Calibration-free in vivo measurement of analytes using electrochemical sensors

Abstract

To provide a sensor system that can be operated with drift-free or calibration-free measurement.SOLUTION: Typical electrochemical sensors measure target-induced changes in current output. Such measures of target binding are inconsistent across individual sensors, and furthermore, signal will drift over time when the sensor is deployed for long periods. These shortcomings can be avoided by a novel use of chronoamperometry to measure current decay kinetics as the indicator of target binding. Current decay lifetimes will vary in a concentration dependent manner, but remain stable across individual sensors and over time, allowing for calibration-free operation. By these methods, aptamer based electrochemical sensors and other sensor types may be deployed in vivo for extended periods of time and will provide accurate measurement of target binding without calibration.SELECTED DRAWING: Figure 4B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 578,665, entitled "Calibration Free In-Vivo Measurement of Analytes Using Electrochemical Sensors," filed October 30, 2017, the contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. W911NF-09-0001 awarded by the US Army Research Office. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Various types of electrochemical sensors for detecting target molecules are known. Many such sensors produce a current output whose magnitude changes in response to the binding of a target molecule. Electrochemical aptamer-based (EA-B) sensors use an aptamer modified with a redox reporter and bound to an electrode, where binding-induced conformational changes in the aptamer result in a measurable change in electron flow between the redox reporter and the electrode. EA-B sensors have provided the art with a versatile platform for measuring target analytes in complex samples. EA-B sensors have also been demonstrated to be useful in vivo for real-time detection of drugs and other target species in flowing whole blood. However, despite the great potential of this platform, EA-B sensors suffer from certain drawbacks that limit their clinical deployment.

[0004] Specifically, like all complex devices, EA-B sensors suffer from fabrication inconsistencies, with different numbers of recognition elements present on individual sensors, even when manufactured in the same batch. This physical variation between sensors means that the output obtained from different sensors of the same design can vary significantly. Furthermore, even the best E-ABs are prone to drift when placed in complex sample environments such as whole blood, where nonspecific interactions between aptamers and the complex mixture of molecules in the sample result in readings that fluctuate over time. These factors necessitate calibration steps or signal correction procedures in order to interpret the sensor output. For sensors implanted in vivo, where calibration is impractical or often impossible, the above sources of error present significant obstacles to clinical implementation.

[0005] Thus, there remains a need in the art for new electrochemical sensor systems and methods for operating sensors that enable calibration-free measurements. Additionally, there remains a need in the art for means to accurately measure analytes in vivo without being confounded by signal drift. There also remains a need in the art to improve the performance and efficiency of existing sensing platforms. Summary of the Invention

[0006] Prior art electrochemical sensing methods rely on measurements of current output, such as SWV peaks (i.e., absolute current values), to determine target concentrations, and such measurements are strongly affected by sensor-to-sensor variations and sensor drift. The inventors of the present disclosure have advantageously discovered that certain aspects of the signal output kinetics of electrochemical sensors, specifically current decay kinetics, are less susceptible to sensor-to-sensor variations and sensor drift. Similar to absolute currents, these current decay kinetic parameters respond to target binding in a concentration-dependent manner, but unlike absolute currents, they are stable across a given class of sensors and over time, providing a means to avoid the variations observed in absolute currents.

[0007] The inventors of the present disclosure have advantageously developed a novel method for operating electrochemical sensors and interpreting their output, such that signals produced by different sensors, or at different times, can be accurately correlated to target concentrations in a sample. The method of the present invention allows a class of sensors (e.g., using the same recognition element) to be effectively calibrated a priori and deployed for long-term, calibration-free measurements of target species in complex environments such as in vivo whole blood.

[0008] In a first aspect, the scope of the present invention includes methods for acquiring and interpreting sensor output to provide an accurate measurement of target analyte concentration. In another aspect, the scope of the present invention includes sensor systems that can be operated with drift-free or calibration-free measurements. In another aspect, the scope of the present invention includes a class of electrochemical sensors, where the sensor output is correlated to target concentration by a relationship that is stable across all sensors in the class. In another aspect, the scope of the present invention includes computer programs, software, and operations that allow target concentration to be assessed by acquiring and interpreting the output of an electrochemical sensor. [The present invention 1001] 1. A method for measuring the concentration of a target species in a sample by use of an electrochemical sensor, the method comprising the steps of: positioning an electrochemical sensor exposed to a sample, the output of the electrochemical sensor being a faradaic current that varies in a concentration-dependent manner with the concentration of a target species in the sample; applying one or more excitation pulses to the electrochemical sensor, wherein a faradaic current output is generated by each pulse; acquiring time-resolved faradaic current data following each of the one or more excitation pulses; calculating a value of a selected measure of current decay from the acquired time-resolved faradaic current data; Calculating the concentration of the target species by application of a mathematical relationship between the selected measure of current decay and the concentration of the target species in the sample using the calculated value of the measure of current decay. [The present invention 1002] 1001. The method of claim 1001, wherein the electrochemical sensor comprises an electrode functionalized with a plurality of recognition elements that undergo a conformational change upon target binding, each recognition element being functionalized with one or more redox reporters. [The present invention 1003] The method of claim 1002, wherein the recognition element comprises an aptamer. [The present invention 1004] The method of claim 1001, wherein the sample is selected from the group consisting of whole blood, serum, saliva, urine, sweat, interstitial fluid, spinal fluid, cerebrospinal fluid, tissue exudate, macerated tissue sample, cell solution, intracellular compartment, water, wash water, wastewater, groundwater, food, and beverage. [The present invention 1005] 1001. The method of claim 1001, wherein the sample comprises flowing whole blood. [The present invention 1006] The method of the present invention 1004, wherein the sample is not treated prior to measurement. [The present invention 1007] The method of the present invention 1004, wherein the sample is not diluted. [The present invention 1008] The method of claim 1001, wherein the target species is selected from the group consisting of small molecule drugs, metabolites, hormones, peptides, proteins, carbohydrates, nucleic acids, lipids, hormones, metabolites, growth factors, neurotransmitters, nutrients, and pollutants, pathogen-induced or pathogen-derived factors, pathogens, or cells. [The present invention 1009] 1001. The method of claim 1001, wherein the selected measure of target decay is selected from a decay constant, a mean lifetime, a half-life, and a relative amplitude. [The present invention 1010] 1001. The method of claim 1001, wherein the selected measure of current decay is obtained from an exponential fit of the time-resolved current data. [The present invention 1011] The method of the present invention 1010, wherein the selected measure of current decay is obtained from a single exponential fit of the time-resolved current data. [The present invention 1012] 1001. The method of claim 1001, wherein the selected measure of current decay is obtained from a bi-exponential fit of the time-resolved current data. [The present invention 1013] The method of the present invention 1001, wherein a mathematical relationship between a selected measure of current decay and a target concentration has been obtained for a sensor of the same class as the deployed electrochemical sensor. [The present invention 1014] The method of the present invention 1001, wherein no calibration step is performed before or after the measurement. [The present invention 1015] The method of this invention 1001, wherein repeated measurements are achieved over an extended period of time. [The present invention 1016] The method of claim 1014, wherein the electrochemical sensor is deployed in vivo. [The present invention 1017] The method of claim 1015, wherein the electrochemical sensor is placed in a human subject. [The present invention 1018] The method of claim 1015, wherein the electrochemical sensor is placed in a non-human animal. [The present invention 1019] The method of claim 1001, wherein the electrochemical sensor is deployed in a point-of-care system. [The present invention 1020] the electrochemical sensor is configured such that when the electrochemical sensor is placed in a sample, the output of the sensor is a faradaic current that varies in a concentration-dependent manner depending on the concentration of the target species in the sample; and a stable mathematical relationship relating a measure of faradaic current decay to target concentration is known for the class of sensors to which the electrochemical sensor belongs; Electrochemical sensors. [The present invention 1021] 1020 An electrochemical sensor according to the present invention, wherein the electrochemical sensor comprises an electrode functionalized with a plurality of recognition elements that undergo a conformational change upon target binding, each recognition element being functionalized with one or more redox reporters. [The present invention 1022] 1021. An electrochemical sensor of the present invention, wherein said class of sensors includes sensors having the same recognition element type, the same redox reporter type, and the same attachment properties for binding to an electrode. [The present invention 1023] 1021. The electrochemical sensor of claim 1021, wherein the recognition element comprises an aptamer. [The present invention 1024] An electrochemical sensing system, including: the electrochemical sensor, wherein the electrochemical system is configured such that when the electrochemical sensor is placed in a sample, the output of the sensor is a faradaic current that varies in a concentration-dependent manner depending on the concentration of a target species in the sample; hardware components, including apparatus for application of excitation pulses to the electrochemical sensor and for acquisition of time-resolved faradaic current decays from the electrochemical sensor following the application of each pulse; and A non-transitory computer-readable medium storing data and computer programs that enable the electrochemical sensor and hardware components to implement any of the methods of the present invention 1001 to 1019. [The present invention 1025] 1024. An electrochemical sensing system according to claim 1024, wherein the electrochemical sensor comprises an electrode functionalized with a plurality of recognition elements that undergo a conformational change upon target binding, each recognition element being functionalized with one or more redox reporters. [The present invention 1026] 1024. The electrochemical sensing system of claim 1024, wherein the recognition element comprises an aptamer. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1A shows the basic operation of an E-AB sensing platform in which an aptamer (102) bound to an electrode substrate (101) is modified with a methylene blue redox reporter (103). In the absence of target, electron flow (104) between methylene blue and the substrate is slow. Upon target (105) binding to the aptamer, the resulting conformational change alters the proximity of the reporter (103) to the substrate (101), increasing the rate of electron transfer (104). Figure 1B shows a representative square-wave voltammetry trace for this type of E-AB sensor, showing a higher peak current in the presence of target than in its absence. [Figure 2] Figure 2A shows SWV titration curves for a set of six aminoglycoside-detecting sensors using tobramycin as the target. The absolute peak currents generated using SWV depend not only on the concentration of the target molecule but also on the number of redox reporter-modified probes on the sensor surface. Due to differences in the active area of ​​the working electrode and the density of aptamer probes loaded thereon, these absolute peak currents can vary significantly from sensor to sensor, potentially leading to large differences in the raw (uncalibrated) sensor output. Figure 2B shows the output of the sensors in Figure 2A when normalized by performing a calibration step. [Figure 3]Figure 1 shows a log-log plot of the mean current versus time transient measured by chronoamperometry of an aminoglycoside-conjugated E-AB sensor in flowing whole blood. The current decay is appropriately described as the sum of two logarithmic phases. From left to right, the first phase, to the left of the dotted vertical line, is the decay of the double layer charging current, where the sensor is unresponsive to the target. Toward the right of the vertical line, the transient current is highly target-dependent. In transients obtained in the absence of target, a slower exponential decay of the faradaic current is observed. In the presence of target (here, tobramycin), the mean current lifetime is substantially reduced. The illustrated transient was recorded by stepping the potential from -0.1 V to -0.3 V (full potential reported vs. Ag / AgCl) and sampling the resulting current every 10 μs. The solid line is a multiexponential fit of the experimental data. [Figure 4A]Figure 4A shows the average transient current measured by chronoamperometry for an aminoglycoside-binding E-AB sensor (using tobramycin as the target), with decaying transient currents that decrease with increasing target concentration. The solid line represents a monoexponential fit of the two transient currents. Figure 4B shows the relationship between current lifetime and target concentration in buffer and whole blood established for a set of aminoglycoside-binding E-AB sensors. The difference in lifetime between the two sample types is likely due to changes in electrolyte composition and viscosity, which affect electron transfer from methylene blue. Figure 4C shows the tobramycin concentration measured by five sensors of the same class as used to generate the standard curve in Figure 4B, but not used to generate the standard curve. Using these independent sensors to estimate the concentration of tobramycin in flowing whole blood using the curve generated for the sensor class provided accurate and precise measurements of tobramycin concentration over a wide range of concentrations without the need to calibrate each individual sensor. When tested in undiluted whole blood, the measured concentrations are within 10% of the actual (spiked) concentrations of tobramycin over the range of 1 μM to 1 mM. In Figures 4B and 4C, the error bars (which are so small in Figure 4B that they are difficult to see) represent the standard deviation. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5] Figure 5A shows the output of an E-AB sensor in flowing whole blood (no target here), where the average peak current recorded from SWV drifts significantly over the course of several hours (uncorrected signal). A dynamic differential measurement correction technique applied to the measurements provides a corrected signal. Figure 4B shows the average current amplitude and average current lifetime for the same sensor placed in whole blood (no target) for an extended period. The amplitude of the chronoamperometric current decay drifts very significantly over the test period. In contrast, however, the current lifetime is stable over time and does not drift over the test period. [Figure 6A] Sensor placement in the jugular vein of a live, anesthetized rat is shown. The sensor was placed within a 22-gauge catheter for structural support and placed within the external jugular vein at a depth of 2 cm. An infusion line was implanted on the contralateral side to allow for drug infusion. [Figure 6B] Figure 1 shows real-time nonlinear regression analysis of current transients generated by chronoamperometry to extract current lifetimes and convert them to target concentrations in real time. Traces are 20-point running averages. The dotted rectangles indicate the time frames during which tobramycin was injected. [Figure 6C] Lifetime versus time traces (20-point running average) at 300 ms per time point are shown, where the temporal resolution of the technique is sufficient to monitor not only the drug infusion but also the subsequent tens of seconds of "mixing" phase associated with drug homogenization within the circulatory system. This panel corresponds to the zoomed region marked with a dash from Figure 6B. [Figure 6D] Mean concentration versus time is shown, where unprecedented time resolution allows minute-scale distribution phase measurements of the drug with over 1000 experimental points, resulting in ultra-high precision estimates of relevant pharmacokinetic parameters. DETAILED DESCRIPTION OF THE INVENTION

[0010] Detailed Description of the Invention Operating principle The invention disclosed herein was made possible by extensive study of the operation of electrochemical sensors and the discovery that certain outputs are less susceptible to sensor fabrication variations and signal drift. In standard electrochemical sensor implementations, current flow is altered by target binding and assessed by voltammetric methods, including cyclic voltammetry, alternating current voltammetry, and square-wave voltammetry. These methods measure the peak current, which is related to the proportion of bound recognition elements modified with redox reporters and is also highly dependent on the total number of active recognition elements (e.g., aptamers) present on the sensor.

[0011] The present inventors have developed an alternative approach for monitoring changes in electron transfer induced by target binding. Rather than measuring voltammetric peak currents, which are indirectly related to binding-dependent electron transfer kinetics, electron transfer kinetics can be measured directly, and advantageously, the present inventors have discovered that these values ​​are less susceptible to sources of error that affect peak current measurements. The present inventors have confirmed that electron transfer kinetics, measured by chronoamperometric current decay, is primarily determined by target binding and, unlike peak currents, is independent of the number of active recognition units on each sensor. Thus, a relationship between target concentration and electron transfer kinetics can be determined for a selected sensor configuration, and this relationship is stable and applicable to all similar sensors operating under similar conditions. Furthermore, this predictive relationship is stable for individual sensors operating over long periods of time.

[0012] This novel operating principle of the present invention eliminates the need to calibrate individual sensors and provides a means for drift-free operation in challenging environments such as in vivo. Measurements can also be obtained on extremely fine time scales, allowing for millisecond time scale resolution of biological processes with electrochemical sensors.

[0013] The various elements of the present invention are now described.

[0014] Electrochemical Sensors Various aspects of the present invention are directed to methods of using electrochemical sensors. As used herein, an electrochemical sensor is any sensor capable of measuring the concentration of a target species in a sample, where binding of the target species to a recognition element of the sensor induces a measurable change in the current output by the sensing element, such that the output of the sensor can be used to infer the concentration of the target in the sample.

[0015] EAB sensor In a first implementation, the electrochemical sensor utilized in the method of the present invention comprises an E-AB sensor. Any E-AB sensor design or configuration known in the art can be used. In an E-AB sensor, the recognition element comprises an aptamer, as known in the art. The aptamer can include DNA aptamers, RNA aptamers, or aptamers containing non-natural nucleic acids, as well as hybrids of the foregoing. Variations of the E-AB concept in which the recognition element is other than nucleic acid, such as sensors using proteins, chemical species, and other molecules, are also within the scope of the present invention.

[0016] In the E-AB sensor, one or more selected portions of the working electrode are functionalized with aptamers. The aptamers can be bound or otherwise associated with the surface of the electrode by any suitable chemical property, such as covalent bonding, chemical adsorption, or adsorption. The aptamers can also be bound to the surface of the electrode using an alkanethiol monolayer, which is particularly suitable for the surface of a gold electrode.

[0017] Each aptamer is functionalized with one or more redox reporters. Binding of the target species causes the aptamer to change its conformation, such that the location (or electrode accessibility) of the one or more redox reporters is detectably altered. Redox species include any composition of matter that interacts with an electrode such that a change in electrode accessibility or proximity to the electrode causes a change in electron transfer kinetics. Typical redox species include methylene blue, ferrocene, viologen, anthraquinone or any other quinone, ethidium bromide, daunomycin, organometallic redox labels such as porphyrin complexes or crown ether rings or linear ethers, ruthenium, bispyridine, trispyridine, bisimidazole, ethylenetetraacetic acid-metal complexes, cytochrome C, plastocyanin, and cytochrome C'.

[0018] In some implementations, the E-AB sensor is a signal-on type sensor, where target binding enhances the signal, while in other implementations, the E-AB sensor may include a signal-off configuration. In one embodiment, the E-AB sensor is a double-stranded sensor, where the redox species is present on a separate strand, a portion of which is complementary to or can otherwise reversibly bind to a portion of the aptamer. In the presence of the target species, the strand of the redox species is released from the aptamer, allowing the target species to bind to the aptamer and the redox species to contact or approach the electrode.

[0019] Sensor Components Electrochemical sensors have multiple recognition elements (e.g., 0.1 × 10 11 ~1×10 13 molecule / cm 2 The working electrode may comprise any electrode material suitable for electrochemical sensing, including, for example, any metal surface that forms bonds with thiols or amines; gold; any gold-coated metal (e.g., titanium, tungsten, platinum, carbon, aluminum, copper, etc.), bare palladium electrodes, carbon electrodes, etc.

[0020] The working electrode can be configured in any desired shape or size. For example, paddle-shaped electrodes, rectangular electrodes, wire electrodes, electrode arrays, screen-printed electrodes, and other configurations can be used. For in vivo measurements, thin wire configurations are advantageous because they can be inserted into veins, arteries, tissues, or organs without obstructing blood flow within the vessels or causing substantial tissue damage. For example, wires having diameters of 1 to 500 μm, e.g., 100 μm, can be used.

[0021] The electrochemical sensing system of the present invention further includes an auxiliary or counter electrode, such as a platinum auxiliary electrode. The electrochemical sensing element may be used in conjunction with a reference electrode, such as an Ag / AgCl electrode, or other reference electrodes known in the art. The electrochemical sensor of the present invention may be configured in a two-electrode or three-electrode system appropriately configured for performing chronoamperometric measurements. The cell system containing the electrode may also include a mixing chamber or other container in which the electrode resides and contacts the sample.

[0022] The sensor and electrode system may include an assembly for obtaining a measurement of faradaic current when placed in or exposed to a sample. The assembly may include a housing. For example, for placement within a living body, the housing may include a needle, catheter, or other implantable structure. For ex vivo applications, the housing may include a well, microfluidic tube, or other structure such as found in lab-on-a-chip devices.

[0023] The electrochemical sensors of the present invention are operatively connected to appropriate components for performing chronoamperometric measurements. The chronoamperometric components may comprise two or more devices, or may comprise a single integrated device, electrically connected and / or networked to one another.

[0024] The first component for performing chronoamperometric measurements includes a device or combination of devices capable of delivering excitation voltage pulses of desired magnitude, frequency, and waveform to the sensing element. The chronoamperometric components may include a potentiostat or other voltage source and a voltage regulator for applying voltage steps to the working electrode.

[0025] The second component for performing chronoamperometric measurements involves a device or combination of devices capable of acquiring the time-resolved Faradaic current output from the sensing element. These components include circuitry for reading the sensor output and storing such output or sending the output to other devices, including analog-to-digital converters, amplifiers, and storage media. Most sensor systems require resolution on very fine time scales, e.g., microsecond to millisecond time scales, to measure current decay kinetics.

[0026] Other Sensor Types The scope of the present invention is not limited to E-AB sensors. The scope of the present invention also encompasses any electrochemical sensor in which target binding to a recognition element results in a measurable change in the electron transfer rate, which can be measured by the sensing element. In one aspect, sensors using non-mediated electrochemical sensing, including the use of direct electron transfer and redox equilibrium as a means of generating a signal, can be used. These biochemical sensors include, for example, sensors that detect the consumption or production of species belonging to a redox couple. Other sensors may use mediated electrochemical analysis, i.e., the use of a redox species mediator for electron transfer and the establishment of redox equilibrium. Examples of mediated and non-mediated sensors can be found in Sander et al. 2015, A Review of Nonmediated and Mediated Approaches. Environ. Sci. Technol. 49:5862-5878.

[0027] Additional sensor types include chemically modified electrodes, immunosensors, oligopeptide-based sensors, and sensors that utilize organelles (e.g., chloroplasts, mitochondria), animal and plant tissues, microorganisms, enzymes, tissue sections, peptides, and antibodies.

[0028] Additional sensor types include sensors that measure changes in electron transfer from solution-phase redox reporters, e.g., sensors that measure the redox of ferrocyanate / ferricyanate conjugated to a 17-mer peptide that specifically recognizes cyclic AMP, as described in Katayama et al. 2000, The Design of Cyclic AMP-Recognizing Oligopeptides and Evaluation of Its Capability for Cyclic AMP Recognition Using an Electrochemical System. Anal Chem. 2000;72(19):4671-4.

[0029] Another sensor structure that can be used is a polymeric sensor, such as a sensor that uses a cationic polythiophene having ferrocene substituents as a mediator in an aptamer system, as described in Le Floch, 2006, Label-Free Electrochemical Detection of Protein Based on a Ferrocene-Bearing Cationic Polythiophene and Aptamer. Anal Chem. 2006;78(13):4727-31. doi: 10.1021 / ac0521955.

[0030] Another sensor type that can be used is one based on changes in electron transfer due to ligand displacement induced by target binding, such as hexamethylphosphoramide with samarium(II) iodide, as described by Prasad, 2004, The Role of Ligand Displacement in Sm(II)-HMPA-Based Reductions. J Am Chem Soc. 2004;126(22):6891-4.

[0031] Another typical sensor type is based on a change in the reorganization energy of a redox reporter, e.g., a ferrocenoyl-peptide, as described in Plumb, 2003, Interaction of a Ferrocenoyl-Modified Peptide with Papain: Toward Protein-Sensitive Electrochemical Probes. Bioconj Chem. 2003;14(3):601-6. doi: 10.1021 / bc0256446; or a trinuclear ruthenium cluster, as described in Feld, 2012, Trinuclear Ruthenium Clusters as Bivalent Electrochemical Probes for Ligand-Receptor Binding Interactions. Langmuir. 2012;28(1):939-49. doi: 10.1021 / la202882k.

[0032] Another type of sensor that can be used is one based on sterically induced changes in the efficiency with which a scaffold-attached redox reporter approaches the underlying electrode surface, e.g., duplex DNA, quadruplex DNA, and DNA nanoswitches, as described in Ge 2010, “A Robust Electronic Switch Made of Immobilized Duplex / Quadruplex DNA.” Angew Chem Int Ed. 2010;49(51):9965-7. doi: 10.1002 / anie.201004946, or DNA containing small molecule recognition elements, as described in Cash 2009, “An Electrochemical Sensor for the Detection of Protein-Small Molecule Interactions Directly in Serum and Other Complex Matrices.” J Am Chem Soc. 2009;131(20):6955-7. doi: 10.1021 / ja9011595.

[0033] Sensor Class The various methods of the present invention are based on the discovery that the output, i.e., current decay kinetics, of a sensor is stable among sensors of a given type. As used herein, a "class" refers to multiple sensors that share one or more common characteristics. Sensor characteristics can include a variety of factors, including electrode configuration and materials, the type of sample being analyzed, the recognition element type, the redox reporter type and its placement, the packing density of the recognition element on the working electrode, the electrode functionalization chemistry, and other sensor parameters that affect the sensor output. Another class parameter can be the sensor production lot, in which sensors of a certain class are fabricated with the same allocation.

[0034] In one embodiment, a class of sensors includes multiple sensors having substantially identical sensor architecture, identical recognition elements and redox reporter elements, identical chemistries for attachment of the recognition elements to the working electrode, identical manufacturing methods, and similar packing densities of the recognition elements (e.g., packing densities in terms of moles of probe per square centimeter that vary 1-20% between sensors within a batch).

[0035] target species The sensors used in the methods of the present invention are directed to the detection of target species. Target species can include any inorganic or organic molecule, such as a small molecule drug, metabolite, hormone, peptide, protein, carbohydrate, nucleic acid, lipid, hormone, metabolite, growth factor, neurotransmitter, or nutrient. Targets can include pollutants or contaminants. Targets can include toxins. Targets can include pathogen-induced or pathogen-derived factors, or viruses or cells. In some embodiments, target species include drugs with significant side effects, such as chemotherapeutic drugs, or drugs with a narrow therapeutic index, where accurate measurement of blood concentrations is essential to ensure safe dosing or minimal side effects.

[0036] Operating conditions The sensor is utilized under selected operating conditions that encompass various aspects of the detection process. The operating conditions may encompass any combination of factors that affect the operation and output of the sensor.

[0037] In a first aspect, the operating conditions include the sample type to be analyzed. The target species of the present invention are evaluated in the sample. The sample includes a liquid. The sample may include whole blood, serum, saliva, urine, sweat, interstitial fluid, spinal fluid, cerebrospinal fluid, tissue exudate, macerated tissue sample, cell solution, intracellular compartment, water, lavage water, wastewater, groundwater, food, beverage, or other biological or environmental sample. In some embodiments, the sample is obtained from a subject, e.g., a human patient or a non-human animal such as a veterinary subject or test animal. In one embodiment, the sample includes flowing whole blood, i.e., blood sampled by a sensing system including a sensor implanted in vivo (e.g., in the circulatory system) of the subject. In another embodiment, the method is used to measure the target species in a gas equilibrated with the liquid sample.

[0038] In one embodiment, the sample is processed before measurement. Examples of processing include filtration, dilution, buffering, centrifugation, and applying other materials or processes to the sample before analysis. In some embodiments, the sample is not processed before measurement, for example, the sample is not diluted, filtered, or concentrated.

[0039] In a second aspect, the operating conditions include assay conditions. General assay conditions refer to the reaction conditions for an assay, such as sample volume, temperature, pH, etc.

[0040] In a third aspect, the operating conditions may be defined by the operating parameters used to obtain the sensor measurements, for example, the shape and frequency of the applied voltage waveform, the voltage step value, and the sampling interval are some of the variables that make up the operating parameters.

[0041] Measurement of target concentration by current decay In a first aspect, the present invention includes a general method for measuring the concentration of a target species in a sample by use of an electrochemical sensor. The method includes the following general steps: (A) Develop a mathematical relationship between a selected measure of current decay and target concentration for a selected class of electrochemical sensors operating under a selected set of operating conditions; (B) placing a selected class of electrochemical sensor in a sample of unknown target concentration under selected operating conditions and obtaining values ​​for the selected measure of current decay; and (C) Applying the current decay vs. concentration relationship established in step (A), the current decay values ​​observed in step (B) are used to determine the concentration of the target species in the sample.

[0042] For example, in one embodiment, the present invention encompasses a method for determining the concentration of a target species in a sample by use of an electrochemical sensor, the method comprising the steps of: positioning an electrochemical sensor exposed to a sample, the output of the electrochemical sensor being a faradaic current that varies in a concentration-dependent manner with the concentration of a target species in the sample; applying one or more excitation pulses to the electrochemical sensor, each pulse producing a time-dependent faradaic current output; acquiring time-resolved faradaic current data following each of the one or more excitation pulses; calculating a value of a selected measure of current decay from the acquired time-resolved faradaic current data; Calculating the concentration of the target species by application of a mathematical relationship between the selected measure of current decay and the concentration of the target species in the sample using the calculated value of the measure of current decay.

[0043] Measure of current decay The inventors of the present disclosure have advantageously demonstrated that the decay rate of a transient current generated in response to an excitation stimulus is related to the concentration of the target species and is stable across sensors of the same class. Thus, the methods of the present invention rely on measurements of current decay, the relationship between current decay rate and target concentration, and the remarkable stability of this relationship over time across similar sensors. The chronoamperometric decay lifetime depends only on the relative total number of bound and unbound recognition elements (e.g., aptamers), not the absolute number of bound and unbound recognition elements, thus providing a means of measuring target concentration that is independent of factors that can vary significantly between individual sensors of the same class.

[0044] For example, in an electrochemical sensor containing an electrode substrate functionalized with multiple recognition elements, each recognition element functionalized with one or more redox reporters, electrical excitation of the redox reporters induces a transient flow of current between the redox reporters and the electrode substrate (or between the electrode substrate and the redox reporters, depending on the system configuration). Stepping the voltage of a working electrode, such as the substrate of an E-AB sensor, makes the working electrode either a stronger reducing agent (if stepped to a more negative potential) or a stronger oxidizing agent (if stepped to a more positive potential). Within the appropriate range (near or above the redox potential of the redox reporter), this voltage step induces a faradaic current flow between the redox reporter of the sensor's recognition element and the electrode substrate. As current flows, the pool of electrons mobilized by excitation is depleted, causing the current to decay exponentially (or multiexponentially) at a rate and amplitude that depends on the binding state of the sensor's recognition elements, where target binding induces faster or slower current movement by, for example, varying the proximity of the redox reporter to the working electrode. Thus, the ratio of target-bound to target-unbound recognition elements, which is proportional to the concentration of target species in the sample, determines the rate and amplitude of the current decay observed for the sensor as a whole.

[0045] As used herein, "current" refers to the flow of electrons measured by a sensor placed in a sample. For example, the current may include the flow of electrons from a redox reporter to an electrode, or may include the flow of electrons from an electrode to a redox reporter. As used herein, "current decay" refers to the behavior of the transient current measured by a sensor over time in response to the application of an excitation stimulus to the sensor or sample.

[0046] Measurement of current decay can be achieved using chronoamperometry, as known in the art. Decay parameters can be measured by applying an excitation to the sensor and / or sample and measuring the current response over a period of time following the excitation. Therefore, measuring the decay parameter requires a sensor or sample environment associated with a component to (1) deliver an excitation pulse of a desired voltage, frequency, and waveform, and (2) measure the current response over a time scale corresponding to the duration of the transient current.

[0047] The excitation pulse can be any type of stimulus that induces a transient current, such as stepping the potential of the sensor electrode to a value at which the redox reporter is substantially (e.g., fully) oxidized or reduced. Appropriate excitation waveforms can be selected as known in the art. For example, voltage steps in the range of ±0.1 V to 0.5 V can be utilized at repetition rates of 1 to 10,000 Hz, e.g., 5 Hz, 10 Hz, 20 Hz, 50 Hz, 100 Hz, and intermediate repetition rates between 1 Hz and 10,000 Hz.

[0048] Obtaining time-resolved current measurements on microsecond to millisecond time scales is commonly required. Typical current transients have durations in the 10-100 millisecond range and can be resolved by sampling at shorter time intervals, e.g., every 1 μs, 2 μs, 3 μs, 5 μs, or 10 μs.

[0049] Once time-resolved current data have been acquired, these data can then be analyzed to obtain any number of mathematical parameters that describe the kinetics of current decay. The selected measure of decay can be any parameter of the transient current that varies in a concentration-dependent manner depending on the concentration of the target species in the sample. The selected measure of current decay can be evaluated as any measure of decay kinetics, such as the rate constant, lifetime, half-life, or any other quantification of the current decay. For example, as described below, in one embodiment, the measure of decay is obtained by fitting the entire decay curve to a function and utilizing the lifetime (inverse rate constant) or half-life for that decay curve. As described below, in another embodiment, decay is obtained by fitting the time-resolved data to a function that derives the sum of two or more exponential functions and utilizing the relative amplitudes of the exponential components, or a similar measure.

[0050] A selected mathematical analysis is applied to the acquired time-resolved current data to obtain a selected measure of decay. Any regression analysis may be applied to obtain a selected measure of current decay to account for the simultaneous electron transfer of bound and unbound recognition elements. In one embodiment, a measure of current decay is obtained using a mono-exponential fit to the sensor current trace. In one embodiment, a measure of current decay is obtained using a multi-exponential fit. In one embodiment, a measure of current decay is obtained using a bi-exponential fit. In one embodiment, a measure of current decay is obtained using a tri-exponential fit. Current decay values ​​may be evaluated from a single data point or by averaging multiple data points. For example, sampled decay values ​​may include average values ​​observed over a selected number of excitation-decay cycles ranging from 1 to 100 cycles, e.g., 5, 10, 20, 50, 75, or 100 cycles, or any intermediate value.

[0051] In some sensor systems, the kinetics of interconversion between the bound and unbound states of the recognition element are more rapid than the electron transfer events measured by the sensor. Therefore, the observed current transient reflects a population-weighted average of the bound and unbound states. This can be analyzed by estimating the current decay lifetime using a single-exponential fit, as shown in Figure 4A, for example. The lifetime of this "best fit" single-exponential function is monotonically related to the target concentration. That is, a current decay trace best described as the sum of two exponential processes (unbound decay and bound decay) can be fitted to a single-exponential curve, whose parameters (e.g., decay constant, half-life, etc.) provide a means of measuring the target concentration.

[0052] In one embodiment, the selected measure of decay can be obtained by fitting time-resolved current data to a function that leads to the sum of two exponents, and using the relative amplitude of exponential components as a measure of target binding.For example, because the observed current decay is a combination of binding and non-binding decay, a bi-exponential fit can be used to draw a decay curve, where the faster phase represents target binding decay, and the slower phase represents non-binding decay.The relative amplitude of either decay, i.e., its amplitude relative to the total amplitude of the two phases combined, or the ratio of the amplitudes of the two phases, can be used as a measure of the proportion of target-bound recognition element.In another implementation, decay can be described by three or more exponential functions, such as a tri-exponential fit.

[0053] For example, a typical method utilizing an exponential fit is described in Kamman et al., C., "Multi-exponential relaxation analysis with MR imaging and NMR spectroscopy using fat-water systems," in Magnetic Resonance Imaging, Volume 5, Issue 5, 1987, Pages 381-392.

[0054] For example, in one implementation, the current decay from a chronoamperometric sensor is displayed on a log i (current) vs. log t (time) plot and a nonlinear regression analysis is performed using the following equation: For a single exponential system, according to Equation 1: TIFF0007744694000001.tif7128For the biexponential system, according to Equation 2: TIFF0007744694000002.tif7128For the tri-exponential system, according to Equation 3: TIFF0007744694000003.tif7128With reference to Equations 1-3, t is the time following excitation, τ is the time constant, i is the current, and C is the constant background current, if any.

[0055] The relative amplitude is calculated as the ratio of one selected amplitude to the sum of all amplitudes. For example, for a bi-exponential fit: TIFF0007744694000004.tif26128

[0056] Figure 3 provides an illustration of current decay kinetics for an electrochemical sensor. In this example, a pulsed voltage is applied to an E-AB sensor containing a methylene blue redox reporter, and the current is measured following the pulse. Two curves are presented: one representing the current kinetics for a sample containing no target, and the other representing the current decay for a sample containing a saturating concentration of target (i.e., the concentration at which all of the sensor's recognition elements, the aptamers, are bound to the target). Applying a voltage step at a sufficiently negative potential achieves complete reduction of all of the methylene blue reporter. These electron flows are measured over time following the end of the pulse. In the first phase, typically about 0.1 milliseconds after application of the pulse in aqueous solution, decay is rapid. This phase is due to charging of the electrical double layer (i.e., the movement of aqueous ions, which has a microsecond timescale) that forms on the electrode surface at this potential bias. The decay of the double layer charge is largely unaffected by the target concentration in the sample and is generally not useful for predicting target concentration. Following this phase, a second phase is observed, in which the decay kinetics are strongly related to target binding, corresponding to faradaic reduction of the reporter, e.g., reduction of methylene blue to leucomethylene blue. In Figure 2, for the recognition element saturated with target, a rapid exponential decay of current is observed, with a lifetime of 100 ± 30 microseconds. For the recognition element in the target-free sample, a slower decay is observed, with a lifetime of 6.5 ± 0.5 milliseconds. The approximately 5-fold decrease in lifetime (comparing the saturated sample to the sample lacking target) corresponds to a change in the proximity of the redox reporter to the electrode surface and reflects the total number of aptamers bound to the target, which transfer electrons faster than aptamers without target.

[0057] Class Calibration In one aspect, the scope of the present invention is directed to a method for calibrating a class of sensors using a representative subset of sensors from that class. The purpose of the calibration process is to generate a standard curve for interpreting sensor output for all sensors in that class. Accordingly, calibration of a given class of sensors is performed by assaying a representative sample using a representative sensor of the same design under operating conditions similar to and / or representative of the operating conditions under which the sensors will be placed. Once such calibration has been performed using the representative subset, other sensors from that class can be used to perform calibration-free measurements of target species under similar operating conditions.

[0058] Class calibration is achieved by establishing a "decay-concentration relationship" for a selected sensor type in a representative sample under selected operating conditions. The decay-concentration relationship is the predicted relationship between the target concentration in a sample and the observed current decay resulting from a voltage step at the working electrode. The decay-concentration relationship can be calculated using any suitable regression analysis. Because the signal output is the combined sum of bound and unbound decays, a model that estimates a monotonic function based on the sum of two exponential decay curves is well suited for the calculation process. For example, if current lifetime is the selected measure of current decay, the relationship between current lifetime and concentration is determined. In another embodiment, the calibration data is fitted to extract a multi-exponential process, so that a relationship between relative amplitude and concentration is established, and the relative amplitude calculated for each exponential phase is a measure of decay.

[0059] The calibration measurements used to establish the relationship in step (A) are performed using a set of standards of various known target concentrations. Measurements should be made within the dynamic range of the sensor, i.e., across a range of target concentrations from zero target to saturated target levels, e.g., by using spiked samples. Any number of data points may be generated, e.g., 2-1000 data points may be sampled to generate calibration data. For example, 25-100, e.g., 50-75 data points may be used in the calibration.

[0060] The class calibration process uses a representative set of sensors. The representative set includes a sufficient number of sensors to provide a calibration curve that accurately predicts other sensors in the class. The number of sensors in the representative set can be established by methods known in the art, for example, for determining a sample average based on sub-sample values. The representative set can include, for example, 1, 3, 5, 10, 20, 50, 100, or more sensors, and intermediate values ​​therebetween.

[0061] Figure 3B shows the calibration curves of the present invention. Current lifetimes were evaluated for five E-AB aminoglycoside detection sensors over a range of concentrations in both whole blood and buffer. A "best fit" single exponential function was monotonically related to target concentration by nonlinear regression of lifetime versus concentration to the Langmuir isotherm. These results demonstrate the effect of different operating conditions on sensor output, with different blood and buffer calibration curves.

[0062] Multiple sensors of a given class operated under similar operating conditions have sufficiently similar decay outputs to allow prediction of the target using a standard curve generated for all sensors of that class. Sensors of the same class, when exposed to similar samples containing a given target concentration, may have decay outputs with values ​​on a selected current decay scale that differ by less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%, depending on manufacturing rigor, the signal-to-noise inherent in the system, and other variables.

[0063] In one embodiment, the scope of the present invention encompasses class-calibrated sensors. Class-calibrated sensors refer to a selected class of sensors having, for example, selected recognition elements, selected redox reporters, selected electrode attachment properties, selected electrode configurations, and other parameters that define the performance of the sensor, where the decay-concentration relationship between a selected measure of current decay and the concentration of a selected target is known for that class of sensors. In one embodiment, the class-calibrated sensors are aptamer-based electrochemical sensors.

[0064] Calibration-free measurements In one aspect, the scope of the present invention includes a method for calibration-free measurement of the concentration of a target species. As used herein, calibration-free operation refers to the operation of a selected class of electrochemical sensor in which the sensor output (e.g., current decay lifetime or relative amplitude) is directly converted to a target concentration value by use of a calibration curve specific to the selected class. In calibration-free operation, the sensor is not necessarily calibrated before, during, or after measurement.

[0065] Drift-free operation In one aspect, the scope of the present invention encompasses drift-free operation of electrochemical sensors operated for extended periods of time, e.g., hours, days, months, or longer. As shown in Figure 5A, an E-AB sensor placed directly in flowing whole blood has an output that drifts significantly when the sensor output is measured as amplitude by square-wave voltammetry. This significant baseline drift is eliminated when measuring current lifetime instead, as in Figure 5B, where the absolute sensor output drifts significantly over the test period, while the current lifetime remains stable for 8 hours.

[0066] Subsecond time resolution The methods of the present invention enable direct, in vivo, calibration-free, subsecond-resolved measurement of molecules. As described in the Examples, the methods of the present invention enable real-time resolution of target concentrations in blood or other body compartments, e.g., on time scales of 100 to 500 milliseconds. The unprecedented temporal resolution of this approach allows for the measurement of rapidly fluctuating target kinetics during physiological events, such as drug uptake and distribution, hormone and neurotransmitter release, and other physiological events occurring over short time scales. The fine time scale resolution enabled by chronoamperometry makes the methods of the present invention particularly well suited for pharmacokinetic analysis. In one embodiment, the present invention encompasses real-time calculation of pharmacokinetic parameters from calibration-free measurements of drugs, metabolites, excretion products, or other species involved in drug metabolism.

[0067] Sensor placement and operation The inherent stability of electron transfer kinetics across a class of sensors operated under similar operating conditions enables the use of electrochemical sensors in a variety of settings. In various implementations, the methods of the present invention enable sensors to provide accurate measurements when deployed in settings where calibration is impossible, cumbersome, or expensive. The calibration-free, drift-free methods of the present invention are particularly suited for in vivo measurements. For example, the sensing element or housing of a sensing system may be inserted, implanted, or otherwise placed within a living organism. The sensor element of a sensing system may be implanted in the circulatory system, subcutaneously, intraperitoneally, within an organ, or other body compartment, where the sensing element is exposed to in vivo fluids, such as interstitial fluid, blood, e.g., flowing whole blood. Implanted sensing systems of the present invention may include an implanted sensing element connected (e.g., by leads, wires, or wireless communication means) to components outside the body, where the external components perform pulse generation, data acquisition, or processing. Alternatively, one or more auxiliary components of the sensing element, or even the entire sensing system of the present invention, may be implanted within the body along with a means of communication (e.g., by leads, wires, or wireless communication device) with an external device for data collection.

[0068] In one embodiment, the method of the present invention is implemented in a feedback-controlled drug administration system, as known in the art, in which a drug is administered to maintain blood concentrations within a therapeutic or safety index. For example, in such methods and systems, the method of the present invention is applied by using an electrochemical sensing element implanted in a subject to measure the concentration of a target species, where the target species is a drug, a drug metabolite, or a biomarker that indicates that a drug should be administered. If the detected level of the target species indicates that the subject requires administration of a drug or other agent, an implanted pump or other drug delivery device administers a metered dose of the drug or agent to maintain the drug or agent concentration within the desired range.

[0069] In other situations, the sensors of the present invention are utilized for long-term and / or continuous monitoring of environmental or industrial sites, such as rivers, oceans, water treatment plants, industrial facilities, food processing facilities, and the like.

[0070] The sensor of the present invention can also be used in ex vivo diagnostic applications. In one embodiment, the method of the present invention includes removing a sample from a living body and measuring the concentration of a target species in the sample by calibration-free measurement. In one embodiment, the sensor of the present invention is used in a point-of-care testing system. For example, in one embodiment, the sample is a blood sample, such as a self-drawn, pinprick, or finger-prick blood sample, or a urine, sweat, or saliva sample. In such an embodiment, the electrochemical sensor may be disposed in a housing such as a well, a slide, a lab-on-a-chip, a microfluidic chamber, or other device.

[0071] Chronoamperometry with calibration The scope of the present invention is not limited to calibration-free chronoamperometry. In one implementation, the accuracy of concentration measurements achieved by electrochemical sensors performing chronoamperometry is enhanced by performing one or more calibration steps, i.e., steps in which the deployed sensor is exposed to one or more samples of known target concentrations to examine the deviation between the observed and expected concentrations and to correct any such deviation. This embodiment further involves the improved time resolution that chronoamperometry offers for interrogating this class of sensors compared to established electrochemical methods.

[0072] In one embodiment, the attenuation-concentration relationship of a deployed sensor is established by calibration: the deployed sensor may be exposed to one or more samples of known target concentrations to establish the attenuation-concentration relationship for the sensor under current operating conditions.

[0073] Computer processes and programs The scope of the present invention encompasses various embodiments, including software, computer programs, and program devices. In one embodiment, the scope of the present invention encompasses a non-transitory computer-readable recording medium storing data and / or a coded program that causes a computer to perform a series of operations. A computer may include any general-purpose computer, processor, embedded processor, mobile device, or other computing device. A computer may also include hardware elements, including input devices such as a keyboard, mouse, touchscreen, and other input means. A computer may also include hardware elements for data output, storage, or display, including a graphical user interface, display, and storage device.

[0074] In one embodiment, the invention includes a non-transitory computer-readable recording medium storing data and / or a coded program that causes a computer to perform a series of operations, where the data and / or the series of operations cause operation of an electrochemical sensing system to carry out a method of the invention. In various embodiments, the non-transitory computer-readable recording medium can carry out operations such as: controlling a potentiostat or equivalent device to deliver a stepped series of voltage pulses to a deployed electrochemical sensor; controlling a data acquisition device to record the sensor current output following delivery of the voltage pulses; controlling a processor to perform calculations to obtain one or more selected measures of current decay from the sensor output data; storing a calibration curve relating the selected measures of current decay to target concentrations; controlling a processor to calculate the target concentration based on the sensor output and the stored calibration curve; storing instructions for carrying out the method of the invention; and other operations of the invention.

[0075] In one embodiment, the scope of the present invention includes devices programmed to perform the operations of the present invention, such as devices that include or are in connection (e.g., networked) with the non-transitory computer-readable medium described above.

[0076] Kits and Systems The scope of the present invention extends to assemblies of components configured for carrying out the analyte concentration measurements of the present invention. In various embodiments, the scope of the present invention encompasses assemblies comprising two or more members for carrying out the methods of the present invention, the two or more components being selected from the group consisting of: an electrochemical sensor of a class in which the relationship between a measure of decay obtained by the sensor under selected operating conditions and a target concentration is known; a sensor housing for placing the sensor in a selected sample type; a potentiostat and its controller capable of delivering an electrical signal to generate a transient current; data acquisition and computer-readable storage medium, and / or processing means for calculating a selected measure of current decay from the sensor current data; a computer-readable storage medium, and / or processing means for calculating a target concentration from the current decay data; and instructions for carrying out the methods of the present invention.

[0077] Typical aspects In one embodiment, the present invention is a method for determining the concentration of a target species in a sample by use of an electrochemical sensor, the method comprising the steps of: positioning an electrochemical sensor exposed to a sample, the output of the electrochemical sensor being a faradaic current that varies in a concentration-dependent manner with the concentration of a target species in the sample; applying one or more excitation pulses to the electrochemical sensor, wherein a faradaic current output is generated by each pulse; acquiring time-resolved faradaic current data following each of the one or more excitation pulses; calculating a value of a selected measure of current decay from the acquired time-resolved faradaic current data; Calculating the concentration of the target species by application of a mathematical relationship between the selected measure of current decay and the concentration of the target species in the sample using the calculated value of the measure of current decay.

[0078] In one embodiment, the electrochemical sensor comprises an electrode functionalized with multiple recognition elements that undergo a conformational change upon target binding, where each recognition element is functionalized with one or more redox reporters. In one embodiment, the recognition elements comprise aptamers.

[0079] In one embodiment, the sample is selected from the group consisting of whole blood, serum, saliva, urine, sweat, interstitial fluid, spinal fluid, cerebrospinal fluid, tissue exudate, macerated tissue sample, cell solution, intracellular compartment, water, wash water, wastewater, groundwater, food, and beverage. In one embodiment, the sample is not processed before measurement.

[0080] In one embodiment, the target species is selected from the group consisting of a small molecule drug, a metabolite, a hormone, a peptide, a protein, a carbohydrate, a nucleic acid, a lipid, a hormone, a metabolite, a growth factor, a neurotransmitter, a nutrient, and a pollutant, a pathogen-induced or pathogen-derived factor, a pathogen, or a cell.

[0081] In one embodiment, the selected measure of target decay is selected from a decay constant, a mean lifetime, a half-life, and a relative amplitude. In one embodiment, the selected measure of current decay is obtained from an exponential fit of the time-resolved current data. In one embodiment, the selected measure of current decay is obtained from a single exponential fit of the time-resolved current data. In one embodiment, the selected measure of current decay is obtained from a bi-exponential fit of the time-resolved current data.

[0082] In one embodiment, a mathematical relationship between a selected measure of current decay and target concentration is obtained for a sensor of the same class as the electrochemical sensor deployed.

[0083] In one embodiment, no calibration step is performed before or after the measurement. In one embodiment, repeated measurements are achieved over an extended period of time.

[0084] In one embodiment, the electrochemical sensor is deployed in vivo. In one embodiment, the electrochemical sensor is deployed in a human subject. In one embodiment, the electrochemical sensor is deployed in a non-human animal. In one embodiment, the electrochemical sensor is deployed ex vivo. In one embodiment, the ex vivo deployment is in a point-of-care system.

[0085] In one embodiment, the electrochemical sensor is configured such that when the electrochemical sensor is placed in a sample, the output of the sensor is a faradaic current that varies in a concentration-dependent manner depending on the concentration of the target species in the sample; and wherein a stable mathematical relationship relating a measure of faradaic current decay to target concentration is known for the class of sensors to which the electrochemical sensor belongs.

[0086] In one embodiment, the electrochemical sensor comprises an electrode functionalized with multiple recognition elements that undergo a conformational change upon target binding, where each recognition element is functionalized with one or more redox reporters. In one embodiment, the class of sensors comprises sensors that have the same recognition element type, the same redox reporter type, and the same attachment properties for binding to the electrode. In one embodiment, the recognition element comprises an aptamer.

[0087] In one embodiment, the present invention is a sensing system comprising: the electrochemical sensor is configured such that when the electrochemical sensor is disposed in a sample, the output of the sensor is a faradaic current that varies in a concentration-dependent manner depending on the concentration of a target species in the sample; hardware components, including apparatus for application of excitation pulses to the electrochemical sensor and for acquisition of time-resolved faradaic current decays from the electrochemical sensor following the application of each pulse; and A non-transitory computer-readable medium storing data and computer programs that enable the electrochemical sensor and hardware components to perform the methods described herein. In one embodiment of the system, the electrochemical sensor comprises an electrode functionalized with multiple recognition elements that undergo a conformational change upon target binding, where each recognition element is functionalized with one or more redox reporters. In one embodiment, the recognition elements comprise an aptamer. In one embodiment, the electrochemical sensing system is programmed to perform chronoamperometric current decay analysis to derive the concentration of a target species in a sample by the methods disclosed herein. In one embodiment, the sensor element of the sensing system is implanted into an animal and communicates wirelessly or via wires with ex vivo hardware components. In one embodiment, one or more auxiliary hardware elements for delivering voltage pulses, collecting time-resolved current data, and calculating target concentrations are implanted with the sensor. In one embodiment, in addition to a power source (e.g., a battery), the sensor and auxiliary elements, including elements for delivering voltage pulses and elements for collecting and analyzing time-resolved current data, are implanted into the animal. In one embodiment, the system further comprises a drug delivery means for administering a drug to the animal in response to a threshold level of the target species being detected by the methods of the present invention, or a means for issuing an alert that drug administration is necessary. In one embodiment, the drug delivery means is a pump. In one embodiment, the means for providing an alert that medication administration is required comprises a wearable or mobile device in wireless or wired communication with components of the implanted sensor system.

[0088] In one embodiment, the invention includes a non-transitory storage medium having computer-readable instructions for operating a chronoamperometric electrochemical sensing system to perform chronoamperometric current decay analysis to derive the concentration of a target species in a sample according to the systems and methods disclosed herein. [Example]

[0089] Example 1 Real-time, subsecond-resolved measurement of specific molecules directly in vivo using E-AB sensors interrogated by chronoamperometry Electrochemical aptamer-based sensors offer a modular approach for continuous, real-time measurement of specific molecular targets, regardless of their chemical reactivity. Platforms consisting of aptamer "probes" modified with redox-active "reporters" and attached to interrogating electrodes (Figure 1A) signal through binding-induced conformational changes that alter electron transfer from the reporter, resulting in a readily measurable electrochemical output (Figure 1B). Because their signaling mechanism mimics the structure-linked signaling used by naturally occurring in vivo reporters, E-AB sensors are particularly insensitive to nonspecific binding and readily support continuous, long-term measurements directly in flowing, undiluted serum. Furthermore, while E-AB sensors often exhibit significant drift when tested in undiluted whole blood, the present inventors have recently shown that when combined with a protective film, improved surface passivation properties, and / or active drift-correction mechanisms, E-AB sensors support continuous, real-time measurement of specific molecules in whole blood and even in situ over several hours.

[0090] E-AB signaling is driven by binding-induced changes in the electron transfer kinetics of the aptamer-bound redox reporter. Previously, cyclic current, alternating current, or square-wave voltammetry has been used to "read" this change by observing the peak current. Square-wave voltammetry (SWV), the most commonly used of these, accomplishes this transduction by subjecting the sensor to a series of potential pulses and sampling the resulting faradaic current after a delay defined by the square-wave frequency. Therefore, the magnitude of the observed current is dependent on the electron transfer rate (which determines how much the current has decayed by the time it is measured), which in turn depends quantitatively on the target concentration. Specifically, when an E-AB sensor is interrogated by SWV, the resulting peak current either increases or decreases monotonically (depending on the square-wave frequency) with increasing target concentration (Figure 1B). The relative magnitude of this binding-induced change (ie, signal gain) is dependent on the aptamer used in the sensor and can be maximized by optimizing the frequency and amplitude of the square wave.

[0091] While SWV has proven to be a particularly convenient and reliable means of converting binding-induced changes in electron transfer kinetics into a readily measurable output, the technique is not without limitations. First, the peak current (measured in amperes) produced by SWV depends not only on the presence or absence of target but also on the number of redox reporter-modified aptamers on the sensor surface, which can vary from device to device due to fabrication differences (Figure 2A). Previously, this difference was addressed by calibrating each device in a reference sample of known (typically zero) target concentration before use, which is effective (Figure 2B), but adds complexity. Second, although E-AB sensors interrogated by SWV are selective enough to be placed directly in undiluted serum, they exhibit significant drift when placed in whole blood either in vitro or in vivo, a problem previously overcome using various drift-correction and drift-avoidance mechanisms. Finally, the time required to scan the required hundreds of millivolt potential window of SWV limits its time resolution to a few seconds. Demonstrated herein is the ability to overcome these limitations by replacing voltammetry, which indirectly measures changes in electron transfer rates, with chronoamperometry, which directly measures them, thereby achieving calibration-free, subsecond-resolved measurements of specific molecules in situ in vivo.

[0092] Results and Discussion Unlike SWV, which converts changes in electron transfer rate into changes in peak current and therefore indirectly reports on transfer kinetics, chronoamperometry directly measures electron transfer kinetics. It does so by determining the lifetime of the transient current generated in response to a step in electrode potential to a value at which the redox reporter is fully oxidized or reduced. For E-AB sensors bound to aminoglycoside antibiotics, we applied a sufficiently negative potential to completely reduce all of the methylene blue reporter, and measured the resulting current (Figure 3). We observed a current decay trace that is best described as the sum of two exponential processes. In the absence of their target, for example, these sensors exhibit one fast logarithmic phase with a lifetime of 100 ± 30 μs (throughout this document, errors represent standard deviation from five independently fabricated sensors) and a slower logarithmic phase with a lifetime of 6.5 ± 0.5 ms. The faster phase is due to charging of the double layer formed on the electrode surface at this potential bias (i.e., the transfer of aqueous ions with a timescale of microseconds) and remains unaffected by changes in target concentration. In contrast, the slower phase corresponds to the faradaic reduction of methylene blue to leucomethylene blue. Upon addition of a saturating target concentration, the second phase becomes faster with a lifetime of 1.20 ± 0.01 ms. This approximately 5-fold decrease in lifetime (compared to samples lacking target) is consistent with a change in the proximity of the redox reporter to the electrode surface and likely reflects the total number of aptamers bound to the target, which transfer electrons faster than aptamers without the target.

[0093] In theory, if the structural dynamics of an aminoglycoside-binding aptamer follow a two-state model, the relative amplitude of the exponential phase (reflecting the total number of bound and unbound aptamers) will vary monotonically with target concentration. However, the two-state model assumes that the two exponential phases can be measured independently. This is not the case here, because the lifetimes of the two exponential phases are quite similar at any given target concentration, making it difficult to extract their amplitudes with sufficient precision. That is, if the kinetics of the interconversion between the bound and unbound states of the aptamer are more rapid than the electron transfer events, as is the case here, the structural equilibrium cannot be sampled as separate static populations. Instead, the measured lifetime reflects a population-weighted average of the bound and unbound states. This limitation is overcome by estimating the current decay lifetime using a single-exponential fit (Figure 4A).

[0094] The monotonic relationship between the chronoamperometric lifetime of an E-AB sensor and its target concentration provides a calibration-free approach to E-AB measurements. Unlike the absolute SWV peak current, which depends on the total number of aptamers on the sensor, the chronoamperometric decay lifetime depends only on the relative total number of bound and unbound aptamers. Therefore, once the lifetime-concentration relationship is established for a given type of sensor, it can be used to determine target concentration without the need to calibrate each individual sensor. Lifetime-concentration was the established relationship for aminoglycoside-detecting E-AB sensors when used to detect tobramycin in vitro in flowing whole blood. Specifically, a nonlinear regression of lifetime versus concentration onto the Langmuir isotherm was performed (red line in Figure 4B) and analyzed for concentration. A batch of five new E-AB sensors (i.e., sensors not in the initial training set) was challenged with tobramycin in whole blood, and the observed chronoamperometric lifetimes were converted to estimated concentrations using a pre-determined Langmuir isotherm (Figure 4C), which successfully determined drug concentrations over the range of 1 μM to 1000 μM with precision and accuracy better than 10%.

[0095] The use of chronoamperometrically determined current decay lifetimes as a means of defining target concentrations is not only calibration-free, but also resistant to drift. Furthermore, while E-AB sensors interrogated by SWV are selective enough to function well in undiluted serum, they often exhibit significant baseline drift when placed directly in flowing whole blood (Figure 5A). This has previously been corrected using square-wave voltammetry techniques, with measurements made at multiple frequencies (Figure 5A). In contrast, chronoamperometric lifetime measurements are inherently resistant to such drift; as noted above, although the total amplitude of the transient current drifts significantly (presumably due to the monolayer structure), 22 surface reorganization), the lifetime of its exponential decay is independent of its amplitude and therefore exhibits little drift (Figure 5B).

[0096] The drift resistance of E-AB sensors interrogated by chronoamperometry is sufficient to support continuous, real-time measurements directly in situ in the blood of live animals. An aminoglycoside-conjugated E-AB sensor was fabricated on a 75 μm diameter gold microwire, placed in a 22-gauge catheter for structural support, and placed directly in the jugular vein of a live rat (Figure 6A). Following intravenous administration of 30 mg / kg, the plasma pharmacokinetics of the antibiotic tobramycin was monitored by continuous chronoamperometry measurements for a total of 2 h (Figure 6B). Continuous measurements were achieved by sequentially pulsing the E-AB sensor potential between -0.1 V and -0.3 V, with each pulse lasting only 100 ms. Next, nonlinear regression analysis of the transient current generated at -0.3 V was performed to derive the current decay lifetime in real time, and these lifetimes were converted to concentration using a predefined Langmuir isotherm (Figure 6C). With a time resolution of 300 ms (limited by aptamer-target binding kinetics, data acquisition, and computation time), chronoamperometry measurements resolve not only the duration of drug infusion, but also the time it takes for the drug to "mix" and become homogenous in the bloodstream after the infusion has ended (Figure 6C).

[0097] The subsecond time resolution of the chronoamperometrically interrogated E-AB sensor provides unprecedented pharmacokinetic measurements. The distribution α, elimination β, and lifetime of tobramycin were determined by fitting in vivo data to a two-compartment pharmacokinetic model. The 300-millisecond time resolution of the chronoamperometrically interrogated E-AB sensor supports the measurement of the drug's distribution phase, α = 3.74 ± 0.04 min, with over 1,000 measurement points and a calculated standard error of approximately 1% from the fit. Similarly, the drug's elimination phase was measured as β = 69 ± 2 min with 14,000 measurement points, reducing the standard error of the fit to only 3%; most of this small deviation likely arises from metabolic fluctuations in the animals during the course of the experiment (i.e., β is not exactly constant). This precision represents an order of magnitude improvement over previous in vivo E-AB measurements achieved using square wave voltammetry, resulting in a significant improvement over previous measurements using blood sampling and ex vivo analysis.

[0098] Herein, we achieved calibration-free, subsecond-resolved measurements of specific small molecules directly in vivo using chronoamperometric interrogation of an E-AB sensor. The unprecedented temporal resolution of this technique suggests it may improve our understanding of rapidly fluctuating physiological events such as drug uptake, hormone and neurotransmitter release, and the movement of drugs and metabolites within the central nervous system. In applications ranging from therapeutic drug monitoring to long-term, feedback-controlled drug delivery, the ability to perform real-time, in vivo, calibration-free measurements of specific molecules may also enhance the efficiency and accuracy of drug administration.

[0099] E-AB sensors are not the only class of biosensors that rely on binding-induced changes in electron transfer kinetics for analyte detection. Other examples include sensors that measure changes in electron transfer from solution-phase redox reporters, changes in electron transfer due to binding-induced ligand displacement, changes in reporter reorganization energy, or sterically induced changes in the efficiency with which a scaffold-attached redox reporter approaches the underlying electrode surface. In this regard, we hypothesize that the ability of chronoamperometry to directly measure electron transfer kinetics may also be valuable in interrogating these other platforms.

[0100] method The E-AB sensors were fabricated as follows: 12 cm long, 11.5 cm long, and 11 cm long sections of pure gold, platinum, and silver wires were cut to form the sensors. Approximately 2 cm of insulation was removed from both ends of these wires using a surgical blade to allow electrical contact. These wires were then soldered to one of the three ends of a connector cable using 60% tin / 40% lead rosin solder (0.8 mm diameter). The wires were then attached to each other by applying heat to the shrinkable tubing surrounding the wires, except for a small window approximately 5 mm long at the end of each wire. The wires were attached in a layered fashion, first insulating only the gold wire, then both the gold and platinum wires together, and finally all three wires together. The purpose of this triple-layer insulation was to provide mechanical strength for the malleable probe body. To prevent electrical shorting between the wires, different lengths were used for each wire, as described above. The sensor window (i.e., the area without insulation) in the gold wires was cut to approximately 3 mm in length. A silver wire was used as the reference electrode by first soaking it in bleach overnight to form a silver chloride film. To increase the surface area of ​​the gold working electrode (to obtain a larger peak current), the sensor surface was immersed in 0.5 M sulfuric acid, followed by 16,000 pulses of potential vs. Ag / AgCl (E = 0.01). initial = 0.0V to E highThe surface was electrochemically roughened by stepping back and forth between 0 V and 2.0 V. Each potential step was 20 ms in duration with no "rest period." To fabricate the sensor, an aliquot of DNA construct was reduced with a 1000-fold molar excess of tris(2-carboxyethyl)phosphine for 30 minutes at room temperature. The newly roughened probe was then rinsed with deionized water and immersed in a 200 nM solution of the reduced DNA construct in PBS for 1 hour at room temperature. Following this, the sensor was immersed overnight in 20 mM 6-mercapto-1-hexanol in PBS for 12 hours at 4°C to coat the remaining gold surface. After this, the sensor was rinsed with deionized water and stored in PBS.

[0101] For SWV measurements, the sensor was interrogated from 0.0 V to -0.5 V vs. Ag / AgCl using a 50 mV amplitude, a potential step size of 1 to 5 mV, and a variation frequency ranging from 10 Hz to 500 Hz. All SWV measurements were performed using a three-electrode setup on a CH Instruments™ electrochemical workstation (Austin, TX, Model 660D) with a commercially available Ag / AgCl reference electrode and a platinum counter electrode filled with saturated KCl solution. For chronoamperometry, the sensor potential was stepped continuously from -0.1 V to -0.3 V, with each step lasting 100 ms. Current sampling was performed every 10 μs for in vitro measurements and every 100 μs for in vivo measurements (to reduce the number of experimental points and rate data acquisition). All chronoamperometric measurements were performed using the three-electrode E-AB sensor described above and recorded on a GAMRY™ Reference 600+ Potentiostat / Galvanostat / ZRA (Warminster, PA).

[0102] To study the chronoamperometric current decay behavior, measure aptamer affinity, and relate signal gain to target concentration, the sensors were interrogated by either square-wave voltammetry or chronoamperometry with increasing concentrations of the corresponding target, first in flowing PBS and then in flowing heparinized bovine blood. These experiments were performed in a closed flow system intended to mimic the type of blood transport found in veins. Blood flow was achieved using a magnetic gear pump, and the flow rate was set at 1–10 mL per minute, as measured by a flow meter. To generate binding curves (titration of the aptamer with the target), stock solutions of tobramycin were prepared immediately before measurement in PBS buffer or blood, respectively. Sensor challenges to demonstrate calibration-free behavior were performed by testing new batches of aminoglycoside-bound E-AB sensors against stock solutions prepared from tobramycin standards.

[0103] In vivo measurements were performed in anesthetized rats with a silastic catheter inserted for infusion or an E-AB sensor placed in the jugular vein for measurements. All in vivo measurements were performed using a three-electrode setup, with the reference electrode being a silver wire coated with a silver chloride film as described above and the counter electrode being a platinum wire. Recordings were taken for up to 3 hours with a sampling rate of one point every 300 milliseconds. To obtain pharmacokinetic profiles from our real-time data, nonlinear regression analysis was performed using a two-compartment model to fit the intravenous injection. The equation used in the regression was: TIFF0007744694000005.tif7128In formula, C P is the measured plasma concentration, and A and B are the maximum concentrations A + B = C MAX where α is the contribution of each pharmacokinetic compartment to σ, α is the first-order time constant of drug distribution, and β is the elimination time constant of the drug. During the regression analysis, all variables were kept constant to determine the best fit by minimizing the squared error.

[0104] All patents, patent applications, and publications cited in this specification are herein incorporated by reference to the same extent as if each individual patent application or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. Although the present invention has been described with reference to described embodiments thereof, it will be understood by those skilled in the art that changes can be made in the structure and elements of the present invention without departing from the spirit and scope of the invention as a whole.

Claims

1. 1. A method of operating an electrochemical sensing system for measuring the concentration of a target species in a sample, the electrochemical sensing system comprising: The method includes an electrochemical sensor having an electrode functionalized with a plurality of recognition elements that undergo a conformational change upon binding of a target species in a sample, the method comprising: applying one or more excitation pulses to the electrochemical sensor, wherein a faradaic current output is generated by each pulse and varies with the conformational change of the recognition element; acquiring time-resolved faradaic current data following each of the one or more excitation pulses; calculating a value of a selected measure of current decay from said time-resolved faradaic current data; applying a mathematical relationship between the selected measure of current decay and the concentration of the target species to the calculated value of the selected measure of current decay, thereby calculating the concentration of the target species; The method wherein the electrochemical sensors are not individually calibrated prior to exposure to the sample.

2. 10. The method of claim 1, wherein each recognition element is functionalized with one or more redox reporters.

3. The method of claim 2 , wherein the recognition element comprises an aptamer.

4. 2. The method of claim 1, wherein the sample is selected from the group consisting of whole blood, serum, saliva, urine, sweat, interstitial fluid, spinal fluid, cerebrospinal fluid, tissue exudate, macerated tissue sample, cell solution, subcellular compartment, water, washwater, wastewater, groundwater, food, and beverage.

5. 5. The method of claim 4, wherein the sample is not processed prior to measurement.

6. 5. The method of claim 4, wherein the sample is an undiluted sample.

7. 2. The method of claim 1, wherein the target species is selected from the group consisting of a small molecule drug, a metabolite, a hormone, a peptide, a protein, a carbohydrate, a nucleic acid, a lipid, a hormone, a metabolite, a growth factor, a neurotransmitter, a nutrient, and a pollutant, a pathogen-induced or pathogen-derived factor, a pathogen, or a cell.

8. 10. The method of claim 1, wherein the selected measure of current decay is selected from a decay constant, a rate constant, a mean lifetime, a half-life, and a relative amplitude.

9. 10. The method of claim 1, wherein the selected measure of current decay is obtained from an exponential fit of time-resolved current data.

10. 10. The method of claim 9, wherein the selected measure of current decay is obtained from a single exponential fit of time-resolved current data.

11. 10. The method of claim 1, wherein the selected measure of current decay is obtained from a bi-exponential fit of time-resolved current data.

12. 10. The method of claim 1, wherein the mathematical relationship between the selected measure of current decay and the target concentration is a relationship that has been determined and is applicable for sensors of the same class as the electrochemical sensor.

13. 10. The method of claim 1, wherein repeated measurements are achieved over an extended period of time.

14. The method of claim 1 , wherein the electrochemical sensor is deployed ex vivo in a point-of-care system.

15. An electrochemical sensor, the electrochemical sensor comprising: an electrode functionalized with a plurality of recognition elements that undergo a conformational change upon target binding, each recognition element functionalized with one or more redox reporters; the electrochemical sensor, under conditions in which it is placed in a sample, outputs a faradaic current that varies in a concentration-dependent manner with the concentration of a target species in the sample; The output of the sensor is correlated to the concentration of the target species by a mathematical relationship between a measure of faradaic current decay and the concentration of the target species for the class of sensors to which the electrochemical sensor belongs; and the class of sensors to which the electrochemical sensor belongs includes sensors having the same recognition element type, the same redox reporter type, and the same attachment properties for binding to an electrode; Electrochemical sensors.

16. 16. The electrochemical sensor of claim 15, wherein each recognition element is functionalized with one or more redox reporters.

17. 17. The electrochemical sensor of claim 16, wherein the recognition element comprises an aptamer.

18. An electrochemical sensing system comprising: The electrochemical sensor of any one of claims 15 to 17; hardware components, including apparatus for applying excitation pulses to the electrochemical sensor and for acquiring time-resolved faradaic current decays from the electrochemical sensor following the application of each pulse; and A non-transitory computer readable medium storing data and computer programs that enable the electrochemical sensor and hardware components to perform the method of any one of claims 1 to 14.

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