CN117783227A - Electrochemical sensor for detecting Alzheimer disease markers and preparation method thereof - Google Patents
Electrochemical sensor for detecting Alzheimer disease markers and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an electrochemical sensor for detecting Alzheimer disease markers and a preparation method thereof, wherein the electrochemical sensor comprises a substrate, a working electrode, a counter electrode, a reference electrode, an insulating layer, metal dendrite, an antibody connector and an antibody; the method comprises the steps of manufacturing a working electrode, a counter electrode and a reference electrode on a substrate, arranging an insulating layer on the counter electrode and the reference electrode, sequentially coating a metal dendrite, an antibody connector and an antibody on the working electrode, wherein the metal dendrite is a metal dendrite or a silver dendrite, the antibody connector is used for connecting and fixing the antibody, the antibody is used for specifically recognizing a target antigen in a sample to be detected, and the target antigen is A beta-40 monomer, A beta-40 oligomer, A beta-42 monomer or A beta-42 oligomer. The specific surface area of the working electrode is enlarged by electrochemical deposition of metal dendrites, more attachment sites are provided for antibodies, the sensitivity is higher, the detection precision is higher, and the manufactured portable early detection system for Alzheimer's disease is convenient to carry and has lower detection cost.
Description
Technical Field
The invention belongs to the technical field of detection of electrochemical sensors, and particularly relates to an electrochemical sensor for detecting Alzheimer disease markers (A beta-40 monomer, A beta-42 monomer, A beta-40 oligomer and A beta-42 oligomer) and a preparation method thereof.
Background
Alzheimer's Disease (AD) is an irreversible neurodegenerative disease, threatening the health of the elderly over the globe tens of millions, and this figure is rising every year. At present, alzheimer's disease cannot be cured, but the probability of suffering from Alzheimer's disease can be reduced through early prevention, so early detection of Alzheimer's disease is particularly important.
Recent studies have shown that the levels of amyloid beta 1-40 (aβ40), amyloid beta 1-42 (aβ42), phosphorylated tau (p-tau) protein and total tau (t-tau) protein are altered during the development and progression of AD. Beta-amyloid is a marker for early diagnosis of AD. Therefore, a high-sensitivity detection device is urgently needed to detect the contents of human biomarkers A beta-40 monomer, A beta-42 monomer, A beta-40 oligomer and A beta-42 oligomer so as to discover and prevent Alzheimer's disease in time. The following are related art sensors for detecting beta-amyloid:
patent application No. CN202010050552.9 discloses a preparation method of novel composite material for detecting beta-amyloid, and g-C 3 N 4 Mixing the suspension with Hemin solution, adding ammonia water and hydrazine hydrate under high-speed stirring, rapidly stirring, standing in water bath at 60deg.C for 4 hr, standing in refrigerator at 4deg.C overnight, centrifuging, washing, and re-dispersing in secondary deionized water to obtain g-C 3 N 4 -Heme suspension. The novel composite material for detecting amyloid peptide can be used for constructing a biosensor for detecting beta-amyloid. The sensor is favorable for the combination effect of heme and amyloid polypeptide, realizes the detection of amyloid behavior, has better specificity on amyloid, and can rapidly and sensitively realize the early detection of the amyloid.
Patent application number CN202310833365.1 discloses an electrode for detecting amyloid content of alzheimer disease marker, its preparation method, its use method and its detection device, the electrode comprises: the screen printing electrode comprises a working electrode, a reference electrode and a counter electrode; the biological recognition layer is arranged on the surface of the working electrode in the screen printing electrode and comprises a conductive agent, an electromechanical chemistry probe and a first antibody; the second mixed solution comprises a second antibody modified metal nano material marker and is used for being modified on the surface of the working electrode after a sample to be detected is added, wherein the first antibody and the second antibody respectively comprise an Abeta 40 antibody or an Abeta 42 antibody. The electrode can be subjected to electrochemical oxidation on the surface of the electrode through antigen-antibody recognition, and the organic electrochemical probe and the metal nano material marker can generate a detectable current signal, and the formed current ratio is positively correlated with the concentration of the antigen, so that the ratio-type high-sensitivity detection of the Alzheimer disease marker beta amyloid can be realized. It enables detection of amyloid content, however the preparation method is complex.
Disclosure of Invention
In order to solve the problems, the invention aims to provide an electrochemical sensor for detecting an Alzheimer disease marker and a preparation method thereof, wherein the marker is at least one of Abeta-40 monomer, abeta-42 monomer, abeta-40 oligomer and Abeta-42 oligomer.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an electrochemical sensor for detecting Alzheimer disease markers comprises a substrate, a working electrode, a counter electrode, a reference electrode, an insulating layer, metal dendrites, an antibody connector and an antibody; the method comprises the steps of manufacturing a working electrode, a counter electrode and a reference electrode on a substrate, arranging an insulating layer on the counter electrode and the reference electrode, sequentially coating a metal dendrite, an antibody connector and an antibody on the working electrode, wherein the metal dendrite is a metal dendrite or a silver dendrite, the antibody connector is used for connecting and fixing an antibody, the antibody is used for specifically recognizing a target antigen in a sample to be detected, and the target antigen is at least one of an A beta-40 monomer, an A beta-40 oligomer, an A beta-42 monomer and an A beta-42 oligomer.
Specifically, the antibody is an Abeta-40 or Abeta-42 specific antibody.
In particular, the antibody linker is polypyrrole-3-carboxylic acid (Py-3-COOH), polypyrrole-2-carboxylic acid (Py-2-COOH), or polythiophene acetic acid (PTAA).
Specifically, the substrate is a PET, PDMS, PI or SEBS substrate, the working electrode and the counter electrode are gold, silver, carbon nano tube or graphene electrodes, and the reference electrode is a silver and silver chloride electrode.
Preferably, the substrate is a PET substrate, the working and counter electrodes are gold electrodes, the reference electrode is a silver and silver chloride electrode, and the antibody linker is polypyrrole-3-carboxylic acid (Py-3-COOH).
Furthermore, the electrochemical sensor for detecting the Alzheimer disease marker further comprises a chitosan hydrogel layer, wherein the chitosan hydrogel layer is coated on the antibody, and the chitosan hydrogel layer is formed by mixing chitosan, glycerol and acetic acid, and the surface of the chitosan hydrogel layer is provided with micro-pores and can filter macromolecular substances.
The invention relates to a preparation method of an electrochemical sensor for detecting Alzheimer disease markers, which specifically comprises the following steps:
(1) Manufacturing an electrode layer on a substrate, wherein the electrode layer comprises a working electrode, a counter electrode and a reference electrode, an insulating layer is prepared on the electrode layer, and the insulating layer covers the areas except a reaction area and a grounding area in the working electrode, the counter electrode and the reference electrode, and exposes the reaction area and the grounding area of the working electrode, the counter electrode and the reference electrode;
(2) Manufacturing three-dimensional metal dendrite on the surface of the working electrode through electrochemical deposition, wherein the three-dimensional metal dendrite comprises three-dimensional silver dendrite and three-dimensional gold dendrite;
(3) Dropwise adding a solution containing potassium ferricyanide, potassium ferrocyanide and an antibody connector on the surface of the dendrite, carrying out electrochemical polymerization by a cyclic voltammetry to deposit the antibody connector on the surface of the dendrite, washing with deionized water, and naturally airing for later use; antibody linkers are used to link the antibodies;
(4) The antibody is dripped on an antibody connector, incubated at room temperature, residual antibody solution is slowly washed by PBS solution, bovine serum albumin solution is dripped on a working electrode and is preserved at-4 ℃ for standby, and the antibody is used for specifically recognizing Abeta-40 monomer, abeta-40 oligomer, abeta-42 monomer or Abeta-42 oligomer, and the optional materials are Abeta-40 and Abeta-42 specific antibodies.
Specifically, in the step (1), first paste is printed at the corresponding positions of a working electrode and a counter electrode on a substrate, and then dried to obtain the working electrode and the counter electrode, wherein the first paste is made of gold paste, silver paste, carbon nanotubes or graphene; printing a second sizing agent at the reference electrode position on the substrate, and then drying to obtain a reference electrode, wherein the second sizing agent is a silver and silver chloride mixed material; and finally, covering the insulating slurry on the electrode layer to form an insulating layer, wherein the insulating layer covers the outer parts of the working electrode and the counter electrode and one half of the working electrode close to the counter electrode and the reference electrode, drying, and cleaning with 75% ethanol solution and deionized water.
Specifically, in the step (2), the working electrode 2 manufactured in the step (1) is immersed in Na 2 SO 4 And AuCl 3 Preparing three-dimensional gold dendrite on a working electrode by using a timing ampere method in an aqueous solution, then flushing with deionized water to remove surface residues, and obtaining the three-dimensional gold dendrite on the surface of the working electrode, wherein the deposition potential is preferably-0.65 v in the process, and the deposition time is 100s; or immersing the working electrode manufactured in the step (1) into Na 2 SO 4 And AgNO 3 In the aqueous solution, preparing three-dimensional gold dendrite on the working electrode by using a chronoamperometry method, then flushing with deionized water to remove surface residues, and obtaining the three-dimensional silver dendrite on the surface of the working electrode.
The portable early detection system for the Alzheimer's disease comprises a micro controller, an electrochemical sensor chip for early detection of the Alzheimer's disease, a Bluetooth module and a power supply module, wherein the electrochemical sensor chip for early detection of the Alzheimer's disease, the Bluetooth module and the power supply module are all connected with the micro controller, the electrochemical sensor chip for early detection of the Alzheimer's disease is used for monitoring an electric signal of a target to be detected and transmitting the electric signal to the micro controller, the micro controller analyzes and processes the electric signal data and transmits an analysis result to the Bluetooth module, the electric signal data is transmitted to a mobile phone and other general equipment through the Bluetooth module for display, and the power supply module provides electric energy for the whole portable early detection system for the Alzheimer's disease.
Compared with the prior art, the invention has the following beneficial effects: (1) The specific surface area of the working electrode is enlarged by electrochemical deposition of metal dendrite, more attachment sites are provided for the antibody, the sensitivity is higher, the detection precision is higher, and the detection lower limit reaches 4.5 multiplied by 10 -8 pg/mL; (2) The chitosan hydrogel is adopted to remove macromolecular substances in the sample, so that non-specific adsorption is prevented, and the detection accuracy is higher; (3) Pyrrole-3-carboxylic acid (Py-3-COOH), pyrrole-2- (Py-2-COOH) or thiophene acetic acid (PTAA) is used as a linker between the antibody and the electrode, which has a higher conductivity; (4) The method is used for detecting the Abeta-42 and Abeta-40 content in serum and saliva, realizing the noninvasive detection of Alzheimer's disease, and (5) manufacturing the portable sensor, and has the advantages of convenient carrying and lower detection cost.
Drawings
Fig. 1 is a schematic structural diagram of an electrochemical sensor for detecting an alzheimer's disease marker according to the present invention.
Fig. 2 is a process flow diagram of a method for preparing an electrochemical sensor for detecting an alzheimer's disease marker according to the present invention.
FIG. 3 is a graph showing the peak current values at different potentials detected by cyclic voltammetry in example 1.
FIG. 4 is an SEM image of three-dimensional gold dendrites obtained at a voltage of-0.65V in example 1.
FIG. 5 is a plot of i-t for different electrochemical deposition times in example 1.
FIG. 6 is a graph showing the impedance of human serum samples with different concentrations of Abeta-42 antigen solution.
FIG. 7 is a graph showing the impedance gradients of samples of artificial saliva with different concentrations of Abeta-42 antigen solution.
Detailed Description
The invention will be further described with reference to specific examples and figures. They are not to be construed as limiting the scope of the invention.
As shown in fig. 1, the electrochemical sensor for detecting the alzheimer disease marker comprises 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 connector 7 and an antibody; the method comprises the steps of manufacturing a working electrode 2, a counter electrode 3 and a reference electrode 4 on a substrate 1, arranging an insulating layer 5 on the counter electrode 3 and the reference electrode 4, and sequentially coating a metal dendrite 6, an antibody connector 7 and an antibody on the working electrode 2, wherein the metal dendrite 6 is a gold dendrite or a silver dendrite, the antibody connector 7 is used for connecting and fixing an antibody, the antibody is used for specifically recognizing a target antigen in a sample to be detected, and the target antigen is at least one of an A beta-40 monomer, an A beta-40 oligomer, an A beta-42 monomer and an A beta-42 oligomer.
Specifically, the antibody is an Abeta-40 or Abeta-42 specific antibody.
Specifically, the antibody connector 7 is an antibody connector which is polypyrrole-3-carboxylic acid (Py-3-COOH), polypyrrole-2-carboxylic acid (Py-2-COOH) or polythiophene acetic acid (PTAA).
Furthermore, the electrochemical sensor for detecting the Alzheimer disease marker further comprises a chitosan hydrogel layer, wherein the chitosan hydrogel layer is coated on the antibody, and the chitosan hydrogel layer is formed by mixing chitosan, glycerol and acetic acid, and the surface of the chitosan hydrogel layer is provided with micro-pores and can filter macromolecular substances.
As shown in fig. 2, the preparation method of the electrochemical sensor for detecting the alzheimer disease marker in this embodiment specifically includes the following steps:
(1) An electrode layer is manufactured on a clean substrate 1, and comprises a working electrode 2, a counter electrode 3 and a reference electrode 4.
As shown in fig. 2a, first, printing first slurry on the corresponding positions of the working electrode and the counter electrode on the substrate 1, and then drying to obtain the working electrode 2 and the counter electrode 3, wherein as shown in fig. 2b, the first slurry is selected from gold slurry, silver slurry, carbon nanotubes or graphene, and the materials used in the substrate 1 include, but are not limited to, PET, PDMS, PI or SEBS;
as shown in fig. 2c, a second slurry is printed on the substrate at the reference electrode position, and then dried to obtain a reference electrode 4, wherein the second slurry is a mixed material of silver and silver chloride;
as shown in fig. 2d, an insulating paste is finally coated on the electrode layer to form an insulating layer 5, and the insulating layer 5 covers the outer parts of the working electrode 2 and the counter electrode 3 and one half of the working electrode 2 near the counter electrode 3 and the reference electrode 4, followed by drying and washing with 75% ethanol solution and deionized water. The insulating layer 5 covers part of the areas of the working electrode 2, the counter electrode 3 and the reference electrode 4, and exposes the reaction areas and the electric connection areas of the working electrode 2, the counter electrode 3 and the reference electrode 4. The distances between any two of the working electrode 2, the counter electrode 3 and the reference electrode 4 are equal.
(2) As shown in fig. 2e, three-dimensional metal dendrites are manufactured on the surface of the working electrode 2 by electrochemical deposition, and the three-dimensional metal dendrites include three-dimensional silver dendrites and three-dimensional gold dendrites;
specifically, the working electrode 2 manufactured in the step (1) is immersed in Na 2 SO 4 And AuCl 3 In the aqueous solution, three-dimensional gold dendrite (AuD) is prepared on the working electrode 2 by using a timing ampere method (CA), surface residues are removed by washing with deionized water, the three-dimensional gold dendrite is obtained on the surface of the working electrode, and the three-dimensional gold dendrite is stored at the temperature of-4 ℃ for standby.
Or immersing the working electrode 2 manufactured in the step (1) into Na 2 SO 4 And AgNO 3 In the aqueous solution, three-dimensional gold dendrite (AuD) is prepared on the working electrode 2 by using a timing ampere method (CA), surface residues are removed by washing with deionized water, the three-dimensional silver dendrite is obtained on the surface of the working electrode, and the three-dimensional silver dendrite is stored at the temperature of-4 ℃ for standby.
(3) As shown in fig. 2f, a solution containing potassium ferricyanide, potassium ferrocyanide and antibody connector is dripped on the surface of dendrite, the antibody connector 7 is deposited on the surface of metal dendrite through electrochemical polymerization by Cyclic Voltammetry (CV), and the dendrite is rinsed with deionized water and naturally dried for later use; such antibody linkers include, but are not limited to, polypyrrole 3-carboxylic acid (Py-3-COOH), polypyrrole 2-carboxylic acid (Py-2-COOH), polythiophene acetic acid (PTAA).
The antibody linker is used to attach an antibody while it also improves the conductivity of the sensor.
(4) As shown in FIG. 2g, the antibody used for specifically recognizing Abeta-40 monomer, abeta-40 oligomer, abeta-42 monomer or Abeta-42 oligomer, the optional materials are Abeta-40 and Abeta-42 specific antibodies is added dropwise onto an antibody linker, incubated at room temperature, the residual antibody solution is slowly rinsed with PBS solution, the bovine serum albumin solution is added dropwise onto a working electrode and is preserved at-4 ℃ for standby.
Example 1
As shown in fig. 2, the preparation method of the electrochemical sensor for early detection of alzheimer's disease according to the embodiment specifically includes the following steps:
(1) As shown in fig. 2a, first, printing a first slurry on a substrate according to a pattern, and then drying at 160 ℃ to obtain a working electrode 2 and a counter electrode 3, wherein as shown in fig. 2b, the first slurry is gold slurry, and the substrate 1 is a PET substrate;
as shown in fig. 2c, a second paste is then printed on the substrate according to a pattern, and then dried at 130 ℃ to obtain a reference electrode 4, wherein the second paste is a silver and silver chloride mixed material;
as shown in fig. 2d, finally, insulating paste is coated on the electrode layer, the insulating paste covers the outer parts of the working electrode 2 and the counter electrode 3, and one half of the working electrode 2, and then the insulating layer 4 is obtained by drying at 100 ℃, and then washed with 75% ethanol solution and deionized water.
(2) As shown in FIG. 2e, the working electrode manufactured in the step (1) is immersed in Na 2 SO 4 And AuCl 3 In aqueous solution, electrochemical deposition is carried out, three-dimensional gold dendrites (AuD) are prepared on the working electrode 2 by using a Chronoamperometry (CA), and then surface residues are removed by washing with deionized water and stored at-4 ℃ for later use.
Specifically, the working electrode manufactured in the step (1) is immersed in Na 2 SO 4 And AuCl 3 In the aqueous solution, electrochemical deposition is carried out under different voltages of-0.2V, -0.35V, -0.5V, -0.65V and-0.8V respectively, and the three-dimensional golden dendrite (AuD) is prepared on the working electrode 2.
FIG. 3 shows current peaks of electrochemical sensors for detecting three-dimensional gold dendrites deposited at different potentials by cyclic voltammetry, and shows that: the peak current reached a maximum at-0.65 v potential and a downward trend occurred after-0.65 v. The resistance of the deposited sensor at a potential of 0.8v becomes large because the deposited gold dendrites are agglomerated due to the excessive potential. With further research, it is found that the deposition potential exceeds-0.65 v under the experimental conditions, and the phenomenon of gold dendrite agglomeration occurs on the surface of the sensor, so that the conductivity of the sensor is reduced. Thus eventually-0.65 v was chosen as the optimal deposition voltage for the gold dendrites. Fig. 4 is an SEM image of a three-dimensional gold dendrite obtained at-0.65 v voltage, and it can be understood that the specific surface area of the working electrode is enlarged by using a metal dendrite deposition method, so that more adsorption sites are provided, the sensitivity of the sensor is higher, and besides the gold dendrite of the embodiment, a silver dendrite can be prepared.
Further, immersing the substrate with the electrode layer into Na 2 SO 4 And AuCl 3 Electrochemical deposition was performed in aqueous solution at a voltage of-0.65 v, with observations and impedance tests performed every 100s.
FIG. 5 is an i-t plot of different electrochemical deposition times, it being understood that the height of the gold dendrites increases with time and the impedance of the sensor decreases, however, after the deposition time reaches 200s, the low frequency region of the impedance spectrum begins to be chaotic. Based on the observation, the optimal deposition time was finally selected to be 100s. The growth of the gold dendrites was stable during this period and performed well in the impedance test.
(3) As shown in fig. 2f, a solution containing potassium ferricyanide solution, potassium ferrocyanide and polypyrrole 3-carboxylic acid is dropwise added on the surface of dendrite, polypyrrole 3-carboxylic acid (Py-3-COOH) is deposited on the surface of dendrite through Cyclic Voltammetry (CV), and the dendrite is rinsed with deionized water and dried naturally for later use;
(4) As shown in FIG. 2g, the antibody is dripped on the antibody connector, incubated at room temperature, the residual antibody solution is slowly washed by PBS solution, bovine serum albumin solution is dripped on the surface of an electrode and then is preserved at-4 ℃ for standby, and the antibody can be used for specifically recognizing Abeta-40 monomer and oligomer of Abeta-40, abeta-42 monomer and oligomer of Abeta-42, and the optional materials are Abeta-40 and Abeta-42 specific antibodies.
(5) As shown in FIG. 2h, a human is employedSerum samples are respectively added with Abeta-42 antigen solutions with different concentrations to respectively prepare 4.5X10 -5 pg/ml、4.5×10 -4 pg/ml、4.5×10 -3 pg/ml、4.5×10 -2 pg/ml and 4.5X10 -1 pg/ml of aβ -42 serum solution, impedance changes were detected after incubation on the sensor, wherein fig. 6 is an impedance plot of the serum samples. The results show that different concentrations of the A.beta. -42 peptide can still be distinguished significantly in serum. The linear relationship of serum sample impedance with increasing antigen concentration can be expressed as Δr=a+b×lgc, where a= 3735.38 ±93.13, b= 740.36 ±27.28, and the determination coefficient R 2 ＝0.994。
Similarly, different concentrations of Abeta-42 antigen solution are respectively added into human saliva, and impedance change is detected after incubation on a sensor, wherein FIG. 7 is an impedance diagram of saliva samples.
The electrochemical active area and the electron transfer rate constant of the developed electrode were analyzed by CV (cyclic voltammetry). Electroactive surface area of electrode was measured at 20mM K by Randes-Sevcik equation 3 [Fe(CN) 6 ] 3-/4- CV determination in solution. Wherein i is p Is the peak current, n is the number of electrons transferred, A is the electroactive area (cm 2 ) D is [ Fe (CN) 6 ] 3- V is the sweep rate (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.
i P ＝ 0.4463 nFAC(nFvD/RT) 1/2 (1)
From (1), it can be deduced that the bare electrode A is 551.90 ×10 2 mm 2 Sensor A after deposition of three-dimensional structure gold dendrites was increased to 1103.80 ×10 2 mm 2 Sensor a after PPY-3-COOH deposition was further expanded to 1324.56 x 10 2 mm 2 。
The target antigen can be selected from Abeta-40 monomer, abeta-42 monomer, abeta-40 oligomer and Abeta-42 oligomer, and various detection substances can improve the expansibility of the electrode.
Further, a chitosan hydrogel layer is manufactured on the antibody, the chitosan hydrogel layer is formed by mixing chitosan, glycerol and acetic acid, and tiny pores are formed on the surface of the chitosan hydrogel layer for filtering macromolecular substances.
Further, the portable early detection system for Alzheimer's disease comprises a micro controller, an electrochemical sensor chip for early detection of Alzheimer's disease, a Bluetooth module and a power supply module, wherein the electrochemical sensor chip for early detection of Alzheimer's disease, the Bluetooth module and the power supply module are all connected with the micro controller, the electrochemical sensor chip for early detection of Alzheimer's disease is used for monitoring an electric signal of a target to be detected and transmitting the electric signal to the micro controller, the micro controller analyzes and processes the electric signal data and transmits an analysis result to the Bluetooth module, the electric signal is transmitted to a main terminal device such as a mobile phone through the Bluetooth module for display, and the power supply module provides electric energy for the whole portable early detection system for Alzheimer's disease. The surface impedance of the electrochemical sensor chip for early detection of Alzheimer's disease is scanned by adopting the voltage with the frequency of 0.1HZ-1000KHZ, and the obtained impedance signal and gradient are compared to obtain the detection result, so that the electrochemical sensor chip has the advantages of low power consumption, large detection range, strong expansibility and the like. In addition, the electrochemical sensor chip for early detection of the Alzheimer's disease can normally work in an environment of-40 ℃ to +85 ℃, so that the early detection system of the Alzheimer's disease is guaranteed to have good stability, and can normally work in a severe environment.
Claims (10)
1. An electrochemical sensor for detecting Alzheimer disease markers, which is characterized by comprising a substrate, a working electrode, a counter electrode, a reference electrode, an insulating layer, metal dendrites, an antibody connector and an antibody; the method comprises the steps of manufacturing a working electrode, a counter electrode and a reference electrode on a substrate, arranging an insulating layer on the counter electrode and the reference electrode, sequentially coating a metal dendrite, an antibody connector and an antibody on the working electrode, wherein the metal dendrite is a metal dendrite or a silver dendrite, the antibody connector is used for connecting and fixing an antibody, the antibody is used for specifically recognizing a target antigen in a sample to be detected, and the target antigen is at least one of an A beta-40 monomer, an A beta-40 oligomer, an A beta-42 monomer and an A beta-42 oligomer.
2. The electrochemical sensor for detecting alzheimer's disease marker according to claim 1, characterized in that said antibody is an aβ40 or aβ42 specific antibody.
3. The electrochemical sensor for detecting alzheimer's disease markers according to claim 1, characterized in that the antibody linker is polypyrrole-3-carboxylic acid (Py-3-COOH), polypyrrole-2-carboxylic acid (Py-2-COOH) or polythiophene acetic acid (PTAA).
4. The electrochemical sensor for detecting alzheimer's disease markers according to claim 1, characterized in that the substrate is a PET, PDMS, PI or SEBS substrate, the working and counter electrodes are gold, silver, carbon nanotubes or graphene electrodes, and the reference electrode is a silver and silver chloride electrode.
5. The electrochemical sensor for detecting alzheimer's disease markers according to claim 1, characterized in that the substrate is a PET substrate, the working and counter electrodes are gold electrodes, the reference electrode is a silver and silver chloride electrode, and the antibody linker is polypyrrole-3-carboxylic acid (Py-3-COOH).
6. The electrochemical sensor for detecting the Alzheimer's disease marker according to claim 1, further comprising a chitosan hydrogel layer, wherein the chitosan hydrogel layer is coated on the antibody, and the chitosan hydrogel layer is formed by mixing chitosan, glycerol and acetic acid, and has micro-pores on the surface for filtering macromolecular substances.
7. A method for preparing an electrochemical sensor for detecting a marker of alzheimer's disease according to any of claims 1-6, comprising the steps of:
(1) Manufacturing an electrode layer on a substrate, wherein the electrode layer comprises a working electrode, a counter electrode and a reference electrode, an insulating layer is prepared on the electrode layer, and the insulating layer covers partial areas of the working electrode, the counter electrode and the reference electrode, and exposes a reaction area and a power connection area of the working electrode, the counter electrode and the reference electrode;
(2) Manufacturing three-dimensional metal dendrite on the surface of the working electrode through electrochemical deposition, wherein the three-dimensional metal dendrite comprises three-dimensional silver dendrite and three-dimensional gold dendrite;
(3) Dropwise adding a solution containing potassium ferricyanide, potassium ferrocyanide and an antibody connector on the surface of the dendrite, carrying out electrochemical polymerization by a cyclic voltammetry to deposit the antibody connector on the surface of the dendrite, washing with deionized water, and naturally airing for later use; antibody linkers are used to link the antibodies;
(4) The antibody is dripped on an antibody connector, incubated at room temperature, residual antibody solution is slowly washed by PBS solution, bovine serum albumin solution is dripped on a working electrode and is preserved at-4 ℃ for standby, and the antibody is used for specifically recognizing Abeta-40 monomer, abeta-40 oligomer, abeta-42 monomer or Abeta-42 oligomer and is Abeta-40 and Abeta-42 specific antibody.
8. The method for preparing an electrochemical sensor for detecting an Alzheimer's disease marker according to claim 7, wherein in the step (1), first a first slurry is printed on the substrate at the corresponding positions of the working electrode and the counter electrode, and then dried to obtain the working electrode and the counter electrode, wherein the first slurry is selected from gold slurry, silver slurry, carbon nanotubes or graphene; printing a second sizing agent at the reference electrode position on the substrate, and then drying to obtain a reference electrode, wherein the second sizing agent is a silver and silver chloride mixed material; and finally, covering the insulating slurry on the electrode layer to form an insulating layer, wherein the insulating layer covers the outer parts of the working electrode and the counter electrode and one half of the working electrode close to the counter electrode and the reference electrode, drying, and cleaning with 75% ethanol solution and deionized water.
9. The method for producing an electrochemical sensor for detecting Alzheimer's disease marker according to claim 7, wherein in step (2), the working electrode 2 produced in step (1) is immersed in Na 2 SO 4 And AuCl 3 Preparing three-dimensional gold dendrite on a working electrode by using a timing ampere method in an aqueous solution, then flushing with deionized water to remove surface residues, and obtaining the three-dimensional gold dendrite on the surface of the working electrode, wherein the deposition potential is-0.65 v in the process, and the deposition time is 100s; or immersing the working electrode manufactured in the step (1) into Na 2 SO 4 And AgNO 3 In the aqueous solution, preparing three-dimensional gold dendrite on the working electrode by using a chronoamperometry method, then flushing with deionized water to remove surface residues, and obtaining the three-dimensional silver dendrite on the surface of the working electrode.
10. The portable early detection system for the Alzheimer's disease is characterized by comprising a micro controller, the electrochemical sensor chip for early detection of the Alzheimer's disease, a Bluetooth module and a power supply module, wherein the electrochemical sensor chip for early detection of the Alzheimer's disease, the Bluetooth module and the power supply module are all connected with the micro controller, the electrochemical sensor chip for early detection of the Alzheimer's disease is used for monitoring an electric signal of a target to be detected and sending the electric signal to the micro controller, the micro controller analyzes and processes the electric signal data and sends an analysis result to the Bluetooth module, the analysis result is sent to a general terminal device such as a mobile phone through the Bluetooth module for display, and the power supply module provides electric energy for the whole portable early detection system for the Alzheimer's disease.
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