Electrochemical impedance system and method for real-time monitoring of biochemical interactions and / or sensing of biomarkers

Abstract

The invention provides an electrochemical impedance system (1) and method for monitoring biochemical interactions and / or sensing biomarkers in a biological fluid. The system (1) comprises at least one electrochemical cell (2) having electrodes configured to be in contact with the biological fluid, and a potentiostat (3) for controlling the operating conditions of the electrochemical cell (2) and for measuring the reactions in the electrochemical cell (2). The potentiostat (3) applies an alternating voltage or current input having a constant frequency to the electrochemical cell (2) during one experiment and measures the output signal of the electrochemical cell (2), thereby measuring at least the initial impedance value of the electrochemical cell (2) at the beginning of the reaction occurring in the electrochemical cell (2) and the final impedance value of the electrochemical cell (2) at the end of the reaction to measure the change in impedance of the electrochemical cell (2) due to the reaction.

Description

Technical Field

[0001] This invention relates to a method for real-time monitoring of biochemical interactions and / or sensing of biomarkers using an electrochemical impedance spectroscopy system, wherein a constant-frequency alternating voltage or current is applied to one or more working electrodes. The invention also relates to an electrochemical impedance spectroscopy system configured for real-time monitoring of biochemical interactions and / or real-time sensing of biomarkers. Background Technology

[0002] Biosensors are analytical devices that convert biological interactions into digital signals. In recent years, biosensors have demonstrated their potential applications in medical diagnostics, food quality and safety, environmental monitoring, and biodefense. According to the International Union of Pure and Applied Chemistry (IUPAC), a biosensor is an analytical device that uses biological or biologically derived biorecognition elements to provide quantitative or semi-quantitative information. These biorecognition elements target specific bioanalytes and come into contact with physicochemical transducers. Biorecognition elements can be made of biological materials (e.g., enzymes, antibodies, nucleic acids, or cell receptors), biologically derived materials (e.g., aptamers or recombinant antibodies), or even biomimetic materials (molecularly imprinted polymers and synthetic catalysts). The biorecognition interaction between the biorecognition element and the analyte is converted into a digital signal by the transducer, which is then interpreted by a computer.

[0003] Over the past few years, efforts have been focused on developing biosensors capable of detecting disease biomarkers and applying them to health monitoring, disease diagnosis, and treatment at the point of healthcare. These biosensors have emerged as alternatives to traditional, laboratory-based diagnostic techniques: chromatography, spectroscopy, immunology-based techniques (e.g., enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay), and nucleic acid-based techniques (polymerase chain reaction (PCR), real-time PCR), among others. In particular, electrochemical biosensors have been continuously studied, and their reported applications cover a wide range of healthcare fields, including the detection of foodborne pathogens, bacterial infections, respiratory diseases, cancer, and clinical diagnosis. Electrochemical biosensors are categorized into potential sensors, current sensors, and impedance sensors. Impedance sensors, in particular, can detect biorecognition events occurring on the sensing surface through changes in interfacial properties.

[0004] Few publications report the application of impedance biosensors in the analysis of clinically relevant samples. Most electrochemical impedance spectroscopy (EIS)-based sensors rely on Faraday impedance and require the presence of redox probes; therefore, these sensors are automatically classified as labeled sensors. Lasserre et al., in their publication "SARS-CoV-2 Aptamer Sensor Based on Electrochemical Impedance Spectroscopy and Low-Cost Gold Electrode Substrate," Anal Chem. 94, 2126-2133 (2022), employed an impedance biosensor to detect the SARS-CoV-2 spike protein using a truncated SARS-CoV-2 aptamer. Ferricyanide was used as the redox pair, and EIS measurements were performed after 15 minutes of incubation of the SARS-CoV-2 protein with the aptamer. Vasantham et al., in their publication "Paper-Based Point-of-Sensation Immunosensor for Impedance Detection of Cardiac Troponin I Biomarker," Biomed Microdevices 22, 6 (2019), also used a ferricyanide redox pair. They reported a detection limit (LOD) of 0.05 ng / mL for cardiac troponin I (cTnI) using an EIS biosensor. Despite the low reported LOD, the fact that these biosensors require redox probes complicates their application in real-life, point-of-care testing environments outside of traditional laboratory settings.

[0005] On the other hand, non-Faraday EIS sensors do not require redox probes; therefore, these sensors are considered label-free. The vast majority of publications employing non-Faraday EIS sensors utilize Nyquist plots, which are extracted by measuring impedance over a wide frequency range to describe impedance changes that occur when biorecognition interactions take place on the sensing surface. Although this type of measurement is considered label-free, signal amplification is used to increase detection sensitivity according to numerous publications. Signal amplification can employ nanotechnology-based and biotechnology-based strategies, such as nanotags, nanocatalysis, nanocarriers, assembly-based, and polymerase-based DNA amplification strategies.

[0006] Existing multi-step EIS-based biosensors are typically very complex, with limitations in integration and miniaturization. Research has been conducted on simplified non-Radida EIS biosensors. Aydin et al., in their publication "An impedance immunosensor for highly sensitive detection of IL-8 in human serum and saliva samples: a novel surface modification method of 6-phosphonohexanoic acid for biosensing applications," Anal Biochem. 554, 44-52 (2018), employed an EIS sensor and scanned the frequency from 50,000 Hz to 0.05 Hz during measurement to detect interleukin-8 (IL-8) in human serum and saliva. Without signal amplification or sample pre-concentration, the reported LOD was 6 fg / mL. However, the incubation time of IL-8 with the sensor was 45 minutes, which limits its application in point-of-care diagnostic devices. The same group, in the publication "Selective and Ultrasensitive Electrochemical Immunoassay of NSE Cancer Biomarker in Human Serum Using a Disposable ITO Electrode Modified with Epoxy-Substituted Poly(pyrrole) Polymer," Sens Actuators B Chem., 306, 127613 (2020), proposed using the same type of biosensor to detect the standard biomarker for lung cancer, namely neuron-specific enolase (NSE), and achieved the same LOD (6.1 fg / mL). This time, the incubation time of the sensor was reduced to 30 minutes, which still precludes its use as a point-of-care biosensor. Summary of the Invention

[0007] One object of the present invention is to overcome at least some of the aforementioned problems related to sensing biomarkers and / or monitoring biochemical interactions in electrochemical cells.

[0008] According to a first aspect of the present invention, a method is provided for real-time monitoring of biochemical interactions and / or sensing of biomarkers in biological fluids using an electrochemical impedance system according to claim 1.

[0009] The proposed method offers several advantages. Specifically, the ability to operate at a constant frequency facilitates real-time monitoring of biochemical interactions. The system sensitivity can be adjusted based on the operating frequency to match the biochemical reaction. The shortened incubation time is a unique feature of this invention, distinguishing it from conventional EIS-based methods that utilize Nyquist and / or Bode plots to illustrate impedance changes. Therefore, this method is characterized by its speed (incubation time less than 5 minutes) and the generation of impedance-time plots, providing real-time impedance data. Furthermore, the sensors used in this method can be label-free or labeled.

[0010] According to a second aspect of the present invention, an electrochemical impedance system for monitoring biochemical interactions and / or sensing biomarkers in biological fluids is provided according to claim 9.

[0011] Other aspects of the invention are set forth in the appended dependent claims. Attached Figure Description

[0012] Referring to the accompanying drawings, other features and advantages of the invention will become apparent from the following description of non-limiting exemplary embodiments, in which: Figure 1 A first example electrochemical impedance system forming a single sensor, a two-electrode system, is schematically shown, in which the teachings of the present invention can be implemented; Figure 2 A portion of a second example electrochemical impedance system, forming a single sensor and a three-electrode system, is schematically shown, in which the teachings of the present invention can be implemented; Figure 3 A portion of a third example electrochemical impedance system is schematically shown forming a dual-sensor system, each sensor having two electrodes, wherein the sensors form separate electrochemical cells, wherein the teachings of the present invention can be realized; Figure 4 A portion of a fourth example electrochemical impedance system is schematically shown forming a dual-sensor system, each sensor having three electrodes, wherein the sensors form separate electrochemical cells, wherein the teachings of the present invention can be realized; Figure 5 A portion of a fifth example electrochemical impedance system is schematically shown forming a dual-sensor system, each sensor having two electrodes, wherein the sensors form a single electrochemical cell, and the teachings of the present invention can be realized therein; Figure 6 A portion of a sixth example electrochemical impedance system is schematically shown forming a dual-sensor system, each sensor having three electrodes, wherein the sensors form a single electrochemical cell, and the teachings of the present invention can be realized therein; Figure 7 It shows Figure 1 The example Bode plot and circuit model of the electrochemical cell are shown; and Figure 8a and Figure 8b A flowchart illustrating an example method for monitoring biochemical interactions and / or sensing biomarkers in biological fluids using an electrochemical impedance system is shown. Detailed Implementation

[0013] Some embodiments of the invention will now be described in detail with reference to the accompanying drawings. As used herein, “and / or” refers to any one or more items in the list connected by “and / or”. For example, “x and / or y” represents any element in the three-element set {(x), (y), (x, y)}. In other words, “x and / or y” means “one or both of x and y”. Similarly, “x, y and / or z” represents any element in the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, “x, y and / or z” means “one or more of x, y and z”. Furthermore, the term “comprising” is used herein as an open-ended term. This means that the object includes all listed elements, but may also include other unnamed elements. Therefore, the word “comprising” is interpreted in a broader sense as “including,” “containing,” or “covering”. The same or corresponding functional and structural elements appearing in different figures are given the same reference numerals. It should be noted that, unless explicitly stated or implied in the context, the use of terms such as “first,” “second,” and “third” may not imply any particular order or rank. Furthermore, the term “signal” in this specification may be interpreted as a function that conveys information about a phenomenon, but does not necessarily include any coded information unless explicitly or implicitly indicated in the context.

[0014] The electrochemical impedance spectroscopy system shown is a single-cell or dual-cell system developed for real-time or near-real-time monitoring and detection of biochemical interactions, employing electrodes as transducers. Depending on specific requirements, systems like the examples below include 2 to 6 electrodes, which can be made of various materials, such as metals, oxides, or two-dimensional (2D) materials, i.e., monolayer materials, such as graphene. At least some of these electrodes are designed to undergo functionalization to enhance the ability to detect specific target biomarkers or one or more target analytes. Target analytes can include molecular biomarkers, such as proteins, hormones, metabolites, nucleic acids, and / or whole cells. Measurements will be performed using a potentiostat, also known as a galvanometer.

[0015] Figure 1A first example electrochemical impedance system 1 is schematically illustrated, in which the teachings of the present invention can be implemented. The system in this first configuration includes an electrochemical cell 2 coupled to a control and measurement device 3, in this case, a potentiostat. However, any device or system that can provide an AC input signal and accurately measure the AC signal can be used as the control and measurement device. The potentiostat 3 is operatively coupled to the electrochemical cell 2 to control the operating conditions of the electrochemical cell and to measure the reactions within the electrochemical cell. The electrochemical cell in this example includes: a first electrode E1, which in this example is a working electrode (WE); and a second electrode, which in this example is a counter electrode (CE), which can optionally operate as a combined counter electrode and reference electrode. The electrodes in the electrochemical cell serve as conductive interfaces connecting the electronic circuitry and the electrolyte. The device allows monitoring of electrochemical reactions occurring on the electrode surface by evaluating the electrochemical and / or electrochemical properties of the electrodes. In this example, the working electrode E1 is functionalized with a biorecognition element or layer 4 to capture a target analyte 5 from the electrolyte, also referred to as a biofluid or solution, in this case being a liquid. At least in operation, the electrochemical impedance system therefore includes a biofluid. The biorecognition element 4 comprises or is composed of biological materials, or comprises or is composed of biologically derived materials, or comprises or is composed of biomimetic materials, or is composed of any combination of biological materials, biologically derived materials, and / or biomimetic materials. Biological materials include one or more antibodies, enzymes, nucleic acids, and / or cell receptors; biologically derived materials include one or more aptamers and / or recombinant antibodies; or biomimetic materials include one or more molecularly imprinted polymers and / or synthetic catalysts.

[0016] The working electrode E1 is the electrode where the target electrochemical reaction occurs in this example. The substance under study binds, reacts, oxidizes, or is reduced at the electrode. The working electrode is typically the electrode in a study where the electrochemical process is to be observed or controlled. The reference electrode is used to control the potential of the electrochemical cell. A stable and known electrochemical potential is provided, to which the potential of the working electrode can be compared. The counter electrode E2, also known as the auxiliary electrode, provides a pathway for current to flow into or out of the working electrode E1. This completes the circuitry in electrochemical cell 2.

[0017] like Figure 1 As shown, the potentiostat includes a voltage source 6, which is an AC voltage source V. AC It is used to provide input voltage to electrochemical cells, although AC power supply I can also be used. ACInstead of providing input current to the electrochemical cell, this is used. In this example, the potentiostat includes a direct current (DC) power supply voltage 7 to establish a DC bias voltage on the working electrode. The primary function of this DC bias source is to maintain a predetermined potential on the working electrode.

[0018] Figure 1 The right-hand side illustrates the operation of electrochemical cell 2. As shown, target analyte 5 is captured by biorecognition element 4. Biochemical interactions on the electrode surface alter the capacitance and / or resistivity of the electrochemical cell under test, which is measured by a potentiostat.

[0019] In operation, a continuous AC signal at a fixed frequency is applied to either the working electrode E1 or the counter electrode E2 during at least one experiment, and the resulting signal, whether current or voltage, reflects the impedance of the electrochemical cell under test relative to the input signal. This method is used to monitor biochemical interactions occurring on functionalized electrode surfaces in real time. A fixed frequency AC signal is selected based on the sensor design. This specific frequency is chosen based on the type of target analyte and / or biochemical reaction to optimize the capacitive and / or resistive response of the cell, thereby maximizing system sensitivity. The process of optimizing and selecting the working frequency will be detailed later. Any interactions / reactions occurring on the electrode surface result in mass adsorption or charge generation on the electrode, which leads to changes in the cell's capacitive and / or resistive response. These changes can be used to monitor and detect target biomolecules and / or biochemical interactions.

[0020] Figure 2 A second example of an electrochemical cell configuration is schematically shown, in which the teachings of the present invention can be implemented. According to... Figure 2 The second configuration shown is a three-electrode system, comprising: a first electrode E1, which is the working electrode in this case; a second electrode E2, which is the counter electrode in this case; and a third electrode E3, which is the reference electrode in this case.

[0021] In such Figure 1 and Figure 2In the single-sensor setup shown, a biometric element 4 is applied to the working electrode E1 and / or the counter electrode E2. In this electrochemical cell setup, a DC signal is used to establish a reference potential. An AC signal, as voltage or current, is applied to the working electrode E1 or the counter electrode E2 at a constant frequency, and subsequently, a synthesized signal, as current or voltage, is recorded on the counter electrode E2 or the working electrode E1, respectively. The phase and amplitude difference between the input signal and the measured signal is used to characterize or measure the impedance of the electrochemical cell 2. Depending on the specific target analyte, the real part or imaginary part of the impedance, or both, can be considered as the sensing signal. In this case, the roles of the working electrode E1 and the counter electrode E2 are interchangeable. Although various methods exist for extracting the impedance of an electrochemical cell, the results obtained by these methods are essentially similar. Electrochemical cells can use methods such as... Figure 1 The common counter electrode and reference electrode shown, or having, as Figure 2 The individual reference electrode is shown.

[0022] Figure 3 A third example of an electrochemical cell configuration is schematically illustrated, in which the teachings of the present invention can be implemented. In this third configuration, the system forms a dual-sensor configuration, including: a first electrochemical cell 2 a The first electrochemical cell serves as an active or target sensor in this case; and the second electrochemical cell 2 r In this case, the second electrochemical cell serves as a reference sensor. The active sensor exhibits sensitivity to the target analyte, while the reference sensor remains insensitive to the target. First electrochemical cell 2 a Includes a first working electrode E1 and a first counter electrode E2, which also operates as a first reference electrode in this case, while the second electrochemical cell 2 r It includes: a second working electrode E1r; and a second counter electrode E2r, which in this case also operates as a second reference electrode. The electrochemical cell according to this configuration is a dual-electrode cell and forms a separate dual-electrode sensor.

[0023] Figure 4 A fourth example of an electrochemical cell configuration is schematically illustrated, in which the teachings of the present invention can be implemented. In this fourth configuration, the system forms a dual-sensor configuration, including: a first electrochemical cell 2 a The first electrochemical cell serves as an active or target sensor in this case; the second electrochemical cell 2 r The second electrochemical cell serves as a reference sensor in this case. The first electrochemical cell 2 a It includes a first working electrode E1, a first counter electrode E2, and a first reference electrode E3, while the second electrochemical cell 2 rIt includes a second working electrode E1r, a second counter electrode E2r, and a second reference electrode E3r. The electrochemical cell configured this way is a three-electrode cell, and a separate three-electrode sensor is formed.

[0024] The third and fourth configurations, as well as other electrochemical cell configurations presented in this specification, are primarily used for detecting target biomolecules, such as proteins and hormones. To mitigate parasitic (non-specific) signals, simplify data analysis, and minimize errors, the active sensor 2... a A reference sensor, i.e., a second electrochemical cell, is introduced alongside. These setups consist of two parallel electrochemical cells, both exposed to or in contact with the same biofluid. In one cell (in this case, the first electrochemical cell), a biofunctional element is applied to the first working electrode E1 and / or the first counter electrode E2, making it sensitive to the target biomolecule (i.e., the receptor of the biorecognition element is able to capture the target analyte). In the other cell (in this case, the second electrochemical cell), a non-specific biofunctional element or layer is applied to the second working electrode E1r and / or the second counter electrode E2r, making it insensitive to the target (i.e., the receptor of the biorecognition element cannot capture the target analyte). The sensing signal will be ΔZ. real and / or ΔZ imaginary : ΔZ imaginary = Z imaginary_active - Z imaginary_reference ; and / or Δz real = Z real_active - Z real_reference , Among them, Z imaginary_active Z represents the virtual impedance value of an active (first) electrochemical cell. imaginary_reference Z represents the virtual impedance value of the reference (second) electrochemical cell. real_active Z represents the actual impedance value of an active (first) electrochemical cell. real_reference This indicates the actual impedance value of the reference (second) electrochemical cell.

[0025] Figure 5 A fifth example of an electrochemical cell configuration is schematically shown, in which the teachings of the present invention can be implemented. In this fifth configuration, the system forms a dual-sensor comprising only one electrochemical cell 2. The electrochemical cell 2 includes a first working electrode E1, a second working electrode E1r, and a counter electrode E2, which optionally also functions as a reference electrode. The electrochemical cell according to this configuration is a three-electrode cell. In this case, the active sensor comprises the first working electrode E1 and the counter electrode E2, while the reference sensor comprises the second working electrode E1r and the counter electrode E2.

[0026] Figure 6A sixth example of an electrochemical cell configuration is schematically shown, in which the teachings of the present invention can be implemented. In this sixth configuration, the system forms a dual-sensor system comprising only one electrochemical cell 2. The electrochemical cell 2 includes a first working electrode E1, a second working electrode E1r, a counter electrode E2, and a reference electrode E3. The electrochemical cell according to this configuration is a four-electrode cell. In this case, the active sensor includes the first working electrode E1, the counter electrode E2, and the reference electrode E3, while the reference sensor includes the second working electrode E1r, the counter electrode E2, and the reference electrode E3.

[0027] The main objectives and basic principles of the fifth and sixth configurations are very consistent with those of the third and fourth configurations, but the fifth and sixth configurations differ from the third and fourth configurations in the following ways: Both the active sensor and the reference sensor are immersed in the same biological fluid (a single electrochemical cell). The counter electrode E2 and the reference electrode E3 are shared between the active sensor and the reference sensor, thereby reducing complexity and minimizing the managed surface area. The first working electrode E1 is modified with a specific biological functional element, while the second working electrode E2r is coated with a non-specific biological functional element (although this arrangement is also possible in the third and fourth configurations, in which the two sensors are in different electrochemical cells). Neither the counter electrode E2 nor the reference electrode E3 underwent any biofunctionalization (although this also applies to the electrodes of the reference sensor in the third and fourth configurations).

[0028] The operating frequency of the real-time electrochemical impedance spectroscopy system 1 is measured by... Figure 7 The Bode plot shown on the right is used for optimization. The Bode plot is the frequency response of the tested electrochemical cell, that is, the amplitude and phase changes as a function of frequency, taking into account the following fact: Z imaginary =|Z|×sin(φ) and Z real =|Z|×cos(φ), where φ represents the phase of the impedance. To improve the sensitivity of an electrochemical impedance system, especially with respect to the imaginary part of the signal, it is important to choose an operating frequency that maximizes the system's capacitive response. It is worth noting that at extremely low frequencies, low-frequency noise becomes dominant. Therefore, there is a trade-off between maximizing the capacitive signal and managing low-frequency noise to achieve an optimized signal-to-noise ratio (SNR), such as... Figure 7 As shown visually.

[0029] In such Figure 7 In the circuit model of electrochemical cell 2 shown on the left, R S It is the ohmic resistance of the solution (i.e., the biological fluid), R CT It is the charge transfer resistance of each electrode, C DLThis refers to the capacitance of the electrochemical double layer, a structure that appears on the surface of an object when it is exposed to a fluid. Therefore, in this case, the electrochemical double layer refers to the charge-separated region formed at the interface between the electrode and the electrolyte solution. The presence of this interface is due to the potential difference between the electrode and the electrolyte. Therefore, the total impedance of the electrode can be calculated as follows: (1) Where τ=R CT C DL Assuming the electrochemical cell is used to detect biomolecular interactions in a liquid solution, and according to Equation 1, the real part of the impedance monitors the effect of the bulk solution on the total electrode impedance (i.e., the effect of the electrolyte only); any biochemical interactions occurring on the sensing surface of the electrode, whether it be charge transfer on the surface (R0) CT ) or mass adsorption (C DL All of these factors will affect the imaginary part of the impedance. To monitor Z in real time... imaginary and / or Z real The potentiostat 3 will apply a fixed-frequency AC signal to the working electrode, depending on the electrode design and its surface area, measurement settings, and surface functionalization. The selected frequency optimizes the cell's capacitance / resistance response, thereby maximizing the system's sensitivity. The response type depends on the biometric element used and / or the target analyte. Therefore, the operator of the electrochemical impedance system 1 is generally aware of the response type. The recommended frequency range for sensing based on the sensing signal is as follows: Capacitor response only: 1 mHz to 1 kHz Capacitive and resistive response: 1 kHz to 100 kHz Resistance response only: >100 kHz.

[0030] It is worth noting that the shape of the Bode plot (and therefore, the selection of the optimized operating frequency) is highly dependent on the electrode design and associated surface functionalization. Therefore, the selection of the operating frequency is significantly influenced by a variety of factors, including the measurement setup, electrical connections, the potentiostat's internal low-pass filter, and the overall noise level of the system. While the electrode impedance is indeed independent of the amplitude of the input AC signal, it is worth noting that using a higher signal amplitude can be advantageous. This is particularly beneficial when the amplitude of the impedance measurement signal is close to the detection limit of the measurement setup, as this can lead to a lower measurement noise level and an improved signal-to-noise ratio (SNR). Furthermore, the amplitude of the input signal should not be too high, as the system should operate under small signals to extract the impedance parameters. Therefore, a recommended range for the AC signal amplitude is below 100 mV peak-to-peak.

[0031] Figure 8a and Figure 8bThe flowchart summarizes the proposed method for monitoring biochemical interactions and / or sensing biomarkers in biological fluids using an electrochemical impedance system 1, which in this example comprises two electrochemical cells 2. a 2 r That is, the first active electrochemical cell 2 a Second reference electrochemical cell 2 r In this example, the first electrochemical cell forms the first sensor, which is an active sensor, while the second electrochemical cell forms the second sensor, which is a reference sensor. In this example, the first electrochemical cell is controlled by a first control and measurement device 3, and the second electrochemical cell is controlled by a second control and measurement device 3, for controlling the operating conditions of the respective electrochemical cells and measuring the reactions within them. However, it is possible to use a single control and measurement device to control both electrochemical cells.

[0032] In step 11, a biofluid is obtained or collected. The biofluid is at least partially obtained via a needle (in this case, the biofluid may be interstitial fluid or blood), or obtained through excretion (in this case, the biofluid may be sweat, urine, or tears), or produced as a result of a pathological process (in this case, the biofluid may be vesicular fluid). It should be noted that the sensor can be stored in a dry state or immersed in a humid environment prior to the experiment. When wet storage is chosen, it is important to replace the solution completely covering the sensor with the target-specific biofluid.

[0033] In step 12, the obtained or collected biofluid is applied to or added to the first and second electrochemical cells 2. a 2 r In this example, it is necessary to ensure that the biofluid completely covers all electrode surfaces within the electrochemical cell. In step 13, the first and second control and measurement devices 3 apply an AC voltage or current input with a substantially constant frequency to the first and second electrochemical cells, respectively. The applied input signals can be substantially the same for both the first and second electrochemical cells. The AC signal is present throughout the experiment, enabling real-time monitoring of biochemical interactions. In step 14, the impedance values ​​of the first and second sensors at the start of the reaction are determined by the control and measurement devices 3. More specifically, the first control and measurement device determines the initial impedance value of the first sensor, and the second control and measurement device determines the initial impedance value of the second sensor, which is used as an initial reference impedance value. The initial impedance values ​​of the first and second sensors are obtained simultaneously or substantially simultaneously. In step 15, the first electrochemical cell 2... aThe first impedance value is determined as the difference between the impedance values ​​of the first and second sensors at the start of the reaction. More specifically, the initial impedance value of the second sensor is subtracted from the initial impedance value of the first sensor. This determination can be performed by one or both of the control and measurement devices, or by another computing unit (assuming the unit calculating the difference has already received the initial impedance values ​​of the first and second sensors). Thus, the first impedance value of the first electrochemical cell is determined before or at the time of the reaction between the biometric element and one or more target analytes.

[0034] In step 16, a set of intermediate impedance values ​​for the first and second sensors are obtained, and a set of intermediate impedance values ​​for the first electrochemical cell are obtained based on these values ​​to continuously measure the impedance of the first electrochemical cell for real-time monitoring of the biochemical reaction. In other words, the corresponding intermediate impedance value of the first electrochemical cell is obtained as the difference between the corresponding intermediate impedance value of the first sensor and the corresponding intermediate impedance value of the second sensor.

[0035] In step 17, the first and second sensor impedance values ​​are determined by the control and measurement device 3 at the end of the reaction. More specifically, the first control and measurement device determines the first sensor terminal impedance value, and the second control and measurement device determines the second sensor terminal impedance value, which is used as the terminal or final reference impedance value. The first and second sensor terminal impedance values ​​are obtained simultaneously or substantially simultaneously. In step 18, the second impedance value of the first electrochemical cell is determined as the difference between the first and second sensor impedance values ​​at the end of the reaction. More specifically, the second sensor terminal impedance value is subtracted from the first sensor terminal impedance value. This determination can be performed by one or both of the control and measurement devices, or by another calculation unit (assuming the unit calculating the difference has already received the first and second sensor terminal impedance values). The second impedance value of the first electrochemical cell is determined after substantially all or part of the target analyte in the biofluid has reacted with the biorecognition element, or the biofluid has reached equilibrium (in which case the number of idle receptors of the biorecognition element remains substantially constant) or saturation (in which case there is more analyte than the receptors of the biorecognition element can capture), while maintaining a substantially constant input frequency. In this case, the time interval between the start of the reaction (or the moment when the first or initial impedance value is taken) and the end of the reaction (or the moment when the second or final impedance value is taken) defines the length of the experiment, during which the frequency of the input signal remains constant.

[0036] In step 19, the difference between the first and second impedance values ​​is calculated by one or two of the control and measurement devices or by another calculation unit to obtain the impedance change of the first electrochemical cell attributable to the reaction (assuming the unit calculating the impedance change has received the first and second impedance values). In step 20, the concentration of one or more target analytes in the biofluid is derived from the impedance change by one or two of the control and measurement devices or by another calculation unit (assuming the unit calculating the difference has received the impedance change). This is possible assuming the volume or estimated volume of the biofluid in the first electrochemical cell is known. To obtain the concentration of one or more target analytes in the biofluid, a calibration or mapping curve or table is used to map the impedance change value to a specific concentration value. It should be noted that in step 16, one or more intermediate impedance values ​​of the first and second sensors can be measured to obtain the intermediate impedance value of the first electrochemical cell, thereby continuously monitoring how the impedance value of the first electrochemical cell evolves. The above method is also applicable to single-sensor electrochemical impedance systems. If the electrochemical impedance system is a single-sensor system, steps 14 and 17 are not required, and in steps 15 and 18, the first and second impedance values ​​are measured directly without calculating the difference between the first and second sensor impedance values.

[0037] To summarize the above teachings of the present invention, an electrochemical impedance system 1 and method are provided for monitoring biochemical interactions and / or sensing biomarkers in a biological fluid. System 1 includes: at least one electrochemical cell 2 having electrodes configured to contact the biological fluid; and a potentiostat 3 for controlling the operating conditions of the electrochemical cell 2 and for measuring reactions within the electrochemical cell 2. The potentiostat 3 applies an alternating voltage or current input with a constant frequency to the electrochemical cell 2 during at least one experiment and measures the output signal of the electrochemical cell 2, thereby measuring at least the initial impedance value of the electrochemical cell 2 at the start of a reaction occurring in the electrochemical cell 2 and the final impedance value of the electrochemical cell 2 at the end of the reaction, to measure the impedance change of the electrochemical cell 2 attributed to the reaction. The electrochemical impedance system 1 can be configured as a wearable device, in which case the wearable device will be a modular, miniaturized sensing platform that will provide unique health insights (e.g., during critical health events) through continuous and real-time analysis of one or more target analytes (e.g., proteins and hormones) on the skin surface, thereby improving the standard of care in a simple and minimally invasive manner.

[0038] While the invention has been detailed and described in the accompanying drawings and the foregoing description, such description is to be considered illustrative or exemplary, and not restrictive, and the invention is not limited to the disclosed embodiments. Based on a study of the drawings, the disclosure, and the appended claims, those skilled in the art will understand and implement other embodiments and variations in carrying out the claimed invention. Further variations can be obtained by combining the teachings of any of the foregoing designs.

[0039] In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. The fact that different features are referenced in mutually different dependent claims does not imply that combinations of these features cannot be used advantageously. Any reference numerals in the claims should not be construed as limiting the scope of the invention.

Claims

1. A method for monitoring biochemical interactions and / or sensing biomarkers in a biological fluid using an electrochemical impedance system (1), said electrochemical impedance system comprising at least a first electrochemical cell (2, 2... a ) and for controlling the first electrochemical cell (2, 2) a The first control and measuring device (3) is used to measure the operating conditions of the first electrochemical cell (2) and to measure the reaction in the first electrochemical cell (2), the control and measuring device (3) being coupled to the first electrochemical cell (2, 2) including a first working electrode (E1) and a first counter electrode (E2). a Both the first working electrode and the first counter electrode are configured to contact a biofluid comprising one or more target analytes (5), and at least one electrode (E1, E2) is functionalized with a biorecognition element (4) sensitive to the one or more target analytes, the method comprising: To the first electrochemical cell (2, 2) a Apply (13) an AC voltage or current input with a substantially constant frequency; Before or at the time of the reaction between the biometric element (4) and the one or more target analytes, determine (15) the first electrochemical cell (2, 2) a The first impedance value; Determine (18) the first electrochemical cell (2, 2) a The second impedance value of ) while maintaining the substantially constant frequency after substantially all or one of the target analytes in the biofluid have reacted with the biorecognition element (4), or after the biofluid has reached equilibrium or saturation; The first electrochemical cell (2, 2) attributable to the reaction can be calculated from the first and second impedance values ​​(19). a The impedance change; and The concentration of one or more target analytes (5) in the biofluid is derived from the impedance change (20).

2. The method according to claim 1, wherein, The reaction is an electrochemical reaction, and / or the biorecognition element (4) is specific to the one or more target analytes (5), and / or wherein the one or more target analytes are molecular biomarkers, such as proteins, hormones, metabolites, nucleic acids and / or whole cells.

3. The method according to claim 1 or 2, wherein, The frequency of the electrochemical impedance system (1) is only between 1 mHz and 1 kHz for the capacitor response, between 1 kHz and 100 kHz for both capacitor and resistor responses, or only greater than 100 kHz for the resistor response.

4. The method according to any one of the preceding claims, wherein, The biofluid is at least partially obtained through needles, or through excretion, or is produced as a result of a pathological process.

5. The method according to any one of the preceding claims, wherein, The first and second impedance values ​​are real impedance values, or virtual impedance values, or a combination of real impedance values ​​and virtual impedance values.

6. The method according to any one of the preceding claims, wherein, The first working electrode (E1) and the first counter electrode (E2) in the first electrochemical cell (2), together with an optional first reference electrode (E3), form a first sensor sensitive to the one or more target analytes. The electrochemical impedance system (1) further includes a second sensor insensitive to the one or more target analytes. The method further includes: At the start of the reaction, the impedance values ​​of the first and second sensors are determined (14), wherein the first impedance value of the first electrochemical cell is obtained as the difference between the impedance values ​​of the first and second sensors at the start of the reaction; and At the end of the reaction, the impedance values ​​of the first and second sensors are determined (17), wherein the second impedance value of the first electrochemical cell is obtained as the difference between the impedance values ​​of the first and second sensors at the end of the reaction.

7. The method according to claim 6, wherein, The first electrochemical cell (2) further includes a second working electrode (E1r) and an optional first reference electrode (E3), wherein the second sensor includes the second working electrode (E1r), the first counter electrode (E2), and the optional first reference electrode (E3).

8. The method according to claim 6, wherein, The electrochemical impedance system (1) also includes components connected to the first electrochemical cell (2). a The second electrochemical cell (2) separated r ), wherein the second electrochemical cell (2 r The second sensor includes a second working electrode (E1r), a second reverse electrode (E2r), and an optional second reference electrode (E3r).

9. An electrochemical impedance system (1) for monitoring biochemical interactions and / or sensing biomarkers in biological fluids, said electrochemical impedance system (1) comprising: First electrochemical cell (2, 2) a The device includes a first working electrode (E1) and a first counter electrode (E2), both configured to contact a biological fluid, and at least one of the first working electrode (E1) and the first counter electrode (E2) is functionalized by a biorecognition element (4) for capturing one or more target analytes (5) in the biological fluid; and First control and measurement device (3), used to control the first electrochemical cell (2, 2) a The operating conditions of the first electrochemical cell (2, 2) were used to measure the operating conditions of the first electrochemical cell (2, 2). a The reaction in the control and measurement device (3) is coupled to the first electrochemical cell (2, 2) a The control and measuring device (3) is configured to apply an AC voltage or current input with a substantially constant frequency to one of the first working electrode (E1) and the first counter electrode (E2) during at least one experiment. The control and measuring device (3) is also configured to measure the output signal at the other of the first working electrode (E1) and the first counter electrode (E2), thereby measuring at least the reaction start of the first electrochemical cell (2, 2) between the biorecognition element (4) and the one or more target analytes (5). a The initial impedance value of the first electrochemical cell (2, 2) and the initial impedance value of the first electrochemical cell at the end of the reaction. a The terminal impedance value of the first electrochemical cell (2, 2) was measured to measure the terminal impedance value of the first electrochemical cell (2, 2). a The impedance change is attributed to the impedance change in the first electrochemical cell (2, 2). a The reaction that occurs in ).

10. The electrochemical impedance system (1) according to claim 9, wherein, The electrochemical impedance system (1) is a single electrochemical cell system, and wherein the first electrochemical cell (2, 2) a Optionally includes a first reference electrode (E3).

11. The electrochemical impedance system (1) according to claim 9 or 10, wherein, The first electrochemical cell (2, 2) a The system also includes a second working electrode (E1r), wherein the first working electrode (E1), the first counter electrode (E2), and optionally the first reference electrode (E3) together form a first sensor sensitive to the one or more target analytes (5), and the second working electrode (E1r), the first counter electrode (E2), and optionally the first reference electrode (E3) together form a second sensor insensitive to the target analytes, wherein the first electrochemical cell (2, 2) is obtained. a The corresponding impedance value of the first sensor is used as the difference between the corresponding impedance value of the first sensor and the corresponding impedance value of the second sensor.

12. The electrochemical impedance system (1) according to claim 9, wherein, The electrochemical impedance system (1) also includes a second electrochemical cell (2) r The second electrochemical cell includes a second working electrode (E1r) and a second counter electrode (E2r), wherein at least the first working electrode (E1) and the first counter electrode (E2) together form a first sensor sensitive to one or more target analytes (5), and at least the second working electrode (E1r) and the second counter electrode (E2r) together form a second sensor insensitive to the one or more target analytes (5), and wherein the first electrochemical cell (2, 2) is obtained. a The corresponding impedance value of the first sensor is used as the difference between the corresponding impedance value of the first sensor and the corresponding impedance value of the second sensor.

13. The electrochemical impedance system (1) according to claim 12, wherein, The first electrochemical cell (2) a It also includes a first reference electrode (E3), and the second electrochemical cell (2) r The sensor also includes a second reference electrode (E3r), wherein the first working electrode (E1), the first reverse electrode (E2), and the first reference electrode (E3) together form a first sensor that is sensitive to the one or more target analytes (5), and the second working electrode (E1r), the second reverse electrode (E2r), and the second reference electrode (E3r) together form a second sensor that is not sensitive to the one or more target analytes (5).

14. The electrochemical impedance system (1) according to any one of claims 9 to 13, wherein, The biometric element (4) includes or is composed of biological materials, or the biometric element (4) includes or is composed of biologically derived materials, or the biometric element (4) includes or is composed of biomimetic materials, or the biometric element (4) includes or is composed of any combination of biological materials, biologically derived materials and / or biomimetic materials.

15. The electrochemical impedance system (1) according to claim 14, wherein, The biomaterials include one or more antibodies, enzymes, nucleic acids and / or cell receptors, the bio-derived materials include one or more aptamers and / or recombinant antibodies, or the biomimetic materials include one or more molecularly imprinted polymers and / or synthetic catalysts.

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