Carbon nanotube fiber microfilament electrode and preparation method and application thereof

Abstract

The invention discloses a carbon nanotube fiber microfilament electrode and a preparation method and application thereof, and belongs to the technical field of electrode materials. The carbon nanotube fiber microfilament electrode provided by the invention comprises a carbon nanotube fiber bundle and an insulating layer, the surface of the carbon nanotube fiber bundle is coated with the insulating layer, the end part of the carbon nanotube fiber bundle is not coated with the insulating layer, and the end part is a sensing area. The carbon nanotube fiber microfilament electrode has good sensing performance, can achieve a good sensing detection effect in vitro and in vivo, particularly has good in-vitro selective recognition capability on neuroactive substances such as dopamine, uric acid and ascorbic acid, can be used for accurate detection in complex biological samples, and has good application prospects. The synchronous detection capability on neuroactive substances such as dopamine, uric acid, ascorbic acid and the like is also shown in an in-vivo environment.

Description

Technical Field

[0001] The invention belongs to the technical field of electrode materials, and in particular relates to a carbon nanotube fiber microfilament electrode and a preparation method and application thereof. Background Art

[0002] Neuroactive substances regulate the brain's neural activity through different mechanisms to maintain brain health and normal function. Among them, dopamine (DA) plays an important role in regulating emotions, motivation, reward mechanisms, motor control and cognitive functions. Uric acid (UA) and ascorbic acid (AA) have antioxidant properties and can scavenge free radicals, thereby protecting the brain from oxidative stress damage. Clinical detection of neuroactive substances plays an important role in evaluating brain health and function.

[0003] Microdialysis technology and molecular fluorescent probes are two important methods for in vivo detection of neuroactive substances, each with its own unique advantages and applications. Microdialysis technology is to implant a microdialysis probe with a semipermeable membrane into a specific area of ​​the brain. The perfusate in the probe flows continuously, and the neuroactive substances between the perfusate and the brain tissue diffuse and exchange through the semipermeable membrane. The perfusate from the probe is collected, and the liquid contains the neuroactive substances diffused from the brain tissue. The collected samples are analyzed by methods such as high performance liquid chromatography (HPLC) or mass spectrometry to determine the type and concentration of the neuroactive substances therein. Molecular fluorescent probe technology designs and synthesizes fluorescent probes that can specifically recognize and bind to target neuroactive substances. Then, the fluorescent probes are injected into specific brain areas of animals or humans. The fluorescent probes are excited by light of a specific wavelength to emit fluorescent signals. The fluorescent signals are detected and recorded by a fluorescent microscope, a fluorescent imaging system, or other fluorescent detection equipment. The intensity and location of the fluorescent signal are analyzed to determine the concentration and distribution of the target neuroactive substances. Electrochemical detection methods have gradually attracted widespread attention from researchers due to their advantages such as high sensitivity, rapid response, and easy operation. Electrochemical detection technology is based on the current changes generated when neuroactive substances undergo redox reactions on the electrode surface. By measuring these current changes, the concentration of specific neuroactive substances can be quantitatively analyzed. Electrochemical detection technology selects appropriate electrode materials (such as platinum, gold, and carbon) and modifies them to improve the selectivity and sensitivity of the electrode. Then the electrode surface is specifically modified (such as enzyme modification, nanomaterial modification) so that it can specifically detect the target neuroactive substance. Next, the prepared electrode microprobe is implanted into a specific brain area of ​​an animal or human body to ensure that the concentration changes of the target neuroactive substance can be monitored in real time. Finally, electrochemical detection techniques such as linear sweep voltammetry (LSV), square wave voltammetry (SWV), and differential pulse voltammetry (DPV) are used to detect the signal response of the target neuroactive substance. The current signal is recorded using a data acquisition system and combined with an electrochemical workstation for real-time data analysis.

[0004] Microdialysis technology and molecular fluorescent probes can realize in vivo biomarker detection. However, the sampling rate of microdialysis technology is low, and it is difficult to capture the rapid fluctuations of biomarkers in a short period of time. Molecular fluorescent probes are prone to light quenching, which affects the accuracy and reliability of the test results. Microdialysis technology and molecular fluorescent probes can realize in vivo biomarker detection, but they need to be combined with quantitative detection instruments, making it difficult to achieve in situ, real-time and continuous monitoring of biomarkers.

[0005] Gold electrodes and platinum electrodes have shown excellent performance in the electrochemical detection of substances such as dopamine, uric acid and ascorbic acid, demonstrating their great potential in the field of biosensing. However, in practical applications, they still face some challenges. First, the content of dopamine, uric acid and ascorbic acid in the body is low, and the detection limit of existing electrochemical methods can hardly meet the needs of simultaneous detection of multiple substances in the body. Secondly, the coexistence of substances such as dopamine, uric acid and ascorbic acid in the body environment will cause interference with the detection signal, affecting the accuracy of the detection results. Summary of the invention

[0006] In order to overcome at least one of the problems existing in the above-mentioned prior art, one of the objectives of the present invention is to provide a carbon nanotube fiber microwire electrode, which has good sensing performance and can be particularly used for detecting neuroactive substances.

[0007] A second object of the present invention is to provide a method for preparing the above-mentioned carbon nanotube fiber microfilament electrode.

[0008] The third object of the present invention is to provide an application of the above-mentioned carbon nanotube fiber microfilament electrode in detecting neuroactive substances.

[0009] A fourth object of the present invention is to provide a device for detecting neuroactive substances.

[0010] A fifth object of the present invention is to provide a method for detecting neuroactive substances.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides a carbon nanotube fiber microfilament electrode, comprising a carbon nanotube fiber bundle and an insulating layer, wherein the insulating layer is coated on the surface of the carbon nanotube fiber bundle, and the end of the carbon nanotube fiber bundle is not coated by the insulating layer, and the end is a sensing area.

[0013] The carbon nanotube fiber bundle of the present invention is composed of a plurality of carbon nanotube fibers. The carbon nanotube fiber bundle is prepared into an electrode material, and exhibits good sensing performance for the detection of neuroactive substances, and can meet the demand for simultaneous detection of multiple substances in vitro and in vivo, and can especially solve the problem of interference of detection signals caused by the coexistence of substances such as dopamine, uric acid and ascorbic acid in the in vivo environment, which affects the accuracy of the detection results. The carbon nanotube fiber bundle is coated with an insulating layer, so that only the sensing area of ​​the electrode is exposed during detection, and the surface area of ​​the electrode immersed in the detection solution is equal, so as to ensure the accuracy of the detection.

[0014] Preferably, the raw material for preparing the insulating layer includes polyimide.

[0015] Preferably, the diameter of the carbon nanotube fiber bundle is 50 to 200 μm; more preferably, it is 80 to 180 μm; and even more preferably, it is 100 to 150 μm.

[0016] In the present invention, the length of the sensing area is smaller than the length of the carbon nanotube fiber bundle.

[0017] The length of the carbon nanotube fiber bundle of the present invention can be selected according to specific requirements, and only needs to be longer than the length of the sensing area, that is, to ensure that at least a portion of the surface of the carbon nanotube fiber bundle is covered with an insulating layer.

[0018] Preferably, the length of the sensing area is 0.1 to 2 mm; more preferably, it is 0.5 to 1.5 mm; and even more preferably, it is 0.8 to 1.2 mm.

[0019] The second aspect of the present invention provides a method for preparing the carbon nanotube fiber microfilament electrode described in the first aspect of the present invention, comprising the following steps: reacting a carbon source and a catalyst, spinning to obtain a carbon nanotube fiber bundle, preparing an insulating layer on the surface of the carbon nanotube fiber bundle, and obtaining the carbon nanotube fiber microfilament electrode.

[0020] Preferably, the carbon source comprises at least one of methane, ethylene or propylene.

[0021] Preferably, at least one of iron, cobalt or nickel.

[0022] Under the action of the catalyst, the carbon source will crack and reorganize to grow into the precursor of carbon nanotube fibers, which can be formed into carbon nanotube fiber bundles through spinning and drawing.

[0023] Preferably, no insulating layer is prepared on the end surface of the carbon nanotube fiber to form a sensing area.

[0024] Preferably, the insulating layer is prepared by mixing polyimide with a solvent to obtain an insulating layer solution, coating the insulating layer solution on the surface of the carbon nanotube fiber bundle, and drying to obtain the insulating layer.

[0025] Preferably, in the method for preparing the insulating layer, the solvent is selected from N,N-dimethylformamide (DMF).

[0026] Preferably, in the method for preparing the insulating layer, the drying temperature is 30 to 50°C; more preferably 35 to 45°C.

[0027] Preferably, in the method for preparing the insulating layer, the drying time is 4 to 8 hours; more preferably, 5 to 7 hours.

[0028] Preferably, in the carbon nanotube fiber microfilament electrode, a step of preparing a hardening layer is further included after preparing the insulating layer.

[0029] Preferably, the raw material for preparing the hardening layer includes polyvinyl pyrrolidone (PVP).

[0030] Preferably, the hardening layer is prepared by mixing polyvinyl pyrrolidone with a solvent to obtain a hardening layer solution, coating the hardening layer solution on the surface of the carbon nanotube fiber bundle, and drying to obtain the hardening layer.

[0031] Preferably, in the method for preparing the hardened layer, the hardened layer solution is applied 4 to 6 times.

[0032] Preferably, in the method for preparing the hardening layer, the concentration of polyvinyl pyrrolidone in the hardening layer solution is 5 to 15% (w / v); more preferably, it is 8 to 12% (w / v).

[0033] Preferably, in the method for preparing the hardened layer, the drying temperature is 10 to 40°C; more preferably, 20 to 30°C.

[0034] Preferably, in the method for preparing the hardened layer, the drying time is 1 to 3 hours.

[0035] The third aspect of the present invention provides a use of the carbon nanotube fiber microfilament electrode described in the first aspect of the present invention in detecting neuroactive substances.

[0036] Preferably, the neuroactive substance includes at least one of dopamine, ascorbic acid or uric acid.

[0037] The fourth aspect of the present invention provides a device for detecting neuroactive substances, comprising a control module and the carbon nanotube fiber microfilament electrode according to the first aspect of the present invention, wherein the carbon nanotube fiber microfilament electrode is connected to the control module.

[0038] Preferably, in the device, the neuroactive substance includes at least one of dopamine, ascorbic acid or uric acid.

[0039] Preferably, the control module comprises a printed circuit board, an electrochemical workstation or a combination thereof.

[0040] Preferably, the printed circuit board is integrated with an electrochemical method module, through which different electrochemical methods can be used to control the carbon nanotube fiber microwire electrode to detect the sample.

[0041] Preferably, the electrochemical method that can be implemented by the electrochemical method module includes at least one of linear sweep voltammetry (LSV), square wave voltammetry (SWV) or differential pulse voltammetry (DPV).

[0042] Preferably, the device further comprises a flow control module, a wireless communication module or a combination thereof connected to the control module.

[0043] Preferably, the flow control module includes a microfluidic chip. The flow control module is used to realize automatic processing and injection of samples, thereby improving the efficiency and accuracy of detection. For example, samples are separated and transported through a microfluidic chip to realize multi-parameter synchronous detection.

[0044] Preferably, the wireless communication module includes a Bluetooth module, a Wi-Fi module or a combination thereof. Through the wireless communication module, real-time data transmission and remote monitoring can be achieved. For example, a Bluetooth module is integrated to achieve wireless data transmission; or a Wi-Fi module is integrated to achieve remote data monitoring and control.

[0045] The fifth aspect of the present invention provides a method for detecting neuroactive substances, comprising the following steps: using the carbon nanotube fiber microfilament electrode described in the first aspect of the present invention, or the device described in the fourth aspect of the present invention, measuring the relationship between the concentration of neuroactive substances in the standard sample and the oxidation peak current value of the test curve, and then obtaining a linear regression equation of the relationship, substituting the oxidation peak current value of the test curve of the sample to be tested into the linear regression equation to obtain the concentration of neuroactive substances in the sample to be tested.

[0046] Preferably, in the method, the neuroactive substance comprises at least one of dopamine, ascorbic acid or uric acid.

[0047] Preferably, the test curve comprises at least one of a DPV curve obtained by differential pulse voltammetry, a SWV curve obtained by square wave voltammetry, or an LSV curve obtained by linear sweep voltammetry; further preferably, the test curve is selected from a DPV curve obtained by differential pulse voltammetry.

[0048] Preferably, the method is used for detecting neuroactive substances in vivo.

[0049] The beneficial effects of the present invention are as follows: the carbon nanotube fiber microfilament electrode of the present invention has good sensing performance and can achieve good sensing detection effects in vitro and in vivo. In particular, it has good in vitro selective recognition ability for neuroactive substances such as dopamine, uric acid and ascorbic acid, and can be used for accurate detection in complex biological samples. Moreover, it also exhibits the ability to synchronously detect neuroactive substances such as dopamine, uric acid and ascorbic acid in the in vivo environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of the electrode structure and SEM image of electrode example 1.

[0051] Figure 2Schematic diagram of in vitro detection of electrodes of electrode example 1 and electrode comparison example 1.

[0052] Figure 3 This is a graph showing the electrochemical detection performance of dopamine by the Au microwire electrode of electrode comparison example 1.

[0053] Figure 4 This is a graph showing the electrochemical detection performance of dopamine by the CNTF microwire electrode of electrode example 1.

[0054] Figure 5 This is the electrochemical detection performance diagram of the Au microwire electrode of electrode comparison example 1 for ascorbic acid.

[0055] Figure 6 This is a graph showing the electrochemical detection performance of the CNTF microwire electrode of electrode example 1 for ascorbic acid.

[0056] Figure 7 This is the electrochemical detection performance diagram of uric acid by the Au microwire electrode of electrode comparison example 1.

[0057] Figure 8 This is a graph showing the electrochemical detection performance of uric acid by the CNTF microwire electrode of electrode example 1.

[0058] Fig. 9 This is a schematic diagram of the device embodiment 1.

[0059] Fig.10 It is a schematic diagram of the in vivo experiment and the DPV sensing flow chart for detecting Examples 1 to 3.

[0060] Fig.11 To detect the in vivo DA sensing results of the CNTF microwire electrode in Example 1.

[0061] Fig.12 To detect the in vivo AA sensing results of the CNTF microwire electrode in Example 2.

[0062] Fig.13 To detect the in vivo UA sensing results of the CNTF microwire electrode in Example 3. DETAILED DESCRIPTION

[0063] The content of the present invention is further described in detail below through specific examples. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the principles set forth in the present invention all belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific data exemplified below. The raw materials, reagents or devices used in the following examples and comparative examples, unless otherwise specified, can all be obtained from conventional commercial sources, or can be obtained by existing known methods.

[0064] Electrode Example 1

[0065] A carbon nanotube fiber microfilament electrode comprises the following preparation steps:

[0066] (1) Liquid carbon source (methane, ethylene or propylene) / catalyst (iron, cobalt or nickel) is injected into the ultrasonic atomizer through an injection pump according to the pre-set ratio and flow rate. With the assistance of the atomizer, the solution is atomized into micron-sized droplets and transported to the vaporization chamber. Then, we heat the vaporization chamber through a heating device to vaporize the micron-sized droplets into a gas state. At the same time, the first carrier gas (hydrogen or helium) is input into the chamber through the first carrier gas input port to mix it evenly with the gaseous carbon source / catalyst, so as to provide a continuous, stable, controllable and uniform mixed reaction gas for the next step of carbon nanotube fiber (CNTFs) growth; wherein the inflow rate of the carrier gas can be controlled by adjusting the flow controller. Next, the mixed gas is transported to the synthesis reaction device through a delivery pipeline, and after a series of chemical reactions such as cracking and catalytic growth, the precursor of CNTFs is formed. Finally, after the CNTFs precursor is shrunk and densified in the water tank, it can be fiberized by pulling with the cooperation of rollers, and then the CNTFs are collected by a fiber winding collection device to obtain a CNTF electrode.

[0067] (2) Insulation of electrodes: To ensure that the surface area of ​​the electrodes immersed in the solution is equal, the CNTF electrodes are insulated so that only the sensing area of ​​about 1 mm in length at the top of the electrode is exposed. Polyimide (PI) is used to insulate the non-sensing area. First, 1 mL of DMF is mixed with 0.6 g of PI powder and stirred thoroughly to form a dark red solution. Subsequently, the mixture is slowly applied to the electrode surface using a dust-free cotton swab to expose an area of ​​about 1 mm in length at the tip of the electrode for sensing. Finally, the electrode is placed in a 40°C oven and dried for 6 hours to obtain an insulated electrode.

[0068] (3) Hardening of the electrode: Use ethanol as solvent to prepare a 10% W / V PVP solution. Use a spray bottle to evenly spray the PVP solution on the electrode surface for a total of 5 times. After the electrode is dried (2 h at room temperature), a hardened CNTF microwire electrode that can penetrate brain tissue is obtained.

[0069] Electrode Comparative Example 1

[0070] A microwire electrode, which differs from electrode embodiment 1 in that the CNTF electrode obtained in step (1) is replaced by an Au wire electrode, and steps (2) and (3) are the same as those in electrode embodiment 1 to obtain an Au microwire electrode.

[0071] Electrode performance characterization

[0072] (1) Morphology characterization: The electrode obtained in Electrode Example 1 was characterized using a scanning electron microscope. Figure 1 Schematic diagrams and SEM images of the electrode structure of electrode embodiment 1, wherein (a) is a schematic diagram of the structure, (b) is a SEM image, and the image in the lower left corner is a zoomed-out image of the SEM image.

[0073] (2) In vitro electrochemical characterization: Different concentrations of dopamine (DA), ascorbic acid (AA), and uric acid (UA) were continuously added to PBS buffer, and the LSV response, SWV response, and DPV response of the gold microwire electrode and CNTF microwire electrode were then recorded using an electrochemical workstation. In addition, to ensure the accuracy of the experiment, commercial Ag / AgCl electrode and platinum wire electrode were selected as the reference electrode and counter electrode, respectively. Figure 2 Schematic diagram of in vitro detection of the electrodes of electrode example 1 and electrode comparison example 1. For CNTFs microwire electrodes and gold microwire electrodes, the sensitivity and detection range of LSV, SWV and DPV in detecting DA, AA and UA were evaluated.

[0074] Figure 3 The graph shows the electrochemical detection performance of the Au microwire electrode of electrode comparison example 1 for dopamine (DA), wherein (a1), (b1), and (c1) are the LSV response curve, SWV response curve, and DPV response curve of the Au microwire electrode at different DA concentrations, respectively; (a2), (b2), and (c2) are the relationship curves between the oxidation peak current and the DA concentration in the LSV response curve, SWV response curve, and DPV response curve of the Au microwire electrode, respectively. Figure 4Figure 1 is the electrochemical detection performance diagram of the CNTF microwire electrode of electrode example 1 for dopamine (DA), wherein (a1), (b1), (c1), and (c3) are the LSV response curve, SWV response curve, DPV response curve of low DA concentration (0.2-5 μM), and DPV response curve of high DA concentration (10-300 μM) of the CNTF microwire electrode, respectively; (a2), (b2), (c2), and (c4) are the relationship curves between the oxidation peak current and DA concentration in the LSV response curve, SWV response curve, DPV response curve of low DA concentration, and DPV response curve of high DA concentration of the CNTF microwire electrode, respectively.

[0075] Figure 5 Graph showing the electrochemical detection performance of the Au microwire electrode of electrode comparison example 1 for ascorbic acid (AA), wherein (a1), (b1), and (c1) are respectively the LSV response curve, SWV response curve, and DPV response curve of the Au microwire electrode at different AA concentrations, and (a2), (b2), and (c2) are respectively the relationship curves between the oxidation peak current and the AA concentration in the LSV response curve, SWV response curve, and DPV response curve of the Au microwire electrode. Figure 6 Graph showing the electrochemical detection performance of the CNTF microwire electrode of electrode example 1 for ascorbic acid (AA), wherein (a1), (b1), and (c1) are respectively the LSV response curve, SWV response curve, and DPV response curve of the CNTF microwire electrode at different AA concentrations, and (a2), (b2), and (c2) are respectively the relationship curves between the oxidation peak current and the AA concentration in the LSV response curve, SWV response curve, and DPV response curve of the CNTF microwire electrode.

[0076] Figure 7 The graph shows the electrochemical detection performance of uric acid (UA) by the Au microwire electrode of electrode comparison example 1, wherein (a1), (b1), and (c1) are the LSV response curve, SWV response curve, and DPV response curve of the Au microwire electrode at different UA concentrations, respectively; (a2), (b2), and (c2) are the relationship curves between the oxidation peak current and the UA concentration in the LSV response curve, SWV response curve, and DPV response curve of the Au microwire electrode, respectively. Figure 8 Figure 1 is the electrochemical detection performance diagram of uric acid (UA) of the CNTF microwire electrode of electrode example 1, wherein (a1), (b1), and (c1) are the LSV response curve, SWV response curve, and DPV response curve of the CNTF microwire electrode at different UA concentrations, respectively, and (a2), (b2), and (c2) are the relationship curves between the oxidation peak current and the UA concentration in the LSV response curve, SWV response curve, and DPV response curve of the CNTF microwire electrode, respectively.

[0077] Figures 3 to 8The results showed that in the detection of dopamine (DA), compared with SWV and LSV, the DPV method significantly optimized the detection limit of the electrode in low-concentration DA sensing; in the detection of ascorbic acid (AA), DPV showed a narrower oxidation peak horizontal coordinate difference and a lower peak potential shift than SWV and LSV, thereby achieving higher resolution and analytical accuracy; in the detection of uric acid (UA), compared with SWV and LSV, DPV also showed better detection sensitivity.

[0078] Device Example 1

[0079] A device for detecting neuroactive substances in vivo, comprising a printed circuit board and the CNTF microfilament electrode of electrode embodiment 1, wherein the carbon nanotube fiber microfilament electrode is connected to the printed circuit board. The schematic diagram of the device is shown in FIG. Fig. 9 In this example, the printed circuit board is a multifunctional printed circuit board that can realize three electrochemical methods: linear sweep voltammetry (LSV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). These three electrochemical methods can be used selectively.

[0080] In addition, the device in this example can also:

[0081] 1. Integrated microfluidic system can realize automatic processing and injection of samples, improve the efficiency and accuracy of detection. For example: integrate microfluidic chip on printed circuit board, separate and transport samples through microfluidic chip, and realize multi-parameter synchronous detection.

[0082] 2. Adding a wireless communication module can realize real-time data transmission and remote monitoring. For example: integrating a Bluetooth module on a printed circuit board to realize wireless data transmission; integrating a Wi-Fi module on a printed circuit board to realize remote data monitoring and control.

[0083] Detection Example 1

[0084] A method for detecting DA levels in vivo, comprising the following steps:

[0085] The in vivo brain experiment of SD rats in this case was mainly carried out with the help of brain stereotaxic technology. This technology provides accurate spatial positions for target points / areas under the cerebral cortex by analyzing the brain structure and skull surface landmarks (such as anterior / posterior fontanelles, sagittal suture, external auditory canal, and incisor position, etc.) of animals, and with the help of Cartesian coordinate system (X, Y, Z axis). This method allows the operator to perform precise operations on the brain without completely exposing the brain tissue, including implanting microelectrodes / optical fibers / injection needles, etc., and further conducting targeted stimulation, drug injection, sample collection or lesion resection, and electrical signal recording.

[0086] The animal experimental test procedures in this example have been approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. All SD mice were cared for and handled in accordance with the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee of Sun Yat-sen University (No.: SYSU-IACUC-2023-001854). The SD rats used in the experiment were purchased from the Animal Center of Sun Yat-sen University and needed to be kept for one week after purchase to adapt to the surrounding environment. All SD mice were placed in rat cages with a constant temperature and humidity barrier system in an alternating cycle of 12h light / 12h dark, and provided with a full-price pellet feed supply, and food and water were available for free. In order to detect DA, AA and UA in specific brain areas of rats, it is necessary to perform a craniotomy on the living SD rats after anesthesia, and bury the CNTF microwire electrodes of electrode example 1 in specific brain areas. Fig.10 The in vivo experiment schematic diagram and DPV sensing flow chart of detection embodiments 1 to 3, wherein (a) is a schematic diagram of the in vivo experiment site, (b) is a photo of the in vivo experiment site, and (c) is a DPV sensing flow chart.

[0087] The specific procedures for live animal experiments are as follows:

[0088] 1) Rat anesthesia and fixation

[0089] Before performing craniotomy on SD rats, anesthesia preparation is required first. Shutai is used as an anesthetic. The drug is prepared by thoroughly mixing and dissolving the freeze-dried powder containing the active ingredient with the injection water solvent that comes with the product to prepare a solution with a concentration of 50 mg / mL for standby use. Subsequently, Shutai is injected intramuscularly into SD rats at a dose of 50 mg / kg to ensure that the anesthetic effect is achieved. When the rat enters a state of deep anesthesia (generally by checking for no reaction by gently pinching the toes as a standard), its head is fixed on a stereotaxic instrument. During the operation, the rat's incisors are first placed on a special adapter and the bridge of the nose is gently pressed to ensure that the head is stable; then the adapter is adjusted forward and backward so that no matter from which direction the rat's head is pushed, it does not move, so as to ensure the accuracy of subsequent operations. During the entire experiment, the rat's anesthesia state must be checked regularly (such as once every 20 minutes) to ensure that it continues to be in deep anesthesia; and erythromycin eye ointment should be applied to the rat's eyes in an appropriate amount to protect its eyes from light stimulation.

[0090] 2) Expose the rat skull

[0091] After the rat is fixed, the rat's skull needs to be exposed to facilitate the subsequent electrode implantation. During the operation, first make a small incision of about 2 cm in length along the sagittal suture from the rat's eye to the rear end of the skull, then separate the subcutaneous tissue, and remove the fascia and muscles on the surface of the skull. Next, use saline and cotton balls to clean the surgical area to ensure that the skull surface is clean and dry. After this operation is completed, the landmarks on the skull, such as the bregma, lambdoid suture tip, and sagittal suture should be clearly visible (see Fig.10 (a)).

[0092] 3) Zero point positioning and craniofacial leveling

[0093] After the skull is exposed, the rat's cranial face needs to be adjusted horizontally to ensure the accuracy of subsequent operations. First, move the glass electrode clamped on the stereotaxic instrument so that its tip touches lightly, and set this position as the coordinate origin. Next, by adjusting the height of the ear bar, ensure that the height difference of the skull at about 2.3mm on both sides of the Bregma point is less than 0.03mm to ensure the horizontal symmetry of the skull. At the same time, the height of the nose clip also needs to be adjusted so that the front-to-back height difference between Bregma and Lambda is also less than 0.03mm to ensure the front-to-back balance of the cranial face. It should be noted that if the position of the skull shifts during the adjustment process, the position of Bregma must be recalibrated to ensure the accuracy of positioning.

[0094] 4) Locating the target brain area and drilling

[0095] After the skull is leveled, first determine the three-dimensional coordinates of the target brain area (striatum) according to the stereotaxic atlas. Select appropriate coordinate points (AP: 0.7mm, ML: 3mm, DV: 4mm), and use a marker to make corresponding marks on the skull. These marks represent the position of the striatum in a specific coordinate system. Then, select a cranial drill of appropriate size and drill holes at the marked points. Physiological saline can be used to cool the holes to reduce the temperature during the drilling process. After drilling, carefully use a fine needle to gently open the dura mater. If bleeding occurs during this process, use a thin cotton ball pulled into a long strip to gently absorb the blood.

[0096] 5) Embedded fiber electrodes

[0097] Before preparing to embed the fiber electrode, first fill the micro-syringe with a certain amount of DA solution and fix it on the stereotaxic instrument to ensure that the needle of the syringe is initially positioned at the origin of the coordinates, that is, the Bregma point. Then, according to the predetermined coordinates of the target brain area, adjust the syringe so that its needle moves to the position of the drilling window, and then slowly implant the injection needle until it reaches the specific brain area at a predetermined depth. Then, set the micro-injection pump parameters (speed of 1μL / min, total injection dose of 20μL), and start to slowly inject the DA solution. After the injection is completed, keep the needle position unchanged and let it stand for 10 minutes to allow the solution to fully diffuse and absorb, and then slowly withdraw the needle. Next, slowly implant the pre-prepared CNTF microwire electrode into the target brain area along the original needle hole path and connect it to the external electrochemical workstation for subsequent DPV sensing experiments.

[0098] 6) DPV sensing experiment:

[0099] Different concentrations of DA (see Fig.10 (c)) was sensed, and the corresponding differential pulse voltammetry curves of DA were obtained at different concentrations. Subsequently, the response current value on the curve was correlated with its corresponding concentration to evaluate the detection performance. All experiments were carried out at room temperature using an electrochemical workstation. The specific differential pulse voltammetry test parameters were set as follows: the potential scanning range was set to -0.3V~+0.6V, the amplitude was 0.05V, the pulse width was set to 0.05s, the sample sampling interval was 0.02s, the pulse period was 0.5s, and the static time was set to 2s. These parameters ensure sufficient electrochemical reaction and data acquisition to obtain accurate monitoring results. The specific experimental steps of DA sensing are as follows: In the initialization stage of the experiment, PBS buffer was first used as the base solvent, and low (marked as C1: 1mM), medium (marked as C2: 10mM) and high (marked as C3: 50mM) concentrations of DA solutions were prepared in turn according to the pre-calculated formula. The experiment will be carried out in order from low to high concentrations, and the test of each concentration level will be regarded as an independent cycle. Take the DA sensing experiment at the C1 concentration level as an example: In the experiment, 20 μL of DA solution was slowly injected into the target brain area (striatal nuclei) using a microinjection pump. After the injection was completed, it was left to stand for 10 minutes to ensure the sufficient distribution and absorption of DA in the brain area. For each concentration of DA, a total of 6 repeated tests were performed, and the DPV signal was collected every 5 minutes. The entire measurement process lasted about 30 minutes. After completing the DA measurement at a certain concentration level, rinse the test area with PBS buffer or saline to remove any residual DA. Subsequently, continue to inject and test the DA solution at the next concentration level using the same operating procedure.

[0100] In the DA sensing experiment in the striatum of living SD rats, a DA solution of a predetermined concentration was first slowly injected into the target brain area through a microinjection pump. Subsequently, the prefabricated CNTF microwire electrodes were buried in the striatum of three SD rats marked as Rat#1, Rat#2, and Rat#3 along the original pinhole path, and the electrodes were connected to an external electrochemical workstation.

[0101] Detection Example 2

[0102] A method for detecting AA levels in vivo, which differs from Detection Example 1 in that DA is replaced by AA and the DPV sensing experiment is different, and the other steps are the same as Detection Example 1. In this example, the specific experimental steps of AA sensing in the DPV sensing experiment are shown in Fig.10 (c), as follows: First, PBS buffer was used as the basic solvent for the experiment, and AA solutions of different concentration levels were prepared according to the pre-set formula: low concentration (marked as C1: 1mM), medium concentration (marked as C2: 10mM) and high concentration (marked as C3: 100mM). The experimental plan was carried out from low to high concentration levels to systematically evaluate the performance of AA sensing. For each concentration of AA solution, 20μL was injected into the striatal nucleus by a microinjection pump and left to stand for 10min after the injection. In order to enhance the reliability of the experimental results, 6 independent tests were performed in the measurement cycle of each concentration level, and DPV data were collected every 5min. The entire measurement cycle lasted about 30min. After each concentration level measurement, the experimental area was cleaned with PBS buffer or saline to remove all possible residual AA, and then the same test process was repeated for the next concentration level of AA solution.

[0103] Detection Example 3

[0104] A method for detecting UA levels in vivo, which differs from Detection Example 1 in that DA is replaced by UA and the DPV sensing experiment is different, and the other steps are the same as Detection Example 1. In this example, the specific experimental steps of UA sensing in the DPV sensing experiment are shown in Fig.10 (c) is as follows: First, PBS buffer is used as the base solution, and a UA solution with a concentration of 1mM is prepared according to a predetermined formula. Then, 20μL of UA solution is injected into the striatal nucleus through a microinjection pump for subsequent sensing analysis. During the entire measurement cycle, DPV signals are collected every 5min, for a total of 6 tests. After the measurement, the experimental area is cleaned with PBS / saline.

[0105] Test results such as Figures 11 to 13 shown. Fig.11To detect the in vivo DA sensing results of the CNTF microwire electrode in Example 1, (a), (b), and (c) are the DA concentration values ​​detected in Rat#1, Rat#2, and Rat#3 under different DA concentrations, respectively, and (d) is the average DA concentration value of different rats under different DA concentration conditions. The data are expressed as mean ± SEM. Fig.12 To detect the in vivo AA sensing results of the CNTF microwire electrode in Example 2, (a), (b), and (c) are the AA concentration values ​​detected by Rat#1, Rat#2, and Rat#3 under different AA concentrations, respectively, and (d) is the average AA concentration value of different rats under different AA concentration conditions. The data are expressed as mean ± SEM. Fig.13 To detect the in vivo UA sensing results of the CNTF microwire electrode in Example 3, (a), (b), and (c) are the UA concentration values ​​detected in Rat#1, Rat#2, and Rat#3, respectively, and (d) is the average UA concentration value detected in different rats. The data are expressed as mean ± SEM.

[0106] In detection example 1, the experiment adopts the DPV method to measure the ampere current signals generated by different concentrations of DA (C1, C2, C2) in the order of increasing concentration. After the CNTF microwire electrodes were respectively embedded in the striatal nuclei of three SD rats, Rat#1, Rat#2 and Rat#3 and fixed, different concentration levels (C1, C2 and C3) of DA were injected into the target brain area, and the DPV signals were collected. The response current values ​​were converted into corresponding concentrations for correlation analysis, and the average DA concentrations of different concentration levels (C1, C2 and C3) in the striatal nuclei of all rats during the test were statistically analyzed. The data are expressed as mean ± SEM. The results are as follows Fig.11 As shown, Fig.11 (a) Fig.11 (b) Fig.11 (c) shows the DA sensing results in the striatum of Rat#1, Rat#2 and Rat#3, respectively. The light gray circle, dark gray circle and medium gray circle in the figure represent the DA concentration levels measured by the CNTFs electrode bundle under the conditions of C1 (1mM), C2 (10mM) and C3 (50mM) DA concentrations, respectively. The arrows indicate the injection process of DA solutions of different concentrations. Fig.11(d) The average DA concentration data of all SD rats under different DA concentration conditions are summarized. By comparing and analyzing with the in vitro DA amperometric current-concentration standard curve, the experimentally measured amperometric response current values ​​were converted into the corresponding target analyte concentrations, which facilitated further data analysis. Specifically, the measurement results showed that the DA concentrations of Rat#1 under C1, C2, and C3 concentration conditions were 1.53±0.10μM, 3.62±0.51μM, and 13.87±5.14μM, respectively; the DA concentrations of Rat#2 under C1, C2, and C3 concentration conditions were 2.92±0.85μM, 3.24±0.65μM, and 8.33±1.43μM, respectively; the DA concentrations of Rat#3 under C1, C2, and C3 concentration conditions were 13.28±2.27μM, 30.57±2.17μM, and 89.63±17.03μM, respectively. These results reflect that when using CNTF microwire electrodes for DA sensing, at the same DA concentration, the sensor response concentration in the brain tissue of a single SD rat shows little difference, while the measurement results between different individuals show more obvious differences. This is mainly because the SD rat models selected are all in a healthy state and do not involve models of local inflammation or specific disease states. Therefore, the overall biological indicators of the striatum region of the same SD rat are relatively uniform. The differences between different SD rats may be due to the physiological state between individuals (such as differences in DA absorption or metabolic rate) or inherent individual differences, which leads to certain differences in experimental results.

[0107] In detection example 2, the average values ​​of AA concentrations measured during the test at different AA concentration levels (C1, C2 and C3) in the striatal nuclei of all SD rats were statistically analyzed. Fig.12 (a) Fig.12 (b) Fig.12 (c) shows the detection results of Rat#1, Rat#2 and Rat#3 under different AA concentrations after CNTF microwire electrodes were implanted. The light gray, dark gray and medium gray circles in the figure correspond to the AA concentration levels measured by the CNTF microwire electrodes under the conditions of C1 (1mM), C2 (10mM) and C3 (100mM) AA concentrations, respectively. At the same time, the arrow indicates the injection process of the AA solution. Fig.12(d) The average AA concentrations of all SD experimental rats at each AA concentration were summarized. According to the known AA amperometric current-concentration standard curve, the amperometric current values ​​measured in the experiment were converted into the corresponding target analyte concentrations for further analysis. The measurements showed that the AA concentrations of Rat#1 under C1, C2, and C3 concentrations were 32.38±0.91μM, 37.80±2.12μM, and 183.20±82.59μM, respectively; the AA concentrations of Rat#2 under C1, C2, and C3 concentrations were 181.82±15.70μM, 158.12±16.20μM, and 838.16±106.35μM, respectively; the AA concentrations of Rat#3 under C1, C2, and C3 concentrations were 65.17±4.17μM, 81.04±6.39μM, and 266.71±13.55μM, respectively. The measurement results showed that the AA concentration values ​​of all SD rats showed an increasing trend with the increase of AA concentration, but the response differences between different rats were significant. Specifically, the AA concentration of Rat#1 showed an increasing trend from low concentration to high concentration, from 32.38±0.91μM to 183.20±82.59μM, reflecting that the electrode's detection response to AA increased when the AA concentration increased. However, this response increase was not linear, especially the variability at high concentrations (±82.59μM), indicating that in the high concentration range, the sensing system may be affected by other factors, such as sensor saturation or interference from the biological matrix. The data of Rat#2 increased significantly in AA concentrations from C1 to C3, jumping from 181.82±15.70μM to 838.16±106.35μM. This significant increase indicates that the CNTF microwire electrode is extremely sensitive to AA detection at high concentrations, but at the same time, the large standard deviation at high concentrations also suggests volatility in the measurement process or inherent heterogeneity in biological samples. For Rat#3, the trend of AA concentration was similar to that of the other two rats, but the overall concentration level and increase were lower, from 65.17±4.17μM to 266.71±13.55μM. This difference may reflect physiological differences between individuals, such as differences in metabolic rates or individual variations in the clearance mechanism of AA. Overall, these results highlight the effective sensing ability of CNTF microwire electrodes for AA at different concentrations, showing its potential for AA detection in the striatal nuclei of living rats.

[0108] In detection example 3, the same UA concentration (1 mM UA) was used to test the striatal nuclei of all SD rats, and the average UA concentrations measured during the test were statistically analyzed. Fig.13As shown, the UA concentrations of all rats under the same concentration conditions did not exceed 30 μM. The results also showed that as the detection time increased, the UA concentration detected in the rat brain tended to decrease. Fig.13 (d) The average UA concentration data of three SD rats under 1 mM UA concentration were further integrated, which were 6.92±2.94μM (Rat#1), 12.58±0.60μM (Rat#2) and 15.23±2.60μM (Rat#3). These data show that although all tested individuals received injections of the same concentration of UA solution, the measured UA concentrations showed significant differences between individuals, which may reflect the individual differences in biological responses between different SD rats. This difference may be related to the ability of the rat striatum to absorb and metabolize UA, or the difference in the interaction between the electrode and the tissue microenvironment.

[0109] As can be seen from the above embodiments, the present invention explores the electrochemical detection performance of CNTF microwire electrodes and Au microwire electrodes for dopamine, uric acid and ascorbic acid under different electrochemical detection methods in response to the needs of different detection methods in different situations. Based on these studies, the potential of CNTFs microwire electrodes in practical applications was further verified in vivo according to the detection results in the embodiments of the present invention. The specific research results are summarized as follows: 1) For CNTF microwire electrodes, the sensitivity and detection range of LSV, SWV and DPV in detecting dopamine, uric acid and ascorbic acid were evaluated. The results show that compared with SWV and LSV, the DPV method significantly optimizes the detection limit of the electrode in low-concentration DA sensing. In the detection of AA, DPV exhibits a narrower oxidation peak horizontal coordinate difference and a lower peak potential shift than SWV and LSV, thereby achieving higher resolution and analysis accuracy. In the detection of UA, DPV also exhibits better detection sensitivity than SWV and LSV. 2) For Au microwire electrodes, the effects of different electrochemical voltammetric methods in the detection of dopamine, uric acid and ascorbic acid were investigated. The results show that the Au microwire electrode has poor performance in the detection of low-concentration DA, especially the large difference in the horizontal coordinates of the oxidation peak of the LSV method, which is not conducive to accurate sensing. For the detection of high-concentration DA, SWV and DPV show better applicability. In the detection of AA and UA, although the Au microwire electrode shows a good linear response, due to its large oxidation peak width and high peak potential, it may be interfered by other substances in practical applications. 3) Comparison of the performance differences between CNTF microwire electrodes and Au microwire electrodes under different electrochemical methods. The results show that the DA sensing ability of the Au microwire electrode is limited in the low concentration range, and its sensing sensitivity in the entire concentration range is significantly lower than that of the CNTF microwire electrode. In the detection of AA and UA, although the Au microwire electrode showed a good "concentration-current" linear relationship, its oxidation peak was too wide, the peak potential was too high, and the peak potential values ​​of UA and AA were too close to be distinguished. These characteristics may limit its application in complex biological systems. In contrast, the CNTF microwire electrode performed better overall in the detection of dopamine, uric acid and ascorbic acid, and is more suitable for actual biosensing applications. 4) In the living rat brain experiment, the CNTF microwire electrode successfully detected different concentrations of dopamine, uric acid and ascorbic acid in the striatum brain region. The experimental results show that the CNTF microwire electrode can effectively monitor these bioactive molecules in complex biological systems, demonstrating its wide application in biomedical research and clinical applications.

[0110] In summary, the carbon nanotube fiber microfilament electrode of the present invention has good sensing performance and can achieve good sensing detection effects in vitro and in vivo. In particular, it has good in vitro selective recognition ability for neuroactive substances such as dopamine, uric acid and ascorbic acid, and can be used for accurate detection in complex biological samples. Moreover, it also exhibits the ability to synchronously detect neuroactive substances such as dopamine, uric acid and ascorbic acid in the in vivo environment.

Claims

1. A carbon nanotube fiber microfilament electrode, characterized in that: The invention comprises a carbon nanotube fiber bundle and an insulating layer, wherein the insulating layer is coated on the surface of the carbon nanotube fiber bundle, and the end of the carbon nanotube fiber bundle is not coated by the insulating layer, and the end is a sensing area.

2. The carbon nanotube fiber microfilament electrode according to claim 1, characterized in that: The raw material for preparing the insulating layer includes polyimide; and / or, the diameter of the carbon nanotube fiber bundle is 50 to 200 μm; And / or, the length of the sensing area is 0.1-2 mm.

3. The method for preparing the carbon nanotube fiber microfilament electrode according to claim 1 or 2, characterized in that: The following steps are involved: The carbon source and the catalyst are reacted to obtain a carbon nanotube fiber bundle through spinning, and an insulating layer is prepared on the surface of the carbon nanotube fiber bundle to obtain the carbon nanotube fiber microfilament electrode.

4. Use of the carbon nanotube fiber microfilament electrode according to claim 1 or 2 in detecting neuroactive substances.

5. The use according to claim 4, characterized in that: The neuroactive substance includes at least one of dopamine, ascorbic acid or uric acid.

6. A device for detecting neuroactive substances, characterized in that: It comprises a control module and the carbon nanotube fiber microfilament electrode according to claim 1 or 2, wherein the carbon nanotube fiber microfilament electrode is connected to the control module.

7. The device according to claim 6, characterized in that The control module includes a printed circuit board, an electrochemical workstation or a combination thereof; preferably, the device also includes a flow control module, a wireless communication module or a combination thereof connected to the control module.

8. A method for detecting a neuroactive substance, comprising the following steps: Using the carbon nanotube fiber microfilament electrode described in claim 1 or 2, or the device described in claim 6 or 7, the relationship between the concentration of neuroactive substances in the standard sample and the oxidation peak current value of the test curve is measured, and then a linear regression equation of the relationship is obtained, and the oxidation peak current value of the test curve of the sample to be tested is substituted into the linear regression equation to obtain the concentration of neuroactive substances in the sample to be tested.

9. The method according to claim 8, characterized in that The test curve includes at least one of a DPV curve obtained by differential pulse voltammetry, a SWV curve obtained by square wave voltammetry, or a LSV curve obtained by linear sweep voltammetry.

10. The method according to claim 8 or 9, characterized in that: The method is used for detecting neuroactive substances in vivo.

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