Intracerebral dopamine selective microelectrode and preparation method thereof

Abstract

The invention relates to an intracerebral dopamine selective microelectrode and a preparation method thereof, the intracerebral dopamine selective microelectrode comprises a working electrode, the working electrode comprises a carbon fiber electrode, and the carbon fiber electrode is filamentous; the modification layer comprises a carbon nanomaterial and a stable compound, the carbon nanomaterial is used for improving stability and promoting dopamine oxidation reduction, and the modification layer wraps at least part of the outer surface of the carbon fiber electrode wire. According to the invention, the appearance and physical and chemical properties of the dopamine electrode are improved, so that the purpose of detecting the dopamine with long-time sober activity can be achieved, and the research of neurochemistry closer to the real situation can be met.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical detection, and in particular relates to a brain dopamine selective microelectrode, a preparation method and an application thereof. Background Art

[0002] The current mainstream dopamine detection method is an offline detection method based on electrochemical electrodes, such as detecting the release function of dopamine at the level of brain slices or nerve cells. The real-time in situ dopamine detection at the living body level is mainly aimed at the detection of dopamine release under anesthesia. Generally, fast scanning cyclic voltammetry is used for dopamine detection. This method uses carbon fiber electrodes implanted in brain areas rich in dopaminergic neurons. Using specific scanning parameters, dopamine can undergo a rapid oxidation-reduction process on the electrode surface, thereby obtaining the concentration change process of dopamine during neurotransmission.

[0003] CN111307899B describes a method for detecting vitamin C in the brain, which uses carbon balls modified on the surface of carbon fiber electrodes to achieve real-time detection of vitamin C in the brain.

[0004] CN116297749A discloses a method for preparing a flexible neuroelectrochemical electrode, which uses a flexible hydrogel electrode as an anode and a platinum electrode in a glass tube as a cathode to construct an electrochemical electrode with an intracerebral potential, which can effectively reduce damage to brain tissue and is used for the detection of neurochemical substances in the brain. However, since dopamine detection has strict requirements on electrode materials, electrodes based on carbon fiber materials have not been able to be used for long-term brain implantation.

[0005] There is a difference between the ideal working pH value of the electrode and the detection environment in the brain. At the same time, at a microscopic scale, the diffusion effect of the substance itself has a greater impact on the surface of the electrochemical electrode and the effective ingredients fixed on the surface of the electrode. The current structural design of carbon fiber electrodes and the physical and chemical properties of the carbon fiber surface cannot support long-term brain implantation and scanning. Summary of the invention

[0006] In view of the above technical problems, the present invention improves the appearance and physicochemical properties of dopamine electrodes to achieve the purpose of dopamine detection during long-term waking activities and meet the needs of neurochemical research that is closer to the real situation.

[0007] The first aspect of the present invention provides: a brain dopamine selective microelectrode, comprising: a working electrode, the working electrode comprising: a carbon fiber electrode; a conductive inner core, the conductive inner core is electrically connected to the carbon fiber electrode; a modification layer, the modification layer comprises a carbon nanomaterial and a stability compound for improving stability and promoting dopamine oxidation-reduction, the modification layer is wrapped around at least a portion of the outer surface of the carbon fiber electrode.

[0008] Furthermore, the working electrode also includes: a support member and an adapter; the support member is a hollow structure, which is fixed to the outside of the carbon electrode; the conductive core is fixed inside the support member; the support member, the conductive core and the adapter are fixedly connected.

[0009] Furthermore, the microelectrode also includes a reference electrode, which includes: a conductive core; an electroplating layer, the electroplating layer is wrapped around the outer surface of the conductive core; a stabilizing layer, the stabilizing layer is wrapped around the outer surface of the electroplating layer, and the stabilizing layer contains a stabilizing compound; and a reference electrode adapter, the reference electrode adapter is electrically connected to the conductive core.

[0010] The second aspect of the present invention provides: a method for preparing a dopamine-selective microelectrode in the brain, comprising: covering a carbon electrode with an adhesive layer; placing the carbon electrode into a support, and bonding the support, the carbon electrode and the adapter with ultraviolet-sensitive glue to obtain a basic electrode body; providing a 1 mg / ml carbon nanotube solution and a 10% perfluorosulfonic acid resin aqueous solution, mixing them in a 1:1 ratio to obtain a mixed solution; immersing the basic electrode body into the mixed solution for 5 times, with an interval of 10 minutes each time, and then drying the electrode at 37°C for 24 hours to obtain a working electrode.

[0011] Further: including the reference electrode preparation steps: applying a voltage of 1.5V for 60S to the conductive core in a 3M potassium chloride solution for electroplating to obtain a conductive core with surface modified by silver chloride; immersing the conductive core with surface modified by silver chloride in a 10% perfluorosulfonic acid resin aqueous solution for 5 times, each time with an interval of 10 minutes, and then drying at 37°C for 24 hours; bonding the dried conductive core and the electrode adapter with ultraviolet-sensitive glue to obtain a reference electrode.

[0012] The third aspect of the present invention provides: a brain dopamine selective microelectrode coating composition, comprising perfluorosulfonic acid resin and carbon nanotubes, wherein the mass ratio thereof is 80:1 to 120:1, preferably 100:1.

[0013] The fourth aspect of the present invention provides: a method for applying the brain dopamine-selective microelectrode coating composition provided in the third aspect of the present invention, wherein the coating composition is applied to dopamine detection.

[0014] The beneficial effect of the present invention is that the structure and volume of the dopamine detection electrode are restricted, and the surface of the electrode is modified to improve stability, so as to achieve the purpose of long-term placement in the brain to detect dopamine. The reason is that, under normal circumstances, the electrode will be contaminated by protein when implanted in the brain for a long time, which will induce an inflammatory reaction and eventually lead to the performance degradation of the electrode. In the present invention, hydrophilic molecular materials such as perfluorosulfonic acid resin are modified to the electrode surface to improve the hydrophilicity of the electrode, reduce the adsorption of protein on the electrode surface, and further reduce the trauma of implanting the electrode through redesigned structural miniaturization, thereby reducing the inflammatory reaction in the brain. Compared with ordinary electrodes, the excellent effect of detecting dopamine in the brain for up to 30 days is finally achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the working electrode structure of an embodiment of the present invention;

[0016] Figure 2 Schematic diagram of the overall structure of the carbon fiber electrode and the modified layer according to the embodiment of the present invention;

[0017] Figure 3 Schematic diagram of carbon fiber electrode and conductive inner core structure according to an embodiment of the present invention;

[0018] Figure 4 A schematic diagram of the reference electrode structure of an embodiment of the present invention;

[0019] Figure 5 Cross-sectional view of the layered structure of the conductive core, electroplating layer and stabilizing layer according to an embodiment of the present invention;

[0020] Figure 6 Flow chart of the preparation of working electrode according to the embodiment of the present invention;

[0021] Figure 7 Flow chart of reference electrode preparation according to an embodiment of the present invention;

[0022] Figure 8 The original current curve of the fast scanning cyclic voltammetry of the dopamine electrode modified with carbon nanotubes and nafion in accordance with the embodiment of the present invention in response to dopamine;

[0023] Fig. 9 A fast scanning cyclic voltammetry curve of the dopamine electrode modified with carbon nanotubes and nafion in accordance with an embodiment of the present invention in response to dopamine and the background current;

[0024] Fig.10 The rapid scanning cyclic voltammetry original current curve corresponding to the electrode of the embodiment of the present invention placed in vitro for 30 days;

[0025] Fig.11 The rapid scanning cyclic voltammetry original current curve corresponding to the electrode of the embodiment of the present invention placed in the brain for 30 days;

[0026] Fig.12 The original current curve of the rapid scanning cyclic voltammetry corresponding to the unmodified electrode of the embodiment of the present invention placed in the brain for 30 days;

[0027] Fig.13 A fast scanning cyclic voltammetry background current curve corresponding to the electrode of the embodiment of the present invention under different dopamine concentration detection environments;

[0028] Fig.14 Linearity fitting diagram of the electrode according to the embodiment of the present invention under different dopamine concentration detection environments;

[0029] Fig.15 Schematic diagram of placement of electrodes in the brain according to an embodiment of the present invention;

[0030] Fig.16 Micrograph of the reference electrode silver chloride wire without a modified layer in the embodiment of the present invention;

[0031] Fig.17 Micrograph of the modified layer of the silver chloride wire of the reference electrode of the embodiment of the present invention;

[0032] Fig.18 Micrograph of the appearance of the working electrode modified carbon tube nafion without the modified layer in the embodiment of the present invention;

[0033] Fig.19 The working electrode of the embodiment of the present invention is modified with a carbon tube nafion, and the appearance has a micrograph of the modified layer;

[0034] The meanings of the numbers in the figure are: working electrode 1, carbon fiber electrode 11, conductive inner core 12, modified layer 13, carbon nanomaterial 131, support 14, adapter 15, reference electrode 2, conductive core 21, electroplating layer 22, stabilizing layer 23, reference electrode adapter 24. DETAILED DESCRIPTION

[0035] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0036] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0037] Terminology explanation:

[0038] Working electrode: It is a key element in the electrochemical reaction process, usually referring to the electrode where the reaction occurs in electrochemical equipment such as batteries and electrolyzers.

[0039] Nafion perfluorosulfonic acid resin: It is the strongest known solid superacid with a series of unique physical and chemical properties, making it widely used in many fields.

[0040] Reference electrode: Its electrode potential remains unchanged under the measurement conditions, and is used to observe and measure the electrode potential of the indicator electrode or working electrode. The reference electrode is usually used as a reference comparison electrode when measuring various electrode potentials. By forming a battery with the electrode being measured and measuring the electromotive force value of the battery, the electrode potential of the working electrode can be calculated.

[0041] Rapid scanning cyclic voltammetry curve: It is a commonly used analytical method for electrochemical detection of changes in neurotransmitters in the brain. It changes the electrode potential at an ultra-high scanning voltage speed and measures the current response on the electrode to obtain a characteristic curve. This curve uses potential as the horizontal coordinate and current as the vertical coordinate, and is mainly used to reveal information on the properties, mechanism and kinetics of electrochemical reactions.

[0042] In these embodiments:

[0043] The detection of chemical substances in the brain is of great significance for the study of the molecular mechanisms of physiological and pathological processes related to the nervous system. It has a huge driving force for studying and understanding the brain at the molecular level. Dopamine, in particular, is an important neurotransmitter in the process of neural excitation in the brain and is crucial for the transmission of neural signals. Currently, most neurological diseases have been shown to be related to the apoptosis and dysfunction of dopaminergic neurons.

[0044] By implanting carbon fiber electrodes into brain regions rich in dopaminergic neurons and using specific scanning parameters, dopamine can undergo a rapid oxidation-reduction process on the electrode surface, thereby obtaining the concentration change process of dopamine during neurotransmission.

[0045] Existing carbon fiber electrodes all use a hollow glass tube with a length of about 10 cm and a diameter of about 1.5 mm as the protective body of the electrode. Then, a 7-micron carbon fiber is bonded to the surface of the conductive wire through copper wire or other conductive materials. The glass tube is drawn into a conical glass tip structure using a drawing instrument, and the carbon fiber is passed through the glass tube. The electrode is then sealed with glue to obtain the dopamine electrode currently being tested. However, the glass tube used in this electrode is too large, which will cause great damage to the animal's brain, and the protrusion on the brain surface is too long, resulting in it being only used for short-term dopamine detection under anesthesia. Therefore, for long-term detection scenarios of awake animals that are closer to actual dopamine detection, the stability of the electrode's long-term detection needs to be improved.

[0046] Therefore, the present invention redesigns the appearance of the electrode and modifies the physical and chemical properties of the carbon fiber surface, and further obtains a dopamine detection electrode that can be implanted in the brain for a long time. The present invention describes a new design and production method of a neuroelectrochemical electrode that can be used for dopamine detection implanted in the brain for a long time.

[0047] The preparation of working electrode can be divided into two parts: basic electrode preparation and basic electrode surface modification.

[0048] In some embodiments of the present invention, Figure 1-3 As shown:

[0049] A brain dopamine selective microelectrode, comprising: a working electrode 1, wherein the working electrode comprises:

[0050] Carbon fiber electrode 11;

[0051] A conductive inner core 12, wherein the conductive inner core 12 is electrically connected to the carbon fiber electrode 11;

[0052] The modification layer 13 includes carbon nanomaterials and stability compounds for improving stability and promoting dopamine oxidation-reduction. The modification layer 13 is wrapped around at least a portion of the outer surface of the carbon fiber electrode 11 .

[0053] In some embodiments of the present invention, Figure 1-3 As shown, the difference from the above embodiment is that:

[0054] The working electrode further comprises: a support member 14 and an adapter 15;

[0055] The support member 14 is a hollow structure, which is fixed to the outside of the carbon electrode 11;

[0056] The conductive inner core 12 is fixed inside the support member 14;

[0057] The support member 14 , the conductive inner core 12 and the adapter 15 are fixedly connected.

[0058] In these embodiments:

[0059] 1.1 Basic Electrode:

[0060] like Fig.15 As shown, a hollow polyimide-coated glass tube with a length of about 1 cm and a diameter of about 0.3 mm is used as the supporting body of the electrode, and a silver wire with a diameter of about 0.05-0.15 mm, preferably 0.1 mm, is used as the bonding wire and conductive inner core of the carbon fiber to prepare an ultrafine fiber electrode. Subsequently, a fine support piece is cut by a mold or 3D printed to prepare an adapter of the ultrafine electrode. The electrode adapter, the glass tube and the carbon fiber wire are bonded to each other with ultraviolet-sensitive glue to prepare an electrode with an overall implant size of 0.3 mm and an implant depth of about 1-6 mm as an intracerebral implantable wearable electrode, which can realize long-term detection in a free-moving state of the animal without affecting the animal.

[0061] In the above basic electrodes:

[0062] The overall size of the electrode is about 1.2 cm. At the same time, when implanted in the brain, some electrodes (such as adapters, wires and other components) will not be implanted in the brain, and their implanted size is smaller than the overall size. According to the experimental measurement depth, the electrode implantation depth can be between 0.1 and 0.8 cm, preferably: 0.1 cm, 0.15 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.75 cm. In this embodiment, the electrodes with an implantation size of 0.3 mm and an implantation depth of about 1-6 mm are selected;

[0063] The electrode has a small implant volume, which effectively reduces the impact on brain tissue in the implant area and significantly reduces the discomfort of the subjects, making it suitable for long-term wear.

[0064] 1.1.1 Support

[0065] Such as hollow glass tube or flexible substrate

[0066] The use of a flexible substrate in a dopamine electrode can closely fit the surface of the carbon electrode, reduce the gap between the electrode and the detection object, and improve the accuracy and stability of the detection.

[0067] Hollow glass tube: As the supporting body of the electrode, it can block the internal material of the glass tube from the external environment, and at the same time prevent the material from rapidly diffusing, reacting quickly with the detected object or other substances, and losing the detection ability, reducing the speed of detection performance decline caused by the diffusion of materials in the glass tube, and improving the working cycle and working stability of the electrode.

[0068] At the same time, one end of the hollow glass tube is made into a conical opening, or a hemispherical shape with an opening, or other curved shapes that are continuously narrowed from the cylindrical surface of the hollow glass tube toward the central axis.

[0069] Take the conical opening as an example: part of the outer surface of the conical opening and the carbon fiber electrode 11 are also wrapped with a waterproof layer that improves stability and has water-isolating ability, and the conductive core is sealed in the support, so that the internal environment of the electrode support and the environment in contact with the conductive core are more stable, thereby improving the stability of the electrode during long-term implantation.

[0070] 1.1.2. Carbon electrode: carbon fiber

[0071] Carbon fiber: As an electrode, it does not directly participate in the electrochemical reaction, but carbon fiber can be used as a base material for current collectors or composite electrodes to improve the conductivity and cycle stability of the electrode.

[0072] In some embodiments of the present invention: the diameter of the carbon fiber used in the carbon fiber electrode 11 is 5-10um, preferably 7um, and the exposed length of the electrode is 200um. A certain length can be retained in the support, and the modified layer can be fully wrapped, or the retained part in the support can be unwrapped with the modified layer; the conductivity and stability of the carbon fiber are used as the electrode substrate. The overall size of the working electrode prepared in this way can reach 0.8-2.0cm, and the preferred overall size of the working electrode is 1.2cm.

[0073] 1.1.3. Conductive core: bonding wire

[0074] The bonding wire comprises a conductive material, such as a silver wire or a silver wire and a silver paste;

[0075] Silver wire: As the bonding wire of the carbon fiber electrode, a silver wire with a diameter of about 0.1 mm is used as the bonding wire of the carbon fiber, and it is used as a conductive inner core and connected to the carbon fiber electrode through silver paste or other conductive materials. The silver wire as the bonding wire electrode of the carbon fiber has excellent mechanical properties and can improve the electrochemical performance.

[0076] 1.2. Modification layer:

[0077] The modification layer includes carbon nanomaterials and a stabilizing compound.

[0078] After the basic electrode is prepared, the electrode surface is modified by mixing 1 mg / ml carbon nanotube solution and 10% nafion (perfluorosulfonic acid resin) aqueous solution in a 1:1 ratio. After mixing, the electrode carbon fiber is immersed in the solution. The electrode is immersed in the solution using a leaching machine for 5 seconds each time, then the electrode is lifted above the liquid surface and dried for 10 minutes. The immersion is repeated 5 times, and then the electrode is dried at 37°C for 24 hours.

[0079] The thickness of the modified layer is about 5-20um. The modified layer improves the conductivity of the electrode and its catalytic performance to dopamine. The negatively charged functional groups on the surface of the carbon nanotubes and the negative charge of perfluorosulfonic acid can better enrich the positively charged dopamine and adsorb it to the electrode surface for dopamine detection.

[0080] 1.2.1 Carbon nanomaterials

[0081] Such as carbon nanotubes and carbon balls: They mainly play the following roles outside the dopamine electrode: improving the conductivity of the electrode. Due to its unique structural morphology, carbon nanotubes exhibit metal or semiconductor properties and have excellent conductivity. When carbon nanotubes are applied to the outside of the dopamine electrode, they can significantly improve the conductivity of the electrode, making electron transfer faster and more efficient.

[0082] Improve the catalytic performance of the electrode. In electrochemical detection, the performance of dopamine electrodes is often affected by the catalytic ability of the electrode material. Carbon nanotubes, as a modified material for the electrode, can significantly enhance the electrochemical catalytic effect of the electrode on dopamine and other biological molecules. This catalytic effect makes the oxidation reaction of dopamine on the electrode easier, thereby improving the sensitivity and response speed of the electrode.

[0083] Increase the effective reaction area of ​​the electrode: Carbon nanotubes have an extremely high specific surface area, and when they are modified to the outside of the dopamine electrode, they can significantly increase the effective reaction area of ​​the electrode. This means that more dopamine molecules can react with the electrode surface at the same time, thereby improving the current response and detection sensitivity of the electrode.

[0084] Enhance the stability and durability of the electrode, stabilize the structure, carbon nanotubes have a stable chemical structure and excellent mechanical properties, which allows them to form a solid protective layer on the surface of the electrode. This protective layer can not only prevent the electrode surface from being corroded or contaminated, but also reduce the wear and tear and shedding of the electrode during use, thereby extending the service life of the electrode and adapting to long-term monitoring of dopamine changes in the brain environment.

[0085] Carbon Ball:

[0086] It is a spherical carbon material with a fullerene-like cage structure composed of multiple layers of concentrically opened graphene sheets. In order to improve the application performance of carbon spheres, it is often necessary to modify their surface. By coating the surface of the carbon sphere with another material, the surface modification is achieved to form a composite functional material with a core-shell structure. For example, a modified layer that can promote the redox reaction of dopamine is covered to adapt to long-term environmental monitoring of dopamine changes in the brain.

[0087] 1.2.2 Stable compounds,

[0088] Such as perfluorosulfonic acid resin, betaine, or acetylcholine: perfluorosulfonic acid is negatively charged and can greatly increase the hydrophilicity of the electrode. Betaine and acetylcholine are zwitterions that can also increase the hydrophilicity of the electrode, thereby improving the long-term biocompatibility of the electrode in the body.

[0089] Perfluorosulfonic acid resin: Nafion is a perfluorosulfonic acid type solid strong acid polymer with good chemical stability and thermal stability. After the electrode is immersed in Nafion solution and dried, Nafion will form a protective film on the electrode surface. This protective film not only enhances the corrosion resistance and oxidation resistance of the electrode, but also improves its electrochemical stability in various environments.

[0090] The conductivity is maintained. The Nafion membrane itself has a low conductivity, but its coverage on the electrode surface does not significantly affect the conductivity of the silver wire itself. Because the Nafion membrane is porous, it allows ions to pass through while maintaining good insulation to electrons. Therefore, in electrochemical measurements, this modified electrode can still maintain good conductivity.

[0091] Betaine: Its chemical name is N,N,N-trimethylglycine. It is an alkaloid and belongs to the quaternary ammonium base class.

[0092] Betaine has good conductivity and can show good conductivity at lower concentrations. Therefore, using betaine or its derivatives as electrode modification materials may help improve the conductivity of the electrode, reduce the resistivity, and thus improve the sensitivity of electrochemical detection.

[0093] In the electrochemical reaction of dopamine detection, the rate of electron transfer is a key factor affecting the reaction efficiency. The special chemical structure of betaine may help promote the electron transfer between the electrode surface and the reactants, thereby improving the rate and efficiency of the electrochemical reaction.

[0094] As a recognition element, betaine or its derivatives may have specific recognition ability and can interact specifically with the target analyte. Therefore, using betaine as a modification material for electrochemical electrodes may help achieve highly selective detection of dopamine.

[0095] Acetylcholine: Acetylcholine is an electrode modification material in electrochemical electrode modification. Although acetylcholine itself is not directly used as an electrode modification material, its combination with enzymes (such as acetylcholinesterase) can be used to construct enzyme-modified electrodes. This electrode combines the enzyme with a carrier by chemical or physical means and fixes it on the surface of a solid phase carrier, so that the enzyme is restricted to the electrode surface and maintains enzyme activity and its catalytic ability under continuous operation. This electrode modification method can improve the sensitivity and selectivity of electrochemical detection. The application of acetylcholine in electrochemical electrode modification is mainly reflected in its use as a reaction substrate, a component of electrode modification materials, and a tool for studying neurotransmitter transmission.

[0096] In some embodiments of the present invention, Figure 4-5 As shown, the difference from the above embodiment is that: the microelectrode further includes a reference electrode 2, and the reference electrode 2 includes:

[0097] Conductive core 21;

[0098] An electroplating layer 22, wherein the electroplating layer 22 is wrapped around the outer surface of the conductive core 21;

[0099] A stabilizing layer 23, the stabilizing layer is wrapped around the outer surface of the electroplating layer 33, and the stabilizing layer contains a stabilizing compound;

[0100] A reference electrode adapter 24 , wherein the reference electrode adapter 24 is electrically connected to the conductive core 21 .

[0101] In some embodiments of the present invention, Figure 1-5 As shown, the difference from the above embodiment is that the carbon nanomaterial is a carbon nanotube or a carbon sphere.

[0102] In some embodiments of the present invention, Figure 1-3 As shown in , 18 and 19, the difference from the above embodiments is that the diameter of the conductive inner core is 0.05 to 0.15 mm.

[0103] In some embodiments of the present invention, Figure 1-7 , 16, 17, which are different from the above embodiments in that: the stabilizing compound is perfluorosulfonic acid resin, or betaine, or acetylcholine

[0104] In some embodiments of the present invention, Figure 4-5 , 16, 17, the difference from the above embodiments is that: the conductive core 21 has a diameter of 0.1-0.3 mm and a length of 0.8-1.2 cm;

[0105] The stabilizing layer comprises perfluorosulfonic acid resin, betaine, or acetylcholine.

[0106] 2. Reference electrode

[0107] like Fig.13 As shown: a silver wire with a diameter of 0.1-0.3 mm, preferably 0.2 mm, and a length of 0.8-1.2 cm, preferably 1 cm, is taken as a conductive core, polished, and then the silver wire is electroplated in a 3M potassium chloride solution at a voltage of 1.5 V for 60 seconds to obtain a silver wire with surface silver chloride modification, and the silver wire is further immersed in a 10% nafion solution for 5 times, each time with an interval of 10 minutes, and then the electrode is dried at 37°C for 24 hours. The obtained perfluorosulfonic acid film layer has a thickness of 5-20 um and is a transparent film layer.

[0108] The dried silver wire is cut through a mold or 3D printed with a fine support to prepare an ultra-microelectrode adapter. The electrode adapter and the silver wire are bonded with UV-sensitive glue to prepare a reference electrode with an overall size of 1 cm for intracerebral implantation.

[0109] The reference electrode and the working electrode can be integrated into the same structural component to achieve unified plug-in and unplug connection of instrument lines.

[0110] 2.1 Reference electrode:

[0111] The reference electrode usually consists of two parts: one is a substance with a stable standard potential, such as a saturated calomel electrode (SCE), a hydrogen electrode, etc.; the other is a conductive medium, such as an electrolyte solution.

[0112] 2.23M potassium chloride solution:

[0113] That is, a potassium chloride solution with a concentration of 3 mol / L, concentration: 3 mol / L, composition: mainly composed of potassium chloride (KCl) and deionized water.

[0114] 2.3 Silver wire modified with silver chloride:

[0115] Silver chloride modified silver wire can still maintain good conductivity in a solution containing chloride ions, which is suitable for detection in the human body. Stability: Silver chloride film is relatively stable in general environment and can protect the silver wire from corrosion or oxidation. Silver chloride modified silver wire has certain advantages in electrical properties, such as stable potential and good reproducibility.

[0116] 2.4 Modification by immersion in Nafion solution;

[0117] The silver wire with silver chloride surface modification is further immersed in 10% Nafion solution and after specific immersion and drying steps: Nafion will form a protective film on the surface of the silver wire. This protective film not only enhances the corrosion resistance and oxidation resistance of the silver wire, but also improves its electrochemical stability in various environments.

[0118] Conductivity is maintained. Although the Nafion membrane itself has a low conductivity, its coverage on the surface of the silver wire does not significantly affect the conductivity of the silver wire itself. Because the Nafion membrane is porous, it allows ions to pass through while maintaining good insulation to electrons. Therefore, in electrochemical measurements, this modified silver wire can still maintain good conductivity.

[0119] Reference electrode performance: Silver wire modified with silver chloride on the surface is one of the commonly used reference electrode materials. After being immersed in Nafion solution and dried, its performance as a reference electrode may be further improved. This is because the Nafion membrane can reduce the impact of impurities in the solution on the surface of the silver wire and improve the stability and reproducibility of the electrode.

[0120] After being soaked in Nafion solution and dried, the morphology of the silver wire surface will change. The Nafion membrane forms a uniform covering layer on the surface of the silver wire, making the surface of the silver wire smoother and flatter, which is beneficial to reduce the contact resistance between the electrode and the solution and improve the accuracy of electrochemical measurement.

[0121] The Nafion-modified silver wire electrode can be used in environments that require long-term stable operation due to its good stability and conductivity.

[0122] The preparation process of this modification method is relatively simple, and the materials and equipment required are relatively common. At the same time, since the Nafion membrane has good self-repairing ability, the modified silver wire electrode is also relatively easy to maintain.

[0123] In summary, the silver wire modified with silver chloride on the surface will acquire a series of unique properties and application characteristics after being further immersed in 10% Nafion solution and dried. These properties include improved electrochemical stability, maintained conductivity, improved reference electrode performance, surface morphology changes, and enhanced mechanical strength.

[0124] 2.5 Perfluorosulfonic acid resin

[0125] Excellent heat resistance, high chemical stability, high mechanical strength, etc.

[0126] Chemical properties: Due to the introduction of the fluorine atom with the highest electronegativity into the molecule, a strong field effect and induction effect are generated, which greatly increases its acidity. As a green solid acid catalyst, it has good catalytic activity and selectivity for many reactions.

[0127] Physical properties: It has good thermal stability and can remain stable for a long time at a temperature of about 200°C. The initial decomposition temperature is mostly above 310°C.

[0128] 2.6 The application of silver paste in the field of dopamine detection is mainly reflected in the preparation of electrochemical sensors, especially as the preparation material of reference electrodes.

[0129] In some embodiments of the present invention, Figure 6 As shown, the difference from the above embodiment is that: a method for preparing a dopamine-selective microelectrode in the brain, characterized by comprising:

[0130] The carbon electrode is placed in the support 14, and the support 14, the carbon electrode 11 and the adapter 15 are bonded by ultraviolet-sensitive glue to obtain a basic electrode body;

[0131] Providing a 1 mg / ml carbon nanotube solution and a 10% perfluorosulfonic acid resin aqueous solution, mixing them in a ratio of 1:1 to obtain a mixed solution;

[0132] The basic electrode body is immersed in the mixed solution for 5 times, with an interval of 10 minutes each time, and then the electrode is dried at 37° C. for 24 hours. The surface of the basic electrode body is covered with a modification layer 13 to obtain a working electrode 1.

[0133] A brain dopamine selective microelectrode coating composition comprises perfluorosulfonic acid resin and carbon nanotubes; the mass ratio thereof is 80:1 to 120:1. 85:1, 90:1, 95:1, 100:1, 105:1, 110:1, 115:1, preferably 100:1.

[0134] In some embodiments of the present invention, Figure 7 As shown, the difference from the above embodiment is that: the reference electrode 2 preparation steps are included:

[0135] Applying a voltage of 1.5V for 60S to the conductive core 21 in a 3M potassium chloride solution for electroplating, the surface of the conductive core 21 is covered with an electroplating layer 22, and a conductive core 21 with a surface modified with silver chloride is obtained;

[0136] The conductive core 21 with surface modified with silver chloride is immersed in a 10% perfluorosulfonic acid resin aqueous solution for 5 times, each time with an interval of 10 minutes, and then dried at 37° C. for 24 hours, so that the electroplating layer 22 is wrapped with the stabilizing layer 23;

[0137] The dried conductive core 21 and the reference electrode adapter 24 are bonded together with ultraviolet-sensitive glue to obtain the reference electrode 2 .

[0138] In some embodiments of the present invention, Figure 1-19 As shown, the difference from the above embodiments is: a method for applying a dopamine-selective microelectrode in the brain as described in any one of the above embodiments, wherein the microelectrode is applied to dopamine detection.

[0139] In these embodiments:

[0140] 3 The working process of this electrode for detecting dopamine

[0141] The dopamine working electrode and the corresponding reference electrode are implanted into the brain area for monitoring dopamine through animal brain surgery. After the animal regains consciousness, the two electrodes are connected to the real-time brain chemistry monitoring system. The dopamine monitoring parameters are set, the working voltage is -400mv to 1296mv, the scanning speed is 400v / s, and then the constant potential is -400mv, and the duration is 91.5ms. Then the instrument working electrode line is connected to the working microelectrode, and the reference / counter electrode two-in-one instrument line is connected to the reference microelectrode, and continuous monitoring of dopamine in the brain can be achieved. The secretion amount and secretion rate of dopamine are observed and calculated through the CV curve.

[0142] like Figure 8 and Fig. 9 As shown: Figure 8 The original current curve and dopamine response of the dopamine electrode modified with carbon nanotubes and nafion Fig. 9 The corresponding background-subtracted current curve. It can be concluded that the dopamine electrode modified with carbon nanotubes and nafion maintains relatively stable electrochemical properties, stabilizes the working stability of the working electrode in the brain environment, and its small size facilitates long-term implantation in the brain to detect dopamine changes. The above experimental data show that the working electrode and reference electrode modified with the modified layer have higher sensitivity for dopamine detection in the brain, with a 2-fold increase in sensitivity and improved potential stability. The CV curves of the dopamine electrode detected over 30 days are basically the same.

[0143] like Fig.10 , Fig.11 and Fig.12 As shown: Fig.10 The corresponding fast scanning cyclic voltammetry curve is obtained when the working electrode is placed in a non-detection environment and scanned in a phosphate buffer solution on the 30th day; Fig.11 The corresponding fast scanning cyclic voltammetry curve is obtained after the described working electrode is implanted in the brain for 30 days and scanned on the 30th day. Fig.12 The working electrode without any modification was implanted in the brain for 30 days and scanned on the 30th day. The corresponding fast scanning cyclic voltammetry curve obtained showed that the peak of the carbon functional group on the surface of the unmodified working electrode shifted from 150mV in vitro to 500mV, indicating that the electrode was contaminated. In contrast, the peak of the carbon functional group of the modified working electrode shifted from 150mV in vitro to 200mV, indicating that the modification layer can effectively reduce the contamination of the electrode and improve the stability of the electrode in the brain.

[0144] In addition, the electrode has excellent linearity and can be used for the detection of dopamine in the brain, such as Fig.13 As shown, after adding 0.05 micromolar, 0.5 micromolar, 1 micromolar, and 5 micromolar dopamine successively, the electrode response to dopamine was tested using rapid scanning voltammetry. The background-subtracted curve response is shown in Fig.14 As shown, the sensitivity of the electrode response is about 96nA / uM, and the linearity is greater than 0.98, indicating that the modified electrode has a good response to dopamine.

[0145] 4. Test results, such as Figure 8 , 9 , 10, 11, 12, 13, 14

[0146] Since the redox potential of dopamine may be similar to that of other substances in the solution (such as ascorbic acid), appropriate measures need to be taken during the measurement to eliminate the influence of these interfering substances and modify a layer of specific material (such as Nafion membrane) on the electrode surface to block the adsorption and reaction of interfering substances.

[0147] The relationship between potential (or current) and dopamine concentration may be established by using standard addition method, internal standard method or external standard method in actual operation. Fig.14 As shown, this embodiment adopts the external standard method, by preparing a series of dopamine standard solutions of known concentrations, measuring their potentials (or currents), and then plotting the relationship between potential (or current) and concentration (i.e., calibration curve). Once the calibration curve is established, the concentration of dopamine in an unknown sample can be estimated by measuring the potential (or current) generated by dopamine.

[0148] The beneficial effects of the present invention are as follows: by limiting the structure and volume of the dopamine detection electrode, it is easy for the detection subject to wear it for a long time; at the same time, the electrode surface is modified to improve stability and improve the detection sensitivity under long-term monitoring conditions, thereby achieving the purpose of being suitable for long-term placement in the brain to detect dopamine.

[0149] The embodiments and functional operations of the subject matter described in this specification may be implemented in: digital electronic circuitry, tangibly implemented computer software or firmware, computer hardware, including the structures disclosed in this specification and their structural equivalents, or a combination of more than one of the above. The embodiments of the subject matter described in this specification may be implemented as one or more computer programs, i.e., one or more modules of computer program instructions encoded on one or more tangible non-transitory program carriers, for execution by a data processing device or to control the operation of a data processing device.

[0150] Specific implementations of the subject matter have been described. Other implementations are within the scope of the following claims. For example, the activities recited in the claims can be performed in a different order and still achieve the desired results. As an example, the processes described in the accompanying drawings do not necessarily require the specific order or sequential order shown in order to achieve the desired results. In certain implementations, multitasking and parallel processing may be advantageous.

[0151] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the presence of other identical elements in the process, method, article or device including the elements.

[0152] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0153] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A dopamine-selective microelectrode in the brain, characterized in that: include: A working electrode (1), comprising: A carbon fiber electrode (11), the carbon fiber electrode (11) is in a filament shape; A modified layer (13), the modified layer (13) comprising a carbon nanomaterial (131) and a stability compound for improving stability and promoting dopamine oxidation-reduction, the modified layer (13) being wrapped around at least a portion of the outer surface of the carbon fiber electrode wire (11).

2. The dopamine-selective microelectrode in the brain according to claim 1, characterized in that: The working electrode further comprises: a conductive inner core (12), a support member (14) and an adapter (15); A conductive inner core (12), the conductive inner core (12) is in a filamentous shape and is fixedly connected end to end to the carbon fiber electrode (11); The support member (14) is a hollow structure, the conductive inner core (12) is fixed inside the support member, and the carbon fiber electrode (11) is fixed outside the support member; The conductive inner core (12) is fixed inside the support member (14); The support member (14), the conductive inner core (12) and the adapter (15) are fixedly connected; The carbon fiber electrode (11), the conductive inner core (12), and the adapter (15) are electrically connected in sequence.

3. The dopamine-selective microelectrode in the brain according to claim 1, characterized in that: The microelectrode further comprises a reference electrode (2), wherein the reference electrode (2) comprises: Conductive core (21); An electroplating layer (22), the electroplating layer (22) wrapping around the outer surface of the conductive core (21); A stabilizing layer (23), the stabilizing layer wrapping the outer surface of the electroplating layer (33), the stabilizing layer comprising a stabilizing compound; A reference electrode adapter (24), wherein the reference electrode adapter (24) is electrically connected to the conductive core (21).

4. The dopamine-selective microelectrode in the brain according to claim 1, characterized in that: The carbon nanomaterial of the modified layer (13) is carbon nanotubes or carbon spheres.

5. The dopamine-selective microelectrode in the brain according to claim 1, characterized in that: The conductive inner core (12) has a diameter of 0.05 to 0.15 mm.

6. The dopamine-selective microelectrode in the brain according to claim 1, characterized in that: The stabilizing compound is perfluorosulfonic acid resin, betaine, or acetylcholine.

7. The dopamine-selective microelectrode in the brain according to claim 3, characterized in that: The conductive core (21) has a diameter of 0.1 to 0.3 mm and a length of 0.8 to 1.2 cm; The stabilizing layer comprises perfluorosulfonic acid resin, betaine, or acetylcholine.

8. A method for preparing a dopamine-selective microelectrode in the brain, characterized in that: include: Electrically connecting the carbon fiber electrode (11) to the conductive inner core (12); Placing a conductive inner core (12) electrically connected to the carbon fiber electrode (11) into a support member (14), and exposing the carbon fiber electrode (11) to the support member (14); The support member (14), the carbon fiber electrode (11) and the adapter (15) are bonded together by using ultraviolet-sensitive glue to obtain a basic electrode body; Providing a 1 mg / ml carbon nanotube solution and a 10% perfluorosulfonic acid resin aqueous solution, mixing them in a ratio of 1:1 to obtain a mixed solution; The basic electrode body is immersed in the mixed solution, taken out and dried, and a modified layer (13) is formed on the surface of the carbon fiber electrode (11) of the basic electrode body to obtain a working electrode (1).

9. A brain dopamine-selective microelectrode coating composition, characterized in that: It comprises perfluorosulfonic acid resin and carbon nanotubes; the mass ratio thereof is 80:1 to 120:

1.

10. A method for using the brain dopamine selective microelectrode coating composition as claimed in claim 9, characterized in that: The coating composition is applied to dopamine detection.

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