Electrochemical biosensor for alpha-synuclein detection

Abstract

The invention provides an electrochemical biosensor used for alpha-synuclein detection. The sensor comprises a magnetic particle capture probe coated by a monoclonal antibody, a SiO2 nanoparticle signal probe for detecting antibody modification and a nano porous membrane loaded glassy carbon electrode modified by graphene. During detection, alpha-synuclein is specifically combined with the monoclonal antibody-coated magnetic particle capture probe and the detection antibody-modified cadmium-doped SiO2 nanoparticle signal probe to form a compound with a sandwich structure, the compound is directly precipitated in an external magnetic field, uncombined substances are removed through cleaning, and the detection sensitivity is high. And then re-dispersing the precipitate (compound) in the electrolyte, and immersing the glassy carbon electrode loaded by the graphene-modified nano porous membrane. After electrification, the cadmium ions in the compound are subjected to redox reaction to generate a current signal, and the current intensity of the current signal is in direct proportion to the content of alpha-synuclein. The sensor has the advantages of simplicity in operation, rapidness in detection, high sensitivity, high specificity and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical biosensors, and particularly relates to an electrochemical biosensor for detecting α-synuclein. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease of the nervous system. Its prevalence rate has been increasing year by year and it is the second most common neurodegenerative disorder after Alzheimer's disease. Its typical clinical symptoms mainly include motor symptoms such as resting tremor, bradykinesia, muscle rigidity, and postural instability, as well as non-motor symptoms such as autonomic dysfunction, sleep disorders, and mood disorders. Currently, the number of Parkinson's disease patients in China has exceeded 3 million, and there are approximately 4.5 million Parkinson's disease patients globally. By 2050, the number of Parkinson's disease patients globally will reach 25.2 million.

[0003] α-synuclein (α-Syn) is a soluble protein widely expressed in the central nervous system. It consists of 140 amino acids with a molecular weight of approximately 14 kDa and is located on human chromosome 4. Under pathological conditions, α-synuclein is prone to misfolding and forms insoluble fibrous aggregates, which are called Lewy bodies and are the hallmark pathological features of neurodegenerative diseases such as Parkinson's disease (PD) and multiple system atrophy (MSA). Since the abnormal aggregation of α-synuclein is an early pathological marker of diseases such as Parkinson's disease, its detection may contribute to the early diagnosis of the disease. Therefore, it is of great significance to develop an electrochemical biosensor to achieve rapid and accurate monitoring of α-synuclein.

[0004] Currently, traditional detection methods mainly include techniques such as enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and fluorescence detection. However, these methods have obvious disadvantages such as cumbersome operation, low automation level, and long time consumption, which are not conducive to popularization in hospitals. As a cutting-edge biological detection technology, electrochemical biosensors have significant clinical application potential and broad development prospects in the field of protein immunoassay based on the advantages of high efficiency, sensitivity, time saving, small error, and low cost. By using the unique recognition and binding function of antibodies to corresponding antigens, a detection device composed of an antibody or antigen and an electrode can achieve efficient and specific determination of target biomarkers. Summary of the Invention

[0005] One of the objectives of the present invention is to provide an electrochemical biosensor for detecting α-synuclein. The electrochemical biosensor is easy to operate and sensitive in response, and is suitable for in vitro clinical diagnosis. It can provide key technical support for the effective diagnosis and dynamic monitoring of neurological diseases, realize the accurate detection of disease monitoring and drug treatment, provide an important basis for clinical decision-making, and also greatly promote the innovation and development of electrochemical detection technology in the field of α-synuclein detection.

[0006] The present invention provides an electrochemical biosensor for detecting α-synuclein. The sensor includes a magnetic particle capture probe coated with monoclonal antibody, a SiO 2 nanoparticle signal probe modified with detection antibody, and a glassy carbon electrode loaded with a graphene-modified nanoporous membrane; the monoclonal antibody coated on the magnetic particle capture probe component of the sensor is 5G6, and the sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region of the monoclonal antibody are: SEQ ID NO.3: KGQYWN, SEQ ID NO.4: YLSEDGSHNNNPTLKN, SEQ ID NO.5: AEVF; the sequences of the three complementarity-determining regions CDR4, CDR5, and CDR6 in the light chain variable region are SEQ ID NO.6: NSSQSLLESDGKSYIQ, SEQ ID NO.7: LVTKIDS, SEQ ID NO.8: YQASQFPNT.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO.1: ELQLQESGPGIVKPSQSISLTCSVSGYSITKGQYWNWIRQFPGQKLDWMGYLSEDGSHNNNPTLKNHISLTRETSHNQFFIKINSVTAEESATYYCVRAEVFYGQATIVTVSA; the amino acid sequence of the light chain variable region is SEQ ID NO.2: DVVMTQTPLTISVTLGQPGSLTCNSSQSLLESDGKSYIQWILQRPAQSPKHIIWLVTKIDSGVPDRFTASGSATDFTIDISRLEAEDIAIYYCYQASQFPNTFAGASKIEIK.

[0008] Furthermore, the method for coating the magnetic particle capture probe with the monoclonal antibody is: (1) Mix 50-100 µL of 10 mg / mL Fe 3 O 4Magnetic particles and 50 - 100 µg of monoclonal antibody are added to 1 mL of 0.1 - 0.3 M MES (pH 5.5) buffer; (2) While vortexing and mixing the magnetic particles, 100 - 200 µL of 10 - 15 mg / mL EDC solution is added; (3) Vortex and mix for 1 min, disperse by ultrasonic bath for 30 s, rotate and incubate on a shaking mixer for 4 h - 6 h, magnetically separate, and add 1 mL of 0.2 M glycine solution; (4) Incubate at room temperature for 1 h - 3 h, magnetically separate, add 1 mL of 1% - 5% BSA, and incubate overnight; (5) Magnetically separate, add 1 mL of TBS-T buffer and wash twice, magnetically separate, aspirate the liquid, add 1 mL of PBS buffer to prepare a monoclonal antibody-coated magnetic particle capture probe.

[0009] Further, the method for the detection antibody-modified SiO 2 nanoparticle signal probe is as follows: (1) Add 50 - 100 µL of 100 nm - 500 nm silica nanoparticles to ethanol and 3-aminopropyltriethoxysilane, stir at room temperature for 8 h - 12 h, then centrifuge and wash to obtain amino-functionalized silica nanoparticles (SiO 2 -NH 2 ); (2) Add the amino-functionalized silica nanoparticles to a cadmium nitrate solution with a concentration of 5 - 10 mg / mL, react at room temperature for 4 h - 8 h, centrifuge, collect the complex, and wash with PBS buffer (pH 7.2 - 7.4) to obtain Cd 2+ / SiO 2 -NH 2 nanoparticle complex; (3) While vortexing and mixing, add 100 - 200 µg of the detection antibody to 100 - 200 µL of 10 - 15 mg / mL EDC solution, after reacting at room temperature for 1 h - 4 h, add 1 - 5 mg / mL NHS and Cd 2+ / SiO 2 -NH 2 nanoparticle complex, incubate overnight at room temperature, centrifuge, and wash three times with PBS buffer; (4) Discard the supernatant, add 2 mL of 1% - 5% BSA to block unreacted sites, wash with PBS buffer to prepare a detection antibody@Cd 2 + / SiO 2 nanoparticle signal probe.

[0010] Furthermore, the method for loading the graphene-modified nanoporous membrane on the glassy carbon electrode is as follows: (1) Add 0.3 - 0.6 g of graphene oxide (GO) to ultrapure water, sonicate for 1 - 2 h, immerse the porous membrane with a nanopore diameter of 10 nm - 100 µm into the dispersion liquid, add ascorbic acid with a concentration of 10 - 20 mg / mL, and react with shaking at room temperature for 2 - 4 h; (2) Take out the functionally modified porous membrane, wash it with ultrapure water multiple times, and dry it in an oven for 6 - 8 h; (3) Load the functionally modified nanoporous membrane on the surface of the glassy carbon electrode as the working electrode.

[0011] The technical principle of the present invention: The electrochemical biosensor of the present invention includes a monoclonal antibody-coated magnetic particle capture probe, a detection antibody-modified SiO 2 nanoparticle signal probe, and a graphene-modified nanoporous membrane-loaded glassy carbon electrode. During detection, α-synuclein in the sample to be detected specifically binds to the monoclonal antibody-coated magnetic particle capture probe and the detection antibody-modified cadmium-doped SiO 2 nanoparticle signal probe respectively, forming a "sandwich"-structured complex, which directly precipitates in an external magnetic field. Unbound substances are removed by washing, and then the precipitate (complex) is redispersed in the electrolyte and added to the electrolytic cell. Subsequently, the glassy carbon electrode loaded with the graphene-modified nanoporous membrane is immersed in the above electrolyte. Cadmium ions in the complex will undergo redox reactions after being energized, thus increasing the current signal. An electrochemical workstation is used to record the change in the electrical signal. The current intensity is proportional to the content of α-synuclein in the sample to be detected, thereby realizing the detection of α-synuclein in the sample to be detected.

[0012] Compared with the prior art, the beneficial effects of the present invention are at least as follows:

[0013] The graphene-modified nanoporous membrane-loaded glassy carbon electrode effectively enhances the electrochemical response of the electrode, increases the specific surface area of the electrode, enables more target molecules to come into contact with the electrode surface, and thus improves the detection sensitivity.

[0014] By specifically recognizing the antigen to be detected through the capture probe and the signal probe, based on the ultra-high sensitivity of the electrochemical biosensor, the detection of low-concentration target biomarkers can be realized, which is helpful for early diagnosis.

[0015] It is achieved through the synergistic effect of magnetic particles and nanoparticles. Without enzyme assistance, rapid separation and enrichment are realized by an external magnetic field, reducing background interference, avoiding complex washing and separation steps, enabling rapid and efficient detection, promoting portable applications, and meeting the requirements of clinical diagnosis.

[0016] This sensor has a simple design, is easy to operate, does not require professionals and complex equipment, and is suitable for wide application in primary medical institutions. Brief Description of the Drawings

[0017] Figure 1 It is a detection principle diagram of an electrochemical biosensor for detecting α-synuclein.

[0018] Figure 2 It is an electron micrograph of a porous membrane.

[0019] Figure 3 It is the optimization of (A) incubation time and (B) pH during the construction of the electrochemical biosensor.

[0020] Figure 4 It is a standard curve of the current change in response to different concentrations.

[0021] Figure 5 It is a diagram for stability study.

[0022] Figure 6 It is a diagram for specificity. Detailed Embodiments

[0023] The following will describe the implementation embodiments of the present invention in detail in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0024] According to one aspect of the present invention, the present invention provides an electrochemical biosensor for detecting α-synuclein, which includes a magnetic particle capture probe coated with a monoclonal antibody, a SiO 2 nanoparticle signal probe modified with a detection antibody, and a glassy carbon electrode loaded with a graphene-modified nanoporous membrane; the monoclonal antibody coated on the magnetic particle capture probe component of the sensor is 5G6, and the sequences of the three complementarity-determining regions CDR1, CDR2, and CDR3 in the heavy chain variable region of this monoclonal antibody are: SEQ ID NO.3: KGQYWN, SEQ ID NO.4: YLSEDGSHNNNPTLKN, SEQ ID NO.5: AEVF; the sequences of the three complementarity-determining regions CDR4, CDR5, and CDR6 in the light chain variable region are SEQ ID NO.6: NSSQSLLESDGKSYIQ, SEQ ID NO.7: LVTKIDS, SEQ ID NO.8: YQASQFPNT.

[0025] The monoclonal antibody used in the present invention is a self-developed α-synuclein-specific antibody with excellent specificity and sensitivity, and is very suitable for the detection of α-synuclein.

[0026] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO.1: ELQLQESGPGIVKPSQSISLTCSVSGYSITKGQYWNWIRQFPGQKLDWMGYLSEDGSHNNNPTLKNHISLTRETSHNQFFIKINSVTAEESATYYCVRAEVFYGQATIVTVSA; the amino acid sequence of the light chain variable region is SEQ ID NO.2: DVVMTQTPLTISVTLGQPGSLTCNSSQSLLESDGKSYIQWILQRPAQSPKHIIWLVTKIDSGVPDRFTASGSATDFTIDISRLEAEDIAIYYCYQASQFPNTFAGASKIEIK.

[0027] In a preferred embodiment of the present invention, the method for the monoclonal antibody to coat the magnetic particle capture probe is as follows: (1) Add 50 - 100 μL of 10 mg / mL magnetic particles with a particle size of 0.1 - 1 μm of Fe 3 O 4 and 50 - 100 μg of the monoclonal antibody into 1 mL of 0.1 - 0.3 M MES (pH 5.5) buffer; (2) While vortexing and mixing the magnetic particles, add 100 - 200 μL of 10 - 15 mg / mL EDC solution; (3) Vortex and mix for 1 min, disperse by ultrasonic bath for 30 s, rotate and incubate on a shaking mixer for 4 h - 6 h, perform magnetic separation, and add 1 mL of 0.2 M glycine solution; (4) Incubate at room temperature for 1 h - 3 h, perform magnetic separation, add 1 mL of 1% - 5% BSA, and incubate overnight; (5) Perform magnetic separation, add 1 mL of TBS-T buffer to wash twice, perform magnetic separation, aspirate the liquid, and add 1 mL of PBS buffer to prepare the magnetic particle capture probe coated with the monoclonal antibody.

[0028] In a typical but non-limiting embodiment of the present invention, the dosage of Fe 3 O 4 magnetic particles is, for example, 50, 60, 70, 80, 90 or 100 μL; Fe 3 O 4The particle size of the magnetic particles is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 µm; the dosage of the monoclonal antibody is 50, 60, 70, 80, 90 or 100 µg; the dosage of the EDC solution is 100, 120, 140, 160, 180 or 200 µL; the concentration of the EDC solution is 10, 11, 12, 13, 14 or 15 mg / mL; in step (3), the incubation time is 4, 5 or 6 h; in step (4), the incubation time is 1, 2 or 3 h; the concentration of BSA is 1%, 2% or 3%.

[0029] The detection antibody used in the present invention is a commercially available product, which has high specificity and high sensitivity and is suitable for the detection of α-synuclein.

[0030] In a preferred embodiment of the present invention, the method for detecting the antibody-modified SiO 2 nanoparticle signal probe is as follows: (1) Add 50-100 µL of 100 nm-500 nm silica nanoparticles to ethanol and 3-aminopropyltriethoxysilane, stir at room temperature for 8 h-12 h, and then centrifuge and wash to obtain amino-functionalized silica nanoparticles (SiO 2 -NH 2 ); (2) Add the amino-functionalized silica nanoparticles to a cadmium nitrate solution with a concentration of 5-10 mg / mL, react at room temperature for 4 h-8 h, centrifuge, collect the complex, and wash with PBS buffer (pH 7.2-7.4) to obtain Cd 2+ / SiO 2 -NH 2 nanoparticle complex; (3) While vortexing and mixing, add 100-200 µg of the detection antibody to 100-200 µL of 10-15 mg / mL EDC solution, react at room temperature for 1 h-4 h, then add 1-5 mg / mL NHS and Cd 2+ / SiO 2 -NH 2 nanoparticle complex, incubate overnight at room temperature, centrifuge, and wash three times with PBS buffer; (4) Discard the supernatant, add 2 mL of 1%-5% BSA to block the unreacted sites, wash with PBS buffer, and prepare the detection antibody@Cd 2+ / SiO 2 nanoparticle signal probe.

[0031] In a typical but non-limiting embodiment of the present invention, the dosage of the silica nanoparticles is, for example, 50, 60, 70, 80, 90 or 100 µL; the particle size of the silica nanoparticles is, for example, 100, 200, 300, 400 or 500 nm; the dosage of the detection antibody is, for example, 100, 120, 140, 160, 180 or 200 µg; the dosage of the EDC solution is, for example, 100, 120, 140, 160, 180 or 200 µL; the concentration of the EDC solution is, for example, 10, 11, 12, 13, 14 or 15 mg / mL; in step (3), the incubation time is, for example, 1, 2, 3 or 4 h; in step (4), the concentration of BSA is, for example, 1%, 2% or 3%.

[0032] In a preferred embodiment of the present invention, the method for loading a graphene-modified nanoporous membrane on a glassy carbon electrode is as follows: (1) Add 0.3 - 0.6 g of graphene oxide (GO) to ultrapure water, sonicate for 1 - 2 h, immerse a porous membrane with a nanochannel pore diameter of 10 nm - 100 µm into the dispersion, add ascorbic acid with a concentration of 10 - 20 mg / mL, and react with shaking at room temperature for 2 - 4 h; (2) Take out the functionalized porous membrane, wash it with ultrapure water multiple times, and dry it in an oven for 6 - 8 h; (3) Load the functionalized nanoporous membrane on the surface of the glassy carbon electrode as the working electrode.

[0033] In a typical but non-limiting embodiment of the present invention, the dosage of graphene oxide is, for example, 0.3, 0.4, 0.5 or 0.6 g, the pore diameter of the nanoporous membrane is, for example, 100 nm, 1 µm, 20 µm, 50 µm, 100 µm; the concentration of ascorbic acid is, for example, 10, 20 mg / mL. Example 1

[0034] A preparation method of an electrochemical biosensor for detecting α-synuclein, the specific implementation manner is as Figure 1 、 Figure 2 shown, including the following steps:

[0035] (1) Add 0.3 g of graphene oxide (GO) to ultrapure water, sonicate for 1 h, immerse the porous membrane into the dispersion, add ascorbic acid with a concentration of 10 mg / mL, and react with shaking at room temperature for 2 h; then take out the functionalized porous membrane, wash it with ultrapure water, and dry it in an oven for 6 h; subsequently, load the functionalized nanoporous membrane on the surface of the glassy carbon electrode as the working electrode.

[0036] (2) Subsequently, add 50 µL of α-synuclein with different concentrations to 50 µL of a capture probe solution with a concentration of 2 mg / mL, incubate at room temperature for 40 min, magnetically separate and collect the capture probe, redisperse it with 1 mL of PBS buffer, and then add 50 µL of a detection antibody-Cd with a concentration of 2 mg / mL2+ / SiO 2 The nanoparticle signal probe was incubated at room temperature for 40 min ( Figure 3 A), and the unbound substances were removed by washing to obtain the specifically bound α-Syn antibody@silica@cadmium ion@α-Syn@α-Syn antibody@magnetic microsphere precipitate (biocomplex). It was redispersed with 2 mL of acetic acid-sodium acetate buffer electrolyte with a concentration of 250 ng / mL Bi 3+ and added to the electrolytic cell ( Figure 3 B). Example 2

[0037] The application of an electrochemical biosensor for detecting α-synuclein as described above includes the following steps:

[0038] The glassy carbon electrode loaded with the graphene-modified nanoporous membrane in Example 1 was immersed in Bi 3+ acetic acid-sodium acetate buffer electrolyte, enriched at a voltage of -1.2 V for 180 s, then left standing for 30 s. Square wave voltammetry (SWV) was used to detect the biocomplex on the electrode, and the current response was recorded by an electrochemical workstation to quantify the concentration of the biomarker, and a standard curve of the concentration and current change was plotted ( Figure 4 ).

[0039] Under the same conditions, the stability of the electrochemical biosensor was tested on the 0th, 3rd, 6th, 9th, and 12th days. The detected electrical signals showed no significant changes ( Figure 5 ), and its relative standard deviation was about 1.05%. The results indicate that the electrochemical biosensor has good stability. Example 3

[0040] To study the specificity of the detection method for α-synuclein, standard solutions of neurofilament light chain protein (NfL), phosphorylated tau protein 217 (p-tau217), Tau protein, and glial fibrillary acidic protein (GFAP) were used as controls, and experiments were carried out according to the detection method of Example 2. The results are as Figure 6 shown. Compared with all other biomarkers, the target analyte was significantly higher than other interfering substances. The experimental results show that the present detection method has high specificity for α-synuclein and can effectively distinguish α-synuclein from other non-related biomarkers, providing a solid foundation for the accurate diagnosis of diseases.

[0041] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention, rather than to restrict it. Although the present invention has been described in detail in combination with specific embodiments, those of ordinary skill in the art can, according to the actual situation, modify the technical solutions of the present invention or adopt equivalent replacement methods without exceeding the essence and scope of the technical solutions of the present invention.

Claims

1. An electrochemical biosensor for detecting α-synuclein, characterized in that: The sensor includes a monoclonal antibody-coated magnetic particle capture probe, a detection antibody-modified SiO2 nanoparticle signal probe, and a graphene-modified nanoporous membrane-loaded glassy carbon electrode; the monoclonal antibody coated with the magnetic particle capture probe component of the sensor is 5G6, and the sequences of the three complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the monoclonal antibody are respectively: SEQ ID NO.3: KGQYWN, SEQ ID NO.4: YLSEDGSHNNNPTLKN, SEQ ID NO.5: AEVF; the sequences of the three complementary determining regions CDR4, CDR5, and CDR6 of the light chain variable region are respectively SEQ ID NO.6: NSSQSLLESDGKSYIQ, SEQ ID NO.7: LVTKIDS, SEQ ID NO.8: YQASQFPNT.

2. An electrochemical biosensor for α-synuclein detection according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is SEQ ID NO.1: ELQLQESGPGIVKPSQSISLTCSVSGYSITKGQYWNWIRQFPGQKLDWMGYLSEDGSHNNNPTLKNHISLTRETSHNQFFIKINSVTAEESATYYCVRAEVFYGQATIVTVSA; the amino acid sequence of the light chain variable region is SEQ ID NO.2: DVVMTQTPLTISVTLGQPGSLTCNSSQSLLESDGKSYIQWILQRPAQSPKHIIWLVTKIDSGVPDRFTASGSATDFTIDISRLEAEDIAIYYCYQASQFPNTFAGASKIEIK.

3. An electrochemical biosensor for α-synuclein detection according to claim 1, characterized in that: The method for preparing the monoclonal antibody-coated magnetic microparticle capture probe is as follows: (1) adding 50-100 µL of 10 mg / mL Fe3O4 magnetic microparticles with a particle size of 0.1-1 µm and 50-100 µg of the monoclonal antibody described in claim 1 to 1 mL of 0.1-0.3 M MES (pH 5.5) buffer; (2) adding 100-200 µL of 10-15 mg / mL EDC solution while vortexing the magnetic microparticles; (3) vortexing for 1 min, ultrasonically dispersing in a water bath for 30 s, rotating and incubating on a shaking mixer for 4-6 h, magnetically separating, and adding 1 mL of 0.2 M glycine solution; (4) incubating at room temperature for 1 h to 3 h, magnetically separating, adding 1 mL of 1%-5% BSA, and incubating overnight; (5) magnetically separating, adding 1 mL of TBS-T buffer to wash twice, magnetically separating, absorbing the liquid, and adding 1 mL of PBS buffer to prepare the monoclonal antibody-coated magnetic microparticle capture probe.

4. An electrochemical biosensor for α-synuclein detection according to claim 1, characterized in that: The method for detecting the antibody-modified SiO2 nanoparticle signal probe is as follows: (1) adding an appropriate amount of 100-500 nm silica nanoparticles to ethanol and 3-aminopropyltriethoxysilane, stirring at room temperature for 8 h to 12 h, and then centrifuging and washing to obtain amino-modified silica nanoparticles (SiO2-NH2); (2) adding the amino-modified silica nanoparticles to a 5-10 mg / mL cadmium nitrate solution, reacting at room temperature for 4 h to 8 h, centrifuging, collecting the complex, and washing with PBS buffer (pH 7.2-7.4) to obtain Cd 2+ / SiO2-NH2 nanoparticle complex; (3) 100-200 µg of detection antibody was added to 100-200 µL of 10-15 mg / mL EDC solution while vortexing, and reacted at room temperature for 1 h to 4 h, and then 1-5 mg / mL NHS and Cd were added. 2 + / SiO2-NH2 nanoparticle complex, incubate at room temperature overnight, centrifuge, and wash three times with PBS buffer; (4) discard the supernatant, add 2mL 1%-5% BSA to block unreacted sites, wash with PBS buffer, and prepare detection antibody@Cd 2+ / SiO2 nanoparticle signal probe.

5. An electrochemical biosensor for α-synuclein detection according to claim 1, characterized in that: The method for loading a glassy carbon electrode with a graphene-modified nanoporous membrane is as follows: (1) adding 0.3-0.6 g of graphene oxide (GO) into ultrapure water, ultrasonicating for 1-2 h, immersing a porous membrane with a nanochannel pore size of 10 nm-100 µm into the dispersion, adding ascorbic acid with a concentration of 10-20 mg / mL, and shaking the reaction at room temperature for 2-4 h; (2) taking out the functionally modified porous membrane, washing it with ultrapure water for multiple times, and drying it in a drying oven for 6-8 h; (3) loading the functionally modified nanoporous membrane on the surface of a glassy carbon electrode as a working electrode.

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