Parkinson's disease blood biomarker detection method and system based on self-driven magnesium-based micron motor

Abstract

The invention discloses a Parkinson's disease blood biomarker detection method based on a self-driven magnesium-based micron motor, and belongs to the technical field of biomedical sensing and detection. The detection method disclosed by the invention comprises the following steps: preparing a self-driven magnesium-based micron motor; then putting the magnesium-based micron motor into the whole blood sample subjected to red blood cell lysis treatment to capture the biomarker to obtain a motor for capturing the biomarker, separating the motor for capturing the biomarker from the whole blood sample by adopting a magnetic attraction method, and then carrying out electrochemical detection to obtain the biomarker. And during electrochemical detection, an enzyme reaction substrate is added to amplify an electrochemical detection signal, so that quantitative determination of the Parkinson's disease blood biomarker is realized. The self-driven magnesium-based micron motor introduced by the method effectively improves the capture efficiency of target molecules, and solves the technical problem that the existing detection method cannot directly detect alpha-syn at high sensitivity in a whole blood environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical sensing and detection technology, and particularly relates to a Parkinson's disease blood biomarker detection method and system based on a self-driven magnesium-based micromotor. BACKGROUND

[0002] Parkinson's disease is a common neurodegenerative disease, and its incidence rate is increasing year by year with the global population aging. The disease mainly manifests as motor function and cognitive dysfunction, which seriously affects the quality of life of patients and brings heavy burden to families and society. Due to the lack of specific early diagnostic methods, clinical diagnosis of Parkinson's disease is usually made by excluding other diseases through imaging examinations such as single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI) or positron emission tomography (PET). However, these methods cannot directly and accurately diagnose Parkinson's disease, resulting in many patients failing to receive timely and targeted treatment at an early stage. Therefore, there is an urgent need to develop a high-sensitivity method for directly detecting Parkinson's disease-related biomarkers to achieve early and accurate diagnosis of the disease.

[0003] α-syn is one of the important blood biomarkers of Parkinson's disease. α-syn is composed of 140 amino acids and is expressed in the brain and peripheral blood of Parkinson's disease patients. Studies have shown that during the onset of Parkinson's disease, α-syn gradually spreads from the central nervous system to the peripheral blood. Therefore, accurate quantification of the content of α-syn in blood has an indicative effect on the early diagnosis of Parkinson's disease. However, due to the complexity of blood composition, which contains a large number of blood cells and proteins, direct detection of α-syn in whole blood faces great challenges. The current commonly used method requires pretreatment of the blood sample, i.e., first separating the plasma from the red blood cells by centrifugation and then detecting the content of α-syn in the plasma. For example, there are reports on the use of a self-assembled monolayer electrode-based label-free electrochemical immunosensor for the detection of α-syn in diluted serum, and a method for measuring α-syn in serum using a magnetically modified electrochemical sensor. These technologies have promoted the development of methods for detecting α-syn in blood to some extent. However, it should be noted that the concentration of α-syn in red blood cells (about 40 μg / mL) is much higher than that in plasma (about 3.6×103-1.8×106 pg / mL). The α-syn contained in red blood cells accounts for the vast majority of the total amount of blood, so detecting only α-syn in serum / plasma may not fully reflect the progression of Parkinson's disease. There is a lack of a method in the prior art that can directly and highly sensitively detect α-syn in the whole blood environment (including plasma and red blood cell components). The existing detection process often requires complex pretreatment such as centrifugal separation, and the overall sensitivity is limited, which makes it difficult to meet the needs of early diagnosis of Parkinson's disease. SUMMARY

[0004] The purpose of this invention is to provide a method and system for detecting blood biomarkers of Parkinson's disease based on a self-driven magnesium-based micromotor, in order to solve the technical problem that existing detection methods cannot directly detect α-syn with high sensitivity in whole blood.

[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for detecting blood biomarkers of Parkinson's disease based on a self-driven magnesium-based micromotor, comprising the following steps: Fabrication of a self-driven magnesium-based micromotor; Subsequently, the magnesium-based micromotor was placed into a whole blood sample that had undergone erythrocyte lysis to capture biomarkers. The motor that captured the biomarkers was then separated from the whole blood sample using a magnetic attraction method. Electrochemical detection was then performed, and the electrochemical detection signal was amplified by adding an enzyme reaction substrate during the electrochemical detection, thereby achieving quantitative determination of blood biomarkers for Parkinson's disease.

[0006] Furthermore, the blood biomarker for Parkinson's disease is α-synuclein; and the enzyme reaction substrate used in the electrochemical detection is glucose solution.

[0007] Furthermore, one side surface of the self-driven magnesium-based micromotor is modified with magnetic Fe3O4 / GOx nanoparticles, an anti-α-synuclein antibody for recognizing blood biomarkers of Parkinson's disease, and bovine serum albumin.

[0008] Furthermore, the fabrication process of the self-driven magnesium-based micromotor includes the following steps: S1: Magnesium spheres are dispersed in ethanol to obtain an ethanol suspension of magnesium spheres; a glass slide coated with a polymethyl methacrylate film is vertically immersed in the ethanol suspension of magnesium spheres, and then the glass slide is removed, so that the magnesium spheres are deposited on the surface of the glass slide coated with the polymethyl methacrylate film. Then the glass slide is placed in anhydrous ethanol / water mixture, APTES is added and the reaction is carried out. After the reaction is completed, the glass slide is removed and glutaraldehyde aqueous solution is dropped on the surface to continue the reaction. After the reaction is completed, magnesium spheres with aldehyde functional groups introduced on the surface are obtained on the glass slide. S2: The enzyme-loaded magnetic nanocomposite Fe3O4 / GOx was dispersed in anhydrous ethanol / water mixture to obtain a nanoparticle dispersion; the nanoparticle dispersion was dropped onto a glass slide with magnesium spheres with aldehyde functional groups introduced on the surface for reaction; after the reaction was completed, the magnesium spheres on the glass slide were released to obtain the asymmetric magnesium-based micromotor initial product. S3: The asymmetric magnesium-based micromotor precursor was dispersed in PBS buffer, followed by incubation with a mixture of EDC and NHS to obtain a reaction system. Then, PBS solution containing anti-α-synuclein antibody and bovine serum albumin, which are used to identify blood biomarkers of Parkinson's disease, was added to the reaction system and incubated again to obtain a self-driven magnesium-based micromotor.

[0009] Further, in S1, the concentration of the ethanol suspension of the magnesium spheres is 4-6 wt%; the polymethyl methacrylate film is obtained by coating a polymethyl methacrylate solution onto the surface of a carrier film and drying it at 60-70 °C for 12-14 h; the polymethyl methacrylate solution is obtained by mixing polymethyl methacrylate with ethyl acetate; the concentration of the polymethyl methacrylate solution is 2.5-3 g / 40 mL; in the anhydrous ethanol / water mixture, the volume ratio of water to anhydrous ethanol is 1:1; the volume ratio of the anhydrous ethanol / water mixture to APTES is 60-65 mL: 100-105 μL; The reaction was carried out at room temperature for 12-14 h. The volume ratio of the glutaraldehyde aqueous solution to APTES was 20 mL:100 μL; the mass concentration of the glutaraldehyde aqueous solution was 2.5-3%; the second reaction was carried out at room temperature for 4-6 h.

[0010] Further, in S2, the concentration of the nanoparticle dispersion is 0.2~0.3 mg / mL; the nanoparticle dispersion is added dropwise at a rate of 1 mL every 30 minutes to a glass slide containing magnesium spheres with aldehyde functional groups introduced on the surface, and the reaction is allowed to proceed at room temperature for 12~14 h. Subsequently, the polymethyl methacrylate film is dissolved and removed with chloroform, and the magnesium spheres on the glass slide are released to obtain the asymmetric magnesium-based micromotor initial product. In S3, the volume ratio of the asymmetric magnesium-based micromotor precursor, PBS buffer, and the mixture of EDC and NHS is 8-10 mL: 1-2 mL: 1-2 mL. The concentrations of EDC and NHS in the EDC and NHS mixture were 20 mg / mL and 15 mg / mL, respectively; the incubation time was 1-2 h; and the re-incubation time was 5-6 h. In the reaction system, the concentration of anti-α-synuclein antibody is 1 μg / mL, and the mass concentration of bovine serum albumin is 0.1%.

[0011] Further, in S2, the preparation method of the enzyme-loaded magnetic nanocomposite Fe3O4 / Gox is as follows: 3-Aminopropyltriethoxysilane was added to an aqueous dispersion of Fe3O4 and reacted under light-protected conditions. After the reaction was completed, the reaction product was collected and then washed with anhydrous ethanol and deionized water in sequence. The product was then redispersed in deionized water for later use to obtain amino-functionalized Fe3O4 nanoparticles. Glucose oxidase was dissolved in deionized water to obtain a Gox solution; N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide were dissolved in deionized water to obtain an EDC / NHS mixture; the Gox solution was added to the EDC / NHS mixture for activation to obtain an activated Gox solution. An aqueous solution of Fe3O4 nanoparticles with amino functional groups on their surface was added to an activated Gox solution for reaction. After the reaction was completed, the reaction product was collected, washed with water, and redispersed in deionized water to obtain the enzyme-loaded magnetic nanocomposite Fe3O4 / Gox.

[0012] Furthermore, the Fe3O4 aqueous dispersion is obtained by mixing Fe3O4 nanoparticles and deionized water; the ratio of Fe3O4 nanoparticles, deionized water and 3-aminopropyltriethoxysilane is 25~30 mg: 5~6 mL: 0.15~0.2 mL; the reaction is carried out under light-protected conditions for 12~13 h. In the Gox solution, the ratio of glucose oxidase to deionized water is 10-12 mg: 1-1.5 mL; in the EDC / NHS mixture, the ratio of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, N-hydroxysuccinimide, and deionized water is 5-6 mg: 5-6 mg: 5-8 mL; the volume ratio of the Gox solution to the EDC / NHS mixture is 100 μL: 6 mL; the activation is performed at room temperature for 30-40 min. The ratio of Fe3O4 nanoparticles with amino functional groups on the surface to deionized water in the aqueous solution is 10~12 mg: 5~6 mL. The ratio of the aqueous solution of Fe3O4 nanoparticles with amino functional groups on the surface to the activated Gox solution is 5~7 mL: 5~7 mL; the reaction time of adding the aqueous solution of Fe3O4 nanoparticles with amino functional groups on the surface to the activated Gox solution is 5~6 h.

[0013] Furthermore, the specific process of the electrochemical detection includes: After the motor that captures the marker is separated from the whole blood sample using a magnetic attraction method, it is transferred and fixed onto the electrochemical sensor, and then electrochemical detection is performed using time-current analysis.

[0014] The present invention also discloses a blood biomarker detection system for Parkinson's disease based on a self-driven magnesium-based micromotor, for implementing the above detection method.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for detecting blood biomarkers of Parkinson's disease based on a self-driven magnesium-based micromotor. When the self-driven magnesium-based micromotor is placed in an aqueous medium, the exposed magnesium reacts with water to produce hydrogen gas. The bubbles continuously released from the exposed side of the magnesium sphere propel the micromotor forward. This self-driven motion significantly increases the chance of contact and collision between the surface antibody of the micromotor and the target protein (α-syn), thereby greatly improving the capture efficiency of the target molecule. This method can directly detect whole blood samples without prior separation of plasma and blood cells, making the operation simple and rapid. It simultaneously acquires all α-syn information in the blood, providing more comprehensive diagnostic significance. Furthermore, the self-driven magnesium-based micromotor introduced in this invention effectively improves the capture efficiency of the target molecule, significantly shortens the capture time, and avoids the cumbersome centrifugation and washing processes through magnetic recovery, solving the technical problem of existing detection methods being unable to directly detect α-syn with high sensitivity in a whole blood environment.

[0016] Furthermore, one side of the self-driven magnesium-based micromotor of the present invention is modified with magnetic Fe3O4 / GOx nanoparticles and an anti-α-synuclein antibody for recognizing blood biomarkers of Parkinson's disease. Bovine serum albumin (BSA) is also modified on the surface of the capture agent (micromotor) to block non-specific adsorption sites and avoid non-specific binding. The asymmetric design of the magnesium spheres causes one side to have the above-mentioned functional components, while the other side is completely exposed. By using the magnesium-based asymmetric micromotor as a capture element and signal amplification element, the capture efficiency of target molecules is improved by the self-driven movement of the micromotor in the liquid, and the electrochemical signal is amplified by the enzyme catalytic reaction, thereby enabling the direct detection of Parkinson's disease biomarkers such as α-synuclein in whole blood samples treated with erythrocyte lysis.

[0017] Furthermore, this invention utilizes a self-driven magnesium-based micromotor for electrochemical time-current electrochemical testing. By integrating an enzyme element (GOx) onto the micromotor to amplify the detection signal, the sensor exhibits extremely high sensitivity and a wide linear detection range. Experiments have demonstrated that the method of this invention achieves a detection limit for α-syn in whole blood (after lysis treatment) at approximately pg / mL, significantly outperforming many methods reported in existing literature.

[0018] Furthermore, the materials used in the detection process of the method of the present invention (such as magnesium, ferrite, and biological enzymes) all have good biocompatibility, and the detection process has high specificity for the recognition of α-syn, making it very suitable for clinical applications. In summary, the novel electrochemical biosensing method provided by the present invention offers a highly sensitive tool for the early diagnosis of neurodegenerative diseases such as Parkinson's disease, and has significant innovative value and clinical application potential. Attached Figure Description

[0019] Figure 1 These are characterization data of functional nanoparticles in the embodiments of the present invention; Among them: A - Transmission electron microscopy (TEM) image of Fe3O4 nanoparticles; B - TEM image of Fe3O4 / GOx nanocomposite; C - UV-Vis-NIR spectrum of Fe3O4 and Fe3O4 / GOx nanocomposite; D - Dynamic light scattering (DLS) particle size distribution of Fe3O4 and Fe3O4 / GOx nanocomposite; E, F: XPS C1s spectrum of Fe3O4 and Fe3O4 / GOx nanocomposite; G - X-ray diffraction (XRD) patterns of different samples; H - Magnetization hysteresis curves of different samples; I - Thermogravimetric analysis (TGA) curves of different samples. Figure 2 This serves as a verification of the morphology and composition of the self-driven magnesium-based micromotor of the present invention. Among them: A- Scanning electron microscope (SEM) images of Mg / Fe3O4 / GOx micromotors; B- Elemental distribution map of Fe on Mg / Fe3O4 / GOx / anti-α-syn / BSA functionalized micromotors; C- Fourier transform infrared (FTIR) spectra of different samples; D- Fluorescence microscope images of Mg / Fe3O4 / GOx / anti-α-syn / BSA micromotors (green represents Fe3O4 / GOx-BSA-FITC fluorescence signal); Figure 3 The motion performance of the self-driven magnesium-based micromotor of the present invention in different media; Wherein: A - Representative motion trajectory of the Mg / Fe3O4 / GOx / anti-α-syn / BSA micromotor in water; B - Motion trajectory in serum; C - Motion trajectory in lysed whole blood samples; D - Comparison of the average motion velocity of the micromotor in the above different media; E - Parabolic fitting curve of the mean square displacement (MSD) of moving particles versus time (y=ax) 2 +bx+c); F - Linear fitting curve of mean square displacement (MSD) of moving particles over time (y=a+bt); Figure 4The results of the electrochemical sensor based on the Mg / Fe3O4 / GOx / anti-α-syn / BSA micromotor of this invention on the detection of α-syn under different media are shown. Wherein: A, B: Current-time (it) curves and calibration curves obtained from PBS; C, D: It curves and calibration curves obtained from serum; E, F: It curves and calibration curves obtained from lysed whole blood. Detailed Implementation

[0020] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0021] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0022] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0023] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0024] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0025] This invention provides a method for detecting Parkinson's disease blood biomarkers based on a self-driven magnesium-based micromotor. The method utilizes a magnesium-based asymmetric micromotor as both a capture element and a signal amplification element. The self-driven motion of the micromotor in the liquid enhances the capture efficiency of target molecules, and the electrochemical signal is amplified by an enzyme-catalyzed reaction. This allows for the direct detection of Parkinson's disease biomarkers such as α-synuclein in whole blood samples treated with erythrocyte lysis. The method includes the following steps: A self-driven magnesium-based micromotor was prepared and added to a whole blood sample that had undergone erythrocyte lysis. The micromotor moved autonomously and captured the target protein. After a period of capture, the target protein was separated and collected from the sample using the magnetic component contained in the micromotor. The collected micromotor product containing the target protein was then transferred and immobilized onto an electrochemical sensor (working electrode). Finally, electrochemical detection was performed, and the target biomarker was quantitatively determined by comparing the changes in current signals before and after the addition of the enzyme substrate.

[0026] The self-driven magnesium-based micromotor of this invention possesses a unique asymmetric structure. Specifically, the self-driven magnesium-based micromotor comprises magnesium microspheres and functional components modified on one side of the magnesium spheres—magnetic Fe3O4 / glucose oxidase (GOx) nanoparticles and specific antibodies for recognizing target biomarkers (such as α-synuclein). In addition, bovine serum albumin (BSA) is modified on the surface of the trapping agent (micromotor) to block non-specific adsorption sites and prevent non-specific binding. The asymmetric design of the magnesium spheres results in one side carrying the aforementioned functional components, while the other side is completely exposed. When the micromotor is placed in an aqueous medium, the exposed magnesium reacts with water to produce hydrogen gas. The bubbles continuously released from the exposed side of the magnesium sphere propel the micromotor forward. This self-driven motion significantly increases the chance of contact and collision between the antibody on the micromotor surface and the target protein (α-syn), thereby greatly improving the capture efficiency of the target molecule. After a period of time, the magnesium spheres are gradually consumed by the reaction, leaving only the magnetic nanocomposite carrying the captured target. Utilizing the Fe3O4 magnetic component contained in the complex, the product capturing α-syn can be rapidly separated and enriched using an external magnetic field. The enriched product containing the target compound is then immobilized on the surface of a magnetic glassy carbon working electrode and allowed to air dry. After drying, electrochemical time-current (it) electrochemical testing is performed: a glucose solution is added to the electrolyte as a substrate for GOx. Glucose reacts with oxygen and water under enzyme catalysis to produce gluconic acid and hydrogen peroxide. The hydrogen peroxide produced significantly improves the electron transfer efficiency at the electrode interface, amplifying the electrochemical current signal. However, due to the poor conductivity of the α-syn protein itself, the more α-syn captured by the micromotor, the more some active sites of GOx are masked, resulting in a corresponding decrease in catalytic amplification efficiency and a weakened amplification effect of the current signal. Therefore, by detecting the change in current signal gain before and after the addition of the substrate, the level of α-syn in the sample can be reflected: the higher the concentration of the target protein, the smaller the current gain generated by enzyme amplification, and the lower the final measured current response. By establishing a standard linear relationship between α-syn concentration and current gain, the concentration of α-syn in unknown samples can be quantitatively analyzed.

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0029] Example 1 A method for detecting blood biomarkers of Parkinson's disease based on a self-driven magnesium-based micromotor, comprising the following steps: Synthesis of Fe3O4 magnetic nanoparticles: 2.7 g FeCl3•6H2O was dissolved in 80 mL ethylene glycol, and 2 g anhydrous sodium acetate and 0.9 g sodium citrate were added under stirring at room temperature. After stirring the mixture for 1 h, it was transferred to a sealed reaction vessel and reacted at 200 ℃ for 20 h, and then naturally cooled to room temperature. The product was washed three times each with distilled water and anhydrous ethanol, and the product was collected by magnetic separation. After vacuum drying, Fe3O4 magnetic nanoparticles were obtained. Amino-functionalized modification of Fe3O4 nanoparticles: 25 mg of the above-prepared Fe3O4 nanoparticles were placed in a centrifuge tube, washed once with deionized water, and then redispersed with 5 mL of deionized water; 0.15 mL of 3-aminopropyltriethoxysilane (APTES) was added to the Fe3O4 aqueous dispersion, and the mixture was allowed to stand in the dark for 12 h; after the reaction was completed, the product was collected with a magnet, washed successively with anhydrous ethanol and deionized water, and the product was redispersed in deionized water for later use, thus obtaining Fe3O4 nanoparticles with amino functional groups on the surface. Enzyme covalently coupled to the surface of aminated Fe3O4 nanoparticles: 10 mg of glucose oxidase (GOx) was weighed and dissolved in 1 mL of deionized water for later use; 5 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were separately dissolved in 5 mL of deionized water; 100 μL of the above GOx solution was added to the EDC / NHS mixture and activated at room temperature for 30 min; 10 mg of aminated Fe3O4 nanoparticles were dispersed in 5 mL of deionized water, and then added to the above activated GOx solution, reacting for 5 h to couple GOx to the Fe3O4 surface via amide bonds; after the reaction was completed, the product was magnetically separated and collected, thoroughly washed with pure water, and then redispersed in deionized water to obtain the enzyme-loaded magnetic nanocomposite Fe3O4 / GOx; Preparation and Functionalization of Magnesium-Based Asymmetric Micromotor: Using magnesium microspheres as the motor body, the asymmetric structure of the micromotor was constructed through the following steps: 2.5 g of polymethyl methacrylate (PMMA) was dissolved in 40 mL of ethyl acetate to prepare a PMMA solution with a mass fraction of approximately 6%; a clean glass slide (after ultrasonic cleaning with ethanol and acetone sequentially) was vertically immersed in the above PMMA solution and then slowly removed, and dried overnight at 60 °C to form a transparent PMMA film coating; 1 g of commercially available magnesium microspheres (micrometer-sized) were added to acetone and ultrasonically cleaned for 30 min, and after centrifugation to remove the acetone, the magnesium microspheres were redispersed to prepare a 4 wt% ethanol suspension; the glass slide coated with the PMMA film was vertically immersed in the magnesium microsphere ethanol suspension and slowly removed to allow the magnesium microspheres to be uniformly deposited on the surface of the PMMA film; subsequently, the glass slide was transferred to a sealed container, approximately 100 μL of chloroform was added along the inner wall of the container, and the container was sealed and allowed to stand at 30 °C for 30 minutes. After min, remove the slide and let it air dry naturally. Then, place the slide in 60 mL of anhydrous ethanol / water mixture with a volume ratio of 1:1, add 100 μL of APTES, and let it stand at room temperature overnight. After the reaction is complete, remove the slide and discard the solution. Then, add 20 mL of 2.5% glutaraldehyde (GA) aqueous solution to the surface of the magnesium microspheres on the slide and react at room temperature for 4 h. After the above treatment, aldehyde functional groups are introduced on the exposed side of the magnesium microspheres. Then, the magnetic nanoparticles were modified: the Fe3O4 / GOx magnetic nanocomposite obtained in Example 1 was dispersed in an anhydrous ethanol / water mixture with a volume ratio of 1:1, at a concentration of approximately 0.2 mg / mL; the nanoparticle dispersion was slowly added to a container containing a glass slide in multiple portions of 1 mL every 30 min, allowing the Fe3O4 / GOx nanoparticles to bind to the aldehyde sites on the surface of the magnesium spheres; after standing at room temperature for 12 h, the glass slide was removed and allowed to air dry naturally; subsequently, the PMMA film was dissolved and removed with chloroform, releasing the magnesium spheres from the film; the asymmetric magnesium-based micromotor initial product (denoted as Mg / Fe3O4 / GOx micromotor) was collected by an external magnetic field. The above-mentioned Mg / Fe3O4 / GOx micromotor was dispersed in PBS buffer, and a mixture of EDC (20 mg / mL) and NHS (15 mg / mL) was added. The mixture was incubated at room temperature for 1 h to activate the surface carboxyl groups. Then, an anti-α-synuclein monoclonal antibody (final concentration 1 μg / mL) and EDC antibody were added to the system. Subsequently, the product was transferred to a PBS solution containing 0.1% bovine serum albumin (BSA) and incubated for 5 h to block the non-specific adsorption sites on the surface of the micromotor. After the above functionalization treatment, a self-driven magnesium-based micromotor (denoted as Mg / Fe3O4 / GOx / anti-α-syn / BSA) was finally obtained. The obtained self-driven magnesium-based micromotor was added to the lysed whole blood sample, and after gentle shaking and mixing, the capture time was started and controlled at 30 min. During this period, the self-driven magnesium-based micromotor could move autonomously in the lysed whole blood sample and capture α-syn target molecules. After the capture process was completed, the container holding the sample was placed in an external magnetic field (e.g., a strong magnet was placed close to the bottom of the container) and left to stand for a few seconds to allow the micromotor product that captured the target molecule to be magnetically enriched at the bottom of the container. The supernatant was carefully removed to obtain a precipitate containing the captured product. The precipitate was resuspended with PBS buffer and gently washed once to remove non-specifically adsorbed impurities. The enriched capture product was then collected again with a magnet, and the washing solution was discarded. The enriched capture product was transferred to a magnetic glassy carbon working electrode for iterative electrochemical detection. Specifically, the capture product was resuspended in a small amount of PBS and drop-coated onto the surface of the magnetic glassy carbon electrode, then air-dried in a fume hood to form a sensing electrode modified with the capture product. A three-electrode system was then assembled (Ag / AgCl electrode as the reference electrode, platinum wire as the counter electrode, and the magnetic glassy carbon electrode modified with the capture product as the working electrode). Iterative electrochemical measurements were performed at a constant potential, recording the change in current over time for a total test time of 100 s. At the start of the reaction (50 s), a glucose solution (as a substrate for glucose oxidase) was rapidly added to the electrolyte solution of the electrode system, and the current signal changes were recorded for the remaining time after substrate addition. By comparing the difference in current response before and after substrate addition, the α-syn content in the sample could be determined. In actual testing, the recorded enzyme catalytic current gain gradually decreased with increasing α-syn concentration. A standard curve was plotted based on the current changes corresponding to different α-syn concentrations, allowing for the quantitative determination of the α-syn concentration in unknown samples.

[0030] Experimental results show that the self-driven magnesium-based micromotor electrochemical sensor constructed in this invention can sensitively detect α-syn in PBS; during testing, the addition of 0.1 mL of glucose solution resulted in a significant current signal response in PBS, exhibiting a good concentration dependence and linear calibration curve (e.g., ...). Figure 4 As shown); detect the current signal gain and concentration of α-syn (10 -3 The concentration of -10 μg / mL showed a good linear relationship, with the linear relationship being: y = 19.41x + 52.61 (R² - 10 μg / mL). 2 The detection limit (pg / mL) is 0.9901, demonstrating extremely high sensitivity and reliability. These results fully validate the feasibility and superior performance of the method described in this invention for detecting blood biomarkers in Parkinson's disease.

[0031] Example 2 An electrochemical detection method for blood biomarkers of Parkinson's disease based on a self-driven magnesium-based micromotor includes the following steps: Synthesis of Fe3O4 magnetic nanoparticles: 2.7 g FeCl3•6H2O was dissolved in 80 mL ethylene glycol, and 2 g anhydrous sodium acetate and 0.9 g sodium citrate were added under stirring at room temperature. After stirring the mixture for 1.5 h, it was transferred to a sealed reaction vessel and reacted at 200 ℃ for 18 h, and then naturally cooled to room temperature. The product was washed three times each with distilled water and anhydrous ethanol, and the product was collected by magnetic separation. After vacuum drying, Fe3O4 magnetic nanoparticles were obtained. Amino-functionalized modification of Fe3O4 nanoparticles: 30 mg of the above-prepared Fe3O4 nanoparticles were placed in a centrifuge tube, washed once with deionized water, and then redispersed with 6 mL of deionized water; 0.2 mL of 3-aminopropyltriethoxysilane (APTES) was added to the Fe3O4 aqueous dispersion, and the mixture was allowed to stand in the dark for 12 h; after the reaction was completed, the product was collected with a magnet, washed successively with anhydrous ethanol and deionized water, and the product was redispersed in deionized water for later use, thus obtaining Fe3O4 nanoparticles with amino functional groups on the surface. Enzyme covalently coupled to the surface of aminated Fe3O4 nanoparticles: 10 mg of glucose oxidase (GOx) was weighed and dissolved in 2 mL of deionized water for later use; 5 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were dissolved in 6 mL of deionized water; 100 μL of the above GOx solution was added to the EDC / NHS mixture and activated at room temperature for 30 minutes; 15 mg of aminated Fe3O4 nanoparticles were dispersed in 6 mL of deionized water and then added to the above activated GOx solution, and the reaction was carried out for 6 h, so that GOx was coupled to the Fe3O4 surface through amide bonds; after the reaction was completed, the product was magnetically separated and collected, washed thoroughly with pure water, and then redispersed in deionized water to obtain the enzyme-loaded magnetic nanocomposite Fe3O4 / GOx; Preparation and Functionalization of Magnesium-Based Asymmetric Micromotor: Using magnesium microspheres as the motor body, the asymmetric structure of the micromotor was constructed through the following steps: 2.5 g of polymethyl methacrylate (PMMA) was dissolved in 40 mL of ethyl acetate to prepare a PMMA solution with a mass fraction of approximately 6%; a clean glass slide (after ultrasonic cleaning with ethanol and acetone sequentially) was vertically immersed in the above PMMA solution and then slowly removed, and dried overnight at 60 °C to form a transparent PMMA film coating; 1 g of commercially available magnesium microspheres (micrometer-sized) were added to acetone and ultrasonically cleaned for 20 min, and after centrifugation to remove the acetone, the magnesium microspheres were redispersed to prepare a 4 wt% ethanol suspension; the glass slide coated with the PMMA film was vertically immersed in the magnesium microsphere ethanol suspension and slowly removed to allow the magnesium microspheres to be uniformly deposited on the PMMA film surface; subsequently, the glass slide was transferred to a sealed container, approximately 110 μL of chloroform was added along the inner wall of the container, and the container was sealed and incubated at 30 °C. After standing at ℃ for 40 minutes, the slide was removed and allowed to air dry naturally. Then, the slide was placed in 50 mL of anhydrous ethanol / water mixture with a volume ratio of 1:1, and 110 μL of APTES was added. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the slide was removed and the solution was discarded. Then, 20 mL of 2.5% glutaraldehyde (GA) aqueous solution was added to the surface of the magnesium microspheres on the slide, and the reaction was carried out at room temperature for 5 h. After the above treatment, aldehyde functional groups were introduced into the exposed side of the magnesium microspheres. Then, the magnetic nanoparticles were modified: the Fe3O4 / GOx magnetic nanocomposite obtained in Example 1 was dispersed in an anhydrous ethanol / water mixture with a volume ratio of 1:1, at a concentration of approximately 0.25 mg / mL; the nanoparticle dispersion was slowly added to a container containing a glass slide in multiple portions at a rate of 1 mL every 25 min, allowing the Fe3O4 / GOx nanoparticles to bind to the aldehyde sites on the surface of the magnesium spheres; after standing at room temperature for 12 h, the glass slide was removed and allowed to air dry naturally; subsequently, the PMMA film was dissolved and removed with chloroform, releasing the magnesium spheres from the film; the asymmetric magnesium-based micromotor initial product (denoted as Mg / Fe3O4 / GOx micromotor) was collected by an external magnetic field. The above-mentioned Mg / Fe3O4 / GOx micromotor was dispersed in PBS buffer, and a mixture of EDC (25 mg / mL) and NHS (20 mg / mL) was added. The mixture was incubated at room temperature for 1 h to activate the surface carboxyl groups. Then, an anti-α-synuclein monoclonal antibody (final concentration 1 μg / mL) and EDC antibody were added to the system. Subsequently, the product was transferred to a PBS solution containing 0.1% bovine serum albumin (BSA) and incubated for 4 h to block the non-specific adsorption sites on the surface of the micromotor. After the above functionalization treatment, a self-driven magnesium-based micromotor (denoted as Mg / Fe3O4 / GOx / anti-α-syn / BSA) was finally obtained. The obtained self-driven magnesium-based micromotor was added to the lysed whole blood sample, and after gentle shaking and mixing, the capture time was started and controlled at 30 min. During this period, the micromotor could move autonomously in the lysed whole blood sample and capture α-syn target molecules. After the capture process was completed, the container holding the sample was placed in an external magnetic field (e.g., a strong magnet was placed close to the bottom of the container) and left to stand for a few seconds to allow the micromotor product that captured the target to be magnetically enriched at the bottom of the container. The supernatant was carefully removed to obtain a precipitate containing the captured product. The precipitate was resuspended with PBS buffer and gently washed once to remove non-specifically adsorbed impurities. The enriched capture product was then collected again with a magnet, and the washing solution was discarded. The enriched capture product was transferred to a magnetic glassy carbon working electrode for iterative electrochemical detection. Specifically, the capture product was resuspended in a small amount of PBS and drop-coated onto the surface of the magnetic glassy carbon electrode, then air-dried in a fume hood to form a sensing electrode modified with the capture product. A three-electrode system was then assembled (Ag / AgCl electrode as the reference electrode, platinum wire as the counter electrode, and the magnetic glassy carbon electrode modified with the capture product as the working electrode). Iterative electrochemical measurements were performed at a constant potential, recording the current change over time for a total test time of 100 s. At the start of the reaction (50 s), a glucose solution (as a substrate for glucose oxidase) was rapidly added to the electrolyte solution of the electrode system, and the current signal changes were recorded for the remaining time after substrate addition. By comparing the difference in current response before and after substrate addition, the α-syn content in the sample could be determined. In actual testing, the recorded enzyme-catalyzed current gain gradually decreased with increasing α-syn concentration. A standard curve was plotted based on the current changes corresponding to different α-syn concentrations, allowing for the quantitative determination of the α-syn concentration in unknown samples.

[0032] The micromotor electrochemical sensor constructed in this invention can sensitively detect α-syn in serum. During testing, the addition of 0.1 mL of glucose solution resulted in a significant current signal response in serum, exhibiting a good concentration dependence and linear calibration curve (e.g., ...). Figure 4 As shown). Among them, the current signal gain and concentration of α-syn in lysed whole blood were detected (10). -3 The concentration of -10 μg / mL showed a good linear relationship, with the linear relationship being: y = 12.64x + 58.04 (R² - 10 μg / mL). 2 The detection limit (pg / mL) is 0.9948, demonstrating extremely high sensitivity and reliability. These results fully validate the feasibility and superior performance of the method described in this invention for detecting blood biomarkers in Parkinson's disease.

[0033] Example 3 An electrochemical detection method for blood biomarkers of Parkinson's disease based on a self-driven magnesium-based micromotor includes the following steps: Synthesis of Fe3O4 magnetic nanoparticles: 2.7 g of Fe3O4•6H2O was dissolved in 80 mL of ethylene glycol. 2 g of anhydrous sodium acetate and 0.9 g of sodium citrate were added under stirring at room temperature. After stirring the mixture for 1.5 h, it was transferred to a sealed reaction vessel and reacted at 200 ℃ for 18 h, then allowed to cool naturally to room temperature. The product was washed three times each with distilled water and anhydrous ethanol, and the product was collected by magnetic separation. After vacuum drying, Fe3O4 magnetic nanoparticles were obtained. Amino-functionalized modification of Fe3O4 nanoparticles: 30 mg of the above-prepared Fe3O4 nanoparticles were placed in a centrifuge tube, washed once with deionized water, and then redispersed with 6 mL of deionized water; 0.2 mL of 3-aminopropyltriethoxysilane (APTES) was added to the Fe3O4 aqueous dispersion, and the mixture was allowed to stand in the dark for 12 h; after the reaction was completed, the product was collected with a magnet, washed successively with anhydrous ethanol and deionized water, and the product was redispersed in deionized water for later use, thus obtaining Fe3O4 nanoparticles with amino functional groups on the surface. Enzyme covalently coupled to the surface of aminated Fe3O4 nanoparticles: 10 mg of glucose oxidase (GOx) was weighed and dissolved in 2 mL of deionized water for later use; 5 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS) were dissolved in 6 mL of deionized water; 100 μL of the above GOx solution was added to the EDC / NHS mixture and activated at room temperature for 30 min; 15 mg of aminated Fe3O4 nanoparticles were dispersed in 6 mL of deionized water and then added to the above activated GOx solution, and reacted for 6 h to couple GOx to the Fe3O4 surface via amide bonds; after the reaction was completed, the product was magnetically separated and collected, thoroughly washed with pure water, and then redispersed in deionized water to obtain the enzyme-loaded magnetic nanocomposite Fe3O4 / GOx; Preparation and Functionalization of Magnesium-Based Asymmetric Micromotor: Using magnesium microspheres as the motor body, the asymmetric structure of the micromotor was constructed through the following steps: 2.5 g of polymethyl methacrylate (PMMA) was dissolved in 40 mL of ethyl acetate to prepare a PMMA solution with a mass fraction of approximately 6%; a clean glass slide (after ultrasonic cleaning with ethanol and acetone sequentially) was vertically immersed in the above PMMA solution and then slowly removed, and dried overnight at 70 °C to form a transparent PMMA film coating; 1 g of commercially available magnesium microspheres (micrometer-sized) were added to acetone and ultrasonically cleaned for 20 min, and after centrifugation to remove the acetone, the magnesium microspheres were redispersed to prepare a 4 wt% ethanol suspension; the glass slide coated with the PMMA film was vertically immersed in the magnesium microsphere ethanol suspension and slowly removed to allow the magnesium microspheres to be uniformly deposited on the PMMA film surface; subsequently, the glass slide was transferred to a sealed container, approximately 110 μL of chloroform was added along the inner wall of the container, and the container was sealed and incubated at 30 °C. After standing at ℃ for 40 minutes, the slide was removed and allowed to air dry naturally. Then, the slide was placed in 50 mL of anhydrous ethanol / water mixture with a volume ratio of 1:1, and 110 μL of APTES was added. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the slide was removed and the solution was discarded. Then, 20 mL of 2.5% glutaraldehyde (GA) aqueous solution was added to the surface of the magnesium microspheres on the slide, and the reaction was carried out at room temperature for 5 h. After the above treatment, aldehyde functional groups were introduced into the exposed side of the magnesium microspheres. Then, the magnetic nanoparticles were modified: the Fe3O4 / GOx magnetic nanocomposite obtained in Example 1 was dispersed in an anhydrous ethanol / water mixture with a volume ratio of 1:1, at a concentration of approximately 0.25 mg / mL; the nanoparticle dispersion was slowly added to a container containing a glass slide in multiple portions at a rate of 1 mL every 25 min, allowing the Fe3O4 / GOx nanoparticles to bind to the aldehyde sites on the surface of the magnesium spheres; after standing at room temperature for 12 h, the glass slide was removed and allowed to air dry naturally; subsequently, the PMMA film was dissolved and removed with chloroform, releasing the magnesium spheres from the film; the asymmetric magnesium-based micromotor initial product (denoted as Mg / Fe3O4 / GOx micromotor) was collected by an external magnetic field. The above-mentioned Mg / Fe3O4 / GOx micromotor was dispersed in PBS buffer, and a mixture of EDC (25 mg / mL) and NHS (20 mg / mL) was added. The mixture was incubated at room temperature for 1 h to activate the surface carboxyl groups. Then, an anti-α-synuclein monoclonal antibody (final concentration 1 μg / mL) and EDC antibody were added to the system. Subsequently, the product was transferred to a PBS solution containing 0.1% bovine serum albumin (BSA) and incubated for 6 h to block the non-specific adsorption sites on the surface of the micromotor. After the above functionalization treatment, a self-driven magnesium-based micromotor (denoted as Mg / Fe3O4 / GOx / anti-α-syn / BSA) was finally obtained. The obtained self-driven magnesium-based micromotor was added to the lysed whole blood sample, and after gentle shaking and mixing, the capture time was started and controlled at 30 min. During this period, the micromotor could move autonomously in the lysed whole blood sample and capture α-syn target molecules. After the capture process was completed, the container holding the sample was placed in an external magnetic field (e.g., a strong magnet was placed close to the bottom of the container) and left to stand for a few seconds to allow the micromotor product that captured the target to be magnetically enriched at the bottom of the container. The supernatant was carefully removed to obtain a precipitate containing the captured product. The precipitate was resuspended with PBS buffer and gently washed once to remove non-specifically adsorbed impurities. The enriched capture product was then collected again with a magnet, and the washing solution was discarded. The enriched capture product was transferred to a magnetic glassy carbon working electrode for iterative electrochemical detection. Specifically, the capture product was resuspended in a small amount of PBS and drop-coated onto the surface of the magnetic glassy carbon electrode, then naturally dried in a fume hood to form a sensing electrode modified with the capture product. A three-electrode system was then assembled (Ag / AgCl electrode as the reference electrode, platinum wire as the counter electrode, and the magnetic glassy carbon electrode modified with the capture product as the working electrode). Iterative electrochemical measurements were performed at a constant potential, recording the change in current over time for a total test time of 100 s. At the start of the reaction (50 s), a glucose solution (as a substrate for glucose oxidase) was rapidly added to the electrolyte solution of the electrode system, and the current signal changes were recorded for the remaining time after substrate addition. By comparing the difference in current response before and after substrate addition, the α-syn content in the sample could be determined. In actual testing, the recorded enzyme catalytic current gain gradually decreased with increasing α-syn concentration. A standard curve was plotted based on the current changes corresponding to different α-syn concentrations, allowing for the quantitative determination of the α-syn concentration in unknown samples.

[0034] Experimental results show that the micromotor electrochemical sensor constructed in this invention can sensitively detect α-syn in whole blood samples; during testing, the addition of 0.1 mL of glucose solution resulted in a significant current signal response in the lysed whole blood matrix, exhibiting a good concentration (10) -3 -10 μg / mL) dependence and linear calibration curve (e.g.) Figure 4 As shown); among them, the current signal gain for detecting α-syn in lysed whole blood showed a good linear relationship with concentration, the linear relationship being: y = 11.01x + 55.68 (R0). 2 The detection limit was as low as 224 pg / mL (0.9980), demonstrating extremely high sensitivity and reliability. These results fully validate the feasibility and superior performance of the method described in this invention for the detection of blood biomarkers for Parkinson's disease.

[0035] Experimental results show that the micromotor electrochemical sensor constructed in this invention can sensitively detect α-syn in whole blood samples. During testing, the addition of 0.1 mL of glucose solution resulted in a significant current signal response in the lysed whole blood matrix, exhibiting a good concentration (10⁻⁶). -3 -10 μg / mL) dependence and linear calibration curve (e.g.) Figure 4 (As shown). Among them, the current signal gain for detecting α-syn in lysed whole blood showed a good linear relationship with concentration, the linear relationship being: y = 11.01x + 55.68 (R²). 2The detection limit was as low as 224 pg / mL (0.9980), demonstrating extremely high sensitivity and reliability. These results fully validate the feasibility and superior performance of the method described in this invention for the detection of blood biomarkers for Parkinson's disease.

[0036] Figure 1 The preparation process and material characterization of Fe3O4 / GOx are shown. (A) TEM images of Fe3O4 and (B) Fe3O4 / GOx. Fe3O4 consists of uniformly dispersed and regularly shaped spherical nanoparticles. After modifying Fe3O4 with GOx, the size of the nanoparticles slightly increases. (C) UV-Vis-NIR spectra of Fe3O4 and Fe3O4 / GOx. The modified particles show a significant enhancement of the UV absorption peak near 450 nm, indicating the presence of GOx. (D) Hydrated particle size of Fe3O4 and Fe3O4 / GOx (a: Fe3O4, b: Fe3O4 / GOx). DLS results show that the hydrated particle size of Fe3O4 is 264.5 nm, consistent with the TEM results. After GOx modification, the hydrated particle size of the nanoparticles increased to 291.5 nm. This may be because GOx, as a protein, can fully expand in aqueous solution, so the hydrated particle size is larger than that obtained by TEM. (E) C 1s XPS spectra of Fe3O4 and (F) Fe3O4 / GOx. In the C 1s spectrum of Fe3O4, the peak at 284.76 eV indicates the presence of C-C bonds in Fe3O4. In the C 1s spectrum of Fe3O4 / GOx, the peaks at 284.52, 285.78, and 287.78 eV correspond to different carbon environments, including C-C bonds, C=N bonds, and C=O / CO bonds, respectively; (G) X-ray diffraction patterns of Fe3O4 and Fe3O4 / GOx show that the crystal structure of Fe3O4 remains unchanged after GOx modification; (H) hysteresis loops of Fe3O4 and Fe3O4 / GOx show that the magnetic strength of the nanoparticles is weakened after GOx modification, but Fe3O4 / GOx still has good magnetism and can be quickly collected by a magnet; (I) Thermogravimetric curves of Fe3O4 and Fe3O4 / GOx show that the GOx content in Fe3O4 / GOx is about 2.9%.

[0037] Figure 2Characterization of magnesium-based micromotors. (A) SEM image of Mg / Fe3O4 / GOx / anti-α-syn / BSA. One side of the magnesium sphere is relatively smooth, while the other side is covered with a large number of nanoparticles. (B) SEM-mapping spectrum of Fe on Mg / Fe3O4 / GOx / anti-α-syn / BSA. After asymmetric modification, the surface of the magnesium sphere shows a clear distribution of Fe, with the Fe concentrated on one side of the Mg sphere. (C) FTIR spectra of different samples during motor fabrication. After sequentially modifying Fe3O4 / GOx, anti-α-syn, and BSA on Mg-CHO, the FTIR spectra at 1600–1700 cm⁻¹... -1 The absorption within the range is obvious and can be attributed to the amide I band of the protein; (D) Fluorescence image of Mg / Fe3O4 / GOx / anti-α-syn / BSA (Fe3O4 / GOx is labeled with FITC, green fluorescence), the fluorescently labeled Fe3O4 / GOx nanoparticles are concentrated on one side of the Mg spheres, further indicating that Mg / Fe3O4 / GOx / anti-α-syn / BSA is an asymmetric structure.

[0038] Figure 3 To characterize the motion performance of the motor. The motion trajectories of Mg / Fe3O4 / GOx / anti-α-syn / BSA in (A) H2O, (B) serum, and (C) whole blood after lysis; (D) the average velocity of Mg / Fe3O4 / GOx / anti-α-syn / BSA in different media and its corresponding (E) parabolic fit (y = ax). 2 The linear fitting of (y = a + bx + c) and (F) indicates that the micromotors still maintain strong mobility in the blood, which may increase their likelihood of capturing α-syn in complex blood environments.

[0039] Figure 4 Chorometric and fitted curves of the micromotor Mg / Fe3O4 / GOx / anti-α-syn / BSA for electrochemical detection of α-syn in three media: PBS (A, B), serum (C, D), and lysed whole blood (E, F). With increasing α-syn concentration, the current signal amplification of the complex obtained in all three media was gradually suppressed. Furthermore, in all three media, the change in current signal intensity showed a good linear relationship with the concentration of α-syn with the addition of the GOx substrate glucose.

[0040] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor, characterized in that, Includes the following steps: Fabrication of a self-driven magnesium-based micromotor; Subsequently, the magnesium-based micromotor was placed into a whole blood sample that had undergone erythrocyte lysis to capture biomarkers. The motor that captured the biomarkers was then separated from the whole blood sample using a magnetic attraction method. Electrochemical detection was then performed, and the electrochemical detection signal was amplified by adding an enzyme reaction substrate during the electrochemical detection, thereby achieving quantitative determination of blood biomarkers for Parkinson's disease.

2. The method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 1, characterized in that, The blood biomarker for Parkinson's disease is α-synuclein; the enzyme reaction substrate used in the electrochemical detection is glucose solution.

3. The method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 1, characterized in that, One side surface of the self-driven magnesium-based micromotor is modified with magnetic Fe3O4 / GOx nanoparticles, an anti-α-synuclein antibody for recognizing blood biomarkers of Parkinson's disease, and bovine serum albumin.

4. The method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 3, characterized in that, The fabrication process of the self-driven magnesium-based micromotor includes the following steps: S1: Magnesium spheres are dispersed in ethanol to obtain an ethanol suspension of magnesium spheres; a glass slide coated with a polymethyl methacrylate film is vertically immersed in the ethanol suspension of magnesium spheres, and then the glass slide is removed, so that the magnesium spheres are deposited on the surface of the glass slide coated with the polymethyl methacrylate film. Then the glass slide is placed in anhydrous ethanol / water mixture, APTES is added and the reaction is carried out. After the reaction is completed, the glass slide is removed and glutaraldehyde aqueous solution is dropped on the surface to continue the reaction. After the reaction is completed, magnesium spheres with aldehyde functional groups introduced on the surface are obtained on the glass slide. S2: The enzyme-loaded magnetic nanocomposite Fe3O4 / GOx was dispersed in anhydrous ethanol / water mixture to obtain a nanoparticle dispersion; the nanoparticle dispersion was dropped onto a glass slide with magnesium spheres with aldehyde functional groups introduced on the surface for reaction; after the reaction was completed, the magnesium spheres on the glass slide were released to obtain the asymmetric magnesium-based micromotor initial product. S3: The asymmetric magnesium-based micromotor precursor was dispersed in PBS buffer, followed by incubation with a mixture of EDC and NHS to obtain a reaction system. Then, PBS solution containing anti-α-synuclein antibody and bovine serum albumin, which are used to identify blood biomarkers of Parkinson's disease, was added to the reaction system and incubated again to obtain a self-driven magnesium-based micromotor.

5. A method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 4, characterized in that, In S1, the concentration of the ethanol suspension of the magnesium spheres is 4-6 wt%; the polymethyl methacrylate film is obtained by coating the surface of the carrier film with a polymethyl methacrylate solution and drying it at 60-70 °C for 12-14 h; the polymethyl methacrylate solution is obtained by mixing polymethyl methacrylate with ethyl acetate; the concentration of the polymethyl methacrylate solution is 2.5-3 g / 40 mL; in the anhydrous ethanol / water mixture, the volume ratio of water to anhydrous ethanol is 1:1; the volume ratio of the anhydrous ethanol / water mixture to APTES is 60-65 mL: 100-105 μL; The reaction was carried out at room temperature for 12-14 h. The volume ratio of the glutaraldehyde aqueous solution to APTES was 20 mL:100 μL; the mass concentration of the glutaraldehyde aqueous solution was 2.5-3%; the second reaction was carried out at room temperature for 4-6 h.

6. A method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 4, characterized in that, In S2, the concentration of the nanoparticle dispersion is 0.2~0.3 mg / mL; the nanoparticle dispersion is added dropwise at a rate of 1 mL every 30 minutes to a glass slide containing magnesium spheres with aldehyde functional groups introduced on the surface, and the reaction is allowed to proceed at room temperature for 12~14 h. Subsequently, the polymethyl methacrylate film is dissolved and removed with chloroform, and the magnesium spheres on the glass slide are released to obtain the asymmetric magnesium-based micromotor initial product. In S3, the volume ratio of the asymmetric magnesium-based micromotor precursor, PBS buffer, and the mixture of EDC and NHS is 8-10 mL: 1-2 mL: 1-2 mL. The concentrations of EDC and NHS in the EDC and NHS mixture were 20 mg / mL and 15 mg / mL, respectively; the incubation time was 1-2 h; and the re-incubation time was 5-6 h. In the reaction system, the concentration of anti-α-synuclein antibody is 1 μg / mL, and the mass concentration of bovine serum albumin is 0.1%.

7. The method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 4, characterized in that, In S2, the preparation method of the enzyme-loaded magnetic nanocomposite Fe3O4 / Gox is as follows: 3-Aminopropyltriethoxysilane was added to an aqueous dispersion of Fe3O4 and reacted under light-protected conditions. After the reaction was completed, the reaction product was collected and then washed with anhydrous ethanol and deionized water in sequence. The product was then redispersed in deionized water for later use to obtain amino-functionalized Fe3O4 nanoparticles. Glucose oxidase was dissolved in deionized water to obtain a Gox solution; N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide were dissolved in deionized water to obtain an EDC / NHS mixture; the Gox solution was added to the EDC / NHS mixture for activation to obtain an activated Gox solution. An aqueous solution of Fe3O4 nanoparticles with amino functional groups on their surface was added to an activated Gox solution for reaction. After the reaction was completed, the reaction product was collected, washed with water, and redispersed in deionized water to obtain the enzyme-loaded magnetic nanocomposite Fe3O4 / Gox.

8. A method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 7, characterized in that, The Fe3O4 aqueous dispersion was obtained by mixing Fe3O4 nanoparticles and deionized water; the ratio of Fe3O4 nanoparticles, deionized water and 3-aminopropyltriethoxysilane was 25~30 mg: 5~6 mL: 0.15~0.2 mL; the reaction was carried out under light-protected conditions for 12~13 h. In the Gox solution, the ratio of glucose oxidase to deionized water is 10-12 mg: 1-1.5 mL; in the EDC / NHS mixture, the ratio of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, N-hydroxysuccinimide, and deionized water is 5-6 mg: 5-6 mg: 5-8 mL; the volume ratio of the Gox solution to the EDC / NHS mixture is 100 μL: 6 mL; the activation is performed at room temperature for 30-40 min. The ratio of Fe3O4 nanoparticles with amino functional groups on the surface to deionized water in the aqueous solution is 10~12 mg: 5~6 mL. The ratio of the aqueous solution of Fe3O4 nanoparticles with amino functional groups on the surface to the activated Gox solution is 5~7 mL: 5~7 mL; the reaction time of adding the aqueous solution of Fe3O4 nanoparticles with amino functional groups on the surface to the activated Gox solution is 5~6 h.

9. The method for detecting blood biomarkers for Parkinson's disease based on a self-driven magnesium-based micromotor according to claim 1, characterized in that, The specific process of the electrochemical detection includes: After the motor that captures the marker is separated from the whole blood sample using a magnetic attraction method, it is transferred and fixed onto the electrochemical sensor, and then electrochemical detection is performed using time-current analysis.

10. A blood biomarker detection system for Parkinson's disease based on a self-driven magnesium-based micromotor, characterized in that, Used to implement the detection method according to any one of claims 1 to 9.

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