A wraparound multi-neurotransmitter recognition biohybrid synaptic system

Abstract

The present invention belongs to the field of biomedical detection technology and specifically discloses a wraparound multi-neurotransmitter identification biohybrid synapse system, which 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. The flexible biosynapse device enables direct interaction between biological tissue and equipment, and utilizes the redox reaction of neurotransmitters at the gate to simulate long-term changes in synaptic weights, thereby improving the system's dynamic adaptability and information processing capabilities. When neurons generate spike signals containing different neurotransmitters, the system can accurately distinguish and identify these signals and efficiently transmit them. This makes the biohybrid synapse system of the present invention an ideal multi-neurotransmitter monitoring tool that can be widely used in 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 biohybrid 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 synaptic system comprising a flexible biosynaptic device, a microfluidic channel, and neurotransmitters transmitted and diffused in the microfluidic channel;

[0007] The flexible biological synaptic 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, which is provided with a bowl-shaped groove, a main channel connected to the bottom of the bowl-shaped groove, several 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 open bottom end, 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 the 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 surround 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 top source electrode and the top drain electrode are both made of 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 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: Expose the pattern of the microfluidic channel on the SU-8 epoxy resin through a mask;

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

[0025] Step 4: PDMS and cross-linking agent were mixed in a weight ratio of 10:1, placed in a vacuum desiccator for degassing, and poured onto the silicon wafer containing the 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 demolding to obtain the microfluidic channel;

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

[0028] In step 7, a small amount of uncured PDMS was coated on the bottom surface of the microfluidic channel, placed on a silicon substrate and baked at 80° C. for 1 h.

[0029] Compared with the existing technology, the surround-type multi-neurotransmitter recognition biohybrid synapse system of the present invention consists of a flexible biosynaptic 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, it can accurately simulate the binding / detachment process of neurotransmitters on the receptor; the flexible biosynaptic device realizes direct interaction between biological tissue and equipment, and uses the redox reaction of neurotransmitters at the gate to simulate long-term changes in synaptic weights, thereby improving the dynamic adaptability and information processing capabilities 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 that 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 synaptic system of the present invention.

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

[0032] Figure 3 It is a patterned gate electrode structure of the 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 solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0035] like Figures 1-4 As shown, this embodiment exemplarily shows a surround-type 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 disposed on the upper end surface of the flexible substrate layer 101, and the gate dielectric layer 103 is disposed 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. The structure of the bottom patterned gate electrode layer 102 is as follows: Figure 3 As shown, it is composed of a number of electrode sheets 1021 distributed in a surrounding manner; the organic channel layer 104 is provided 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 comprises a PDMS base mold, which is provided with a bowl-shaped recess 201, a main channel 202 connected to the bottom of the recess 201, several primary sub-channels 203 connected to the ends of the main channel 202, and two secondary sub-channels 204 connected to the ends of the primary sub-channels 203. The ends of the secondary sub-channels 204 are connected to a first box body 205 with an open bottom. 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 pipe 207, and the end of the pipe 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 a redox reaction. The resulting solution (waste liquid) continues to flow along the pipe 207 and is ultimately discharged from the second box body 206.

[0038] The specific preparation process is as follows:

[0039] First, soft lithography was used to fabricate circular microchannels on a polydimethylsiloxane (PDMS) substrate. During the fabrication process, a layer of SU-8 epoxy resin was evenly coated on a silicon wafer. The desired microchannel pattern was then exposed to light through photolithography. Development was then performed to remove the resin from the unexposed areas, forming a mold for the microchannels. Next, PDMS and a crosslinker were mixed in a 10:1 weight ratio, degassed, and poured into the mold. After pre-curing at room temperature, the mixture was heated and cured at 80°C for 24 hours, forming PDMS microchannels with excellent biocompatibility and flexibility. The cured PDMS microchannels were then pierced with a punch to create inlet and outlet holes. Uncured PDMS was then used to bond the microchannels to the silicon substrate, completing the fabrication of the microfluidic channel. This method not only ensures the precise dimensions of the microchannels but also leverages the properties of the PDMS material, providing a high-performance microfluidic platform for biomedical research and microfluidic applications.

[0040] Secondly, a microelectronic device simulating 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 treated 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, this ensures a more uniform spin-coated surface in the active area of ​​the device, ensuring device stability. Subsequently, PMMA was used as the gate dielectric layer, and a 5% mass fraction PMMA solution was prepared using DMF as the solvent. After spinning at 3000 rpm for 45 seconds, the solution was spin-coated onto the ITO film surface through a nitrogen atmosphere. This step prevented the influence of air on the film formation. The film was then annealed at 100°C for 5 minutes to improve surface flatness. The device's organic channel layer, made of PEDOT:PSS, was solidified by spinning at 2000 rpm for 30 seconds and annealing at 100°C for 5 minutes, forming a channel layer approximately 300 nm thick. Finally, to form the top source and drain electrodes, a mask was fixed to the device surface, and Au was sputtered onto the organic channel layer using magnetron sputtering again, forming top source and drain electrodes approximately 100 nm thick.

[0041] In actual use, the Au of the top source and drain electrodes can be modified to enable recognition of multiple neurotransmitters. For dopamine recognition, a composite modification of 3-mercaptopropionic acid (3-MPA) and polydopamine (PDA) is used. 3-MPA provides thiol groups that bind to Au, and PDA has a specific adsorption capacity for dopamine.

[0042] For serotonin recognition, molecularly imprinted polymer (MIP) technology is 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, the potassium ion-selective crown ether valinomycin is used for modification. Valinomycin forms a pore of a specific size, selectively binding to K+ and exhibiting extremely high selectivity for K+.

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

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

[0047] First, artificial cerebrospinal fluid (ASF) was prepared, consisting of 100 nM dopamine, 1 nM serotonin, 0.1 mM calcium chloride (CaCl2), and 1 mM potassium chloride (KCl). The ASF was then continuously perfused into the annular microchannel at a constant flow rate of 5 μL / min using a microfluidic system. Next, cyclic voltammetry was performed on the flexible biosynaptic device, specifically measuring changes in source-drain current at a scan rate of 10 mV / s over a gate voltage range of -0.4 V to 0.8 V.

[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 spike signals 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 changes in source-drain current over time were measured and recorded, with particular 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 signals caused by different neurotransmitters and convert them into amplified electrical output signals. This method can systematically study the effects of different neurotransmitter releases on neuronal electrical signal transmission, providing an experimental basis for further understanding the information processing mechanisms of the nervous system.

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

[0053] First, using the above-mentioned simulated neuronal signal, 100 stimulations were applied continuously at intervals of 10 seconds. Second, the peak current after each stimulation was recorded, and a curve of the change in peak current versus 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] The above examples demonstrate that the encircling biohybrid synaptic system for multi-neurotransmitter recognition described in this invention exhibits the following characteristics: accurate recognition of multiple neurotransmitters, efficient transmission of neural signals, and the ability to simulate synaptic plasticity. This system provides a novel, highly integrated tool for neuroscience research and clinical diagnosis, contributing to a deeper understanding of the information processing mechanisms of the nervous system and the principles of synaptic plasticity, and opening 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. Any 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 synaptic system, comprising a flexible biosynaptic device, a microfluidic channel, and neurotransmitters transported and diffused in the microfluidic channel; characterized by: The flexible biological synaptic 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 and is a surround type that diverges around the center; 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, which is provided with 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 open bottom end, 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 the second box body; Specific functionalized modified electrodes are used on the flexible biological synaptic device for different neurotransmitters to achieve specific detection of different neurotransmitters, thereby realizing simultaneous detection of multiple neurotransmitters.

2. The surround-type multi-neurotransmitter recognition biohybrid synaptic system according to claim 1, characterized in that: The material of the flexible substrate layer is 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, and the thickness of the bottom patterned gate electrode layer is 150-250 nm.

4. The surround-type multi-neurotransmitter recognition biohybrid synaptic system according to claim 1, characterized in that: 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.

5. The surround-type multi-neurotransmitter recognition biohybrid synaptic 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 top source electrode and the top drain electrode are both made of 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 synaptic 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 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 synaptic 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: Expose the pattern of the microfluidic channel on the SU-8 epoxy resin through a mask; Step 3, development: removing the epoxy resin outside the exposed area to form a mold for the microfluidic channel; Step 4: PDMS and cross-linking agent were mixed in a weight ratio of 10:1, placed in a vacuum desiccator for degassing, and poured onto the silicon wafer containing the 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 demolding to obtain the microfluidic channel; Step 6, using a puncture punch to make the inlet and outlet holes, which are made of Teflon tubes; In step 7, a small amount of uncured PDMS was coated on the bottom surface of the microfluidic channel, placed on a silicon substrate and baked at 80° C. for 1 h.

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