Preparation method of flexible sensor and monitoring system

Abstract

The invention discloses a preparation method of a flexible sensor and a monitoring system. The method comprises the following steps: preparing a microelectrode array and a friction nano-generator; assembling the microelectrode array, the first flexible film and the PCB from top to bottom, and arranging a hollow area on the PCB; the first flexible film corresponding to the hollow area is used for placing biological cells to be detected; and packaging the first flexible thin film and the second flexible thin film into a first cavity, placing the friction nano generator in the first cavity, and adjusting the pressure of the first cavity to be less than standard atmospheric pressure to obtain the flexible sensor. According to the embodiment of the invention, through the negative pressure design of the first cavity, the friction nanometer generator can sensitively measure a mechanical signal; the operation is simple, no influence is caused on cells, and the sensitivity of the flexible sensor is improved. The method can be widely applied to the technical field of noninvasive detection.

Description

Technical Field

[0001] The present invention relates to the technical field of non-invasive detection, and in particular to a preparation method of a flexible sensor and a monitoring system. Background Art

[0002] As the core organ of life activities, the normal operation of the heart is crucial for maintaining systemic blood supply and oxygen supply. Cardiomyocytes are the basic units that play the main functions in the heart, and one of their main characteristics is the excitation-contraction coupling mechanism. Electrophysiological signals (such as action potentials) are direct reflections of the excitability and conductivity of cardiomyocytes, and can provide information about cell excitatory characteristics, electrical conductivity, and the effects of drug action on ion channels. Mechanical pulsation signals are direct manifestations of the contraction and relaxation activities of cardiomyocytes, and can provide information about systolic function, energy metabolism status, and pharmacological positive or negative inotropic effects. Therefore, electrophysiological signals and mechanical pulsation signals respectively reflect the functional states of cardiomyocytes from different perspectives. Synchronously monitoring these two signals can more comprehensively evaluate the functional state of cardiomyocytes. In the research of cardiovascular diseases and the process of new drug development, understanding and evaluating the comprehensive function of cardiomyocytes is crucial. The monitoring platform for isolated cardiomyocytes provides an important means for the research of cardiovascular diseases and new drug development, helps to better understand the complex pathological mechanisms of heart diseases, and provides key data for developing more effective treatment strategies.

[0003] In the related art, non-invasive microelectrode arrays (MEAs) and multi-transistor arrays can achieve high-throughput extracellular electrical signal detection. These methods only focus on the electrical signals of single cardiomyocytes and ignore the mechanical contraction signals. Therefore, other methods are needed to synchronously detect pulsation signals, including optical methods, bioimpedance methods, piezoelectric / piezoresistive sensors, and crack and cantilever sensors. However, the above methods have problems such as complex operation, affecting cell growth, and poor sensitivity. Summary of the Invention

[0004] The purpose of the present invention is to solve at least to some extent one of the technical problems existing in the prior art.

[0005] For this reason, the purpose of the present invention is to provide a preparation method of a highly sensitive flexible sensor and a monitoring system.

[0006] To achieve the above technical objectives, on the one hand, an embodiment of the present invention provides a method for preparing a flexible sensor, including the following steps: preparing a microelectrode array and a triboelectric nanogenerator; assembling the microelectrode array, a first flexible film, and a PCB from top to bottom, and setting a hollowed-out area on the PCB; the first flexible film corresponding to the hollowed-out area is used to place biological cells to be measured; encapsulating the first flexible film and a second flexible film into a first cavity, placing the triboelectric nanogenerator in the first cavity, and adjusting the pressure in the first cavity to be less than the standard atmospheric pressure to obtain a flexible sensor. Through the negative pressure design of the first cavity in this application embodiment, the triboelectric nanogenerator can sensitively measure mechanical signals; the operation is simple, has no influence on cells, and is beneficial to improving the sensitivity of the flexible sensor.

[0007] In some embodiments, preparing the triboelectric nanogenerator includes:

[0008] Preparing a first friction layer and a second friction layer, where the first friction layer is close to the PCB, and the second friction layer is close to the bottom of the second flexible film;

[0009] Setting a micro-structure array on the first friction layer and between the first friction layer and the second friction layer to realize the contact and separation of the friction layers by combining the biological cells.

[0010] In some embodiments, the micro-structure array includes a micro-column array, and the micro-column array is prepared through the following steps:

[0011] Covering a PET porous filter membrane on a substrate and performing curing treatment;

[0012] Spin-coating a composite material on the PET porous filter membrane and performing curing treatment;

[0013] Etching the PET porous filter membrane by oxygen plasma etching to obtain the micro-column array.

[0014] In some embodiments, the composite material includes CNT and PDMS, and the method further:

[0015] Setting the multiple of the mass of the PDMS to the mass of the CNT;

[0016] Adjusting the column height by oxygen plasma etching.

[0017] In some embodiments, the flexible sensor measures mechanical signals through the following steps:

[0018] Adjusting the pressure by removing the gas in the first cavity;

[0019] Combining with the elastic deformation characteristics of the first flexible thin film, the contraction force of the biological cells is converted into a field potential signal, and then the mechanical signal is determined.

[0020] In some embodiments, the method further includes:

[0021] Performing a surface sputtering gold layer treatment on the microstructure array and performing a corona charging treatment on the second friction layer;

[0022] Electrically connecting the two friction layers and the microelectrode array electrodes of the triboelectric nanogenerator to the PCB electrodes through conductive paint;

[0023] A quartz tube is provided on the PCB; the quartz tube is used for culturing the biological cells.

[0024] In some embodiments, the preparation of the microelectrode array includes:

[0025] Using a spin coating method to obtain a first flexible thin film;

[0026] Obtaining the electrodes and wire patterns of the microelectrode array by sputter depositing a conductive metal;

[0027] Insulating the wires and exposing part of the electrodes to obtain the microelectrode array.

[0028] On the other hand, an embodiment of the present invention proposes a monitoring system based on a flexible sensor, including:

[0029] A flexible sensor prepared by the preparation method of the flexible sensor as described above, a first amplification module, a second amplification module, and a control module;

[0030] The first amplification module is used to receive the field potential signal of the flexible sensor, and the second amplification module is used to receive the mechanical signal of the flexible sensor.

[0031] In some embodiments, the first amplification module includes:

[0032] A conditioning circuit and an amplification circuit; the field potential signal is transmitted to the amplification circuit through the conditioning circuit.

[0033] In some embodiments, the second amplification module includes a transimpedance amplification circuit.

[0034] The embodiments of the present application at least include the following beneficial effects: The method provided by the embodiments of the present invention includes: preparing a microelectrode array and a triboelectric nanogenerator; assembling the microelectrode array, a first flexible film and a PCB from top to bottom, and providing a hollowed-out area on the PCB; the first flexible film corresponding to the hollowed-out area is used to place the biological cells to be measured; packaging the first flexible film and a second flexible film into a first cavity, placing the triboelectric nanogenerator in the first cavity, and adjusting the pressure in the first cavity to be less than the standard atmospheric pressure to obtain a flexible sensor. Through the negative pressure design of the first cavity, the triboelectric nanogenerator in the embodiments of the present application can sensitively measure mechanical signals; the operation is simple and has no influence on cells, which is beneficial to improving the sensitivity of the flexible sensor. Description of the Drawings

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following introduces the relevant technical solution drawings in the embodiments of the present invention or the prior art. It should be understood that the drawings introduced below are only for conveniently and clearly presenting some embodiments of the technical solutions in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative efforts.

[0036] Figure 1 Structural and design schematic diagram of an embodiment of the flexible sensor provided by the present invention;

[0037] Figure 2 Schematic diagram of the sensor state in an embodiment of the cell detection process of the flexible sensor provided by the present invention;

[0038] Figure 3 Structural schematic diagram of an embodiment of the nanowire array provided by the present invention;

[0039] Figure 4 Physical circuit schematic diagram of an embodiment of the monitoring system provided by the present invention;

[0040] Figure 5 Circuit principle schematic diagram of an embodiment of the monitoring system provided by the present invention;

[0041] Figure 6 Waveform schematic diagram of an embodiment of the mechanical signal and the field potential signal provided by the present invention;

[0042] Figure 7 Influence relationship schematic diagram of the pressure in the first cavity on the mechanical signal provided by the present invention;

[0043] Figure 8 Synchronous signal waveform schematic diagram of cardiomyocytes provided by the present invention;

[0044] Figure 9 Schematic diagram of signal change waveforms after intervention with different concentrations of drugs provided by the present invention;

[0045] Figure 10 is Figure 9 Schematic diagram of the amplified waveform of the signal in the embodiment;

[0046] Figure 11 Schematic diagram of the comparison of typical waveforms of signals after intervention with different concentrations of drugs provided by the present invention;

[0047] Figure 12 Schematic diagram of signal change waveforms of the control group and interventions with different concentrations provided by the present invention;

[0048] Figure 13 Schematic diagram of signal duration change waveforms of the control group and interventions with different concentrations provided by the present invention;

[0049] Figure 14 Schematic diagram of columnar signal changes after intervention with different concentrations of drugs provided by the present invention;

[0050] Figure 15 Schematic diagram of waveform for long-term stability test of sensors for cardiomyocytes provided by the present invention;

[0051] Figure 16 Schematic diagram of waveform of another embodiment of long-term stability test of the sensor provided by the present invention;

[0052] Figure 17 Schematic diagram of signal-to-noise ratio statistics for synchronous signals provided by the present invention. Detailed implementation manners

[0053] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention. For the step numbers in the following embodiments, they are only set for the convenience of explanation and illustration, and no limitation is imposed on the order between the steps. The execution order of each step in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0054] As the core organ of life activities, the normal functioning of the heart is crucial for maintaining systemic blood supply and oxygen supply. Cardiomyocytes are the basic units that perform the main functions in the heart, and one of their main characteristics is the excitation-contraction coupling mechanism. Electrophysiological signals (such as action potentials) are the direct reflections of the excitability and conductivity of cardiomyocytes, and can provide information about cell excitatory characteristics, electrical conductivity, drug effects on ion channels, etc. Mechanical pulsation signals are the direct manifestations of the contraction and relaxation activities of cardiomyocytes, and can provide information such as contractile function, energy metabolism status, and pharmacological positive or negative inotropic effects. Therefore, electrophysiological signals and mechanical pulsation signals respectively reflect the functional states of cardiomyocytes from different perspectives, and synchronous monitoring of these two signals can more comprehensively evaluate the functional state of cardiomyocytes. In the research of cardiovascular diseases and the development of new drugs, understanding and evaluating the comprehensive function of cardiomyocytes is crucial. The monitoring platform for isolated cardiomyocytes provides an important means for the research of cardiovascular diseases and the development of new drugs, helps to better understand the complex pathological mechanisms of heart diseases, and provides key data for developing more effective treatment strategies. Therefore, developing a sensing platform that can simultaneously monitor the electrophysiological signals and mechanical pulsation signals of cardiomyocytes is of great significance for studying the function of cardiomyocytes and further applying it to drug research.

[0055] Cell-based biosensing technologies include invasive / marker-based detection techniques and non-invasive / marker-free detection techniques. The invasive patch clamp technique is the gold standard method for detecting the electrical signals of cardiomyocytes, which can directly measure the ion channel current inside a single cell and provide high-resolution data. However, its complex operation, low throughput, and invasiveness limit the long-term monitoring of cellular electrical signals. Non-invasive microelectrode arrays (MEAs) and multi-transistor arrays can achieve high-throughput extracellular electrical signal detection. Thanks to the high affinity between cells and electrodes, the MEA system can continuously monitor electrical signal changes for a long time, with high temporal and spatial resolution, and can accurately capture the field potential signals of multi-channel cardiomyocytes. However, these methods only focus on the electrical signals of individual cardiomyocytes and ignore the mechanical contraction signals. Therefore, other methods are needed to synchronously detect the pulsation signals, including optical methods, bioimpedance methods, piezoelectric / piezoresistive sensors, and crack and cantilever sensors. Optical methods (such as video microscopy and Ca2+ fluorescence imaging) allow simultaneous recording of the mechanical activities of multiple cells and have the advantage of non-contact measurement. Although it has the characteristics of high throughput and non-invasiveness, its operation is complex, the requirements for data acquisition and analysis are high, the long-term labeling of chemical dyes can cause cytotoxicity, and the measurement time is limited. The bioimpedance method measures the impedance changes in the attachment state of cardiomyocytes by applying a small-amplitude alternating current or voltage, thereby reflecting the mechanical activity. This method has strong real-time monitoring ability, but the continuously applied alternating signal may affect cell growth and function, and the signal processing is complex, and the contractile force cannot be directly measured. Piezoelectric / piezoresistive sensors, as well as crack and cantilever sensors, obtain pulsation signals by detecting the mechanical deformation of cardiomyocytes and have high sensitivity and response speed. However, these sensors are usually complex in design, defective in repeated use, and require an external excitation power supply and a complex signal processing circuit.

[0056] In recent years, as an emerging active sensing technology, the triboelectric nanogenerator (TENG) has been widely used in monitoring physiological signals such as human motion, respiration, and pulse due to its high energy conversion efficiency and sensitive response to tiny mechanical stimuli, showing great potential in the fields of wearable devices and health monitoring. Flexible TENG has the characteristics of material diversity, self-power supply, high sensitivity, and low energy consumption, making it perform superior to traditional methods in real-time monitoring of cell mechanical activities. Combining MEA with TENG to construct a hybrid sensing platform for contact / non-contact sensing of cardiomyocytes to achieve synchronous recording of electrical signals and mechanical pulsation signals is a very promising research direction. However, compared with the deformation of the body and tissues, the contraction deformation of cardiomyocytes is very small (micrometer level), and the contraction force is extremely weak (millinewton level). Therefore, the currently commonly used TENG technology still faces challenges in detecting such weak mechanical forces. One embodiment proposed a mesh TENG device composed of a PDMS wrapping layer and a copper core layer (with a gap of about 3 μm) for measuring the contraction of cardiomyocytes, but the signal-to-noise ratio of its detected signal is relatively low, and it needs to rely on the spectral components of the fast Fourier transform (FFT) to distinguish drug information. Similarly, one embodiment proposed a bridge structure (B-TENG) flexible strain sensor for measuring the contraction stress of cardiomyocytes and drug testing, but the signal-to-noise ratio of this B-TENG is only 12.1 dB. There is still room for improvement in these TENG sensors in terms of detection lower limit, signal-to-noise ratio, and synchronous detection of electrical signals. Therefore, developing a flexible sensing platform for electromechanical synchronous sensing of cardiomyocytes that can meet the requirements of ultra-high mechanical sensitivity, high signal-to-noise ratio, and high durability remains an urgent challenge to be solved.

[0057] The preparation method and system of the flexible sensor proposed according to the embodiment of the present invention will be described in detail below with reference to the accompanying drawings. First, the preparation method of the flexible sensor proposed according to the embodiment of the present invention will be described with reference to the accompanying drawings.

[0058] Refer to Figure 1, in the embodiments of the present invention, a preparation method of a flexible sensor is provided. The preparation method of the flexible sensor in the embodiments of the present invention can be applied to a terminal, can also be applied to a server, or can also be software running on a terminal or a server, etc. The terminal can be a tablet computer, a notebook computer, a desktop computer, etc., but is not limited thereto. The server can be an independent physical server, can also be a server cluster or a distributed system composed of multiple physical servers, or can also be a cloud server providing basic cloud computing services such as cloud services, cloud databases, cloud computing, cloud functions, cloud storage, network services, cloud communications, middleware services, domain name services, security services, CDN, and big data and artificial intelligence platforms. The preparation method of the flexible sensor in the embodiments of the present invention mainly includes the following steps:

[0059] S100: Prepare a microelectrode array and a triboelectric nanogenerator;

[0060] S200: Assemble the microelectrode array, the first flexible film and the PCB from top to bottom, and set a hollow area on the PCB; the first flexible film corresponding to the hollow area is used to place biological cells to be measured;

[0061] S300: Package the first flexible film and the second flexible film into a first cavity, place the triboelectric nanogenerator in the first cavity, and adjust the pressure in the first cavity to be less than the standard atmospheric pressure to obtain a flexible sensor.

[0062] In some possible implementation manners, the first flexible film and the second flexible film can be PDMS, or can also be composed of similar materials. The present application does not specifically limit the materials of the flexible films. Refer to Figure 2 As shown, the first flexible film is a film on the PCB, the second flexible film is a film under the PCB, the thickness of the second flexible film is greater than that of the first flexible film, and the first flexible film and the second flexible film are hermetically packaged through the flexible film to obtain the first cavity.

[0063] The present application has developed a flexible sensing platform (TENG-MEA) based on a negative pressure TENG (Negative Pressure TENG, NP-TENG) and a microelectrode array and its measuring device ( Figure 1) Among them, the NP-TENG increases the contact area by adopting a nanocolumn structure on the surface of the friction layer, thereby improving the performance. At the same time, a negative pressure is applied to the contact area of the friction layer, enabling the friction layer to come into contact in advance and achieve smoother contact and separation during cell contraction. This platform can stably synchronously record the electrophysiological signals and mechanical pulsation signals of neonatal rat cardiomyocytes for a long time, with high sensitivity (1.146 pA / Pa), high signal-to-noise ratio (the pulsation signal is 43.1 dB, and the field potential signal is 41.0 dB), and high stability (exceeding 300 million cycles). The experimental results show that this platform can stably obtain high-quality synchronous signals during a 20-day cell culture cycle. When the same TENG-MEA is reused multiple times (>3 times), the changes in the signal-to-noise ratio are 2.2 dB and 3.2 dB respectively. In the drug intervention experiment with E-4301 (1 μM), we observed that the pulsation frequency of cardiomyocytes decreased significantly. Although the change in the amplitude of the field potential was not obvious, the contractility of cardiomyocytes decreased to 31% of the original. In the experiment of treating cardiomyocytes with three drugs, the TENG-MEA sensitively captured the changes in electrical and mechanical activities, which were consistent with the effects of the drugs, demonstrating its great application potential in drug screening and cardiac toxicity assessment. These experimental results show that the TENG-MEA and its measurement device can stably synchronously record the changes in the field potential and mechanical pulsation of cardiomyocytes for a long time, contributing to the in-depth study of the excitation-contraction coupling mechanism of cardiomyocytes. At the same time, it provides a powerful tool for new drug development and cardiac disease research and is expected to play an important role in future cardiovascular research and clinical applications.

[0064] The present invention provides a flexible sensing platform (TENG-MEA), which realizes the long-term synchronous monitoring of the mechanical pulsation and electrical signals of cardiomyocytes by integrating a negative-pressure TENG and an MEA. Its technical solutions include:

[0065] Negative-pressure cavity design: The friction layer is pre-contacted by negative pressure (-20 Pa to -80 Pa), and micron-scale separation of the friction layer is achieved in combination with the contractility of cardiomyocytes, improving the sensitivity (the detection limit <1 Pa, and the sensitivity reaches 1.146 pA / Pa).

[0066] Microcolumn array: Carbon nanotube (CNT) / PDMS composite microcolumns with a height of 3 - 5 μm (i.e., the height of the microstructural array is 3 - 5 μm), increasing the friction area and enhancing the charge transfer efficiency.

[0067] Signal synchronous acquisition: The MEA detects the field potential signal, and the NP-TENG detects the mechanical signal. In combination with a dedicated amplifier circuit, high signal-to-noise ratio (the pulsation signal is 43.1 dB, and the field potential is 41.0 dB) acquisition of dual-mode signals is achieved.

[0068] Long-term stability: The PDMS substrate has excellent biocompatibility, supports the survival of cardiomyocytes for 20 days, and can be reused more than 4 times. The durability of the sensor exceeds 3 million cycles.

[0069] In some embodiments, preparing a triboelectric nanogenerator, comprising:

[0070] Preparing a first friction layer and a second friction layer, the first friction layer being close to the PCB, and the second friction layer being close to the bottom of the second flexible film;

[0071] Setting a micro-structure array on the first friction layer and between the first friction layer and the second friction layer to combine the biological cells to achieve contact and separation of the friction layers.

[0072] In some embodiments, the micro-structure array includes a micro-column array, and the micro-column array is prepared by the following steps:

[0073] Covering a PET porous filter membrane on a substrate and performing a curing treatment;

[0074] Spin-coating a composite material on the PET porous filter membrane and performing a curing treatment;

[0075] Etching the PET porous filter membrane by an oxygen plasma etching method to obtain the micro-column array.

[0076] In some embodiments, the composite material includes CNT and PDMS, and the method further:

[0077] Setting the multiple of the mass of the PDMS to the mass of the CNT;

[0078] Adjusting the column height by an oxygen plasma etching method.

[0079] In some embodiments, the flexible sensor measures mechanical signals by the following steps:

[0080] Adjusting the pressure by removing the gas in the first cavity;

[0081] Combining the elastic deformation characteristics of the first flexible film, converting the contraction force of the biological cells into a field potential signal, and then determining the mechanical signal.

[0082] In some embodiments, the method further includes:

[0083] Performing a surface sputtering gold layer treatment on the micro-structure array and performing a corona charging treatment on the second friction layer;

[0084] Electrically connecting the two friction layers and the micro-electrode array electrodes of the triboelectric nanogenerator to the PCB electrodes through a conductive paint;

[0085] A quartz tube is provided on the PCB; the quartz tube is used for culturing the biological cells.

[0086] In some embodiments, the preparation of the microelectrode array includes:

[0087] Obtaining a first flexible film by spin coating;

[0088] Obtaining the electrode and wire patterns of the microelectrode array by sputter depositing a conductive metal;

[0089] Insulating the wires and exposing some of the electrodes to obtain the microelectrode array.

[0090] On the other hand, an embodiment of the present invention provides a monitoring system based on a flexible sensor, including:

[0091] A flexible sensor prepared by the preparation method of the flexible sensor as described above, a first amplification module, a second amplification module, and a control module;

[0092] The first amplification module is used to receive the field potential signal of the flexible sensor, and the second amplification module is used to receive the mechanical signal of the flexible sensor.

[0093] In some embodiments, the first amplification module includes:

[0094] A conditioning circuit and an amplification circuit; the field potential signal is transmitted to the amplification circuit through the conditioning circuit.

[0095] In some embodiments, the second amplification module includes a transimpedance amplification circuit.

[0096] It can be seen that the content in the above method embodiments is applicable to the system embodiments of the present invention. The functions specifically implemented in the system embodiments of the present invention are the same as those in the above method embodiments, and the beneficial effects achieved are also the same as those in the above method embodiments.

[0097] The following takes specific embodiments to introduce in detail the preparation method of the flexible sensor and the monitoring system provided by the present application:

[0098] The present application provides a flexible sensing platform based on a negative pressure TENG and MEA, including:

[0099] 1.1 Microelectrode array (MEA), with a PDMS film with a thickness of 10 ± 1 μm as the substrate, a gold electrode array is sputtered on the surface, the size of the electrode exposure area is 200 μm (length) × 50 μm (width), used for detecting myocardial cell field potential signals with high spatial resolution (at least 16 channels), and the flexible characteristics of the PDMS film ensure that the electrode is in close contact with the cell membrane, reducing the interface impedance to 4.05 kΩ @ 1 kHz;

[0100] 1.2 Negative pressure triboelectric nanogenerator (NP-TENG), which includes a microcolumn array composed of carbon nanotube (CNT) / PDMS composite material (i.e., the microstructural array in this application). The height of the column is 3 - 5 μm, the diameter is 1 μm, and the array gap matches the micron-level contraction displacement of cardiomyocytes. Combining with a negative pressure chamber (i.e., the first chamber in this application, with a pressure range of -20 Pa to -80 Pa) to pre-tighten the friction layer, the friction layer is separated by the millinewton-level (mN) contraction force of cardiomyocytes to achieve high-sensitivity detection of mechanical pulsation signals (sensitivity ≥ 1.146 pA / Pa, detection limit < 1 Pa);

[0101] 1.3 Seal the negative pressure chamber (i.e., the first chamber), which is integrated between the PDMS substrate and the PCB. By dynamically adjusting the negative pressure to balance the cell contraction resistance, it ensures the stable separation and contact of the friction layer under millinewton-level external forces;

[0102] 1.4 Dual-mode signal processing module, including: a field potential signal processing unit, which uses a band-pass filter with a bandwidth of 0.5 - 800 Hz and a 300-fold gain amplification circuit, with a signal-to-noise ratio ≥ 41.0 dB; a mechanical signal processing unit, which uses a transimpedance amplifier (TIA) to amplify the pA-level current signal of the NP-TENG by 10^10 times, with a signal-to-noise ratio ≥ 43.1 dB.

[0103] In this application, the mass ratio of CNT to PDMS in the microcolumn array is 1:9. By regulating the column height through oxygen plasma etching, the contact area of the friction layer is maximized, and the response current is positively correlated with the CNT content (R 2 > 0.99).

[0104] In this application, the pressure regulation of the negative pressure chamber is achieved by dynamically controlling the gas volume with a syringe. For every 1 μL of gas removed, the pressure drops by 8.217 Pa. Combining with the elastic deformation characteristics of the PDMS film, the cardiomyocyte contraction force (0.1 - 5 mN) is linearly converted into an electric current signal (4.4 - 48.35 pA).

[0105] In this application, the 10-μm PDMS film is obtained by spin coating at 4000 r / min (i.e., the first flexible film is obtained by spin coating). Its ultra-thin and flexible characteristics reduce the mechanical constraint on the growth of cardiomyocytes. At the same time, the cell adhesion rate is enhanced by surface fibronectin modification, ensuring the long-term stability of the field potential signal (amplitude fluctuation < 9.3% within 20 days).

[0106] In this application, the signal processing module integrates a 24-bit high-precision ADC with a sampling rate of 1950 Hz, supporting synchronous acquisition of pulsation signals (0.4 - 16 Hz) and field potential signals (0.4 - 800 Hz), and eliminating signal delay through a zero-phase filtering algorithm, with a time resolution < 1 ms.

[0107] The present application also provides a method for synchronous monitoring of myocardial cell mechano-electric signals, including the following steps:

[0108] a) Cell culture: Neonatal rat cardiomyocytes (density 2200 cells / mm 2 ) are inoculated on the surface of the PDMS film, and the initial pressure is adjusted to -20 Pa through the negative pressure chamber to make the microcolumn array pre-contact with the FEP friction layer;

[0109] b) Signal synchronous acquisition: The amplitudes of myocardial cell field potentials (0.06 - 0.33 mV) and mechanical pulsation currents (0.01 - 9.34 pA) are recorded in real time, and parameters such as pulsation frequency (1.8 - 4.6 Hz), contraction time (64 - 255 ms), and field potential duration (111 - 216 ms) are extracted;

[0110] c) Drug response analysis: By adding ion channel drugs (such as E-4031, quinidine, isoproterenol hydrochloride), the changes in mechanical contractile force (such as an 84% decrease in amplitude) and the prolongation of electroactivity duration (such as a 22% increase in field potential duration) are quantified, and a mechano-electric coupling pharmacodynamic evaluation model is established.

[0111] The physical object of the TENG-MEA is as shown in A of Figure 1 , and its structure is jointly composed of a quartz tube above the PCB, a flexible MEA, and a negative pressure triboelectric nanogenerator (NP-TENG) below. The electrode pattern of the flexible MEA layer is fabricated on the PDMS film through microfabrication technology (as shown in B of Figure 2 ).

[0112] First, we obtain 10-μm-thick PDMS by spin coating. Then, a mask plate is placed on the PDMS surface (the second step of B in Figure 1 ), and titanium / gold with a thickness of 5 nm / 100 nm is deposited by sputtering to form the electrode and wire patterns of the MEA (the third step of B in Figure 1 ). Subsequently, the wire part is insulated by PDMS. These electrodes are evenly distributed in a circular pattern with a diameter of 12 mm, and the length of the exposed electrodes after insulation is 200 μm and the width is 50 μm (Fig.S1-1C). Finally, the flexible MEA layer is cut into a 20-mm square and peeled off on the substrate for further use.

[0113] First, spin-coat PDMS on the acrylic substrate at a speed of 4000 rpm, and then cure it at 40 °C for 10 hours. Next, cover the PDMS substrate with a mask template and deposit gold (Au) by sputtering to form the electrode pattern of the MEA. Subsequently, insulate the outside of the electrodes with PDMS. The characterization results show that these electrodes are uniformly distributed in a circular structure on the circumference with a diameter of 12 mm. After insulation, the exposed electrode length is about 200 μm and the width is about 50 μm. Then, use absolute ethanol to peel the PDMS film from the substrate and cut it into 20-mm squares for standby.

[0114] The friction layer of the NP-TENG consists of a PET porous membrane substrate containing a CNT / PDMS microcolumn structure (i.e., the first friction layer) and an FEP film with a thickness of 50 μm (i.e., the second friction layer). The preparation method of the microcolumns is as Figure 1 shown in C of [reference], which is similar to our previous report, using the microporous template method and oxygen etching method. First, PDMS is coated on the acrylic substrate as an adhesive. Immediately afterwards, a PET porous membrane (pore diameter 1 μm, thickness 11 μm) is covered on the surface of the PDMS and cured at 80 °C. Then, a CNT / PDMS mixture with a mass ratio of 1:9 is spin-coated onto the PET porous membrane, and the excess mixture on the surface of the PET porous membrane is removed and cured at 40 °C for 10 hours. Subsequently, the PET membrane is partially etched by the oxygen plasma etching method to expose the CNT / PDMS microcolumn structure. SEM characterization shows that the diameter of the microcolumns is about 1 μm and the height is 3 - 5 μm ( Figure 1 the fifth step of C in [reference]). The height of the microcolumns can be adjusted by controlling the oxygen etching time. These microcolumns provide microscopic support for the three-dimensional contact between the two friction layers. Since the surface morphology of the friction layer directly affects the output performance of the TENG, the microcolumn structure increases the contact area and surface charge density of the friction layer, significantly improving the electrical output performance of the TENG. In addition, we found that the electrical output performance of the TENG increases with the increase in the proportion of CNT in the CNT / PDMS composite material. When the mass ratio of CNT is 1:9, the output amplitude is about 10 times that of the PDMS control group, which indicates that the addition of CNT significantly improves the output performance of the TENG, as Figure 3 shown in A and B of [reference]. However, too high a CNT ratio will increase the hardness of the composite material, which may not be conducive to detecting the weak beating signals of cardiomyocytes. Before device assembly, we perform corona charging treatment on the upper surface of the FEP film to increase its surface electron density and further improve the output performance during friction.

[0115] As Figure 2As shown, the two friction layers, PCB, MEA substrate and glass tube of the NP-TENG are assembled from bottom to top to form the TENG-MEA. We electrically connected the MEA electrodes and the two friction layers of the TENG to the PCB electrodes using conductive silver paint, and then encapsulated them with PDMS. The PDMS substrate of the MEA and the PDMS plate at the bottom together form a transparent sealed cavity to encapsulate the NP-TENG. This design not only protects the friction layer of the TENG and forms a negative pressure cavity, but also allows the growth of cardiomyocytes on the chip to be observed through an optical microscope. Finally, the glass tube is bonded to the PCB with PDMS as the cell culture.

[0116] Furthermore, we used COMSOL Multiphysics software to simulate the contact and separation processes of the TENG friction layer by finite element analysis and tested the dependence of the initial spacing on the output voltage. The contact and separation processes under external force were simulated by programmatically controlling the distance between the friction layers. Figure 6 The simulation results showing the electric field intensity distribution and potential changes in four key stages are presented. The results show that the output voltage of the NP-TENG increases linearly with the separation distance of the friction layer, indicating its good linear output ability in displacement detection and making it very suitable for measuring the contraction signals of cardiomyocytes. These research results show that by optimizing the material composition and microstructure design of the friction layer, the NP-TENG can achieve efficient electrical signal output and sensitive mechanical signal detection, providing reliable technical support for the monitoring of the mechanical activities of cardiomyocytes.

[0117] After fabricating the TENG-MEA, we adjusted the air pressure in the sealed cavity to form a negative pressure cavity of about -20 Pa. Under the action of the negative pressure, when the cells are in a resting state, the distance between the nanowire friction layer and the FEP friction layer is compressed. When the cardiomyocytes growing on the PDMS film contract, under the combined action of the supporting force of the microcolumn array and the contractile force of the cardiomyocytes, the micron-scale friction layer is effectively separated ( Figure 2 as shown in A), causing electrons to flow from the microcolumn electrode to the FEP electrode and form a current in the external circuit. When the cardiomyocytes relax, the negative pressure forces the PDMS film to rebound downward, causing the separated friction layers to come into contact again ( Figure 2In Figure B), the flowing electrons return to the micro-column electrode through the external circuit. This design of the negative pressure cavity with the upper and lower double-layer functions enables the TENG-MEA to simultaneously sense the pulsation signals of cardiomyocytes and the field potential signals with a high spatial resolution of 16 channels. The TENG-MEA is connected to the signal amplification and acquisition device through round pins with a 2.54 mm pitch. The cardiomyocytes are cultured in a quartz tube with a height of 15 mm on the PCB. 1 mL of Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) is added to the tube, and the measurement is carried out under the conditions of 5% CO2, constant temperature of 37 °C, and asepsis.

[0118] The design of this application will be introduced in detail with reference to the accompanying drawings:

[0119] Figure 1 It is a schematic diagram of the overall structure of the TENG-MEA, where B is the design process of the MEA layer and C is the design process of the NP-TENG micro-column friction layer. Figure 2 It is the design of the negative pressure cavity in the TENG-MEA and the working schematic diagrams during the mechanical contraction A and relaxation B of cardiomyocytes. Figure 3 It is the SEM image (A) of the micro-column array and the curve (B) of the relationship between the CNT content and the current response.

[0120] Figure 4 It is a 3D schematic diagram of the circuit board of the signal amplification and acquisition device. Figure 5 It is a circuit structure block diagram. In order to record the mechanical pulsation signals and field potential signals of cardiomyocytes obtained by the TENG-MEA, this application designs and manufactures a set of signal amplification and acquisition devices. The hardware circuit of this device mainly includes the following modules: the TENG-MEA interface 1, the conditioning circuits 2 and 3 for the field potential signals and the signal amplification circuit 4, the transimpedance amplification circuit 5 for the mechanical pulsation signals, the ADC circuit 6, the MCU circuit module 7, the data transmission modules 8 and 9, and the power supply module 10. The layout of each module on the circuit board is as Figure 4As shown in the figure. In circuit design, the current amplification factor of the NP-TENG amplifier circuit is 200 dB (10,000,000,000 times), and the designed bandwidth is 16 Hz; the voltage amplification factor of the field potential signal amplifier circuit is 49.5 dB (300 times), and the designed bandwidth is 0.5 - 800 Hz. To optimize the PCB design, we separate the digital circuit and the analog circuit for layout, and use a low dropout regulator to provide a low-noise power supply for analog devices. At the same time, the AD chip is independently powered by a separate low-noise low dropout regulator power supply, and an external high-precision low-temperature-drift low-noise reference source is used to provide a reference voltage for signal conversion. These improvement measures effectively reduce the noise of the system itself and enhance the resistance to external interference, thus significantly improving the quality of signal acquisition. The overall size of the acquisition device is 120 mm × 66 mm, with a compact circuit layout and modular design, suitable for long-term signal acquisition inside a cell incubator.

[0121] Figure 6 Figure (A) shows the typical waveforms of the pulsation signal and the field potential signal of a single channel based on TENG-MEA synchronous acquisition, and figure (B) shows the time correlation between the pulsation signal and the field potential. Further analysis reveals that there are time differences between the field potential signals captured by different MEA channels and the pulsation signal. Taking the peak point of the pulsation signal as a reference, the delay time (Tad) between different channels and the pulsation signal is calculated. The delay time ranges from +8.7 ms (ch5) to -86.1 ms (ch14), indicating that there are differences in the propagation of electrical signals between cardiomyocytes at different positions. The analysis of the relationship between the electrode position and the delay time shows that this difference is related to the position of the beating focus in the cardiomyocyte cluster. It is worth noting that TENG-MEA also detected a bimodal contraction curve of cardiomyocytes, which was not found by optical imaging. This difference may be due to the incomplete synchronization of cardiomyocytes at the bottom of the sensor, which may form two independent beating foci in contact with the sensor. The optical imaging system relies on analyzing the pixel displacement at the local cell edge and cannot detect these two independent beating foci.

[0122] Figure 7 Figures (A), (B), and (C) show the influence of the pressure in the negative pressure chamber on the signal of the perceived cardiomyocyte contractile force, and figure (D) shows a bar chart. The test shows that when the initial negative pressure is -40 Pa, adding 1 mL of PBS solution can increase the pressure in the negative pressure chamber to 0 Pa. Too low negative pressure may increase the contraction burden of cardiomyocytes attached to the PDMS film, resulting in a decrease in the amplitude of the detected signal. This pressure offset phenomenon helps to reduce the pulsation resistance of cardiomyocytes growing in an attached manner, ensuring the stable separation of the NP-TENG friction layer, and laying a foundation for TENG-MEA to accurately capture the weak contractile force of cardiomyocytes.

[0123] Figure 8 It is a waveform diagram of synchronous signals of cardiomyocytes, waveforms of different frequencies, and the beating signal curves of cardiomyocytes in different states.

[0124] We cultured neonatal rat cardiomyocytes in the TENG-MEA device and simultaneously recorded their beating signals and field potential signals to evaluate their potential applications in monitoring cardiomyocyte physiological signals. By synchronously monitoring the cardiomyocytes cultured in TENG-MEA through an optical microscope and a self-made acquisition device, we observed that the beating signals and field potential signals recorded by TENG-MEA were consistent with the beating frequencies of the cells in the optical microscope.

[0125] Statistical analysis of the signal-to-noise ratio of the synchronous signals found that the highest SNR of the field potential signal could reach 41.0 dB, and the highest SNR of the beating signal could reach 43.1 dB( Figure 17 ). These results indicate that with the synergistic effect of the self-made acquisition device, TENG-MEA has excellent sensing ability and anti-interference ability, and can record high-quality mechano-electric coupling signals of cardiomyocytes. To further evaluate the dynamic stability and reliability of TENG-MEA in long-term signal monitoring, we analyzed 10 hours of continuously recorded data. By statistically analyzing the periods of the two signals and plotting an XY scatter plot, the linear fitting results showed that the beating signals and field potential signals obtained by TENG-MEA were highly consistent in terms of period (frequency). In addition, taking the peak points of the signals as the time reference points, we extracted 0.5 seconds of data for coincidence analysis. These signals showed a high degree of consistency on the time axis, indicating that the electrical activity and mechanical activity of cardiomyocytes in the stable state had good repeatability and synchrony.

[0126] Figures 9 to 14 They are continuous change curves of signals and parameters under drug interventions of different concentrations. Analysis of the changes in the parameters of the beating signals and field potential signals of cardiomyocytes under quinidine interventions at concentrations of 10 μM, 20 μM, and 40 μM.

[0127] Figure 9 They are field potential curves (A) and beating signal curves (B) within 5 minutes before quinidine intervention and 30 minutes after intervention at different concentrations (10 μM, 20 μM, 40 μM).

[0128] Figure 10 They are enlarged displays of the waveforms of the field potential (A) and beating signal (B) of cardiomyocytes under quinidine interventions at different concentrations.

[0129] Figure 11 They are comparisons of the typical waveform characteristics of the field potential signal (A) and beating signal (B) at different concentrations.

[0130] Figure 12In A, it is the curve of the change of the amplitude of the field potential signal over time under the control group and different concentrations of intervention. Figure 12 In B, it is the curve of the change of the amplitude of the pulsation signal over time under the control group and different concentrations of intervention. Figure 13 In A, it is the curve of the change of the pulsation frequency of cardiomyocytes over time under the control group and different concentrations of intervention. Figure 13 In B, it is the curve of the change of the duration of the field potential over time under the control group and different concentrations of intervention. Figure 13 In C, it is the curve of the change of the systolic time over time under the control group and different concentrations of intervention. Figure 13 In D, it is the curve of the change of the diastolic time over time under the control group and different concentrations of intervention.

[0131] Figure 14 In A, it is the statistical bar chart of the amplitudes of the pulsation signal and the field potential signal. Figure 14 In B, it is the statistical bar chart of the frequencies of the pulsation signal and the field potential signal. Figure 14 In C, it is the statistical bar chart of the systolic time and the diastolic time. Figure 14 In D, it is the statistical bar chart of the duration of the field potential. The statistical chart analyzes 800 data points for each group of samples and is expressed as mean±SD. One-way ANOVA shows significant differences, and **** in the figure indicates p<0.0001.

[0132] We continuously intervened in cardiomyocytes with different doses of Quinidine to further evaluate the ability of TENG-MEA in drug screening and cardiac toxicity assessment. Quinidine is a classic class I antiarrhythmic drug that affects the electrical and mechanical activities of cardiomyocytes by blocking the fast sodium channel (I_Na) and the rapid delayed rectifier potassium channel (I_Kr) of cardiomyocytes, prolonging the action potential duration and the effective refractory period. In the experiment, cardiomyocytes were continuously intervened with doses of 10 μM, 20 μM, and 40 μM, and the field potential signals ( Figure 9 In A) and the pulsation signals ( Figure 9 In B) before intervention (control group) and during intervention were continuously recorded using TENG-MEA. Figure 10 A and B in it show the comparison of the waveforms of the field potential and the pulsation signal for 2 consecutive seconds at the 30th minute after different-dose interventions with those before intervention. The field potential ( Figure 11 In A) and the pulsation signal ( Figure 11The typical waveforms in [B] intuitively reflect the responses of cardiomyocytes in terms of time and amplitude at different doses. At a concentration of 40 μM, the initial rapid depolarization spike of the field potential signal disappears, which may be related to the inhibition of depolarization caused by sodium channel block. As the concentration increases, the amplitude of the pulsation signal significantly weakens, the turning point at the junction of the diastolic signal and the systolic signal gradually elongates, and the signal period significantly prolongs. The amplitude of the field potential changes less at different doses, being 0.17 mV, 0.18 mV, 0.16 mV, and 0.11 mV respectively ( Figure 12 in [A]). Different from the relative stability of the field potential, the amplitude of the pulsation signal generally shows a downward trend, being 9.34 pA, 7.81 pA, 6.91 pA, and 5.70 pA respectively ( Figure 12 in [B]), reflecting the weakening of mechanical contractility.

[0133] Under normal physiological conditions, cardiomyocytes have a stable pulsation frequency, which reflects the normal operation of their ion channels, calcium handling, and sarcoplasmic reticulum function, enabling effective electro-mechanical coupling. In the control group, the pulsation frequency of cardiomyocytes was 3.8 Hz. After adding different concentrations of quinidine, the pulsation frequency of cardiomyocytes rapidly decreased. Subsequently, the pulsation frequency of the cells slowly recovered and reached 3.4 Hz, 2.8 Hz, and 2.5 Hz respectively at 30 minutes. The duration of the field potential in the initial control group was basically stable at 126 ms. Under quinidine intervention, the duration of the field potential showed an increasing trend, and the higher the concentration, the greater the increase. At 30 minutes after intervention, the durations of the field potential were 131 ms, 139 ms, and 171 ms respectively ( Figure 13 in [B]). This is consistent with the mechanism of action of quinidine in blocking potassium channels and prolonging the action potential duration. For the systolic time, it remained stable at 148 ms in the control group. It rapidly decreased in the early stage of intervention and then tended to be stable. At 30 minutes after intervention, the systolic times were 189 ms, 232 ms, and 255 ms respectively ( Figure 13 in [C]). The diastolic time showed a similar change trend to the systolic time. It was stable at 101 ms in the control group and was 108 ms, 122 ms, and 148 ms respectively at 30 minutes after drug intervention ( Figure 13 in [D]).

[0134] We plotted the amplitudes of the pulsation signal and the field potential signal of cardiomyocytes before drug administration and at 30 minutes after intervention at different doses ( Figure 14 in [A]), the pulsation frequency ( Figure 14 in [B]), the systolic time and the diastolic time ( Figure 14 in [C]), and the duration of the field potential ( Figure 14Bar graph in D). By calculating the Pearson Correlation Coefficient between parameters at different doses, we found that the significance level p < 0.0001, indicating significant differences between these data. These results suggest that the signals recorded by TENG-MEA reflect that the mechanical contraction time is affected by the electrical activity duration and ion channel activity. This is consistent with the mechanism of action of quinidine, further verifying the sensitivity and accuracy of TENG-MEA in evaluating the effects of drugs. These experiments have verified the potential of TENG-MEA in evaluating cardiac safety and the effects of ion channel blocking drugs, providing a powerful tool for future drug development and safety testing.

[0135] Figure 15 Results of the long-term stability test of the sensor (signal display of cardiomyocytes incubated once for 20 days of growth). Thanks to the excellent biocompatibility of TENG-MEA, cardiomyocytes exhibit a survival cycle of up to 20 days on its surface. We recorded the beating signals and field potential signals of cardiomyocytes for multiple consecutive days and conducted a systematic analysis of the data. The results showed that the beating frequency of cardiomyocytes gradually decreased from 4.6 Hz on the 2nd day to 2.3 Hz on the 9th day and stabilized at 1.8 Hz after the 15th day. This decrease in frequency may be closely related to the dynamic changes in cell activity and metabolic levels. In terms of the beating signal, its peak gradually increased from 0.4 pA on the 2nd day, reached a maximum of 2.9 pA on the 9th day, then gradually decreased to 1.8 pA on the 12th day, and dropped to 0.2 pA on the 20th day. This changing trend reflects the process of the mechanical contraction ability of cardiomyocytes gradually decaying after reaching its peak on the 8th - 10th day. In terms of the field potential signal, its amplitude gradually increased from 0.09 mV on the 2nd day, reached a maximum of 0.33 mV on the 8th day, then slightly decreased to 0.25 mV and remained at this level until the 16th day, and finally dropped to 0.06 mV on the 20th day. At the same time, the duration of the field potential gradually increased from 140 ms on the 1st day, reached 162 ms on the 9th day, and reached a maximum of 216 ms on the 16th day, remaining at a high level of 207 ms until the 20th day. These data indicate that although the mechanical contraction ability of cardiomyocytes gradually weakens over time, their electrical activity characteristics remain relatively stable for a long time, further verifying the reliability and stability of TENG-MEA in long-term cardiomyocyte culture and signal monitoring.

[0136] Figure 16This is the test result of the long-term stability of the sensor. To verify the reusability of the TENG-MEA, we used the same sensor to conduct multiple independent cell culture experiments (A)(B)(C), and calculated the SNR of the recorded signals in 3 of these experiments (D). The results showed that the SNR change of the pulsation signal was less than 2.2 dB (<8.8%) and the SNR change of the field potential signal was less than 3.2 dB (<9.3%) in the 3 experiments, indicating the reusability of the TENG-MEA.

[0137] The following describes the implementation steps of the present invention in detail in combination with embodiments and drawings:

[0138] 1. Device preparation;

[0139] 1.1 Preparation of the MEA layer (i.e., microelectrode array);

[0140] Step 1: Molding of the PDMS substrate;

[0141] Mix PDMS and the curing agent at a mass ratio of 10:1, spin-coat it on an acrylic substrate (diameter 5 cm, thickness 2 mm), at a rotation speed of 4000 rpm for 60 s. Cure at 40 °C for 10 hours to form a PDMS film with a thickness of 10 μm.

[0142] Step 2: Patterning of the gold electrodes;

[0143] Cover the surface of the PDMS film with a photolithography mask, sputter a gold layer (100 nm Au), with a sputtering power of 300 W for 120 s. Remove the mask to form a microelectrode array with an annular distribution (outer diameter 12 mm), the size of a single electrode is 200 μm in length × 50 μm in width, and the width of the lead-out wire is 200 μm.

[0144] Step 3: Electrode insulation and peeling;

[0145] Spin-coat PDMS to cover the non-contact area of the electrodes, and form an insulating layer after curing. Immerse the PDMS film in absolute ethanol, cut it into a 20 mm × 20 mm square according to the electrode edge for standby.

[0146] 1.2 Preparation of the NP-TENG layer (i.e., triboelectric nanogenerator);

[0147] Step 4: Preparation of CNT / PDMS micro-columns;

[0148] Mix carbon nanotubes (CNT) and PDMS at a mass ratio of 1:9, and ultrasonically disperse for 30 minutes to form a homogeneous slurry. Spin-coat the slurry onto the surface of a PET porous membrane (pore size 1 μm, thickness 11 μm) at a rotation speed of 1000 rpm for 200 s. After scraping off the excess slurry, cure at 40 °C for 10 hours. Use oxygen plasma etching (power 100 W, oxygen flow rate 80 sccm) for 1.5 hours to form a microcolumn array with a height of 3 - 5 μm and a diameter of 1 μm.

[0149] Step 5: Conductive treatment of the friction layer;

[0150] Sputter a gold layer (thickness 50 nm, sputtering power 300 W, time 60 s) on the surface of the microcolumns to enhance the charge transfer efficiency. Perform corona charging treatment on the FEP film (thickness 10 - 50 μm is acceptable) to increase the surface charge density. Then sputter a gold layer (thickness 50 nm, sputtering power 300 W, time 60 s) on the FEP surface.

[0151] 1.3 Sensor assembly;

[0152] Step 6: Integration of MEA and PCB;

[0153] Attach the prepared MEA layer to the front side of the PCB substrate, and use conductive silver paste (model Agar 2900) to connect the electrodes to the PCB pads, and cure at 80 °C for 2 hours.

[0154] Step 7: Construction of the negative pressure chamber;

[0155] Fix a quartz tube (inner diameter 15 mm, height 15 mm) at the center of the PCB, and coat the surrounding with PDMS for sealing. Stack the microcolumn friction layer (CNT / PDMS side up) and the FEP friction layer (gold side up) in sequence, and adjust the gap between the two to 3 - 5 μm through negative pressure. Use PDMS to encapsulate the outer ring to form a sealed cavity, and extract air through a 32G syringe to adjust the negative pressure to -20 Pa.

[0156] Step 8: Biocompatibility treatment;

[0157] Rinse the cavity with 75% ethanol and sterilize with ultraviolet light for 2 hours. Drop a 10 ng / mL fibronectin solution onto the PDMS surface and incubate at 37 °C for 1 hour to promote the adhesion of cardiomyocytes.

[0158] 2. Signal acquisition system setup;

[0159] 2.1 Circuit design;

[0160] Step 9: TENG signal amplification module;

[0161] A transimpedance amplifier circuit (TIA) is constructed using an LTC6269 operational amplifier, with a parallel feedback resistor (10 GΩ) and capacitor (1 pF), achieving a current gain of 10^10 times and a bandwidth of 0.4 - 16 Hz.

[0162] Step 10: Field potential signal processing module;

[0163] An AD8244 high-input-impedance buffer is used, in conjunction with a band-pass filter (0.5 - 800 Hz) and an inverting amplifier (gain 300 times), to suppress high-frequency noise.

[0164] Step 11: Data acquisition and transmission;

[0165] A 24-bit ADC chip (AD7124) is used for analog-to-digital conversion (sampling rate greater than twice the signal, here it is 1950 Hz). The data is transmitted to a computer in real time through an STM32 MCU. On the software side, signal filtering (Butterworth zero-phase filtering) and parameter extraction are implemented based on MATLAB.

[0166] 3. Application implementation example;

[0167] 3.1 Myocardial cell culture and signal recording;

[0168] Step 12: Myocardial cell isolation and seeding;

[0169] Myocardial cells are isolated from 1 - 3-day-old Sprague-Dawley rats (provided by the Experimental Animal Center of Sun Yat-sen University). Before the experiment, the rats are disinfected with 75% ethanol, and the ventricular tissue is quickly excised and washed in ice-cold DMEM medium (SH30022.01b, Thermo Fisher Scientific Inc.). The ventricular tissue is placed in Hanks balanced salt solution (HBSS, 14175095, Thermo Fisher Scientific Inc.) and cut into pieces approximately 1 mm 3Tissue fragments. Then, digestion was performed 10 times at 37 °C for 8 minutes each time using HBSS containing 0.07% trypsin (27250 - 018, Thermo Fisher Scientific Inc.). After each digestion, the digestive enzyme was diluted with 5 volumes of DMEM medium, and the supernatant was collected. The supernatant was centrifuged at 1000 rpm for 5 minutes to collect the precipitated cardiomyocytes. The cardiomyocytes were further purified by differential adhesion twice for 45 minutes in a 37 °C, 5.0% CO2 incubator. The purified cardiomyocytes were resuspended in DMEM medium containing 10% fetal bovine serum (10099141, Thermo Fisher Scientific Inc.), and cell counting was performed. The cell density was adjusted to approximately 2200 cardiomyocytes / mm 2 . Then, the cells were seeded into the glass ring of the TENG - MEA. The seeded TENG - MEA was placed in a 37 °C, 5% CO2 cell culture incubator, and the medium was changed every 24 hours to ensure normal cell growth and function.

[0170] Step 13: Signal synchronization monitoring;

[0171] After 24 hours of culture, the sensor was connected to the acquisition device to record the pulsation signal and field potential signal in real - time. The contact state of the friction layer was dynamically adjusted through the negative pressure chamber to ensure signal stability.

[0172] 3.2 Drug intervention experiment;

[0173] Step 14: Drug response test;

[0174] 1 μM E - 4031 (hERG potassium channel blocker) was added, and it was monitored that the pulsation frequency decreased by 44% and the contractility decreased by 84% within 30 minutes. 10 μM isoproterenol hydrochloride (β - receptor agonist) was added, and the frequency increased by 190% and the contraction time shortened by 41%.

[0175] In the quinidine (Macklin, CAS number 56 - 54 - 2) drug intervention experiment, we used the method of gradually increasing the drug concentration to observe its effects on the electrophysiology and mechanical activities of cardiomyocytes. First, a cell culture medium containing 10 μM concentration of quinidine was prepared and used for the experiment. After recording the 30 - minute data of cardiomyocytes at this concentration, a small amount of high - concentration quinidine solution was added to the medium to gradually increase the concentration of the medium to 20 μM and 40 μM. This method allows us to gradually increase the drug concentration without changing the entire medium, thereby reducing the perturbation of the cell environment and ensuring the continuity and comparability of the experimental conditions.

[0176] Step 15: Long - term stability verification;

[0177] The signal was continuously recorded for 20 days, and the pulsation frequency decreased from 4.6 Hz to 1.8 Hz. When the same device was used to culture cells three times, the signal-to-noise ratio of the mechanical pulsation signal and the field potential remained stable (fluctuation < 9.3%), demonstrating the durability of the device.

[0178] This application proposes the synergistic effect of microcolumn arrays and negative pressure:

[0179] The height of the micro-columns (3 - 5 μm) is controlled by the plasma etching time, and the friction layer is adaptively separated in combination with a negative pressure chamber (-20 Pa to -80 Pa), with a sensitivity of 1.146 pA / Pa.

[0180] High-fidelity acquisition of dual-modal signals:

[0181] The impedance of the MEA electrode is as low as 4.05 kΩ @ 1 kHz. In combination with a TENG transimpedance amplifier circuit (gain of 10^10 times), the signal-to-noise ratio breaks through 43.1 dB (pulsation signal) and 41.0 dB (field potential signal).

[0182] Long-term biocompatibility design:

[0183] The PDMS substrate and fibronectin modification support the survival of cardiomyocytes for 20 days, and the SNR fluctuation is < 3.2 dB after the sensor is reused three times.

[0184] Through the above steps, those skilled in the art can reproduce the device according to the content of the present invention and achieve high-precision synchronous monitoring of the mechano-electric signals of cardiomyocytes.

[0185] The method involved in this application includes:

[0186] Manufacture of the MEA component of TENG-MEA: Spin-coat PDMS (4000 rpm, 60 s) on an acrylic plate with a diameter of 5 cm and a thickness of 2 mm, and then cure it at 40 °C for 10 hours. Use a mask to sputter a layer of gold (Au) about 100 nm thick on the cured PDMS film, with sputtering conditions of 300 W for 120 seconds. Cut the sputtered PDMS film into a square, peel it off with absolute ethanol, attach it to the front of the sensor PCB, and dry it at 80 °C for 10 minutes to fix it. Electrically connect the PCB and the Au electrode with conductive silver paste, and then dry it at 80 °C for 2 hours to solidify it. Finally, cover the outer part of the electrode with PDMS to protect the electrode, and then dry it at 80 °C for 2 hours for curing.

[0187] Preparation of the NP-TENG component of TENG-MEA: Carbon nanotubes (CNTs) and PDMS were mixed at a mass ratio of 1:9. The mixture was filled into a PET porous membrane by spin coating (1000 rpm, 200 s), and the surface mixture was scraped off and then cured at 40 °C for 10 hours. Then, an oxygen plasma etching method (100 W, 90 min) was used to expose a columnar structure of 3–5 μm to form an MC substrate. After that, gold (Au) was sputtered on the surface of the microcolumns under the sputtering conditions of 300 W for 90 seconds, and it was cut into a cuboid of 8 mm × 12 mm for standby. Gold (Au) was sputtered on the surface of a 10-μm-thick FEP film under the sputtering conditions of 300 W for 60 seconds and cut into a cuboid of 10 mm × 12 mm for standby.

[0188] Assembly of TENG-MEA: A quartz tube with an inner diameter of 15 mm and a height of 15 mm was placed in the middle of the sensor PCB with the MEA part pasted. PDMS was used to bond between the glass tube and the PCB, and then it was dried at 80 °C for 2 hours for curing. It was flipped so that the quartz tube was at the bottom and the PDMS film was on the top. The back of the MC friction layer (with the microcolumns facing up) was closely attached to the PDMS film. An FEP friction layer was placed above the MC friction layer. Conductive silver paste was used to fix one end of the two friction layers at the electrode of the sensor PCB, and then it was dried at 80 °C for 60 minutes to fix and form an electrical connection. The outside of the friction layer was protected with the cured PDMS, and the outer ring part was encapsulated with PDMS to form a sealed negative pressure cavity, which was dried at 80 °C for 30 minutes. Finally, a 32G injection needle was used to extract the air in the cavity to form a negative pressure. After being rinsed with 75% ethanol, TENG-MEA was placed under ultraviolet light in a biosafety cabinet for disinfection for 2 hours. Before cell seeding, a small amount of 10 ng / mL fibronectin solution (F0895, Sigma) was added to TENG-MEA and incubated in an incubator for 1 hour.

[0189] Scanning electron microscope imaging: Imaging was performed using a Zeiss SUPRA60 field emission gun (FEG) scanning electron microscope (SEM). The samples were imaged at an acceleration voltage of 10 kV and a working distance of 10 mm. Before imaging, the samples were coated with a conductive coating. ImageJ software was used to evaluate the diameter and height of the nanocolumns. High-resolution (2048×1768 pixels) SEM images were used for analysis.

[0190] Fabrication of the signal amplification and acquisition system: According to the parameters such as the internal resistance, frequency, and amplitude of the measured signal, a dedicated signal amplification and acquisition circuit board was designed and fabricated.

[0191] Field potential signal amplification module: The field potential signal of cardiomyocytes first enters a band-pass filter composed of a high-input-impedance buffer ADC8244 for signal filtering, then enters an inverting amplifier OPA2377 for 300-fold amplification, and is restored by a 1-fold inverting amplifier. Then, it enters the pre-anti-aliasing filter of the ADC chip synchronously with the reference voltage.

[0192] NP-TENG signal amplification module: The weak current generated by the contraction signal of cardiomyocytes driving the TENG enters two TIA conversion circuits composed of LTC6269 for synchronous amplification respectively, and then enters the pre-anti-aliasing filter of the ADC chip in the form of a differential signal.

[0193] Analog-to-digital conversion and data processing: Use a high-precision 24-bit ADC chip ADC7124 to convert analog signals into digital signals, and process and transmit the data to a computer through a microcontroller. The reference voltage of the ADC is provided by +2.5V generated by an external chip ADR4525.

[0194] Power supply circuit: The 5V power input from the USB interface is filtered and divided into three paths after filtering: one path enters LM27762 to provide ±2.5V low-noise voltage for the operational amplifier; another path enters ME6210A33 to provide 3.3V voltage for the MCU and other digital circuits; the third path enters REF3033 to provide 3.3V low-noise and low-drift voltage for ADC7124. In addition, a stable power supply module provides positive and negative low-noise voltages to ensure the stable operation of the circuit.

[0195] Cyclic voltammetry measurement of MEA: In order to evaluate the electrochemical performance of MEA, cyclic voltammetry measurement was carried out. The measurement was carried out using an electrochemical workstation (CHI760E, CH Instruments, USA) in a standard three-electrode configuration. A single electrode in MEA was used as the working electrode, a platinum wire was used as the counter electrode, and an Ag / AgCl electrode was used as the reference electrode. The electrolyte was 0.1M PBS (pH 7.4). The potential window was scanned at a scanning rate of 100 mV / s in the range of -0.4V to 0.4V. All measurements were carried out in a Faraday cage at room temperature to minimize external noise interference.

[0196] Electrochemical impedance spectroscopy measurement of MEA: The impedance characteristics of microelectrodes were studied by electrochemical impedance spectroscopy. The measurement used the same electrochemical workstation, with a three-electrode configuration. The selected microelectrode was used as the working electrode, a platinum wire was used as the counter electrode, and an Ag / AgCl electrode was used as the reference electrode. The electrolyte was 0.1M PBS (pH 7.4). The impedance spectrum was recorded in the frequency range of 1Hz to 100kHz, and the amplitude of the AC perturbation was 5mV. All measurements were carried out in a Faraday cage at room temperature to minimize external noise.

[0197] Isolation and culture of cardiomyocytes: All operations complied with relevant regulations. Cardiomyocytes were isolated from 1- to 3-day-old Sprague-Dawley rats (provided by the Experimental Animal Center of XX University). Before the experiment, the rats were disinfected with 75% ethanol, and the ventricular tissue was quickly excised and washed in ice-cold DMEM medium (SH30022.01b, Thermo Fisher Scientific Inc.). The ventricular tissue was placed in Hanks' balanced salt solution (HBSS, 14175095, Thermo Fisher Scientific Inc.) and cut into tissue fragments of about 1 mm 3 in size. Then, HBSS containing 0.07% trypsin (27250-018, Thermo Fisher Scientific Inc.) was used to digest the tissue 10 times at 37 °C, with each digestion lasting 8 minutes. After each digestion, the digestive enzyme was diluted with 5 volumes of DMEM medium, and the supernatant was collected. The supernatant was centrifuged at 1000 rpm for 5 minutes to collect the precipitated cardiomyocytes. The cardiomyocytes were further purified by differential adhesion twice for 45 minutes in an incubator at 37 °C and 5.0% CO2. The purified cardiomyocytes were resuspended in DMEM medium containing 10% fetal bovine serum (10099141, Thermo Fisher Scientific Inc.), and cell counting was performed. The cell density was adjusted to approximately 2200 cardiomyocytes / mm 2 . Then, the cells were seeded into the glass ring of the TENG-MEA. The seeded TENG-MEA was placed in a cell incubator at 37 °C and 5% CO2, and the medium was changed every 24 hours to ensure the normal growth and function of the cells.

[0198] Recording of cardiomyocyte signals: In cardiomyocyte experiments, usually after 24 hours of culture, the cardiomyocytes will adhere to the bottom of the culture medium and start to beat spontaneously. This is because after the cells adapt to the culture environment, they will gradually form functional connections and exhibit autonomous electrical and mechanical activities. To record the beating signals and field potential signals of cardiomyocytes, we connected the TENG-MEA containing cardiomyocytes to a self-made acquisition device to monitor and record the electrophysiological activities and mechanical beats of cardiomyocytes in real time.

[0199] Drug intervention: Prepare 1 μM of E-4031 (MCE, CAS number 113559-13-0) and 10 μM of isoproterenol hydrochloride (aladdin, CAS number 51-30-9) using cell culture medium respectively. Before adding the drugs, record the pulsation signal and field potential signal for 5 minutes as the control. Remove the original culture medium in the TENG-MEA, add 1 mL of the culture medium containing the drugs, and continuously record the pulsation signal and field potential signal for 30 minutes. In the quinidine (Macklin, CAS number 56-54-2) drug intervention experiment, we used the method of gradually increasing the drug concentration to observe its effects on the electrophysiology and mechanical activities of cardiomyocytes. First, prepare a cell culture medium containing 10 μM of quinidine and use it for the experiment. After recording the data of cardiomyocytes at this concentration for 30 minutes, add a small amount of high-concentration quinidine solution to the culture medium and gradually increase the concentration of the culture medium to 20 μM and 40 μM. This method allows us to gradually increase the drug concentration without replacing the entire culture medium, thereby reducing the perturbation to the cell environment and ensuring the continuity and comparability of the experimental conditions.

[0200] Data processing and analysis: Use a program written in MATLAB (MATLAB Inc., Version 2024a) to filter, denoise, and extract feature points from the collected signals. The parameters are set as the sampling rate of 1950 Hz, the field potential frequency of 0.4 - 800 Hz, and the pulsation signal frequency of 0.4 - 16 Hz. The filtering coefficients are generated by a Butterworth filter, and the zero-phase filtering function filtflit is used to process the data. Extract the position information of the peak, trough, and the end point of the field potential duration of the field potential signal, and the position information of the peak, trough, the starting point of contraction, and the end point of diastole of the pulsation signal from the filtered signals as needed. According to the sampling rate of the signal being 1950 Hz, calculate the corresponding key parameters such as signal frequency, signal amplitude, field potential duration, contraction time, and diastole time based on these feature points. Conduct a statistical analysis on the data under different drug intervention conditions, calculate the mean, standard deviation, and correlation, and further plot the change curves and bar charts.

[0201] In some alternative embodiments, the functions / operations mentioned in the block diagrams may not occur in the order mentioned in the operation diagrams. For example, depending on the functions / operations involved, two consecutively shown blocks can actually be executed substantially simultaneously or the blocks can sometimes be executed in the reverse order. Additionally, the embodiments presented and described in the flowcharts of the present invention are provided by way of example for a more comprehensive understanding of the technology. The disclosed methods are not limited to the operations and logical flows presented herein. Alternative embodiments are anticipated where the order of various operations is changed and where the sub-operations described as part of a larger operation are executed independently.

[0202] In addition, although the present invention has been described in the context of functional modules, it should be understood that, unless otherwise stated to the contrary, one or more of the functions and / or features may be integrated in a single physical device and / or software module, or one or more functions and / or features may be implemented in separate physical devices or software modules. It should also be understood that a detailed discussion of the actual implementation of each module is not necessary for an understanding of the present invention. Rather, given the attributes, functions, and internal relationships of the various functional modules in the devices disclosed herein, the actual implementation of the modules will be understood within the ordinary skill of an engineer. Thus, those skilled in the art can implement the present invention as set forth in the claims without undue experimentation. It should also be understood that the particular concepts disclosed are merely illustrative and are not intended to limit the scope of the present invention, which is determined by the full scope of the appended claims and their equivalents.

[0203] If the functions are implemented in the form of software function units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or a part of this technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several programs for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of the present invention. The aforementioned storage medium includes: various media such as USB flash drives, mobile hard disks, read-only memories (ROMs), random access memories (RAMs), magnetic disks, or optical discs that can store program codes.

[0204] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a sequenced list of executable programs for implementing logical functions, and can be specifically implemented in any computer-readable medium for use by a program execution system, apparatus, or device (such as a computer-based system, a system including a processor, or other systems that can retrieve and execute programs from the program execution system, apparatus, or device), or in conjunction with these program execution systems, apparatus, or devices. For the purposes of this specification, a "computer-readable medium" can be any device that can contain, store, communicate, propagate, or transport a program for use by or in conjunction with a program execution system, apparatus, or device.

[0205] In the foregoing description of this specification, the descriptions referring to the terms "one embodiment / example", "another embodiment / example" or "certain embodiments / examples", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0206] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0207] The above is a specific description of the preferred embodiments of the present invention, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent deformations or substitutions without departing from the spirit of the present invention, and these equivalent deformations or substitutions are all included within the scope defined by the claims of the present invention.

Claims

1. A preparation method of a flexible sensor, characterized in that, Including the following steps: Preparing a microelectrode array and a triboelectric nanogenerator; Assembling the microelectrode array, the first flexible film, and the PCB from top to bottom, and providing a hollowed-out area on the PCB; The first flexible film corresponding to the hollowed-out area is used to place the biological cells to be measured; Encapsulating the first flexible film and the second flexible film into a first cavity, placing the triboelectric nanogenerator in the first cavity, and adjusting the pressure in the first cavity to be less than the standard atmospheric pressure to obtain a flexible sensor.

2. The preparation method of the flexible sensor according to claim 1, characterized in that, Preparing a triboelectric nanogenerator, including: Preparing a first friction layer and a second friction layer, the first friction layer being close to the PCB, and the second friction layer being close to the bottom of the second flexible film; Providing a micro-structure array between the first friction layer and between the first friction layer and the second friction layer to combine the biological cells to achieve contact and separation of the friction layers.

3. The preparation method of the flexible sensor according to claim 2, wherein The micro-structure array includes a micro-column array, and the micro-column array is prepared through the following steps: Covering a PET porous filter membrane on a substrate and performing curing treatment; Spin-coating a composite material on the PET porous filter membrane and performing curing treatment; Etching the PET porous filter membrane through an oxygen plasma etching method to obtain the micro-column array.

4. The method for preparing a flexible sensor according to claim 3, characterized in that, The composite material includes CNT and PDMS, and the method further: Setting the multiple of the mass of the PDMS to the mass of the CNT; Adjusting the column height through an oxygen plasma etching method.

5. The preparation method of the flexible sensor according to claim 1, wherein, The flexible sensor measures mechanical signals through the following steps: Adjusting the pressure by removing the gas in the first cavity; Combining the elastic deformation characteristics of the first flexible film, converting the contraction force of the biological cells into a field potential signal, and then determining the mechanical signal.

6. The preparation method of the flexible sensor according to claim 1, wherein The method further includes: Performing surface sputtering gold layer treatment on the micro-structure array and performing corona charging treatment on the second friction layer; Electrically connecting the two friction layers of the triboelectric nanogenerator and the microelectrode array electrodes to the PCB electrodes through conductive paint; A quartz tube is provided on the PCB; the quartz tube is used for culturing the biological cells.

7. The preparation method of the flexible sensor according to claim 1, wherein, The preparation of the microelectrode array includes: Using a spin-coating method to obtain a first flexible film; Obtaining the electrode and wire patterns of the microelectrode array through sputter deposition of a conductive metal; Insulating the wires and exposing part of the electrodes to obtain the microelectrode array.

8. A monitoring system based on a flexible sensor, characterized in that, Including: A flexible sensor prepared through the preparation method of the flexible sensor according to any one of claims 1 to 7, a first amplification module, a second amplification module, and a control module; The first amplification module is used to receive the field potential signal of the flexible sensor, and the second amplification module is used to receive the mechanical signal of the flexible sensor.

9. The monitoring system based on a flexible sensor according to claim 8, characterized in that The first amplification module includes: A conditioning circuit and an amplification circuit; the field potential signal is transmitted to the amplification circuit through the conditioning circuit.

10. The monitoring system based on a flexible sensor according to claim 8, wherein The second amplification module includes a transimpedance amplification circuit.

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