Double synergistic recognition neural probe based on acupuncture needle extended grid and preparation method and application thereof

Abstract

The application discloses a kind of based on acupuncture needle extension grid double synergic recognition nerve probe and preparation method and application, the nerve probe is based on medical acupuncture needle extension grid field effect transistor AN-EGFET, and its sensing interface is formed by gold nanoparticles, 4-mercapto phenylboric acid and polyphenylene diamine molecular imprinting polymer modification.The nerve probe of the application, 4-mercapto phenylboric acid is used as recognition molecule, and the space matching of molecular imprinting cavity is combined, and specific double synergic recognition to dopamine is generated, and the concentration of dopamine in biological sample can be quantitatively detected by detecting the change rate of source-drain current.The dopamine real-time monitoring method constructed by the nerve probe can be used to in situ track the dopamine release level change in in vitro cell line or deep brain tissue, evaluate the regulation effect of related pharmacological drugs on dopamine release, and also can be used to evaluate the abnormal depletion condition of dopamine in Parkinson's disease and other neurodegenerative disease models.

Description

Technical Field

[0001] This invention relates to the fields of bioelectronics and neurochemical sensing technology, specifically to an implantable neural probe based on an acupuncture needle extended gate field-effect transistor (AN-EGFET), its preparation method, and its application. Background Technology

[0002] Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to severe depletion of dopamine (DA) in the striatum. Precise quantification of neurochemical changes is crucial for elucidating the functional mechanisms of the brain. Traditional ectopic analysis techniques, due to inherent limitations, lack the ability to continuously monitor neurochemical dynamics in situ. Extended gate field-effect transistors (EGFETs) offer a highly sensitive, label-free, real-time monitoring architecture. However, current EGFET sensing interfaces are mostly constructed on large planar substrates, causing significant tissue displacement and severe local trauma when performing in-situ tracking of deep tissues. On the other hand, ultra-miniature sensors based on nanopipettes face inherent mechanical fragility, easily breaking when penetrating tissue. Therefore, developing an implantable sensing platform that combines excellent mechanical toughness, low damage, and real-time monitoring remains a significant challenge. Summary of the Invention

[0003] To address the aforementioned technical problems in the prior art, the purpose of this invention is to provide a dual-synergistic recognition neural probe AN-EGFET based on acupuncture needle extension grid, its preparation method, and its application. The technical problem to be solved by this invention is that: implantable probes in the prior art are prone to causing significant tissue displacement and trauma when performing in-situ tracking of deep tissues, and are prone to breakage due to insufficient mechanical toughness when penetrating tissues, and are difficult to achieve high temporal resolution real-time continuous in vivo monitoring in complex physiological environments.

[0004] The specific technical solution for achieving the objective of this invention is as follows:

[0005] A dual-cooperative recognition neural probe based on an acupuncture needle extended gate is characterized in that the neural probe is a field-effect transistor neural probe based on an extended gate of a medical acupuncture needle, which includes an n-type metal-oxide-semiconductor field-effect transistor, a reference electrode, and a medical acupuncture needle as the extended gate.

[0006] The surface of the medical acupuncture needle is sequentially modified with a gold nanoparticle layer, a 4-mercaptophenylboronic acid layer, and a poly(o-phenylenediamine) molecularly imprinted polymer layer; the 4-mercaptophenylboronic acid layer is connected to the gold nanoparticle layer through gold-thiol bonds, and the phenylboronic acid groups in the 4-mercaptophenylboronic acid are used to form boric acid affinity recognition with dopamine molecules; the poly(o-phenylenediamine) molecularly imprinted polymer layer has a molecularly imprinted cavity that matches the dopamine molecule.

[0007] The conductive needle body of the medical acupuncture needle is electrically connected to the gate terminal of the n-type metal-oxide-semiconductor field-effect transistor. The source and drain of the n-type metal-oxide-semiconductor field-effect transistor are respectively connected to the current detection unit. The reference electrode is placed in the test environment and connected to the external gate bias terminal, thereby forming a liquid gate-controlled extended gate field-effect detection circuit composed of the reference electrode, the test environment, the extended gate of the medical acupuncture needle, and the n-type metal-oxide-semiconductor field-effect transistor.

[0008] In this process, dopamine molecules undergo dual synergistic recognition with the 4-mercaptophenylboronic acid layer and the molecularly imprinted cavity, causing a change in the potential of the extended gate surface of the medical acupuncture needle, and further modulating the source and drain current of the n-type metal oxide semiconductor field-effect transistor.

[0009] A method for preparing the above-mentioned dual-synergistic recognition neural probe includes the following specific steps:

[0010] (1) The extended gate AN of the acupuncture needle was placed in an 8 mmol / L HAuCl4 solution. Cyclic voltammetry was used with a voltage scan range of -1.5-0.5 V and a scan rate of 100 mV / s for 5 cycles. Gold nanoparticles AuNPs were electrodeposited on its surface to form the extended gate AuNPs / AN of the acupuncture needle.

[0011] (2) The acupuncture needle extended gate AuNPs / AN described in (1) is immersed in a 5 mg / mL solution of 4-mercaptophenylboronic acid (4-MPBA) and incubated for 6 hours to obtain the acupuncture needle extended gate 4-MPBA / AuNPs / AN;

[0012] (3) The acupuncture needle extended gate 4-MPBA / AuNPs / AN described in (2) was placed in a PBS buffer solution with a concentration of 0.01 M and pH 7.4 containing 6 mmol / L o-phenylenediamine (OPD) and 2 mmol / L dopamine (DA). Cyclic voltammetry was used to perform in-situ electropolymerization with a voltage scan range of 0-0.8 V, a scan rate of 50 mV / s, and 8 cycles to generate a molecularly imprinted polymer (MIP) layer encapsulating dopamine, resulting in unwashed acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN.

[0013] (4) The acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN described in (3) is placed in a mixture of 95% ethanol and 0.1 M hydrochloric acid and eluted for 10 minutes to remove dopamine molecules from the polymer network, thereby obtaining the acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN; wherein the volume ratio of 95% ethanol to 0.1 M hydrochloric acid is 6:1;

[0014] (5) The acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN described in (4) is coupled with an n-type metal oxide semiconductor field-effect transistor MOSFET and an Ag / AgCl reference electrode to obtain the dual collaborative recognition neural probe, denoted as AN-EGFET.

[0015] An application of the aforementioned dual-synergistic recognition neural probe in dopamine detection and dynamic evaluation of dopamine release, the application comprising at least one of the following:

[0016] Detecting the concentration or dynamic changes of dopamine in in vitro biological samples and in vitro cell systems;

[0017] In situ monitoring of the dynamic concentration of dopamine in the local microenvironment of living tissues of experimental animals;

[0018] To evaluate the regulatory effects of test compounds and candidate drugs on dopamine release in cells or in vivo brain regions of experimental animals;

[0019] Used to assess abnormal dopamine depletion or differences in dopamine signaling in different brain regions in experimental animal models of neurodegenerative diseases.

[0020] In the aforementioned application, dopamine molecules are specifically bound to 4-mercaptophenylboronic acid on the neural probe AN-EGFET via covalent borate bonds, and spatially matched with the cavity generated by the molecularly imprinted polymer, forming a dual synergistic recognition mechanism. This specific binding causes a change in the extended gate interface potential. By detecting the rate of change of the source-drain current of the neural probe AN-EGFET, the concentration or dynamic changes of dopamine in the local microenvironment of in vitro biological samples, in vitro cell systems, or living tissues of experimental animals can be evaluated. Specifically, this includes: using the neural probe AN-EGFET, immersing or implanting the extended gate of the neural probe into the test object under physiological conditions, so that the extended gate is in contact with the test environment; recording the changes in the source-drain current of the field-effect transistor under set gate voltage and source-drain voltage conditions, thereby quantitatively assessing the concentration or release dynamics of dopamine in the test object.

[0021] Furthermore, the source / drain current signal response generated by the dual synergistic recognition of the molecularly imprinted polymer and 4-mercaptophenylboronic acid is superior to the signal response generated by using 4-mercaptophenylboronic acid alone, or by using the molecularly imprinted polymer alone, or by the control modification of the molecularly imprinted polymer / mercaptosalicylic acid TSA, exhibiting a synergistic enhancement effect; the binding of dopamine molecules to the recognition cavity causes a detectable change in source / drain current in the field-effect transistor under a constant gate voltage.

[0022] Furthermore, the test objects include dopamine-containing buffer solutions, cell culture media, in vitro cell systems with dopamine release function, and local microenvironments in the brain regions of experimental animals.

[0023] Furthermore, the evaluation described in the application specifically includes: for in vitro cell systems with dopamine release function, contacting or co-incubating the in vitro cell system with the test compound or candidate drug; for live brain regions of experimental animals, administering the test compound or candidate drug to the experimental animals before detection; subsequently applying a stimulus that can induce dopamine release; using the neural probe AN-EGFET to record the source and leakage currents before and after stimulation in real time, and comparing them with a control group that has not been treated with the test compound, candidate drug, or external stimulus, to obtain the promoting or inhibiting effect of the evaluated factor on dopamine release.

[0024] Furthermore, the assessment of the abnormal dopamine depletion state or the difference in dopamine signals in different brain regions involves selecting in vitro cell models with dopamine release function, normal experimental animal models, or neurodegenerative disease animal models used to compare the abnormal dopamine depletion state between the normal group and the disease group; the neurodegenerative disease animal models include MPTP-induced Parkinson's disease models.

[0025] Compared with existing technologies, the technological advancements of this invention are significant. Using the dual-synergistic recognition implantable neural probe AN-EGFET described in this invention to detect dopamine has the following advantages:

[0026] (1) High specificity and significant synergistic effect: The sensing design of this invention is not only an optimization of a single material, but also demonstrates the difference between the individual and combined effects of each component. Through the spatial selectivity of the molecularly imprinted polymer and the covalent affinity of 4-mercaptophenylboronic acid (linked by gold-thiol bonds and with two hydroxyl groups clearly present on the boron atom), a synergistic enhancement effect of 1+1>2 is achieved. In the specificity evaluation, this probe can effectively eliminate interference from various in vivo structural analogs such as ascorbic acid and uric acid.

[0027] (2) High sensitivity and affinity: Driven by the synergistic effect of dual recognition, the recognition cavity of this AN-EGFET has a high affinity for dopamine molecules and has an ultra-low detection limit at the picomolar (pM) level, which can accurately capture minute changes in dopamine concentration in the neural microenvironment.

[0028] (3) Minimal trauma and excellent mechanical stability: Using medical acupuncture needles as the extended grid substrate balances the ultra-fine size required for microscopic detection with the mechanical toughness required for tissue puncture. Compared with traditional glass nanopipette probes, the neural probe is less prone to breakage during tissue puncture; compared with planar microelectrode substrates, the neural probe has a smaller implantation cross-section, which helps reduce brain tissue displacement and local trauma.

[0029] (4) Real-time in-situ continuous detection and high stability in complex environments: With its specific sensing interface constructed from a dual recognition network, AN-EGFET maintains high stability and reliable response in complex physiological matrices (such as cell culture media containing serum and living brain tissue). It can achieve real-time, continuous monitoring and record dynamic changes in dopamine release in deep living brain tissue or extracellular fluid without relying on extraction methods with significant time lags, such as microdialysis or isotope tracing.

[0030] (5) The preparation process is reliable and the model evaluation capability is strong: the construction and modification process of the sensing interface is stable. The in vivo monitoring method established using this probe can effectively distinguish the concentration difference between abnormal dopamine depletion in Parkinson's disease pathology and normal physiological state, and has application potential in the assessment of neurodegenerative diseases. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the preparation process of the medical acupuncture needle with an extended gate according to the present invention.

[0032] Figure 2 The morphology and chemical composition characterization diagram of the medical acupuncture needle with extended gate of the present invention;

[0033] Figure 3 This is a characterization diagram of the electrical performance of the neural probe AN-EGFET of the present invention;

[0034] Figure 4 This is a diagram illustrating the synergistic effect of interface modification on the neural probe AN-EGFET of this invention.

[0035] Figure 5 This is a diagram showing the concentration response of the neural probe AN-EGFET of this invention to dopamine.

[0036] Figure 6 This is a specificity analysis diagram of the neural probe AN-EGFET of the present invention;

[0037] Figure 7 This is a real-time measurement of drug-induced dopamine release in PC12 cells using the neural probe AN-EGFET of this invention.

[0038] Figure 8 This is a real-time measurement of dopamine release in different brain regions of mice using the neural probe AN-EGFET of this invention.

[0039] Figure 9 This is a real-time measurement of dopamine release in mice under drug intervention using the neural probe AN-EGFET of this invention.

[0040] Figure 10This is a real-time measurement of dopamine release in mice under normal and Parkinson's disease conditions using the neural probe AN-EGFET of this invention.

[0041] Figure 11 This is a schematic diagram of the structure of the neural probe AN-EGFET of the present invention. Detailed Implementation

[0042] This invention provides a dual-synergistic recognition implantable neural probe for real-time dopamine detection, which is an extended-gate field-effect transistor (AN-EGFET) based on a medical acupuncture needle. The probe includes an n-type metal-oxide-semiconductor field-effect transistor (MOSFET), a reference electrode, and a medical acupuncture needle serving as the extended gate. The surface of the acupuncture needle is sequentially modified with gold nanoparticles (AuNPs), 4-mercaptophenylboronic acid (4-MPBA), and a poly(o-phenylenediamine) molecularly imprinted polymer (MIP) layer. The 4-MPBA is linked to the gold nanoparticles via gold-thiol bonds, and the two hydroxyl groups on its boron atoms serve as recognition groups. The molecularly imprinted cavities together form a recognition network with high affinity.

[0043] This invention also provides a method for preparing the above-mentioned implantable neural probe, which uses a medical acupuncture needle as a substrate, expands the active specific surface area by electrodepositing gold nanoparticles, completes monolayer self-assembly by immersion in a 4-mercaptophenylboronic acid solution, and finally performs in-situ electropolymerization in a solution containing o-phenylenediamine monomer and dopamine template molecules to generate a molecularly imprinted polymer layer; a subsequent elution process removes dopamine molecules, thereby obtaining the dual-recognition neural probe.

[0044] The present invention also provides the use of the above-mentioned neural probe in detecting the dynamic concentration of dopamine in biological samples or living tissues and in assessing the characteristics of dopamine release and regulation.

[0045] Furthermore, the dopamine molecule is specifically captured by 4-mercaptophenylboronic acid on the neural probe through the formation of a covalent borate bond, and binds to the shape-matched cavity of the molecularly imprinted polymer to form a dual synergistic recognition. This specific binding leads to a change in the potential of the extended gate surface, and the concentration of dopamine in the biological sample is quantitatively detected by measuring the rate of change of the source-drain current of the field-effect transistor before and after binding. This dual recognition mechanism effectively superimposes the chemical affinity of 4-mercaptophenylboronic acid with the spatial selectivity of the molecularly imprinted cavity. Compared with a single recognition component, the combination of the two achieves complementary advantages, thereby significantly amplifying the electrical response signal induced by dopamine binding and producing a significant synergistic recognition effect.

[0046] Furthermore, after dopamine binds to the dual recognition interface on the neural probe AN-EGFET, the dopamine binds to the borate groups of 4-mercaptophenylboronic acid within the imprinted cavity, causing a change in the probe interface potential. This results in electrical characteristics different from the unbound state under a constant gate voltage, leading to a significant change in source-drain current. This invention also provides a method for detecting dopamine concentration in biological samples. Using the aforementioned implantable neural probe AN-EGFET, an acupuncture needle with an extended gate is implanted into the subject under physiological conditions. After incubation (e.g., 5 minutes) to reach dynamic equilibrium, the rate of change of source-drain current is detected using a semiconductor source-drain current system, thereby quantitatively assessing the dopamine concentration in the microenvironment.

[0047] Furthermore, the test subject includes, but is not limited to, extracts of secretions from in vitro cultured nerve cells (such as PC12 cells), or specific dopamine-rich brain tissues from live animals (such as the dorsal striatum).

[0048] The present invention also provides a method for evaluating dopamine regulators, using the above-mentioned implantable neural probe to determine the regulatory effect (including inhibition and promotion) of any test compound on dopamine release activity in cells or living brain regions.

[0049] Furthermore, using the aforementioned neural probe AN-EGFET as a detection tool, for in vitro cell systems with dopamine release function, the in vitro cell system is contacted or co-incubated with the test compound or candidate drug; for the live brain region of experimental animals, the test compound or candidate drug is administered to the experimental animals before detection, and then stimulation (such as high potassium stimulation) is applied to trigger dopamine release; the source-leakage current changes of the neural probe AN-EGFET are recorded and compared with the group without the test compound (control group) to obtain the regulatory ability of the test compound on dopamine release.

[0050] Furthermore, the biological sample containing dopamine secretion function can be selected from in vitro cell models (such as the PC12 cell line) or normal experimental animal models with release function. In animal experiments targeting neurodegenerative diseases, this probe can be used to continuously monitor the difference in dopamine release between normal physiological models and disease models, thereby quantitatively reflecting the abnormal depletion of dopamine molecules.

[0051] The compound to be evaluated is a dopamine release regulator (such as the receptor antagonist pimozide or the dopamine precursor levodopa). The measurement focuses on the regulator's ability to control dopamine release (inhibition or promotion). The target molecule, dopamine, enters the imprinted cavity and binds to a borate group, causing a change in the probe interface potential and generating a change in the source-leakage current signal. The rate of change in the source-leakage current after stimulation is measured and compared with the signal of the group without the compound to be evaluated to obtain the regulatory ability of the compound (regulator) on dopamine release.

[0052] The dual-synergistic recognition neural probe AN-EGFET of this invention can specifically capture dopamine molecules and convert them into electrical signals of a field-effect transistor. As a specific monitoring tool for dopamine, the neural probe AN-EGFET of this invention can determine the dynamic level of dopamine in different physiological or pathological microenvironments by detecting the decrease in source leakage current after stimulation.

[0053] This invention can be used to evaluate the dynamic changes in dopamine release levels in in vitro neural cell systems or in vivo brain regions of experimental animals, thereby providing a detection tool for in situ monitoring of dopamine-related neurochemical processes and evaluation of experimental animal models. This invention can also determine the regulatory effects (including inhibitory and promoting effects) of any compound to be evaluated on dopamine release activity in biological systems such as in vitro cells or in vivo brain tissue. These biological systems include, but are not limited to, various in vitro cultured neural cell models, specific brain regions of normal experimental animals, and neurodegenerative disease models such as Parkinson's disease used for comparison between normal and abnormal exhaustion states.

[0054] The following embodiments will further illustrate the present invention, but are not intended to limit the invention.

[0055] Example 1: Construction of the neural probe AN-EGFET and fabrication of the extended gate sensing interface of acupuncture needle

[0056] This embodiment provides a method for constructing a neural probe, AN-EGFET. For example... Figure 1 As shown, the neural probe AN-EGFET consists of a field-effect transistor module, an acupuncture needle extended gate, and a reference electrode. First, the acupuncture needle extended gate is progressively modified with gold nanoparticles, 4-mercaptophenylboronic acid, and a poly(o-phenylenediamine) molecularly imprinted polymer layer. Then, the modified acupuncture needle extended gate is electrically connected to the gate terminal of the field-effect transistor module, and together with the reference electrode, a complete neural probe AN-EGFET is constructed.

[0057] Figure 11 This is a schematic diagram of the structure of the neural probe AN-EGFET of the present invention. Figure 11 As shown, the neural probe AN-EGFET includes a field-effect transistor module, an extended gate for acupuncture needles, and a reference electrode. The extended gate for acupuncture needles is used to construct a dopamine dual-synergistic recognition sensing interface and is electrically connected to the gate terminal G of the field-effect transistor module. The source terminal S and drain terminal D of the field-effect transistor module are used to connect to a current detection unit to record source-drain current signals. The reference electrode is placed in the test environment and connected to the external gate bias terminal V. Ref A connection is established to provide a stable reference potential. Thus, the acupuncture needle extended gate, reference electrode, and field-effect transistor module together constitute the AN-EGFET neural probe for real-time dopamine monitoring.

[0058] The fabrication process of the extended gate electrode of the acupuncture needle is as follows:

[0059] (1) The extended gate of a medical acupuncture needle (AN) was placed in an 8 mmol / L HAuCl4 solution, and gold nanoparticles (AuNPs) were electrodeposited on its surface using cyclic voltammetry (-1.5 to 0.5 V, 100 mV / s, 5 cycles) to obtain the extended gate AuNPs / AN of the acupuncture needle. See Figure 1 Step 1.

[0060] (2) The modified acupuncture needle extended gate AuNPs / AN was immersed in a 5 mg / mL solution of 4-mercaptophenylboronic acid (4-MPBA) and incubated for 6 hours. A monomolecular self-assembled layer was formed through gold-thiol bonds to obtain the acupuncture needle extended gate 4-MPBA / AuNPs / AN. See Figure 1 Step 2.

[0061] (3) The acupuncture needle extended gate 4-MPBA / AuNPs / AN was electropolymerized in PBS buffer (0.01 M, pH 7.4) containing 6 mmol / L o-phenylenediamine (OPD) and 2 mmol / L dopamine (DA) in cyclic voltammetry (0 to 0.8 V, 50 mV / s, 8 cycles) to generate a molecularly imprinted polymer (MIP) layer encapsulating dopamine, resulting in uneluted acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN. See Figure 1 Step 3.

[0062] (4) The uneluted extended gate acupuncture needle MIP / 4-MPBA / AuNPs / AN was treated in a mixture of 95% ethanol and 0.1 M hydrochloric acid (volume ratio 6:1) for 10 minutes to elute dopamine molecules from the polymer network, yielding the extended gate acupuncture needle MIP / 4-MPBA / AuNPs / AN. See Figure 1 Step 4.

[0063] (5) During detection, dopamine molecules re-enter the molecularly imprinted cavity and form a covalent borate bond with 4-mercaptophenylboronic acid through their catechol structure; simultaneously, the dopamine molecules and the molecularly imprinted cavity match in size, configuration, and action site, thus forming a dual synergistic recognition. See Figure 1 Step 5.

[0064] Example 2: Chemical structure and morphological characterization of the extended gate sensing interface of acupuncture needles

[0065] (1) Morphology and elemental characterization: The surface morphology changes of the acupuncture needle base before and after modification were observed using scanning electron microscopy (SEM). Figure 2A in the image is a low-magnification scanning electron microscope image of the extended gate of the modified acupuncture needle. It can be seen that a continuous and relatively uniform modification layer is formed on the surface of the acupuncture needle, indicating that the sensing interface can stably cover the surface of the acupuncture needle substrate. Figure 2 Image B in the image is a further magnified scanning electron microscope image, showing that the surface of the acupuncture needle exhibits a distinctly roughened structure. Figure 2 Image C in the image is a high-magnification scanning electron microscope image, which reveals that the surface is composed of a large number of nanoparticles and clustered structures, exhibiting a rough micro-nano structure similar to cauliflower, indicating that gold nanoparticles and the subsequent polymer layer were successfully constructed on the surface of the acupuncture needle. Elemental mapping analysis was performed using energy-dispersive X-ray spectroscopy (EDS) to confirm the uniform distribution of key elements such as Au, S, B, and N at the interface. Figure 2 In the diagram, D represents the elemental distribution at the sensing interface. Au, S, N, and B elements are all relatively uniformly distributed on the surface of the acupuncture needle. Au is derived from electrodeposited gold nanoparticles, S and B from 4-mercaptophenylboronic acid, and N from the poly(o-phenylenediamine) molecularly imprinted polymer layer. These results indicate that gold nanoparticles, 4-mercaptophenylboronic acid, and the poly(o-phenylenediamine) molecularly imprinted polymer have been successfully assembled onto the extended gate surface of the acupuncture needle.

[0066] (2) Chemical structure and electronic state analysis: X-ray photoelectron spectroscopy (XPS) was used to detect the surface electronic state of the final sensing interface. Figure 2 The EH values ​​in the figure represent the high-resolution X-ray photoelectron spectra of Au 4f, S 2p, N 1s, and B 1s at the sensing interface. Figure 2 The characteristic peak of Au 4f in E in the image proves that gold nanoparticles were successfully modified on the surface of acupuncture needles; Figure 2 The characteristic peak of F in S 2p indicates that 4-mercaptophenylboronic acid is anchored on the surface of gold nanoparticles through gold thiol bonds; Figure 2 The characteristic peak of N 1s in G in the figure proves that o-phenylenediamine undergoes electropolymerization and forms a poly-o-phenylenediamine molecularly imprinted polymer layer; Figure 2 The characteristic peak of B1s in H indicates that the phenylboronic acid group in 4-mercaptophenylboronic acid is retained in the sensing interface. Conclusion: Morphological characterization, elemental distribution, and XPS results jointly demonstrate that the molecularly imprinted polymer layer of gold nanoparticles, 4-mercaptophenylboronic acid, and poly(o-phenylenediamine) has been successfully constructed on the extended gate surface of the acupuncture needle, forming a dual synergistic sensing interface for dopamine recognition.

[0067] Example 3: Electrical performance testing of the neural probe AN-EGFET;

[0068] In this embodiment, the electrical properties of the constructed neural probe AN-EGFET were tested using a semiconductor source meter system. The extended gates of the acupuncture needles at each modification stage were placed in 10 mmol / L, pH 7.4 PBS buffer solution.

[0069] Output characteristic test: The source-drain voltage was scanned in the range of 0 to 2 V under different gate voltage conditions, and the source-drain current (I) was recorded. DS ).like Figure 3 As shown in Figure A, as the source-drain voltage increases, the source-drain current gradually increases and tends to saturate, exhibiting obvious field-effect transistor output characteristics; at the same time, as the gate voltage increases, the source-drain current increases overall, indicating that the extended gate can effectively regulate the MOSFET channel current.

[0070] Transfer characteristic test: Set source-drain voltage V DS The gate voltage V is 0.2 V. G Scan within the range of 0 to 2 V and record the source-drain current I. DS .like Figure 3 As shown in B, the source-drain current increases with the increase of the gate voltage, and the source-drain current is larger at higher source-drain voltages, indicating that the constructed device has typical n-type enhancement-mode field-effect modulation behavior.

[0071] Conclusion: The device exhibits standard n-type field-effect modulation behavior with a clear saturation region and good electrochemical performance, making it suitable as an electrical signal conversion platform for subsequent dopamine detection. (See...) Figure 3 ).

[0072] Example 4: Synergistic Effect Analysis of Interface Modification of the Neural Probe AN-EGFET

[0073] The preparation process of the extended gate modification interface for the four types of acupuncture needles used in this experiment is as follows:

[0074] (1) MIP / 4-MPBA / AuNPs / AN dual-modified interface: prepared entirely by the method described in Example 1;

[0075] (2) MIP / AuNPs / AN single molecular imprint recognition interface: Refer to the method described in Example 1, except that the self-assembly process of 4-MPBA in step (2) is omitted, and the electropolymerization of the MIP layer is performed directly on the surface of AuNPs / AN.

[0076] (3) 4-MPBA / AuNPs / AN single boric acid affinity recognition interface: referring to the method described in Example 1, after completing the 4-MPBA self-assembly in step (2), the electropolymerization and elution processes in steps (3) and (4) are omitted;

[0077] (4) MIP / TSA / AuNPs / AN control identification interface: Refer to the method described in Example 1, except that in step (2), 4-MPBA is replaced with thiosalicylic acid (TSA) of the same concentration for self-assembly, and the remaining electropolymerization and elution steps are exactly the same.

[0078] The four types of acupuncture needles prepared above were coupled to a field-effect transistor module and an Ag / AgCl reference electrode, respectively, and then placed in a dopamine (DA) solution of the same concentration (e.g., 100 nmol / L) for detection. The constant voltage source drain voltage V... DS = 2 V, gate voltage V G = At 2 V, continuously record the rate of change of source and drain current of each group of field-effect transistors ΔI / I0 (where I0 is the initial baseline current before the introduction of dopamine, and ΔI is the change in current after the introduction of dopamine).

[0079] Conclusion: Modification of MIP or 4-MPBA alone, as well as the MIP / TSA control modification interface, all produce a certain current response. However, the current response produced by the dual modification interface of MIP / 4-MPBA / AuNPs / AN is the most significant, far exceeding that of the other three groups, demonstrating that the dual modification interface achieves a synergistic enhancement effect (see...). Figure 4 ).

[0080] Example 5: Dopamine Concentration Response of the Neural Probe AN-EGFET

[0081] The neural probe AN-EGFET prepared according to Example 1 was immersed in PBS buffer containing different concentrations of dopamine (DA) (ranging from 1 nmol / L to 10 μmol / L) and incubated for 5 minutes. A constant source-drain voltage V was maintained. DS = 2 V, gate voltage V G The steady-state source-drain current response was recorded at 2 V. A linear regression was performed on the logarithm of the dopamine concentration (lg[DA]) using the source-drain current change rate ΔI / I0. Figure 5 As shown, a linear fit was performed with the source-drain current change rate ΔI / I0 as the ordinate and the logarithm of dopamine concentration as the abscissa. The results show that in the concentration range of 1 nmol / L to 10 μmol / L, the source-drain current change rate has a good linear relationship with the logarithm of dopamine concentration, and the correlation coefficient R² is 0.96.

[0082] Conclusion: Within the range of 1 nmol / L to 10 μmol / L, the source-drain current change rate ΔI / I0 exhibits an excellent linear relationship with the logarithm of dopamine concentration (see...). Figure 5 ).

[0083] Example 6 Specificity analysis of the neural probe AN-EGFET

[0084] The neural probe AN-EGFET was placed in PBS solution containing 100 nmol / L DA and in solutions containing common in vivo interfering agents at concentrations ten times higher (1 μmol / L). These interfering agents included ascorbic acid (AA), glutamate (Glu), serotonin (5-HT), norepinephrine (NE), and uric acid (UA). The source-leakage current change rate ΔI / I0 of the neural probe was measured in different solutions. The results showed that, as Figure 6 As shown, the neural probe AN-EGFET exhibits a significant source-drain current rate of change response to 100 nmol / L dopamine, while producing only a weak response to interfering substances such as ascorbic acid, glutamate, serotonin, norepinephrine, and uric acid at concentrations as high as 1 μmol / L. These results indicate that even when the concentration of interfering substances is 10 times that of dopamine, the neural probe AN-EGFET still exhibits a significantly higher electrical response to dopamine, demonstrating that this neural probe possesses good dopamine recognition specificity and anti-interference ability.

[0085] Conclusion: The neural probe AN-EGFET exhibits good specificity for dopamine detection and can effectively distinguish the differences in electrical responses between dopamine and common in vivo interfering agents (see...). Figure 6 ).

[0086] Example 7 Real-time measurement of drug-induced dopamine release in PC12 cells using the neural probe AN-EGFET;

[0087] (1) Cell culture and drug intervention: PC12 cell lines with dopamine secretion function were randomly divided into three groups for culture. The control group received no additional treatment; the calcium channel inhibitor group was pretreated with medium containing 10 μmol / L nifedipine for 30 minutes; and the dopamine precursor group was pretreated with medium containing 10 μmol / L levodopa for 30 minutes.

[0088] (2) Neural probe preparation and baseline stabilization: The extended gate of the fully modified and eluted neural probe AN-EGFET was immersed in the cell culture medium of each of the above groups, and connected to the Ag / AgCl reference electrode. A constant test voltage (V) was applied. G = 2 V, V DS = 0.1 V), until the source-drain current I of the field-effect transistor. DS To reach a stable baseline state;

[0089] (3) Stimulation triggering and real-time monitoring: After the baseline stabilized, 10 mmol / L potassium chloride (KCl) solution was added to the cell culture medium. The source-leakage current I was then continuously recorded. DS And the rate of change over a certain period of time (ΔI / I0);

[0090] like Figure 7 As shown in Figure A, after stimulation with potassium chloride, the source-leakage current decreased to varying degrees in the control group, nifedipine group, and levodopa group, indicating that dopamine released from PC12 cells can be recognized in real time by the neural probe AN-EGFET and converted into an electrical signal. The decrease in source-leakage current in the nifedipine group was smaller than that in the control group, indicating that the dopamine exocytosis process was blocked after calcium ion channel inhibition. The decrease in source-leakage current in the levodopa group was greater than that in the control group, indicating that levodopa increased intracellular dopamine reserves, thereby enhancing high-potassium stimulation-induced dopamine release.

[0091] like Figure 7 As shown in Figure B, statistical analysis of the source-leakage current change rate in different treatment groups revealed that the levodopa group had the highest current change rate, followed by the control group, while the nifedipine group had the lowest. This result further demonstrates that the neural probe AN-EGFET can distinguish the differences in dopamine release levels in PC12 cells under different drug intervention conditions and can be used to evaluate the regulatory effect of drugs on dopamine release.

[0092] Conclusion: The neural probe AN-EGFET possesses excellent real-time monitoring capabilities of the in vitro cellular microenvironment, enabling highly sensitive in-situ tracking and quantification of dynamic changes in dopamine release in cell models under different pharmacological interventions (see...). Figure 7 ).

[0093] Example 8: Real-time measurement of dopamine release in different brain regions of mice using the neural probe AN-EGFET

[0094] (1) Mouse anesthesia and in situ implantation: Normal mice were anesthetized and fixed on a stereotaxic apparatus. The extended gate of the neural probe AN-EGFET was precisely implanted into the dopamine-rich dorsal striatum (DS) or the forelimb area of ​​the primary somatosensory cortex (S1FL) with relatively low dopamine levels. A reference electrode was connected and a constant test voltage (V) was applied. G = 2 V, V DS =0.1 V), maintain until the baseline current stabilizes;

[0095] (2) Chemical stimulation: After the current stabilizes, a 70 mmol / L potassium chloride solution is precisely micro-injected into the local microenvironment of the brain tissue around the neural probe, triggering the release of local dopamine in the region through chemical depolarization.

[0096] like Figure 8As shown in Figure A, after potassium chloride stimulation, the AN-EGFET neural probe implanted in the dorsal striatum (DS) region recorded a significant decrease in source-leakage current response, indicating that this region releases a higher level of dopamine after high potassium stimulation. In contrast, the AN-EGFET neural probe implanted in the forelimb region of the primary somatosensory cortex (S1FL) recorded only a weaker change in source-leakage current, indicating that the dopamine release level in this brain region is relatively low.

[0097] like Figure 8 As shown in Figure B, statistical analysis of the current change rate in the two brain regions DS and S1FL showed that the current change rate in the DS region was significantly higher than that in the S1FL region, further demonstrating that the neural probe AN-EGFET can distinguish the differences in dopamine release levels in the local microenvironment of different brain regions.

[0098] Conclusion: The neural probe AN-EGFET can record potassium chloride stimulation-induced dopamine release responses in real time within the brain regions of experimental animals, and can distinguish the differences in dopamine release levels in the local microenvironment of the dorsal striatum and the forelimb area of ​​the primary somatosensory cortex (see...). Figure 8 ).

[0099] Example 9: Real-time measurement of dopamine release in mice under drug intervention using the neural probe AN-EGFET

[0100] (1) Test preparation: Anesthetize the mice, precisely implant the extended gate of the neural probe AN-EGFET into the dorsal striatum of the mice, and apply a constant test voltage (V). G = 2 V, V DS = 0.1 V). Thirty minutes before the test, mice in the drug intervention group were intraperitoneally injected with either the dopamine receptor antagonist pimozide (0.002 mg / g) or the dopamine precursor levodopa (0.13 mg / g), while the control group was injected with an equal volume of physiological saline and maintained at baseline homeostasis.

[0101] (2) Stimulation triggering: After the baseline current of the neural probe reaches stability, a 70 mmol / L potassium chloride solution is injected into the brain in situ to induce high potassium stimulation and initiate the release of dopamine.

[0102] (3) Continuous monitoring and analysis: Source-leakage current I after stimulation DS Continuous recording was performed. Based on the difference between the rate of change of source-leakage current (ΔI / I0) after stimulation in the drug intervention group and the rate of change in the control group, the regulatory intensity of different drugs on dopamine release was calculated.

[0103] like Figure 9As shown in Figure A, after potassium chloride stimulation, the source-leakage current decreased to varying degrees in the control group, pimozide group, and levodopa group, indicating that potassium chloride stimulation can induce local dopamine release in the dorsal striatum of mice, which is captured in real time by the neural probe AN-EGFET. The decrease in source-leakage current in the pimozide group was significantly smaller than that in the control group, indicating that the dopamine-related release response was inhibited after pimozide intervention; the decrease in source-leakage current in the levodopa group was greater than that in the control group, indicating that levodopa can enhance the dopamine release response under potassium chloride stimulation.

[0104] like Figure 9 As shown in B, statistical analysis of the current change rate in different treatment groups revealed that the current change rate in the pimozide group was lower than that in the control group, while the current change rate in the levodopa group was higher than that in the control group. This further demonstrates that the neural probe AN-EGFET can distinguish the differences in the regulation of dopamine release levels in mouse brain regions by drug intervention.

[0105] Conclusion: The neural probe AN-EGFET can record changes in dopamine release response under drug intervention in situ and in real time in the brain regions of experimental animals, and can distinguish the different regulatory effects of inhibitors and promoters on dopamine release levels, indicating that this neural probe can be used for in vivo evaluation of dopamine release regulators or candidate drugs (see...). Figure 9 ).

[0106] Example 10 Real-time measurement of dopamine release in Parkinson's mice using the neural probe AN-EGFET

[0107] (1) Disease model construction: Male C57 mice were randomly divided into two groups. The Parkinson's disease (PD) model group was injected intraperitoneally with probenecid (0.1 mg / g), followed by intraperitoneal injection of the neurotoxin MPTP (0.03 mg / g) 30 minutes later, for 7 days; the normal control group was injected with an equal volume of physiological saline.

[0108] (2) Implantation and detection of neural probes: Two groups of mice were anesthetized and fixed on a stereotaxic instrument. The extended gate of the neural probe AN-EGFET was precisely implanted into the dorsal striatum, and a constant test voltage (V) was applied. G = 2 V, V DS = 0.1 V).

[0109] (3) In vivo in situ stimulation and measurement: After the baseline current stabilized, in situ stimulation was performed using 70 mmol / L potassium chloride solution to trigger dopamine release, and the source-leakage current I was recorded over a certain period of time. DS And the rate of change of response ΔI / I0.

[0110] like Figure 10As shown in A, after stimulation with potassium chloride, the source-leakage current of normal mice decreased significantly, while the source-leakage current of Parkinson's disease model mice showed a smaller change, indicating that the level of dopamine that can be released in the dorsal striatum of Parkinson's disease model mice was reduced.

[0111] like Figure 10 As shown in B, the rate of change of current in the Parkinson's disease model group was significantly lower than that in the normal group, indicating that the neural probe AN-EGFET can effectively distinguish the difference in dopamine release levels in the brain region between the normal state and the Parkinson's disease model state.

[0112] Conclusion: The neural probe AN-EGFET can be used as an in vivo sensing tool to differentiate between abnormal dopamine depletion in normal states and neurodegenerative disease models (see...). Figure 10 ).

Claims

1. A dual-synergistic recognition neural probe based on an acupuncture needle extension grid, characterized in that, The neural probe includes an n-type metal-oxide-semiconductor field-effect transistor, a reference electrode, and a medical acupuncture needle as an extended gate. The surface of the medical acupuncture needle is sequentially modified with a gold nanoparticle layer, a 4-mercaptophenylboronic acid layer, and a poly(o-phenylenediamine) molecularly imprinted polymer layer; the 4-mercaptophenylboronic acid layer is connected to the gold nanoparticle layer through gold-thiol bonds, and the phenylboronic acid groups in the 4-mercaptophenylboronic acid are used to form covalent borate ester bonds with dopamine molecules; the poly(o-phenylenediamine) molecularly imprinted polymer layer has a molecularly imprinted cavity that matches the dopamine molecule. The conductive needle body of the medical acupuncture needle is electrically connected to the gate terminal of the n-type metal-oxide-semiconductor field-effect transistor. The source and drain of the n-type metal-oxide-semiconductor field-effect transistor are respectively connected to the current detection unit. The reference electrode is placed in the test environment and connected to the external gate bias terminal, thereby forming a liquid gate-controlled extended gate field-effect detection circuit composed of the reference electrode, the test environment, the extended gate of the medical acupuncture needle, and the n-type metal-oxide-semiconductor field-effect transistor. In this process, dopamine molecules undergo dual synergistic recognition with the 4-mercaptophenylboronic acid layer and the molecularly imprinted cavity, causing a change in the potential of the extended gate surface of the medical acupuncture needle, and further modulating the source and drain current of the n-type metal oxide semiconductor field-effect transistor.

2. A method for preparing the dual-synergistic recognition neural probe according to claim 1, characterized in that, The method includes the following specific steps: (1) The extended gate AN of the acupuncture needle was placed in an 8 mmol / L HAuCl4 solution. Cyclic voltammetry was used with a voltage scan range of -1.5-0.5 V and a scan rate of 100 mV / s for 5 cycles. Gold nanoparticles AuNPs were electrodeposited on its surface to form the extended gate AuNPs / AN of the acupuncture needle. (2) The acupuncture needle extended gate AuNPs / AN described in (1) is immersed in a 5 mg / mL 4-mercaptophenylboronic acid 4-MPBA solution and incubated for 6 hours to obtain the acupuncture needle extended gate 4-MPBA / AuNPs / AN; (3) The acupuncture needle extended gate 4-MPBA / AuNPs / AN described in (2) was placed in a PBS buffer solution containing 6 mmol / L o-phenylenediamine OPD and 2 mmol / L dopamine at pH 7.

4. Cyclic voltammetry was used to perform in-situ electropolymerization to generate a molecularly imprinted polymer (MIP) layer encapsulating dopamine, resulting in unwashed acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN. (4) The acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN described in (3) is placed in a mixture of 95% ethanol and 0.1 M hydrochloric acid and eluted for 10 minutes to remove dopamine molecules from the polymer network, thereby obtaining the acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN; wherein the volume ratio of 95% ethanol to 0.1 M hydrochloric acid is 6:1; (5) The acupuncture needle extended gate MIP / 4-MPBA / AuNPs / AN described in (4) is coupled with an n-type metal oxide semiconductor field-effect transistor MOSFET and an Ag / AgCl reference electrode to obtain the dual collaborative recognition neural probe, denoted as AN-EGFET.

3. The application of the dual synergistic recognition neural probe of claim 1 in dopamine detection and dynamic evaluation of dopamine release, characterized in that, The applications include detecting the concentration or dynamic changes of dopamine in in vitro biological samples and in vitro cell systems; the test objects include dopamine-containing buffer solutions, cell culture media, and in vitro cell systems with dopamine release function.

4. The application according to claim 3, characterized in that, Dopamine molecules specifically bind to 4-mercaptophenylboronic acid on the neural probe AN-EGFET via covalent borate bonds, and spatially match the cavity generated by the molecularly imprinted polymer, forming a dual synergistic recognition mechanism. This specific binding causes a change in the extended gate interface potential. By detecting the rate of change of the source-drain current of the neural probe AN-EGFET, quantitative assessment of the concentration or dynamic changes of dopamine in in vitro biological samples and in vitro cell systems can be achieved. Specifically, the process includes: using the neural probe AN-EGFET, immersing the extended gate of the neural probe into the test object under physiological conditions, so that the extended gate is in contact with the test environment; recording the changes in the source-drain current of the field-effect transistor under set gate voltage and source-drain voltage conditions, thereby quantitatively assessing the concentration or dynamic changes of dopamine in the test object.

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