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CN-122016968-A - P-Tau181 protein electrochemical immunosensor and preparation method and application thereof

CN122016968ACN 122016968 ACN122016968 ACN 122016968ACN-122016968-A

Abstract

The invention discloses an electrochemical immunosensor for ultra-sensitively detecting an Alzheimer's marker p-Tau 181 and a preparation method thereof. Firstly, the catalytic performance of a Bi 2 O 3 /Co 3 O 4 heterojunction composite material modified by three noble metals (Ag, au and Pt) is designed and compared, and the electrocatalytic reduction activity of the Ag modified composite material (Ag/Bi 2 O 3 /Co 3 O 4 ) to hydrogen peroxide (H 2 O 2 ) is found to be optimal. Based on the method, the Ag/Bi 2 O 3 /Co 3 O 4 composite material is used as a signal amplification label, and is connected with a detection antibody in a covalent coupling mode, so that the sandwich type electrochemical immunosensor is constructed. The sensor realizes ultrasensitive detection of p-Tau 181 by utilizing the high-efficiency catalysis of Ag/Bi 2 O 3 /Co 3 O 4 on H 2 O 2 . The invention provides a sensitive and convenient detection method for the key biomarker p-Tau 181 of the Alzheimer disease, and simultaneously provides a powerful new tool for early diagnosis of the Alzheimer disease.

Inventors

  • YUAN MIN
  • XU FEI
  • ZHANG SIYAO

Assignees

  • 上海理工大学

Dates

Publication Date
20260512
Application Date
20260304

Claims (8)

  1. 1. The p-Tau 181 protein electrochemical immunosensor and the preparation method and the application thereof are characterized by comprising the following steps: S1, preparing a g-C 3 N 4 material, namely placing melamine into a crucible, placing the crucible into a muffle furnace for roasting, cooling to obtain light yellow powder, and grinding to obtain g-C 3 N 4 . S2, preparing and testing an M/Bi 2 O 3 /Co 3 O 4 (M=Ag, au and Pt) composite material, namely dissolving cobalt nitrate hexahydrate and urea in deionized water, adding bismuth nitrate pentahydrate, uniformly stirring, heating in a reaction kettle, centrifuging, repeatedly washing with absolute ethyl alcohol and deionized water, and vacuum drying and calcining the obtained precipitate to obtain Bi 2 O 3 /Co 3 O 4 . Bi 2 O 3 /Co 3 O 4 is weighed and added into deionized water, and dispersion liquid I is obtained through ultrasonic dispersion. And (3) respectively adding the silver nitrate solution, chloroplatinic acid hexahydrate and tetrachloroauric acid trihydrate serving as precursors into the dispersion liquid I, and uniformly stirring, wherein the mass of metals in the dispersion liquid is the same (the mass of Ag, au and Pt is 1 mg). Slowly adding sodium borohydride, stirring, centrifuging, repeatedly washing precipitate with absolute ethyl alcohol and deionized water, and vacuum drying to obtain three composite materials. The three composite materials are dripped on the surface of a glassy carbon electrode and placed in a PBS solution containing H 2 O 2 for electrochemical detection, and the material with the best catalytic performance on H 2 O 2 is selected through comparing current values. Adding the selected optimal metal precursor solution into Bi 2 O 3 /Co 3 O 4 dispersion liquid, respectively stirring at different temperatures by using sodium borohydride as a reducing agent, centrifuging, repeatedly washing by using absolute ethyl alcohol and deionized water, and carrying out vacuum drying to obtain a composite material and carrying out electrochemical test. The material with the best performance is dispersed in absolute ethyl alcohol by ultrasonic, and APTES is slowly added and stirred in an oil bath. And (3) washing the mixture by using absolute ethyl alcohol after centrifugation, and drying the mixture in vacuum to obtain M/Bi 2 O 3 /Co 3 O 4 -NH 2 . S3, ab 2 /M/Bi 2 O 3 /Co 3 O 4 -NH 2 preparation, namely adding EDC/NHS solution into Ab 2 (detection antibody) solution of p-Tau 181 to activate carboxyl, adding M/Bi 2 O 3 /Co 3 O 4 -NH 2 , stirring overnight at 4 ℃, adding BSA and stirring for 1 h. The pellet was dispersed in PBS solution by centrifugation and stored at 4℃until use. S4, preparing an electrochemical immunosensor, namely firstly dripping g-C 3 N 4 solution onto a glassy carbon electrode for drying, and then dripping 5% Glutaraldehyde (GA) solution for incubation. The capture antibody of p-Tau 181 (Ab 1 ) was added dropwise to the electrode surface and incubated overnight at 4 ℃. BSA was added dropwise to block the remaining active sites, followed by incubation at 37 ℃ with p-Tau 181 and Ab 2 /M/Bi 2 O 3 /Co 3 O 4 -NH 2 , respectively, the electrode was slowly rinsed with PBS solution and dried to give an electrochemical immunosensor electrode. The electrode was immersed in a PBS solution containing H 2 O 2 for DPV detection.
  2. 2. The p-Tau 181 protein electrochemical immunosensor, and the preparation method and the application thereof, according to claim 1, wherein in the step S2, 30 mg Bi 2 O 3 /Co 3 O 4 is weighed and added into 15 mL deionized water, and dispersion liquid I is obtained after ultrasonic dispersion. 200 mu L of silver nitrate solution, chloroplatinic acid hexahydrate and tetrachloroauric acid trihydrate are respectively added into the dispersion liquid I as precursors to be stirred uniformly, and meanwhile, the quality of metals (Ag, au and Pt) in the dispersion liquid is the same. 150. Mu.L of 30 mg/mL sodium borohydride solution was then slowly added and stirred for 1: 1 h. And (3) repeatedly washing the mixture by using absolute ethyl alcohol and deionized water after centrifugation, and drying the mixture in vacuum at 60 ℃ overnight to obtain the composite material.
  3. 3. The p-Tau 181 protein electrochemical immunosensor, and the preparation method and the application thereof, according to claim 1, wherein in S2, 200 μl of 0.610 mM silver nitrate solution is added to dispersion I and stirred for 30 min. 150. Mu.L of 30 mg/mL sodium borohydride solution was then slowly added and stirred in an oil bath at 25-70℃for 1 h. After centrifugation, repeated washing with absolute ethanol and deionized water, and vacuum drying at 60 ℃ overnight, ag/Bi 2 O 3 /Co 3 O 4 is obtained.
  4. 4. The electrochemical immunosensor for p-Tau 181 protein, and the manufacturing method and application thereof, according to claim 1, wherein in S2, 10 mg Ag/Bi 2 O 3 /Co 3 O 4 is added to 5 mL and then to absolute ethanol for ultrasonic dispersion of 30min, and then 500 μl APTES is slowly added and stirred in an oil bath at 70 ℃ for 1.5 h. After centrifugation, the mixture was washed with absolute ethanol and dried under vacuum at 60℃overnight to give Ag/Bi 2 O 3 /Co 3 O 4 -NH 2 .
  5. 5. An M/Bi 2 O 3 /Co 3 O 4 (m=ag, au, pt) composite material prepared according to the method of any one of claims 1 to 4 and its use in the preparation of electrochemical immunosensors.
  6. 6. The p-Tau 181 protein electrochemical immunosensor, and the preparation method and the application thereof, according to claim 1, wherein in the S2, ag/Bi 2 O 3 /Co 3 O 4 -NH 2 is weighed and added into PBS solution for dispersion treatment to form 2-6mg/mL of suspension. It was added to Ab 2 solution and stirred overnight at 4 ℃. BSA was then added to the solution and stirred for 1h. After centrifugation, the precipitate is dispersed in PBS solution, so that the mass concentration of Ag/Bi 2 O 3 /Co 3 O 4 -NH 2 contained in the solution is 2-6mg/mL.
  7. 7. An electrochemical sensor, characterized in that the electrochemical immunosensor containing the M/Bi 2 O 3 /Co 3 O 4 (m=ag, au, pt) composite material according to any one of claims 1 to 6 comprises a glassy carbon electrode or a screen printed electrode. The method is characterized in that 2 mg/mL g-C 3 N 4 solution is dripped on a GCE electrode for drying, and then 5% GA solution is dripped for incubation 25 min. Then 10. Mu.L of 20. Mu.g/mL Ab 1 to p-Tau 181 was added dropwise to the electrode surface and incubated overnight at 4 ℃. BSA was then added dropwise to block the remaining active sites. Then 10. Mu.L of p-Tau 181 was added dropwise to the electrode surface and incubated at 37℃for 30 min. Finally, ab 2 /Ag/Bi 2 O 3 /Co 3 O 4 -NH 2 is dripped on the surface of the electrode, and 45 min is incubated at 37 ℃ to obtain the immunosensor.
  8. 8. The electrochemical immunosensor of claim 7, wherein the capture antibody and the detection antibody are p-Tau 181 monoclonal antibodies, and the modified electrode or the electrochemical sensor are both used in p-Tau 181 detection.

Description

P-Tau181 protein electrochemical immunosensor and preparation method and application thereof Technical Field The invention belongs to the technical field of electrochemical sensor preparation and detection, and particularly relates to a sandwich type electrochemical immunosensor for detecting p-Tau 181 protein and application thereof. Background Alzheimer's Disease (AD) is a common and irreversible neurodegenerative disease characterized clinically by progressive memory loss and cognitive dysfunction. The early and accurate diagnosis is important for delaying the disease development and improving the life quality of patients. Currently, a variety of biomarkers have been applied to diagnosis of AD, such as beta amyloid (aβ40, aβ42) and phosphorylated Tau protein (p-Tau). Among these biomarkers, phosphorylated Tau 181 protein was confirmed as a highly specific biomarker for early diagnosis of AD. Currently, AD biomarkers are detected primarily by imaging techniques and cerebrospinal fluid (CSF) analysis. However, the imaging technique is costly, while CSF requires lumbar puncture, which is invasive and complicated. Compared with the traditional CSF markers, the blood markers have the remarkable advantages of convenience in sampling, low invasiveness and the like. However, the concentration of p-Tau 181 in blood is generally low, on the order of picograms per milliliter (pg/mL), and therefore needs to be achieved by means of highly sensitive detection techniques. At present, quantitative detection methods of p-Tau 181 protein comprise enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence (ECL), single-molecule immune array (Simoa) and the like, but the methods are difficult to popularize and apply due to the problems of low sensitivity, dependence on large-scale instruments, higher detection cost, complex operation and the like. The electrochemical immunosensor combines the advantages of high specificity of immunoassay and high sensitivity of electrochemical detection, simple and convenient operation, easy microminiaturization and the like, and provides a very promising technical platform for rapid detection of AD biomarkers. The detection performance of the sandwich immunosensor is characterized in that a signal transduction and amplification strategy, and a material with high catalytic activity is used as an effective means for an antibody marker. Therefore, the development of the composite material with high catalytic performance and the application of the composite material in the signal amplification label of the high-sensitivity immunosensor are of great significance in promoting the detection technology development of the AD biomarker p-Tau 181. Disclosure of Invention The invention aims to solve the technical problems in the prior art, and provides a sandwich type electrochemical immunosensor for detecting p-Tau 181 protein, which is prepared from a composite material with higher catalytic activity in M/Bi 2O3/Co3O4 (M=Ag, au, pt) composite material and is used as a signal amplification label. In order to achieve the above purpose, the present invention provides the following technical solutions: a p-Tau 181 protein electrochemical immunosensor and a preparation method and application thereof comprise the following steps: S1, preparation of g-C 3N4 materials: And placing melamine into a crucible, placing the crucible into a muffle furnace, roasting, cooling to obtain light yellow powder, and grinding to obtain g-C 3N4. Preparation and testing of S2, M/Bi 2O3/Co3O4 (m=ag, au, pt) composite: Dissolving cobalt nitrate hexahydrate and urea in deionized water, adding bismuth nitrate pentahydrate, stirring uniformly, heating in a reaction kettle, centrifuging, washing repeatedly with absolute ethyl alcohol and deionized water, and vacuum drying and calcining the obtained precipitate to obtain Bi 2O3/Co3O4. Bi 2O3/Co3O4 is weighed and added into deionized water, and dispersion liquid I is obtained after ultrasonic dispersion. And respectively adding the silver nitrate solution, chloroplatinic acid hexahydrate and tetrachloroauric acid trihydrate serving as precursors into the dispersion liquid I, and uniformly stirring, wherein the masses of metals (Ag, au and Pt) in the dispersion liquid are the same. Slowly adding sodium borohydride serving as a reducing agent, stirring, centrifuging, repeatedly washing with absolute ethyl alcohol and deionized water, and vacuum drying to obtain three composite materials. The three composite materials are dripped on the surface of GCE and placed in PBS solution containing H 2O2 for electrochemical detection, and the material with the best catalytic performance on H 2O2 is selected by comparing current values. Adding the selected optimal metal precursor solution into Bi 2O3/Co3O4 dispersion liquid, respectively stirring at different temperatures by using sodium borohydride as a reducing agent, centrifuging, repeatedly washing by using absolute ethyl alcohol and