Preparation method and application of an optoelectronic neural probe

Abstract

The application provides a preparation method of an optoelectronic neural probe and application thereof, and comprises the following steps: S1: adding a flexible material precursor and a curing agent into a polyvinyl alcohol polymer through a syringe, mixing and dispersing, and then curing to obtain flexible material microbeads; S2: mixing the flexible material microbeads after impurity removal with a dopamine solution, and then plating conductive fillers to obtain flexible material microspheres coated with conductive fillers; and S3: mixing the flexible material microspheres coated with conductive fillers, conductive fillers, a flexible material precursor and a diluent, and then shaping and curing. The flexible material precursor solution is prepared into microbeads, a layer of conductive material is attached to the microbeads, the microbeads are mixed with conductive fillers and diluted flexible material which is not cured, and then the flexible neural probe with controllable diameter and length is prepared by injecting the mixture into a mold through a mold method.

Description

Technical Field

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

[0002] Optogenetics is a powerful technique that genetically modulates the expression of light-sensitive ion channel proteins on the cell membranes of specific neurons. This allows precise control of the activity of neurons expressing light-sensitive opsins and allows for specific spatiotemporal manipulation of neuronal activity, making it a powerful tool for dissecting the neural circuits that mediate specific behaviors and for therapeutic intervention in neurological diseases. The success of optogenetics is largely due to the invention of optoelectronic neural probes, which bridge the gap between the complex nervous system and the outside world. Optoelectronic neural probes have two primary functions: first, they can stimulate light, and second, they can simultaneously record neuronal activity during stimulation. To meet these two requirements, numerous scientists have devoted considerable effort. Early studies used silica optical fibers combined with filament electrodes to fabricate neural probes. These probes, with the electrode filament and optical fiber kept within a minimal distance, allowed simultaneous recording of neuronal signals during light stimulation. However, the average Young's modulus of silica optical fibers is at least six orders of magnitude greater than that of neural tissue. The elastic mismatch between silica optical fibers and the biological host can cause damage to host tissue, leading to neuronal death in the implanted environment and poor signal quality, or even the inability 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, artificial rubber, hydrogels, etc., but the neural probes made from them also record neural signals by placing electrode wires around the optical fiber, and are not truly integrated into one. In order to achieve better recording effects and cause less damage to tissues, scientists have made an integrated neural probe, leaving 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 address at least one of the aforementioned technical problems existing in the prior art. To this end, the present invention provides a method for preparing an optoelectronic neural probe. The method comprises forming microbeads from an uncured precursor solution of a flexible material, attaching a layer of conductive material thereto, then mixing the microbeads with a conductive filler and diluted uncured flexible material. The mixture is then injected into a mold using a mold method to produce 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 curing agent to the polyvinyl alcohol polymer through a syringe, mixing, dispersing and curing to obtain flexible material microbeads;

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

[0008] S3: The flexible material microspheres coated with the conductive filler, the conductive filler, the flexible material precursor and the diluent are mixed and then molded and cured.

[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. After demolding, a flexible stretchable photoelectrode can be obtained. 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 the peripheral nervous system with more intense movements.

[0011] According to some embodiments of the present 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 dispersion method includes: ultrasonic dispersion.

[0018] The flexible material liquid precursor is injected into a polyvinyl alcohol (PVA) polymer using a syringe. The polymer is stirred and ultrasonicated to break the precursor droplets into microspheres. Uncured PDMS (precursor and curing agent) is added to the PVA (2-3 wt%) using a syringe pump system. The concentration of PVA prevents the microspheres from breaking easily. The injection rate (1-3 mL / min) and needle size (16-20 Gauge) ensure the microsphere size. During the injection process, magnetic stirring is applied at a high speed to allow the droplets to drip into the PVA polymer solution. The PVA is then ultrasonically sonicated for 1-2 hours to completely break the millimeter-long flexible material precursor droplets into microspheres. The PVA polymer solution is placed in an ice bath during injection to prevent the flexible material precursor from solidifying.

[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 amino 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 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 isopropyl alcohol 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 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 clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described 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, reference to terms such as "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 exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[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 those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[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 10:1 mass ratio) was added to polyvinyl alcohol (PVA 3 wt%) using a syringe pump system. The injection rate was 1 mL / min and the needle size was 18G. Magnetic stirring was performed at 1000 rpm during the injection process. 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. The solid PDMS microbeads were then collected by centrifugation at 5000 rpm and washed three times with Tris buffer.

[0044] S2: Next, the collected PDMS microbeads were mixed with 200 mL of a 2 mg / mL dopamine solution (the concentration of the dopamine solution can be changed, which affects the adhesion of silver to the microbead surface). The solution was magnetically stirred at 400 rpm at room temperature 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 PDMS@PDA surface using an electroless 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 three times with isopropanol. Finally, they were dried in an 80°C oven to obtain PDMS@Ag microspheres.

[0045] S3: The core-shell PDMS@Ag microspheres were mixed with conductive filler Ag (1 g of Ag nanoparticles and 1 g of silver nanowire ethanol solution), uncured PDMS matrix (5 g) and PDMS diluent (3%) in a planetary mixer to obtain a mixed solution, which was then injected into a Teflon mold with a diameter of 300 μm and a length of 3 cm and heated for curing. After demolding, a flexible stretchable photoelectrode was 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 μm (from right to left).

[0047] The 500 μm flexible neural probe prepared in Example 1 was implanted into the brain of a transgenic VGAT-ChR2 mouse. Before light stimulation, the neural signals of neurons in the mouse brain could be recorded. 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 shown in FIG. 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 achieves 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. Through surface chemical modification and optimization of the silver plating, the probe can produce an efficient signal activation response under light stimulation and can accurately record neural signals. By adjusting the dopamine concentration, the thickness and surface smoothness of the silver layer can be controlled experimentally. At low dopamine concentrations, the silver layer is relatively smooth, which helps to maintain good conductivity. Higher concentrations 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 Example 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 pump system with an injection rate of 1 mL / min and an 18G needle. Magnetic stirring was performed at 1000 rpm during the injection process. 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. The solid PDMS microbeads were then collected by centrifugation at 5000 rpm and 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 400 rpm at room temperature 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 PDMS@PDA surface using an electroless 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 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 and then rinsed with isopropanol three times. Finally, they were dried in an 80°C oven to obtain PDMS@Ag microspheres.

[0053] S3: The core-shell PDMS@Ag microspheres were mixed evenly with the conductive filler Ag, uncured PDMS matrix, and PDMS diluent in a planetary mixer. The mixture was then injected into a Teflon mold with a diameter of 300 μm 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 smooth. The dopamine concentration used in Example 2 is relatively high, and the Ag formed 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 Example 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) in a 10:1 ratio) was added to polyvinyl alcohol (PVA 3 wt%) using a syringe pump system with an injection rate of 1 mL / min and an 18G needle. Magnetic stirring was applied at 1000 rpm during the injection process. 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. The solid PDMS microbeads were then collected by centrifugation at 5000 rpm and washed three times with Tris buffer.

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

[0059] S3: The core-shell PDMS@Ag microspheres were mixed evenly with the conductive filler Ag, uncured PDMS matrix, and PDMS diluent in a planetary mixer. The mixture was then injected into a Teflon mold with a diameter of 300 μm 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) to curing agent (SYLGARD 184) ratio of 10:1) was added to polyvinyl alcohol (PVA 3wt%) using a syringe pump system with an injection rate of 1 mL / min and an 18G needle. Magnetic stirring was performed at 1000 rpm during the injection process. 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. The solid PDMS microbeads were then collected by centrifugation at 5000 rpm and 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 PDMS@PDA surface using an electroless 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 three times with isopropanol, and finally dried in an 80°C oven to obtain PDMS@Ag microspheres.

[0064] S3: The core-shell PDMS@Ag microspheres were mixed evenly with the conductive filler Ag, uncured PDMS matrix, and PDMS diluent in a planetary mixer. The mixture was then injected into a Teflon mold with a diameter of 300 μm 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 is used to replace 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 curing agent to the polyvinyl alcohol polymer through a syringe, mixing, dispersing and curing to obtain flexible material microbeads; S2: removing impurities from the flexible material microbeads, mixing the mixture with a dopamine solution, and then plating the mixture with a conductive filler to obtain flexible material microbeads coated with the conductive filler; S3: mixing the flexible material microspheres coated with the conductive filler, the conductive filler, the flexible material precursor and the diluent, and then forming and curing the mixture; The flexible material includes polydimethylsiloxane; In step S2, the concentration of the dopamine solution is 1-5 mg / mL.

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

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

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

5. The preparation method according to claim 1, characterized in that In step S2, the step of plating the conductive filler includes: mixing the flexible material microspheres and the dopamine solution to obtain the dopamine-coated flexible material microspheres, a conductive filler source, and a reducing agent for reaction.

6. The preparation method according to claim 5, characterized in that The reducing agent includes at least one of sodium citrate, sodium ascorbate and ascorbic acid.

7. The preparation method according to claim 1, characterized in that The molding and curing method comprises: heating and curing the molding in a Teflon mold.

8. Use of a neural probe prepared by the method according to any one of claims 1 to 7 in the biomedical field.

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