Method and system for interrogating electrochemical sensor

Abstract

Systems and methods for determining the amount of an analyte in a sample using an electrochemical sensor are described. Many embodiments provide methods for interrogating an electrochemical sensor to determine an amount of an analyte without calibrating the sensor. Determining the amount of analyte may be performed at an increased temporal resolution.

Description

[0001] Cross-reference related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 496,257, filed April 14, 2023, entitled "An Improved Method for Interrogating Electrochemical Sensors". The disclosure of U.S. Provisional Patent Application No. 63 / 496,257 is hereby incorporated herein by reference in its entirety for all purposes.

[0003] Federally Funded Research Statement

[0004] This invention was completed with government funding from the National Institutes of Health (NIH) under project EB022015. The government holds certain rights to this invention.

[0005] sequence list

[0006] This application contains a sequence list, which has been electronically submitted in XML format and is hereby incorporated herein by reference in its entirety. The XML copy was created on April 8, 2024, named 08510PCT.xml, and is 3KB in size. Technical Field

[0007] This invention generally relates to electrochemical sensors for determining the amount of an analyte in a sample. More specifically, this invention provides a method for interrogating an electrochemical sensor that allows for the determination of the amount of an analyte without a sensor calibration step and has high temporal resolution. Background Technology

[0008] Electrochemical sensors have found applications in a wide range of fields, including the detection of target analytes in the environment, manufacturing process flows, and biological fluids. Advantageously, these sensors are able to provide continuous quantitative output, allowing for ongoing monitoring of the analyte.

[0009] Many types of electrochemical sensors operate based on changes in electron transfer kinetics in response to the selective binding of a target analyte. In some sensors, the electron transfer kinetics between the sensor's redox reporter molecule and its electrode increase, and vice versa; the average change in the transfer rate depends on the concentration of the target.

[0010] Electrochemical aptamer-based (EAB) sensors are a type of electrochemical sensor. The aptamer is an oligonucleotide with a defined base sequence known to selectively interact with a target analyte. In one version, the aptamer is coupled to the surface of the working electrode, and a redox reporter molecule is coupled to the free end of the aptamer. The binding of the analyte to the aptamer causes a conformational change in the aptamer, resulting in the redox reporter molecule moving closer to the electrode. This movement, in turn, affects the electron transfer rate (kJ) between the redox reporter molecule and the electrode. et (Increase. k) et This change reflects the concentration of the target analyte in real time, without the need to add exogenous reagents.

[0011] EAB sensors can measure the levels of clinically relevant analytes in in vivo and in vitro biological fluids. EAB sensors can routinely provide clinicians with vital information about individuals to assist in diagnosing new medical conditions, managing existing conditions, and informing prognoses.

[0012] Interrogating an EAB sensor to detect a target analyte requires an input of electrical energy; the current output from the working electrode is used to determine the amount of the target analyte in solution. Square wave voltammetry (SWV) is commonly used for in vivo analyte detection because it is sensitive to changes in electron transfer rates and can correct for current output drift, as often seen in in vivo sensor placement. To explain, the amount of analyte reported by an EAB sensor tends to drift downwards over time due to the continuous loss of functional aptamers in contact with bodily fluids. This signal drift is a problem when continuously measuring the amount of analyte, such as in in vivo monitoring applications. However, measuring consecutive square wave voltammeters at two different frequencies allows for drift correction in a method called kinetic differential measurement (KDM). KDM uses the difference between SWV measurements performed at the two frequencies to subtractively eliminate the drift.

[0013] The problem with SWV is that it requires two square-wave voltammetry plots for each measurement point, which in turn reduces the temporal resolution of such tests. For many in vivo examples, the temporal resolution of this approach is between 6 and 22 seconds. This low temporal resolution can be detrimental in applications that require more continuous, real-time data, such as in monitoring rapid physiological processes. Neurotransmitter release is an example of a physiological process that occurs much faster than this timescale.

[0014] Another issue is that SWV-based queries are highly sensitive to manufacturing variations from sensor to sensor, caused by differences in the number of identification elements on each working electrode. Therefore, SWV-based sensors must be individually calibrated before use to correlate peak current with the amount of analyte.

[0015] One aspect of the present invention is to provide an improvement to existing electrochemical sensor interrogation methods. Another aspect of the present invention provides a useful alternative to existing electrochemical sensor interrogation methods.

[0016] The discussions of documents, actions, materials, devices, articles, etc., included in this specification are for the purpose of providing background information on the invention. They do not imply or imply that any or all of these matters constitute part of the prior art or common general knowledge in the field related to the invention, existing prior to the priority date of each provisional claim. Invention Summary

[0017] Some implementation schemes include a method for determining the amount of an analyte in a sample, comprising:

[0018] The working electrode of the electrochemical sensor is interrogated by applying a voltage perturbation to it, the voltage perturbation being the sum of two or more sinusoidal waveforms, each having a different frequency; simultaneously measuring the voltage and / or current values ​​across the working electrode and the counter electrode at each different frequency; generating an impedance spectrum by applying an integral transform method to the measured voltage and / or current values ​​by determining the impedance at the frequencies of the two or more sinusoidal waveforms; and using the impedance spectrum to determine the amount of analyte in the sample.

[0019] In some implementations, the integral transform method is selected from Fourier transform, fast Fourier transform, Laplace transform, Merlin transform, Hartley transform, and Chirplet transform.

[0020] In some implementations, the two or more sine waveforms are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sine waveforms.

[0021] In some implementations, the frequencies of the two or more sinusoidal waveforms define a frequency range that includes frequencies that provide information on electron transfer kinetics between redox reporter molecules and the working electrode surface of the electrochemical sensor.

[0022] In some implementations, the frequencies of the two or more sinusoidal waveforms define a frequency range that excludes frequencies that do not provide information on electron transfer kinetics between redox reporter molecules and the working electrode surface for electrochemical sensors.

[0023] In some implementations, the frequencies of the two or more sinusoidal waveforms define a frequency range that excludes frequencies that could cause drift errors in the measured analyte concentration.

[0024] In some embodiments, the frequencies of the two or more sinusoidal waveforms are each less than 2000Hz, 1900Hz, 1800Hz, 1700Hz, 1600Hz, 1500Hz, 1400Hz, 1300Hz, 1200Hz, 1100Hz, 1000Hz, 900Hz, 800Hz, 700Hz, 600Hz, 500Hz, 400Hz, 300Hz, 200Hz, or 100Hz.

[0025] In some embodiments, the frequencies of the two or more sinusoidal waveforms are each 1Hz-2000Hz, or each 1Hz-1000Hz, or each 10Hz-100Hz.

[0026] In some embodiments, the frequencies of the two or more sinusoidal waveforms include a first frequency, a second frequency, and a third frequency, wherein the first frequency provides information on the electron transfer dynamics between the redox reporter molecules of the electrochemical sensor and the working electrode surface; the second frequency is higher than the first frequency; and the third frequency is lower than the first frequency.

[0027] In some embodiments, the frequencies of the two or more sinusoidal waveforms include a lower frequency and one or more frequencies higher than the lower frequency, and each of the one or more frequencies higher than the lower frequency is a multiple of the lower frequency.

[0028] In some implementations, the lower frequency is the lowest frequency among the frequencies of the two or more sinusoidal waveforms.

[0029] In some embodiments, the step of determining the amount of analyte using the impedance spectroscopy includes comparing the impedance spectrum or a portion thereof from the test sample with the impedance spectrum or a portion thereof from a control sample that does not contain the analyte.

[0030] In some implementations, the impedance spectra from the control sample and the impedance spectra from the test sample are both presented in the form of frequency versus phase.

[0031] In some embodiments, the impedance spectrum from the control sample and the impedance spectrum from the test sample each contain features at a first frequency and a second frequency, respectively, and the amount of analyte is determined by reference to the difference between the first frequency and the second frequency.

[0032] In some implementations, the features are peaks or maximum values, valleys or minimum values, upward-sloping portions of the spectrum, or downward-sloping portions of the spectrum.

[0033] In some embodiments, the step of determining the amount of analyte using the impedance spectroscopy includes using the spectrum to determine the electron transfer kinetics between the redox reporter molecule of the electrochemical sensor and the working electrode surface.

[0034] In some embodiments, the electron kinetics is the rate of electron transfer between the redox reporter molecule of the electrochemical sensor and the working electrode surface.

[0035] In some implementations, the method can repeatedly determine the concentration of the analyte at intervals of less than 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, or 1 second.

[0036] In some embodiments, the method includes repeatedly determining the concentration of the analyte at intervals of less than 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, or 1 second.

[0037] In some embodiments, the method includes repeatedly determining the concentration of the analyte for at least 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours.

[0038] In some implementations, the method does not require any frequency scanning steps across a frequency range to determine the amount of analyte in the sample.

[0039] In some implementations, the method does not require any calibration steps to determine the amount of analyte in the sample.

[0040] In some implementations, the method does not require any drift adjustment steps to determine the amount of analyte in the sample.

[0041] In some implementations, the sample is bodily fluids inside or around the subject's body.

[0042] In some embodiments, the body fluid is selected from: interstitial fluid (ISF), blood, saliva, lacrimal gland secretions, lactational secretions, nasal secretions, tracheal secretions, bronchial secretions, alveolar secretions, gastric secretions, gastric contents, glandular secretions, vaginal secretions, uterine secretions, prostatic secretions, semen, urine, sweat, cerebrospinal fluid, glomerular filtrate, liver secretions, bile, and intraocular fluid.

[0043] In some implementations, the working electrode is a wire, needle, or microneedle.

[0044] In some embodiments, the electrochemical sensor includes a recognition element configured to specifically identify a target analyte.

[0045] In some implementations, the recognition element is associated with a redox reporter molecule.

[0046] In some embodiments, the identification element and / or the redox reporter molecule undergoes a change in the presence of the target analyte, and the change alters the electron transfer rate between the redox reporter molecule and the working electrode surface.

[0047] In some implementations, the change in the recognition element is a conformational change.

[0048] In some implementations, the conformational change of the recognition element alters the distance between the redox reporter molecule and the working electrode surface.

[0049] In some embodiments, the conformational change of the recognition element alters the recombination energy of the redox reporter molecule.

[0050] In some embodiments, the rate at which the redox reporter molecule approaches the working electrode surface is altered in the presence of the target analyte.

[0051] In some implementations, the proportion of time the redox reporter molecule is near rather than away from the working electrode surface is altered in the presence of the target analyte.

[0052] In some implementations, the change in rate or time ratio is associated with changes in the spatial volume parameters, biomolecular rigidity parameters, electrostatic parameters, or hydrodynamic radius of the redox reporter molecule associated with the target analyte.

[0053] In some embodiments, the change in the recognition element and / or the redox reporter molecule is the dissociation of the recognition element from the target analyte.

[0054] In some embodiments, the conformational change alters the electron transfer rate between the redox reporter molecule associated with the recognition element and the working electrode surface.

[0055] In some embodiments, the recognition element is associated with the working electrode surface, and the redox reporter molecule is associated with the recognition element, and a conformational change of the recognition element alters the distance between the redox reporter molecule and the working electrode surface, which in turn alters the electron transfer rate between the redox reporter molecule and the working electrode surface.

[0056] In some implementations, the identification element is a biopolymer.

[0057] In some implementations, the biopolymer is a nucleic acid.

[0058] In some implementations, the biopolymer is an aptamer.

[0059] In some implementations, the electrochemical sensor is configured as a wearable device.

[0060] Some embodiments include an apparatus for detecting the amount of an analyte in a test sample, the apparatus comprising: an electrochemical sensor having a working electrode and a counter electrode; a power supply configured to apply a voltage perturbation to the working electrode, the voltage perturbation being the sum of two or more sinusoidal waveforms, each of the two or more sinusoidal waveforms having a different frequency; a voltage and / or current measurement circuit connected between the working electrode and the counter electrode; and a processor configured to perform an integral transformation on the measured voltage and / or current to generate an impedance spectrum, and to use the impedance spectrum to determine the amount of the analyte in the test sample.

[0061] In some embodiments, the processor is able to access program instructions configured to perform the method of any one of claims 1 to 42.

[0062] In some embodiments, the electrochemical sensor includes a redox reporter molecule, and the processor has access to program instructions configured to perform the methods described in some embodiments.

[0063] In some implementations, the working electrode is a wire, needle, or microneedle.

[0064] In some embodiments, the electrochemical sensor includes a recognition element configured to specifically identify a target analyte.

[0065] In some implementations, the recognition element is associated with a redox reporter molecule.

[0066] In some embodiments, the identification element and / or the redox reporter molecule undergoes a change in the presence of the target analyte, and the change alters the electron transfer rate between the redox reporter molecule and the working electrode surface.

[0067] In some implementations, the change in the recognition element is a conformational change.

[0068] In some embodiments, a conformational change in the recognition element alters the distance between the redox reporter molecule and the working electrode surface or a coupling constant describing electron transfer through the recognition element.

[0069] In some embodiments, the conformational change of the recognition element alters the recombination energy of the redox reporter molecule.

[0070] In some implementations, the rate or time proportion during which the redox reporter molecule approaches the working electrode surface is altered in the presence of the target analyte.

[0071] In some implementations, the proportion of time that the redox reporter molecule is close to rather than far from the working electrode surface is altered in the presence of the target analyte.

[0072] In some implementations, the change in rate or time ratio is associated with changes in the spatial volume parameters, biomolecular rigidity parameters, electrostatic parameters, or hydrodynamic radius of the redox reporter molecule related to the target analyte.

[0073] In some embodiments, the change in the recognition element and / or the redox reporter molecule is the dissociation of the recognition element from the target analyte.

[0074] In some embodiments, a conformational change in the recognition element alters the electron transfer rate between the redox reporter molecule associated with the recognition element and the working electrode surface.

[0075] In some embodiments, the recognition element is associated with the working electrode surface, and the redox reporter molecule is associated with the recognition element, and a conformational change of the recognition element alters the distance between the redox reporter molecule and the working electrode surface, which in turn alters the electron transfer rate between the redox reporter molecule and the working electrode surface.

[0076] In some implementations, the identification element is a biopolymer or a biomimetic polymer.

[0077] In some implementations, the biopolymer is a nucleic acid.

[0078] In some implementations, the biopolymer is an aptamer.

[0079] In some implementations, the electrochemical sensor is configured as a wearable device.

[0080] In some implementations, the non-transient computer-readable medium contains computer-executable program instructions configured to perform the methods described in many implementations.

[0081] In some implementations, the program instructions are provided by a non-transient computer-readable medium.

[0082] Other embodiments and features are set forth in part in the description which follows, and in part will be apparent to those skilled in the art upon viewing this specification, or may be learned by practice of this disclosure. The nature and advantages of this disclosure can be further understood by reference to the remainder of this specification and the accompanying drawings, which form part of this disclosure.

[0083] Brief description of the attached figures

[0084] The description will be more fully understood with reference to the following accompanying drawings, which are presented as embodiments of the invention and should not be construed as a complete set of the scope of the invention, wherein:

[0085] Figures 1A to 1E The illustration shows some mechanisms by which the binding of a target analyte can alter the electron transfer rate in an electrochemical biosensor according to one embodiment. Among other mechanisms, the interaction between the target analyte and the redox reporter molecule-modified acceptor can alter the electron transfer rate between the redox reporter molecule and the working electrode surface in ways such as: Figure 1A As shown, this is combined with induced changes in the shape of the reporter molecule; or as... Figure 1B As shown, by preventing redox reporter molecules from approaching the electrode; or as... Figure 1C As shown, by altering the recombination energy of the reporter molecule; or as... Figure 1D As shown, changing the rigidity of the receptor or the coupling constant describing electron transfer through the receptor; or as... Figure 1E As shown, the hydrodynamics or spatial radius of the receptor / target complex can be altered.

[0086] Figure 2 An implementation scheme is shown, applied to Figure 1A Impedance measurement of the sensor.

[0087] Figures 3A to 3D A series of graphs are shown demonstrating that, according to one implementation, when applied to an EAB sensor, Fast Fourier Transform Electrochemical Impedance Spectroscopy (FFT-EIS) can rapidly and monotonically correlate electrochemical impedance with target molecule concentration. (A) The response of a vancomycin-detecting EAB sensor when immersed in whole bovine blood and challenged with increasing numbers of target molecules. (B) Analysis of FFT-EIS data using equivalent circuit modeling. (C) Challenge of the EAB sensor with increasing concentrations of vancomycin reduces electron transfer resistance. (D) The electron transfer rate approximated using Equation 5. The resulting binding curves conform to the Hill-Langmuir isotherm, with a binding constant K. D =144±31μM (the latter reflects the 95% confidence interval of the estimate).

[0088] Figures 4A to 4DThis illustrates how FFT-EIS supports high-frequency real-time in vivo measurements according to one implementation. (A) The middle figure shows the placement of the sensor electrode in a live rat in vivo experiment. (B) The middle figure shows that impedance spectra can be measured approximately every 1.8 seconds using FFT-EIS. Some downward drift is evident. (C) A graph showing the fitted spectra reveals the drift shown in (A) with an immediate, steady increase in charge transfer resistance (Rct) and redox reporter molecule (C... ads The corresponding decrease in pseudocapacitance was correlated with this. However, only Rct showed a response after injection of 30 mg / kg vancomycin. (D)k et Calculations revealed that this parameter is stable before drug infusion, indicating that the intrinsic electron transfer rate constant of the aptamer unbound state is unaffected by the factors leading to Rct and C. ads The influence of any factors on drift.

[0089] Figures 5A to 5C A series of graphs are shown demonstrating that using the FFT-EIS interrogation EAB sensor according to one implementation scheme can generate rapid time-resolved molecular measurements in vivo. Experiments in three separate animals (A, B, and C, respectively) confirmed the absence of drug prior to infusion and show the expected peak concentration after infusion (error bars reflect 95% confidence intervals).

[0090] Figure 6A and Figure 6B A series of graphs are shown demonstrating the use of an FFT-EIS-interrogated phenylalanine detection EAB sensor to monitor the analyte in the jugular vein of live rats at a resolution of 1.8 seconds, for fasted animals (A) and non-fasted animals (B). Fasted animals were infused with two consecutive doses of phenylalanine, and in both cases, the concentration of free phenylalanine in the blood rapidly declined back to the pre-infusion baseline. In contrast, for non-fasted animals, the recovery to the post-infusion baseline was slower, consistent with previous studies on phenylalanine homeostasis in such animals.

[0091] Figure 7 A table is displayed that details the frequency, amplitude, and phase used in the embodiments to construct the multi-frequency perturbation waveform according to the implementation plan.

[0092] Figures 8A to 8C A series of cyclic voltammograms of a vancomycin detection EAB sensor according to one embodiment are shown. Cyclic voltammograms were recorded prior to all EIS experiments to determine E... 1 / 2 This potential is used as a DC bias during electrochemical impedance spectroscopy (EIS). (A) EAB sensor in 1X PBS + 2mM MgCl2. The voltammogram was recorded at a scan rate of 100mV / s at 25°C. Half-wave potential E 1 / 2That is, the average voltage between the reduction peak and the oxidation peak potential, which is -285mV relative to Ag|AgCl. (B) In vivo EAB sensor, E 1 / 2 (Black dashed line) -324mV; the difference is due to the use of anodic silver wire as a reference electrode. (C) In vivo EAB sensor after vancomycin administration. Peak area decreased (presumably due to monolayer loss), E 1 / 2 Maintain at -324mV.

[0093] Figure 9A and Figure 9B The diagram shows (A) frequency domain and (B) time domain representations of the multisine EIS waveform used in an embodiment according to one implementation.

[0094] Figure 10 The diagram shows a Bode |Z| plot of a vancomycin detection EAB sensor in blood at 37°C, according to one embodiment, when challenged with increasing concentrations of vancomycin.

[0095] Figure 11A and Figure 11B The phase (blue) and modulus (green) Bode plots of a 10kΩ resistor according to one embodiment are shown. (A) Uncorrected spectrum, showing non-zero phase at higher frequencies, and (B) Corrected spectrum, showing zero phase across the entire frequency spectrum.

[0096] Figure 12 The binding curve of the EAB sensor for detecting phenylalanine is shown, demonstrating that in fresh rat blood, the present invention can be used based on k derived from FFT-EIS. et The concentration of phenylalanine is determined by value. According to one implementation scheme, k... et The values ​​(black dots, error bars representing the standard deviation of four independently manufactured and tested sensors) fall on the Langmuir isotherm.

[0097] Figure 13A and Figure 13B This paper describes how an FFT-EIS query of a lactate detection EAB sensor according to one embodiment supports real-time monitoring of plasma lactate concentration in live rats. (A) shows the calibration of the lactate detection sensor in undiluted bovine blood at 37°C using FFT-EIS query. Before calibration, the blood lactate concentration was increased from 6 mM to 106 mM. During calibration, the blood with 106 mM lactate was gradually diluted by adding blood without additional lactate and PBS + 25 mg / mL BSA to reduce the lactate concentration. kJ was measured after each dilution step. et(B) The FFT-EIS query of the EAB sensor supports real-time in vivo plasma lactate monitoring using a rat animal model. A stable baseline concentration was observed before intravenous infusion of 1000 mg / kg lactate. Plasma concentrations rose sharply after intravenous infusion of lactate and then returned to their original levels.

[0098] Unless otherwise stated herein, features in different figures that bear the same numerical designations are considered to be the same features or at least functionally similar features.

[0099] The accompanying drawings are not prepared to any particular scale or size and are not intended to represent the full and accurate representation of the various embodiments. Detailed Implementation

[0100] After considering this description, those skilled in the art will understand how the invention can be practiced in various alternative embodiments and applications. However, although various embodiments of the invention have been described herein, it should be understood that these embodiments are presented by way of example only and not limitation. Therefore, the description of various alternative embodiments should not be construed as limiting the scope or breadth of the invention. Furthermore, statements of advantages or other aspects apply to specific exemplary embodiments and not necessarily to all embodiments or any embodiments actually covered by the claims.

[0101] In the description and claims of this specification, the word "comprise" and its variations, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers, or steps.

[0102] The reference to "one embodiment" or "implementation" in this specification means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places in this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment.

[0103] This invention is based, at least in part, on the inventors' discovery that electrochemical impedance spectroscopy (EIS), using measurements performed simultaneously at multiple frequencies, can be used for electrochemical sensor interrogation methods. This method eliminates the need for any prior sensor calibration and also achieves excellent temporal resolution.

[0104] In this method, impedance is simultaneously measured within a frequency range (using voltage and / or current measurements across the working and counter electrodes). At higher frequencies, impedance reflects rapid processes, such as the formation of an electrochemical bilayer. Conversely, impedance measured at lower frequencies is typically associated with slower processes, such as electron transfer, adsorption and intercalation events, and mass transport. These lower frequencies are particularly interesting because the ability to track changes in electron transfer between the sensor's redox reporter molecules and the sensor's working electrode surface can be used to determine the amount of the target analyte present in the test sample.

[0105] In some implementations, Fast Fourier Transform Electrochemical Impedance Spectroscopy (FFT-EIS) is used to simultaneously measure impedance at multiple frequencies, resulting in both high time resolution and the inclusion of information depth from the complete impedance spectrum. In this way, k can be evaluated. et (Electron transfer kinetics) are used to determine the concentration of the target analyte. The concentration can be output very quickly (e.g., every few seconds), providing a method for querying electrochemical sensors that ideally offers both higher time resolution and requires no calibration.

[0106] In many implementations, exemplary electrochemical sensors are EAB sensors. These sensors are capable of measuring the concentration of specific target analytes in the body in real time and have the potential to revolutionize health monitoring as well as the diagnosis and treatment of diseases. For example, by providing a real-time observation window of drug and biomarker concentrations in plasma, interstitial fluid, or another bodily fluid, EAB sensors can significantly improve the personalization of drug therapy.

[0107] refer to Figure 1A The diagram illustrates an EAB sensor (10) comprising a gold electrode (15) on which a sub-monolayer of a target recognition, redox reporter molecule (20) modified nucleic acid aptamer (25) is deposited via a thiol-gold self-assembled monolayer. In some embodiments, the introduction of the target analyte (30) triggers a conformational change in the aptamer (25), altering the electron transfer rate (ket) between the redox reporter molecule (20) and the electrode (15). In some embodiments, the displacement of the binding-induced redox reporter molecule from the binding pocket may also play a role.

[0108] k et The changes reflect the target analyte concentration in real time, without the need for exogenous reagents. Notably, this signal transduction mechanism is independent of the chemical transformation of the target analyte, making the method versatile. Consistent with this, EAB sensors have been used to measure a variety of drugs, metabolites, neurotransmitters, hormones, toxins, and protein biomarkers in real time, both in vitro and in vivo.

[0109] EAB sensors have been used in many non-clinical applications, such as environmental monitoring and for monitoring manufacturing processes.

[0110] It should be understood that this invention is applicable to electrochemical sensors other than EAB sensors. This invention is feasible for any type of electrochemical sensor that relies on changes in electron kinetics during the detection of a target analyte. Examples include... Figure 1B The sensor (10) shown is based on a binding-induced change related to the diffusion change of the redox reporter molecule (20) in the solution phase, which leads to a change in the electron transfer rate (ET becomes ET').

[0111] Another example is Figure 1C As shown, there is a sensor (10) based on the shift induced by ligand binding on the reporter molecule (20) or the change in the redox recombination energy of the reporter molecule induced by binding.

[0112] Figure 1D An example is a sensor (10) that is based on a binding-induced change in the flexibility of the reporter molecule or on a change in the coupling constant that defines how fast electrons transfer between the electrode (15) and the reporter molecule (20).

[0113] Figure 1E Another example shown is a sensor (10) which is based on the change in the efficiency of a space- or hydrodynamically induced scaffold-attached redox reporter molecule (20) approaching the surface of the underlying electrode (15).

[0114] By reference Figure 2 The illustrated scheme further describes the invention, using only the EAB sensor as an exemplary form of the sensor. A DC voltage is applied to the sensor's working electrode, and a voltage perturbation is applied to this DC voltage. The voltage perturbation is the sum of multiple sinusoidal waveforms, each with a different frequency. The sensor current (shown as the leftmost trace in the lower left figure) is processed by FFT-EIS to simultaneously measure impedance at multiple frequencies. The lower right figure shows a graph of phase versus frequency, with a significant phase shift in the presence of the target analyte. The simultaneous nature of the impedance measurement eliminates the need to scan multiple frequencies, allowing for rapid, continuous readings and thus improving time resolution. Furthermore, the electron transfer rate, and consequently the sensor response, is independent of the number of functional aptamers on the working electrode surface, thus avoiding the need for sensor calibration.

[0115] In EIS, a sinusoidal oscillating voltage above a set DC bias is applied to the working electrode, and the (sinusoidal) current response is recorded. The impedance Z at a specific frequency ω is defined as the ratio of voltage to current at that frequency (Equation 1), and the "hysteresis" between the voltage disturbance and the current response is quantified as the phase shift Φ.

[0116]

[0117] Here, |V| and |I| are the amplitudes of voltage and current, respectively, ω is the frequency, i is the square root of -1, and |Z| is the magnitude of the impedance. The main advantage of EIS (and its "spectroscopy" label) stems from the measurement of Z over a wide frequency range (e.g., millihertz to kilohertz). Specifically, frequency-dependent impedance measurements can be used to characterize processes ranging from rapid charging of the electric double layer at high frequencies to electron transfer reactions and molecular diffusion occurring on longer timescales.

[0118] In applications such as real-time measurements of specific molecules in vivo, a limitation of EIS is its typically poor temporal resolution. Specifically, for conventional "frequency-scanning" EIS, each interrogated frequency f requires at least 1 / f of the measurement time. Therefore, measuring spectra down to approximately 1 Hz requires tens of seconds or longer of total acquisition time. In this invention, FFT-EIS preserves information contained across the entire frequency range while significantly reducing acquisition time. It achieves this by simultaneously measuring impedance at multiple frequencies.

[0119] In one application of the FFT-EIS according to the invention, the applied voltage perturbation is a superposition of 18 sine waves spanning the desired frequency range. It should be understood that other numbers of sine waves may be feasible, for example, at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 sine waves.

[0120] Because the electrochemical system is approximately linear under small voltage changes, the resulting current response is a superposition of current responses at each applied frequency. Therefore, a fast Fourier transform (FFT) of the recorded voltage and current data can produce an impedance spectrum. Using this method, an impedance spectrum can be generated over a timescale defined by the slowest applied frequency (t...). spectrum ≥1 / f min Collect complete impedance spectra.

[0121] Electron transfer kinetics can be extracted from impedance spectroscopy. For example, the spectrum can be first fitted to an equivalent circuit to extract parameters describing the circuit, such as the adsorption pseudocapacitance C. ad Solution resistance R s Double-layer capacitor C dl and charge transfer resistance R ct Then the electron transfer kinetics can be evaluated from its relationship with these parameters.

[0122] The invention will now be described more fully with reference to the following non-limiting embodiments.

[0123] Example 1: Materials and methods used in the experimental work described in Examples 2 to 6

[0124] Example 1.1 Materials

[0125] The in vitro sensors are fabricated using 0.2 mm diameter gold wire (99.99% purity) and insulated with polyolefin heat shrink tubing (0.05", 0.017", 0.007"). For in vitro testing, commercial Ag|AgCl(s) reference electrodes (and commercial platinum reference electrodes) are used. However, it is noted that such tests can be performed using any reference electrode suitable for both in vitro and in vivo experiments. The intravenous sensors for in vivo measurements are fabricated using 0.2 mm diameter gold wire, 0.005 inch diameter platinum wire (99.99% purity), and 0.005 inch diameter silver wire (99.99% purity). The insulation material used for these sensors is polytetrafluoroethylene heat shrink tubing (HS). Sub-Lite-Wall (0.02, 0.005, 0.003 ± 0.001 inches, black). Sodium hydroxide, 6-mercapto-1-hexanol, tris(2-carboxyethyl)phosphine, sulfuric acid, phenylalanine, and phenylalanine assay kits were obtained. Phosphate-buffered saline (PBS) was prepared by diluting 20x stock solution. Vancomycin-HCl and methylene blue- and HO-C6S-S-C6-modified DNA sequences were purchased from commercially available sources; their sequences are listed below.

[0126]

[0127] Example 1.2 Sensor Manufacturing

[0128] The in vitro sensors were fabricated by heat-shrinking gold wire with polyolefin, leaving 3 mm of bare wire exposed. These sensors were prefabricated and required no additional steps before electrochemical cleaning.

[0129] The intravenous sensor is constructed using gold (for the working electrode), platinum (for the counter electrode), and silver (for the reference electrode). These wires are individually heat-shrinkable insulated with PTFE and bundled together in an interleaved manner, with the gold wire at the bottom, followed by the platinum wire, and then the silver wire. The exposed lengths of each wire are 3 mm, 6 mm, and 1 cm, respectively. After bundling, the intravenous three-electrode sensor is immersed overnight in household bleach (7.5% sodium hypochlorite) to chlorinate the silver electrode. The three electrodes are then rinsed with Millipore water before electrochemical cleaning.

[0130] Prior to aptamer deposition, the gold working electrode was electrochemically cleaned in NaOH and then roughened using a CH1040C regulator in H2SO4. Cleaning involved immersing the electrode in 0.5M NaOH and cycling the potential between -1.0V and -2V at 2V / s for 1000 cycles. Roughening was then performed in 0.5M H2SO4 while applying 20ms pulses at 0V and 2.2V for 32,000 cycles, as previously described, to increase the electrode's microsurface area. The electrodes were then analyzed by cyclic voltammetry in 0.5M H2SO4 (1.5 to -0.35V, 1V / s) to determine their electroactive surface area. At this point, an intravenous sensor for in vivo was inserted into a 20G catheter.

[0131] To functionalize the working electrode, the alkylthiols and disulfide bonds in the methylene blue-modified aptamer in the stock solution were first reduced by binding 14 μL of 10 mM tris(2-carboxyethyl)phosphine with 2 μL of 100 μM DNA in the dark for 1 hour. The gold electrode was electrochemically cleaned and roughened with Millipore water, then immersed in 500 nM reduced DNA in PBS for 1 hour. The electrode was then transferred to PBS solution of 10 mM 6-mercapto-1-hexanol and stored overnight before use.

[0132] Example 1.3 Electrochemical Measurement

[0133] All electrochemical measurements were performed using a three-electrode setup. In vitro experiments used an Ag|AgCl (saturated KCl) reference electrode and a platinum wire counter electrode. In vivo experiments used a silver wire coated with silver chloride (as described above) as the reference electrode and a platinum wire counter electrode. Any other reference electrode suitable for in vivo use may be substituted, if desired.

[0134] All electrochemical measurements were performed using an Autolab PGStat128N regulator. The regulator was configured in "high stability mode" with a current range of ±1 μA, which affected filter characteristics. For FFT-EIS measurements, multiple sinusoidal waveforms were generated using a DG812 arbitrary waveform generator (Rigol Technologies) and fed into the external voltage input of the regulator via a BNC connection. The voltage waveform consisted of 18 superimposed sine waves with logarithmically spaced frequencies from 1 Hz to 1 kHz. The amplitude and phase of each sinusoidal oscillation were optimized for maximum signal-to-noise ratio, as described in the supporting information for Example 7, and the summed waveform was scaled to a peak-to-peak amplitude of 25 mV. The apparent potential of methylene blue was set using the regulator's built-in software (NOVA) and measured by cyclic voltammetry over AC disturbances. Voltage and current were recorded at 70 kHz using an SDS1202X-E oscilloscope. After collecting each oscilloscope frame (1.4 seconds), the current and voltage data are transmitted to the host computer, subjected to Fast Fourier Transform, saved, and displayed on the GUI for real-time monitoring. Data logging and processing are controlled by a custom Python program. Further details regarding the selected waveforms and manual corrections are described in the support information for Example 7. Figure 7 (Figures 9 and 11).

[0135] After each experiment, the impedance spectrum was fitted to the equivalent circuit model using MEISP 3.0. The adsorption pseudocapacitance C was then... ads The model is performed as a constant-phase element, given by Equation 2 (where i is the imaginary number, ω is the frequency, and n is the constant-phase parameter). The parameter n is fixed at 0.84 for all fits to improve the fit C. ads Consistency of values.

[0136]

[0137] Example 1.4 In vivo measurement

[0138] All in vivo experiments were conducted on male Sprague-Dawley rats (4-5 months old). The rats weighed 350-500g and were housed in pairs in a standard photoperiod room (12:12 regular photoperiod, lights turned on at 8:00 AM). They were allowed free access to food and water, and the experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Santa Barbara, in accordance with NIH guidelines for laboratory animal care and use.

[0139] Prior to measurement, rats were anesthetized in an plexiglass anesthesia chamber using 4% isoflurane. Anesthesia was then maintained throughout the experiment using 2-3% isoflurane via a nasal cone. The necks were shaved and dissected to surgically separate the left and right jugular veins. After separating the two jugular veins, each vein was ligated using sterile 6-0 silk sutures. Before measurement, the leads in the sensor were adjusted so that the counter and working electrodes were exposed outside the 20G catheter entering the vein, as previously described. A small incision was then made in each vein using spring-loaded microscissors, allowing insertion of the catheter containing the sensor into the right jugular vein and the infusion tubing into the left jugular vein. The sensor and drug infusion tubing were anchored in place using two sterile 6-0 silk sutures. Immediately after sensor insertion and before any recording, 30 units of heparin were infused via the infusion tubing. To administer the rats intravenously at a dose of 30 mg / kg, a pre-calculated volume of 0.05 M vancomycin solution was injected using an infusion pump.

[0140] Example 2: FFT-EIS can rapidly correlate the electrochemical impedance of an EAB sensor with the concentration of target molecules.

[0141] Referring to Figure 3, in (A), the vancomycin detection EAB sensor is immersed in whole bovine blood at 37°C and challenged by an increased number of target molecules. This induces a conformational change in the aptamer, resulting in a shift in the impedance spectrum, as resolved by FFT-EIS.

[0142] In (B), equivalent circuit modeling is used to analyze the FFT-EIS data. The circuit model used includes a representation of the bulk solution resistance (R0). s ) and Faraday electron transfer (R) between the electrode and the methylene blue portion ct Resistors representing electrochemical double layers (C) and resistors representing electrochemical double layers (C) dl The pseudocapacitance (C) between the electrode surface and the surface-bound methylene blue. ads The capacitor was used. Kirchhoff's laws were used to calculate the transfer function of the circuit, and the experimental measurements (blue dots) were fitted to this function (dashed line). The data presented in this paper were collected from a vancomycin detection sensor immersed in whole bovine blood at 37°C in the absence of vancomycin.

[0143] In (C), using vancomycin at increased concentrations to target the EAB sensor showed that increasing the target concentration primarily affected (in this case, decreased) R. ct This is because a low electron transfer resistance corresponds to a high electron transfer rate constant, which corresponds to the target binding state of the aptamer (the error bars in this figure represent the standard deviation of four independently manufactured and queried sensors).

[0144] In (D), the electron transfer rate k et Formula 5 can be used to extract from R ct and C adsThe approximate result is obtained. The resulting binding curve conforms to the Hill-Langmuir isotherm, and the dissociation constant (K) is... D The value was 144 ± 31 μM (the latter reflects the 95% confidence interval of the estimate).

[0145] The impedance characteristics of the EAB sensor are sensitive to the target concentration and can be rapidly measured using FFT-EIS, enabling high-time-resolution molecular measurements. To demonstrate this, the half-wave potential (E0) of the methylene blue redox reporter molecule from the sensor was measured. 1 / 2 ) is applied as a DC bias voltage (before each experiment; E 1 / 2 The impedance spectra were determined using cyclic voltammetry, typically approximately -0.285 V relative to Ag|AgCl (Figure 8). Then, FFT-EIS was used to record the impedance spectra while the sensor was immersed in whole bovine blood at 37°C. When the spectra obtained in the absence of the target molecule are presented as Bode plots (phase relative to frequency),... Figure 3A When displayed as a black trajectory, a local maximum was observed around 10 Hz, reflecting the electron transfer rate between methylene blue and the electrode surface. As expected (considering the increased electron transfer rate between methylene blue and the electrode upon target binding), this peak steadily shifted to higher frequencies as the sensor was exposed to increased concentrations of vancomycin. To determine the origin of this concentration-dependent shift, the surface-immobilized redox material (SMR) previously used to represent what is seen in EAB sensors was analyzed. Figure 3B A simple four-element equivalent circuit is presented, modeled using the equivalent circuit method. This equivalent circuit includes resistors to simulate the bulk solution resistance (R0). s ), which is connected in series with three other components: a capacitor, representing the interface double-layer capacitance (C dl The resistor connected in parallel represents the Faraday charge transfer resistance (R0). ct ), and capacitor (C) ads ), representing methylene blue adhering to the surface. To better explain the rough, non-ideal surface of the EAB sensor, the latter was modeled as a phase-constant element rather than a true capacitor. This model fits the FFT-EIS data very well (x 2 ~0.015 (Figure 3, B), indicating that this four-component equivalent circuit is sufficient to describe the physical characteristics of the sensor.

[0146] As expected, the components of the equivalent circuit responded to the sensor being challenged by the target object. For example, R s C dl and C ads In fact, it is unrelated to vancomycin concentration. In contrast, R ct The rate decreases with increasing vancomycin concentration (Figure 3, C). This may be due to the increased electron transfer rate between the electrode and the bound, folded aptamer, because R...ct With k et Inversely proportional:

[0147]

[0148] Here, R is the gas constant, T is the temperature, F is the Faraday constant, A is the electrochemical surface area of ​​the working electrode, and Γ is the surface coverage of the oxidizing active molecules. Except for k... et Furthermore, each of these variables is constant during a given experiment, therefore R0 ct The reduction is entirely attributed to k et The increase of C. Therefore, conversely, C ads It is given by the following formula:

[0149]

[0150] k et It can be generated by R ct and C ads calculate

[0151]

[0152] Given that the electron transfer rate from the redox reporter molecule depends on whether the aptamer binds to the target, when k et When plotting vancomycin concentrations, the Langmuir-Hill isotherm was drawn. As expected, it was indeed so (Figure 3, D). The resulting monotonic relationship can be used to calculate k... et This is converted to an estimated vancomycin concentration; this method requires no calibration. Specifically, k et The absolute Faraday current is independent of the number of surface-bound, methylene blue-modified aptamers, and therefore independent of important sensor-to-sensor source fabrication changes, such as variations in electrode microsurface area or aptamer packing density, which alter the number of methylene blue-modified aptamers and thus the absolute Faraday current. Therefore, the k measured by a single sensor... et The calibration curve for vancomycin concentration can be applied to all other sensors using the same aptamer, thus avoiding the need to calibrate each individual sensor.

[0153] Example 3: FFT-EIS supports high-frequency real-time measurement in vivo.

[0154] Refer to Figure 4. In (A), the in vivo EAB sensor consists of an aptamer-functionalized gold working electrode, a platinum counter electrode, and an Ag|AgCl reference electrode, each bound in a heat-shrink tubing and inserted through a catheter into the right jugular vein of anesthetized rats. (B) Using FFT-EIS, impedance spectra were measured approximately every 1.8 seconds by applying the apparent potential of methylene blue as a DC bias and a necessary multisine waveform as an AC perturbation. The magnitude of the impedance, |Z|, increases during the experiment, particularly at low frequencies. This is presumably due to contamination caused by the non-specific adsorption of proteins, cells, or small molecules onto the sensor surface. Refer to (C). Fitted spectra reveal this drift in relation to R... ct The immediate, steady increase and C ads The corresponding reduction was associated with this. However, after injection of 30 mg / kg vancomycin, only R... ct There is a response. See (D). k et Calculations show that this parameter is stable before drug infusion, indicating that the intrinsic electron transfer rate constant of the aptamer unbound state is not affected by R. ct and C ads The influence of any factors affecting drift. However, after drug infusion, k et A sudden increase indicates a greater number of aptamer target binding states. After infusion, as the drug is excreted by the kidneys, kJ... et The trend declines, and unbound aptamers once again dominate. Here, the raw data (light blue dots) is smoothed using a 13-second rolling average (dark blue trajectory).

[0155] Example 4: FFT-EIS interrogation of EAB sensor supports rapid measurement of specific molecules in vivo.

[0156] An aptamer-modified gold wire working electrode was bundled with a platinum counter electrode and a silver-silver chloride reference electrode in a 20-gauge catheter (Fig. 4, A), and the resulting three-electrode sensor was surgically inserted into the right jugular vein of anesthetized rats. The total impedance spectrum (containing 18 frequencies between 1 Hz and 1 kHz, Fig. 9) was then measured every 1.8 seconds for 2.5 hours. This was compared with the relatively constant impedance observed in whole blood in vitro. Figure 10 In contrast, under these in vivo conditions, the impedance increases over time (Figure 4, B), suggesting that the degradation mechanism of the EAB sensor may differ under the two conditions. Fitting the time-resolved spectra indicates that this impedance change is related to R... ct The steady increase and C ads The corresponding reduction is related to (Figure 4, C). This may occur due to the non-specific adsorption of proteins and cells to the electrode, which is expected to increase R by reducing the number of methylene blue reporter molecules capable of contacting the electrode surface. ct And reduce C ads In contrast, k etNo drift (Figure 4, D), indicating that any factor causing impedance drift does not affect the electron transfer kinetics of maintaining the electrochemically accessible aptamer. However, after infusion of 30 mg / kg vancomycin, as the aptamer transforms to its target binding conformation, k et Rapidly rising (Figure 4, D). After the infusion ends, as the drug is cleared from the plasma via the kidneys, k et Return to its baseline value.

[0157] Example 5: Using FFT-EIS interrogation of the EAB sensor enables rapid time-resolved molecular measurements in vivo.

[0158] Refer to Figure 5. Experiments in three independent animals confirmed the absence of drug prior to infusion and showed the expected peak concentration. Here, the raw data (light dots) are smoothed using a 13-second rolling average (darker trajectory). After each administration, antibiotic concentrations exhibited a single exponential decay (fitted as a black trajectory) with time constants of 33.1 ± 0.5, 37.3 ± 0.7, and 47.4 ± 1.3 minutes (error bars reflect 95% confidence intervals).

[0159] FFT-EIS interrogation of the EAB sensor provides a highly time-resolved window into molecular physiology and pharmacokinetics (Figure 5, A). To visualize this, the sensor was inserted into the jugular vein of three rats. Prior to drug infusion, vancomycin concentrations were measured to fluctuate closely around zero (1.1 ± 1.7 μM). After infusion, concentrations were observed to rise to a maximum of 70–250 μM, followed by an exponential decline over time constants of 32–47 minutes. The varying decay rates observed across the three independent animals reflect the unique physiology of each animal, highlighting the benefits of personalized clinical dosing using the EAB sensor.

[0160] Example 6: Applying FFT-EIS to the EAB sensor for detecting phenylalanine

[0161] To demonstrate the general applicability of FFT-EIS as a querying technique for EAB sensors, this technique was then used to query a sensor targeting the endogenous target phenylalanine (Figure 6). Specifically, after calibrating the phenylalanine detection EAB sensor in vitro, FFT-EIS querying was used to detect the concentration of this molecule in the jugular vein of anesthetized rats at a resolution of 1.8 seconds. In doing so, baseline phenylalanine concentrations were observed to be 41 ± 10 μM in fasted animals (the latter reflecting one standard deviation) and 39 ± 6 μM in non-fasted animals, values ​​consistent with previous reports. Following two intravenous infusions of additional phenylalanine, a rapid rise to a peak concentration of 300–400 μM was observed in fasted animals, followed by a rapid decay back to baseline. Fitting the decay transients to a previously reported bi-exponential model of phenylalanine kinetics yielded time constants of τ1 = 0.2 ± 0.1 min and τ2 = 3.8 ± 0.2 min for the first injection and τ1 = 1.7 ± 0.1 min and τ2 = 20 ± 2 min for the second injection (error bars at 95% confidence intervals), suggesting that the animals' ability to rapidly store additional phenylalanine may have saturated after the first challenge. In contrast, in non-fasted animals, measurements showed higher peak concentrations (~600 μM) and slower decay (τ1 = 3.3 ± 0.1 min and τ2 = 35 ± 7 min) to a higher, slowly decaying baseline after the phenylalanine challenge, differences consistent with previous in vivo measurements of phenylalanine kinetics in fasted and non-fasted rats.

[0162] Referring to Figure 6. This metabolite in vivo in the jugular vein of live rats was monitored in situ at a resolution of 1.8 seconds using an FFT-EIS-interrogated EAB sensor for phenylalanine detection. Reference (A). Two consecutive doses of phenylalanine were infused into fasted animals. In both cases, the concentration of free phenylalanine in the blood rapidly decayed back to the pre-infusion baseline (41 ± 10 μM, 31 ± 9 μM, and 25 ± 10 μM before, after the first injection, and after the final injection, respectively). Reference (B). In contrast, in non-fasted animals, recovery to the post-infusion baseline was slower, consistent with previous reports on phenylalanine steady-state. Here, the raw data (light dots) are smoothed using a 13-second rolling average (darker trajectory). Concentration decay transients were fitted to a two-compartment (i.e., two-exponential) model (black trajectory).

[0163] Example 7: Supporting information related to the experimental work described in Examples 2 through 6

[0164] Example 7.1 Multi-frequency waveform

[0165] Figure 7 The table shown details the frequency, amplitude, and phase used to construct the multi-frequency perturbation waveform.

[0166] Example 7.2 Sensor Cyclic Voltmeter

[0167] Referring to Figure 8(A), the cyclic voltammogram recorded by the vancomycin-detected EAB sensor in 1X PBS + 2mM MgCl is shown. The voltammogram was recorded at a scan rate of 100 mV / s at 25 °C. The half-wave potential (average voltage between the reduction and oxidation peaks), E1 / 2, was measured as -285 mV (relative to Ag|AgCl). Cyclic voltammograms were recorded prior to all EIS experiments to determine E1 / 2. 1 / 2 It is applied as a DC bias during EIS. See reference (B). In vivo, E... 1 / 2 (Black dashed line) represents -324 mV; this difference stems from the use of anodized silver wire as the reference electrode, rather than a sintered single-junction reference electrode used in vitro. Reference (C) shows the cyclic voltammogram recorded in vivo at the end of the vancomycin dosing experiment. Although the peak area is reduced (presumably due to monolayer loss), E... 1 / 2 Maintain at -324mV.

[0168] Referring to Figure 9, (A) shows the frequency domain representation of the multisine EIS waveform used, while (B) shows its time domain representation.

[0169] A set of 18 log-interval frequencies was selected, which are integer multiples of the fundamental frequency (1 Hz) to avoid any second harmonics. The phase was chosen to minimize constructive interference. The amplitude was set to produce a similar current output at each frequency (i.e., V(ω) ∝ |Z|(ω)). This strategy significantly increases the signal-to-noise ratio by applying a higher voltage amplitude at low frequencies, where the current is typically lower than at high frequencies (in electrochemical cells). The listed frequency f, amplitude a, and phase were used. ( Figure 7 The waveform is digitally synthesized into:

[0170]

[0171] The optimized waveforms used in this study are shown in Figure 9(A) in the frequency domain and in Figure 9(B) in the time domain. In the time domain, the peak-to-peak amplitude of the summed waveform was set to 25mV. The digital waveforms were saved to a Rigol DG812 arbitrary waveform generator, which outputs a waveform at 100kHz as the voltage input to an Autolab PGStat128N.

[0172] refer to Figure 10 Bode|Z| plots were collected from a vancomycin detection EAB sensor that was immersed in whole bovine blood at 37°C and challenged with increasing concentrations of vancomycin. Although phase shift was observed, the modulus of impedance did not change significantly with target concentration at any frequency.

[0173] Example 7.3 Filter Correction

[0174] The following experiments were conducted to accurately measure the impedance spectrum by correcting for systematic artifacts in the experimental setup.

[0175] Referring to Figure 11, the phase (blue) and modulus (green) Bode plots of a 10kΩ resistor. (A) shows the spectrum without filter correction. From 1Hz to 1kHz, |Z| is the expected value for 10kΩ, but the phase is non-zero at high frequencies. Referring to (B), after correction, the phase is zero across the entire frequency spectrum.

[0176] The low-pass current filter applied by the regulator may affect the measured impedance spectrum and should be corrected. For this purpose, the impedance spectrum of a 10kΩ resistor was measured (Figure 8, A). Although the resistor was expected to have a constant 10kΩ impedance and a constant 0° phase, a phase shift of 7° was measured at 1kHz. This is caused by the tailing of the low-pass filter, which cannot be directly controlled on the regulator used in this study but is affected by the choice of current range. Subsequently, the impedance spectrum was corrected using this reference spectrum, as shown in Equations S2 and S3:

[0177]

[0178] φ(ω)=φ measured (ω)-φ ref (ω) (S3)

[0179] The impedance spectrum of the same 10kΩ resistor was re-recorded and this correction procedure was applied, yielding the expected 10kΩ|Z| with a phase of 0° across the entire spectrum (Fig. 8, B).

[0180] refer to Figure 12 The binding curve of the EAB sensor for phenylalanine detection using EIS query can be used to determine the binding curve of phenylalanine by k. et The concentration of phenylalanine was determined. To record this curve, the sensor was immersed in freshly collected whole rat blood at 37°C. The endogenous concentration of phenylalanine in the blood (52 μM) was determined using a fluorescence detection kit. Then, an aliquot of phenylalanine dissolved in PBS-BSA was added to the blood to increase the phenylalanine concentration. et The values ​​(black dots, error bars representing the standard deviation of four independently manufactured and tested sensors) fall on the Langmuir isotherm.

[0181]

[0182] Where k et,0 K is the value when phenylalanine is absent. et Value, k et,max It is the k when phenylalanine is saturated. et Value, kD is the dissociation constant, and n is the Hill coefficient. Fitting this equation yields k. et,0 =62.9s -1 k et,max =225.8s -1 k D =6.89mM, and n=0.38.

[0183] Example 8: Using FFT-EIS to interrogate the in vivo EAB sensor to monitor plasma lactate concentration

[0184] FFT-EIS can be used to query in vivo EAB sensors to monitor plasma lactate concentration in live rats in real time. Figure 13A and 13B Lactate is an important clinical biomarker for sepsis and tissue hypoxia during anesthesia, and lactate levels help monitor motor performance. This was achieved by gradually altering lactate levels and measuring the corresponding kJ / L2 levels. et The values ​​were characterized using FFT-EIS to measure lactate levels in undiluted blood at approximately 37°C using the EAB sensor. Figure 13A Electron transfer rate k et It varies with lactate levels and displays the Langmuir binding isotherm. This is achieved by measuring kJ using an EAB sensor for lactate detection. et The changes can be measured in real time by measuring the lactic acid concentration in live rats. Figure 13B A stable concentration was measured before intravenous infusion of approximately 1000 mg / kg sodium lactate. Plasma lactate levels subsequently rose, then gradually returned to the previous baseline as lactate was degraded.

[0185] The in vitro EAB sensor was manufactured by soldering a 4.5cm gold wire to an electrochemical connector for connection to a voltage regulator and using a 3.6cm heat-shrinkable polyolefin tube for insulation. The uncovered gold wire was cut to 6mm.

[0186] To fabricate the electrodes for the intravenous sensor, gold (0.2 μm diameter × 10 cm length; 99.9% purity), platinum (0.125 μm diameter × 10 cm length; 99.95% purity), and silver (0.125 μm diameter × 10 cm length; 99.99% purity) wires were cut and insulated with polytetrafluoroethylene (PTFE) heat-shrink tubing. These wires were bundled together, with physical gaps separating each wire to prevent short circuits. The insulation was then trimmed to produce exposed lengths of 3 mm (gold), 5 mm (platinum), and 1 cm (silver). To convert the silver wire into a reference electrode, it was immersed overnight in 7.5% sodium hypochlorite (a commercial bleach) to form a stable silver chloride film. Finally, the electrodes were rinsed with Milli-Q water to remove any residual bleach.

[0187] To clean the gold wire, it was first immersed in a 0.5M NaOH solution, and electrochemical cleaning was performed using a three-electrode setup with a platinum counter electrode and an Ag / AgCl reference electrode. The potential window ranged from -1.0V to -2.0V (relative to the potential of Ag / AgCl), and the cycle was repeated 1000 times using a CH1040C regulator at a scan rate of approximately 1V / s. Next, the micro-roughness of the gold wire was increased by placing it in a 0.5M HSO solution and pulsating from 0.0V to 2.2V with a pulse width of 0.02 seconds, repeating this process approximately 32,000 times. The degree of surface roughening was verified by measuring the electrode surface area using a potential window of 0V to 1.8V in the 0.5M HSO solution at a scan rate of 1V / s for 10 cycles.

[0188] The lactate aptamer was modified with hexa-thiol and the redox reporter molecule methylene blue. The aptamer was reduced for 1 hour in a solution containing approximately 11.26 μM aptamer and 8.87 mM TCEP, followed by dilution to 500 nM in PBS buffer + 2 mM MgCl₂. The intravenous sensor was inserted through a 20-gauge catheter, and the in vitro sensor was used as is. To form the lactate detection EAB sensor, the aptamer was deposited on a gold electrode by immersing the electrode in a 500 nM solution of reduced lactate aptamer for 1 hour, followed by overnight immersion in 10 mM 6-mercaptohexanol to form a self-assembled monolayer. The fabricated sensor was rinsed with Milli-Q water before use. The catheter was filled with 1×PBS before in vivo use.

[0189] An in vitro sensor was used to calibrate the lactate detection EAB sensor in bovine blood using FFT-EIS. 2.5 mg / mL NaF was added to the blood to inhibit anaerobic glycolysis, and the blood was stored at approximately 37°C for 2 hours prior to calibration. The lactate detection EAB sensor was immersed in the blood for approximately 45 minutes to obtain a stable baseline. FFT-EIS was performed on an Autolab PGStat128N. Endogenous lactate concentration in the blood was determined using a lactate blood test kit prior to calibration. The lactate concentration in the blood sample was increased to 106 mM, and then gradually decreased by diluting the blood lactate level with blood containing no additional lactate and PBS + 25 mg / mL BSA.

[0190] In vivo experiments were conducted using adult male Sprague-Dawley rats (4-5 months old, 300-500 g). These rats were housed in pairs in a temperature- and humidity-controlled animal room under a 12-hour light-dark cycle, with free access to food and water. Anesthesia was induced in a resin-glass anesthesia chamber using 4% isoflurane gas. Anesthesia was maintained during the experiment by administering 2-3% isoflurane gas / oxygen via a nasal cone. Heart rate and SpO2 were measured using a pulse oximeter during the experiment. The rats' fur was shaved, and the skin above the jugular vein was disinfected with 70% ethanol and povidone-iodine. A small incision was made to separate the two jugular veins. A small incision was made in the jugular vein using spring-loaded microscissors. A silicone catheter (consisting of a curved steel cannula and silicone tubing) was inserted into the left jugular vein for infusion. An EAB sensor was inserted into the right jugular vein for intravenous lactate monitoring and stabilized with sterile 6-0 silk sutures. After insertion, 30 units of heparin were infused via an indwelling infusion line to prevent clotting on the electrode surface. Before lactate infusion, wait approximately 45 minutes to establish a stable baseline. For lactate administration, infuse 1× phosphate-buffered saline solution in 3M sodium lactate stock solution via a silicone tubing connected to an electric infusion pump.

[0191] Example 9: Discussion of the experimental results described in Examples 2 to 8

[0192] The experimental work detailed above establishes FFT-EIS as a rapid and reliable interrogation method for EAB sensors, suitable for both in vitro and in vivo applications. Specifically, this work demonstrates FFT-EIS's ability to measure electron transfer rates associated with EAB sensors and utilize these rates to determine the concentration of target analytes with a time resolution of only 1.8 seconds. This is because this method uses k... et As a means of monitoring target concentration (rather than absolute current), it is independent of sensor-to-sensor manufacturing variations and drift caused by biofluid contamination, making the technology suitable for performing calibration-free in vivo measurements. To support this, vancomycin detection EAB sensors and phenylalanine detection EAB sensors have been demonstrated to successfully monitor plasma concentrations of these targets in the veins of live animals with a time resolution better than 2 seconds, without requiring calibration of each individual sensor. When combined with the modularity of aptamers, the impedance interrogation-related benefits of EAB sensors can enhance our understanding of pharmacokinetics, metabolism, disease progression, and neurochemistry, playing a significant role in the future of personalized medicine.

[0193] In this system, any device or server may include a network interface device configured to interface with other components of the system. This network interface device typically routes data to and outputs data from system components.

[0194] The methods and apparatus described herein can be deployed, in whole or in part, via one or more processors that execute computer software, program code, and / or instructions on the processor. The processor can be part of a server, client, network infrastructure, mobile computing platform, fixed computing platform, or other computing platform. The processor can be any type of computing or processing device capable of executing program instructions, code, binary instructions, etc. The processor can be or can include signal processors, digital processors, embedded processors, microprocessors, or any variations thereof, such as coprocessors (mathematical coprocessors, graphics coprocessors, communication coprocessors, etc.) that can directly or indirectly facilitate the execution of program code or program instructions stored thereon. Furthermore, the processor can implement the execution of multiple programs, threads, and code.

[0195] Multiple threads can be executed concurrently to enhance processor performance and facilitate simultaneous operation of applications. Through execution, the methods, program code, program instructions, etc., described herein can be executed in one or more threads. Threads can spawn other threads, which may have priorities assigned to them; the processor can execute these threads based on priority or any other order of instructions provided in the program code. The processor may include memory storing the methods, code, instructions, and programs described herein and elsewhere.

[0196] Any processor, mobile communication device, or server can access a storage medium via an interface, which can store the methods, code, and instructions described herein and elsewhere. Storage media associated with a processor for storing methods, programs, code, program instructions, or other types of instructions executable by a computing or processing device can include, but are not limited to, one or more of the following: CD-ROM, DVD, memory, hard disk, flash drive, RAM, ROM, cache memory, etc.

[0197] The processor may include one or more cores that can improve the speed and performance of the multiprocessor. In some implementations, the processor may be a dual-core processor, a quad-core processor, or other chip-level multiprocessors that combine two or more independent cores (referred to as a die).

[0198] The methods and systems described herein can be deployed, in part or in whole, via one or more hardware components that execute software on servers, clients, firewalls, gateways, hubs, routers, or other such computer and / or network hardware. The software program may be associated with a server, which may include file servers, print servers, domain servers, internet servers, intranet servers, and other variations, such as secondary servers, primary servers, distributed servers, etc. The server may include one or more of the following: memory, processors, computer-readable media, storage media, ports (physical and virtual), communication devices, and interfaces capable of accessing other servers, clients, computers, and devices via wired or wireless media, etc. The methods, programs, or code described herein and elsewhere may be executed by a server. Additionally, other devices required to perform the methods described herein may be considered part of the infrastructure associated with the server.

[0199] The server can provide interfaces to other devices, including but not limited to clients, other servers, printers, database servers, print servers, file servers, communication servers, distributed servers, etc. Furthermore, this coupling and / or connection can facilitate remote execution of programs across networks. Networking of some or all of these devices can facilitate parallel processing of programs or methods at one or more locations without departing from the scope of the invention. Additionally, any device connected to the server via the interface may include at least one storage medium capable of storing methods, programs, code, and / or instructions. A central repository can provide program instructions to be executed on different devices. In this execution, a remote repository can act as a storage medium for program code, instructions, and programs.

[0200] The software program may be associated with a client, which may include a file client, print client, domain client, internet client, intranet client, and other variations, such as a secondary client, primary client, distributed client, etc. The client may include one or more of the following: memory, processor, computer-readable medium, storage medium, port (physical and virtual), communication device, and interface capable of accessing other clients, servers, computers, and devices via wired or wireless media. The methods, programs, or code described herein and elsewhere may be executed by the client. Additionally, other devices required to perform the methods described in this application may be considered part of the infrastructure associated with the client.

[0201] The client can provide an interface to other devices, including but not limited to servers, other clients, printers, database servers, print servers, file servers, communication servers, distributed servers, etc. Furthermore, this coupling and / or connection can facilitate remote execution of programs across networks. Partial or complete networking of these devices can facilitate parallel processing of programs or methods at one or more locations without departing from the scope of the invention. Additionally, any device connected to the client via the interface may include at least one storage medium capable of storing methods, programs, applications, code, and / or instructions. A central repository can provide program instructions to be executed on different devices. In this execution, a remote repository can act as a storage medium for program code, instructions, and programs.

[0202] The methods and systems described herein can be deployed, in part or in whole, through a network infrastructure. This network infrastructure may include elements such as computing devices, servers, routers, hubs, firewalls, clients, personal computers, communication devices, routing devices, and other active and passive devices, modules, and / or components known in the art. Computing and / or non-computing devices associated with the network infrastructure may include, among other components, storage media such as flash memory, buffers, stacks, RAM, ROM, etc. The techniques, methods, program code, and instructions described herein and elsewhere may be executed by one or more of the network infrastructure elements.

[0203] The methods, program code, calculations, algorithms, and instructions described herein can be implemented on a cellular network with multiple cells. The cellular network can be a Frequency Division Multiple Access (FDMA) network or a Code Division Multiple Access (CDMA) network. The cellular network may include mobile devices, cell sites, base stations, repeaters, antennas, transmission towers, etc. The cellular network can be GSM, GPRS, 3G, 4G, EVDO, mesh, or other network types.

[0204] The methods, program code, calculations, algorithms, and instructions described herein can be executed on or through a mobile device. The mobile device may include navigation devices, cellular phones, mobile phones, mobile personal digital assistants, laptops, PDAs, netbooks, pagers, e-book readers, music players, etc. These devices may include, among other components, storage media such as flash memory, buffers, RAM, ROM, and one or more computing devices. The computing devices associated with the mobile device may be enabled to execute the program code, methods, and instructions stored thereon.

[0205] Alternatively, the mobile device may be configured to collaborate with other devices to execute instructions. The mobile device may communicate with a base station connected to the server and be configured to execute program code. The mobile device may communicate on a peer-to-peer network, mesh network, or other communication network. The program code may be stored on a storage medium associated with the server and executed by a computing device embedded within the server. The base station may include a computing device and a storage medium. The storage device may store program code and instructions executed by the computing device associated with the base station.

[0206] Computer software, program code, and / or instructions may be stored and / or accessed on a computer-readable medium, which may include: computer components, devices, and recording media that retain digital data for computation over a time interval; semiconductor storage called random access memory (RAM); mass storage typically used for more permanent storage, such as optical discs, various forms of magnetic storage (e.g., hard disks, magnetic tapes, magnetic drums, magnetic cards, and other types); processor registers, cache memory, volatile memory, and non-volatile memory; optical storage, such as CDs and DVDs; removable media, such as flash memory (e.g., USB flash drives or keys), floppy disks, magnetic tapes, paper tapes, punch cards, stand-alone RAM disks, Zip drives, removable mass storage, offline storage, etc.; and other computer memories, such as dynamic memory, static memory, read / write memory, variable memory, read-only memory, random access, sequential access, location-addressable, file-addressable, content-addressable, network-attached storage, storage area networks, barcodes, magnetic ink, etc.

[0207] The methods and systems 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.

[0208] The elements described and depicted herein, including the flowcharts and block diagrams in all the accompanying drawings, imply logical boundaries between elements. However, in accordance with software or hardware engineering practice, the depicted elements and their functions may be implemented on a computer via a computer-executable medium having a processor capable of executing program instructions stored thereon as a whole software architecture, as a standalone software module, or as a module using external routines, code, services, etc., or any combination thereof, and all such execution is within the scope of this disclosure.

[0209] The methods and / or processes and their steps described above can be implemented in hardware, software, or any combination of hardware and software suitable for a particular application. The hardware may include general-purpose computers and / or special-purpose computing devices, or specific aspects or components of a particular computing device. The processes may be implemented in one or more microprocessors, microcontrollers, embedded microcontrollers, programmable digital signal processors, or other programmable devices, as well as internal and / or external memory. The processes may also be embodied in application-specific integrated circuits, programmable gate arrays, programmable array logic, or any other device or combination of devices configurable to process electronic signals. It will be further understood that the one or more processes can be implemented as computer-executable code capable of executing on a computer-readable medium.

[0210] Application software can be created using structured programming languages ​​(such as C), object-oriented programming languages ​​(such as C++, Python), or any other high- or low-level programming languages ​​(including assembly language, hardware description languages, and database programming languages ​​and techniques). These languages ​​can be stored, compiled, or interpreted to run on one of the aforementioned devices, or on a heterogeneous combination of processors, processor architectures, or combinations of different hardware and software, or on any other machine capable of executing program instructions.

[0211] Therefore, in one aspect, each of the methods described above and combinations thereof can be embodied in computer-executable code, which, when executed on one or more computing devices, performs its steps. In another aspect, the methods can be embodied in a system that performs its steps and can be distributed in various ways within the device, or all functionality can be integrated into a dedicated, stand-alone device or other hardware. In yet another aspect, the means for performing the steps related to the above-described processes can include any of the hardware and / or software described above. All these permutations and combinations are intended to fall within the scope of this disclosure.

[0212] This invention can be embodied in a set of program instructions executable on one or more computers. Such a set of instructions may include any one or more of the following instruction types:

[0213] Data processing and memory operations may include instructions to set a register to a fixed constant value, or to copy data from a memory location to a register, or vice versa (machine instructions are often called moves, but the term is misleading), to store the contents of a register, the result of a calculation, or to retrieve stored data for later calculations, or to read and write data from a hardware device.

[0214] Arithmetic and logical operations, which may include adding, subtracting, multiplying or dividing the values ​​of two registers, placing the result into a register, possibly setting one or more condition codes in a status register, performing bitwise operations, such as taking the conjunction and disjunction of corresponding bits in a pair of registers, inverting each bit in a register, or comparing two values ​​in a register (e.g., checking if one is smaller or if they are equal).

[0215] Control flow operations can include instructions that branch to another location in the program and execute instructions there, conditionally branch to another location if a certain condition is met, indirectly branch to another location, or call another code block, while saving the location of the next instruction as a return point.

[0216] Coprocessor instructions may include instructions for loading / storing data to interact with the coprocessor, exchanging data with CPU registers, or performing coprocessor operations.

[0217] The computer's processor in this system may contain "complex" instructions in its instruction set. A single "complex" instruction may do what would require many instructions on another computer. Typical characteristics of such instructions are that they require multiple steps, control multiple functional units, or are otherwise larger than most simple instructions executed by a given processor. Some examples of "complex" instructions include: saving multiple registers to the stack at once, moving large blocks of memory, complex integer and floating-point operations (sine, cosine, square root, etc.), SIMD instructions, instructions that perform operations on multiple values ​​in parallel, atomic tests and set instructions or other read-modify-write atomic instructions, and instructions that perform ALU operations using operands from memory rather than registers.

[0218] Instructions can be defined based on their components. In more traditional architectures, instructions include an opcode specifying the operation to be performed (e.g., adding the contents of memory to a register) and zero or more operand specifiers, which can specify registers, memory locations, or literal data. Operand specifiers can have addressing modes that define their meaning, or they can be located in fixed fields. In Very Long Instruction Set (VLIW) architectures, which include many microcode architectures, multiple simultaneous opcodes and operands are specified in a single instruction.

[0219] Some instruction sets lack an opcode field (e.g., Transfer Triggered Architecture (TTA) or Forth Virtual Machine), containing only operands. Other unusual "0-operand" instruction sets lack any operand specifier field, such as some stack machines that include NOSC.

[0220] Conditional instructions typically have a predicate field—a few bits that encode a specific condition that causes an operation to be executed rather than not executed. For example, a conditional branch instruction will be executed and the branch will be adopted when the condition is true, causing execution to jump to a different part of the program; it will not be executed and the branch will not be adopted when the condition is false, allowing execution to continue in sequence. Some instruction sets also have conditional moves, such that: if the condition is true, the move will be executed and data will be stored at the target location; if the condition is false, it will not be executed and the target location will not be modified. Similarly, the IBM z / architecture has conditional storage. A few instruction sets include a predicate field in every instruction; this is called branch prediction.

[0221] The instructions that make up a program are rarely specified in their internal numerical form (machine code); they can be specified using assembly language, or more typically, generated by a compiler from a programming language.

[0222] Those skilled in the art will understand that the invention described herein is readily adaptable to other variations and modifications beyond the specific description. It should be understood that the invention includes all such variations and modifications falling within the spirit and scope of the invention.

[0223] Therefore, the spirit and scope of the present invention should not be limited to the foregoing examples, but should be understood in the broadest sense permitted by law.

Claims

1. A method for determining the amount of an analyte in a sample, comprising: The working electrode of the electrochemical sensor is interrogated by applying a voltage perturbation to it, wherein the voltage perturbation is the sum of two or more sinusoidal waveforms, each of which has a different frequency. Simultaneously measure the voltage across the working electrode and the counter electrode and / or the current through the working electrode and the counter electrode at each different frequency; By measuring the impedance at the frequencies of the two or more sinusoidal waveforms, an integral transform method is applied to the measured voltage and / or current values ​​to generate an impedance spectrum. as well as The impedance spectroscopy is used to determine the amount of analyte in the sample.

2. The method as described in claim 1, wherein the integral transform method is selected from Fourier transform, fast Fourier transform, Laplace transform, Merlin transform, Hartley transform, and Chirplet transform.

3. The method of claim 1 or 2, wherein the two or more sine waveforms are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sine waveforms.

4. The method of any one of claims 1 to 3, wherein the frequencies of the two or more sinusoidal waveforms define a frequency range, the frequency range including frequencies that provide information on electron transfer kinetics between redox reporter molecules of the electrochemical sensor and the working electrode surface.

5. The method of any one of claims 1 to 4, wherein the frequencies of the two or more sinusoidal waveforms define a frequency range that excludes frequencies that do not provide information on electron transfer kinetics between redox reporter molecules and the working electrode surface of the electrochemical sensor.

6. The method of any one of claims 1 to 5, wherein the frequencies of the two or more sinusoidal waveforms define a frequency range that excludes frequencies that would cause drift errors in the measured analyte concentration.

7. The method of any one of claims 1 to 6, wherein the frequencies of the two or more sine waveforms are each less than 2000 Hz, 1900 Hz, 1800 Hz, 1700 Hz, 1600 Hz, 1500 Hz, 1400 Hz, 1300 Hz, 1200 Hz, 1100 Hz, 1000 Hz, 900 Hz, 800 Hz, 700 Hz, 600 Hz, 500 Hz, 400 Hz, 300 Hz, 200 Hz, or 100 Hz.

8. The method of any one of claims 1 to 7, wherein the frequencies of the two or more sine waves are each 1Hz-2000 Hz, or each 1Hz-1000 Hz, or each 10Hz-100 Hz.

9. The method of any one of claims 1 to 8, wherein the frequencies of the two or more sinusoidal waveforms include a first frequency, a second frequency, and a third frequency, the first frequency providing information on electron transfer dynamics between redox reporter molecules of the electrochemical sensor and the working electrode surface; the second frequency being higher than the first frequency; and the third frequency being lower than the first frequency.

10. The method of any one of claims 1 to 9, wherein the frequencies of the two or more sinusoidal waveforms include a lower frequency and one or more frequencies higher than the lower frequency, and each of the one or more frequencies higher than the lower frequency is a multiple of the lower frequency.

11. The method of claim 10, wherein the lower frequency is the lowest frequency among the frequencies of the two or more sinusoidal waveforms.

12. The method of any one of claims 1 to 11, wherein the step of determining the amount of the analyte using the impedance spectroscopy comprises: The impedance spectrum or a portion thereof from the test sample is compared with the impedance spectrum or a portion thereof from a control sample that does not contain the analyte.

13. The method of claim 12, wherein the impedance spectrum from the control sample and the impedance spectrum from the test sample are both presented in frequency versus phase form.

14. The method of claim 12 or 13, wherein the impedance spectrum from the control sample and the impedance spectrum from the test sample each contain features at a first frequency and a second frequency, respectively, and wherein the amount of analyte is determined by reference to the difference between the first frequency and the second frequency.

15. The method of claim 14, wherein the feature is a peak or maximum value, a valley or minimum value, an upward sloping portion of the spectrum, or a downward sloping portion of the spectrum.

16. The method of any one of claims 1 to 15, wherein the step of determining the amount of analyte using the impedance spectroscopy comprises using the spectroscopy to determine the electron transfer kinetics between the redox reporter molecule of the electrochemical sensor and the working electrode surface.

17. The method of claim 16, wherein the electron kinetics is the electron transfer rate between the redox reporter molecule of the electrochemical sensor and the working electrode surface.

18. The method of any one of claims 1 to 17, wherein the method is capable of repeatedly determining the concentration of the analyte at intervals of less than 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, or 1 second.

19. The method of any one of claims 1 to 18, wherein the method comprises repeatedly determining the concentration of the analyte at intervals of less than 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds, 9 seconds, 8 seconds, 7 seconds, 6 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, or 1 second.

20. The method of any one of claims 1 to 19, wherein the method comprises repeatedly determining the concentration of the analyte for at least 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours.

21. The method of any one of claims 1 to 20, wherein the method does not require any frequency scanning step across a frequency range to determine the amount of analyte in the sample.

22. The method of any one of claims 1 to 21, wherein the method does not require any calibration step to determine the amount of analyte in the sample.

23. The method of any one of claims 1 to 22, wherein the method does not require any drift adjustment step to determine the amount of analyte in the sample.

24. The method of any one of claims 1 to 23, wherein the sample is bodily fluid within or around the subject.

25. The method of claim 24, wherein the body fluid is selected from: interstitial fluid (ISF), blood, saliva, lacrimal gland secretions, lactational secretions, nasal secretions, tracheal secretions, bronchial secretions, alveolar secretions, gastric secretions, gastric contents, glandular secretions, vaginal secretions, uterine secretions, prostatic secretions, semen, urine, sweat, cerebrospinal fluid, glomerular filtrate, liver secretions, bile, and intraocular fluid.

26. The method of any one of claims 1 to 25, wherein the working electrode is a wire, needle, or microneedle.

27. The method of any one of claims 1 to 26, wherein the electrochemical sensor includes a recognition element configured to specifically identify a target analyte.

28. The method of claim 27, wherein the recognition element is associated with a redox reporter molecule.

29. The method of claim 27 or 28, wherein the identification element and / or the redox reporter molecule changes in the presence of the target analyte, the change altering the electron transfer rate between the redox reporter molecule and the working electrode surface.

30. The method of claim 29, wherein the change in the recognition element is a conformational change.

31. The method of claim 30, wherein the conformational change of the recognition element alters the distance between the redox reporter molecule and the working electrode surface.

32. The method of claim 30 or 31, wherein the conformational change of the recognition element alters the recombination energy of the redox reporter molecule.

33. The method of any one of claims 28 to 32, wherein the rate at which the redox reporter molecule approaches the surface of the working electrode is altered in the presence of the target analyte.

34. The method of any one of claims 28 to 33, wherein the proportion of time the redox reporter molecule is near rather than far from the working electrode surface is altered in the presence of the target analyte.

35. The method of claim 33 or 34, wherein the change in rate or the change in time ratio is associated with a change in the spatial volume parameter, biomolecular rigidity parameter, electrostatic parameter, or hydrodynamic radius of the redox reporter molecule related to the target analyte.

36. The method of any one of claims 29 to 35, wherein the change in the recognition element and / or the redox reporter molecule is the dissociation of the recognition element from the target analyte.

37. The method of any one of claims 30 to 36, wherein the conformational change alters the electron transfer rate between the redox reporter molecule associated with the recognition element and the working electrode surface.

38. The method of any one of claims 27 to 37, wherein the identification element is associated with the working electrode surface, and the redox reporter molecule is associated with the identification element, and a conformational change of the identification element alters the distance between the redox reporter molecule and the working electrode surface, which in turn alters the electron transfer rate between the redox reporter molecule and the working electrode surface.

39. The method of any one of claims 27 to 38, wherein the identification element is a biopolymer.

40. The method of claim 39, wherein the biopolymer is a nucleic acid.

41. The method of claim 39 or 40, wherein the biopolymer is an aptamer.

42. The method of any one of claims 1 to 41, wherein the electrochemical sensor is configured as a wearable device.

43. An apparatus for detecting the amount of an analyte in a test sample, the apparatus comprising: An electrochemical sensor having a working electrode and a counter electrode; A power supply configured to apply a voltage perturbation to the working electrode, the voltage perturbation being the sum of two or more sinusoidal waveforms, each of the two or more sinusoidal waveforms having a different frequency; A voltage and / or current measurement circuit connected to the working electrode and the counter electrode; as well as A processor configured to perform an integral transformation on the measured voltage and / or current to generate an impedance spectrum, and to use the impedance spectrum to determine the amount of analyte in the test sample.

44. The apparatus of claim 43, wherein the processor is capable of accessing program instructions configured to perform the method of any one of claims 1 to 42.

45. The device of claim 43 or 44, wherein the electrochemical sensor comprises a redox reporter molecule, and the processor is capable of accessing program instructions configured to perform the method of claim 4 or 5.

46. ​​The device according to any one of claims 43 to 45, wherein the working electrode is a wire, needle, or microneedle.

47. The device of any one of claims 43 to 46, wherein the electrochemical sensor includes a recognition element configured to specifically identify a target analyte.

48. The device of claim 47, wherein the identification element is associated with a redox reporter molecule.

49. The device of claim 47 or 48, wherein the identification element and / or the redox reporter molecule changes in the presence of the target analyte, the change altering the electron transfer rate between the redox reporter molecule and the working electrode surface.

50. The device of claim 49, wherein the change in the identification element is a conformational change.

51. The device of claim 50, wherein the conformational change of the recognition element alters the distance between the redox reporter molecule and the surface of the working electrode or a coupling constant describing electron transfer through the recognition element.

52. The device of claim 50 or 51, wherein the conformational change of the recognition element alters the recombination energy of the redox reporter molecule.

53. The apparatus of any one of claims 48 to 52, wherein the rate or time proportion during which the redox reporter molecule approaches the surface of the working electrode is altered in the presence of the target analyte.

54. The apparatus of any one of claims 48 to 53, wherein the proportion of time the redox reporter molecule is near rather than far from the working electrode surface is altered in the presence of the target analyte.

55. The device of any one of claims 49 to 54, wherein the change in rate or time ratio is associated with a change in the spatial volume parameter, biomolecular rigidity parameter, electrostatic parameter, or hydrodynamic radius of the redox reporter molecule related to the target analyte.

56. The device of any one of claims 49 to 55, wherein the change in the recognition element and / or the redox reporter molecule is the dissociation of the recognition element from the target analyte.

57. The device of any one of claims 50 to 56, wherein the conformational change of the recognition element alters the electron transfer rate between the redox reporter molecule associated with the recognition element and the working electrode surface.

58. The device of any one of claims 47 to 57, wherein the identification element is associated with the working electrode surface, and the redox reporter molecule is associated with the identification element, and a conformational change of the identification element alters the distance between the redox reporter molecule and the working electrode surface, which in turn alters the electron transfer rate between the redox reporter molecule and the working electrode surface.

59. The device of any one of claims 47 to 58, wherein the identification element is a biopolymer or a biomimetic polymer.

60. The device of claim 59, wherein the biopolymer is a nucleic acid.

61. The device of claim 59 or 60, wherein the biopolymer is an aptamer.

62. The device of any one of claims 43 to 61, wherein the electrochemical sensor is configured as a wearable device.

63. A non-transient computer-readable medium comprising computer-executable program instructions configured to perform the method of any one of claims 1 to 42.

64. The device of any one of claims 43 to 62, wherein the program instructions are provided by the non-transient computer-readable medium of claim 63.

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