阅读说明：本技术 一种休眠检测调控集成化植入式柔性神经电极及测试系统 (Dormancy detection, regulation and control integrated implanted flexible neural electrode and test system ) 是由 王怡丁 蔡新霞 宋轶琳 徐声伟 李欣蓉 谢精玉 戴玉川 杨固成 徐世弘 邢宇 于 2021-09-10 设计创作，主要内容包括：本发明涉及一种休眠检测调控集成化植入式柔性神经电极及测试系统,包括微电极阵列,引线,焊盘；所述微电极阵列通过引线与长方形的焊盘连接,每根引线对应一个焊盘；所述的神经电极的焊盘由FPC接口引出；微电极阵列的每个电极的针头上分布有多个检测位点,且每两个相邻检测位点的位点间直线距离d,满足神经元在脑中的排布方式又满足检测位点的2倍宽电学要求；所述的检测位点包括神经电生理检测位点和神经电化学检测位点。所述柔性神经电极的微电极阵列的每个电极依次包括：绝缘层、微阵列导电层、柔性基底层、可溶性增强层。(The invention relates to a dormancy detection, regulation and control integrated implantation type flexible neural electrode and a test system, which comprise a microelectrode array, a lead and a bonding pad; the microelectrode array is connected with the rectangular bonding pads through leads, and each lead corresponds to one bonding pad; the bonding pad of the nerve electrode is led out from the FPC interface; a plurality of detection sites are distributed on the needle head of each electrode of the microelectrode array, and the linear distance d between the sites of every two adjacent detection sites meets the arrangement mode of neurons in the brain and also meets the 2-time wide electrical requirement of the detection sites; the detection sites comprise neuroelectrophysiology detection sites and neuroelectrochemistry detection sites. Each electrode of the microelectrode array of the flexible neural electrode comprises in sequence: insulating layer, microarray conducting layer, flexible stratum basale, soluble enhancement layer.)

1. A dormancy detection, regulation and control integrated implantation type flexible neural electrode is characterized by comprising a microelectrode array, a lead and a bonding pad;

the microelectrode array is connected with the rectangular bonding pads through leads, and each lead corresponds to one bonding pad; the bonding pad of the nerve electrode is led out from the FPC interface;

a plurality of detection sites are distributed on the needle head of each electrode of the microelectrode array, and the linear distance d between the sites of every two adjacent detection sites meets the arrangement mode of neurons in the brain and also meets the 2-time wide electrical requirement of the detection sites;

the detection sites comprise neuroelectrophysiology detection sites and neuroelectrochemistry detection sites.

2. The sleep detection, regulation and integration implantation type flexible neural electrode according to claim 1,

the microelectrode array comprises 2 x 8 circular neuro-electrophysiological detection sites and 2 rectangular neuro-electrochemical detection sites; all the nerve electrophysiology detection sites form a 2 x 8 microelectrode array and are distributed on the needle heads of 2 probes, 8 nerve electrophysiology detection sites and 1 nerve electrochemistry detection site are arranged on the needle head of each probe, and the linear distance between the detection sites is 70 mu m.

3. The sleep detection, regulation and integration implantable flexible neural electrode according to claim 1, wherein each electrode of the microelectrode array of the flexible neural electrode sequentially comprises: insulating layer, microarray conducting layer, flexible stratum basale, soluble enhancement layer, wherein:

an insulating layer covering the lead, exposing the microelectrode array and the pad;

a microelectrode array conducting layer, the microelectrode array comprising 18 detection sites; wherein, the 18 detection sites are divided into 2 groups which are distributed in a dormancy-related double brain area in a needle shape and are used for detecting neuro-electrophysiological and electrochemical signals of the hypothalamus;

the flexible substrate layer is used as a carrier of the implanted flexible neural electrode array;

the soluble enhancement layer is composed of PEG sucrose and is bonded with the back of the implanted flexible neural array.

4. The dormancy detection, regulation and integration implantable flexible neural electrode according to claim 3, wherein the flexible substrate layer comprises a Parylene (Parylene) material; the solubility enhancing layer comprises polyethylene glycol (PEG) and sucrose.

5. The dormancy detection, regulation and integration implantation flexible neural electrode according to claim 3, wherein the microelectrode array conducting layer comprises detection sites, and the diameter of the electrophysiological detection sites comprises 4-20 μm; the size of the electrochemical detection site is 16 multiplied by 28 mu m; the bonding pad is 2950 multiplied by 300 mu m; the lead wires are 4 micrometers, and the distance between every two adjacent lead wires is 8 micrometers or more; the distribution characteristics comprise that the tip distribution range of the detection sites is needle-like, the width is 51-120 mu m, the length is 134-314 mu m, the linear interval between two adjacent detection sites is 30-70 mu m, and the requirement of 2-time interval arrangement without mutual interference is met; the electrophysiological detection site and the electrochemical detection site are distributed on the two detection probes, and the horizontal interval between the two detection probes is 200-250 mu m.

6. The sleep detection, regulation and integration implantable flexible neural electrode according to claim 3, wherein the insulating layer covers the lead and exposes the electrophysiology detection site, the electrochemical detection site and the bonding pad.

7. The dormancy detection, regulation and integration implantation flexible neural electrode according to claim 3, wherein the electrophysiological detection site and the electrochemical detection site are modified by nano materials such as platinum black nanoparticles and/or carbon nanotube nanoparticles; the electrochemical detection site is modified by oxidase corresponding to neurotransmitter on the basis of the modification of the nano material.

8. A dormancy detection regulation apparatus integrated with an implantable flexible neural electrode according to any one of claims 1 to 7, comprising a flexible drug tube, an FCB plate, an insulating silica gel, and a flexible neural electrode;

the flexible medicine tube is made of an elastic fused capillary quartz sleeve;

the FCB board is provided with a flexible nerve electrode, and a flexible medicine tube is arranged on the flexible nerve electrode; the distance between the flexible medicine tube and the flexible nerve electrode is smaller than the preset distance, and the flexible medicine tube and the flexible nerve electrode are fixed by insulating silica gel.

9. The sleep detection modulation device according to claim 8, wherein the implanted flexible neural electrode is led out through an FPC interface; the elastic fused capillary quartz sleeve comprises an outer tube and an inner tube; the elastic fused capillary quartz sleeve is bonded with the implanted flexible neural electrode array through insulating silica gel.

10. A preparation method of an integrated implanted flexible nerve electrode is characterized by comprising the following steps:

step 1, evaporating a flexible substrate layer on a silicon wafer by adopting a vapor deposition method;

step 2, preparing a metal conducting layer on the flexible substrate layer through negative photoresist photoetching, electron beam evaporation and stripping technologies, wherein the metal conducting layer takes chromium as an adhesion layer;

step 3, evaporating a flexible material as an insulating layer on the conducting layer, removing the insulating layer on the surfaces of the microelectrode array and the bonding pad through positive glue and plasma etching, and only reserving the insulating layer covering the lead;

step 4, using SU-8 photoresist to carry out photoetching development to form a mask required by etching the flexible substrate, and forming the profile of the flexible substrate probe after oxygen ion etching;

step 5, removing the photoresist to enable the flexible micro-nano electrode to be separated from the silicon wafer, and obtaining a complete implanted flexible neural electrode array;

and 6, preparing the soluble enhancement layer by adopting a mould casting method, and sticking the implanted flexible neural array and the soluble enhancement layer.

11. A preparation method of a soluble enhancement layer of an integrated implanted flexible neural electrode is characterized by comprising the following steps:

step 1, evaporating a flexible substrate layer on a silicon wafer by adopting a vapor deposition method;

step 2, manufacturing a mold groove with the same size as the implanted flexible neural array by using a photoetching technology and an oxygen plasma etching technology;

step 3, pouring the heated and prepared liquid PEG into a groove of a mold, standing, cooling and hardening to obtain a PEG mold;

and 4, sticking the back of the implanted flexible neural array on the PEG of the mould through sucrose.

12. A test system applying the integrated implantable flexible neural electrode as claimed in any one of claims 1-7, comprising:

the integrated implanted flexible nerve electrode is used for long-term in-vivo detection and regulation;

the transparent columnar dormancy cabin is provided with a water bottle at a preset height away from the bottom;

a dormancy cabin net cover is arranged above the transparent columnar dormancy cabin and is used as an experimental object moving and dormancy space, and meanwhile, the experimental object moving and dormancy space is in a limited test area;

the outside of the transparent columnar dormant cabin comprises a noise shielding box used for reducing external noise interference;

the elastic traction rope is used for suspending a data wire, and the data wire is connected with the implanted flexible nerve electrode;

the tripod is adjustable in height and is used for controlling the elastic traction rope.

Technical Field

The invention relates to the field of micromachining of biosensors, the electrochemical field of nano material modification and the field of neural information detection, in particular to a dormancy detection, regulation and control integrated implanted flexible neural electrode and a test system.

Background

According to the existing research, GABAergic and glutamatergic neurons play an important role in dormancy. At present, the commonly used regulation and control means of neurons comprise drug regulation and control, but the neuron is limited to be used in an experimental mouse under the anesthesia state, and a hard injection needle tube has great damage to the brain. In addition, under the condition of long-term implantation, the conventional silicon-based micro-nano electrode array is not matched with the Young modulus, namely the rigidity, of brain tissues, so that inflammatory reaction in the brain is easily caused, and further, the detection performance of the electrode is reduced or the electrode falls off and other adverse effects are brought. Compared with the prior art, the implanted flexible nerve electrode array is more preferable, but due to the flexibility of the implanted flexible nerve electrode array, an auxiliary tool is needed when the implanted flexible nerve electrode array is implanted, and the conventional mode easily causes implantation displacement and cannot ensure an accurate implantation position; or the brain tissue is damaged too much due to the overlarge auxiliary tool, and the signal detection is not easy to be carried out, and the like. In addition, the dormant experimental mouse is agile in action, signal interference is easy to generate in the action process, and the influence on the experimental result is great, so that a special test system is required to be formulated for the experimental mouse, so that long-term regulation and detection are convenient.

In conclusion, the integrated implanted flexible neural electrode array aiming at dormancy and the test system thereof are developed for neuroelectrophysiology and neuroelectrochemistry detection and regulation, and the characteristics of high density, flexibility, small damage and the like of the integrated implanted flexible neural electrode array are of great significance to the exploration and research of the dormancy mechanism.

Disclosure of Invention

In view of the above, the present invention provides a dormancy detection, regulation and control integrated implantation type flexible neural electrode, which is formed by using a mold combined with PEG to perform in-vivo implantation, using an FPC interface and a flexible drug tube to perform integrated packaging, and then building a dormancy regulation and test system suitable for a long-term in-vivo laboratory mouse, so as to at least partially solve at least one of the above mentioned technical problems.

In order to achieve the purpose, the technical scheme of the invention is as follows: a dormancy detection, regulation and control integrated implantation type flexible neural electrode comprises a microelectrode array, a lead and a bonding pad;

the microelectrode array is connected with the rectangular bonding pads through leads, and each lead corresponds to one bonding pad; the bonding pad of the nerve electrode is led out from the FPC interface;

a plurality of detection sites are distributed on the needle head of each electrode of the microelectrode array, and the linear distance d between the sites of every two adjacent detection sites meets the arrangement mode of neurons in the brain and also meets the 2-time wide electrical requirement of the detection sites;

the detection sites comprise neuroelectrophysiology detection sites and neuroelectrochemistry detection sites.

Further, the microelectrode array comprises 2 × 8 circular neuro-electrophysiological detection sites and 2 rectangular neuro-electrochemical detection sites; all the nerve electrophysiology detection sites form a 2 x 8 microelectrode array and are distributed on the needle heads of 2 probes, 8 nerve electrophysiology detection sites and 1 nerve electrochemistry detection site are arranged on the needle head of each probe, and the linear distance between the detection sites is 70 mu m.

Further, each electrode of the microelectrode array of the flexible neural electrode comprises in sequence:

an insulating layer covering the lead, exposing the micro-electrode array and the pad;

a microelectrode array conducting layer, the microelectrode array comprising 18 detection sites; wherein the 18 detection sites are distributed in groups of 2 in a needle shape in both brain areas related to dormancy and are used for detecting neuroelectrophysiological and electrochemical signals of hypothalamus (dorsal nucleus and arcuate nucleus);

the flexible substrate layer is used as a carrier of the implanted flexible neural electrode array;

the soluble enhancement layer is composed of PEG sucrose and is bonded with the back of the implanted flexible neural array;

packaging the position of the FPC board electrode and the flexible medicine tube together by adopting insulating silica gel;

further, the flexible substrate layer comprises a Parylene (Parylene) material, preferably having a thickness of 19 μm; the solubility enhancing layer is characterized by comprising polyethylene glycol (PEG) and sucrose.

Further, the microelectrode array conducting layer comprises detection sites, and the diameter of each electrophysiological detection site is 4-20 μm; the size of the electrochemical detection site is 16 multiplied by 28 mu m; the bonding pad is 2950 multiplied by 300 mu m; the lead wires are 4 micrometers, and the distance between every two adjacent lead wires is 8 micrometers or more; the distribution characteristics comprise that the tip distribution range of the detection sites is needle-like, the width is 51-120 mu m, the length is 134-314 mu m, the linear interval between two adjacent detection sites is 30-70 mu m, and the 2-time interval distribution requirement of non-mutual interference is met; the electrophysiological detection site and the electrochemical detection site are distributed on the two detection probes, and the horizontal interval between the two detection probes is 200-250 mu m.

Further, the insulating layer covers the lead, and exposes the electrophysiological detection site, the electrochemical detection site, and the bonding pad.

Furthermore, the electrophysiological detection site and the electrochemical detection site are modified by nano materials such as platinum black nano particles and/or carbon nano tube nano particles; the electrochemical detection site is modified by oxidase corresponding to neurotransmitter on the basis of the modification of the nano material.

According to another aspect of the present invention, a sleep detection and regulation device integrated with the implanted flexible neural electrode is provided, which includes: the flexible drug tube, the FCB board, the insulating silica gel and the flexible nerve electrode;

the flexible medicine tube is made of an elastic fused capillary quartz sleeve;

the FCB board is provided with a flexible nerve electrode, and a flexible medicine tube is arranged on the flexible nerve electrode; the distance between the flexible medicine tube and the flexible nerve electrode is smaller than the preset distance, and the flexible medicine tube and the flexible nerve electrode are fixed by insulating silica gel.

Furthermore, the implanted flexible nerve electrode is led out through an FPC interface; the elastic fused capillary quartz sleeve comprises an outer tube with the inner diameter of 250 multiplied by 350 mu m and an inner tube with the inner diameter of 100 multiplied by 200 mu m; the elastic fused capillary quartz sleeve is bonded with the implanted flexible neural electrode array through insulating silica gel.

According to another aspect of the invention, a method for preparing an integrated implantable flexible neural electrode is provided, which comprises the following steps:

evaporating a flexible substrate layer on a silicon wafer by adopting a vapor deposition method;

preparing a metal conducting layer on the flexible substrate layer by negative photoresist lithography, electron beam evaporation and stripping technologies, wherein the metal conducting layer is preferably made of gold, and chromium is used as an adhesion layer;

evaporating a flexible material as an insulating layer on the conducting layer, removing the insulating layer on the surfaces of the microelectrode array and the bonding pad through positive glue and plasma etching, and only reserving the insulating layer covering the lead;

using SU-8 photoresist to carry out photoetching development to form a mask required by etching the flexible substrate, and forming the profile of the flexible substrate probe after oxygen ion etching;

removing the photoresist to separate the flexible micro-nano electrode from the silicon wafer to obtain a complete implanted flexible neural electrode array;

and preparing a soluble enhancement layer by adopting a mould casting method, and sticking the implanted flexible neural array and the soluble enhancement layer.

According to another aspect of the present invention, a method for preparing a soluble enhancement layer of an integrated implantable flexible neural electrode is provided, which comprises the following steps:

evaporating a flexible substrate layer on a silicon wafer by adopting a vapor deposition method;

manufacturing a mold groove with the same size as the implanted flexible neural array by using a photoetching technology and an oxygen plasma etching technology;

pouring the heated and prepared liquid PEG into a groove of a mould, standing, cooling and hardening to obtain a PEG mould;

the back side (side without exposed sites) of the implanted flexible neural array was attached to the mold PEG through sucrose.

According to another aspect of the present invention, a testing system using an integrated implantable flexible neural electrode is provided, including:

the integrated implanted flexible nerve electrode is used for long-term in-vivo detection and regulation;

a transparent columnar dormancy cabin with the diameter of 30cm and the height of 40cm, wherein a water bottle is arranged at the height of 10cm away from the bottom;

the diameter of the dormancy cabin net cover is 30cm, so that the experimental mouse is in a limited test area while the activity and dormancy space of the experimental mouse are ensured;

the length, the width and the height of the noise shielding box are respectively 50 multiplied by 60cm, so that the external noise interference is reduced;

the elastic traction rope is 60cm in length and is used for suspending the data line;

the tripod is adjustable in height and is used for controlling the elastic traction rope.

Further, a micro-nano electrode processing technology is used, and a flexible substrate layer, a microelectrode array conducting layer, an insulating layer and a soluble enhancement layer are sequentially formed from bottom to top;

the design condition of the implanted flexible neural electrode array is fully considered for forming each layer, the existing process preparation method is improved, and a proper photoresist and MEMS (micro-electromechanical systems) process is selected until the actually usable implanted flexible neural electrode array with optimal neural signal detection performance is obtained;

and preparing a soluble enhancement layer by adopting a mould casting method, and bonding the implanted flexible neural array with the soluble enhancement layer.

Has the advantages that:

compared with the prior art, the invention has the following beneficial effects:

(1) the implanted flexible neural electrode array provided by the invention has good Parylene biocompatibility, and the thinner Parylene is used as the substrate, so that the damage of the electrode to brain tissue is reduced, and the requirement of long-term detection of a laboratory mouse is met; the microelectrode array comprises a plurality of detection sites, and the detection sites are distributed in a needle shape and fit with the size of a brain area to be detected;

(2) the microelectrode array is provided with an array arrangement of micron-sized electrode sites comprising 16 electrophysiological detection sites with the diameter of 4-20 mu m and 2 electrochemical detection sites with the diameter of 16 multiplied by 28 mu m, and the linear interval between two adjacent detection sites can be 30-70 mu m; the small size of the site greatly improves the neural signal detection capability of the brain area, and meanwhile, high density distribution can be realized, and the signal-to-noise ratio is greatly improved;

(3) the microelectrode array is directly implanted into a dorsal nucleus and an arcuate nucleus of a hypothalamus, and simultaneously records electrophysiological activity of neural signals and electrochemical information transformed by neurotransmitter, so that the influence of intermediate tissues on electroencephalogram activity recording is eliminated, and the sensitivity is improved;

(4) the design of the flexible FCB and the integrated flexible medicine tube package can combine regulation and control of injected medicines with nerve signal detection, and can greatly improve the experimental efficiency; compared with the traditional medicine injection regulation and control mode, the method is not limited by the state of the experimental mouse, and can carry out medicine regulation and control and synchronous detection in the waking state of the experimental mouse according to the requirement of the experimental situation, thereby providing a convenient method for the research of the nerve experiment; meanwhile, due to the adoption of the flexible circuit board, the electrodes can be bent to a specific angle, so that the electrodes can be conveniently inserted and pulled out when the body of the laboratory mouse is curled after the laboratory mouse enters dormancy; moreover, due to the flexible material, the electrode is not easy to be pulled down by a laboratory mouse, thereby being beneficial to long-term monitoring;

(5) the testing system designed aiming at the agility and the uncontrolled behavior of the experimental mouse not only provides a comfortable environment for the experimental mouse to be favorable for the experimental mouse to enter dormancy, but also does not influence the regulation and the detection of long-term signals as much as possible;

(6) all of the above designs provide new approaches for the study of neural mechanisms in this behavioral mode of dormancy.

Drawings

Fig. 1 is a schematic structural diagram of an implanted flexible neural electrode array according to an embodiment of the present invention;

FIG. 2 is a partially enlarged schematic view of a micro-electrode array according to an embodiment of the present invention;

fig. 3 is a process flow chart of a method for manufacturing an implanted flexible neural electrode array according to an embodiment of the present invention;

fig. 4 is a process flow of manufacturing a soluble enhancement layer of an implanted flexible neural electrode according to an embodiment of the present invention;

fig. 5(a) is a schematic front view of a packaging diagram of a neural electrode array integrated with a flexible drug tube according to an embodiment of the present invention, and fig. 5(b) is a schematic structural diagram of the packaging diagram of the neural electrode array integrated with the flexible drug tube.

Fig. 6 is a schematic view of the construction of the overall test system for the laboratory mouse according to the embodiment of the invention.

In the above figures, the reference numerals have the following meanings:

1. a microelectrode array; 2. a lead wire; 3. a pad; 4. a neuroelectrophysiological detection site; 5. a neuroelectrochemical detection site; 6. an FPC board; 7. a medicine tube; 8. a flexible neural electrode; 9. insulating silica gel; 10. a noise shielding box; 11. a transparent columnar dormancy compartment; 12. a dormancy cabin net cover; 13. a tripod stand; 14. an elastic hauling rope.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings by referring to specific embodiments, but it should be noted that the following embodiments are only for illustrating the technical solutions of the present invention, but the present invention is not limited thereto.

According to one embodiment of the invention, the dormancy detection, regulation and control integrated implantable flexible neural electrode and the testing system thereof are designed, and can be used for dormant travel and 'cryotherapy' of astronauts. The site design of the multichannel is used for realizing the real-time electrophysiological and electrochemical signal detection for accurately detecting the dorsal nucleus and the arcuate nucleus of the hypothalamus. Because the Young modulus of the Parylene flexible material is close to that of the brain, the flexibility can improve the biocompatibility of the electrode, and further the nerve state of the experimental mouse in the long-term dormant state for 2-3 months can be monitored. On the other hand, the neural signals acquired through the micro-nano electrode array can be subjected to data analysis to acquire characteristics, so that the experimental rat cranial nerve mechanism in a dormant state is researched. In addition, electrical stimulation or drug regulation can be carried out on one nucleus, and the change of the nerve signal of the other nucleus can be detected, so that the nerve regulation and control of the experimental mouse in the dormant state can be realized.

As shown in fig. 1 and fig. 2, fig. 1 is a schematic diagram of an integrated implantable flexible neural electrode structure according to the present invention, including a microelectrode array 1, a lead 2, and a pad 3;

the microelectrode array 1 refers to an array structure at a needle head; the microelectrode array 1 is connected with rectangular bonding pads 3 through leads 2, and each lead corresponds to one bonding pad. The size of the single pad 3 is 2950 multiplied by 300 mu m, the pad interval is 1mm, and the standard size of the FPC interface is met. The bonding pad 3 of the neural electrode can be led out by an FPC interface and then is connected with a rear-end signal data acquisition instrument by a designed interface circuit board.

The width of the whole structure of the nerve electrode is 13.0mm, the length of the two probes is respectively 10.0mm and 13.0mm, the detection area of the probes comprises the hypothalamus dorsal nucleus and the hypothalamus arcuated nucleus of the key brain area of the experimental mouse, the width of the front end microelectrode array 1 part is 126 micrometers, and the length is 313.5 micrometers; a plurality of detection sites are distributed on the needle head, and the linear distance between every two adjacent detection sites is 70 mu m, so that the arrangement mode of neurons in the brain is considered, and the electrical requirement of the sites with the width being 2 times of that of the neurons is met.

The detection sites comprise a neuroelectrophysiology detection site 4 and a neuroelectrochemistry detection site 5;

as shown in FIGS. 1 and 2, FIG. 2 is a partially enlarged view of the micro-electrode array. The microelectrode array 1 comprises 2 × 8 circular neuro-electrophysiological detection sites 4 and 2 rectangular neuro-electrochemical detection sites 5. All the neuro-electrophysiological detection sites 4 form a 2 x 8 microelectrode array and are distributed on the needle heads of 2 probes, 8 neuro-electrophysiological detection sites 4 and 1 neuro-electrochemical detection site 5 are arranged on the needle head of each probe, the linear distance between the detection sites is 70 mu m, and the interval is selected, so that the mutual crosstalk between the detection sites and the leads in the leads 2 can be avoided, and the distribution interval of neuron cell bodies can be satisfied.

FIG. 2 is a schematic enlarged view of a part of a microelectrode array, which shows the arrangement of detection sites and the sizes of the detection sites, and comprises 4 neuroelectrophysiological detection sites with a diameter of 4-20 μm and 1 neuroelectrochemical detection site 5 with a diameter of 16X 28 μm.

The size of the neuron electrochemical detection site 5 is designed to facilitate regional neuroelectrochemical detection;

the diameter of the site 4 of the neuro-electrophysiological detection site is smaller than that of the prior art site, the process difficulty is higher, but the sensitivity of detecting action potential and the distribution density of the electrode can be improved, and the arrangement range of the site covers an important brain area, so that the neuro-electrophysiological signal change of the brain area can be detected as much as possible, and the performance of the implanted flexible neural electrode array is greatly improved; combining the neuroelectrophysiology and neuroelectrochemistry conditions, processing and analyzing neuron signals in the dormant state by adopting algorithms such as clustering, ICA (independent component analysis), GAN (gas evolution) and the like so as to predict the neural mechanism in the dormant state.

In this embodiment, referring to fig. 3, a specific process for manufacturing the implantable flexible neural electrode array specifically includes the following steps:

1. evaporating Parylene on the surface of the silicon wafer subjected to the surface cleaning treatment to obtain a 19-micron substrate layer (shown as (a) in FIG. 3);

2. spin-coating an inverse photoresist AZ5214 with the thickness of 3 μm on a silicon wafer of the thermal oxide layer cleaned by the oxygen plasma; and photoetching and developing to obtain a structural pattern of the microelectrode array, the lead and the bonding pad. Then evaporated to a thickness ofCr seed layer of to add Au conductive filmAdhesion of layer to Parylene substrate, followed by sputteringA gold thin film layer (shown as (b) in fig. 3);

3. a stripping process is used for constructing a conducting layer, the silicon wafer with the metal layer evaporated is placed in an acetone solution, the photoresist is dissolved in acetone, and then the redundant Cr/Au thin film layer can be removed, and the needed microelectrode array, the lead and the bonding pad are left (as shown in (c) in fig. 3);

4. depositing Parylene with a thickness of 2 μm on the surface of the metal film having the conductive layer as an insulating layer of the electrode (as shown in fig. 3 (d));

5. spin-coating a positive photoresist AZ4620 on the surface of the insulating layer for second photoetching, exposing electrode sites (including detection sites and grounding sites) and pad parts after development, and reserving the photoresist of the lead parts (as shown in (e) in FIG. 3);

6. etching the exposed electrode sites and the Parylene on the surface of the pad by an oxygen plasma etching process until the metal layer is exposed on the electrode sites and the pad portion, and simultaneously, the Parylene insulating layer on the surface of the lead is remained (as shown in (f) in fig. 3);

7. spin-coating a negative photoresist SU-8 on the surface of the insulating layer for a third photolithography, and leaving the photoresist on the surface of the electrode after development to serve as a protective layer (as shown in (g) in FIG. 3);

8. etching the substrate layer by multiple long-time oxygen plasma etching processes to expose the shape of the electrode (as shown in (h) of fig. 3);

9. cleaning SU-8 photoresist on the surface of the silicon wafer, soaking the silicon wafer in water, and completely releasing the implanted flexible neural microelectrode array (as shown in (i) in FIG. 3);

10. connecting the electrode to an electrochemical workstation, and depositing the nano particles with the improved electrode detection capability on the surface of the electrode site by means of electrochemical deposition to obtain a high-sensitivity microelectrode array (shown as (j) in figure 3).

11. Glutamate oxidase is modified on a site for neuroelectrochemical detection by means of electrochemical modification, and a flexible nerve array electrode capable of detecting the concentration of glutamate is obtained (such as (k) in figure 3).

Fig. 4 is a manufacturing process of the solubility-enhanced layer of the implanted flexible neural electrode according to an embodiment of the present invention, which includes the following steps.

1. Evaporating Parylene on the surface of the silicon wafer subjected to the surface cleaning treatment to obtain a 25-micron substrate layer (shown as (a) in FIG. 4);

2. spin-coating positive photoresist AZ4903 on the silicon wafer of the thermal oxidation layer cleaned by the oxygen plasma, wherein the thickness is 30 mu m; and photoetching and developing to obtain the overall shape of the micro-nano electrode. (as shown in (b) of fig. 4);

3. etching away the electrode shape with the thickness of 15 μm by a plurality of long-time plasma etching processes to form an electrode shape recess with the depth of 15 μm (as shown in (c) of fig. 4);

4. cleaning off the photoresist, drying, and pouring the diluted and heated PEG into a groove (as shown in fig. 4 (d));

5. scribing the electrode profile against the shape of the mold with an art designer knife, soaking the prepared silicon wafer in water, standing for the mold to fall off, and obtaining the soluble enhancement layer of the PEG material (as shown in (e) in FIG. 4);

6. coating sucrose on one side of the mold PEG, i.e., the solubility-enhanced layer (as shown in fig. 4 (f));

7. the flexible nerve electrode and the PEG mould are well adhered to each other, and the flexible nerve electrode meeting the requirement of hard implantation can be obtained.

Fig. 5(a) is a schematic front view of a packaging diagram of a neural electrode array integrated with a flexible drug tube, and fig. 5(b) is a schematic structural diagram of the packaging diagram of the neural electrode array integrated with the flexible drug tube. The FCB board 6 is provided with a flexible nerve electrode 8, and the flexible nerve electrode 8 is provided with a flexible medicine tube 7. The depth positions of the flexible medicine tube 7 and the flexible nerve electrode 8 can be determined according to experimental requirements, the distance between the flexible medicine tube 7 and the flexible nerve electrode 8 is smaller than 100 micrometers, and the flexible medicine tube 7 and the flexible nerve electrode 8 are fixed by insulating silica gel 9.

Fig. 6 is a schematic diagram of an overall testing system adapted to a laboratory mouse according to the present invention, which includes a noise shielding box 10, a transparent cylindrical sleeping compartment 11, a sleeping compartment mesh cover 12, a tripod support 13, and an elastic hauling rope 14. The experimental mouse is embedded with an electrode and then placed in a transparent columnar dormancy cabin 11, a data line connected with the electrode passes through a dormancy cabin net cover 12 and is suspended by an elastic traction rope 14, the elastic traction rope 14 is fixed at a tripod support 13, and the data line is connected with a data detection instrument at the rear end. The transparent columnar dormant cabin 11 is placed in the noise shielding box 10, and a round hole with the diameter of 1cm is formed below the transparent columnar dormant cabin 11 and used for placing a drinking bottle. The design of the dormant cabin net cover 12 can provide a moving space for the laboratory mouse and ensure that the laboratory mouse is in a limited test area. The data line can be prevented from being bitten by a laboratory mouse after the data line is suspended by the elastic traction rope 14 and the tripod support 13, and meanwhile, a comfortable activity space is provided for the laboratory mouse due to the telescopic effect of the elastic traction rope 14.

The nerve electrode can be implanted into brain areas related to dormancy in the brain of an experimental animal for a long time, drug regulation and control can be carried out on an experimental mouse in a dormant state at any time and scene by utilizing the design of the integrated flexible drug tube, brain damage is small, and the implanted flexible nerve electrode can be used for detecting the electrophysiological signals of neurons and the electrochemical signals of neurotransmitters before and after the regulation of the two brain areas in real time and is combined with the reaction of macroscopic dormancy behaviors.

In addition, a test system is set up and reasonably optimized on the premise of fully considering the habit of the laboratory mouse, and the long-term in-vivo nerve regulation and detection are realized by the technical means, so that the activity condition of neurons in a dormant state and whether nervous mechanisms such as nerve triggering exist can be presumed.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.