CN109115855B - Preparation method and application of electrochemical immunosensor for detecting Alzheimer's disease marker - Google Patents

Preparation method and application of electrochemical immunosensor for detecting Alzheimer's disease marker Download PDF

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CN109115855B
CN109115855B CN201811315614.3A CN201811315614A CN109115855B CN 109115855 B CN109115855 B CN 109115855B CN 201811315614 A CN201811315614 A CN 201811315614A CN 109115855 B CN109115855 B CN 109115855B
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李月云
高增强
张春燕
张栓
李新进
徐振
刘青
王平
陈志伟
董云会
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Shandong University of Technology
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Abstract

The invention relates to a preparation method and application of an electrochemical immunosensor for detecting Alzheimer's disease markers, and belongs to the field of novel technologyThe field of nano functional materials, immunoassay and biosensors. The prepared gold nanoparticle functionalized reduced graphene oxide is used as a substrate material, so that the solid loading capacity of the Alzheimer's disease marker capture antibody is improved, and the electron transfer rate on the surface of the electrode is effectively accelerated; preparation of AuCuxO@CeO2The composite nano material is used as a signal amplification label to immobilize a detection antibody; by Au NPs, CeO2And CuxThe composite material further improves the catalytic efficiency under the synergistic action of O; the prepared electrochemical immunosensor can be used for detecting Alzheimer disease related markers. Has strong specificity, high sensitivity and low detection limit, and has important scientific significance and application value.

Description

Preparation method and application of electrochemical immunosensor for detecting Alzheimer's disease marker
Technical Field
The invention belongs to the technical field of immunoassay and biosensing, and provides a preparation method and application of an electrochemical immunosensor for detecting Alzheimer's disease markers.
Background
Alzheimer's Disease (AD), a degenerative disorder of the central nervous system characterized by progressive cognitive dysfunction and behavioral impairment, is the most common form of senile dementia, accounting for more than about 50% of senile dementia. Clinically manifested as changes in memory impairment, aphasia, disuse, agnosia, visual impairment, abstract and computational impairment, personality and behavior. The clinical symptoms of AD can be roughly divided into two categories, namely cognitive decline and the accompanying decline in living ability, and non-cognitive neuropsychiatric symptoms. Beta-amyloid (Abeta) has a molecular weight of about 4kDa, is hydrolyzed from beta amyloid precursor protein, is secreted by cells, and has a strong neurotoxic effect after accumulation of cell matrix precipitates. Neurotoxic effects of a β plays a major role in the progression of alzheimer's disease. The increase of plasma Abeta can be used as an index for early diagnosis of Alzheimer's disease, and how to efficiently and accurately realize the detection of Abeta is an urgent problem to be solved at present.
A beta is one of the best markers for diagnosing the Alzheimer disease at present, and has important significance for early diagnosis of the Alzheimer disease. At present, many methods for detecting Alzheimer's disease markers, such as radioimmunoassay, immunoradiometric method, electrochemiluminescence assay and the like, are available, but most detection methods are complicated, complex to operate, expensive in cost and high in detection limit, so that the establishment of a rapid, simple, convenient and sensitive detection method is of great significance.
At present, the electrochemical immunosensor is widely used for detecting disease markers, and the sandwich type electrochemical immunosensor combines a high-specificity immunoassay technology and a high-sensitivity electrochemical analysis technology, has the advantages of high sensitivity, high detection speed and the like, and has important application values in the fields of clinical examination, environmental monitoring, food safety control, biological monitoring and the like.
The electrochemical immunosensor constructed by the invention is a detection device for determining the concentration of an object to be detected based on the strength of an electric signal, and has the following advantages: firstly, biomolecules such as antigen and antibody are fixed on the surface of an electrode by a simple, rapid and effective method; and secondly, the synergistic effect between materials is utilized to improve the efficiency of the catalytic capability and further amplify the sensing signal.
The gold nanoparticle functionalized reduced graphene oxide has large specific surface area and good biocompatibility, and can remarkably improve the Ab of the capture antibody1The solid loading amount of the electrode is reduced, and the electron transfer rate of an electrode interface is effectively accelerated; au with good conductivity and Cu with excellent catalytic performance under room temperature conditionxO grows uniformly on mesoporous CeO with large specific surface2Above, make AuCuxThe shape of the O composite material is further optimized, and Au and CeO are simultaneously utilized2And CuxAnd the signal amplification is realized by the synergistic action of O, so that the kit is applied to sensitive detection of the Alzheimer's disease marker.
Disclosure of Invention
The invention aims to provide a preparation method of an electrochemical immunosensor which has high sensitivity, strong specificity and quick detection and can be used for detecting Alzheimer's disease markers, and the prepared sensor can be used for the quick and sensitive detection of the Alzheimer's disease markers and the like. Based on the purpose, the prepared gold nanoparticle functionalized reduced graphene oxide material is adopted to modify the glassy carbon electrode, so that the specific surface area of the electrode is effectively improved, the loading capacity of an antibody is increased, and the electronic response speed of an electrode interface is greatly improved; then using AuCuxO@CeO2Immobilizing an alzheimer's disease marker detection antibody; and AuCuxO@CeO2The synergistic effect between the two obviously improves the catalytic capability of the composite material, and the signal amplification type electrochemical immunosensor constructed by the composite material realizes the sensitive detection of the Alzheimer's disease marker.
The technical scheme of the invention is as follows:
1. a preparation method of an electrochemical immunosensor for detecting Alzheimer's disease markers comprises the following steps:
1) preparation of gold nanoparticle functionalized reduced graphene oxide
Dissolving 5 ~ 20 mg of graphene oxide in 10 mL of ultrapure water, performing ultrasonic treatment for 60min, and adding 200 muL of 1% HAuCl4And 4 mL of 50 wt% thionine solution, stirring at room temperature for 12 h, carrying out centrifugal washing with ultrapure water for three times, and drying the obtained solid in a 60 ℃ vacuum freeze dryer for 10 ~ 14 h to obtain gold nanoparticle functionalized reduced graphene oxide;
2) Ab2-AuCuxO@CeO2preparation of hatching solution
0.4 ~ 0.6.6 g of Ce (NO) was taken3)3·6H2Dissolving O in 1 mL of mixed solvent, adding 15 mL of ethylene glycol, stirring for 60min, transferring the mixed solution into a 30 mL polytetrafluoroethylene reaction kettle, heating to 150 ~ 200 deg.C, and reactingThe time is 200 min; cooling to room temperature, centrifuging, washing the obtained precipitate with ultrapure water and anhydrous ethanol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h to obtain mesoporous CeO2；
The mixed solvent is prepared by mixing ultrapure water and absolute ethyl alcohol according to the volume ratio of 1: 1;
1 ~ 2 mg of the above-prepared CeO was taken2Dispersed in 20 mL of ultrapure water, sonicated for 1 h, and 200 uL of 1wt% HAuCl was added4Stirring for 3min, then adding 200 uL of 1wt% sodium citrate solution into the mixed solution, stirring for 5min, adding 15 ~ 25 mg of copper acetate, stirring for 15 min, adding 200 ~ 600 uL of 0.075 wt% sodium borohydride solution, stirring for 3 ~ 6h, washing the obtained precipitate with ultrapure water three times, drying for 12 h in a vacuum drying oven at 45 ℃ to obtain the AuCuxO@CeO2；
The 0.075 wt% sodium borohydride solution is prepared by dissolving 6 mg of sodium borohydride in 8 mL of 1wt% sodium citrate solution;
2 ~ 6 mg of AuCuxO@CeO2Dispersed into 2 mL of phosphate buffer solution with pH of 7.4, and added with 2 mL of Alzheimer's disease marker detection antibody Ab of 20 mug/mL2Carrying out shake incubation on the solution in a constant-temperature shake incubator at 4 ℃ for 12 h; ab was prepared by centrifugation, washed, and dispersed in 4 mL of 1.0 wt% bovine serum albumin solution having a pH of 7.02-AuCuxO@CeO2Detecting the antibody hatching substance solution, and storing the antibody hatching substance solution in a refrigerator at 4 ℃ for later use;
3) preparation of electrochemical immunosensor
(1) Al for glassy carbon electrode with diameter of 3 ~ 5 mm2O3Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 6 muL of gold nanoparticle functionalized reduced graphene oxide solution of 0.5 ~ 2.0.0 mg/mL to the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the antibody Ab with the disease marker of 6 muL and 5 ~ 15 mug/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding a 3 mu L and 0.1 ~ 1.0.0% BSA solution to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(5) dropwise adding 6 muL of disease marker antigen solution of 0.0001 ~ 50ng/mL, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) detecting antibody hatching Ab with markers of 6 muL and 1 ~ 3 mg/mL2-AuCuxO@CeO2Dripping the solution on the surface of the electrode, placing the electrode in a refrigerator at 4 ℃ for incubation for 60min, washing the surface of the electrode with ultrapure water, and drying the electrode in the refrigerator at 4 ℃ to prepare the electrochemical immunosensor for detecting the Alzheimer's disease marker.
The preparation method of the electrochemical immunosensor for detecting the Alzheimer's disease marker comprises AuCuxO@CeO2Is a composite nano material for amplifying the immobilized detection antibody signal.
According to the preparation method of the electrochemical immunosensor for detecting the Alzheimer's disease marker, the gold nanoparticle functionalized reduced graphene oxide is used as a substrate material and is synthesized by adopting an in-situ reduction method.
Detecting disease markers by the following steps:
(1) an electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the test is carried out in 10 mL of 50 mmol/L phosphate buffer solution with the pH value of 5.10 ~ 7.98.98;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tended to be stable, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution with pH = 7.4 every 50 s, and the change in current was recorded.
(4) The beta-amyloid serum solution to be detected replaces a beta-amyloid antigen standard solution for detection, and the electrochemical immunosensor for detecting the Alzheimer's disease marker is used for detecting the beta-amyloid, the detection range is 0.0001-50ng/mL, and the detection limit is 36 fg/mL.
Advantageous results of the invention
(1) The gold nanoparticle functionalized reduced graphene oxide synthesized by the invention is a layered nano composite material, has large specific surface area and more excellent conductivity, contains a large amount of hydrophilic groups on the surface, can be effectively combined with amino groups on an antibody to realize immobilization of the antibody, the gold nanoparticles have good conductivity and good biocompatibility, a large number of antibodies can be combined through coordination bonds, the gold nanoparticle functionalized reduced graphene oxide can accelerate electron transfer and immobilize a large number of antibodies, has important function for improving the sensitivity of the sensor, and during the reduction of gold, the weak reduction electroactive substance thionine can ensure that the gold is uniformly distributed on the surface of the graphene, meanwhile, the conductivity of the material can be further improved, and the method has important significance for realizing high sensitivity and low detection limit of the sensor;
(2) the invention synthesizes nano composite material AuCuxO@CeO2Constructing a sandwich type electrochemical immunosensor as a detection antibody marker; mesoporous CeO2Has large surface area and good catalytic performance, and the existence of the catalyst can limit the AuCuxGrowth of O nanoparticles to avoid AuCuxIrregular growth of O;
(3) AuCu synthesized by the inventionxO@CeO2The nano material has good synergistic effect, effectively promotes electron transfer, and improves the catalytic performance of the material, thereby realizing high detection sensitivity;
(4) using nanocomposite material AuCuxO@CeO2Directly combining with a marker detection antibody to construct an enzyme-free immunosensor, thereby avoiding detection errors caused by inactivation or leakage of enzyme; meanwhile, the high catalytic performance of the composite material is utilized to realize signal amplification, so that the detection sensitivity of the electrochemical sensor is greatly improved, and the method has important scientific significance and application value;
(5) the detection range of the sandwich type electrochemical immunosensor prepared by the invention on the Alzheimer's disease marker beta-amyloid is 100 fg/mL ~ 50ng/mL, and the detection limit is 36 fg/mL.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
Embodiment 1 a method for preparing an electrochemical immunosensor for detecting alzheimer's disease markers, comprising the following steps:
1) preparation of gold nanoparticle functionalized reduced graphene oxide
Dissolving 5 mg of graphene oxide in 10 mL of ultrapure water, performing ultrasonic treatment for 60min, and adding 200 muL of 1% HAuCl4And 4 mL of a thionine solution with the concentration of 50 wt%, stirring at room temperature for 12 h, carrying out centrifugal washing with ultrapure water for three times, and drying the obtained solid in a 60 ℃ vacuum freeze dryer for 10 h to obtain gold nanoparticle functionalized reduced graphene oxide;
2) Ab2-AuCuxO@CeO2preparation of hatching solution
0.4 g of Ce (NO) was taken3)3·6H2Dissolving O in 1 mL of mixed solvent, adding 15 mL of ethylene glycol, stirring for 60min, transferring the mixed solution into a 30 mL polytetrafluoroethylene reaction kettle, heating to 150 ℃, and reacting for 200 min; cooling to room temperature, centrifuging, washing the obtained precipitate with ultrapure water and anhydrous ethanol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h to obtain mesoporous CeO2；
The mixed solvent is prepared by mixing ultrapure water and absolute ethyl alcohol according to the volume ratio of 1: 1;
1 mg of the above-obtained CeO was taken2Dispersed in 20 mL of ultrapure water, sonicated for 1 h, and 200 uL of 1wt% HAuCl was added4Stirring for 3min, then adding 200 uL of 1wt% sodium citrate solution into the mixed solution, stirring for 5min, adding 15 mg of copper acetate, stirring for 15 min, adding 200 uL of 0.075 wt% sodium borohydride solution into the mixed solution, stirring for 3h, and washing the obtained precipitate with ultrapure water for three times; drying in a vacuum drying oven at 45 ℃ for 12 h to obtain AuCuxO@CeO2；
The 0.075 wt% sodium borohydride solution is prepared by dissolving 6 mg of sodium borohydride in 8 mL of 1wt% sodium citrate solution;
2 mg of AuCuxO@CeO2Dispersed into 2 mL of phosphate buffer solution with pH of 7.4, and added with 2 mL of Alzheimer's disease marker detection antibody Ab of 20 mug/mL2Carrying out shake incubation on the solution in a constant-temperature shake incubator at 4 ℃ for 12 h; ab was prepared by centrifugation, washed, and dispersed in 4 mL of 1.0 wt% bovine serum albumin solution having a pH of 7.02-AuCuxO@CeO2Detecting the antibody hatching substance solution, and storing the antibody hatching substance solution in a refrigerator at 4 ℃ for later use;
3) preparation of electrochemical immunosensor
(1) Al for glassy carbon electrode with diameter of 3 mm2O3Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding a gold nanoparticle functionalized reduced graphene oxide solution of 6 mu L and 0.5 mg/mL to the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the antibody Ab with the disease marker of 6 muL and 5 mug/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding 3 mu L of 0.1% BSA solution to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(5) dropwise adding 6 muL of disease marker antigen solution of 0.0001 ~ 50ng/mL, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) detection of antibody hatching Ab with 6 muL and 1 mg/mL markers2-AuCuxO@CeO2Dripping the solution on the surface of the electrode, placing the electrode in a refrigerator at 4 ℃ for incubation for 60min, washing the surface of the electrode with ultrapure water, and drying the electrode in the refrigerator at 4 ℃ to prepare the electrochemical immunosensor for detecting the Alzheimer's disease marker.
Embodiment 2 a method for preparing an electrochemical immunosensor for detecting alzheimer's disease markers, comprising the following steps:
1) preparation of gold nanoparticle functionalized reduced graphene oxide
Dissolving 10 mg of graphene oxide in 10 mL of ultrapure water, performing ultrasonic treatment for 60min, and adding 200 muL of 1% HAuCl4And 4 mL of a thionine solution with the concentration of 50 wt%, stirring at room temperature for 12 h, carrying out centrifugal washing with ultrapure water for three times, and drying the obtained solid in a 60 ℃ vacuum freeze dryer for 12 h to obtain gold nanoparticle functionalized reduced graphene oxide;
2) Ab2-AuCuxO@CeO2preparation of hatching solution
0.5 g of Ce (NO) was taken3)3·6H2Dissolving O in 1 mL of mixed solvent, adding 15 mL of ethylene glycol, stirring for 60min, transferring the mixed solution into a 30 mL polytetrafluoroethylene reaction kettle, heating to 180 ℃, and reacting for 200 min; cooling to room temperature, centrifuging, washing the obtained precipitate with ultrapure water and anhydrous ethanol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h to obtain mesoporous CeO2；
The mixed solvent is prepared by mixing ultrapure water and absolute ethyl alcohol according to the volume ratio of 1: 1;
1.5 mg of the above-obtained CeO was taken2Dispersed in 20 mL of ultrapure water, sonicated for 1 h, and 200 uL of 1wt% HAuCl was added4Stirring for 3min, then adding 200 uL of 1wt% sodium citrate solution into the mixed solution, stirring for 5min, adding 20 mg of copper acetate, stirring for 15 min, adding 400 uL of 0.075 wt% sodium borohydride solution into the mixed solution, stirring for 4.5h, and washing the obtained precipitate with ultrapure water for three times; drying in a vacuum drying oven at 45 ℃ for 12 h to obtain AuCuxO@CeO2；
The 0.075 wt% sodium borohydride solution is prepared by dissolving 6 mg of sodium borohydride in 8 mL of 1wt% sodium citrate solution;
4 mg of AuCuxO@CeO2Dispersed into 2 mL of phosphate buffer solution with pH of 7.4, and added with 2 mL of Alzheimer's disease marker detection antibody Ab of 20 mug/mL2Carrying out shake incubation on the solution in a constant-temperature shake incubator at 4 ℃ for 12 h; centrifuged and dispersed in 4 mL of 1.0 wt% pAb is prepared from H7.0 bovine serum albumin solution2-AuCuxO@CeO2Detecting the antibody hatching substance solution, and storing the antibody hatching substance solution in a refrigerator at 4 ℃ for later use;
3) preparation of electrochemical immunosensor
(1) Al for glassy carbon electrode with diameter of 4mm2O3Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 6 mu L of gold nanoparticle functionalized reduced graphene oxide solution to the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the antibody Ab with the disease marker of 6 mu L and 10 mu g/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding 3 mu L of 0.6% BSA solution to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(5) dropwise adding 6 muL of disease marker antigen solution of 0.0001 ~ 50ng/mL, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) detection of antibody hatching Ab with 6 muL and 2 mg/mL markers2-AuCuxO@CeO2Dripping the solution on the surface of the electrode, placing the electrode in a refrigerator at 4 ℃ for incubation for 60min, washing the surface of the electrode with ultrapure water, and drying the electrode in the refrigerator at 4 ℃ to prepare the electrochemical immunosensor for detecting the Alzheimer's disease marker.
Embodiment 3 a method for preparing an electrochemical immunosensor for detecting alzheimer's disease markers, comprising the following steps:
1) preparation of gold nanoparticle functionalized reduced graphene oxide
Dissolving 20 mg of graphene oxide in 10 mL of ultrapure water, performing ultrasonic treatment for 60min, and adding 200 muL of 1% HAuCl4And 4 mL of 50 wt% thionine solution, stirring at room temperature for 12 h, carrying out centrifugal washing with ultrapure water for three times, drying the obtained solid in a 60 ℃ vacuum freeze dryer for 14 h, and obtaining the gold nanoparticle functionalized reduced oxidized stone(ii) a graphene;
2) Ab2-AuCuxO@CeO2preparation of hatching solution
0.6 g of Ce (NO) was taken3)3·6H2Dissolving O in 1 mL of mixed solvent, adding 15 mL of ethylene glycol, stirring for 60min, transferring the mixed solution into a 30 mL polytetrafluoroethylene reaction kettle, heating to 200 ℃, and reacting for 200 min; cooling to room temperature, centrifuging, washing the obtained precipitate with ultrapure water and anhydrous ethanol for three times, and drying in a vacuum drying oven at 50 ℃ for 12 h to obtain mesoporous CeO2；
The mixed solvent is prepared by mixing ultrapure water and absolute ethyl alcohol according to the volume ratio of 1: 1;
2 mg of the above-obtained CeO was taken2Dispersed in 20 mL of ultrapure water, sonicated for 1 h, and 200 uL of 1wt% HAuCl was added4Stirring for 3min, then adding 200 uL of 1wt% sodium citrate solution into the mixed solution, stirring for 5min, adding 25 mg of copper acetate, stirring for 15 min, adding 600 uL of 0.075 wt% sodium borohydride solution into the mixed solution, stirring for 6h, and washing the obtained precipitate with ultrapure water for three times; drying in a vacuum drying oven at 45 ℃ for 12 h to obtain AuCuxO@CeO2；
The 0.075 wt% sodium borohydride solution is prepared by dissolving 6 mg of sodium borohydride in 8 mL of 1wt% sodium citrate solution;
6 mg of AuCuxO@CeO2Dispersed into 2 mL of phosphate buffer solution with pH of 7.4, and added with 2 mL of Alzheimer's disease marker detection antibody Ab of 20 mug/mL2Carrying out shake incubation on the solution in a constant-temperature shake incubator at 4 ℃ for 12 h; ab was prepared by centrifugation, washed, and dispersed in 4 mL of 1.0 wt% bovine serum albumin solution having a pH of 7.02-AuCuxO@CeO2Detecting the antibody hatching substance solution, and storing the antibody hatching substance solution in a refrigerator at 4 ℃ for later use;
3) preparation of electrochemical immunosensor
(1) Al for glassy carbon electrode with diameter of 5 mm2O3Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 6 mu L of gold nanoparticle functionalized reduced graphene oxide solution to the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the antibody Ab with the disease marker of 6 muL and 15 mug/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding 3 mu L of 1.0% BSA solution to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(5) dropwise adding 6 muL of disease marker antigen solution of 0.0001 ~ 50ng/mL, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) detection of antibody hatching Ab with markers of 6 muL and 3 mg/mL2-AuCuxO@CeO2Dripping the solution on the surface of the electrode, placing the electrode in a refrigerator at 4 ℃ for incubation for 60min, washing the surface of the electrode with ultrapure water, and drying the electrode in the refrigerator at 4 ℃ to prepare the electrochemical immunosensor for detecting the Alzheimer's disease marker.
Example 4 detection of disease markers, the procedure was as follows:
(1) an electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the test is carried out in 10 mL of 50 mmol/L phosphate buffer solution with the pH value of 5.10 ~ 7.98.98;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tends to be stable, 10 mu L of 5 mol/L hydrogen peroxide solution is injected into 10 mL of 50 mmol/L phosphate buffer solution with pH = 7.4 every 50 s, and the current change is recorded;
(4) and (3) replacing the beta-amyloid antigen standard solution with the beta-amyloid serum solution to be detected for detection, and determining that the linear range of the beta-amyloid is 0.0001-50ng/mL and the detection limit is 36 fg/mL by adopting a standard curve method.

Claims (5)

1. A preparation method of an electrochemical immunosensor for detecting Alzheimer's disease markers is characterized by comprising the following preparation steps:
1) preparation of gold nanoparticle functionalized reduced graphene oxide
Dissolving 5 ~ 20 mg of graphene oxide in 10 mL of ultrapure water, performing ultrasonic treatment for 60min, and adding 200 muL of 1% HAuCl4And 4 mL of 50 wt% thionine solution, stirring at room temperature for 12 h, carrying out centrifugal washing with ultrapure water for three times, and drying the obtained solid in a 60 ℃ vacuum freeze dryer for 10 ~ 14 h to obtain gold nanoparticle functionalized reduced graphene oxide;
2) Ab2-AuCuxO@CeO2preparation of hatching solution
0.4 ~ 0.6.6 g of Ce (NO) was taken3)3·6H2Dissolving O in 1 mL of mixed solvent, adding 15 mL of ethylene glycol, stirring for 60min, transferring the mixed solution into a 30 mL polytetrafluoroethylene reaction kettle, heating to 150 ~ 200 ℃, reacting for 200min, cooling to room temperature, performing centrifugal separation, washing the obtained precipitate with ultrapure water and absolute ethyl alcohol for three times in sequence, and drying in a vacuum drying oven at 50 ℃ for 12 h to obtain the mesoporous CeO2；
The mixed solvent is prepared by mixing ultrapure water and absolute ethyl alcohol according to the volume ratio of 1: 1;
1 ~ 2 mg of the above-prepared CeO was taken2Dispersed in 20 mL of ultrapure water, sonicated for 1 h, and 200 uL of 1wt% HAuCl was added4Stirring for 3min, then adding 200 uL of 1wt% sodium citrate solution into the mixed solution, stirring for 5min, adding 15 ~ 25 mg of copper acetate, stirring for 15 min, adding 200 ~ 600 uL of 0.075 wt% sodium borohydride solution, stirring for 3 ~ 6h, washing the obtained precipitate with ultrapure water three times, drying for 12 h in a vacuum drying oven at 45 ℃ to obtain the AuCuxO@CeO2；
The 0.075 wt% sodium borohydride solution is prepared by dissolving 6 mg of sodium borohydride in 8 mL of 1wt% sodium citrate solution;
2 ~ 6 mg of AuCuxO@CeO2Dispersed into 2 mL of phosphorus at pH 7.42 mL of 20. mu.g/mL of Alzheimer's disease marker detection antibody Ab was added to the acid salt buffer solution2Carrying out shake incubation on the solution in a constant-temperature shake incubator at 4 ℃ for 12 h; ab was prepared by centrifugation, washed, and dispersed in 4 mL of 1.0 wt% bovine serum albumin solution having a pH of 7.02-AuCuxO@CeO2Detecting the antibody hatching substance solution, and storing the antibody hatching substance solution in a refrigerator at 4 ℃ for later use;
3) preparation of electrochemical immunosensor
(1) Al for glassy carbon electrode with diameter of 3 ~ 5 mm2O3Polishing the polishing powder, and cleaning with ultrapure water;
(2) dropwise adding 6 muL of gold nanoparticle functionalized reduced graphene oxide solution of 0.5 ~ 2.0.0 mg/mL to the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the antibody Ab with the Alzheimer's disease marker of 6 muL and 5 ~ 15 mug/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding a 3 mu L and 0.1 ~ 1.0.0% BSA solution to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(5) dropwise adding 6 muL of Alzheimer's disease marker antigen solution of 0.0001 ~ 50ng/mL, washing the surface of the electrode with ultrapure water, and drying in a refrigerator at 4 ℃;
(6) detecting antibody hatching Ab with 6 mu L and 1 ~ 3 mg/mL of Alzheimer's disease marker2-AuCuxO@CeO2Dripping the solution on the surface of the electrode, placing the electrode in a refrigerator at 4 ℃ for incubation for 60min, washing the surface of the electrode with ultrapure water, and drying the electrode in the refrigerator at 4 ℃ to prepare the electrochemical immunosensor for detecting the Alzheimer's disease marker.
2. The method of claim 1, wherein the AuCu is AuCu, and the method comprises the step of preparing an electrochemical immunosensor for detecting the Alzheimer's disease markerxO@CeO2Is a composite nano material for amplifying the immobilized detection antibody signal.
3. The method for preparing an electrochemical immunosensor for detecting Alzheimer's disease markers according to claim 1, wherein the gold nanoparticle functionalized reduced graphene oxide is synthesized by an in-situ reduction method as a substrate material.
4. The electrochemical immunosensor for detecting alzheimer's disease markers, prepared by the preparation method according to claim 1, wherein the electrochemical immunosensor is used for detecting disease markers, and the detection steps are as follows:
(1) an electrochemical workstation is used for testing in a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the test is carried out in 10 mL of 50 mmol/L phosphate buffer solution with the pH value of 5.10 ~ 7.98.98;
(2) detecting the analyte by a time-current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tended to be stable, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution with pH = 7.4 every 50 s, and the change in current was recorded.
5. The electrochemical immunosensor for detecting an alzheimer's disease marker, prepared according to the preparation method of claim 1, wherein the electrochemical immunosensor is used for detecting a disease marker, and the disease marker is amyloid beta.
CN201811315614.3A 2018-11-07 2018-11-07 Preparation method and application of electrochemical immunosensor for detecting Alzheimer's disease marker Expired - Fee Related CN109115855B (en)

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