Construction method based on nerve probe and array culture chip and nerve electrophysiological toxicity assessment method and equipment

Abstract

The invention relates to a construction method based on a nerve probe and an array culture chip and a nerve electrophysiological toxicity evaluation method and equipment, and the construction method comprises the steps: obtaining a substrate material, a probe material and a chip circuit layout; according to a substrate material, a probe material and a chip circuit layout, a traditional micro-electro-mechanical system manufacturing stripping process and a micro-pattern seal technology are adopted for processing, and a chip substrate with array nerve probes is constructed; and constructing a multi-channel nerve cell culture room corresponding to the array nerve probe on the chip substrate to obtain the multi-channel chip with the nerve probe and array culture. According to the construction method, an ultrathin nerve probe array and an array type culture room layer are integrated to form a complete multi-channel chip, and the multi-channel chip can realize high-throughput nerve electrophysiological toxicity detection; full-process automation and high throughput from nerve cell culture to toxicity detection can be realized on a multi-channel chip, so that the test cost, time and complexity are remarkably reduced.

Description

Technical Field

[0001] The present application relates to the field of chip technology, and in particular to a construction method based on neural probes and array culture chips, and a method and device for evaluating neuroelectrophysiological toxicity. Background Art

[0002] At present, the models used in in vitro neurotoxicity testing mainly include mammalian models, non-mammalian models and in vitro cell models. Mammalian models are expensive, time-consuming, difficult to construct, and not suitable for high-throughput testing; non-mammalian models such as zebrafish and fruit fly models have short cycles and low costs, but the evaluation results are limited in accuracy and difficult to repeat; in vitro cell models mostly rely on the biological behavior of nerve cells for evaluation, and the results are relatively single and limited, making it difficult to detect the toxic effects of chemicals on the brain's neuroelectric physiological signals. Summary of the invention

[0003] The present application provides a construction method based on neural probes and array culture chips and a neuroelectrophysiological toxicity assessment method and device, which are used to solve the technical problems of existing neurotoxicity test models, such as high cost, long time consumption, and complex construction.

[0004] In order to achieve the above objectives, this application provides the following technical solutions:

[0005] In one aspect, a method for constructing a neural probe and array culture chip is provided, comprising the following steps:

[0006] Obtaining a substrate material, a probe material, and a chip circuit layout, and processing the substrate material, the probe material, and the chip circuit layout using a conventional MEMS manufacturing peeling process and a micro-pattern stamping technology to construct a chip substrate with an array of neural probes;

[0007] Constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate to obtain a multi-channel chip with neural probes and array culture; the multi-channel neural cell culture chamber is connected to the electrode of the chip substrate;

[0008] Among them, the contents of constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate include: using the traditional micro-electromechanical system manufacturing peeling process to process the chip substrate to obtain a multi-channel neural cell culture chamber; the multi-channel neural cell culture chamber includes an entrance and exit, a cell planting layer, a gradient layer and a plurality of culture chamber layers corresponding to the array neural probe, the entrance and exit are connected through the gradient layer, the cell planting layer is arranged above each of the culture chamber layers, the gradient layer is arranged below all the culture chamber layers, and the gradient layer is connected to the electrode of the chip substrate.

[0009] Preferably, the substrate material, the probe material and the chip circuit layout are processed by using a conventional MEMS manufacturing peeling process and a micro-pattern stamping technology to construct a chip substrate with an array of neural probes, comprising:

[0010] According to the chip circuit layout, a conventional MEMS manufacturing stripping process is used on the substrate material to perform n×n array electrode circuit routing layout to obtain a substrate to be modified with a neural probe assembly position; and a solution method is used to synthesize the probe material to obtain a neural probe;

[0011] Modifying the substrate to be modified by a silanization coupling agent to obtain a substrate to be printed;

[0012] The micro-pattern stamp technology is used to print the neural probes at each neural probe assembly position of the substrate to be printed, thereby obtaining a chip substrate with array neural probes.

[0013] Preferably, the cell planting layer is used to plant cells and limit the range of the culture solution; and / or, the inlet and outlet are used to introduce chemical molecules into the gradient layer; the gradient layer is used to diffuse the introduced chemical molecules to construct a horizontal gradient in the XY plane, so as to provide each culture chamber layer with a gradually decreasing concentration of input chemical molecules.

[0014] Preferably, the culture chamber layer is made of a scaffold or a hydrogel material, and the thickness of the culture chamber layer is 250 µm; and / or the thickness of the gradient layer is 400 µm.

[0015] In another aspect, a method for evaluating neuroelectrophysiological toxicity based on a neural probe and an array culture chip is provided, comprising the following steps:

[0016] Obtaining a multi-channel chip constructed by the above-mentioned construction method based on the neural probe and array culture chip;

[0017] Constructing a gradient target compound in the multi-channel chip, collecting and classifying the neurophysiological signals generated by the neural probe of the gradient target compound at different gradient exposure concentrations to obtain collected data; the neurophysiological signals include action potential signals and local field potential signals;

[0018] A neurotoxicity standard is obtained, and the collected data is analyzed according to the neurotoxicity standard to obtain a toxicity grade and a neuroelectrophysiological toxicity assessment standard corresponding to the gradient target compound.

[0019] Preferably, constructing a gradient target compound in the multi-channel chip comprises:

[0020] Establishing an array neural network in the multi-channel chip;

[0021] Obtaining the test compound and setting parameters of different gradient exposure concentrations, and inputting the test compound into the array neural network according to the setting parameters to obtain a gradient target compound;

[0022] Among them, establishing an array neural network in the multi-channel chip includes: adding at least 80,000 neurons to the cell planting layer for seeding, and the cells of the cell planting layer will fall into the multi-channel independent culture chamber layers respectively through gravity. After at least 3 weeks of cell inoculation / induction in the culture chamber layer, an array neural network is obtained.

[0023] Preferably, the test compound includes heavy metal lead, nitric oxide and / or neurotoxin chemical molecules of tetrodotoxin.

[0024] Preferably, the neuroelectrophysiological signals generated by the neural probe of the gradient target compound at different gradient exposure concentrations are collected and classified to obtain the collected data including:

[0025] Amplify and bandpass filter the collected neural electrophysiological signals to obtain processed data;

[0026] Using the EM algorithm to perform spike sorting on the action potential signal of the processed data to obtain sorted signal data;

[0027] Acquiring a signal classification standard, and performing peak classification on the signal data according to the signal classification standard to obtain classified data;

[0028] Electrophysiological features are extracted for each type of spike in the classified data to obtain collected data consisting of electrophysiological features of all spike classifications.

[0029] Preferably, the signal classification criteria include the peak value, rise duration and fall duration of the spike; and / or the electrophysiological characteristics include frequency, amplitude, resonance, duration, rise time, decay time, peak-to-peak interval, peak energy, signal center frequency and information entropy.

[0030] In another aspect, a terminal device is provided, comprising a processor and a memory;

[0031] The memory is used to store program codes and transmit the program codes to the processor;

[0032] The processor is used to execute the above-mentioned neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips according to the instructions in the program code.

[0033] The method for constructing a neural probe and array culture chip and the method and device for evaluating neuroelectrophysiological toxicity include obtaining substrate material, probe material and chip circuit layout, processing the substrate material, probe material and chip circuit layout using a conventional micro-electromechanical system manufacturing stripping process and a micro-pattern stamping technology to construct a chip substrate with an array neural probe; constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate to obtain a multi-channel chip with neural probe and array culture; the multi-channel neural cell culture chamber is connected to the electrode of the chip substrate; wherein, the content of constructing the multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate includes: processing the chip substrate using a conventional micro-electromechanical system manufacturing stripping process to obtain a multi-channel neural cell culture chamber; the multi-channel neural cell culture chamber includes an inlet and outlet, a cell planting layer, a gradient layer and a plurality of culture chamber layers corresponding to the array neural probe, the inlet and outlet are connected to the gradient layer, the cell planting layer is arranged above each culture chamber layer, the gradient layer is arranged below all culture chamber layers, and the gradient layer is connected to the electrode of the chip substrate.

[0034] It can be seen from the above technical scheme that the present application has the following advantages: the construction method based on neural probes and array culture chips constructs a multi-channel neural cell culture chamber on a chip substrate composed of array neural probes, thereby integrating an ultra-thin neural probe array with an array culture chamber layer to form a complete multi-channel chip. The multi-channel chip can realize high-throughput neuroelectrophysiological toxicity detection, and the full process automation and high-throughput quantification from neural cell culture to toxicity detection can be realized on the multi-channel chip, thereby significantly reducing the test cost, time and complexity, and solving the technical problems of existing neurotoxicity test models such as high cost, long time consumption and complex construction.

[0035] The neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips uses the array neural probes and array culture layer chambers of a multi-channel chip to construct gradient target compounds for the test compound, thereby reducing experimental material consumption and animal use, and lowering testing costs; the neural probes of the multi-channel chip output neuroelectrophysiological signals of the test compound at different gradient exposure concentrations for collection, and the collected data are analyzed to achieve rapid assessment of the neurotoxicity of a large number of compounds, thereby improving test efficiency and shortening the test cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0037] Figure 1 This is a flowchart of the steps of the method for constructing a neural probe and array culture chip according to an embodiment of the present application;

[0038] Figure 2 This is a schematic diagram of the structure of a multi-channel neural cell culture chamber in the construction method based on neural probes and array culture chips described in the embodiments of the present application;

[0039] Figure 3 A schematic diagram of a multi-channel chip for conducting a neurotoxicity test in a method for constructing a neural probe and array culture chip according to an embodiment of the present application;

[0040] Figure 4 This is a schematic structural diagram of a chip substrate having an array neural probe in the method for constructing a neural probe and array culture chip described in an embodiment of the present application;

[0041] Figure 5 A schematic diagram of a neural probe collecting signals in a construction method based on a neural probe and an array culture chip described in an embodiment of the present application;

[0042] Figure 6 A schematic diagram of a neural probe collecting signals in a construction method based on a neural probe and an array culture chip described in an embodiment of the present application;

[0043] Figure 7 This is a flowchart of the steps of the neuroelectrophysiological toxicity assessment method based on the neural probe and array culture chip described in the embodiments of the present application;

[0044] Figure 8 This is a flow chart of the neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips described in the embodiments of the present application;

[0045] Fig. 9 A schematic diagram of the construction of an array neural network in the neuroelectrophysiological toxicity assessment method based on a neural probe and an array culture chip described in an embodiment of the present application;

[0046] Fig.10 A schematic diagram showing that the neuroelectrophysiological response in the neuroelectrophysiological toxicity assessment method based on a neural probe and an array culture chip described in an embodiment of the present application has ultra-sensitivity and specificity;

[0047] Fig.11 A schematic diagram of a terminal device described in an embodiment of the present application. DETAILED DESCRIPTION

[0048] In order to make the purpose, features, and advantages of the invention of this application more obvious and easy to understand, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the embodiments described below are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0049] In the description of the embodiments of the present application, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present application, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

[0050] In the embodiments of the present application, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral one; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0051] At present, the methods for in vitro neurotoxicity testing are mainly based on three categories: mammalian models, non-mammalian models and in vitro cell models.

[0052] The mammalian model is the standard model in the current neurotoxicity assessment of chemicals, with high and wide recognition. Its standards include traditional neurodevelopmental toxicity test methods based on experimental animals (such as OPPTS 870.6300) and TG426 issued by the Organization for Economic Cooperation and Development (OCED). These standards generally require the use of living mammals for neurotoxicity assessment, such as mice, rabbits, dogs, monkeys, etc. However, neurotoxicity assessment based on mammalian models has disadvantages such as long experimental cycle, high cost, and low throughput, which makes it difficult to meet the needs of neurotoxicity detection of a wide range of high-throughput compounds.

[0053] Due to the limitations of mammalian experimental models, various alternative non-mammalian models have begun to emerge in recent years, such as zebrafish models and fruit fly models. One of the most important advantages of non-mammalian models is that they develop quickly, have short cycles, and are small in size, which can significantly shorten the neurotoxicity assessment cycle and reduce costs. However, neurotoxicity assessment based on non-mammalian models also has some disadvantages, such as short time windows, limited and difficult to repeat assessment results, and difficult to observe samples, which limits the development of related detection and assessment technologies.

[0054] Compared with other models, in vitro cell models have the advantages of being simple, efficient, and easy to operate, and can be used to model and evaluate key events in the neural development process, including evaluating the toxic effects of compounds on early neuronal cell proliferation and metabolism, neural differentiation, cell migration, axonal dendrite growth and synaptogenesis, glial cell differentiation, and myelination. Reasonable selection of cell models can not only evaluate the neurotoxic mechanism of compounds from different dimensions, but also simplify the evaluation process, making it low-cost and high-throughput. Therefore, for drug synthesis and large-scale neurotoxicity screening, cell experimental models are low-cost, easy to observe, and have rich modeling types. They are suitable for the development of high-throughput array technology and are one of the better candidates. However, in vitro cell models only reflect the local physiological functional characteristics of the nervous system and cannot reflect the comprehensive toxicity level of the compound to the nervous system.

[0055] Most current neurotoxicity assessment technologies generally use the biological behavior of the nervous system as the benchmark for toxicity assessment, but the assessment results have the problem of single parameters and insensitive toxicity results. As a functional feature of the brain neural network, the electrical physiological signals of nerve cells are extremely sensitive to the environment and the state of cells, and can be used to assess the overall health of the nervous system. It has been found that a variety of neurotoxic substances will affect the electrical physiological signals of the nervous system. For example, beta amyloid protein can cause changes in neural electrical physiological signals, including the number, frequency, amplitude, phase, etc. of nerve action potentials, and produce toxicity to the brain neural network system. However, the current related technologies focus more on the exploration of the toxicity mechanism of compounds, and lack neurotoxicity assessment research based on the corresponding characteristics of neural electrical physiological signals. Moreover, as the terminal functional signal of the brain neural network, the neural electrical physiological signal is sensitive and reliable to environmental changes, but it is limited by the development of related technologies and lacks effective detection technology and evaluation standards. In general, the current existing neurotoxicity detection technology is difficult to meet the current needs of large-scale neurotoxicity detection of new compounds and preclinical neurotoxicity safety assessment due to problems such as difficulty in high-throughput quantification and limited evaluation parameters.

[0056] In recent years, due to the rapid development of micro-nano processing technology and microelectronics technology, various probe systems have been designed to detect neural electrophysiological signals. At present, the most common neural probe technology is to use metal materials / silicon materials as electrodes to record physiological electrical activities in the extracellular environment. Such electrodes still have problems such as easy interference, poor selectivity, poor sensitivity and complex signal separation and processing. Therefore, a new ultra-thin neural probe technology based on the synthesis of two-dimensional monolayer molecular materials has been developed, which can achieve the response amplification of neurotransmitters, transmit and record parameters such as cell action potential frequency, amplitude, waveform and signal propagation speed between cell networks. Combining new probe materials and field effect tube technology, a new "neural probe system" has been developed. This neural probe technology has technical advantages such as small size, high throughput, low detection limit, good biocompatibility, and convenient synthesis, and is suitable for the development of new neurotoxicity detection chips. In terms of high-throughput neural cell culture technology, most of the current research is to use microfluidic technology to achieve the construction of multi-channel culture chambers, which can accurately control, monitor and manipulate the extracellular microenvironment.

[0057] In this field, a multi-channel neural tropism research chip platform can detect the chemotaxis of thousands of 3D neural network microsystems at one time, realizing high-throughput evaluation of the effects of compounds on the nervous system.

[0058] Therefore, neuroelectrophysiological characteristics are the terminal evaluation criteria for the health of nervous system function, but the evaluation results are single parameters and not sensitive enough. For chemical neurotoxicity that is difficult to detect with conventional detection methods, its neurotoxicity can only be discovered through analysis of its electrical characteristic parameters by professional electrophysiological detection technology, but there is still no relevant evaluation technology available.

[0059] Multi-electrode array MEA technology is an advanced method for recording and analyzing the electrophysiological activity of neurons or neural networks. This technology can simultaneously monitor electrical signals at multiple sites, including action potentials (APs) and local field potentials (LFPs), by integrating multiple tiny electrodes on a culture dish or chip. Its advantage is that it can simultaneously record a group of cells and support observations for up to several days without the need for auxiliary equipment such as micromanipulation. Since the neuronal network cultured on the multi-electrode array MEA can survive for up to several months and the multi-electrode array MEA is a non-invasive technology, acute and chronic toxicity and developmental neurotoxicity can be studied. Another advantage of the multi-electrode array MEA is that it can be applied to routine screening work at the industrial level. In fact, with the advancement of high-throughput multi-electrode array MEA technology, it is now possible to place microelectrode arrays at the bottom of each well in a multi-well plate, making it possible to evaluate 200 drugs per day. In 2018, Jenna DStrickland et al. evaluated the effects of 1055 chemicals in the EPAI phase ToxCast library on neural function and cell health in order to demonstrate the practicality of the microelectrode array (MEA)-based method as a rapid neurotoxicity screening tool. The results suggest that large-scale functional screening using neural networks on multi-electrode array MEAs can fill a critical gap in the ToxCast assay results for assessing potential neurotoxicity. The popularity of multi-electrode array MEAs has grown rapidly over the past decade. Multi-electrode array MEA devices have been widely used to measure the activity of neuronal cultures, primarily derived from rodents. Rodent neuronal cultures on multi-electrode array MEAs have been used to investigate physiological mechanisms, study the effects of chemicals in neurotoxicity screening, and model the electrophysiological phenotypes of neuronal networks under different pathological conditions.

[0060] Patent terms:

[0061] MEMS manufacturing processes mainly include surface micromachining technology, bulk micromachining technology, LIGA technology, etc. Surface micromachining technology uses thin film deposition, photolithography, etching and other methods to construct microstructures by adding materials layer by layer on the substrate and finally removing the sacrificial layer.

[0062] ‌Micro-pattern stamp technology‌ is a technology that uses elastic stamp technology to print micro-patterns on the surface of the stamp onto a flat substrate to form a pattern. This technology usually uses an elastic stamp with surface viscosity to transfer the pattern to the target substrate through the micro-patterns on its surface. The elastic stamp is usually composed of a thin layer of silicone, and the back is encapsulated with low-expansion quartz glass to provide rigid support.

[0063] The EM algorithm is an optimization algorithm based on the Maximum Likelihood Estimation (MLE) theory. Given independent observation data and a probability model containing latent variables and parameters, according to the MLE theory, the optimal single-point estimate of the parameter is given when the likelihood of the probability model reaches a maximum value.

[0064] The peak-to-peak interval refers to the time from the maximum value of a peak to the maximum value of the next peak.

[0065] The peak energy refers to the energy integral from the extreme point on the left side of the peak to the extreme point on the right side.

[0066] SEM images are images acquired by a scanning electron microscope (SEM).

[0067] Titanium aluminum carbide Ti3AlC2 is a ceramic material belonging to the MAX phase (M represents a metal element, A represents a main group element, and X represents carbon or nitrogen).

[0068] The embodiments of the present application provide a construction method based on neural probes and array culture chips and a neuroelectrophysiological toxicity assessment method and device, which solve the technical problems of existing neurotoxicity test models such as high cost, long time consumption, and complex construction.

[0069] Embodiment 1:

[0070] Figure 1 This is a flowchart of the steps of the method for constructing a neural probe and array culture chip described in an embodiment of the present application.

[0071] like Figure 1 As shown, the present application embodiment provides a construction method based on a neural probe and an array culture chip, comprising the following steps:

[0072] S1. Obtain substrate materials, probe materials and chip circuit layout, and use traditional MEMS manufacturing peeling process and micro-pattern stamp technology to process the substrate materials, probe materials and chip circuit layout to construct a chip substrate with array neural probes.

[0073] It should be noted that in step S1, the material for making the neural probe and array culture chip is first obtained, and then the obtained material is made into a chip substrate with array neural probe by using the traditional MEMS manufacturing peeling process and micro-pattern stamping technology. In this embodiment, the substrate material can be selected from glass, silicon wafer, etc. The probe material can be selected from MXene, a two-dimensional semiconductor material with a wide band gap, high sensitivity, and good biocompatibility.

[0074] Figure 2This is a schematic diagram of the structure of a multi-channel neural cell culture chamber in the construction method based on neural probes and array culture chips described in an embodiment of the present application.

[0075] S2. Construct a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate to obtain a multi-channel chip with neural probes and array culture; the multi-channel neural cell culture chamber is connected to the electrodes of the chip substrate. Figure 2 As shown, the contents of constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate include: using a traditional micro-electromechanical system manufacturing peeling process to process the chip substrate to obtain a multi-channel neural cell culture chamber; the multi-channel neural cell culture chamber includes an inlet and outlet INLET / DRAIN, a cell planting layer SOURCE, a gradient layer STEPNESS and a plurality of culture chamber layers Cell corresponding to the array neural probe, the inlet and outlet INLET / DRAIN is connected through the gradient layer STEPNESS, the cell planting layer SOURCE is arranged above each culture chamber layer Cell, the gradient layer STEPNESS is arranged below all culture chamber layer Cells, and the gradient layer STEPNESS is connected to the electrode of the chip substrate.

[0076] It should be noted that in step S2, a multi-channel neural cell culture chamber is constructed on the chip substrate constructed in step S1, and an ultra-thin neural probe array is integrated with an arrayed culture chamber layer to form a complete multi-channel chip. The multi-channel chip can realize high-throughput neuroelectrophysiological toxicity detection, and the full process automation and high-throughput quantification from neural cell culture to toxicity detection can be realized on the multi-channel chip. Compared with the shortcomings of existing neurotoxicity testing technologies such as high cost, long time consumption, and complex construction, the construction method based on neural probes and array culture chips can significantly reduce the test cost, time and complexity by constructing a multi-channel chip to perform high-throughput neuroelectrophysiological toxicity detection.

[0077] Figure 3 This is a schematic diagram of conducting neurotoxicity testing on a multi-channel chip in the construction method based on neural probes and array culture chips described in an embodiment of the present application. Figure 3 The a in it refers to the construction of a high-throughput concentration gradient of the compound to be tested; Figure 3 The b in this part refers to the high-throughput measurement of the effects of compounds on neural migration, axons, dendrites, and other characteristics.

[0078] like Figure 2As shown, in the embodiment of the present application, the cell planting layer SOURCE is used to plant cells and limit the range of the culture solution; and / or, the inlet and outlet INLET / DRAIN is used to introduce chemical molecules into the gradient layer STEPNESS; the gradient layer STEPNESS is used to diffuse the introduced chemical molecules to construct a horizontal gradient in the XY plane, so as to provide each culture chamber layer Cell with a gradually decreasing concentration of input chemical molecules. The culture chamber layer Cell is made of a scaffold or a hydrogel material, and the thickness of the culture chamber layer Cell is 250µm; and / or, the thickness of the gradient layer STEPNESS is 400µm.

[0079] It should be noted that the multi-channel neural cell culture chamber includes an array cell culture chamber layer Cell corresponding to the array neural probe, a gradient layer STEPNESS of the target chemical, a cell planting layer SOURCE, and an inlet / outlet INLET / DRAIN. Each layer of the multi-channel neural cell culture chamber is mainly processed into a specific dimethylsiloxane PDMS structure through the micro-electromechanical system MEMS process, and the multi-channel neural cell culture chamber is assembled on the chip substrate of the arrayed neural probe to complete the construction of the electrophysiological array chip (such as a multi-channel chip) of the multi-channel independent neural microsystem. In this embodiment, Figure 3As shown, first, according to the reserved position of the ultra-thin neural probe in the chip substrate, an n×n (such as 8×8) matrix culture chamber layer CELL is designed. The arrayed culture chamber layer CELL is composed of 64 hollow cylinders with a diameter of 2000µm, and the PDMS structure is formed by replicating the micro-patterned SU-8 (Microchem) layer group on the chip substrate (such as a silicon wafer). The cell planting layer SOUCE is further constructed on the culture chamber layer CELL for planting cells and limiting the range of the culture solution. The bottom gradient layer STEPNESS sets the inlet and outlet INLET / DRAIN for introducing chemical molecules. The height of the gradient layer STEPNESS is about 400µm, which can ensure that the molecules only establish a horizontal gradient in the XY plane by diffusion, thereby providing a gradually decreasing input concentration inside each culture chamber layer CELL. The multi-channel chip can be used to test dozens of different gradient steepnesses (along the X-axis) and multiple repetitions (along the Y-axis) through the multi-channel neural cell culture chamber, greatly improving the detection efficiency of compound toxicity detection using multi-channel chips. The high-throughput capability of the multi-channel chip is crucial for screening a wide range of compounds for neurotoxicity, solving the problem that although traditional MEA technology can also perform multi-channel recording, the drug concentration setting is often relatively simple and the gradient setting of concentration is not convenient. The construction method based on the neural probe and array culture chip uses materials such as three-dimensional scaffolds or hydrogels to make the culture chamber layer cells, so that the culture chamber layer cells can simulate the growth environment of neurons in the body, promote neurons to form more complex network structures, and improve the physiological relevance of the test results for the application of the multi-channel chip for compound toxicity detection.

[0080] The present application provides a construction method based on a neural probe and array culture chip, which includes obtaining a substrate material, a probe material and a chip circuit layout, and processing the substrate material, the probe material and the chip circuit layout using a conventional micro-electromechanical system manufacturing stripping process and a micro-pattern stamping technology to construct a chip substrate with an array neural probe; constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate to obtain a multi-channel chip with neural probes and array culture; the multi-channel neural cell culture chamber is connected to the electrodes of the chip substrate; wherein, the content of constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate includes: processing the chip substrate using a conventional micro-electromechanical system manufacturing stripping process to obtain a multi-channel neural cell culture chamber; the multi-channel neural cell culture chamber includes an inlet and outlet, a cell planting layer, a gradient layer and a plurality of culture chamber layers corresponding to the array neural probe, the inlet and outlet are connected through the gradient layer, the cell planting layer is arranged above each culture chamber layer, the gradient layer is arranged below all culture chamber layers, and the gradient layer is connected to the electrodes of the chip substrate. The construction method based on neural probes and array culture chips constructs a multi-channel neural cell culture chamber on a chip substrate composed of arrayed neural probes, integrates an ultra-thin neural probe array with an arrayed culture chamber layer, and forms a complete multi-channel chip. The multi-channel chip can realize high-throughput neuroelectrophysiological toxicity detection, and can realize full-process automation and high-throughput quantification from neural cell culture to toxicity detection on the multi-channel chip, thereby significantly reducing testing cost, time and complexity; and solves the technical problems of existing neurotoxicity test models such as high cost, long time consumption and complex construction.

[0081] Figure 4 This is a schematic structural diagram of a chip substrate having an array neural probe in the method for constructing a neural probe and array culture chip according to an embodiment of the present application, Figure 5 This is a schematic diagram of the neural probe collecting signals in the construction method based on the neural probe and array culture chip described in the embodiment of the present application, Figure 6 This is a schematic diagram of the neural probe collecting signals in the construction method based on the neural probe and array culture chip described in the embodiment of this application. Figure 4 In the figure, a refers to the SEM image of the multilayered MXene; b refers to the XRD pattern of the transformation of titanium Ti3AlC2 to Ti3C2Tx; c refers to the schematic diagram of the biosensor device based on the MXene field effect transistor. Figure 5In the figure, a refers to the schematic diagram of the working principle of using MXene-FET field effect devices to detect action potentials; the left figure of b refers to the fluorescent image of neurons immunostained with βIII-tubulin; the right figure of b refers to the merged image combining bright field and fluorescent channels, showing good compatibility between neuronal cells and MXene micro-patterns. Scale bar, 100μm; c refers to the higher temporal resolution of neural probes arrayed on chip substrates compared with traditional calcium imaging technology.

[0082] like Figures 4 to 6 As shown, in one embodiment of the present application, the conventional MEMS manufacturing peeling process and micro-pattern stamp technology are used to process the substrate material, the probe material and the chip circuit layout, and the chip substrate with array neural probes is constructed, including:

[0083] According to the chip circuit layout, a conventional MEMS manufacturing stripping process is used on the substrate material to perform n×n array electrode circuit routing layout to obtain a substrate to be modified with a neural probe assembly position; and a solution method is used to synthesize the probe material to obtain a neural probe;

[0084] Modifying the substrate to be modified by a silanization coupling agent to obtain a substrate to be printed;

[0085] A neural probe is printed on each neural probe assembly position of a substrate to be printed by using a micro-pattern stamp technology, thereby obtaining a chip substrate with an array of neural probes.

[0086] It should be noted that the chip circuit layout adopts the traditional micro-electro-mechanical system (MEMS) manufacturing lift-off process. The two-dimensional semiconductor material MXene is prepared by solution method (HF and HCl+LiF etching method) for synthesizing the probe body, and the two-dimensional semiconductor material MXene is characterized by scanning electron microscope (SEM) and X-ray Diffraction (XRD), such as Figure 4 As shown in a and b. The neural probe processing first requires the modification of the substrate to be modified by a silanization coupling agent, and then the neural probe array is printed onto the substrate to be printed using a micropattern stamp technology to complete the construction of the array neural probe. MXene-based field effect transistor devices are used to monitor the spike activity in cultured primary hippocampal neurons in real time, such as Figure 5 As shown in a, after the action potential is triggered, the neurotransmitter is released and then binds to the MXene surface to induce electrical signal fluctuations. Due to the ultra-thin thickness of the MXene micropattern (≈5nm), the device is almost transparent, as shown in Figure 5 b, so that conventional microscopy observations are not disturbed. This multichannel chip is able to show good temporal resolution with neural probes because it captures spikes that cannot be observed in calcium imaging due to the slow kinetics of calcium signaling in neuronal cells (about one hundred milliseconds), such as Figure 5 In this embodiment, the multi-channel chip constructed based on the construction method of the neural probe and array culture chip further improves the sensitivity of the chip by using the neural probe designed in the field effect tube mode. n is a natural number not equal to 0.

[0087] like Figure 6 As shown, in the embodiments of the present application, the construction method based on neural probes and array culture chips can select suitable substrate materials (such as glass or silicon wafers) according to the optical requirements of the detection and the performance requirements of the field effect tube, and use the traditional MEMS lift-off process to complete the microelectrode circuit routing layout of the n×n (such as 8×8) array, and reserve about 300×300μm neural probe assembly position. After the surface hydroxyl modification of the substrate to be modified, 3-aminopropyltriethoxysilane (APTES) modification is immediately performed. The substrate to be printed is designed and micro-processed by photolithography technology, and a dimethylsiloxane (PDMS) circuit stamp is reversely molded and printed. The MXene field effect tube probe matrix is ​​printed on the modified substrate to be printed by MXene micro-stamp engraving technology, as shown Figure 6 shown.

[0088] It should be noted that the construction method based on neural probes and array culture chips uses field effect tube design for ultra-thin neural probes and combines it with two-dimensional semiconductor material MXene, which greatly improves the detection sensitivity of multi-channel chips for neural electrophysiological signal detection and can capture weaker and more subtle neural signal changes. The construction method based on neural probes and array culture chips uses two-dimensional semiconductor materials with high conductivity and small size characteristics of carbon nanotubes to make ultra-fine neural probes, further improving the spatial resolution and signal sensitivity of multi-channel chips for detection. By combining the multi-channel chip with optical and / or electrical detection technology, optical signals can be transmitted using optical fibers to monitor neural activity.

[0089] Embodiment 2:

[0090] Figure 7 This is a flowchart of the steps of the neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips described in the embodiments of the present application. Figure 8 This is a flow chart of the neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips described in an embodiment of the present application.

[0091] like Figure 7 and Figure 8As shown, the present application embodiment provides a method for evaluating neuroelectrophysiological toxicity based on a neural probe and an array culture chip, comprising the following steps:

[0092] S01. Obtain a multi-channel chip constructed based on the above-mentioned construction method of the neural probe and array culture chip.

[0093] It should be noted that the content of the construction method based on the neural probe and array culture chip has been described in Example 1, and the content of the construction method based on the neural probe and array culture chip will not be repeated in this embodiment. In step S01, the first is to obtain a multi-channel chip constructed by the construction method based on the neural probe and array culture chip, and the multi-channel chip includes an array neural probe and a multi-channel neural cell culture chamber; assembling the array neural probe and the multi-channel neural cell culture chamber can complete the construction of a multi-channel high-throughput neuroelectrophysiological toxicity detection chip, providing a detection hardware platform for subsequent toxicity detection of the compound to be tested, and realizing that the detection hardware platform for toxicity detection is easy to build and operate, simplifying the experimental process of neurotoxicity testing.

[0094] S02. Construct a gradient target compound in a multi-channel chip, collect, classify and process the neuroelectrophysiological signals generated by the neural probe of the gradient target compound with different gradient exposure concentrations to obtain collected data; the neuroelectrophysiological signals include action potential signals and local field potential signals.

[0095] It should be noted that in step S02, a gradient target compound, such as a gradient curve of the target compound, is constructed in the multi-channel chip constructed in step S01. The frequency, amplitude, phase resonance and other data of the action potential of the neural signal are collected in a high-throughput manner through the gradient curve in the gradient target compound, so as to achieve high-throughput collection of the neuroelectrophysiological signals of the target compound, and provide data for subsequent analysis of the toxicity level of the collected data and the neuroelectrophysiological toxicity assessment standard. The collected data is data based on changes in neuroelectrophysiological signals, which provides accurate data for comprehensively evaluating the effects of the neurotoxicity of the test compound on the function of the nervous system, thereby improving the sensitivity and accuracy of the evaluation.

[0096] S03. Obtain neurotoxicity standards, analyze the collected data according to the neurotoxicity standards, and obtain the toxicity level and neuroelectrophysiological toxicity assessment standard corresponding to the gradient target compound.

[0097] It should be noted that in step S03, one is to obtain neurotoxicity standards, such as traditional neurodevelopmental toxicity test methods based on experimental animals (such as OPPTS 870.6300) and TG426 issued by the Organization for Economic Cooperation and Development (OCED). Second, the neurotoxicity threshold and its grading system are defined according to the neurotoxicity standard, that is, to clarify what level of electrophysiological abnormality can be used as an indication of neurotoxicity of a chemical substance, and to establish standards for quantifying the severity of its toxicity. Secondly, it is based on determining the minimum toxic exposure concentration for common neurotoxic substances to induce toxic effects, and further exploring the variation law of neurotoxicity levels at different concentrations for the collected data of different gradient exposure concentrations collected in step S02, and obtaining the toxicity level and neuroelectrophysiological toxicity evaluation standard corresponding to the gradient target compound. In this embodiment, the neurotoxic substances with known neurotoxicity standards are mainly graded according to their minimum toxic dose or lethal dose, and the electrophysiological toxicity is calibrated according to their electrophysiological response characteristics, thereby constructing a new electrophysiological toxicity evaluation standard and its toxicity grading. For example: by constructing different gradient exposure concentrations of gradient target compounds in a single multi-channel chip, the neuroelectrophysiological responses of different exposure concentration parameters are collected and evaluated in real time with high throughput, and the toxicity level of the substance is determined according to the level intervals into which its electrophysiological damage / changes fall under different gradient exposure concentrations. At the same time, based on the constructed concentration gradient curve analysis, the minimum toxic exposure concentration of the compound and the level of neurotoxicity produced by different concentrations are determined. Among them, the gradient exposure concentration refers to the concentration of the chemical molecules input into the culture chamber layer Cell of the compound to be tested. The exposure concentration parameters include the type of compound and the concentration of the compound.

[0098] In the embodiment of the present application, the neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips refers to and rates known neurotoxic substances, explores and records the relationship between toxic chemicals of different levels and neuroelectrophysiological responses, extracts and constructs new neuroelectrophysiological toxicity assessment and toxicity grading standards. The neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips uses the neuroelectrophysiological physiological signals output by the neural probes of the multi-channel chip as the terminal functional signals of the brain neural network, and provides a basis for neurotoxicity assessment by monitoring and analyzing the frequency, amplitude, phase resonance and other parameters of the neural action potential of the neuroelectrophysiological physiological signals, thereby achieving qualitative and quantitative assessment of the neurotoxicity of the test compound and improving the sensitivity and accuracy of the assessment.

[0099] It should be noted that the neuroelectrophysiological toxicity assessment method based on neural probes and array culture chips uses the array neural probes and array culture layer chambers of a multi-channel chip to construct a gradient target compound for the test compound, thereby reducing experimental material consumption and animal use, and lowering testing costs; the neuroelectrophysiological signals of the test compound with different gradient exposure concentrations are output by the array neural probes of the multi-channel chip for collection, and the collected data are analyzed to achieve rapid evaluation of the neurotoxicity of a large number of compounds, thereby improving test efficiency and shortening the test cycle.

[0100] Fig. 9 This is a schematic diagram of the construction of an array neural network in the neuroelectrophysiological toxicity assessment method based on a neural probe and an array culture chip described in the embodiment of this application. Fig. 9 In the figure, a refers to primary hippocampal neuronal cell culture; b refers to neuroblastoma-induced neural network culture; cf refers to the proven active neural electrophysiological signals of the constructed neural microsystem.

[0101] like Fig. 9 As shown, in one embodiment of the present application, constructing a gradient target compound in a multi-channel chip includes:

[0102] Establishing array neural networks in multi-channel chips;

[0103] Obtaining the test compound and setting parameters of different gradient exposure concentrations, inputting the test compound into the array neural network according to the setting parameters, and obtaining the gradient target compound;

[0104] Among them, establishing an array neural network in a multi-channel chip includes: adding at least 80,000 neurons to the cell planting layer for seeding, and the cells in the cell planting layer will fall into the multi-channel independent culture chamber layers respectively through gravity. After at least 3 weeks of cell inoculation / induction in the culture chamber layer, an array neural network is obtained.

[0105] It should be noted that due to the requirements of multi-channel chips for the electrophysiological characteristics of the nervous system, the subject of neurotoxicity detection selects a neural system with mature neural electrophysiological characteristics. In this embodiment, two sets of schemes are tested as examples. Scheme A uses the primary hippocampal neural network as the array neural network. Fig. 9 As shown in a; Scheme B is based on the neuroblastoma-induced neural cell network with electrophysiological characteristics as the array neural network, such as Fig. 9 As shown in b. Among them, Scheme A uses primary neural cells and retains the biological characteristics of the original tissue in vivo, but the modeling cost is relatively high; Scheme B has the advantage of relatively low cost, but the neurotoxicity detection effect needs to be further verified in experiments. The constructed neural microsystem has active neurophysiological signals and can quickly produce a large number of responses to the test compounds, such as Fig. 9As shown in cf. Scheme A is based on the hippocampus of fetal rats dissected from E18 Sprague Dawley rats under sterile conditions. After washing, cutting, and papain digestion for 15 minutes, the cells were filtered through a 200-mesh filter and cultured for three weeks. Primary neurons were constructed using calcium imaging technology, and their neuroelectrophysiological activity was verified by primary neurons. Scheme B is that neuroblastoma cells were induced in Brainphys culture medium by adding retinoic acid (RA) for three weeks, and a neural cell network was obtained using calcium imaging technology, and neural electrophysiological activity was detected through the neural cell network. The process of constructing an array neural network can be understood as follows: 80,000 neurons are added to the cell planting layer SOURCE and seeded into the culture chamber layer CELL of suspended cells. Through the action of gravity, the cells of the cell planting layer SOURCE will fall into the multi-channel (such as 64) independent culture chamber layer CELL respectively. Three weeks after cell seeding / induction in the culture chamber layer CELL, the neural electrophysiological signals generated by the neural probe can be collected by gradient target compounds with different gradient exposure concentrations.

[0106] In an embodiment of the present application, obtaining the setting parameters of the test compound with different gradient exposure concentrations includes: adding the test compound at the inlet and outlet NLET / DRAIN, and calculating the gradient exposure concentration of each channel according to the canonical diffusion equation. The setting parameters include the gradient exposure concentration of the gradient layer and the gradient exposure concentration of each culture chamber layer.

[0107] It should be noted that the compounds to be tested include heavy metal lead, nitric oxide and / or neurotoxin chemical molecules of tetrodotoxin, etc. The canonical diffusion equation is based on the molecular diffusion coefficient, and the diffusion time and distance are used to calculate the gradient exposure concentration of each channel (such as each culture chamber layer). Calculating the gradient exposure concentration of each channel can determine the concentration of the compound exposed to the cells in each channel, so as to evaluate the neurotoxicity of compounds of different concentrations and find the lowest toxic dose or lethal dose. In this embodiment, relying on the gradient layer STEPNESS to construct different set parameters for multiple channels, and collecting and analyzing the electrophysiological response characteristics of neurotoxicity at different concentrations in its channels. Specifically as follows: After the array neural network is constructed, the compound to be tested is added to the array neural network, and the gradient concentration of each channel is calculated according to the canonical diffusion equation. In the simulation of the 3D diffusion process, the 3D diffusion was simplified into two 1D models with the following assumptions: there is no flux in the Y direction in the bottom horizontal plane (source layer), the concentration variation in the XY plane of the culture chamber layer in the form of a hydrogel cylinder is neglected, and thus there is only a flux in the Z direction in the culture chamber layer of each cylinder. The culture chamber layer (drainage layer) is large enough so that the total concentration in the chamber is not affected by diffusion and remains close to zero during the experiment.

[0108] In the embodiment of the present application, the diffusion in the bottom layer (gradient layer) is controlled by the conventional diffusion equation, which is:

[0109]

[0110] Diffusion in the cylinder of the culture chamber layer is governed by the canonical diffusion equation, which is:

[0111]

[0112] Where x is the distance between the test compound and the entrance of the multi-channel chip, z is the distance between the test compound and the bottom of the culture chamber layer, D0 is the diffusion coefficient of the chemical molecule of the test compound in a low viscosity solution (such as culture medium), n is the number of the culture chamber layer, C n is the gradient exposure concentration of the nth culture chamber layer, C bottom is the gradient exposure concentration of the gradient layer.

[0113] In one embodiment of the present application, the neuroelectrophysiological signals generated by the neural probe of the gradient target compound at different gradient exposure concentrations are collected and classified, and the collected data includes:

[0114] Amplify and bandpass filter the collected neural electrophysiological signals to obtain processed data;

[0115] The EM algorithm is used to perform spike sorting on the action potential signal of the processed data to obtain sorted signal data;

[0116] Acquire a signal classification standard, perform peak classification on the signal data according to the signal classification standard, and obtain classified data;

[0117] Electrophysiological features are extracted for each type of spike in the classified data to obtain collected data consisting of electrophysiological features of all spike classifications.

[0118] It should be noted that the signal classification criteria include the peak value, rise duration and fall duration of the spike; and / or, the electrophysiological characteristics include frequency, amplitude, resonance, duration, rise time, decay time, peak-to-peak interval, peak energy, signal center frequency and information entropy. The neural electrophysiological signals of each channel of the multi-channel chip are amplified in situ through the neural probes of each culture chamber layer to initially improve the neural detection signal-to-noise ratio; the front-end amplification, filtering and recording of the electrical signal rely on a commercial multi-channel neural electrophysiological signal acquisition system (Blackrock Microsystems Inc, Salt Lake City, UT) for processing; the acquired offline data are analyzed by neural signal waveform analysis and processing software. For example: real-time monitoring of neurophysiological signals such as action potential signals APs and local field potential signals LFP in each channel of a multi-channel chip, these neurophysiological signals are sent to the preamplifier of the corresponding channel for amplification and bandpass filtering (1-5000Hz); the t-dist EM algorithm of the Offline Sorter (Plexon, Inc) built into the multi-channel chip is used to perform spike sorting on the action potential signals APs to obtain sorted signal data; then the local field potential signals LFP obtained from the sorted signal data are subjected to spike sorting using a 250Hz offline digital filter to obtain classified data. Further offline analysis of the classified data can be performed to obtain various electrophysiological characteristics of the electrophysiological signals.

[0119] Fig.10 This is a schematic diagram showing that the neuroelectrophysiological response in the neuroelectrophysiological toxicity assessment method based on the neural probe and array culture chip described in the embodiment of this application has ultra-sensitivity and specificity. Fig.10 In this equation, ad refers to the difference in action potential frequency of the nervous system under different conditions; df refers to the difference in gamma oscillation energy of the neural network under different neural conditions.

[0120] In the embodiment of the present application, the neuroelectrophysiological toxicity assessment method based on the neural probe and array culture chip first plants cells in the cell planting layer SOURCE of the multi-channel chip, and records its neuroelectrophysiological signals after three weeks of culture. Select graded neurotoxins such as heavy metal lead, nitric oxide, and tetrodotoxin to wait for the test compound, and introduce the neurotoxin chemical molecules of the test compound into the gradient layer STEPNESS through the inlet INLET to complete the setting of the exposure concentration parameters; obtain the neuroelectrophysiological signals of each channel and analyze the response signals of different exposure concentrations in each channel, and compare them with the healthy nerve background signal to determine its toxicity level; wherein, the neuroelectrophysiological toxicity threshold of the neurotoxicity standard is determined according to the electrophysiological response damage or change amplitude generated at the minimum toxic dose of the neurotoxin (reference to the safety standard concentration of the substance), and is used as the evaluation threshold for defining whether it has neurotoxicity; and for the lowest exposure concentration of the unknown toxic compound to be tested, it is determined by locating the minimum concentration of the electrophysiological response change in each channel that exceeds the toxicity threshold.

[0121] Embodiment three:

[0122] Fig.11 A schematic diagram of a terminal device described in an embodiment of the present application.

[0123] like Fig.11 As shown, an embodiment of the present application provides a terminal device, including a processor and a memory;

[0124] A memory, used for storing program codes and transmitting the program codes to a processor;

[0125] The processor is used to execute the above-mentioned neuroelectrophysiological toxicity evaluation method based on the neural probe and array culture chip according to the instructions in the program code.

[0126] It should be noted that the processor is used to execute the steps in the above-mentioned embodiment of a method for evaluating neuroelectrophysiological toxicity based on a neural probe and an array culture chip according to the instructions in the program code. Alternatively, the processor implements the functions of each module / unit in the above-mentioned system / device embodiments when executing the computer program.

[0127] Exemplarily, the computer program may be divided into one or more modules / units, one or more modules / units are stored in a memory and executed by a processor to complete the present application. One or more modules / units may be a series of computer program instruction segments capable of completing a specific function, and the instruction segments are used to describe the execution process of the computer program in a terminal device.

[0128] The terminal device may be a computing device such as a desktop computer, a notebook, a PDA, or a cloud server. The terminal device may include, but is not limited to, a processor and a memory. Those skilled in the art will appreciate that this does not constitute a limitation on the terminal device, and may include more or fewer components than shown in the figure, or a combination of certain components, or different components. For example, the terminal device may also include input and output devices, network access devices, buses, etc.

[0129] The processor may be a central processing unit (CPU), other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (dSIC), field-programmable gate arrays (FPGAs) or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. A general-purpose processor may be a microprocessor or any conventional processor, etc.

[0130] The memory may be an internal storage unit of the terminal device, such as a hard disk or memory of the terminal device. The memory may also be an external storage device of the terminal device, such as a plug-in hard disk, a smart memory card (SMC), a secure digital (SD) card, a flash card, etc. equipped on the terminal device. Furthermore, the memory may include both an internal storage unit of the terminal device and an external storage device. The memory is used to store computer programs and other programs and data required by the terminal device. The memory may also be used to temporarily store data that has been output or is to be output.

[0131] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0132] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0133] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0134] In addition, each functional unit in each embodiment of the present application may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0135] If the integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present application is essentially or the part that contributes to the prior art or all or part of the technical solution can be embodied in the form of a software product. The computer software product is stored in a storage medium, including several instructions for a computer device (which can be a personal computer, server, or network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present application. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM), random access memory (RAM), disk or optical disk and other media that can store program codes.

[0136] As described above, the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for constructing a neural probe and array culture chip, characterized in that: The following steps are involved: Obtaining a substrate material, a probe material, and a chip circuit layout, and processing the substrate material, the probe material, and the chip circuit layout using a conventional MEMS manufacturing peeling process and a micro-pattern stamping technology to construct a chip substrate with an array of neural probes; Constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate to obtain a multi-channel chip with neural probes and array culture; the multi-channel neural cell culture chamber is connected to the electrode of the chip substrate; Among them, the contents of constructing a multi-channel neural cell culture chamber corresponding to the array neural probe on the chip substrate include: using the traditional micro-electromechanical system manufacturing peeling process to process the chip substrate to obtain a multi-channel neural cell culture chamber; the multi-channel neural cell culture chamber includes an entrance and exit, a cell planting layer, a gradient layer and a plurality of culture chamber layers corresponding to the array neural probe, the entrance and exit are connected through the gradient layer, the cell planting layer is arranged above each of the culture chamber layers, the gradient layer is arranged below all the culture chamber layers, and the gradient layer is connected to the electrode of the chip substrate.

2. The method for constructing a neural probe and array culture chip according to claim 1, characterized in that: According to the substrate material, the probe material and the chip circuit layout, the conventional MEMS manufacturing peeling process and micro-pattern stamp technology are used to construct a chip substrate with array neural probes, including: According to the chip circuit layout, a conventional MEMS manufacturing stripping process is used on the substrate material to perform n×n array electrode circuit routing layout to obtain a substrate to be modified with a neural probe assembly position; and a solution method is used to synthesize the probe material to obtain a neural probe; Modifying the substrate to be modified by a silanization coupling agent to obtain a substrate to be printed; The micro-pattern stamp technology is used to print the neural probes at each neural probe assembly position of the substrate to be printed, thereby obtaining a chip substrate with array neural probes.

3. The method for constructing a neural probe and array culture chip according to claim 1 or 2, characterized in that: The cell planting layer is used to plant cells and limit the range of the culture solution; and / or, the inlet and outlet are used to introduce chemical molecules into the gradient layer; the gradient layer is used to diffuse the introduced chemical molecules to construct a horizontal gradient in the XY plane, so as to provide each culture chamber layer with a gradually decreasing concentration of input chemical molecules.

4. The method for constructing a neural probe and array culture chip according to claim 1 or 2, characterized in that: The culture chamber layer is made of a scaffold or a hydrogel material, and the thickness of the culture chamber layer is 250 µm; and / or the thickness of the gradient layer is 400 µm.

5. A method for evaluating neuroelectrophysiological toxicity based on neural probes and array culture chips, characterized in that: The following steps are involved: Obtain a multi-channel chip constructed based on the construction method of a neural probe and an array culture chip as described in any one of claims 1 to 4; Constructing a gradient target compound in the multi-channel chip, collecting and classifying the neurophysiological signals generated by the neural probe of the gradient target compound at different gradient exposure concentrations to obtain collected data; the neurophysiological signals include action potential signals and local field potential signals; A neurotoxicity standard is obtained, and the collected data is analyzed according to the neurotoxicity standard to obtain a toxicity grade and a neuroelectrophysiological toxicity assessment standard corresponding to the gradient target compound.

6. The method for evaluating neuroelectrophysiological toxicity based on neural probes and array culture chips according to claim 5, characterized in that: Constructing a gradient target compound in the multi-channel chip comprises: Establishing an array neural network in the multi-channel chip; Obtaining the test compound and setting parameters of different gradient exposure concentrations, and inputting the test compound into the array neural network according to the setting parameters to obtain a gradient target compound; Among them, establishing an array neural network in the multi-channel chip includes: adding at least 80,000 neurons to the cell planting layer for seeding, and the cells of the cell planting layer will fall into the multi-channel independent culture chamber layers respectively through gravity. After at least 3 weeks of cell inoculation / induction in the culture chamber layer, an array neural network is obtained.

7. The method for evaluating neuroelectrophysiological toxicity based on neural probes and array culture chips according to claim 6, characterized in that: The test compounds include heavy metal lead, nitric oxide and / or neurotoxin chemical molecules of tetrodotoxin.

8. The method for evaluating neuroelectrophysiological toxicity based on neural probes and array culture chips according to claim 5, characterized in that: The neuroelectrophysiological signals generated by the neural probe of the gradient target compound at different gradient exposure concentrations are collected and classified to obtain the collected data including: Amplify and bandpass filter the collected neural electrophysiological signals to obtain processed data; Using the EM algorithm to perform spike sorting on the action potential signal of the processed data to obtain sorted signal data; Acquiring a signal classification standard, and performing peak classification on the signal data according to the signal classification standard to obtain classified data; Electrophysiological features are extracted for each type of spike in the classified data to obtain collected data consisting of electrophysiological features of all spike classifications.

9. The method for evaluating neuroelectrophysiological toxicity based on neural probes and array culture chips according to claim 8, characterized in that: The signal classification criteria include the peak value, rising duration and falling duration of the spike; And / or, the electrophysiological characteristics include frequency, amplitude, resonance, duration, rise time, decay time, peak-to-peak interval, peak energy, signal center frequency and information entropy.

10. A terminal device, characterized in that: including a processor and a memory; The memory is used to store program codes and transmit the program codes to the processor; The processor is used to execute the neuroelectrophysiological toxicity assessment method based on the neural probe and array culture chip as described in any one of claims 5 to 9 according to the instructions in the program code.

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