An electrochemical sensor for detecting a marker of alzheimer's disease and a method of preparing the same

Abstract

The application discloses an electrochemical sensor for detecting Alzheimer's disease markers and a preparation method thereof. The electrochemical sensor comprises a substrate, a working electrode, a counter electrode, a reference electrode, an insulating layer, a metal dendrite, an antibody linker and an antibody. The working electrode, the counter electrode and the reference electrode are made on the substrate, the insulating layer is arranged on the counter electrode and the reference electrode, and the metal dendrite, the antibody linker and the antibody are sequentially coated on the working electrode. The metal dendrite is a gold dendrite or a silver dendrite. The antibody linker is used for connecting and fixing the antibody. The antibody is used for specifically recognizing target antigens in a sample to be measured. The target antigens are Aβ-40 monomers, Aβ-40 oligomers, Aβ-42 monomers or Aβ-42 oligomers. The specific surface area of the working electrode is expanded by electrochemical deposition of the metal dendrite, more attachment sites are provided for the antibody, the sensitivity is higher, the detection precision is higher, a portable early Alzheimer's disease detection system is prepared, the portable early Alzheimer's disease detection system is convenient to carry and has lower detection cost.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor detection technology, specifically relating to an electrochemical sensor for detecting Alzheimer's disease biomarkers (Aβ-40 monomer, Aβ-42 monomer, Aβ-40 oligomer and Aβ-42 oligomer) and its preparation method. Background Technology

[0002] Alzheimer's disease (AD) is an irreversible neurodegenerative disease that threatens the health of tens of millions of elderly people worldwide, and this number is rising every year. Currently, there is no cure for Alzheimer's disease, but the risk can be reduced through early prevention, making early detection of Alzheimer's disease particularly important.

[0003] Recent studies have shown that the levels of amyloid β1-40 (Aβ40), amyloid β1-42 (Aβ42), phosphorylated tau (p-tau) protein, and total tau (t-tau) protein change during the occurrence and progression of Alzheimer's disease (AD). β-Amyloid is a biomarker for early diagnosis of AD. Therefore, there is an urgent need for highly sensitive detection devices to detect the levels of human biomarkers Aβ-40 monomers, Aβ-42 monomers, Aβ-40 oligomers, and Aβ-42 oligomers to enable timely detection and prevention of Alzheimer's disease. The following are existing sensors for detecting β-amyloid:

[0004] Patent application CN202010050552.9 discloses a method for preparing a novel composite material for detecting β-amyloid protein. The method involves mixing a g-C3N4 suspension and a Hemin solution, adding ammonia and hydrazine hydrate under high-speed stirring, and then rapidly stirring the mixture before placing it in a 60°C water bath for 4 hours. The mixture is then placed in a 4°C refrigerator overnight, centrifuged, washed, and redispersed in deionized water to obtain a g-C3N4-Heme suspension. This novel composite material for detecting amyloid peptides can be used to construct a biosensor for detecting β-amyloid protein. This sensor leverages the binding interaction between heme and amyloid peptides to detect amyloid behavior. The technology exhibits good specificity for amyloid protein, enabling rapid and sensitive early detection.

[0005] Patent application CN202310833365.1 discloses an electrode for detecting the content of amyloid protein, a biomarker of Alzheimer's disease, as well as its preparation method, usage method, and detection device. The electrode comprises: a screen-printed electrode, including a working electrode, a reference electrode, and a counter electrode; a biorecognition layer disposed on the surface of the working electrode, comprising a conductive agent, an organic electrochemical probe, and a first antibody; and a second mixed solution containing a metal nanomaterial marker modified with a second antibody, used to modify the surface of the working electrode after the addition of the test sample. The first and second antibodies respectively include Aβ40 antibody or Aβ42 antibody. The electrode, through antigen-antibody recognition, allows the organic electrochemical probe and the metal nanomaterial marker to undergo electrochemical oxidation on the electrode surface, generating a detectable current signal. The current ratio is positively correlated with the antigen concentration, thereby achieving ratiometric high-sensitivity detection of the Alzheimer's disease biomarker β-amyloid protein. While it can detect amyloid protein content, the preparation method is complex. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide an electrochemical sensor for detecting Alzheimer's disease biomarkers and a method for preparing the same, wherein the biomarker is at least one of Aβ-40 monomer, Aβ-42 monomer, Aβ-40 oligomer, and Aβ-42 oligomer.

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

[0008] An electrochemical sensor for detecting biomarkers of Alzheimer's disease includes a substrate, a working electrode, a counter electrode, a reference electrode, an insulating layer, a metal dendrite, an antibody conjugate, and an antibody. The working electrode, counter electrode, and reference electrode are fabricated on the substrate, with the insulating layer placed on top of the counter electrode and reference electrode. The working electrode is sequentially coated with a metal dendrite, an antibody conjugate, and an antibody. The metal dendrite is either a gold dendrite or a silver dendrite. The antibody conjugate is used to connect and immobilize the antibody. The antibody is used to specifically recognize a target antigen in the test sample. The target antigen is at least one of Aβ-40 monomer, Aβ-40 oligomer, Aβ-42 monomer, and Aβ-42 oligomer.

[0009] Specifically, the antibody is an Aβ-40 or Aβ-42 specific antibody.

[0010] Specifically, the antibody conjugate is polypyrrole-3-carboxylic acid, polypyrrole-2-carboxylic acid, or polythiopheneacetic acid.

[0011] Specifically, the substrate is PET, PDMS, PI or SEBS, the working electrode and counter electrode are gold, silver, carbon, carbon nanotube or graphene electrodes, and the reference electrode is silver and silver chloride electrode.

[0012] Preferably, the substrate is a PET substrate, the working electrode and the counter electrode are gold electrodes, the reference electrode is a silver and silver chloride electrode, and the antibody conjugate is polypyrrole-3-carboxylic acid.

[0013] Furthermore, the electrochemical sensor for detecting Alzheimer's disease biomarkers involved in this invention also includes a chitosan hydrogel layer coated on the antibody. The chitosan hydrogel layer is composed of a mixture of chitosan, glycerol, and acetic acid and has micropores on its surface that can filter macromolecular substances.

[0014] The present invention relates to a method for preparing an electrochemical sensor for detecting biomarkers of Alzheimer's disease, specifically comprising the following steps:

[0015] (1) An electrode layer is fabricated on a substrate. The electrode layer includes a working electrode, a counter electrode, and a reference electrode. An insulating layer is fabricated on the electrode layer. The insulating layer covers the areas of the working electrode, the counter electrode, and the reference electrode except for the reaction area and the charging area, exposing the reaction area and the charging area of ​​the working electrode, the counter electrode, and the reference electrode.

[0016] (2) Three-dimensional metal dendrites are fabricated on the surface of the working electrode by electrochemical deposition. The three-dimensional metal dendrites include three-dimensional silver dendrites and three-dimensional gold dendrites.

[0017] (3) A solution containing potassium ferrocyanide, potassium ferrocyanide and antibody linker is dropped onto the surface of the dendrite. Electrochemical polymerization is carried out by cyclic voltammetry to deposit the antibody linker onto the surface of the metal dendrite. After rinsing with deionized water and air drying, the antibody linker is used to link antibodies.

[0018] (4) Add the antibody to the antibody conjugate, incubate at room temperature, slowly rinse the residual antibody solution with PBS solution, add the bovine serum albumin solution to the working electrode, and store at -4°C for later use. The antibody is used to specifically recognize Aβ-40 monomer, Aβ-40 oligomer, Aβ-42 monomer or Aβ-42 oligomer. The optional materials are Aβ-40 and Aβ-42 specific antibodies.

[0019] Specifically, in step (1), firstly, a first paste is printed on the corresponding positions of the working electrode and the counter electrode on the substrate, and then dried to obtain the working electrode and the counter electrode. The first paste can be selected from materials such as gold paste, silver paste, carbon paste, carbon nanotubes or graphene. Then, a second paste is printed on the reference electrode position on the substrate, and then dried to obtain the reference electrode. The second paste is a mixture of silver and silver chloride. Finally, an insulating paste is covered on the electrode layer to form an insulating layer. The insulating layer covers the outside of the working electrode and the counter electrode, as well as half of the working electrode close to the counter electrode and the reference electrode. Then, it is dried and then cleaned with 75% ethanol solution and deionized water.

[0020] Specifically, in step (2), the working electrode 2 prepared in step (1) is immersed in an aqueous solution of Na2SO4 and AuCl3, and a three-dimensional gold dendrite is prepared on the working electrode using a time-amperometric method. Then, the surface residue is removed by rinsing with deionized water, and a three-dimensional gold dendrite is obtained on the surface of the working electrode. Preferably, the deposition potential is -0.65V and the deposition time is 100s. Alternatively, the working electrode prepared in step (1) is immersed in an aqueous solution of Na2SO4 and AgNO3, and a three-dimensional silver dendrite is prepared on the working electrode using a time-amperometric method. Then, the surface residue is removed by rinsing with deionized water, and a three-dimensional silver dendrite is obtained on the surface of the working electrode.

[0021] A portable early detection system for Alzheimer's disease includes a microcontroller, an electrochemical sensor chip for early detection of Alzheimer's disease, a Bluetooth module, and a power supply module. The electrochemical sensor chip, Bluetooth module, and power supply module are all connected to the microcontroller. The electrochemical sensor chip monitors the electrical signal of the target and sends it to the microcontroller. The microcontroller analyzes and processes the electrical signal data and sends the analysis results to the Bluetooth module. The results are then sent to a mobile phone or other terminal device for display via the Bluetooth module. The power supply module provides power to the entire portable early detection system for Alzheimer's disease.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) By electrochemically depositing metal dendrites to expand the specific surface area of ​​the working electrode, more attachment sites are provided for the antibody, resulting in higher sensitivity, higher detection accuracy, and a detection limit of 4.5 × 10⁻⁶. -8 pg / mL; (2) Chitosan hydrogel is used to remove macromolecular substances in the sample to prevent non-specific adsorption and improve detection accuracy; (3) Polypyrrole-3-carboxylic acid, polypyrrole-2 or thiophene acetic acid are used as the linker between the antibody and the electrode, and the electrode has stronger conductivity; (4) It is used to detect the content of Aβ-42 and Aβ-40 in serum and saliva to achieve non-invasive detection of Alzheimer's disease; (5) Portable sensors are made, which are convenient to carry and have lower detection costs. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of the electrochemical sensor for detecting biomarkers of Alzheimer's disease, which is involved in this invention.

[0024] Figure 2 This is a process flow diagram of the preparation method of the electrochemical sensor for detecting Alzheimer's disease biomarkers, which is involved in this invention.

[0025] Figure 3 The image shows the peak current at different potentials detected by the cyclic voltammetry method in Example 1.

[0026] Figure 4This is a SEM image of the three-dimensional dendrite obtained at a voltage of -0.65V in Example 1.

[0027] Figure 5 The image shows the time (it) curves for different electrochemical deposition times in Example 1.

[0028] Figure 6 Impedance spectroscopy of human serum samples with different concentrations of Aβ-42 antigen solution added.

[0029] Figure 7 Impedance gradient diagrams of samples in artificial saliva with different concentrations of Aβ-42 antigen solution added. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments and accompanying drawings. However, these should not be construed as limiting the scope of protection of the present invention.

[0031] like Figure 1 As shown, the electrochemical sensor for detecting Alzheimer's disease biomarkers according to the present invention includes a substrate 1, a working electrode 2, a counter electrode 3, a reference electrode 4, an insulating layer 5, a metal dendrite 6, an antibody linker 7, and an antibody. The working electrode 2, the counter electrode 3, and the reference electrode 4 are fabricated on the substrate 1, and the insulating layer 5 is placed on the counter electrode 3 and the reference electrode 4. The metal dendrite 6, the antibody linker 7, and the antibody are sequentially coated on the working electrode 2. The metal dendrite 6 is a gold dendrite or a silver dendrite. The antibody linker 7 is used to connect and immobilize the antibody. The antibody is used to specifically recognize the target antigen in the test sample. The target antigen is at least one of Aβ-40 monomer, Aβ-40 oligomer, Aβ-42 monomer, and Aβ-42 oligomer.

[0032] Specifically, the antibody is an Aβ-40 or Aβ-42 specific antibody.

[0033] Specifically, the antibody linker 7 is a polypyrrole-3-carboxylic acid, polypyrrole-2-carboxylic acid, or polythiopheneacetic acid.

[0034] Furthermore, the electrochemical sensor for detecting Alzheimer's disease biomarkers involved in this invention also includes a chitosan hydrogel layer coated on the antibody. The chitosan hydrogel layer is composed of a mixture of chitosan, glycerol, and acetic acid and has micropores on its surface that can filter macromolecular substances.

[0035] like Figure 2 As shown in the figure, this embodiment relates to a method for preparing an electrochemical sensor for detecting Alzheimer's disease biomarkers, which specifically includes the following steps:

[0036] (1) An electrode layer is fabricated on a clean substrate 1. The electrode layer includes a working electrode 2, a counter electrode 3 and a reference electrode 4.

[0037] like Figure 2 As shown in Figure a, firstly, the first paste is printed on the corresponding positions of the working electrode and the counter electrode on substrate 1, and then dried to obtain working electrode 2 and counter electrode 3, as follows. Figure 2 As shown in b, the first slurry can be made of gold paste, silver paste, carbon paste, carbon nanotubes or graphene, etc., and the substrate 1 can be made of materials including but not limited to PET, PDMS, PI or SEBS.

[0038] like Figure 2 As shown in c, a second paste is then printed on the reference electrode position on the substrate, followed by drying to obtain reference electrode 4. The second paste is a mixture of silver and silver chloride.

[0039] like Figure 2 As shown in diagram d, an insulating slurry is finally applied to the electrode layer to form an insulating layer 5. The insulating layer 5 covers the exterior of the working electrode 2 and the counter electrode 3, as well as half of the working electrode 2 closest to the counter electrode 3 and the reference electrode 4. It is then dried and cleaned with a 75% ethanol solution and deionized water. The insulating layer 5 covers a portion of the working electrode 2, the counter electrode 3, and the reference electrode 4, exposing the reaction and contact areas of these electrodes. The distance between any two of the working electrode 2, the counter electrode 3, and the reference electrode 4 is equal.

[0040] (2) such as Figure 2 As shown in e, three-dimensional metal dendrites are fabricated on the surface of working electrode 2 by electrochemical deposition. The three-dimensional metal dendrites include three-dimensional silver dendrites and three-dimensional gold dendrites.

[0041] Specifically, after the working electrode 2 is made in step (1), it is immersed in an aqueous solution of Na2SO4 and AuCl3. Three-dimensional gold dendrites (AuD) are prepared on the working electrode 2 by chronoamperometry (CA). Then, the surface residue is removed by rinsing with deionized water. Three-dimensional gold dendrites are obtained on the surface of the working electrode and stored at -4℃ for later use.

[0042] Alternatively, after the working electrode 2 is prepared in step (1), it is immersed in an aqueous solution of Na2SO4 and AgNO3, and three-dimensional silver dendrites (AuD) are prepared on the working electrode 2 by chronoamperometry (CA). Then, the surface residue is removed by rinsing with deionized water, and three-dimensional silver dendrites are obtained on the surface of the working electrode. The electrode is stored at -4℃ for later use.

[0043] (3) such as Figure 2As shown in f, a solution containing potassium ferrocyanide, potassium ferrocyanide, and antibody linker is dropped onto the surface of the dendrite. Electrochemical polymerization is performed by cyclic voltammetry (CV) to deposit antibody linker 7 onto the surface of the metal dendrite. After rinsing with deionized water and air drying, it is ready for use. The antibody linker includes, but is not limited to, polypyrrole 3-carboxylic acid, polypyrrole 2-carboxylic acid, and polythiopheneacetic acid.

[0044] Antibody linkers are used to connect antibodies and can also improve the conductivity of sensors.

[0045] (4) such as Figure 2 As shown in g, the antibody is dropped onto the antibody conjugate and incubated at room temperature. The residual antibody solution is slowly washed with PBS solution. The bovine serum albumin solution is dropped onto the working electrode and stored at -4°C for later use. The antibody is used to specifically recognize Aβ-40 monomers, Aβ-40 oligomers, Aβ-42 monomers, or Aβ-42 oligomers. The optional materials are Aβ-40 and Aβ-42 specific antibodies.

[0046] Example 1

[0047] like Figure 2 As shown in the figure, the preparation method of an electrochemical sensor for early detection of Alzheimer's disease involved in this embodiment specifically includes the following steps:

[0048] (1) such as Figure 2 As shown in Figure a, firstly, the first paste is printed onto the substrate according to the pattern, and then dried at 160°C to obtain the working electrode 2 and the counter electrode 3, as follows. Figure 2 As shown in b, the first paste is gold paste, and the substrate 1 is a PET substrate;

[0049] like Figure 2 As shown in c, the second paste is then printed on the substrate according to the pattern, and then dried at 130°C to obtain the reference electrode 4. The second paste is a mixture of silver and silver chloride.

[0050] like Figure 2 As shown in d, the insulating slurry is finally applied to the electrode layer, covering the outside of the working electrode 2 and the counter electrode 3, as well as half of the working electrode 2. Then, it is dried at 100°C to obtain the insulating layer 4, which is then cleaned with 75% ethanol solution and deionized water.

[0051] (2) such as Figure 2 As shown in e, the working electrode prepared in step (1) is immersed in an aqueous solution of Na2SO4 and AuCl3 for electrochemical deposition. Three-dimensional gold dendrites (AuD) are prepared on the working electrode 2 using the chronoamperometric method (CA). Then, the surface residue is removed by rinsing with deionized water and stored at -4℃ for later use.

[0052] Specifically, the working electrode prepared in step (1) is immersed in an aqueous solution of Na2SO4 and AuCl3, and electrochemical deposition is performed at different voltages of -0.2V, -0.35V, -0.5V, -0.65V and -0.8V to prepare three-dimensional gold dendrites (AuD) on the working electrode 2.

[0053] Figure 3 To detect the peak current of an electrochemical sensor deposited with three-dimensional gold dendrites at different potentials using cyclic voltammetry, the results showed that the peak current reached its maximum at -0.65 V, and then decreased after -0.65 V. The increased impedance of the sensor deposited at -0.8 V was attributed to dendrite agglomeration caused by the excessively high potential. Further investigation revealed that under the above experimental conditions, dendrite agglomeration occurred on the sensor surface when the deposition potential exceeded -0.65 V, leading to a decrease in the sensor's conductivity. Therefore, -0.65 V was ultimately selected as the optimal deposition voltage for the gold dendrites. Figure 4 The image shows a three-dimensional gold dendrite obtained at -0.65V. It can be seen that the metal dendrite deposition method is used to expand the specific surface area of ​​the working electrode, provide more adsorption sites, and make the sensor more sensitive. In addition to the gold dendrite in this embodiment, silver dendrite can also be prepared.

[0054] Furthermore, the substrate with the electrode layer fabricated was immersed in an aqueous solution of Na2SO4 and AuCl3, and electrochemical deposition was performed at a voltage of -0.65V. Observation and impedance testing were conducted every 100s.

[0055] Figure 5 The impedance spectra for different electrochemical deposition times show that the height of gold dendrites increases with time, while the sensor impedance decreases. However, after 200 s of deposition, the low-frequency region of the impedance spectrum becomes disordered. Based on these observations, the optimal deposition time was ultimately selected as 100 s. Within this timeframe, gold dendrite growth is stable and performs well in impedance testing.

[0056] (3) such as Figure 1 As shown in f, a solution containing potassium ferrocyanide, potassium ferrocyanide and polypyrrole 3-carboxylic acid was dropped onto the dendrite surface. Polypyrrole 3-carboxylic acid was deposited on the dendrite surface by cyclic voltammetry (CV). After rinsing with deionized water, the dendrite was air-dried for later use.

[0057] (4) such as Figure 1As shown in g, the antibody is dropped onto the antibody conjugate and incubated at room temperature. The residual antibody solution is slowly washed with PBS solution. After dropping bovine serum albumin solution onto the electrode surface, it is stored at -4°C for later use. The antibody can be used to specifically recognize Aβ-40 monomers and Aβ40 oligomers, Aβ-42 monomers and Aβ-42 oligomers. The optional materials are Aβ-40 and Aβ-42 specific antibodies.

[0058] (5) such as Figure 1 As shown in h, different concentrations of Aβ-42 antigen solution were added to human serum samples to prepare 4.5 × 10⁻⁶ solutions. -5 pg / ml, 4.5×10 -4 pg / ml, 4.5×10 -3 pg / ml, 4.5×10 -2 pg / ml and 4.5×10 -1 A pg / ml Aβ-42 serum solution was incubated on a sensor, and the impedance change was detected. Figure 6 The results show that different concentrations of Aβ-42 peptide can still be clearly distinguished in serum. The linear relationship between serum sample impedance and antigen concentration can be expressed as ∆R = a + b × lgC, where a = 3735.38 ± 93.13, b = 740.36 ± 27.28, and the coefficient of determination R0 is 1. 2 =0.994.

[0059] Similarly, different concentrations of Aβ-42 antigen solutions were added to human saliva, and after incubation on a sensor, impedance changes were detected. Figure 7 This is an impedance diagram of a saliva sample.

[0060] The electrochemical active surface area and electron transfer rate constant of the fabricated electrode were analyzed using cyclic voltammetry (CV). The electrochemical active surface area of ​​the electrode was determined by the Randles-Sevcik equation at 20 mM K3[Fe(CN)6]. 3- / 4- CV determination in solution. Where, i p This represents the peak current, n is the number of electrons transferred, and A is the electroactive area of ​​the electrode (mm²). 2 D is [Fe(CN)6] 3− The diffusion coefficient, v is the scan rate set by the cyclic voltammetry method (vs). -1 C is the concentration (mol) mL -1 R is the gas constant, T is the absolute temperature, and F is the Faraday constant.

[0061] i P = 0.4463 nFAC(nFvD / RT) 1 / 2 (1)

[0062] From (1), we can deduce that the diameter A of the bare electrode is 551.90 mm. 2 The sensor A, after deposition of three-dimensional gold dendrites, increased to 1103.80 mm. 2 Sensor A, after depositing PPY-3-COOH, further increased to 1324.56 mm². 2 .

[0063] The target antigen can be selected from Aβ-40 monomers, Aβ-42 monomers, Aβ-40 oligomers, and Aβ-42 oligomers, and the variety of detection substances improves the scalability of the electrode.

[0064] Furthermore, a chitosan hydrogel layer is fabricated on the antibody. The chitosan hydrogel layer is composed of a mixture of chitosan, glycerol, and acetic acid, and has micropores on its surface for filtering macromolecular substances.

[0065] Furthermore, the portable Alzheimer's disease early detection system includes a microcontroller, the aforementioned electrochemical sensor chip for early Alzheimer's disease detection, a Bluetooth module, and a power supply module. The electrochemical sensor chip, Bluetooth module, and power supply module are all connected to the microcontroller. The electrochemical sensor chip monitors the electrical signal of the target and sends it to the microcontroller. The microcontroller analyzes and processes the electrical signal data and sends the analysis results to the Bluetooth module for display on a mobile phone or other terminal device. The power supply module provides power to the entire portable Alzheimer's disease early detection system. The surface impedance of the electrochemical sensor chip is scanned using a voltage with a frequency of 0.1Hz-1000kHz. The obtained impedance signal and gradient are compared to obtain the detection result. It exhibits excellent performance such as low power consumption, a large detection range, and strong scalability. In addition, the aforementioned electrochemical sensor chip can operate normally in an environment ranging from -40℃ to +85℃, ensuring the Alzheimer's disease early detection system has good stability and can operate normally in harsh environments.

Claims

1. A method for preparing an electrochemical sensor for detecting biomarkers of Alzheimer's disease, characterized in that, Specifically, the following steps are included: (1) First, the first paste is printed on the corresponding positions of the working electrode and the counter electrode on the substrate, and then dried to obtain the working electrode and the counter electrode. The first paste can be gold paste, silver paste, carbon paste, carbon nanotubes or graphene. Then, the second paste is printed on the reference electrode position on the substrate, and then dried to obtain the reference electrode. The second paste is a mixture of silver and silver chloride. Finally, the insulating paste is covered on the electrode layer to form an insulating layer. The insulating layer covers part of the working electrode, the counter electrode and the reference electrode, exposing the reaction area and the charging area of ​​the working electrode, the counter electrode and the reference electrode. Then, it is dried and then cleaned with 75% ethanol solution and deionized water. (2) Immerse the working electrode prepared in step (1) in an aqueous solution of Na2SO4 and AuCl3, and prepare three-dimensional gold dendrites on the working electrode using a time-amperometric method. Then rinse with deionized water to remove surface residues. Three-dimensional gold dendrites are obtained on the surface of the working electrode. The deposition potential is -0.65V and the deposition time is 100s. Or, immerse the working electrode prepared in step (1) in an aqueous solution of Na2SO4 and AgNO3, and prepare three-dimensional silver dendrites on the working electrode using a time-amperometric method. Then rinse with deionized water to remove surface residues. Three-dimensional silver dendrites are obtained on the surface of the working electrode. (3) A solution containing potassium ferrocyanide, potassium ferrocyanide and antibody linker is dropped onto the dendrite surface. Electrochemical polymerization is carried out by cyclic voltammetry to deposit the antibody linker onto the surface of the metal dendrite. After rinsing with deionized water and air drying, it is ready for use. The antibody linker is used to link antibodies. The antibody linker is polypyrrole-3-carboxylic acid, polypyrrole-2-carboxylic acid or polythiopheneacetic acid. (4) Add the antibody to the antibody conjugate, incubate at room temperature, slowly rinse the residual antibody solution with PBS solution, add the bovine serum albumin solution to the working electrode, and store at -4°C for later use. The antibody is used to specifically recognize Aβ-40 monomer, Aβ-40 oligomer, Aβ-42 monomer or Aβ-42 oligomer, and is an Aβ-40 and Aβ-42 specific antibody.

2. The method for preparing the electrochemical sensor for detecting Alzheimer's disease biomarkers according to claim 1, characterized in that, The substrate is PET, PDMS, PI or SEBS.

3. The method for preparing the electrochemical sensor for detecting Alzheimer's disease biomarkers according to claim 1, characterized in that, A chitosan hydrogel layer is coated on the antibody. The chitosan hydrogel layer is composed of chitosan, glycerol, and acetic acid and has tiny pores on its surface that can filter large molecules.

4. An electrochemical sensor for detecting Alzheimer's disease biomarkers prepared by the method of any one of claims 1-3.

5. A portable early detection system for Alzheimer's disease, characterized in that, The system includes a microcontroller, the electrochemical sensor chip for early detection of Alzheimer's disease as described in claim 4, a Bluetooth module, and a power supply module. The electrochemical sensor chip for early detection of Alzheimer's disease, the Bluetooth module, and the power supply module are all connected to the microcontroller. The electrochemical sensor chip for early detection of Alzheimer's disease is used to monitor the electrical signal of the target and send it to the microcontroller. The microcontroller analyzes and processes the electrical signal data and sends the analysis results to the Bluetooth module. The results are then sent to a mobile terminal device for display via the Bluetooth module. The power supply module provides power to the entire portable early detection system for Alzheimer's disease.

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