Surrounding type multi-neurotransmitter biological mixed synapse recognition system

Abstract

The invention belongs to the technical field of biomedical detection, and particularly discloses a surrounding type multi-neurotransmitter recognition biological mixed synapse system which is composed of a flexible biological synapse device and a microfluidic channel, the microfluidic channel simulates a biological synapse gap and controls transmission and diffusion of different neurotransmitters, and the flexible biological synapse device and the microfluidic channel are used for detecting the biological synapse gap. The releasing and receiving processes of neurotransmitters in a biological synaptic gap are simulated; according to the flexible biological synapse device, direct interaction between biological tissues and equipment is realized, and the change of long-term synapse weight is simulated by utilizing a redox reaction of a neurotransmitter at a grid electrode, so that the dynamic adaptability and the information processing capability of a system are improved; when neurons generate peak signals containing different neurotransmitters, the system can accurately distinguish and identify the signals and efficiently transmit the signals; therefore, the biological mixed synaptic system disclosed by the invention becomes an ideal multi-neurotransmitter monitoring tool and can be widely applied to neuroscience research and clinical diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical detection technology, in particular to a surround-type multi-neurotransmitter recognition biological hybrid synapse system. Background Art

[0002] In neuroscience research, monitoring neurotransmitters is a key step in understanding and analyzing the mechanism of neural signal transmission. Neurotransmitters are chemicals that transmit information between neurons. They are released in the synaptic cleft and then bind to receptors on the postsynaptic membrane, triggering the transmission of neural signals.

[0003] Currently, the methods used for neurotransmitter monitoring include mass spectrometry, immunoassay, and fluorescence imaging technology. However, these methods have shortcomings such as complex equipment, high cost, insufficient sensitivity and specificity, inability to meet the needs of simultaneous monitoring of multiple neurotransmitters, and limited real-time detection. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a surround-type multi-neurotransmitter recognition biohybrid synapse system, which aims to achieve low-power, high-sensitivity, and high-specificity real-time detection of multiple neurotransmitters.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] A wraparound multi-neurotransmitter recognition biohybrid synapse system, comprising a flexible biosynapse device, a microfluidic channel, and neurotransmitters transmitted and diffused in the microfluidic channel;

[0007] The flexible biological synapse device includes, from bottom to top, a flexible substrate layer, a bottom patterned gate electrode layer, a gate dielectric layer, an organic channel layer, a top source electrode, and a top drain electrode; the bottom patterned gate electrode layer is arranged on the upper end surface of the flexible substrate layer, the gate dielectric layer is arranged on the upper end surface of the bottom patterned gate electrode layer and fills the channel region of the patterned gate electrode structure in the bottom patterned gate electrode layer, the organic channel layer is arranged on the upper end surface of the gate dielectric layer, and the top source electrode and the top drain electrode are distributed at both ends of the upper end surface of the organic channel layer;

[0008] The microfluidic channel includes a PDMS bottom mold, on which a bowl-shaped groove, a main channel connected to the bottom of the bowl-shaped groove, a plurality of primary sub-channels connected to the end of the main channel, and two secondary sub-channels connected to the end of the primary sub-channel. The end of the secondary sub-channel is connected to a first box body with an opening at the bottom, and the open end of the first box body is fixedly connected to the upper end surface of the organic channel layer; the rear end of the first box body is connected to a pipeline, and the end of the pipeline is connected to a second box body.

[0009] Furthermore, the material of the flexible substrate layer is one of PET, PI or PDMS.

[0010] Furthermore, the material of the bottom patterned gate electrode layer is ITO, the patterned gate electrode structure is a center-divergent and surrounding type, and the thickness of the bottom patterned gate electrode layer is 150-250 nm.

[0011] Furthermore, the gate dielectric layer is formed by spin coating a PMMA solution with a mass fraction of 5% prepared with DMF as a solvent, and the thickness of the gate dielectric layer is 250-350 nm.

[0012] Furthermore, the organic channel layer is made of PEDOT:PSS conductive polymer.

[0013] Furthermore, the materials of the top source electrode and the top drain electrode are both Au, and the thickness of the top source electrode and the top drain electrode are both 100-200 nm.

[0014] Furthermore, the preparation process of the flexible biological synapse device is as follows:

[0015] Step 1, cleaning the flexible substrate layer to remove surface dirt and impurities;

[0016] Step 2, sputtering a bottom gate electrode on the surface of the flexible substrate layer and performing heat treatment;

[0017] Step 3, etching a designed electrode pattern on the bottom gate electrode to obtain a bottom patterned gate electrode layer;

[0018] Step 4, disposing a gate dielectric layer on the upper end surface of the bottom patterned gate electrode layer and filling the channel region of the patterned gate electrode structure in the bottom patterned gate electrode layer;

[0019] Step 5, spin coating an organic channel layer on the surface of the gate dielectric layer;

[0020] Step 7: Use tape to tightly fix the mask plate to the surface of the organic channel layer, and sputter Au on both sides of the upper surface of the organic channel layer to obtain a top source electrode and a top drain electrode.

[0021] Furthermore, the preparation process of the microfluidic channel is as follows:

[0022] Step 1, coating a layer of SU-8 epoxy resin on the silicon wafer;

[0023] Step 2, photolithography: exposing the pattern of the microfluidic channel on SU-8 epoxy resin through a mask;

[0024] Step 3, development: removing the epoxy resin outside the exposure area to form a mold for the microfluidic channel;

[0025] Step 4, PDMS and a cross-linking agent were mixed in a weight ratio of 10:1, placed in a vacuum desiccator for degassing, and poured onto a silicon wafer containing a SU-8 epoxy resin mold;

[0026] Step 5, pre-curing at room temperature, then heating to 80°C and maintaining for 24 hours to completely cure the PDMS, and then demolding to obtain a microfluidic channel;

[0027] Step 6, using a puncture perforator to make inlet and outlet holes, the holes are made of Teflon tubes;

[0028] Step 7: Coat a small amount of uncured PDMS on the bottom surface of the microfluidic channel, place it on a silicon substrate and bake it at 80° C. for 1 h.

[0029] Compared with the prior art, the wraparound multi-neurotransmitter recognition biohybrid synapse system of the present invention is composed of a flexible biosynapse device and a microfluidic channel. The microfluidic channel simulates the biological synaptic cleft, controls the transmission and diffusion of different neurotransmitters, and simulates the release and reception process of neurotransmitters in the biological synaptic cleft; by regulating the attachment / elution state of these neurotransmitters on the gate surface, the binding / detachment process of neurotransmitters on the receptor can be accurately simulated; the flexible biosynapse device realizes direct interaction between biological tissue and equipment, and utilizes the redox reaction of neurotransmitters at the gate to simulate the change of long-term synaptic weight, thereby improving the dynamic adaptability and information processing capability of the system; when neurons generate spike signals containing different neurotransmitters, the system can accurately distinguish and identify these signals and transmit them efficiently; this makes the biohybrid synapse system of the present invention an ideal multi-neurotransmitter monitoring tool, which can be widely used in neuroscience research and clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the overall structural diagram of the surround-type multi-neurotransmitter recognition biohybrid synapse system of the present invention.

[0031] Figure 2 Schematic diagram of the structure of a flexible biological synapse device.

[0032] Figure 3 A patterned gate electrode structure for a bottom patterned gate electrode layer.

[0033] Figure 4 Schematic diagram of the structure of the microfluidic channel. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0035] like Figure 1-Figure 4 As shown, this embodiment exemplarily shows a wraparound multi-neurotransmitter recognition biohybrid synapse system, including a flexible biosynapse device 1, a microfluidic channel 2, and neurotransmitters transmitted and diffused in the microfluidic channel;

[0036] The flexible biological synaptic device 1 includes, from bottom to top, a flexible substrate layer 101, a bottom patterned gate electrode layer 102, a gate dielectric layer 103, an organic channel layer 104, a top source electrode 105, and a top drain electrode 106; the bottom patterned gate electrode layer 102 is arranged on the upper end surface of the flexible substrate layer 101, the gate dielectric layer 103 is arranged on the upper end surface of the bottom patterned gate electrode layer 102 and fills the channel region of the patterned gate electrode structure in the bottom patterned gate electrode layer, and the bottom patterned gate electrode layer 102 has a structure as shown in FIG. Figure 3 As shown, it is composed of a plurality of electrode sheets 1021 distributed in a surrounding manner; the organic channel layer 104 is arranged on the upper end surface of the gate dielectric layer 103, and the top source electrode 105 and the top drain electrode 106 are distributed at both ends of the upper end surface of the organic channel layer 104;

[0037] The microfluidic channel 2 includes a PDMS bottom mold, on which a bowl-shaped groove 201, a main channel 202 connected to the bottom of the bowl-shaped groove 201, a plurality of primary sub-channels 203 connected to the end of the main channel 202, and two secondary sub-channels 204 connected to the end of the primary sub-channel 203. The end of the secondary sub-channel 204 is connected to a first box body 205 with an opening at the bottom, and the open end of the first box body 205 is fixedly connected to the upper end surface of the organic channel layer 104 at the top of the flexible biological synapse device; the rear end of the first box body 205 is connected to a pipeline 207, and the end of the pipeline 207 is connected to a second box body 206. Neurotransmitters are injected through the microfluidic channel and then contact the organic channel layer 104 of the flexible biological synapse device. During this process, the neurotransmitters undergo an oxidation-reduction reaction, and the solution (waste liquid) after the reaction continues to flow along the pipeline 207 and is finally discharged from the second box body 206.

[0038] The specific preparation process is as follows:

[0039] First, a circular microchannel was fabricated on a polydimethylsiloxane (PDMS) substrate using soft lithography. During the preparation process, a layer of SU-8 epoxy resin was evenly coated on a silicon wafer, and the desired microchannel pattern was exposed on the resin through a photolithography process; then the resin in the unexposed area was removed by development to form a microchannel mold; next, PDMS and a crosslinker were mixed in a weight ratio of 10:1, poured into the mold after degassing, pre-cured at room temperature, and heated and cured at 80°C for 24 hours to form a PDMS microchannel with good biocompatibility and flexibility; the cured PDMS microchannel was used to make inlet and outlet holes through a puncture perforator, and the microchannel was bonded to the silicon substrate using uncured PDMS to complete the preparation of the microfluidic channel. This method not only ensures the precise size of the microchannel, but also utilizes the characteristics of the PDMS material to provide a high-performance microfluidic platform for biomedical research and microfluidic applications.

[0040] Secondly, a microelectronic device that simulates the function of biological synapses is constructed on a flexible substrate. PET is selected as the flexible substrate material and is thoroughly cleaned to ensure that the surface is free of dirt and impurities, which is crucial for the quality of subsequent electrode material deposition. Next, a 200nm thick bottom gate electrode is deposited on the PET substrate using magnetron sputtering technology, and heat treatment is performed to optimize the performance of the ITO film. The required surrounding electrode pattern is etched on the ITO film using laser etching technology. The surrounding structure is as follows: Figure 3 As shown, the spin coating surface of the working area of ​​the device is more uniform, ensuring the stability of the prepared device. Subsequently, PMMA is used as the gate dielectric layer, DMF is used as the solvent, and a 5% mass fraction of PMMA solution is prepared. After rotating at 3000rpm for 45 seconds, nitrogen is passed through the spin coating on the surface of the ITO film. This step avoids the influence of air on film formation, and annealing is performed at 100°C for 5 minutes to improve the surface flatness of the film. The organic channel layer of the device uses PEDOT:PSS material, which is solidified by rotating at 2000rpm for 30 seconds and annealing at 100°C for 5 minutes to form a channel layer with a thickness of about 300nm. Finally, in order to prepare the top source electrode and the top drain electrode, the mask is fixed on the surface of the device, and Au is sputtered on the organic channel layer again using magnetron sputtering technology to form a top source electrode and a top drain electrode with a thickness of about 100nm.

[0041] In actual use, in order to be able to identify a variety of neurotransmitters, the Au of the top source electrode and the top drain electrode can be modified. For dopamine recognition, 3-mercaptopropionic acid (3-MPA) and polydopamine (PDA) are used for composite modification. 3-MPA provides thiol groups to combine with Au, and PDA has specific adsorption for dopamine.

[0042] For serotonin recognition, molecular imprinting polymer (MIP) technology was used with serotonin as the template molecule. This technology has high selectivity and specificity.

[0043] For Ca 2 + recognition, modified with calcium ion selective ionophore ETH129. ETH129 and Ca 2 + Formation of selective complexes to achieve highly selective recognition of Ca 2 +.

[0044] For K+ recognition, potassium ion selective crown ether valinomycin is used for modification. Valinomycin forms a pore of a specific size, selectively binds K+, and has extremely high selectivity for K+.

[0045] The following tests can be performed using the surround-type multi-neurotransmitter recognition biohybrid synapse system of the present invention, and the specific tests are as follows:

[0046] 1. Surround multi-neurotransmitter identification test method

[0047] First, an artificial cerebrospinal fluid was prepared, whose composition was 100nM dopamine, 1nM serotonin, 0.1mM calcium chloride (CaCl2) and 1mM potassium chloride (KCl); then, the artificial cerebrospinal fluid was continuously perfused into the annular microchannel at a constant flow rate of 5μL / min using a microfluidic system; then, a cyclic voltammetry test was performed on the flexible biological synapse device, specifically: a cyclic voltammetry test was performed at a scan rate of 10mV / s in the gate voltage range of -0.4V to 0.8V to measure the change in source-drain current.

[0048] Since specific functionalized modified electrodes are used for different neurotransmitters on the flexible biological synaptic device, these modifiers provide specific binding sites for each neurotransmitter. Therefore, in the cyclic voltammetry test, different neurotransmitters will have oxidation peaks at different potentials. The distinction of these potential windows helps to achieve specific detection of different neurotransmitters and simultaneous detection of multiple neurotransmitters and ions. By measuring the current changes caused by each neurotransmitter, its concentration can be quantitatively analyzed. The current response generated by each neurotransmitter has distinguishable characteristics, such as the size of the peak current, which helps to achieve specific identification. In the case of the simultaneous presence of multiple neurotransmitters in artificial cerebrospinal fluid, the system can distinguish and respond to the target neurotransmitter alone without interference from other components, which shows the high specificity of the present invention.

[0049] 2. Neuronal electrical signal simulation and transmission verification

[0050] First, a function generator was used to generate a spike signal with a frequency of 1 Hz and an amplitude of 100 mV to simulate neuronal action potentials. Second, each time a simulated action potential arrived, the following solutions were injected through the microfluidic channel to simulate the release of different neurotransmitters: 1 μL of 10 μM dopamine solution, 1 μL of 100 nM serotonin solution, 1 μL of 10 mM CaCl2 solution, and 1 μL of 50 mM KCl solution. Subsequently, a signal transmission test was performed, specifically: the gate voltage was fixed at 0.3 V, and the source-drain current was measured and recorded over time, with special attention paid to the amplitude and duration of the current change caused by the release of each simulated neurotransmitter.

[0051] By comparing the amplitude and duration of current changes, the system can accurately distinguish the signals caused by different neurotransmitters and convert them into amplified electrical signal outputs. This method can systematically study the effects of different neurotransmitter releases on neuronal electrical signal conduction, providing an experimental basis for further understanding the information processing mechanism of the nervous system.

[0052] 3. Simulation of long-term synaptic plasticity

[0053] First, using the above simulated neuronal signal, 100 stimulations were applied continuously at intervals of 10 seconds; secondly, the peak current after each stimulation was recorded, and a curve of the change in peak current with the number of stimulations was plotted.

[0054] Subsequently, the results were analyzed and it was observed that the peak current gradually increased with the number of stimulations, stabilized after 50 stimulations, and the final increase was about 150% of the initial value. This change simulated the long-term synaptic potentiation (LTP) process.

[0055] Through the above examples, it is verified that the biohybrid synaptic system for surround multi-neurotransmitter recognition described in the present invention has the following characteristics: it can accurately identify multiple neurotransmitters, efficiently transmit neural signals, and has the ability to simulate synaptic plasticity. The system provides a new and highly integrated tool for neuroscience research and clinical diagnosis, which helps to deeply understand the information processing mechanism of the nervous system and the principle of synaptic plasticity, and opens up new avenues for research and application in related fields.

[0056] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A wraparound multi-neurotransmitter recognition biohybrid synapse system, comprising a flexible biosynapse device, a microfluidic channel, and neurotransmitters transmitted and diffused in the microfluidic channel; characterized in that: The flexible biological synapse device includes, from bottom to top, a flexible substrate layer, a bottom patterned gate electrode layer, a gate dielectric layer, an organic channel layer, a top source electrode, and a top drain electrode; the bottom patterned gate electrode layer is arranged on the upper end surface of the flexible substrate layer, the gate dielectric layer is arranged on the upper end surface of the bottom patterned gate electrode layer and fills the channel region of the patterned gate electrode structure in the bottom patterned gate electrode layer, the organic channel layer is arranged on the upper end surface of the gate dielectric layer, and the top source electrode and the top drain electrode are distributed at both ends of the upper end surface of the organic channel layer; The microfluidic channel includes a PDMS bottom mold, on which a bowl-shaped groove, a main channel connected to the bottom of the bowl-shaped groove, a plurality of primary sub-channels connected to the end of the main channel, and two secondary sub-channels connected to the end of the primary sub-channel. The end of the secondary sub-channel is connected to a first box body with an opening at the bottom, and the open end of the first box body is fixedly connected to the upper end surface of the organic channel layer; the rear end of the first box body is connected to a pipeline, and the end of the pipeline is connected to a second box body.

2. The surround-type multi-neurotransmitter recognition biohybrid synapse system according to claim 1, characterized in that: The material of the flexible substrate layer is one of PET, PI or PDMS.

3. The surround-type multi-neurotransmitter recognition biohybrid synaptic system according to claim 1, characterized in that: The material of the bottom patterned gate electrode layer is ITO, the patterned gate electrode structure is a center-divergent surround type, and the thickness of the bottom patterned gate electrode layer is 150-250nm.

4. The surround-type multi-neurotransmitter recognition biohybrid synaptic system according to claim 1, characterized in that: The gate dielectric layer is made by spin coating a PMMA solution with a mass fraction of 5% prepared with DMF as a solvent, and the thickness of the gate dielectric layer is 250-350nm.

5. The surround-type multi-neurotransmitter recognition biohybrid synapse system according to claim 1, characterized in that: The organic channel layer is made of PEDOT:PSS conductive polymer.

6. The surround-type multi-neurotransmitter recognition biohybrid synaptic system according to claim 1, characterized in that: The materials of the top source electrode and the top drain electrode are both Au, and the thickness of the top source electrode and the top drain electrode are both 100-200 nm.

7. The surround-type multi-neurotransmitter recognition biohybrid synapse system according to any one of claims 1 to 6, characterized in that: The preparation process of the flexible biological synapse device is as follows: Step 1, cleaning the flexible substrate layer to remove surface dirt and impurities; Step 2, sputtering a bottom gate electrode on the surface of the flexible substrate layer and performing heat treatment; Step 3, etching a designed electrode pattern on the bottom gate electrode to obtain a bottom patterned gate electrode layer; Step 4, disposing a gate dielectric layer on the upper end surface of the bottom patterned gate electrode layer and filling the channel region of the patterned gate electrode structure in the bottom patterned gate electrode layer; Step 5, spin coating an organic channel layer on the surface of the gate dielectric layer; Step 7: Use tape to tightly fix the mask plate to the surface of the organic channel layer, and sputter Au on both sides of the upper surface of the organic channel layer to obtain a top source electrode and a top drain electrode.

8. The surround-type multi-neurotransmitter recognition biohybrid synapse system according to any one of claims 1 to 6, characterized in that: The preparation process of the microfluidic channel is as follows: Step 1, coating a layer of SU-8 epoxy resin on the silicon wafer; Step 2, photolithography: exposing the pattern of the microfluidic channel on SU-8 epoxy resin through a mask; Step 3, development: removing the epoxy resin outside the exposure area to form a mold for the microfluidic channel; Step 4, PDMS and a cross-linking agent were mixed in a weight ratio of 10:1, placed in a vacuum desiccator for degassing, and poured onto a silicon wafer containing a SU-8 epoxy resin mold; Step 5, pre-curing at room temperature, then heating to 80°C and maintaining for 24 hours to completely cure the PDMS, and then demolding to obtain a microfluidic channel; Step 6, using a puncture perforator to make inlet and outlet holes, the holes are made of Teflon tubes; Step 7: Coat a small amount of uncured PDMS on the bottom surface of the microfluidic channel, place it on a silicon substrate and bake it at 80° C. for 1 h.

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