Preparation method and application of photoelectric nerve probe

Abstract

The invention provides a preparation method and application of a photoelectric nerve probe. The preparation method comprises the following steps: S1, adding a flexible material precursor and a curing agent into a polyvinyl alcohol polymer through an injector, mixing, dispersing, and curing to obtain flexible material microspheres; s2, removing impurities from the flexible material microspheres, mixing the flexible material microspheres with a dopamine solution, and plating a conductive filler to obtain flexible material microspheres coated with the conductive filler; and S3, mixing the flexible material microspheres coated with the conductive filler, the conductive filler, a flexible material precursor and a diluent, and then molding and curing. The preparation method comprises the following steps: preparing an uncured precursor solution of a flexible material into microbeads, adhering a layer of conductive substance on the microbeads, uniformly mixing the microbeads with a conductive filler and a diluted uncured flexible material, and injecting the mixture into a mold by using a mold method to prepare the flexible nerve probe with controllable diameter and length.

Description

Technical Field

[0001] The invention belongs to the field of probes, and in particular relates to a preparation method of a photoelectric neural probe and an application thereof. Background Art

[0002] Optogenetics is a powerful technology that expresses photosensitive ion channel proteins on the cell membrane of specific neurons through gene editing. It can precisely control the activity of neurons expressing light-sensitive opsins and perform specific spatiotemporal manipulation of neuronal activity, becoming a powerful tool for analyzing neural circuits that mediate specific behaviors and for therapeutic intervention of neurological diseases. The success of optogenetics is largely due to the invention of optoelectronic neural probes, which have built a bridge between the complex nervous system and the outside world. Optoelectronic neural probes have two main functions. One is to perform light stimulation, and the other is to record the activity signals of neurons while performing light stimulation. In order to meet these two conditions, many scientists have made a lot of efforts to this end. The earliest research used silica optical fiber and electrode wire to combine to prepare neural probes, keeping the electrode wire and the optical fiber as small as possible, and recording neuronal signals while performing light stimulation. However, the average Young's modulus of silica optical fiber is at least 6 orders of magnitude larger than that of neural tissue. The elastic mismatch between quartz optical fiber and the organism may cause damage to the host tissue, thereby inducing neuronal death in the implanted environment, making the signal quality poor or failing to record neural signals. To prevent this, stretchable and flexible optoelectronic implants and polymer integrated probes have been developed, and their performance remains good during chronic implantation. Flexible materials include optical plastics, synthetic rubber, hydrogels, etc., but the neural probes made from them also place electrode wires around the optical fiber to record neural signals, and are not truly integrated. In order to achieve better recording effects and cause less damage to tissues, scientists have made an integrated neural probe that leaves a hollow channel when making the optical fiber for the insertion and feeding of metal electrode wires. However, this manufacturing method not only affects the transmission of light, but also fails to achieve true stretchability, which may have a certain impact on the recording of signals from peripheral nerve tissue with more intense activity.

[0003] Therefore, there is an urgent need to provide a stretchable optoelectronic neural probe. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention provides a method for preparing an optoelectronic neural probe, which prepares microbeads by making an uncured precursor solution of a flexible material, attaching a layer of conductive material thereon, and then mixing the microbeads with a conductive filler and a diluted uncured flexible material, and injecting the microbeads into a mold using a mold method to prepare a flexible neural probe with controllable diameter and length.

[0005] A first aspect of the present invention provides a method for preparing a photoelectric neural probe, comprising the following steps:

[0006] S1: adding the flexible material precursor and the curing agent to the polyvinyl alcohol polymer through a syringe, mixing, dispersing and curing to obtain the flexible material microbeads;

[0007] S2: mixing the flexible material microspheres and the dopamine solution and then plating a conductive filler to obtain flexible material microspheres coated with a conductive filler;

[0008] S3: Mix the flexible material microspheres coated with the conductive filler, the conductive filler, the flexible material precursor and the diluent, and then form and solidify them.

[0009] According to the embodiments of the first aspect of the present invention, there are at least the following beneficial effects:

[0010] The stretchable integrated photoelectric neural probe prepared by the present invention has two parts, a flexible light-guiding part and a conductive part, and they are integrated into an integrated neural probe. In step S1, a flexible material liquid precursor is injected into a polyvinyl alcohol polymer using a syringe, mixed and dispersed into microspheres; in step S2, dopamine is agglomerated on the surface of the microspheres to prepare for the surface chemical deposition of conductive substances; in step S3, the flexible material microspheres coated with conductive fillers are mixed with uncured flexible material precursors and diluents, and then molded and cured, and a flexible stretchable photoelectrode can be obtained after demolding. Compared with the combination of independent optical fibers and electrode wires, the present invention can integrate them into one, causing less damage to animals, and is stretchable, and can be applied to peripheral nervous systems with more intense movements.

[0011] According to some embodiments of the invention, the flexible material comprises at least one of polydimethylsiloxane (PDMS) or polystyrene-polyethylene-polystyrene block copolymer (SEBS).

[0012] According to some embodiments of the present invention, in step S1, the concentration of the polyvinyl alcohol is 2-3 wt %.

[0013] According to some embodiments of the present invention, in step S1, the mass ratio of the flexible material precursor to the curing agent is 8 to 10:1.

[0014] According to some embodiments of the present invention, in step S1, the injection rate of the syringe is 1-3 mL / min.

[0015] According to some embodiments of the present invention, in step S1, the size of the syringe needle is 16-20G.

[0016] According to some embodiments of the present invention, in step S1, the reaction temperature of the mixing step is -5 to 0°C.

[0017] According to some embodiments of the present invention, in step S1, the dispersing method includes: ultrasonic dispersion.

[0018] The flexible material liquid precursor is injected into the polyvinyl alcohol polymer using a syringe. The polymer should be stirred and ultrasonicated to break the precursor droplets into microspheres. The uncured PDMS (precursor and curing agent) is added to the polyvinyl alcohol (2-3wt%) using a syringe pumping system. The above polyvinyl alcohol concentration prevents the microspheres from being difficult to break. The injection rate (1-3mL / min) and the needle size (16-20G) ensure the size of the microspheres. During the injection process, magnetic stirring is performed at a relatively high speed so that the droplets can drip into the polyvinyl alcohol polymer solution. Then the polyvinyl alcohol is ultrasonicated for 1h-2h to completely break the millimeter flexible material precursor droplets into microspheres. The polyvinyl alcohol polymer solution is placed in an ice bath during injection to prevent the curing of the flexible material precursor.

[0019] According to some embodiments of the present invention, in step S2, the concentration of the dopamine solution is 1-5 mg / mL.

[0020] Dopamine at the above concentration can provide an appropriate amount of functional groups (such as amine groups and phenolic hydroxyl groups) to facilitate the deposition and adhesion of silver, and does not affect the adhesion of Ag within this range, thereby affecting the conductivity.

[0021] According to some embodiments of the present invention, in step S2, the impurity removal step includes: solidifying the flexible material microbeads, collecting the flexible material microbeads by centrifugation, and then washing them with a buffer solution.

[0022] According to some embodiments of the present invention, in step S2, the curing temperature is 70-80°C.

[0023] According to some embodiments of the present invention, in step S2, the curing time is 5 to 7 hours.

[0024] According to some embodiments of the present invention, in step S2, the mixing parameters include magnetic stirring at a rotation speed of 300-500 rpm for 24 to 30 hours.

[0025] According to some embodiments of the present invention, in step S2, the step of plating the conductive filler includes: mixing the flexible material microspheres coated with polydopamine obtained by mixing the flexible material microspheres with the dopamine solution, an ion source of the conductive filler and a reducing agent.

[0026] According to some embodiments of the present invention, in step S2, the ion source of the conductive filler includes a silver source.

[0027] According to some embodiments of the present invention, in step S2, the silver source includes silver nitrate.

[0028] According to some embodiments of the present invention, step S2 further includes removing impurities from the flexible material microspheres coated with the conductive filler.

[0029] According to some embodiments of the present invention, in step S2, the impurity removal step includes rinsing with deionized water, then rinsing with isopropanol and then drying.

[0030] According to some embodiments of the invention, the reducing agent comprises at least one of sodium citrate, sodium ascorbate and ascorbic acid.

[0031] According to some embodiments of the present invention, the molding and curing method includes: heating and curing the molding in a Teflon mold.

[0032] In step S3, flexible and stretchable photoelectrodes of different sizes (200-800um) and controllable lengths (1-8cm) can be formed in a Teflon mold. The size and length of the photoelectrode are determined by the size and length of the Teflon tube. Different types of Teflon tubes can be selected according to experimental requirements.

[0033] The second aspect of the present invention provides the application of the neural probe prepared by the above preparation method in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The integrated flexible neural probes of different sizes prepared in Example 1;

[0035] Figure 2 The neural signals recorded by the neural probe before and after light stimulation. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0037] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0038] Unless otherwise specified, "room temperature" in the present invention means 25°C±5°C.

[0039] Unless otherwise specified, "about" in the present invention means that the allowable error is within ±2%.

[0040] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0041] Example 1

[0042] This embodiment provides a method for preparing a photoelectric neural probe, specifically:

[0043] S1: Uncured PDMS (polydimethylsiloxane (PDMS) and curing agent (SYLGARD 184) in a mass ratio of 10:1) was added to polyvinyl alcohol (PVA 3wt%) using a syringe pumping system, with an injection rate of 1 mL / min and a needle size of 18G. During the injection process, magnetic stirring was performed at a speed of 1000 rpm. The polyvinyl alcohol was then sonicated at 100% power for 1 h to break the millimeter-sized PDMS droplets into microspheres. The PVA solution was placed in an ice bath during injection to prevent the PDMS from solidifying. The liquid PDMS microspheres were then cured in the PVA solution at 70°C for at least 6 h, and the solid PDMS microbeads were collected by centrifugation at 5000 rpm and then washed three times with Tris buffer.

[0044] S2: Next, the collected PDMS microbeads were mixed with 200 mL of 2 mg / mL dopamine solution (the concentration of the dopamine solution can be changed, which has an effect on the attachment of silver to the microbead surface), and the solution was magnetically stirred at 400 rpm at room temperature for at least 24 h to ensure that dopamine can be coated on the surface of the PDMS microbeads. Finally, a silver layer was plated on the PDMS@PDA surface using a chemical plating process. The PDMS@PDA microspheres and 4 g of silver nitrate (0.04 g / mL) were dispersed in Tris buffer and magnetically stirred for 1 h to form a silver seed layer. Then, ascorbic acid (200 mL) solution was added to promote the reduction of silver to form core-shell PDMS@Ag microspheres. The microspheres were rinsed with deionized water and then rinsed with isopropanol three times. Finally, they were dried in an oven at 80°C to obtain PDMS@Ag microspheres.

[0045] S3: Put the core-shell PDMS@Ag microspheres, conductive filler Ag (1 g of Ag nanoparticles, 1 g of silver nanowire ethanol solution), uncured PDMS matrix (5 g) and PDMS diluent (3%) into a planetary mixer and mix them evenly to obtain a mixed solution, which is then injected into a Teflon mold with a diameter of 300 um and a length of 3 cm and heated to cure. After demolding, a flexible stretchable photoelectrode can be obtained.

[0046] In step S3, flexible neural probes of different sizes can be prepared in molds of different sizes. Figure 1 As shown, flexible neural probes of different sizes can be prepared by injecting the mixed solution in S3 into Teflon tubes of 200, 300, 400, and 500 um (from right to left).

[0047] The 500um flexible neural probe prepared in Example 1 was implanted into the brain of a transgenic VGAT-ChR2 mouse. The neural signals of neurons in the mouse brain could be recorded before light stimulation. During light stimulation, the photosensitive protein was stimulated by light, the neurons were activated, and the neural signals changed. Compared with before light stimulation, the neuronal discharge signals became more intensive. The results are as follows: Figure 2 shown.

[0048] The photoelectric neural probe in Example 1 has the advantages of integration, flexibility, and stretchability. At the same time, through surface modification, silver plating and simple preparation process, it has achieved high conductivity, good biocompatibility and customizability in performance. The probe can be widely used in the recording and regulation of neural signals, and is particularly suitable for long-term implantation and research and treatment of the nervous system in dynamic environments. The prepared photoelectric neural probe has the characteristics of flexibility and stretchability. By using a PDMS substrate and an adjustable mold size, the probe can adapt to different tissue shapes and movements, especially in the peripheral nervous system and parts with more intense movements. The silver plating can improve the signal conduction efficiency between the probe and the neural tissue, enhance its role in neural recording and stimulation, and the stability of the silver layer also improves the long-term service life of the probe. Due to its photoconductive properties, the photoelectric neural probe can accurately activate neurons under light stimulation. The probe can produce an efficient signal activation reaction under light stimulation and can accurately record neural signals through surface chemical modification and optimization of the silver plating. By adjusting the dopamine concentration, the experiment can control the thickness and surface flatness of the silver layer. At low concentrations of dopamine, the silver layer is relatively smooth, which helps to maintain good conductivity. A higher concentration of dopamine may form rougher silver protrusions on the surface. The appropriate concentration can be selected as needed to balance conductivity and surface smoothness.

[0049] Example 2

[0050] This embodiment provides a method for preparing a photoelectric neural probe. The difference between this embodiment and embodiment 1 is that the concentration of the dopamine solution is 5 mg / mL, specifically:

[0051] S1: Uncured PDMS (polydimethylsiloxane (PDMS) and curing agent (SYLGARD 184) ratio of 10:1) was added to polyvinyl alcohol (PVA 3wt%) using a syringe pumping system, with an injection rate of 1 mL / min and a needle size of 18G. During the injection process, magnetic stirring was performed at a speed of 1000 rpm. The polyvinyl alcohol was then sonicated at 100% power for 1 h to break the millimeter-sized PDMS droplets into microspheres. The PVA solution was placed in an ice bath during injection to prevent the PDMS from curing. The liquid PDMS microspheres were then cured in the PVA solution at 70°C for at least 6 h, and the solid PDMS microbeads were collected by centrifugation at 5000 rpm and then washed three times with Tris buffer.

[0052] S2: Next, the collected PDMS microbeads were mixed with 200 mL of 5 mg / mL dopamine solution, and the solution was magnetically stirred at room temperature at 400 rpm for at least 24 h to ensure that dopamine could be coated on the surface of the PDMS microbeads. Finally, a silver layer was plated on the surface of PDMS@PDA using a chemical plating process. PDMS@PDA microspheres and 4 g of silver nitrate (0.04 g / mL) were dispersed in Tris buffer and magnetically stirred for 1 h to form a silver seed layer. Then, ascorbic acid (200 mL) solution was added to promote the reduction of silver to form core-shell PDMS@Ag microspheres. The microspheres were rinsed with deionized water and then rinsed with isopropanol three times, and finally dried in an oven at 80 ° C to obtain PDMS@Ag microspheres.

[0053] S3: The core-shell PDMS@Ag microspheres were mixed evenly with the conductive filler Ag, the uncured PDMS matrix and the PDMS diluent in a planetary mixer, and then injected into a Teflon mold with a diameter of 300 um and a length of 3 cm and heated for curing. After demolding, a flexible and stretchable photoelectrode was obtained.

[0054] The dopamine concentration used in Example 1 is relatively low, and the thickness of the Ag loaded on the surface of the microbeads is relatively thin and relatively smooth. The dopamine concentration used in Example 2 is relatively high, and the formed Ag may have more small protrusions on the surface. In order to ensure that more Ag is loaded and the surface is relatively flat, a dopamine concentration of 2 mg / mL is selected as the best embodiment.

[0055] Example 3

[0056] This embodiment provides a method for preparing a photoelectric neural probe. The difference between this embodiment and embodiment 1 is that the concentration of the dopamine solution is 1 mg / mL, specifically:

[0057] S1: Uncured PDMS (polydimethylsiloxane (PDMS) and curing agent (SYLGARD 184) ratio of 10:1) was added to polyvinyl alcohol (PVA 3wt%) using a syringe pumping system, with an injection rate of 1mL / min and a needle size of 18G. During the injection process, magnetic stirring was performed at a speed of 1000rpm, and then the polyvinyl alcohol was ultrasonicated at 100% power for 1h to break the millimeter-scale PDMS droplets into microspheres. The PVA solution was placed in an ice bath during injection to prevent PDMS from curing. The liquid PDMS microspheres were then cured in the PVA solution at 70°C for at least 6h, and then the solid PDMS microbeads were collected by centrifugation at 5000rpm, and then washed 3 times with Tris buffer;

[0058] S2: Next, the collected PDMS microbeads were mixed with 200 mL of 1 mg / mL dopamine solution, and the solution was magnetically stirred at room temperature at 400 rpm for at least 24 hours to ensure that dopamine could be coated on the surface of the PDMS microbeads. Finally, a silver layer was plated on the surface of the PDMS@PDA using a chemical plating process. The PDMS@PDA microspheres and 5 g of silver nitrate (0.04 g / mL) were dispersed in Tris buffer and magnetically stirred for 1 hour to form a silver seed layer. Then, ascorbic acid (200 mL) solution was added to promote the reduction of silver to form core-shell PDMS@Ag microspheres. The microspheres were rinsed with deionized water, then rinsed with isopropanol three times, and finally dried in an oven at 80°C to obtain PDMS@Ag microspheres;

[0059] S3: The core-shell PDMS@Ag microspheres were mixed evenly with the conductive filler Ag, the uncured PDMS matrix and the PDMS diluent in a planetary mixer, and then injected into a Teflon mold with a diameter of 300 um and a length of 3 cm and heated for curing. After demolding, a flexible and stretchable photoelectrode was obtained.

[0060] Example 4

[0061] This embodiment provides a method for preparing a photoelectric neural probe. The difference between this embodiment and embodiment 1 is that the reducing agent in step S2 is sodium citrate, specifically:

[0062] S1: Uncured PDMS (polydimethylsiloxane (PDMS) and curing agent (SYLGARD 184) ratio of 10:1) was added to polyvinyl alcohol (PVA 3wt%) using a syringe pumping system, with an injection rate of 1 mL / min and a needle size of 18G. During the injection process, magnetic stirring was performed at a speed of 1000 rpm. The polyvinyl alcohol was then sonicated at 100% power for 1 h to break the millimeter-scale PDMS droplets into microspheres. The PVA solution was placed in an ice bath during injection to prevent the PDMS from solidifying. The liquid PDMS microspheres were then cured in the PVA solution at 70°C for at least 6 h, and the solid PDMS microbeads were collected by centrifugation at 5000 rpm, and then washed three times with Tris buffer.

[0063] S2: Next, the collected PDMS microbeads were mixed with a 5 mg / mL dopamine solution, and the solution was magnetically stirred at room temperature at 500 rpm for at least 24 hours to ensure that dopamine could be coated on the surface of the PDMS microbeads. Finally, a silver layer was plated on the surface of the PDMS@PDA using a chemical plating process. The PDMS@PDA microspheres and silver nitrate (0.04 g / mL) were dispersed in Tris buffer and magnetically stirred for 1 hour to form a silver seed layer. Then, sodium citrate (200 mL) solution was added to promote the reduction of silver to form core-shell PDMS@Ag microspheres. The microspheres were rinsed with deionized water, then rinsed with isopropanol three times, and finally dried in an oven at 80°C to obtain PDMS@Ag microspheres;

[0064] S3: The core-shell PDMS@Ag microspheres were mixed evenly with the conductive filler Ag, the uncured PDMS matrix and the PDMS diluent in a planetary mixer, and then injected into a Teflon mold with a diameter of 300 um and a length of 3 cm and heated for curing. After demolding, a flexible and stretchable photoelectrode was obtained.

[0065] Comparative Example 1

[0066] This comparative example provides a method for preparing a photoelectric neural probe. The difference between this comparative example and Example 1 is that polymethyl methacrylate replaces polydimethylsiloxane, and the other conditions are the same.

[0067] PMMA has poor biocompatibility, which affects its use as a biomedical material.

Claims

1. A method for preparing a photoelectric neural probe, characterized in that: The following steps are involved: S1: adding the flexible material precursor and the curing agent to the polyvinyl alcohol polymer through a syringe, mixing, dispersing and curing to obtain the flexible material microbeads; S2: removing impurities from the flexible material microbeads, mixing with the dopamine solution, and then plating with a conductive filler to obtain flexible material microbeads coated with a conductive filler; S3: Mix the flexible material microspheres coated with the conductive filler, the conductive filler, the flexible material precursor and the diluent, and then form and solidify them.

2. The preparation method according to claim 1, characterized in that: The flexible material includes polydimethylsiloxane.

3. The preparation method according to claim 1, characterized in that: In step S1, the concentration of the polyvinyl alcohol is 2-3 wt %.

4. The preparation method according to claim 1, characterized in that: In step S1, the reaction temperature of the mixing step is -5 to 0°C.

5. The preparation method according to claim 1, characterized in that: In step S1, the dispersing method includes: ultrasonic dispersing.

6. The preparation method according to claim 1, characterized in that: In step S2, the concentration of the dopamine solution is 1-5 mg / mL.

7. The preparation method according to claim 1, characterized in that: In step S2, the step of plating the conductive filler comprises: mixing the flexible material microspheres coated with dopamine obtained by mixing the flexible material microspheres with the dopamine solution, a conductive filler source and a reducing agent for reaction.

8. The preparation method according to claim 7, characterized in that: The reducing agent includes at least one of sodium citrate, sodium ascorbate and ascorbic acid.

9. The preparation method according to claim 7, characterized in that: The molding and curing method comprises: heating and curing in a Teflon mold.

10. Use of a neural probe prepared by the method according to any one of claims 1 to 9 in the biomedical field.

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