CN111060569B - Electrochemical aptamer sensor for detecting Alzheimer's disease marker, and preparation method and application thereof - Google Patents
Electrochemical aptamer sensor for detecting Alzheimer's disease marker, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an electrochemical aptamer sensor for detecting Alzheimer's disease markers, and a preparation method and application thereof, and the electrochemical aptamer sensor specifically comprises the following steps: co-depositing a layer of AuPt nanoflower structure on the surface of clean ITO in an electrodeposition mode, wherein the size of AuPt nanoflowers is about 80-800 nm; secondly, by designing a super-hydrophilic micro-well structure, the function of enriching target object amplification signals is achieved; and thirdly, after the aptamer is combined with the detection target object, the structure is changed, so that the surface current of the electrode is changed, and the change of the surface current of the electrode is detected through DPV, so that the concentration of the marker is obtained. The prepared electrochemical aptamer sensor can be used for detecting Alzheimer's disease markers; has the characteristics of strong specificity, high sensitivity, low detection limit and lower price compared with antibody detection.
Description
Technical Field
The invention belongs to the technical field of electrochemical biosensing, and particularly relates to an electrochemical aptamer sensor for detecting Alzheimer's disease markers and a preparation method and application thereof.
Background
Alzheimer's Disease (AD) is one of the most common forms of senile dementia, accounting for about 60% of senile dementia. The clinical symptoms mainly comprise hypomnesis, language disorder, mental retardation, thought retardation and the like, and even death can be caused in severe cases. AD is a very complex neurodegenerative disease, and the aggregation of extracellular β -amyloid peptide (a β) to form amyloid plaques is an important pathological feature of AD. Research has found that aggregation of a β to form oligomers and fibers are important in the pathogenesis of AD. Furthermore, a β oligomers are highly toxic to nerve cells. Therefore, detecting the content of a β oligomers in blood is of great significance for the early diagnosis of AD.
The detection of the content of a β oligomers in blood is of great significance for the prevention and early diagnosis of alzheimer's disease. At present, a plurality of methods for detecting Alzheimer's disease markers are available, such as radioimmunoassay, electrochemiluminescence, colorimetry and the like, but most of the detection methods are complicated, complex to operate, expensive in cost or high in detection limit, so that the establishment of a rapid, simple, convenient and sensitive detection method is of great significance.
At present, electrochemical aptamer sensors are widely used for detecting disease markers, and compared with immunosensors, aptamers have the advantage of high specificity which is equivalent to that of antibodies, and are cheaper and more easily obtained than the antibodies. The aptamer sensor has the advantages of high sensitivity, high specificity, high detection speed and the like, and has important application value in the fields of clinical detection, environmental detection, food safety control, biological analysis and the like.
Disclosure of Invention
The invention aims to provide an electrochemical aptamer sensor for detecting Alzheimer's disease markers, and a preparation method and application thereof.
Based on the purpose, the invention adopts the following technical scheme:
a gold and platinum co-deposited AuPt nanoflower structure is prepared by the following steps:
preparing an AuPt nanoflower structure by an electrodeposition method: respectively preparing sulfuric acid solutions of chloroauric acid and chloroplatinic acid with equal concentrations, mixing the solutions in equal volumes, depositing for 300-1200 s by using a three-electrode system, conductive glass (ITO) as a working electrode, Ag/AgCl as a reference electrode and a platinum sheet electrode as an auxiliary electrode and using a voltage of-0.6V to-0.1V, and thus growing a gold-platinum nanoflower structure on the surface of the conductive glass. Further, the preparation process of the sulfuric acid solution of the chloroauric acid or the chloroplatinic acid is as follows: dissolving chloroauric acid or chloroplatinic acid in 0.5M sulfuric acid to prepare a solution of 1 mg/mL-10 mg/mL. The whole size of the AuPt nanoflower structure is 80-800 nm; and the gold and platinum on the surface of the nanoflower are uniformly distributed.
A method for preparing an electrochemical aptamer sensor for detecting Alzheimer's disease markers by using the AuPt nanoflower structure comprises the following steps:
(1) and (3) cleaning of ITO: cutting the ITO glass into the size of 1cm multiplied by 2cm, then ultrasonically cleaning the ITO glass for 10min by using acetone, ethanol and ultrapure water in sequence, and finally drying the ITO glass by using nitrogen for later use;
(2) washing the conductive glass deposited with the chloroauric acid and the chloroplatinic acid by using ultrapure water, and airing;
(3) soaking the ITO electrode obtained in the step (2) in n-dodecyl mercaptan for 20-30 hours;
(4) then covering the ITO electrode treated in the step (3) by using a photomask plate, and then using O2plasma treatment (100W, 60 s) to obtain a super-hydrophilic micro-well;
(5) dropwise adding an Abeta oligomer aptamer with sulfydryl into the super-hydrophilic microwell on the surface of the ITO electrode obtained in the step (4), reacting for 1h at 37 ℃, washing with phosphate buffer solution, and airing;
(6) dropwise adding a Mercaptohexanol (MCH) solution for sealing, inoculating for 1h, then washing the surface of the electrode by using a phosphate buffer solution, and airing;
(7) 0.05-2000 nM disease marker Abeta oligomer solution is dripped, inoculated for 1h, washed on the surface of an electrode by phosphate buffer solution and dried to prepare the electrochemical aptamer sensor for detecting the Alzheimer's disease marker, and the DNA sequence of the mercapto Abeta oligomer aptamer is 5 ' -SH- (CH2)6-GCCTGTGGTGTTGGGGCGGGTGCG-3 '.
Further, the aperture of the photomask plate in the step (4) is 2 mm; the concentration of the aptamer in the step (5) is 10 mu M, the dosage of the aptamer is 5 mu L, and the concentration of the MCH solution in the step (6) is 10 mu M, and the dosage of the MCH solution is 10 mu L; the dosage of the disease marker A beta oligomer solution in the step (7) is 5 mu L.
Said O is2Performing plasma etching (i.e. O)2plasma treatment), O2The flow rate is 80mL/min, the oxygen pressure is 101325Pa, the etching temperature is room temperature, the power of the plasma cleaner is 100W, and the processing time is 60 s.
Further, the inoculation or incubation means that the incubation is carried out at 37 ℃ for 1h, and the airing is carried out in a refrigerator at 4 ℃.
The application of the electrochemical aptamer sensor in detecting the Alzheimer's disease marker comprises the following steps:
(1) testing by using an electrochemical workstation in a three-electrode system, taking an Ag/AgCl electrode as a reference electrode, taking a platinum sheet electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 20 mL of 10 mM phosphate buffer solution with the pH value of 6.0-7.8;
(2) detecting the concentration of the known series of A beta oligomers by using a square wave voltammetry, wherein the input voltage range is 0.0-0.4V, and recording the change of peak current;
(3) drawing a standard curve by taking LgC as an abscissa and Δ I as an ordinate to obtain a standard curve equation Δ I = 12.008 LgC +153.13, wherein C is the concentration of the a β oligomer, and Δ I is the difference value of the peak current;
(4) and (3) testing the change of the peak current of the sample to be tested according to the step (1) and the step (2), substituting the change into a standard curve equation to obtain the concentration of the A beta oligomer, wherein when the square wave voltammetry is used for detecting different concentrations of the A beta oligomer, the input voltage range is 0.0V-0.4V.
The invention has the following beneficial results:
(1) the AuPt nanoflower structure prepared by the invention has large specific surface area and electron transmission capability, and can be combined with an aptamer through a gold-sulfur bond to capture Abeta oligomers; the AuPt nanoflower has important significance for improving the sensitivity of the sensor.
(2) The size of the AuPt nanoflower structure is about 80-800 nm, and the electrical activity area is greatly increased by the flower-shaped structure.
(3) The sensor adopts the aptamer to capture the target object, and the cost is greatly reduced compared with the sensor using the antibody.
(4) The aptamer electrochemical sensor prepared by the invention has the detection range of 0.05 pg/mL-10 ng/mL on the A beta oligomer of the Alzheimer's disease marker, and the detection limit is 0.05 pg/mL.
(5) The electrochemical aptamer sensor constructed by the invention is a detection device for determining the concentration of a marker based on the change value of the peak current of the DPV, and has the following advantages: firstly, a layer of AuPt nanoflower structure grows on the surface of ITO, so that the specific surface area of a substrate material is greatly increased, and the current is increased; secondly, by designing a super-hydrophilic micro-well, the purpose of enriching and amplifying signals is achieved; thirdly, the detection is carried out through electric signals, and the method is simple and convenient; thirdly, aptamers are more readily available and less expensive than antibodies.
Drawings
Fig. 1 is a scanning electron microscope photograph of AuPt nanoflowers prepared in example 1 of the present invention;
FIG. 2 is a spectrum analysis of the AuPt nanoflower prepared in example 1 of the present invention, wherein the red dots represent the distribution of gold, and the green dots represent the distribution of platinum;
FIG. 3 is a calibration curve of a sensor prepared in example 1 of the present invention.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
The phosphate buffer in the following examples was 7.4.
Example 1
A preparation method of an electrochemical aptamer sensor for detecting Alzheimer's disease markers comprises the following steps:
1. the preparation method of the AuPt nanoflower structure with gold and platinum codeposited comprises the following specific steps:
preparing an AuPt nanoflower structure by an electrodeposition method: preparing sulfuric acid solution (0.5M) of chloroauric acid (10mg/mL) and chloroplatinic acid (10mg/mL), mixing in equal volumes, depositing for 300s by using a three-electrode system, conductive glass (ITO) as a working electrode, Ag/AgCl as a reference electrode and a platinum sheet electrode as an auxiliary electrode and using-0.2V voltage, so that a gold-platinum nanoflower structure can grow on the surface of the ITO, wherein the appearance of the gold-platinum nanoflower structure is shown in figure 1, and the overall size of the nanoflower structure is about 80-800 nm as can be seen from figure 1; according to the energy spectrum analysis chart (figure 2) of the AuPt nanoflower structure, red dots in figure 2 represent the distribution of gold, green dots represent the distribution of platinum, and red green is basically uniformly distributed in the chart, which shows that gold and platinum on the surface of the nanoflower are uniformly distributed.
2. Preparing an aptamer sensor based on an AuPt nanoflower structure:
(1) and (3) cleaning of ITO: cutting ITO glass into a size of 1cm multiplied by 2cm, then sequentially ultrasonically cleaning the ITO glass for 10min by acetone, ethanol and ultrapure water, and finally drying the ITO glass by nitrogen for later use;
(2) then washing the ITO glass deposited with the AuPt nanoflower structure prepared in the step (1) with ultrapure water, and airing;
(3) soaking the ITO electrode obtained in the step (2) in n-dodecyl mercaptan for 24 hours;
(4) then, a photomask plate (the aperture size of the photomask plate is 2 mm) is used for covering the ITO electrode processed in the step (3), and then O is used2plasma in O2The flow rate is 80mL/min, the oxygen pressure is 101325Pa, the power of the plasma cleaning machine is 100W, and the super-hydrophilic microwell is obtained after room temperature treatment for 60 s;
(5) dripping 10 μ L and 10 μ M of A β oligomer (β amyloid protein) aptamer with sulfhydryl (oligomer aptamer sequence shown in figure 1) into the super-hydrophilic microwell on the surface of the ITO electrode obtained in the step (4), reacting for 1h at 37 ℃, washing with phosphate buffer, and drying in the air at 4 ℃ in a refrigerator;
(6) dropwise adding Mercaptohexanol (MCH) solution for sealing, inoculating for 1h at 37 ℃, then washing the surface of the electrode by using phosphate buffer, and drying in the air in a refrigerator at 4 ℃;
(7) 5 mu L of 10 nM disease marker Abeta oligomer solution is dripped, inoculation is carried out for 1h at 37 ℃, the surface of an electrode is washed by phosphate buffer, and the electrode is dried in a refrigerator at 4 ℃ to prepare the electrochemical aptamer sensor for detecting the Alzheimer disease marker.
Example 2
The detection of the Alzheimer's disease marker Abeta oligomer comprises the following steps:
(1) testing by using an electrochemical workstation and a three-electrode system, wherein an Ag/AgCl electrode is used as a reference electrode, a platinum sheet electrode is used as an auxiliary electrode, the prepared sensor is used as a working electrode, and the testing is carried out in 20 mL of 10 mM phosphate buffer solution with the pH value of 7.4;
(2) when square wave voltammetry (DPV) is used for detecting the concentration of different Abeta oligomers, the input voltage range is 0.0-0.4V;
(3) the change in peak current of the DPV was recorded.
Example 3
Performance analysis of the fabricated sensor
To evaluate the performance of the prepared electrochemical aptamer sensors, different concentrations of a β oligomer standards (concentrations of 0.05, 0.1, 1, 10, 100, in order) were prepared in phosphate buffer at pH 7.4,1000. 10000 pg/mL) prepared different aptamer sensors. The electrochemical workstation is used for testing by a three-electrode system, an Ag/AgCl electrode is used as a reference electrode, a platinum sheet electrode is used as an auxiliary electrode, the prepared sensor is a working electrode, the working electrode is tested in 20 mL of 10 mM phosphate buffer solution with the pH value of 7.4, the peak current change values corresponding to different concentration standard products are tested by a DPV method at the input voltage of 0.0-0.4V, and a standard curve is drawn (as shown in figure 3, delta I = 12.008 LgC +153.13, R is shown in the specification of the concentration of the standard products, and the standard product is prepared by the method of the preparation of the electrochemical workstation by using the electrochemical workstation and the method of the electrochemical method2=0.9928, C is the concentration of a β oligomers, Δ I is the difference in peak current) is shown in fig. 3. The detection limit of the sensor on the Alzheimer's disease marker Abeta oligomer is 0.05 pg/mL (the signal-to-noise ratio is 3), and the linear range is 0.05 pg/mL-10 ng/mL.
Example 4
Determination of recovery of Abeta oligomers in goat serum
To goat serum (ThermoFisher Scientific, diluted 1:10 in 10 mM PBS by goat serum to PBS) was added 5. mu.L of A.beta.oligomers (0 pg/mL, 10 pg/mL, 100 pg/mL, 1000 pg/mL), aptamer sensors were constructed according to the method of example 1 and assayed using the method of example 2. The results are shown in table 1, and the concentration of a β oligomers is calculated by substituting the standard curve after averaging in three independent experiments, and the error of the recovery rate obtained by comparing the calculated concentration with the added concentration is within 10%, which indicates that the specificity and recovery rate of the sensor prepared by the present invention are acceptable.
TABLE 1 assay of A β oligomers in goat serum
SEQUENCE LISTING
<110> yellow river science and technology institute
<120> aptamer sensor for detecting Alzheimer's disease marker, and preparation method and application thereof
<130> none
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213> Artificial design
<400> 1
gcctgtggtg ttggggcggg tgcg 24
Claims (8)
1.A method for preparing an electrochemical aptamer sensor for detecting Alzheimer's disease markers by using an AuPt nanoflower structure is characterized by comprising the following preparation steps:
(1) and (3) cleaning of ITO: cutting the ITO glass into a proper size, cleaning, and finally drying by using nitrogen for later use;
(2) washing the conductive glass deposited with the AuPt nanoflower structure with ultrapure water, and airing;
(3) soaking the conductive glass obtained in the step (2) in n-dodecyl mercaptan for 20-30 hours;
(4) then covering the ITO electrode treated in the step (3) by using a photomask plate, and then carrying out O treatment by using a plasma cleaning instrument2Etching with plasma to obtain super-hydrophilic micro-wells;
(5) dropwise adding an Abeta oligomer aptamer with sulfydryl into the super-hydrophilic microwell on the surface of the ITO electrode obtained in the step (4), washing with a phosphate buffer solution after the reaction is finished, and airing;
(6) dropwise adding mercaptohexanol solution for sealing, inoculating, then washing the surface of the electrode by using phosphate buffer solution, and airing;
(7) dropwise adding an Abeta oligomer solution with a certain concentration, inoculating, washing the surface of an electrode by using a phosphate buffer solution, and airing to prepare the electrochemical aptamer sensor for detecting the Alzheimer's disease marker; the DNA sequence of the mercapto-containing A beta oligomer aptamer is 5 '-SH- (CH2) 6-GCCTGTGGTGTTGGGGCGGGTGCG-3'
The preparation process of the AuPt nanoflower structure is as follows: respectively preparing sulfuric acid solutions of chloroauric acid and chloroplatinic acid with equal concentrations, mixing the solutions in equal volumes, depositing for 300-1200 s by using a three-electrode system, conductive glass as a working electrode, Ag/AgCl as a reference electrode and a platinum sheet electrode as an auxiliary electrode and using a voltage of-0.6V to-0.1V, and thus growing a gold-platinum nanoflower structure on the surface of the conductive glass.
2. The method for preparing an electrochemical aptamer sensor for detecting Alzheimer's disease markers by using the AuPt nanoflower structure according to claim 1, wherein the preparation process of the sulfuric acid solution of chloroauric acid or chloroplatinic acid is as follows: dissolving chloroauric acid or chloroplatinic acid in 0.5M sulfuric acid to prepare a solution of 1 mg/mL-10 mg/mL.
3. The method for preparing an electrochemical aptamer sensor for detecting markers of alzheimer's disease using AuPt nanoflower structures according to claim 1, wherein the whole size of the AuPt nanoflower structures is 80-800 nm; and the gold and platinum on the surface of the nanoflower are uniformly distributed.
4. The method for preparing an electrochemical aptamer sensor for detecting Alzheimer's disease markers, according to claim 1, wherein the aperture size of the photomask in the step (4) is 2 mm; the concentration of the aptamer in the step (5) is 10 mu M, the dosage is 10 mu L, and the concentration of the mercaptohexanol solution in the step (6) is 1mM, and the dosage is 10 mu L; the dosage of the disease marker A beta oligomer solution in the step (7) is 5 mu L, and the concentration of the A beta oligomer solution is 0.05 pg/mL-10 ng/mL.
5. The method for preparing an electrochemical aptamer sensor for detecting Alzheimer's disease marker according to claim 1, wherein O is2When plasma etching is performed, O2The flow rate is 80mL/min, the oxygen pressure is 101325Pa, the etching temperature is room temperature, the power of the plasma cleaner is 100W, and the processing time is 60 s.
6. The method for preparing an electrochemical aptamer sensor for detecting Alzheimer's disease markers, according to claim 1, wherein in the step (5), the reaction is kept at a constant temperature of 37 ℃ for 1h, the inoculation or incubation is kept at a constant temperature of 37 ℃ for 1h, and the airing is performed in a refrigerator at 4 ℃.
7. An electrochemical aptamer sensor produced by the production method according to claim 1.
8. Use of the electrochemical aptamer sensor according to claim 7 for detecting markers of alzheimer's disease, characterized by the following steps:
(1) testing by using an electrochemical workstation in a three-electrode system, taking an Ag/AgCl electrode as a reference electrode, taking a platinum sheet electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 20 mL of 10 mM phosphate buffer solution with the pH value of 6.0-7.8;
(2) detecting the concentration of the series of A beta oligomer standard substances by using a square wave voltammetry, wherein the input voltage range is 0.0-0.4V, and recording the change of peak current;
(3) drawing a standard curve by taking lgC as an abscissa and Delta I as an ordinate to obtain a standard curve equation Delta I = 12.008 LgC +153.13, wherein C is the concentration of the Abeta oligomer, and Delta I is the difference value of peak current;
(4) and (3) testing the change of the peak current of the sample to be tested according to the step (1) and the step (2), substituting the change into a standard curve equation to obtain the concentration of the A beta oligomer, wherein when the square wave voltammetry is used for detecting different concentrations of the A beta oligomer, the input voltage range is 0.0-0.4V.
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