Nanoprobe-based electrophysiological signal and biochemical marker detection system

Abstract

This application provides a nanoprobe-based electrophysiological signal and biochemical biomarker detection system, comprising: a patch-clamp amplifier; a working electrode electrically connected to a signal acquisition module, the surface of which, except for the tip, is covered by an insulating protective layer, forming a GΩ-level impedance seal after implantation into the cell; a counter electrode connected to a BATH port, with its active end placed in the matrix environment of the target cell, forming a current loop; a reference electrode connected to a REF port, with its active end placed in the matrix environment of the target cell to provide a potential reference point; the patch-clamp amplifier is configured to operate in voltage-clamp mode, with the REF port connected to the reference electrode as a voltage sensing terminal, and the BATH port connected to the counter electrode as a current output terminal, simultaneously acquiring the electrophysiological signal of the target cell and the current signal corresponding to the concentration of the target biochemical biomarker. This application provides a powerful analytical tool for a deeper understanding of the electrochemical signal coupling mechanism of neurons.

Description

Technical Field

[0001] This application relates to the field of microelectromechanical systems, and more specifically, to a detection system for electrophysiological signals and biochemical biomarkers based on nanoprobes. Background Technology

[0002] Precise detection of electrophysiological signals is central to revealing cellular function and disease mechanisms. Understanding cellular function relies on accurately elucidating the coupling between its electrochemical activity and the secretion of biochemical substances. To study the relationship between cell membrane ion channels and cellular electrophysiological activity, German cell physiologists Evan Neher and Bert Sackmann jointly invented the patch-clamp technique in the 1970s. This technique, by using glass microelectrodes to form a high-impedance seal with the cell membrane, enables the recording and even control of electrophysiological signals such as ion currents and action potentials of individual ion channels or the entire cell with high spatiotemporal resolution, achieving picoampere-level currents and millisecond-level time intervals.

[0003] On the other hand, in addition to the emission of its own electrophysiological signals, the functioning of cells also involves biochemical changes such as neurotransmitter release, hormone secretion, and metabolic product exchange. To detect these cellular biochemical signals, various cellular electrochemical techniques, such as cyclic voltammetry and amperometrics, have been applied and developed. For example, the small size, high sensitivity, and rapid response of conductive electrodes such as carbon fiber microelectrodes are utilized to detect the content and changes of biochemical substances released by cells in real time.

[0004] However, traditional patch-clamp systems typically use nanoparticles for intracellular patch-clamp detection. The nanoparticles are often filled with very high concentrations of KCl solution to minimize total resistance and reduce the filtering effect. A fundamental limitation of this approach is that its signal transduction depends entirely on the ionic current of the high-concentration liquid filling the nanoparticle. Therefore, this approach can only record electrophysiological signals and cannot detect the content and changes of biochemical substances released by cells through Faraday current (i.e., redox current), thus limiting cell research.

[0005] Furthermore, in existing technologies, electrophysiological recording and biochemical detection systems are typically built independently. Electrophysiological signal recording and biochemical signal detection require the separate operation of traditional patch-clamp systems and electrochemical workstations. To understand the electro-chemical signal coupling mechanism of neurons, it is necessary to record both types of cellular signals. Current techniques require implanting the working electrodes of two independent systems sequentially into the same target cell. This process is cumbersome and extremely difficult to coordinate spatially, making it highly susceptible to sealing failure or cell damage due to mechanical interference. It also results in long experimental preparation times, significantly impacts cell viability, and has a very low success rate. In addition, the high-current pulse injected by the traditional patch-clamp system and the scanning voltage applied by the electrochemical system may affect the intracellular fluid environment, thus interfering with the accurate recording of normal cellular signals and reducing data reliability and signal-to-noise ratio.

[0006] A search revealed that the patent application with publication number CN114002131A discloses a device comprising: a detection electrode for detecting electrical signals, including an electrophysiological electrode and an electrochemical electrode; a patch-clamp amplifier for amplifying electrical signals, including an electrophysiological detection unit connected to the electrophysiological electrode and an electrochemical detection unit connected to the electrochemical electrode; a filtering module for filtering electrical signals, including a first band-stop filter, a second band-stop filter, and a low-pass filter; and a control and analysis module for analyzing the electrophysiological and electrochemical conditions of cells based on electrical signals, and for sending control signals to the patch-clamp amplifier to adjust the detection mode of the patch-clamp amplifier. The essence of this prior art is a simple collaboration of two systems, which has the following problems: (1) the equipment structure is complex and occupies a large space; (2) the operation process is cumbersome, requiring separate control of the two systems; (3) it is difficult to achieve precise synchronization of the two signals in time, affecting the reliability of data correlation analysis; and (4) there may be electromagnetic interference or signal crosstalk between the systems, affecting the detection accuracy.

[0007] Therefore, there is an urgent need in this field for a novel device that is highly integrated, easy to operate, has accurate signal synchronization and strong anti-interference capabilities, and can perform controllable research that seamlessly integrates electrophysiological signal recording and cellular biochemical signal detection functions. This would enable seamless switching between real-time cellular physiological signal recording and cellular biochemical signal recording modes, providing a powerful analytical tool for a deeper understanding of the electro-chemical signal coupling mechanism of neurons. Summary of the Invention

[0008] In view of the deficiencies in the prior art, the purpose of this application is to provide a detection system for electrophysiological signals and biochemical biomarkers based on nanoprobes.

[0009] This application provides a detection system for electrophysiological signals and biochemical biomarkers based on nanoprobes, characterized in that it includes: The patch clamp amplifier is equipped with a signal acquisition module, a REF port, a BATH port, and a signal input port; The working electrode is located on the micro-displacement stage and is electrically connected to the signal acquisition module. It also serves as an electrophysiological recording electrode and an electrochemical sensing electrode for the target biochemical marker. The working electrode is a nanoprobe with a nano-diameter tip for implantation into the cell to be tested. The surface of the working electrode, except for the tip, is covered by an insulating protective layer, so that the working electrode forms a GΩ-level impedance seal after implantation into the cell. The counter electrode is connected to the BATH port, and the active end of the counter electrode is placed in the matrix environment where the cell to be tested is located, forming a current loop; A reference electrode is connected to the REF port, and the active end of the reference electrode is placed in the matrix environment where the test cell is located to provide a potential reference point. The patch-clamp amplifier is configured to operate in voltage clamp mode, such that the REF port is connected to the reference electrode to become a voltage sensing terminal, and the BATH port is connected to the counter electrode to become a current output terminal, thereby simultaneously acquiring the electrophysiological signal of the cell under test and the current signal corresponding to the concentration of the target biochemical marker.

[0010] Optionally, the working electrode surface is provided with a conductive layer and a functional layer in sequence, the outer surface of the functional layer is provided with a lead wire connected to the signal acquisition module, and the insulating protective layer is provided on the functional layer in the area other than the tip.

[0011] Optionally, the system has at least one of the following features: - The material of the conductive layer is any one of chromium, gold, silver, nickel, titanium, platinum, aluminum, iridium, carbon nanotubes, graphene, graphite, polypyrrole, alginate and conductive hydrogel; - The material of the functional layer is any one of copper selenide, cobalt copper oxide, platinum, gold, terbium with dual ligands, and enzymes; - The material of the insulating protective layer is any one of paraffin wax, alumina, silicon dioxide, silicon carbide, poly(p-xylene), and phthalates.

[0012] Optionally, the working electrode is made of any one of metallic materials, semiconductor materials, alloy materials, and non-metallic materials.

[0013] Optionally, the coating thickness of the working electrode is 0.1 nm-1 mm, and the impedance is 100 MΩ-10 GΩ.

[0014] Optionally, the working electrode has a length of 10 μm-20 cm and a tip diameter of 1 nm-1 μm.

[0015] Optionally, the system has an input voltage of -800~1000 mV, an input current of -800~1000 pA, a voltage detection range of -20000~20000 mV, and a current detection range of -20000~20000 pA.

[0016] Optionally, the counter electrode is any one of a ring electrode, a wire electrode, and a sheet electrode.

[0017] Optionally, the reference electrode is made of any one of silver / silver chloride, saturated calomel, mercurous sulfate, and mercuric oxide.

[0018] Optionally, the electrophysiological signal includes at least one of action potential signal, postsynaptic potential signal, and postsynaptic current signal; the biochemical marker includes at least one of the neurotransmitter dopamine signal secreted by vesicles, ascorbic acid content signal, uric acid signal, and pH value signal.

[0019] The nanoprobe-based electrophysiological signal and biochemical biomarker detection system provided in this application, through the selective exposure of the nanoscale tip of the working electrode and the design of a full-circumference insulation protection structure, can stably form a GΩ-level high-resistance seal after implantation into the cell. This not only meets the stringent requirements of patch clamp for electrical isolation and mechanical stability, but also ensures that the electrochemical response originates only from the tip active region, significantly improving the spatial resolution of electrochemical detection. By connecting the counter electrode and the reference electrode to the inherent ports of the patch clamp amplifier and redefining their electrical roles in voltage clamp mode, a closed-loop three-electrode measurement system is constructed, which significantly reduces system complexity, eliminates synchronization errors between multiple devices, and achieves for the first time a single system simultaneously records electrophysiological signals and biochemical biomarkers of single-cell activity. It can achieve true synchronous, in-situ, and high-fidelity acquisition of electrophysiological signals and biochemical biomarkers at the single-cell scale, providing a powerful analytical tool for a deeper understanding of the electro-chemical signal coupling mechanism of neurons.

[0020] Other technical effects resulting from the additional features will be further illustrated in the corresponding embodiments. Attached Figure Description

[0021] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of the structure of a nanoprobe-based electrophysiological signal and biochemical marker detection system according to an exemplary embodiment. Figure 2 The circuit diagram illustrates an exemplary embodiment of a nanoprobe-based electrophysiological signal and biochemical marker detection system. Figure 3As shown in an exemplary embodiment: (a) a side view of the working electrode, and (b) a structural diagram of the working electrode tip. Figure 4 This is a schematic diagram illustrating the workflow of a nanoprobe-based electrophysiological signal and biochemical biomarker detection system according to an exemplary embodiment. Figure 5 The image shows the recording results (a) of induced and spontaneous postsynaptic currents in nerve cells in Example 3, and a magnified view (b) of the corresponding part of (a).

[0022] In the figure: 1 is the working electrode, 101 is the electrode material layer, 102 is the conductive layer, 103 is the functional layer, 104 is the insulating protective layer, 2 is the micro-displacement stage clamping device, 3 is the patch clamp amplifier, 4 is the microscope, 5 is the counter electrode, 6 is the matrix environment, 7 is the reference electrode, and 8 is the display. Detailed Implementation

[0023] The present application will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present application, but do not limit the present application in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application, and these all fall within the protection scope of the present application. Parts not described in detail in the following embodiments can be implemented using existing technology.

[0024] In existing technologies, electrophysiological recording and biochemical biomarker detection have long relied on independent hardware systems. This results in a lack of strict synchronization between the two on the timeline, difficulty in precise spatial localization within the same subcellular structure, and a lack of unified calibration benchmarks in signal pathways. In particular, it is impossible to simultaneously maintain high-resistance sealing and specific electrochemical responses in the intracellular microenvironment of a single cell. This fundamental disconnect severely restricts a deeper understanding of the dynamic mechanisms of neuronal electrochemical coupling. Based on the above problems, this application provides an electrophysiological signal and biochemical biomarker detection system based on nanoprobes to address these issues.

[0025] Reference Figure 1In one embodiment of this application, an electrophysiological signal and biochemical biomarker detection system based on nanoprobes includes a patch-clamp amplifier 3, a working electrode 1, a counter electrode 5, and a reference electrode 7. The patch-clamp amplifier 3 is equipped with a signal acquisition module, a REF port, and a BATH port. The working electrode 1 is disposed on a micro-displacement stage and is electrically connected to the signal acquisition module, serving simultaneously as an electrophysiological recording electrode and an electrochemical sensing electrode for the target biochemical biomarker. The working electrode 1 is a nanoprobe with a nanometer-diameter tip for implantation into the target cell. The surface of the working electrode, except for the tip, is covered by an insulating protective layer 104, ensuring the working electrode 1 is securely implanted. After entering the cell, a GΩ-level impedance seal is formed; the counter electrode 5 is connected to the BATH port, and the active end of the counter electrode 5 is placed in the matrix environment 6 where the test cell is located, forming a current loop; the reference electrode 7 is connected to the REF port, and the active end of the reference electrode 7 is placed in the matrix environment 6 where the test cell is located, to provide a stable and constant potential reference point; the operating mode of the patch clamp amplifier 3 is configured as voltage clamp mode, so that the REF port is connected to the reference electrode 7 to become a voltage sensing terminal, and the BATH port is connected to the counter electrode 5 to become a current output terminal, thereby simultaneously acquiring the electrophysiological signal of the test cell and the current signal corresponding to the concentration of the target biochemical marker.

[0026] Specifically, a micro-displacement stage is provided with a micro-displacement stage clamping device 2, and the working electrode 1 is fixed on the micro-displacement stage clamping device 2. The micro-displacement stage has a displacement adjustment function. Under the optical positioning guidance of the microscope 4, the micro-displacement stage drives the working electrode 1 to move and implant the tip into the target cell to be tested.

[0027] The REF port of the patch-clamp amplifier 3 refers to the reference electrode port, which provides a stable reference point relative to ground for the amplifier's input stage. The BATH port refers to the bath / ground port, which connects the bath potential to the system ground, providing a current loop between the recording electrode and the reference electrode 7, and stabilizing the bath potential and suppressing noise. The working electrode 1, the counter electrode 5, and the reference electrode 7 are respectively connected to the signal acquisition module, the BATH port, and the REF port of the patch-clamp amplifier 3, forming a three-electrode system. This system enables multimodal acquisition of single-cell electrophysiological signals (based on the original patch-clamp system) and biochemical signals (based on the newly established three-electrode system), and records data through the display 8.

[0028] The overall circuit structure of the system is referenced. Figure 2 As shown, the interfacial electrochemical behavior at the probe tip can be divided into two typical processes: Faraday processes and non-Faraday processes. Probe resistance. R p The main contributions of charge transfer resistance and mass transfer resistance to the Faraday process are as follows: C pThe equivalent double-layer capacitance is formed at the electrode / solution interface to create a double-layer structure. R s V is the solution resistance that reflects the energy loss caused by impeded ion migration in the bulk solution. m This represents the transmembrane potential, GND represents the ground terminal, and V p For recording intracellular signals, R c Represents the resistance of the cell culture medium. C m 、R m These represent the membrane capacitance and membrane resistance of the cell membrane, respectively. R seal Represents the sealing resistance, due to the diffusion potential. V d and the reverse potential of the leakage current path E m Under solution grounding conditions, this current can be approximated as zero. Therefore, according to Ohm's law, the current involved in intracellular recording is extremely small (typically on the order of pA). This current is at the electrode tip (see electrode tip structure for reference). Figure 3 The potential drop Δ generated on (as shown) V The potential is negligible, thus the electrode potential remains relatively stable. Under these conditions, reference electrodes such as Ag / AgCl are sufficient to serve as dummy reference electrodes, and their potential drift is negligible. Due to the extremely small current, the ion concentration on the surface of reference electrode 7 (e.g., ...) is negligible. Cl The change in concentration is negligible, therefore the potential drift Δ E Approximately zero. Furthermore, by compensating for the liquid junction potential through the potential-clamping function (clamping voltage / clamping current) of the patch clamp, it successfully integrates two detection modes: the patch clamp method for electrophysiological acquisition and the single-cell amperometric method for electrochemical detection. This makes it possible to simultaneously record ion currents and Faraday currents using an all-solid-state connection method. Thus, a multimodal acquisition system based on nanoprobe electrodes that is compatible with both electrophysiological signals and biochemical biomarkers is obtained.

[0029] This system can simultaneously detect electrophysiological signals and biochemical markers. Essentially, when the system voltage is clamped, a redox reaction occurs on the surface of working electrode 1, generating a Faraday current, thereby obtaining the analyte / electrophysiological signal. Taking a platinum electrode clamped at 70mV as an example, the system can simultaneously record the action potential of nerve cells (electrophysiological signal) and the ascorbic acid content signal (electrochemical signal), thus enabling the simultaneous recording of both signals and achieving detection. Therefore, if the electrophysiological signal and the electrochemical analyte are within the sensitive response range of working electrode 1 under the same clamping voltage, simultaneous detection can be achieved, and the corresponding signal can be analyzed based on the signal waveform.

[0030] The multimodal signal acquisition system of this application embodiment can achieve highly integrated, simplified operation, accurate signal synchronization and strong anti-interference capability compatible detection of single-cell electrophysiological signals and biochemical markers, and can achieve stable recording in somatic cells. It provides a powerful analytical tool for a deeper understanding of the electro-chemical signal coupling mechanism of neurons. This means that the brain can be interpreted with unprecedented precision, which helps to promote the development of neuroscience.

[0031] To balance the precision required for high-resistivity sealing and the interface controllability required for electrochemical sensing, in some specific embodiments of this application, reference is made to... Figure 3 The electrode material layer 101 of the working electrode 1 is provided with a conductive layer 102 and a functional layer 103 in sequence. The outer surface of the functional layer 103 is provided with a lead wire connected to the signal acquisition module. The insulating protective layer 104 is provided on the functional layer 103 except for the tip.

[0032] By precisely exposing the GΩ-level impedance at the nanometer-diameter tip and providing external insulation protection, the working electrode 1 achieves high selectivity and sensitivity to target molecules, matching the amplification design of the patch-clamp system for high-resistance sealing. The core of the patch-clamp system is an extremely sensitive current-to-voltage converter. To achieve and record weak ion channel currents (picoampere-pA level) typically greater than 1 GΩ or even reaching 10-100 GΩ in high-resistance sealing, the amplifier's amplification design cannot be singular; instead, a dynamic multi-stage switching strategy must be employed. The core of this strategy is to maintain a strict match with the instantaneous state of the "microelectrode-cell" interface in terms of impedance, noise, and capacitance by switching the feedback resistor.

[0033] The working electrode is designed according to the detection targets of electrophysiological and biochemical signals. In some specific embodiments of this application, the conductive layer 102 is made of any one of chromium, gold, silver, nickel, titanium, platinum, aluminum, iridium, carbon nanotubes, graphene, graphite, polypyrrole, alginate, and conductive hydrogel. The functional layer 103 is made of a material sensitive to one of ions, molecules, and organic matter, and may include any one of copper selenide, cobalt copper oxide, platinum, gold, terbium with dual ligands, and enzymes. The methods for forming the functional layer on the surface of the working electrode include, but are not limited to, material sputtering, atomic layer deposition, chemical vapor deposition, physical vapor deposition, electrochemical deposition, and electrolysis. The insulating protective layer 104 is made of any one of paraffin wax, alumina, silicon dioxide, silicon carbide, poly(p-xylene), and phthalates.

[0034] In some specific embodiments of this application, the material of the working electrode 1 (electrode material layer 101) is any one of metal material, semiconductor material, alloy material and non-metal material.

[0035] For example, metallic materials include, but are not limited to, tungsten, platinum, iridium, copper, or tungsten steel; semiconductor materials include, but are not limited to, silicon, silicon nitride, carbon fiber, silicon carbide, gallium nitride; alloy materials include, but are not limited to, platinum-iridium alloy, stainless steel; and non-metallic materials include, but are not limited to, quartz, borosilicate glass, etc.

[0036] It should be noted that the specific materials of the working electrode itself, and the selection of the conductive layer 102 and functional layer 103 coatings, depend on the type of cells to be recorded and the target electrophysiological or biochemical signals. For example, for high-intensity cell puncture, a tungsten metal electrode with extremely high mechanical strength can be selected; for recording cellular electrophysiological signals and dopamine signals, a sputtered platinum coating can be selected; for recording changes in cellular pH, a sputtered iridium oxide coating can be selected, etc.

[0037] The embodiments described above in this application are based on the target biochemical marker, using specific materials sensitive to the target biochemical substance, and external insulation protection is achieved through multi-step microprocessing. The working electrode with a precisely exposed tip forms a pure solid-state electrical connection with the signal acquisition module. After being implanted into the target area to be tested inside the cell, a high-resistance seal at the level of nearly GΩ is formed, which can ensure the fidelity of signal recording.

[0038] In some specific embodiments of this application, the coating thickness of the working electrode is 0.1 nm-1 mm, and the impedance is 100 MΩ-10 GΩ.

[0039] It should be noted that the coating thickness is the combined thickness of the conductive layer 102, the functional layer 103, the protective layer, etc. This application embodiment does not utilize the core function of patch clamp, namely high-resistance sealing, but achieves a similar effect through modification of the working electrode. This allows for signal acquisition using patch clamp and the clamping of voltage / current, and the acquisition of biochemical marker signals can be achieved through reactions generated by electrode modification.

[0040] In some specific embodiments of this application, the working electrode 1 has a length of 10 μm-20 cm and a tip diameter of 1 nm-1 μm to meet the requirements for intracellular implantation.

[0041] In some specific embodiments of this application, the system input voltage is -800~1000 mV, the input current is -800~1000 pA, the voltage detection range is -20000~20000 mV, and the current detection range is -20000~20000 pA.

[0042] In some specific embodiments of this application, the counter electrode 5 is any one of a ring electrode, a wire electrode, and a sheet electrode, and its material includes, but is not limited to, platinum, stainless steel, copper, and carbon materials such as graphite. During the detection process, the counter electrode 5 is uniformly placed in the matrix environment 6 where the cells are located.

[0043] In some specific embodiments of this application, the material of the reference electrode 7 is any one of silver / silver chloride, saturated calomel, mercurous sulfate, and mercuric oxide.

[0044] In some specific embodiments of this application, the electrophysiological signal includes at least one of action potential signal, postsynaptic potential signal, and postsynaptic current signal; the biochemical marker includes at least one of the neurotransmitter dopamine signal secreted by vesicles, ascorbic acid content signal, uric acid signal, and pH value signal.

[0045] In this embodiment, the simultaneous detection range of electrophysiological signals and biochemical markers includes, but is not limited to, in vitro extracellular, in vitro intracellular, in vivo extracellular, and in vivo intracellular measurements. It can simultaneously collect intracellular electrophysiological signals and biochemical markers down to the subcellular scale to study the coupling mechanism of "electrical signals-chemical signals" in life activities.

[0046] Reference Figure 4 The working process of the system in the above embodiments of this application is as follows: Step 1, System Electrode and Sample Preparation: Provide the target sample to be tested, and prepare specific functionalized working electrodes based on the target biochemical markers; Step 2: Connect to form a multimodal acquisition system: The working electrode is connected to the signal acquisition module of the patch-clamp amplifier to form a pure solid-state electrical connection, the counter electrode is connected to the BATH port, and the reference electrode is connected to the REF port; Step 3, Spatial localization and intracellular implantation of the target cell: The target cell is located using a microscope, and the tip of the working electrode is implanted into the cell; the end of the counter electrode is placed in the matrix environment where the target cell is located to form a current loop; the end of the reference electrode is placed in the matrix environment where the target cell is located to provide a potential reference point. Step 4, Potential clamping and sensing: The diaphragm clamp amplifier is set to voltage clamp mode. The BATH port is connected to the counter electrode to become the current output terminal, and the REF port is connected to the reference electrode to become the voltage sensing terminal. Step 5: Applying stimulation, cell response and data output acquisition: After stimulation is applied, the cell responds electrically and releases chemicals, generating an electric current, and the cell signal is recorded.

[0047] The embodiments described above in this application, by electrically connecting the working electrode to the signal acquisition module, connecting the BATH port of the patch-clamp device to the counter electrode, placing the counter electrode in the matrix environment where the cell resides to form a current loop, and connecting the REF port to the reference electrode with its end submerged in the matrix environment where the cell resides, provide a stable and unchanging potential reference point. This enables seamless integration of intracellular electrophysiological signal recording and biochemical marker detection functions, featuring high integration, simplified operation, accurate signal synchronization, and strong anti-interference capabilities. The embodiments described above in this application can achieve simultaneous recording of electrophysiological and biochemical signals of cellular activity in a single system, integrating two independent and difficult-to-coordinate complex systems traditionally used for studying cellular electrophysiological activity and biochemical secretion processes into a simple, stable, and reliable hardware platform. This provides a physical intersection for interdisciplinary research in electrophysiology and biochemistry, which helps promote basic research in multiple fields such as neuroscience, cardiovascular research, endocrinology, and immunology, lowers the technical threshold for cutting-edge scientific discoveries, and is expected to become a fundamental tool for many important future discoveries in related fields.

[0048] Compared to existing technologies (such as CN114002131A), the embodiments of this application provide a novel device that is highly integrated, simplified in operation, provides accurate signal synchronization, and has strong anti-interference capabilities. It is a controllable research system capable of seamlessly integrating electrophysiological signal recording and cellular biochemical signal detection functions. Single-system control reduces operational complexity, and the integrated structure avoids mutual interference between systems, improving detection reliability. It truly achieves simultaneous, same-point, and same-interface recording of electrophysiological and cellular biochemical signals, providing a more complete perspective for cell function research. Secondly, the electrophysiological signal detection principle of CN114002131A utilizes patch-clamp technology, employing a liquid-filled glass tube for current conduction. This method lacks protection and cannot maintain long-term stability. In contrast, the embodiments of this application employ a purely solid-state connection method, maintaining stability over longer sampling times. The embodiments of this application avoid potential electromagnetic interference or signal crosstalk between systems, thereby improving detection accuracy.

[0049] The preferred features in the above embodiments can be used individually in any embodiment, or in any combination thereof, provided they do not conflict with each other. Furthermore, parts not described in detail in the embodiments can be implemented using existing technologies.

[0050] The following examples and comparative examples will be used to further illustrate this application in order to better understand the above-mentioned technical solutions. It should be understood that the following are only some examples and are not intended to limit this application.

[0051] Application Example 1 This application example provides a multimodal acquisition system based on nanoprobe electrodes that is compatible with the detection of electrophysiological signals and biochemical biomarkers. The steps are as follows: S1. A 5nm titanium layer and a 50nm platinum layer are sputtered onto the surface of the glass electrode, connected by leads, and a 30nm alumina layer is deposited using atomic layer deposition. The working electrode tip has a diameter of approximately 200nm, and the conductive functional layer at the tip is exposed by ion beam etching. The working electrode is fixed to a micro-displacement stage, and the leads form a purely solid-state electrical connection with the signal acquisition module. The tip is then implanted into the target cell to be tested.

[0052] S2, connect the platinum wire to the BATH port of the patch-clamp amplifier, and place the end in the matrix environment where the cell to be tested is located to form a current loop; S3 connects the silver / silver chloride wire to the REF port of the patch-clamp amplifier, with the end submerged in the matrix environment where the target cell is located, providing a stable and unchanging potential reference point; S4, adjust the clamping voltage applied by the multimodal acquisition system to 600mV, and record the dopamine release content of nerve cells.

[0053] It should be noted that the clamping voltage is primarily based on the sensitive potential between the functional layer material on the working electrode surface and the target analyte. This sensitive potential is the characteristic voltage at which the functional layer material produces a specific electrochemical response to the target analyte. Only when this potential is approached or reached can the target analyte undergo a redox reaction on the electrode surface, thereby generating a recordable Faraday current signal. Taking the detection of dopamine with a platinum metal working electrode as an example, the sensitive potential of dopamine is approximately 600 mV (vs. Ag / AgCl). At this clamping voltage, dopamine undergoes a redox reaction on the electrode surface, generating a measurable Faraday current, thus enabling the quantitative detection of dopamine concentration.

[0054] Application Example 2 This application example provides a multimodal acquisition system based on nanoprobe electrodes that is compatible with the detection of electrophysiological signals and biochemical biomarkers. The steps are as follows: S1. A 5nm titanium layer and a 50nm gold layer are deposited on the surface of a glass electrode, connected with leads, oxygen is introduced, and metallic iridium is sputtered to form a 100nm iridium oxide functional layer. The working electrode tip has a diameter of approximately 400nm, and its surface is insulated with paraffin wax so that only the tip is exposed. The working electrode is fixed to a micro-displacement stage, and the leads form a purely solid-state electrical connection with the signal acquisition module. The tip is then implanted into the target cell to be tested.

[0055] S2, connect the graphite rod to the BATH port of the patch clamp amplifier, and place the end in the matrix environment where the cell to be tested is located to form a current loop; S3 connects the silver / silver chloride wire to the REF port of the patch-clamp amplifier, with the end submerged in the matrix environment where the target cell is located, providing a stable and unchanging potential reference point; S4, adjust the clamping voltage applied by the multimodal acquisition system to 0mV and change the solution environment to record changes in cell pH.

[0056] Application Example 3 This application example provides a multimodal acquisition system based on nanoprobe electrodes that is compatible with the detection of electrophysiological signals and biochemical biomarkers, which is implemented according to the following steps: S1. A 5nm titanium layer and a 50nm gold layer are sputtered onto the surface of a tungsten electrode, connected by leads, and the surface is coated with phthalate so that only the tip is exposed. The working electrode tip has a diameter of approximately 400nm. The working electrode is fixed to a micro-displacement stage, and the leads form a pure solid-state electrical connection with the signal acquisition module. The tip is then implanted into the target cell to be tested.

[0057] S2, connect the platinum wire to the BATH port of the patch-clamp amplifier, and place the end in the matrix environment where the cell to be tested is located to form a current loop; S3 connects the silver / silver chloride wire to the REF port of the patch-clamp amplifier, with the end submerged in the matrix environment where the target cell is located, providing a stable and unchanging potential reference point; S4, adjust the clamping voltage applied by the multimodal acquisition system to 0mV, and record the induced postsynaptic current and spontaneous postsynaptic current of nerve cells.

[0058] from Figure 5 The results show completely different waveforms, which are generated by different physiological processes. The downward spike with a large amplitude and obvious width in the figure is an evoked postsynaptic current (ePSC) induced by the firing (action potential) of the upstream neuron. After that, there is a relatively slow decay process to restore the baseline, which is the result of the synchronous release of multiple synaptic vesicles. The regular small current in the figure is a spontaneous spontaneous postsynaptic current (sPSC).

[0059] In the description of the embodiments of this application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0060] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature.

[0061] In the description of the embodiments in this application, "multiple" means two or more, unless otherwise explicitly specified. In this application, unless otherwise explicitly specified and limited, the terms "installed," "connected," "linked," "fixed," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0062] The terms "comprising" and "having," and any variations thereof, in the embodiments of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such processes, methods, products, or devices.

[0063] The foregoing has described some specific embodiments of this application. It should be understood that this application is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the substantive content of this application. The above-described preferred features can be used in any combination without conflict.

Claims

1. A detection system for electrophysiological signals and biochemical biomarkers based on nanoprobes, characterized in that, include: The patch clamp amplifier is equipped with a signal acquisition module, a REF port, a BATH port, and a signal input port; The working electrode is located on the micro-displacement stage and is electrically connected to the signal acquisition module. It also serves as an electrophysiological recording electrode and an electrochemical sensing electrode for the target biochemical biomarker. The working electrode is a nanoprobe with a nano-diameter tip for implantation into the cell to be tested. The surface of the working electrode, except for the tip, is covered by an insulating protective layer, so that the working electrode forms a GΩ-level impedance seal after implantation into the cell. The counter electrode is connected to the BATH port, and the active end of the counter electrode is placed in the matrix environment where the cell to be tested is located, forming a current loop; A reference electrode is connected to the REF port, and the active end of the reference electrode is placed in the matrix environment where the test cell is located to provide a potential reference point. The patch-clamp amplifier is configured to operate in voltage clamp mode, such that the REF port is connected to the reference electrode to become a voltage sensing terminal, and the BATH port is connected to the counter electrode to become a current output terminal, thereby simultaneously acquiring the electrophysiological signal of the cell under test and the current signal corresponding to the concentration of the target biochemical marker.

2. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The working electrode surface is provided with a conductive layer and a functional layer in sequence. The outer surface of the functional layer is provided with a lead wire connected to the signal acquisition module. The insulating protective layer is provided on the functional layer except for the tip.

3. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 2, characterized in that, It has at least one of the following characteristics: - The material of the conductive layer is any one of chromium, gold, silver, nickel, titanium, platinum, aluminum, iridium, carbon nanotubes, graphene, graphite, polypyrrole, alginate and conductive hydrogel; - The material of the functional layer is any one of copper selenide, cobalt copper oxide, platinum, gold, terbium with dual ligands, and enzymes; - The material of the insulating protective layer is any one of paraffin wax, alumina, silicon dioxide, silicon carbide, poly(p-xylene), and phthalates.

4. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The working electrode is made of any one of the following materials: metallic materials, semiconductor materials, alloy materials, and non-metallic materials.

5. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The coating thickness of the working electrode is 0.1 nm-1 mm, and the impedance is 100 MΩ-10 GΩ.

6. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The working electrode has a length of 10 μm-20 cm and a tip diameter of 1 nm-1 μm.

7. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The system has an input voltage of -800~1000 mV, an input current of -800~1000 pA, a voltage detection range of -20000~20000 mV, and a current detection range of -20000~20000 pA.

8. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The counter electrode can be any one of a ring electrode, a wire electrode, or a sheet electrode.

9. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The reference electrode is made of any one of silver / silver chloride, saturated calomel, mercurous sulfate, and mercuric oxide.

10. The electrophysiological signal and biochemical biomarker detection system based on nanoprobes according to claim 1, characterized in that, The electrophysiological signals include at least one of action potential signals, postsynaptic potential signals, and postsynaptic current signals; the biochemical markers include at least one of the neurotransmitter dopamine signals secreted by vesicles, ascorbic acid content signals, uric acid signals, and pH signals.

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