DNA-cho / M-coated ZIF-8 / CFME electrode and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a DNA-ch / M-coated ZIF-8 / CFME electrode as well as a preparation method and application of the DNA-ch / M-coated ZIF-8 / CFME electrode. The DNA-cho / M-coated ZIF-8 / CFME electrode comprises a carbon fiber electrode, and ZIF-8 nanoparticles, a brain cell membrane and an aptamer DNA-cho are sequentially modified on the carbon fiber electrode. According to the invention, ZIF-8 nanoparticles with a dodecahedron structure grow in situ on the carbon fiber electrode, functional stabilization is carried out on ZIF-8 by using cell membrane phospholipid molecules, and meanwhile, a DNA aptamer for specific recognition of DA is inserted, so that the stability, pollution resistance, sensitivity and selectivity of the electrode are remarkably improved. The invention provides an important technical means for exploring the change of dopamine metabolism in the early stage of diabetes.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a DNA-cho / M@ZIF-8 / CFME electrode, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia, caused by absolute or relative insulin deficiency and impaired insulin utilization. Dopamine (DA) is an important catecholamine neurotransmitter secreted by the brain, playing a crucial role in motor control, cognitive function, and mood regulation. Studies have shown that alterations in the dopaminergic system can be observed in diabetic patients, and dopamine is closely related to various diabetic complications such as diabetic retinopathy and diabetic encephalopathy. Therefore, in situ, real-time, dynamic monitoring of changes in dopamine levels in the human body is of great significance for the prevention and treatment of diabetes and its complications.

[0003] Currently, methods for detecting dopamine mainly include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), fluorescence sensors, surface plasmon resonance (SPR), and electrochemical detection. Among these, electrochemical analysis methods typically offer advantages such as miniaturized detection electrodes and high spatiotemporal resolution, making them suitable for in-situ, real-time dynamic monitoring of dopamine in the brain. However, designing sensors to obtain more active sites while ensuring their resistance to contamination to achieve long-term sensitive detection of chemical signals in the brain remains a significant challenge.

[0004] ZIF-8 is a porous nanomaterial composed of metal nodes and organic ligands, providing more active sites for substance detection. Furthermore, ZIF-8 possesses hydrophilic NH groups, and its small pore size and low porosity effectively prevent the adsorption of large protein molecules on the electrode surface. However, ZIF-8 still faces certain challenges in practical applications: 1) Structural instability: Zn in ZIF-8... 2+ 1) Low coordination bond energy with imidazole ligands, making them prone to hydrolysis and dissociation, leading to structural collapse of the material; 2) Poor cell compatibility: Unmodified ZIF-8 may trigger immune or toxic reactions in vivo. These defects greatly limit its application in fields such as biosensors. In previous studies, ZIF-8 has mostly been used as a precursor and sacrificial template, and modified to improve its stability. For example, solid-phase pyrolysis synthesis of nanowires and high-temperature carbonization preparation of Zn-NC catalysts. However, these methods have limitations, requiring high-temperature and high-pressure environments, and the addition of other metals may cause immune inflammatory reactions and cytotoxicity during electrode implantation.

[0005] Therefore, developing a material with excellent sensitivity, specificity, stability and biocompatibility is of great significance for realizing in situ real-time dynamic monitoring of brain adenomas (DA). Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a DNA-cho / M@ZIF-8 / CFME electrode for detecting dopamine.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A DNA-cho / M@ZIF-8 / CFME electrode for detecting dopamine, the electrode comprising a carbon fiber electrode on which ZIF-8 nanoparticles, a brain cell membrane, and an aptamer DNA-cho are sequentially modified.

[0009] Preferably, the nucleotide sequence of the aptamer DNA-cho is as shown in SEQ ID NO.1, and the 3′-end of the aptamer DNA-cho is modified with cholesterol.

[0010] Preferably, the ZIF-8 nanoparticles have a dodecahedral structure.

[0011] Preferably, the functionalization of the cell membrane phospholipid molecules stabilizes ZIF-8, wherein phosphate groups are incorporated into the Zn-N tetrahedron via ligand exchange to enhance the local coordination strength around the metal nodes.

[0012] Preferably, the aptamer DNA-cho is anchored in the cell membrane via cholesterol labeling to enhance the sensitivity and selectivity of the microelectrode.

[0013] Preferably, the phosphate groups (PL-PO4) on the cell membrane phospholipids 3- It can be used as a Lewis base with Zn 2+ Coordination occurs, forming new Zn–O–P bonds.

[0014] The second objective of this invention is to provide a method for preparing a DNA-cho / M@ZIF-8 / CFME electrode.

[0015] To achieve the above objectives, the present invention adopts the following technical solution:

[0016] The preparation method of the DNA-cho / M@ZIF-8 / CFME electrode includes the following steps:

[0017] i) ZIF-8 nanoparticles are grown in situ on the surface of carbon fiber electrodes to obtain ZIF-8 / CFE.

[0018] ii) Soak the ZIF-8 / CFE obtained in step i) in brain cell membrane vesicle solution to obtain M@ZIF-8 / CFE;

[0019] iii) Immerse the M@ZIF-8 / CFE obtained in step ii) in a DNA aptamer solution to obtain a DNA-cho / M@ZIF-8 / CFME electrode.

[0020] Preferably, step i) specifically includes: immersing the carbon fiber electrode in a Zn(NO3)2·6H2O solution, ultrasonically treating it for 10~60 min, adding a 2-methylimidazole solution, magnetically stirring for 40~120 min, washing and drying to obtain ZIF-8 / CFE.

[0021] Preferably, the molar ratio of Zn 2+ :2-MeIM=1:8.

[0022] Preferably, the ultrasonic frequency is 30~60kHz, more preferably 40kHz, and the ultrasonic treatment lasts for 20 minutes.

[0023] Preferably, magnetic stirring is performed for 60 minutes.

[0024] Preferably, the washing solvent is methanol.

[0025] Preferably, vacuum drying is performed at 50-80°C for 4-10 hours, and more preferably at 60°C for 8 hours.

[0026] Preferably, in step ii), the brain cell membrane vesicle solution is homogenized by ultrasound for 10-20 minutes, more preferably 10 minutes.

[0027] Preferably, in step iii), the concentration of the DNA aptamer solution is 1~50 μM, and the soaking time is 30 min~12 h.

[0028] As a preferred embodiment, the concentration of the DNA aptamer solution is 50 μM, and the soaking time is 30 min.

[0029] As a preferred embodiment, the method for preparing the brain cell membrane vesicles includes:

[0030] 1) Separate the brain, wash and cut it into small pieces, and then add it to the membrane protein extraction reagent for processing;

[0031] 2) Grind the processed tissue sample;

[0032] 3) Centrifugation removes cell nuclei and undisturbed cells;

[0033] 4) Centrifuge to precipitate cell membrane fragments.

[0034] As a preferred option, in step 3), centrifuge at 4°C and 700g for 10 minutes.

[0035] As a preferred option, in step 4), centrifugation is performed at 4°C and 14000g for 30 minutes.

[0036] As a preferred embodiment, the carbon fiber electrode pretreatment method is as follows:

[0037] a) Carbon fibers are subjected to ultrasonic treatment and electrochemical activation to obtain activated carbon fiber electrodes;

[0038] b) Prepare the glass capillary sheath;

[0039] c) Connect the carbon fiber to the wire;

[0040] d) Paraffin sealing and curing;

[0041] e) Trim the length of the carbon fiber.

[0042] Preferably, in step a), the carbon fiber is subjected to ultrasonic treatment in acetone, HNO3, KOH and deionized water in sequence.

[0043] More preferably, the ultrasound time is 3 to 10 minutes, and more preferably 5 minutes.

[0044] Preferably, in step a), the electrochemical activation includes: in H2SO4 solution, first applying a potential of +1.5V and maintaining it for 80s by amperometry; then performing cyclic voltammetric scans in the range of -1.0V to +1.0V at a scan rate of 50mV / s until the CV curve stabilizes, and then drying for later use.

[0045] Preferably, in step d), the curing temperature is 50~90℃ for 1~3 hours, more preferably 70℃ for 2 hours.

[0046] The third objective of this invention is to provide a dopamine sensor.

[0047] To achieve the above objectives, the present invention adopts the following technical solution:

[0048] A dopamine sensor comprising the aforementioned DNA-cho / M@ZIF-8 / CFME electrode.

[0049] Preferably, the dopamine sensor further comprises a reference electrode and a counter electrode.

[0050] Preferably, the dopamine sensor uses a platinum wire as the counter electrode and Ag / AgCl as the reference electrode.

[0051] The fourth objective of this invention is to provide an application of the aforementioned DNA-cho / M@ZIF-8 / CFME electrode and / or the aforementioned dopamine sensor in the preparation of products for detecting dopamine content;

[0052] Alternatively, the DNA-cho / M@ZIF-8 / CFME electrode and / or the dopamine sensor may be used in the preparation of products for in situ real-time dynamic monitoring of live brain dopamine (DA).

[0053] Alternatively, the DNA-cho / M@ZIF-8 / CFME electrode and / or the dopamine sensor may be used in the preparation of products for detecting changes in DA release in the striatum of the brain during early diabetes.

[0054] The fifth objective of this invention is to provide a method for detecting dopamine for purposes other than disease diagnosis or treatment.

[0055] To achieve the above objectives, the present invention adopts the following technical solution:

[0056] A method for detecting dopamine for non-disease diagnosis or treatment purposes, using the aforementioned DNA-cho / M@ZIF-8 / CFME electrode or the aforementioned dopamine sensor.

[0057] The sixth objective of this invention is to provide a method for detecting changes in dopamine release in the striatum of the brain in the early stages of diabetes, for purposes other than disease diagnosis or treatment.

[0058] To achieve the above objectives, the present invention adopts the following technical solution:

[0059] A method for detecting changes in dopamine release in the striatum of the brain in the early stages of diabetes, for non-disease diagnosis or treatment purposes, involves using a platinum wire as the counter electrode and Ag / AgCl as the reference electrode. The DNA-cho / M@ZIF-8 / CFME electrode is implanted into the striatum, and KCl is added. Dopamine release kinetics are obtained from the it curve.

[0060] The beneficial effects of this invention are as follows:

[0061] 1. The electrochemical sensor constructed based on the DNA-cho / M@ZIF-8 / CFME electrode of this invention exhibits high sensitivity. This sensor shows a good linear relationship with DA in the range of 0.005-20 μM, with a detection limit as low as 3.25 nM.

[0062] 2. This invention inserts an aptamer specifically recognizing dopamine (DA) onto the cell membrane, significantly improving the sensitivity and selectivity of the electrode for DA detection. Results show that the DNA-cho / M@ZIF-8 / CFME electrode can eliminate interference from most substances in the nervous system, including amino acids (AA), glutamate, dopamine phosphate (DOPAC), amino acids (UA), and glucose.

[0063] 3. This invention modifies the ZIF-8 layer on the carbon fiber electrode using an in-situ growth method and uses cell membrane phospholipid molecules to functionalize and stabilize the ZIF-8, which significantly improves the stability and antifouling properties of the material in the physiological environment, reduces protein adsorption, and extends the service life of the sensor.

[0064] 4. The addition of cell membranes not only enhances the stability of the electrode, but also endows the electrode with good biocompatibility, reduces in vivo immune response and cytotoxicity, making it more suitable for in vivo detection environments.

[0065] 5. The electrochemical sensor developed in this invention successfully tracked changes in dopamine release in the brain of an early diabetic mouse model, revealing the effects of decreased DAT and Girk2 expression on dopamine metabolism, and providing a new molecular mechanism perspective for studying the effects of diabetes on the dopaminergic system. Attached Figure Description

[0066] Figure 1 The diagram shows the structural characterization results of DNA-cho / M@ZIF-8 / CFE. a) shows the construction process of DNA-cho / M@ZIF-8 / CFE; b) shows scanning electron microscope (SEM) images of naked CFE (10 μm and 4 μm); c) shows SEM images of ZIF-8 / CFE (10 μm and 4 μm); d) shows SEM images of M@ZIF-8 / CFE (10 μm and 4 μm); e) shows SEM images of DNA-cho / M@ZIF-8 / CFE (10 μm and 4 μm); f) shows the DiI staining fluorescence of ZIF-8 / CFE; g) shows the HEX staining fluorescence of M@ZIF-8 / CFE; h) shows the DiI staining fluorescence of M@ZIF-8 / CFE; i) shows the HEX staining fluorescence of DNA-cho / M@ZIF-8 / CFE. Scale bar: 100. μm; jo is the elemental (Zn, C, N, O, P, S) energy spectrum image of DNA-cho / M@ZIF-8 / CFE, scale bar, 10 μm.

[0067] Figure 2 Figure 1 shows the physical characterization results of DNA-cho / M@ZIF-8 / CFE. In figure 1, a is a schematic diagram of the incorporation of phosphate groups into the Zn-N tetrahedral structure; b is the Gibbs free energy diagram of the phosphorylation substitution process; c is the density functional theory calculation result of the Zn-N bond strength change; de ​​is the high-resolution Zn 2p spectrum of ZIF-8 and M@ZIF-8; fg is the high-resolution O 1s spectrum of M and M@ZIF-8; and h is the FTIR spectrum of M, ZIF-8, M@ZIF-8 and DNA-cho / M@ZIF-8.

[0068] Figure 3In the diagram, a is a magnified view of the FTIR spectrum of M and M@ZIF-8 (C=O stretching vibration); b is the XRD diffraction pattern of ZIF-8; c is the dynamic light scattering result of ZIF-8; d is the dynamic light scattering result of M@ZIF-8; e is the full XPS spectrum of DNA-cho / M@ZIF-8; f is the high-resolution XPS spectrum of DNA-cho / M@ZIF-8 (Zn 2p); g is the high-resolution XPS spectrum of DNA-cho / M@ZIF-8 (C 1s); h is the high-resolution XPS spectrum of DNA-cho / M@ZIF-8 (O 1s); i is the high-resolution XPS spectrum of DNA-cho / M@ZIF-8 (N 1s); j is the high-resolution XPS spectrum of DNA-cho / M@ZIF-8 (P 2p); and k is the high-resolution XPS spectrum of DNA-cho / M@ZIF-8 (S 2p).

[0069] Figure 4 The figure shows the in vitro electrochemical performance of DNA-cho / M@ZIF-8 / CFE. Figure a shows a typical cyclic voltammetry (CV) curve of DNA-cho / M@ZIF-8 / CFE measured in aCSF containing 10 μM MDA, 20 μM DA, and 50 μM DA, at a scan rate (ν) of 100 mV·s. -1 b shows the electrochemical impedance spectroscopy (EIS) spectra of different modified electrodes: bare CFE, ZIF-8 / CFE, M@ZIF-8 / CFE, and DNA-cho / M@ZIF-8 / CFE in 5 mM [Fe(CN)6] containing 0.1 M KCl. 3- / 4- The results were measured in solution; c is the cyclic voltammetry curve of 20 μM DA on the DNA-cho / M@ZIF-8 / CFE electrode (at different scan rates ν); d is the corresponding current-scan rate curve; e is the amperometric response curve of DNA-cho / M@ZIF-8 / CFE at +0.15 V in aCSF (pH 7.40) to 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20 μM DA; f is the corresponding calibration curve; g is the voltammetric current response curve of DNA-cho / M@ZIF-8 / CFE with the addition of 100 μM AA, 300 μM L-Glu, 20 μM DOPAC, 50 μM UA, 2.5 mM GLu and 20 μM DA; h is the corresponding statistical bar chart (n = 3); i represents the voltammetric current response of DNA-cho / M@ZIF-8 / CFE and ZIF-8 / CFE to 10 μM dopamine, I 0I and I represent the current values ​​at the initial and specified times, respectively; j is the Zata potential detection result of DNA-cho / M@ZIF-8 / CC, M@ZIF-8 / CC and ZIF-8 / CC films (n = 3); k is the image of DNA-cho / M@ZIF-8 / CFE before and after bending; i is the cyclic voltammetry curve of DNA-cho / M@ZIF-8 / CFE in artificial cerebrospinal fluid (aCSF) containing 20 μM dopamine at different bending times.

[0070] Figure 5 The image shows the biocompatibility verification results of DNA-cho / M@ZIF-8 / CFE, with bright-field and fluorescence images of PC12 cells stained with calcein-AM (green) and PI (red).

[0071] Figure 6 The following figures show the biocompatibility and antifouling performance evaluation results of DNA-cho / M@ZIF-8 / CFE. In figure a, it is a schematic diagram of the experimental procedure; in figure b, it is a statistical graph of the survival rate of PC12 cells after incubation with DNA-cho / M@ZIF-8 / CFE or naked CFE for 24 hours (n=3); and in figure c, it is a graph of the static water contact angle test results of CC, ZIF-8 / CC and DNA-cho / M@ZIF-8 / CC.

[0072] Figure 7 The figure shows the results of the antifouling assessment of DNA-cho / M@ZIF-8 / CFE. The current response obtained by chronoamperometry was obtained when dopamine (10 mM each time) was added to artificial cerebrospinal fluid before and after the naked CFE was immersed in the culture medium for 2 hours.

[0073] Figure 8 The figure shows the results of the feasibility assessment of the antifouling properties of DNA-cho / M@ZIF-8 / CFE and the in vivo DA detection. In figure a, a is a schematic diagram of the experimental procedure; and b is the result of the assay after 5 mg / mL... -1 Fluorescence microscopy images of DNA-cho / M@ZIF-8 / CFE and CFE treated with FITC-BSA; c represents the addition of 10 mg / mL. -1Following BSA, typical amperometric response curves of naked CFE (blue) and DNA-cho / M@ZIF-8 / CFE (purple) to 10 μM dopamine (aCSF recording); d is a schematic diagram of the mechanism by which potassium chloride promotes dopamine release and nifedipine inhibits dopamine release; e is a potential response curve recorded by DNA-cho / M@ZIF-8 / CFE in the striatum (local injection of 100 mM KCl or 100 mM KCl + 10 μM nifedipine); f is the corresponding statistical data of dopamine release (n=3); the blue curve marks the time of administration, and the striatal dopamine measurement potential is +0.15 V (relative to Ag / AgCl); the data points are the averages of multiple samples, and the error bars represent the standard deviation; a two-tailed t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons of three or more groups (**p < 0.01, ***p < 0.001, ****p < 0.0001).

[0074] Figure 9 Figure 1 shows the results of early dopamine release detection and specific mechanism study in diabetic rats; where a is a schematic diagram of the animal experimental process and a timeline of the establishment of the diabetic model; b is the striatum of non-diabetic rats and the striatum of STZ-induced diabetic rats (4 weeks) after local micro-injection (4 L·min). -1The dopamine release current response recorded by DNA-cho / M@ZIF-8 / CFE after PBS (NC) and 100 mM KCl; c is the corresponding statistical data of dopamine release (n=3); d is the Western blot analysis results using striatal lysis buffer from non-diabetic or STZ diabetic (4 weeks) rats; e is the grayscale analysis results corresponding to d (n=3); f is the representative immunostaining images of DRD2, Girk2, and DAT in the striatal region of normal rats and STZ diabetic model rats (4 weeks), scale bar = 100 μm; g is the optical density analysis results (n=3); h is a schematic diagram of the possible mechanism of enhanced dopamine-induced release in the early stage of diabetes; i is the dopamine release current response recorded by local micro-injection of saline (NC) and 100 mM KCl (1 μL / min, lasting 20 seconds) in experimental rats after different drug treatments; j is the normal rat injected with saline 30 minutes later and then injected with Vanoxerine (300 mg / min per cerebral hemisphere). The monitoring results are shown in the graphs 30 minutes after injection of saline, quinpirole, and raclopride (10 μg, 4 μL per cerebral hemisphere) in normal rats; k is the corresponding statistical result graph of DA release (n = 3); l is the monitoring result graph of normal rats 15 minutes after injection of saline, 30 minutes after injection of quinpirole, and 30 minutes after co-injection of quinpirole and raclopride (10 μg, 4 μL per cerebral hemisphere); m is the corresponding statistical result graph of DA release (n = 3); n is the monitoring result graph of normal rats 15 minutes after injection of saline, 30 minutes after injection of ifenprodil (300 μM, 4 μL per cerebral hemisphere), and 30 minutes after co-injection of ifenprodil and raclopride (10 μg, 4 μL per cerebral hemisphere); o is the corresponding statistical result graph of DA release (n = 3); data points represent the mean of multiple samples, and error bars represent the standard deviation; one-way ANOVA was used for statistical analysis (*p < 0.05). 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0075] Figure 10 The images show the XPS full spectra of ZIF-8, M, and ZIF-8@M, where A is the XPS full spectrum of ZIF-8, B is the XPS full spectrum of M, and C is the XPS full spectrum of ZIF-8@M.

[0076] Figure 11In the diagram, AB shows the results of optimizing the DNA-cho / M@ZIF-8 / CFE electrode potential using the amperometric method, including +0.10 V, +0.15 V, +0.20 V, +0.25 V, and +0.30 V (relative to Ag / AgCl); CD is a statistical graph of the current ratio of DA to AA at different potentials, including +0.10 V, +0.15 V, +0.20 V, +0.25 V, and +0.30 V (relative to Ag / AgCl, n=3); the data points represent the average values ​​of multiple samples, and all error bars represent the standard deviation.

[0077] Figure 12 The graphs show the selective detection results of dopamine on DNA-cho / M@ZIF-8 / CFE. A shows the differential pulse voltammetry (DPV) results of 20 M dopamine on DNA-cho / M@ZIF-8 / CFE using artificial cerebrospinal fluid; B shows the DPV results of 20 M dopamine on DNA-cho / M@ZIF-8 / CFE using 50 M uric acid; C shows the DPV results of 20 M dopamine on DNA-cho / M@ZIF-8 / CFE using 2.5 mM glutamate; D shows the DPV results of 20 M dopamine on DNA-cho / M@ZIF-8 / CFE using 300 M L-glutamate; and E shows the DPV results of 20 M dopamine metabolites on DNA-cho / M@ZIF-8 / CFE using 20 M glutamate. Differential pulse voltammetry (DPV) plot of M dopamine; F represents 100 M amino acids on DNA-cho / M@ZIF-8 / CFE, and differential pulse voltammetry (DPV) plot of 20 M dopamine.

[0078] Figure 13 The graphs show the results of electrode stability determination by cyclic voltammetry, obtained in a simulated human cerebrospinal fluid containing 50 MDA. In the graphs, A represents the cyclic voltammetry curve of the DNA-cho / M@ZIF-8 / CFE electrode; B represents the cyclic voltammetry curve of the bare CFE electrode, with a scan rate of 50 mV·s. -1 C is a graph showing the cyclic voltammetry curves obtained from the DNA-cho / M@ZIF-8 / CFE electrode for the first cycle (purple) and the 50th cycle (blue); D is a graph showing the cyclic voltammetry curves obtained from the naked CFE electrode for the first cycle (purple) and the 50th cycle (blue).

[0079] Figure 14 Figure 1 shows the blood glucose levels of STZ-induced diabetic rats on day 3, day 7, and week 4.

[0080] Figure 15HE staining image of implanted striatal electrodes.

[0081] Figure 16 In the figure, A shows representative immunostaining images of the striatum region in the brains of normal rats and STZ-induced diabetic rats, with DAPI (blue), DRD2, Girk2, and DAT (green), and a scale bar of 100 μm; B shows the results of optical density analysis (n=3). Statistical analysis was performed using two-way ANOVA combined with Tukey's multiple comparison test, and * P < 0.05 (n = 3).

[0082] Figure 17 HE staining images of brain tissue from normal rats and STZ-induced diabetic rats are shown. In the image, A is the HE staining image of brain tissue from normal rats, and B is the HE staining image of brain tissue from STZ-induced diabetic rats.

[0083] Figure 18 The following figures show the results of the detection related to the optimization of electrode construction conditions: A is a scanning electron microscope image of cell membrane modification at a concentration of 100 v / v%; B is a scanning electron microscope image of cell membrane modification at a concentration of 80% v / v%; C is a scanning electron microscope image of cell membrane modification at a concentration of 50% v / v%; D is a fluorescence image of aptamer at a concentration of 10 M after 30 minutes of modification; E is a fluorescence image of aptamer at a concentration of 10 M after 12 hours of modification; and F is a fluorescence image of aptamer at a concentration of 50 M after 30 minutes of modification. Detailed Implementation

[0084] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0085] In this embodiment of the invention, a ZIF-8 layer is modified onto a carbon fiber (CF) electrode using an in-situ growth method. ZIF-8 is stabilized by functionalization of cell membrane (M) phospholipid molecules, and phosphate groups are incorporated into Zn-N tetrahedra via ligand exchange. This bonding enhances the local coordination strength around the metal nodes, improving the stability of ZIF-8 in physiological environments. Furthermore, the cell membrane itself possesses unique biomimetic functions, endowing the ZIF-8 material with excellent biocompatibility. Further, aptamers specifically recognizing dopamine (DA) are inserted into the cell membrane to enhance the sensitivity and selectivity of the microelectrode. The electrode, with its optimized fabrication process, not only exhibits stability and antifouling properties but also improves the biocompatibility of the ZIF-8 substrate. The electrode constructed in this way lays the foundation for subsequent research into the mechanisms of dopamine metabolism in early diabetic states.

[0086] Current results indicate that decreased DAT expression in early diabetes leads to reduced dopamine reuptake, while increased DA in the synaptic cleft activates the D2R receptor-mediated negative feedback inhibition pathway. Decreased Girk2 expression disrupts the membrane hyperpolarization effect mediated by Girk2 after D2R activation, preventing effective inhibition of dopamine release and resulting in increased dopamine release. These findings provide a new molecular mechanism perspective for elucidating the dysfunction of the dopaminergic system-glucose metabolism axis and offer a theoretical basis for targeting D2R receptor-mediated signaling pathways to intervene in diabetic neurometabolic disorders. Furthermore, this novel real-time in-situ detection platform has significant application potential in the study of disease metabolic pathways and the development of new potential therapeutic strategies.

[0087] In this embodiment of the invention, the cholesterol amphiphilic aptamers Aptamer cholesterol amphiphilies (DNA-cho,5'-GGACGACGCCAGTTTGAAGGTTCGTTCGCAGGTGTGGAGTGACGTCGTCCTTTTTT-TEG-Cho-3', SEQ ID NO.1) were purchased from BBI Life Sinces (Shanghai, China).

[0088] Example 1. Preparation method of brain cell membrane vesicles and electrodes

[0089] 1. Preparation of brain cell membrane vesicles

[0090] (1) The brain was separated and washed in PBS.

[0091] (2) Take about 100 mg of brain tissue and carefully cut it into small tissue fragments with scissors. Just before use, add 1 mL of PMSF membrane protein extraction reagent A (BBI Life Sinces (Shanghai, China)) to gently suspend the tissue fragments and place them in an ice bath for 10-15 minutes.

[0092] (3) Disruption of tissue samples and identification of disruption effect: Transfer the tissue sample to a grinding tube, add a magnetic bead to each tube, and grind in a homogenizer pre-cooled at 4°C. Usually, after homogenizing 30 times, take about 2-3 μL of cell or tissue homogenate and drop it onto a coverslip and observe it under a microscope. If a shiny ring around the nucleus is seen or the cell morphology is intact, it indicates that the cells are still intact. If 70-80% of the cells do not have a shiny ring around the nucleus or intact cell morphology, it indicates that the cells have been sufficiently disrupted, and the next experiment can be carried out.

[0093] (4) Remove cell nuclei and unbroken cells: Centrifuge at 700g for 10 minutes at 4℃, and carefully collect the supernatant into a new centrifuge tube. Do not touch the precipitate when aspirating the supernatant. It is acceptable to leave about 30-50 μL of supernatant behind to ensure that the aspirated supernatant has a high purity.

[0094] (5) Precipitate cell membrane fragments: Centrifuge at 14000g for 30 minutes at 4℃ to precipitate cell membrane fragments.

[0095] 2. Electrode preparation

[0096] (1) Carbon fiber (CF) was sonicated for 5 min each in acetone, 3M HNO3, 1M KOH and deionized water. Before modification, CF was electrochemically activated: in 0.5M H2SO4 solution, a potential of +1.5V was applied and maintained for 80 s by amperometric method (it); then, cyclic voltammetry was performed in the range of -1.0V to +1.0V at a scan rate of 50 mV / s until the CV curve stabilized. The activated carbon fiber electrode was dried for later use.

[0097] (2) Using a needle-drawing device, a borosilicate glass tube with a diameter of 1.5 mm and a length of 10 cm was drawn into two glass microneedles with pointed tips to serve as sheaths for the CFE. A CF was attached to a copper wire with silver conductive paste. After curing at 70°C for 2 hours, the copper wire was carefully inserted into the capillary. Both open ends of the CFE were sealed with molten paraffin and cured at room temperature. The fiber protruding from the glass was cut to a length of 500 μm using a scalpel.

[0098] (3) Preparation of precursor solution: 0.744 g Zn(NO3)2·6H2O + 20 mL water, stir for 10 min; 1.64 g 2-methylimidazole + 20 mL water, stir for 10 min. Molar ratio of Zn... 2+ :2-MeIM=1:8, stability at room temperature for 2h.

[0099] (4) Immerse the pretreated CFE in Zn(NO3)2·6H2O solution and sonicate (40kHz) for 20min to allow the Zn to settle. 2+ Preferentially adsorbed at defect sites; 2-methylimidazole solution was quickly poured in and magnetically stirred for 60 min; the solution gradually turned milky white, indicating ZIF-8 nucleation and growth; CFE was removed and gently rinsed 3 times with methanol to remove loose nanoparticles; vacuum dried at 60°C for 8 h to obtain ZIF-8-CFE.

[0100] (5) Cell membrane coating on ZIF-8-CFE surface using immersion method (M): The electrode was immersed in a membrane vesicle solution homogenized by ultrasound for 15 min; then, taking advantage of the affinity between cholesterol and the membrane, the 3′-terminal cholesterol-modified aptamer (dissolved in a solution containing 5 mM Mg) was coated onto the surface of the ZIF-8-CFE. 2+ The solution was introduced, and M-ZIF-8-CFE was immersed in 1 μM aptamer solution for 12 h. The resulting DNA-cho / M@ZIF-8 / CFME was washed with Milli-Q water and stored in phosphate buffer.

[0101] Example 2. Characterization of the DNA-cho / M@ZIF-8 / CFME electrode

[0102] Figure 1 This invention demonstrates the preparation process of DNA-cho / M@ZIF-8 / CFME, and the electrode morphology is characterized by scanning electron microscopy (SEM). First, a room-temperature crystallization method is used to crystallize the DNA-cho / M@ZIF-8 / CFME on a smooth CFME surface (…). Figure 1 b) In-situ growth of ZIF-8 nanoparticles with a dodecahedral structure ( Figure 1 c). Secondly, by employing a strategy of functionalizing cell membrane (M) phospholipid molecules to stabilize ZIF-8, a membrane structure was observed to coat the surface of ZIF-8. (See details...) Figure 1 d. Finally, by inserting cholesterol-labeled DNA aptamers (DNA-cho) into the cell membrane, specific aptamers with high affinity for dopamine (DA) are anchored to the sensing interface, see details. Figure 1 e. DiI and HEX were used to label the cell membrane and aptamers, respectively. DiI is a lipophilic membrane dye that exhibits strong fluorescence when the cell membrane is permeated and excited by a wavelength of 549 nm. HEX was used to label the 5' end of DNA-cho to characterize aptamer modification. ZIF-8 / CFME was immersed in DiI for 30 min. Figure 1 f) and M-ZIF-8-CFME soaked in HEX for 30 min ( Figure 1 The fluorescence of g) was negligible, while M-ZIF-8-CFME labeled with DiI showed very obvious fluorescence ( Figure 1h), HEX-labeled DNA-cho / M@ZIF-8 / CFME showed pink fluorescence ( Figure 1 i). Element mapping was studied using EDS, and the results are as follows: Figure 1 As shown in jo, Zn (a characteristic element of ZIF-8), C, and O (a characteristic element of M and DNA-cho) are uniformly distributed on the electrode surface. These results preliminarily indicate that the material modification was successful and that the modification process had a significant impact on the chemical structure of the material.

[0103] This unique electrode design offers several significant advantages: First, the cell membrane modification strategy based on biomimetic mineralization principles effectively overcomes the instability of ZIF-8 under physiological conditions by enhancing the Zn-N coordination bond strength. Combined with the porous structure and hydrophobicity of ZIF-8, it provides a stable and anti-fouling sensing interface for substance detection. Second, the biomimetic interface provided by the cell membrane improves the biocompatibility of the ZIF-8 layer while further reducing non-specific adsorption in the brain detection environment. Finally, the DNA aptamer (DNA-cho) inserted into the cell membrane serves as a molecular recognition element, exhibiting extremely high affinity and specificity for DA, effectively distinguishing structural analogs such as ascorbic acid and enhancing detection sensitivity. Therefore, the structural characteristics of the DNA-cho / M@ZIF-8 / CFME sensing platform provide strong support for the highly selective and sensitive detection of DA in complex biological environments.

[0104] Example 3. Mechanism study of DNA-cho / M@ZIF-8 / CFME electrode

[0105] This invention further conducted physical characterization to elucidate the interaction between the cell membrane (M) and ZIF-8. Zn in ZIF-8 2+ The coordination bond between the node and the 2-methylimidazole (2-MIM) ligand is inherently weak and reversible. The phosphate group (PL-PO4) on the M phospholipid... 3- It can be used as a Lewis base with Zn 2+ Coordination forms new Zn–O–P bonds; see details. Figure 2 a and Figure 10 DFT calculations revealed the Zn substitution process during the substitution of phosphate groups. 2+Kinetics of the coordination environment. Free energy diagrams show the free energy changes of the ZIF-8 monomer during coordination substitution: four dimethylimidazolium ligands are gradually replaced by three phosphate groups. The introduction of the first and second phosphate groups resulted in significantly exothermic reactions (ΔG1 = -1.27 eV, ΔG2 = -0.86 eV); however, the introduction of the third phosphate group was endothermic (ΔG3 = 0.16 eV), which can be attributed to the steric hindrance and charge repulsion of the phosphate group. However, considering the large amount of heat released in the first two steps and the extremely low energy barrier, we infer that the phosphate substitution of ZIF-8 can proceed spontaneously and very rapidly at room temperature. When attempting to introduce the fourth phosphate group, the reaction hardly occurred due to the high energy barrier; see [link to relevant documentation]. Figure 2 b.

[0106] X-ray photoelectron spectroscopy (XPS) further verified this conclusion. The increased Zn–N bond binding energy after a single phosphoric acid substitution indicates that the newly formed Zn–O–P bond has a strong electron-donating effect, stabilizing adjacent Zn–N bonds; while the second phosphoric acid substitution is due to PO42-... 3- The electrostatic repulsion between the Zn and O-P bonds significantly weakens the Zn–N bond energy, but the energy released from forming two strong Zn–O–P bonds is sufficient to drive this step. The Zn–N bond energy strengthens again after the third phosphate substitution, indicating that the system has adjusted to a new, more stable coordination conformation. This reveals the dynamic regulation of the Zn coordination environment by phosphate substitution and its crucial role in overall stability; see [link to relevant documentation]. Figure 2 c. XPS display shows Zn 2p 3 / 2 The binding energy increased from 1021.62 eV to 1021.82 eV for Zn 2p. 1 / 2 The voltage increased from 1044.59 eV to 1044.86 eV. See details. Figure 2 d~e indicates Zn 2+ The surrounding electron density decreases; the O 1s level shifts to higher binding energies, see details. Figure 2 f~g reflects the coordination of oxygen atoms to Zn 2+ The electronic contribution is consistent with the newly formed Zn–O coordination bond.

[0107] Fourier transform infrared (FTIR) spectroscopy showed that the M-modified ZIF-8 (M@ZIF-8) was at 1640 cm⁻¹. -1 The C=O stretching vibration peak at this location shows a significant blue shift compared to pure ZIF-8, see details. Figure 2 h and Figure 3 a. This is due to PO4. 3- oxygen atoms and Zn 2+The reduced electron density after coordination, and the weakening of the electron cloud density of adjacent C=O bonds through intramolecular interactions, directly confirms the formation of Zn–O–P bonds. These results collectively demonstrate the existence of coordination interactions between ZIF-8 and M, which contributes to the long-term stability of the electrode sensing interface.

[0108] This invention further characterizes the electrode surface material to confirm the success of modification and synthesis. X-ray diffraction (XRD) shows that ZIF-8 exhibits strong diffraction peaks at 2θ = 7.54°, 10.60°, 12.92°, 14.92°, 16.66°, and 18.22°, corresponding to the (110), (200), (211), (220), (310), and (222) crystal planes, respectively. See details... Figure 3 b. This result confirms the successful synthesis of ZIF-8. Dynamic light scattering (DLS) measured the average particle size of ZIF-8 to be 461 nm, which increased to 650 nm after coating with M. See details. Figure 3 c~d. 421cm in FTIR -1 The peak at 350-1500 cm⁻¹ represents the stretching vibration of the Zn–N bond. -1 Peaks within the range are attributed to imidazole ring vibrations; 1640 and 1233 cm⁻¹ -1 The peaks at these locations correspond to the C=O stretching of M and the vibration of the phosphate group at the phospholipid head, respectively. The peak at 1060 cm⁻¹ corresponds to these vibrations. -1 The peak broadening is attributed to sugar ring vibrations; cholesterol groups cause the peaks at 2925 and 2850 cm⁻¹ to be more pronounced. -1 The intensity of the C–H vibration peaks at these locations increased significantly. Full spectrum and high-resolution XPS (Zn 2p, C 1s, O 1s, N 1s, P 2p, S 2p) of DNA-cho / M@ZIF-8 / CFME were obtained. Figure 3 e~k; Figure 10 All results are consistent with the elemental composition and literature reports, indicating that the material synthesis and modification were successful.

[0109] To achieve the best modification effect and detection performance, the preparation process was optimized in this invention. First, the effect of different concentrations of cell membrane modification on electrode morphology was evaluated using SEM, showing that a 50% cell membrane concentration resulted in a more uniform electrode surface coating. Second, fluorescence experiments were used to evaluate the modification morphology of different aptamer concentrations at different modification times. Finally, 50 μM / 30 min was selected as the final modification concentration and time, as detailed below. Figure 18 Furthermore, the application of potential also has a certain impact on the amperometric measurement of DA. Figure 11AB analysis showed the amperometric response of DNA-cho / M@ZIF-8 / CFME with continuous addition of 10 μM DA at different potentials ranging from 0.1 V to 0.3 V in aCSF (pH 7.4). The current response increased with increasing potential. To more intuitively evaluate the effect of potential on electrode selectivity, we compared the current ratio of DA to AA obtained on DNA-cho / M@ZIF-8 / CFME, as shown below. Figure 11 As shown in CD, the selectivity for DA varies with the potential, reaching a peak at 0.15V, where the signal is sensitive and stable with relatively low noise. Therefore, in subsequent experiments, we chose 0.15V as the detection potential.

[0110] Example 4. In vitro performance evaluation of DNA-cho / M@ZIF-8 / CFME electrode

[0111] Before application to living organisms, this invention first evaluates the electrodes in vitro. Specifically:

[0112] (1) Electrical conductivity

[0113] A simplified analysis of dopamine detection was performed within a potential range of -0.2V to +0.6V. Cyclic voltammetry (CV) showed that the anolyte current increased significantly as the DA concentration increased from 0 μM to 50 μM. (See details...) Figure 4 This is similar to the results reported in DA sensors fabricated using different materials. The charge transfer resistance of Bare CFE, ZIF-8 / CFE, M@ZIF-8 / CFE, and DNA-cho / M@ZIF-8 / CFME was investigated by electrochemical impedance spectroscopy (EIS). An increase in the semicircle diameter of the Nyquist plot corresponds to an increase in electrode resistance (Rct), such as... Figure 4 As shown in b, ZIF-8 / CFME exhibits a very small semicircular diameter, indicating that ZIF-8 modification enhances the electrode's conductivity. As the cell membrane and aptamer gradually encapsulate the modification, the semicircular diameter gradually increases, leading to an increase in impedance. This may be due to the reduced conductivity caused by the biomaterial. The changes in the CV curve and the impedance values ​​show a consistent trend, further illustrating the change in electrode conductivity before and after modification.

[0114] (2) Kinetic study of electrocatalytic reaction

[0115] To further investigate the kinetics of the electrocatalytic reaction, the CV response of DNA-cho / M@ZIF-8 / CFME to 20 μM DA was explored at different scan rates. The results are as follows: Figure 4 As shown in c, the peak current increases with increasing scan rate and is related to v. 1 / 2 The relationship is linear; see details.Figure 4 d indicates that the electrochemical surface interaction is a diffusion-controlled process. DA (5 nM–20 μM) was continuously injected into aCSF solution at +0.15 V, and DA was detected in real-time amperometrically on a DNA-cho / M@ZIF-8 / CFME. Figure 4 As shown in Figure e, the catalytic oxidation current rapidly increased to a stable value after the addition of the standard solution to aCSF. The constructed sensor exhibits a good linear relationship with DA in the range of 0.005-20 μM. (See Figure e for details.) Figure 4 The linear regression equation for f, DA concentration, is I / nA = 0.814C. DA / μM +0.037(R 2 = 0.999). The signal-to-noise ratio (S / N=3) obtained from the 5 nM DA current estimated the limit of detection (LOD) of DNA-cho / M@ZIF-8 / CFME to be 3.25 nM, which is the same as or lower than other DA electrochemical biosensors reported in the literature (Table 1), within the normal range of 2.5–15 nM for DA in the striatum.

[0116] Table 1

[0117]

[0118] The reference information in Table 1 is as follows:

[0119] [1] S. Lu, M. Hummel, K. Chen, et al., “Synthesis of Au@ZIF-8nanocomposites for enhanced electrochemical detection of dopamine,” Electrochemistry Communications, (2020): 114: 106715.

[0120] [2] W. Yao, H. Guo, H. Liu, et al., "Highly electrochemical performance of Ni-ZIF-8 / N S-CNTs / CS composite for simultaneous determination of dopamine, uric acid and L-tryptophan," Microchemical Journal, (2020): 152:104357.

[0121] [3] G. Yu, J. Xia, F. Zhang, et al., “Hierarchical and hybrid RGO / ZIF-8 nanocomposite as electrochemical sensor for ultrasensitivedetermination of dopamine,” Journal of Electroanalytical Chemistry, (2017):801: 496-502.

[0122] [4] M. Nie, S. Lu, D. Lei, et al., “Rapid Synthesis of ZIF-8Nanocrystals for Electrochemical Detection of Dopamine,” Journal of TheElectrochemical Society, (2017): 164(13): H952-H957

[0123] [5] Y. Yuan, J. Xia, F. Zhang, et al., “Nafion / polyaniline / ZeoliticImidazolate Framework-8 nanocomposite sensor for the electrochemicaldetermination of dopamine,” Journal of Electroanalytical Chemistry, (2018):824: 147-152.

[0124] [6] J. Chen, F. Xia, X. Ding, et al., “Universal Covalent GraftingStrategy of an Aptamer on a Carbon Fiber Microelectrode for SelectiveDetermination of Dopamine In Vivo,” Analytical Chemistry, (2024): 96(25):10322-10331.

[0125] [7] J. Chen, F. Xia, X. Ding, et al., “Highly Sensitive andBiocompatible Microsensor for Selective Dynamic Monitoring of Dopamine in RatBrain,” ACS Sensors, (2024): 9(11): 6207-6217.

[0126] [8] T. Ö. Varol, O. Avcı, Ö. Haklı, et al., “An UnsymmetricalPerylene Diimide Dye Modified Carbon Felt Electrode as A NovelElectrochemical Platform for Dopamine Detection,” ChemistrySelect, (2020): 5(37): 11698-11702.

[0127] [9] V. N. Carvalho Da Silva, E. a. O. Farias, A. R. Araújo, et al.,“Rapid and selective detection of dopamine in human serum using anelectrochemical sensor based on zinc oxide nanoparticles, nickelphthalocyanines, and carbon nanotubes,” Biosensors & Bioelectronics, (2022):210: 114211.

[0128]

[10] C. Park, H. Rhyu, S. Jo, et al., “Electrochemical sensor basedon laser-induced graphene and CeO2 for sensitive and selective dopaminedetection,” Journal of Electroanalytical Chemistry, (2025): 977: 118865.

[0129] (3) Selectivity

[0130] This invention investigated the selectivity of DNA-cho / M@ZIF-8 / CFME for DA under physiological conditions by measuring the current response to physiological concentrations of AA, glutamate, DOPAC, UA, glucose, and DA in aCSF. The results are as follows: Figure 4 g~h and Figure 12 As shown, other substances showed almost no significant amperometric response compared to DA, indicating that DNA-cho / M@ZIF-8 / CFME can eliminate interference from most substances in the nervous system.

[0131] (4) Long-term stability

[0132] This invention further investigated long-term stability and found that the current recorded by DNA-cho / M@ZIF-8 / CFME was more stable than that recorded by ZIF-8 / CFE, indicating that the constructed electrode has excellent stability in long-term recording current response. See details... Figure 4 i and Figure 13 The charge properties of the electrode surface were detected by measuring the zeta potential, and the results are as follows: Figure 4 As shown in j, ZIF-8 is positively charged (20±2.19mV); conversely, M is negatively charged (-9.3±1.92mV), and the electrode surface carries more negative charge (-57±16.7mV) after aptamer modification.

[0133] (5) The effect of mechanical deformation on the electrochemical response capability of the electrode

[0134] The effect of mechanically induced deformation on the electrochemical response of the electrode was further evaluated. At 0.15 V (vs. Ag / AgCl), the electrode edge was subjected to three collisions, causing it to bend at approximately 45°. Changes in current were observed in the CV images. The results are as follows: Figure 4 As shown in k~l. Therefore, the stress during implantation will not significantly affect the electrode performance. The excellent sensitivity, stability, and selectivity described above provide strong support for the in vivo application of this sensor.

[0135] Example 5. Feasibility verification of real-time in vivo DA monitoring

[0136] This invention further verifies the biocompatibility, antifouling performance, and feasibility of the electrodes for in vivo experiments. To investigate the biocompatibility of the electrodes, the cytotoxicity of bare carbon cloth (CC), ZIF-8 / CC, M@ZIF-8 / CC, and DNA-cho / M@ZIF-8 / CC was evaluated using a live-dead cell staining method. After co-culturing PC12 cells with these carbon cloth-modified materials in 1640 medium for 12 hours, it was observed that the cell viability on the surface of DNA-cho / M@ZIF-8 / CC was significantly higher than that on ZIF-8 / CC. Figure 5 , Figure 6 a). The cytotoxicity results were further validated by the CCK-8 assay, which detected cell viability after 24 hours of culture on carbon cloth. The results were consistent with the live / dead cell staining assay, indicating that DNA-cho / M@ZIF-8 / CC exhibited superior biocompatibility compared to ZIF-8 / CC. Figure 6 b).

[0137] The hydrophilicity of the electrode surface is crucial for reducing nonspecific protein adsorption. This invention assesses the hydrophilicity of the electrode by measuring the water contact angle before and after modification: from bare CC to ZIF-8 / CC, and then to DNA-cho / M@ZIF-8 / CC, the water contact angle gradually decreases, indicating that biomaterial modification endows the electrode with strong hydrophilicity and resistance to nonspecific protein binding. Figure 6 c). To verify the ability of the electrodes to resist biocontamination and maintain sensing performance in an in vivo detection environment, bovine serum albumin (BSA) was added to artificial cerebrospinal fluid (aCSF) to simulate the biocontamination of microelectrodes in the rat brain, and DNA-cho / M@ZIF-8 / CFE was immersed in PC12 cell culture medium to simulate the biological environment. Figure 7 , Figure 8 a).

[0138] Further comparison of the current response of the DNA-cho / M@ZIF-8 / CFE electrode before and after soaking in waste culture medium for 2 hours: Unlike the bare carbon fiber, the dopamine (DA) detection current response of the DNA-cho / M@ZIF-8 / CFE electrode decreased only slightly after soaking, which may be attributed to the hydrophobic interaction between the DNA-cho / M layer and the nonpolar region of the protein. Figure 7 We also immersed the electrode in a FITC-labeled BSA solution (5 mg / mL) for 4 hours and imaged the electrode surface using a fluorescence microscope. Figure 8 b) As a control, naked CFE was reacted with FITC-BSA under the same conditions. The amount of BSA adsorbed on the surface of DNA-cho / M@ZIF-8 / CFE was significantly lower than that on naked CFE. Figure 8As shown in Figure c, the addition of BSA caused a rapid decrease of approximately 70% in the ampere current response of the bare CFE (blue), while the current response of the DNA-cho / M@ZIF-8 / CFE decreased by only 32% (purple). These experiments collectively demonstrate that the presence of proteins has minimal impact on the DA sensing performance of the DNA-cho / M@ZIF-8 / CFE; the excellent anti-fouling properties of this electrode are further confirmed, indicating its suitability for long-term in vivo monitoring.

[0139] To verify the feasibility of using DNA-cho / M@ZIF-8 / CFE for DA sensing, this invention validated the practicality of the electrode through in vitro experiments. Nifedipine blocks potassium chloride-induced dopamine release by inhibiting calcium ion influx. Figure 8 d). We injected 100 mM KCl into the rat striatum at a distance of 300 μm from the recording electrode, and injected PBS into the striatum as a blank control to eliminate interference from experimental procedures. At the same time, we injected nifedipine as an inhibition test to evaluate the in vivo detection performance of the electrode.

[0140] The results showed that the DA response current released from the striatum of the brain after nifedipine treatment decreased, but did not decay to the initial level, indicating that a small amount of DA was still released; these results confirm that the sensor has good DA response capability and high sensitivity in in vivo physiological environment. Figure 8 In summary, DNA-cho / M@ZIF-8 / CFEs can be used to monitor DA release in vivo, and their excellent performance can support subsequent mechanism exploration and application research.

[0141] Example 6. Effects of diabetic mice on dopamine metabolism

[0142] The experimental results of the foregoing examples demonstrate that the electrochemical sensor developed in this invention possesses high selectivity and high sensitivity, enabling us to track dynamic changes in striatal dopamine (DA). Given that the striatum is one of the earliest regions affected by neurodegenerative changes in the pathological process of diabetes, this invention constructed a streptozotocin (STZ)-induced Sprague-Dawley (SD) rat model of type 1 diabetes (T1D) and monitored changes in striatal DA release during the early stages of diabetes. The results are as follows: Figure 9 a and Figure 14 As shown.

[0143] This invention employs a three-electrode system for real-time monitoring of dopamine (DA) in the rat brain: DNA-cho / M@ZIF-8 / CFME is implanted into the striatum, and a platinum wire electrode and a miniature Ag / AgCl electrode are placed extracranially as the counter electrode and reference electrode, respectively. Figure 15The release kinetics of DA from the striatum of control (Ctrl) and T1D rats was recorded using an amperometric method (+0.15 V vs. Ag / AgCl) after local microinjection of 100 mM KCl at a recording electrode 300 μm. Figure 9 bc). Control group rats showed significant DA release (0.15 ± 0.03 nA); notably, T1D rats showed a three-fold increase in DA release (0.50 ± 0.08 nA, n = 3, p < 0.01 vs. Ctrl). The elevated striatal DA release in early diabetes suggests alterations in striatal DA neurotransmission.

[0144] Western blot analysis of dopaminergic markers in the striatum of STZ-induced diabetic rats ( Figure 9 Immunohistochemistry (DE) showed that D2R expression remained unchanged, but the levels of dopamine transporter (DAT) and Girk2 were significantly reduced, indicating specific alterations in key proteins regulating dopamine neurotransmission. Figure 9 fg、 Figure 16 and Figure 17 Further immunofluorescence experiments confirmed a specific decrease in DAT and Girk2 protein expression in the striatum, consistent with Western blot results. These findings suggest that the dopaminergic system is affected early in diabetes, possibly related to alterations in DA release and reuptake. In summary, we found DA metabolic disorder in early diabetic rats, and this disorder is associated with the D2R-mediated signaling pathway.

[0145] Changes in protein levels in striatal axons led us to hypothesize that diabetes may alter dopamine (DA) neurotransmission in the striatum. Therefore, we combined pharmacological experiments to further explore the potential mechanisms underlying changes in DA release kinetics. Figure 9 i). To investigate the effect of decreased DAT protein expression on DA reuptake, the DAT inhibitor Vanoxerine was injected into the striatum of rats at the same dosage as previously reported. DA release was measured 15 minutes later. Figure 9 The results showed that DA release was significantly increased compared with the control group that did not receive the drug, suggesting that inhibition of DAT function leads to impaired DA reuptake in the synaptic cleft, which in turn causes DA accumulation in the synaptic cleft—a result consistent with the phenotype of downregulated DAT expression in a diabetes model in previous studies.

[0146] The D2R agonist quinpirole and the antagonist raclopride are the most widely used drugs in D2R research. We determined the dosage and duration of drug pretreatment in rats based on previous literature. After 30 minutes of pretreatment with quinpirole via injection into the striatum of rats, dopamine release was induced by potassium chloride stimulation. We found that dopamine release levels decreased after D2R activation, suggesting that D2R receptor activation can inhibit dopamine release. In the central nervous system, D2R is mainly distributed in the presynaptic terminals of dopaminergic neurons; when D2R is activated, it exerts negative feedback regulation on dopamine release through its mediated signal transduction mechanism. We further found that after co-injecting the D2R agonist quinpirole and the D2R inhibitor raclopride into the striatum of rats and pretreating for 30 minutes, dopamine release was restored—this is because raclopride blocks the binding of quinpirole to D2R, thus affecting the inhibitory effect of dopamine release after D2R activation. This further illustrates that the inhibitory effect of dopamine release originates from D2R activation. Figure 9 lm).

[0147] Pretreatment with the Girk2 channel inhibitor ifenprodil for 30 minutes in the rat striatum followed by potassium chloride stimulation to induce dopamine (DA) release showed a significant increase in DA release in the ifenprodil group compared to the control group (p<0.01), confirming that inhibition of the Girk2 channel promotes DA release. However, injection of quinpyrrole activated upstream D2R, leading to changes in the downstream Girk2 channel and consequently reducing DA release. These results indicate that when D2R expression remains constant, decreased Girk2 expression leads to increased striatal stimulation-induced DA release. Figure 9 (no). The above results further explain the mechanism by which decreased DAT and Girk2 expression in early diabetes leads to increased DA release.

[0148] In summary, these results provide new insights into changes in the dopaminergic system in early diabetes. Figure 9 h): Specifically, in the early stages of diabetes, the expression of DAT and Girk2 was significantly reduced. Decreased DAT expression led to reduced DA reuptake and increased DA concentration in the synaptic cleft. DA accumulation in the synaptic cleft activated the D2R receptor-mediated negative feedback inhibition pathway, while decreased Girk2 expression disrupted the Girk2-mediated membrane hyperpolarization effect following D2R activation, preventing effective inhibition of DA release and ultimately leading to increased DA release. This conclusion well explains our experimental results—significantly increased DA release in the striatal synaptic cleft of early-stage diabetic rats.

Claims

1. A DNA-cho / M@ZIF-8 / CFME electrode for detecting dopamine, characterized in that, The electrode includes a carbon fiber electrode, on which ZIF-8 nanoparticles, brain cell membranes, and aptamer DNA-cho are sequentially modified.

2. The electrode according to claim 1, characterized in that, The nucleotide sequence of the aptamer DNA-cho is shown in SEQ ID NO.1, and the 3′-end of the aptamer DNA-cho is modified with cholesterol.

3. A method for preparing a DNA-cho / M@ZIF-8 / CFME electrode, characterized in that, Includes the following steps: i) ZIF-8 nanoparticles are grown in situ on the surface of carbon fiber electrodes to obtain ZIF-8 / CFE; ii) Soak the ZIF-8 / CFE obtained in step i) in brain cell membrane vesicle solution to obtain M@ZIF-8 / CFE; iii) Immerse the M@ZIF-8 / CFE obtained in step ii) in a DNA aptamer solution to obtain a DNA-cho / M@ZIF-8 / CFME electrode.

4. The preparation method according to claim 3, characterized in that, Step i) specifically includes: immersing the carbon fiber electrode in Zn(NO3)2·6H2O solution, sonicating for 10~60 min, adding 2-methylimidazole solution, magnetically stirring for 40~120 min, washing and drying to obtain ZIF-8 / CFE.

5. The preparation method according to claim 3, characterized in that, In step ii), the brain cell membrane vesicle solution is homogenized by ultrasound for 10-20 minutes.

6. The preparation method according to claim 3, characterized in that, In step iii), the concentration of the DNA aptamer solution is 1~50 μM, and the soaking time is 30 min~12 h.

7. A dopamine sensor, characterized in that, The dopamine sensor includes the DNA-cho / M@ZIF-8 / CFME electrode as described in any one of claims 1 to 2.

8. The use of the DNA-cho / M@ZIF-8 / CFME electrode according to any one of claims 1 to 2 and / or the dopamine sensor according to claim 7 in the preparation of products for detecting dopamine content; Alternatively, the DNA-cho / M@ZIF-8 / CFME electrode and / or the dopamine sensor may be used in the preparation of products for in situ real-time dynamic monitoring of live brain dopamine (DA). Alternatively, the DNA-cho / M@ZIF-8 / CFME electrode and / or the dopamine sensor may be used in the preparation of products for detecting changes in DA release in the striatum of the brain during early diabetes.

9. A method for detecting dopamine for non-disease diagnosis or treatment purposes, characterized in that, Detection is performed using the DNA-cho / M@ZIF-8 / CFME electrode as described in any one of claims 1 to 2 or the dopamine sensor as described in claim 7.

10. A method for detecting changes in dopamine (DA) release in the striatum of the brain in early diabetes mellitus for non-disease diagnostic or therapeutic purposes, characterized in that, Using a platinum wire as the counter electrode and Ag / AgCl as the reference electrode, the DNA-cho / M@ZIF-8 / CFME electrode was implanted into the striatum, and KCl was added; dopamine release kinetics were obtained from the it curve.

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