processor architectures, or combinations of different hardware and software, or any other machine capable of executing program instructions.

[0127] 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 another aspect, the apparatus for performing the steps associated with the above process can include any one of the above hardware and / or software. All such arrangements and combinations are intended to fall within the scope of the present disclosure.

[0128] 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:

[0129] Data processing and memory operations, which may 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 for later execution of a calculation on it; or reading from and writing to a hardware device.

[0130] Arithmetic and logical operations, which may 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 bitwise operations, such as conjunctions and disjunctions of corresponding bits in a pair of registers, inverting each bit in a register; or comparing two values in registers (e.g., to determine if one value is smaller, or if they are equal).

[0131] Control flow operations, which may 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.

[0132] Coprocessor instructions, which may include instructions for loading data from / to a coprocessor, or exchanging data with CPU registers, or performing coprocessor operations.

[0133] The processor of a computer of 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 appear on a larger scale than a 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.

[0134] 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 an addressing mode that determines 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.

[0135] Some types of instruction sets do not have an opcode field (such as, for example, a Transport Triggered Architecture (TTA) or a Forth virtual machine), only operands. Other less common "0-operand" instruction sets lack any operand specifier fields, such as some stack machines including NOSC.

[0136] Conditional instructions typically have a predicate field - several bits encoding a particular condition for which the operation is to be performed rather than not performed. For example, a conditional branch instruction is executed, and if the condition is true, the branch is taken, causing execution to proceed to a different part of the program, and is not executed, and if the condition is false, the branch is not taken, causing execution to continue sequentially. Some instruction sets also have conditional move instructions such that if the condition is true, the move is executed and data is stored in the destination location, and if the condition is false, it is not executed 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 known as branch prediction.

[0137] The instructions making up a program are rarely specified using 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.

[0138] The present invention will now be described more fully by reference to the following non-limiting examples.

[0139] Example 1: Square Wave Voltammetry Using Three Different Frequencies

[0140] Reference Figure 1 , which details a preferred embodiment of the method. A potential excitation in the form of a square wave is applied to the working electrode. Square wave potentials are applied using three different frequencies (250 Hz, 25 Hz, and 10 Hz). Then the relationship between the differential current and the potential at each frequency (shown as a voltammogram in Figure 1 ) is derived to define the peak current (ip) in each case. The f value is calculated by inputting the values of ip(250 Hz), ip(25 Hz), and ip(10 Hz) into Equation (3) (derived from Equations (1) and (2) and shown below), i.e., by substituting i(t1), i(t2), i(t3) at t1, t2, and t3 in Equation (3) with ip(250 Hz), ip(25 Hz), ip(10 Hz) respectively.

[0141]

[0142] where r1 is i(t1) / i(t3), and q is given by:

[0143]

[0144] 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:

[0145]

[0146] In equation (3), terms up to n = 10 or even larger can be used to approximate the infinite series of the exponential.

[0147] Multiple separate calculations can be made for f and averaged.

[0148] Now consider in more detail Figure 1 the method of using the peak currents measured at the following three different frequencies to generate a square wave voltammogram: the signal turn-on frequency, the no-response frequency, and the signal turn-off frequency at three time points. The current is sampled just before the square wave flips, which is a recognized practice in the art.

[0149] The peak current from the highest frequency scan (250 Hz) is used to obtain the value corresponding to i(t1), the peak current from the medium frequency scan (25 Hz) is used to obtain the value corresponding to i(t2), and the peak current from the lowest frequency scan (10 Hz) is used to obtain the value corresponding to i(t3). The times t1, t2, and t3 required to calculate the f value are given by 1 / 2F1, 1 / 2F2, and 1 / 2F3 respectively, where F1, F2, and F3 are the three frequencies used to generate the three square wave voltammograms.

[0150] According to this method, the following equations are used to generate the input data for the equation to solve for f:

[0151] i(t1) = ip(F1) and t1 = 1 / 2F1

[0152] i(t2) = ip(F2) and t2 = 1 / 2F2

[0153] i(t3) = ip(F3) and t3 = 1 / 2F3

[0154] where ip(F) represents the square wave voltammetry peak current at the use frequency F.

[0155] For EAB sensors, three frequencies that have been found useful in the present invention are: F1 is in the range where the peak current responds (increases) in one direction when the target analyte is present in the solution; F3 is in the range where the peak current responds (decreases) in the opposite direction when the target analyte is present in the solution; and F2 (which is between F1 and F3) is in the range where the peak current is relatively insensitive to the presence or absence of the target analyte. The frequency at which the peak current increases in the presence of the target analyte is generally referred to in the art as the "signal-on" frequency. This frequency can be a higher or lower frequency, depending on the aptamer used. The frequency at which the peak current decreases in the presence of the target analyte is called the "signal-off" frequency. This frequency can be a higher or lower frequency, depending on the aptamer used, but is always opposite to the signal-on frequency. The frequency that is relatively non-responsive to the presence of the target is called the "non-responsive frequency" or "minimum response frequency". This frequency will be between the signal-on frequency and the signal-off frequency. The present invention is not necessarily limited to using these three frequencies, but will generally use high, medium, and low frequencies. For example, these three frequencies can be determined experimentally by using different frequencies, trial and error, and observing the effectiveness of the obtained f values in determining the analyte concentration.

[0156] For the vancomycin-sensitive aptamer used in this example, the higher frequency is the signal-on frequency, and the lower frequency is the signal-off frequency. An example of a suitable frequency range for an EAB sensor constructed using this aptamer corresponding to the signal-on frequency, signal-off frequency, and non-responsive frequency is as follows: F1 is from 100 to 300 Hz, F2 is from 20 to 50 Hz, and F3 is from 5 to 20 Hz. A suitable potential scan window for this aptamer with methylene blue as the redox reporter molecule is, for example, from -0.45 V to -0.15 V, where the scan can start at -0.45 V or -0.15 V.

[0157] These observations regarding frequency selection and potential scanning can also be applied to methods performed using a single frequency, as detailed in Example 2.

[0158] Reference Figure 2 , which shows the correlation between the f values calculated as described above and the vancomycin concentration. Regardless of the carrier (i.e., PBS at neutral pH, HEPES at pH 8, or TAPS at pH 9), a direct correlation that appears to conform to the Langmuir isotherm is obvious. In addition, it was found that the correlation holds both before and after the sensor was stored for 7 days.

[0159] Reference Figure 3, which shows the results of an experiment for quantifying drift in the f value over a 26-hour period and within the range of vancomycin concentrations. It is readily understood that for each concentration, there is little noticeable drift in the f value determined by the above method. Thus, the method of the present invention can be used to interrogate electrodes placed in situ within the human body for continuous real-time monitoring of an analyte over an extended period of time.

[0160] Example 2: Square wave voltammetry method using a single frequency

[0161] Reference Figure 4 , which outlines a method for determining the f value from peak current values. The difference in this method compared to the method of Example 1 lies in how the i(t1), i(t2), and i(t3) currents are generated. According to this method, a single square wave voltammogram is performed at the F3 frequency. This results in a current transient, as seen in the upper coordinate plot in Figure 4 . Instead of running separate square wave voltammetry at a higher frequency, additional current samples are taken from both the forward and reverse portions of the applied square wave potential. Using the example shown in Figure 4 , the difference in the currents sampled at t1 seconds after the potential steps in the forward and reverse directions is used to construct a voltammogram to obtain the peak current corresponding to i(t1), the difference in the currents sampled at t2 seconds after the potential steps in the forward and reverse directions is used to construct a second voltammogram to obtain the peak current corresponding to i(t2), and the difference in the currents sampled at t3 seconds after the potential steps in the forward and reverse directions is used to construct a third voltammogram to obtain the peak current corresponding to i(t3). The current sampling times selected will typically correspond to 1 / 2F1, 1 / 2F2, and 1 / 2F3, where F1, F2, and F3 are as described above, but can also be determined empirically to obtain the most effective f value.

[0162] Those skilled in the art will realize that the inventions described herein are 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.

[0163] Accordingly, the spirit and scope of the present invention are not limited by the foregoing examples, but are to be understood in the broadest sense permitted by law.

Claims

1. A method for determining the amount of an analyte in a fluid, the method comprising: providing an electrochemical sensor working electrode having a plurality of analyte recognition elements associated therewith, each of the analyte recognition elements having a redox active substance associated therewith; applying a potential to the working electrode according to a periodic waveform; and measuring a current value generated by the application of the potential at one or more time points within a period of the periodic waveform.

2. The method according to claim 1, wherein Each of the plurality of analyte recognition elements is associated with the surface of the working electrode, and the measured current value is used to determine the position or distribution of the redox active substance relative to the surface.

3. The method according to claim 2, wherein The position or the distribution is an initial position or an initial distribution.

4. The method according to claim 2 or claim 3, wherein, The position or the distribution of the redox active substance is used to determine the degree of movement of the redox active substance towards the surface of the working electrode and, in turn, to determine the amount of analyte recognized by the plurality of analyte recognition elements.

5. The method according to claim 1, wherein The measured current value is used to determine the degree of movement of the redox active substance towards the surface of the working electrode and, in turn, to determine the amount of analyte recognized by the plurality of analyte recognition elements.

6. The method according to claim 1 or claim 5, wherein The measured current value is used to generate one or more current-potential relationships.

7. The method according to claim 6, wherein, Each of the one or more current-potential relationships is a differential current-potential relationship.

8. The method according to claim 6 or claim 7, wherein, Each of the one or more current-potential relationships provides a peak current, and the peak current is used to calculate one or more f values, each f value indicating the fraction of the redox active substance at or near the surface of the working electrode.

9. The method according to claim 8, wherein The f value is calculated according to equation (3).

10. The method according to any one of claims 1 to 9, wherein, The periodic waveform has a substantially fixed frequency and is superimposed on a base scan potential.

11. The method according to claim 10, wherein, The base scan potential has a stepped form.

12. The method according to any one of claims 1 to 11, wherein, The periodic waveform has a duty cycle of approximately 50%.

13. The method according to any one of claims 1 to 12, wherein The periodic waveform is shaped to provide a substantially instantaneous potential change.

14. The method according to any one of claims 1 to 13, wherein, The periodic waveform is a substantially square waveform.

15. The method according to any one of claims 1 to 14, wherein, The periodic waveform is applied for a plurality of periods.

16. The method according to claim 15, wherein, The periodic waveform is applied for at least about 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10000 or 100000 periods.

17. The method according to any one of claims 1 to 16, wherein, The periodic waveform has a substantially fixed frequency.

18. The method according to any one of claims 14 to 17, wherein The substantially square waveform is applied according to square wave voltammetry.

19. The method according to any one of claims 1 to 18, wherein, The measured current value is used to provide a time-current relationship.

20. The method according to claim 19, wherein, The time-current relationship defines the variation of current with time.

21. The method according to claim 19, wherein, The variation of current with time is a current transient.

22. The method according to claim 21, wherein, The current transient depends on the rate of electron transfer between the redox active substance and the surface of the working electrode.

23. The method according to claim 22, wherein, The rate of transfer depends on the accessibility of the redox active substance to the surface of the working electrode.

24. The method according to claim 23, wherein, The accessibility of the redox active substance to the surface of the working electrode depends on the proximity of the reporter molecule to the surface of the working electrode.

25. The method according to any one of claims 19 to 24, wherein The time-current relationship is used to determine the distribution of the redox active species related to the proximity of the redox active species to the surface of the working electrode.

26. The method according to any one of claims 1 to 25, comprising measuring the current at 2, 3 or more different time points within the period of the periodic waveform.

27. The method according to any one of claims 1 to 26, comprising measuring the current at 2, 3 or more different frequencies of the periodic waveform.

28. The method according to any one of claims 1 to 27, comprising calculating one or more current ratios based on the measured current.

29. The method according to any one of claims 19 to 28, wherein The time-current relationship is processed to determine the amount of the analyte.

30. The method according to claim 29, wherein, The processing includes determining the rate of current decay that occurs after the application of the potential.

31. The method according to any one of claims 19 to 30, wherein, The time-current relationship is used as an input for a method of determining the amount of an analyte by the amperometric method.

32. The method according to any one of claims 1 to 31, wherein, The periodic waveform has a first frequency, and the method includes: Applying the potential using the periodic waveform at the first frequency, and measuring the current generated by the potential at a later time point within the period of the periodic waveform and at one or two earlier time points.

33. The method according to claim 32, wherein A waveform having only a single frequency is applied to the working electrode.

34. The method according to any one of claims 19 to 33, wherein, The time-current relationship is a current transient or is used to determine the peak current.

35. The method according to any one of claims 1 to 34, wherein Each of the plurality of analyte recognition elements is an aptamer having recognition specificity for the analyte or a functional equivalent thereof.

36. The method according to any one of claims 1 to 35, wherein The redox active species is linked to the analyte recognition element.

37. An electrochemical sensor device or system, comprising: A working electrode having associated therewith (i) an analyte recognition element and (ii) an associated redox active species spatially confined within a layer adjacent to the electrode surface; And A microprocessor-based controller, Wherein the microprocessor-based controller is configured to perform 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-based 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 any one of claims 37 to 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 claim 40, wherein, The program instructions specify the application of the potential in a periodic waveform or a substantially square waveform.

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

43. The electrochemical sensor device or system according to any one of claims 37 to 42, 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.

44. The electrochemical sensor device or system according to any one of claims 37 to 43, wherein, The microprocessor-based controller is configured to process the one or more currents measured by the current measurement circuitry to provide an analyte amount.

45. A computer-readable medium comprising program instructions configured to execute the method according to any one of claims 1 to 36.

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