阅读说明：本技术 基于滚环扩增dna酶和共价有机骨架的电化学-elisa免疫传感器 (electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework ) 是由 庞月红 郭露露 沈晓芳 于 2019-09-19 设计创作，主要内容包括：本发明公开了基于滚环扩增DNA酶和共价有机骨架的电化学-ELISA免疫传感器,属于电化学检测技术领域。本发明将电化学方法与酶联免疫吸附测定相结合,在酶标板上放大核酸信号,用功能纳米材料修饰电极,在0.5ng/mL～80ng/mL范围内还原峰的电信号与黄曲霉毒素M1的浓度具有线性相关性,相关系数为0.993,检测限为0.15ng/mL(S/N＝3)。本发明既能够避免传统电化学分析中电极的钝化,还能够有效地放大ELISA的信号,实现级联信号放大,降低检出限,实现痕量检测,建立了一种灵敏、高选择性、高通量的电化学酶联免疫分析方法。(The invention discloses an electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework, belonging to the technical field of electrochemical detection, wherein an electrochemical method is combined with enzyme-linked immunosorbent assay, a nucleic acid signal is amplified on an enzyme-linked immunosorbent assay, an electrode is modified by a functional nano material, and an electric signal of a reduction peak in the range of 0.5 ng/mL-80 ng/mL has linear correlation with the concentration of aflatoxin M1, the correlation coefficient is 0.993, and the detection limit is 0.15ng/mL (S/N is 3).)

The electrochemical-ELISA immunosensor is characterized in that rolling circle amplification is carried out in an ELISA plate modified by a primer-gold nanoparticle AuNPs-aptamer compound/antigen target/antibody sandwich structure, an amplification product is folded to form DNA enzyme, then an o-aminophenol solution is added into the ELISA plate, and simultaneously an electrode modified by a covalent organic framework nano material is immersed into the solution.

2. The electrochemical-ELISA immunosensor of claim 1, wherein the covalent organic framework nanomaterials are COFs-TpBD.

3. The electrochemical-ELISA immunosensor according to claim 1 or 2, wherein the preparation method of the primer-gold nanoparticle-aptamer complex/antigen target/antibody sandwich structure modified microplate comprises the following steps:

(1) preparation of primer-AuNPs-aptamer complex: preparing a primer-AuNPs-aptamer compound by using AuNPs, a thiol-containing aptamer nucleic acid chain and a thiol-containing primer;

(2) preparing a primer-AuNPs-aptamer/antigen target/antibody sandwich structure modified enzyme label plate: adding an antigen target substance on an ELISA plate containing a corresponding antibody, and then adding a primer-AuNPs-aptamer complex to form the primer-AuNPs-aptamer/antigen target substance/antibody sandwich structure modified ELISA plate.

4. The electrochemical-ELISA immunosensor of any one of claims 1-3 to , wherein the rolling circle amplification comprises steps of adding the circular template, T4DNA ligase, T4DNA ligase buffer and water to the primer-AuNPs-aptamer/antigen target/antibody modified ELISA plate for room temperature incubation, washing the plate after incubation is completed, adding phi29DNA polymerase buffer, dNTPs, BSA, water and phi29DNA polymerase to the ELISA plate for rolling circle amplification reaction to obtain long single-stranded DNA, and finally adding heme and potassium ions to the ELISA plate for folding to form DNase.

5. The electrochemical-ELISA immunosensor of any one of claims 1-4 to , wherein the covalent organic framework nanomaterial-modified electrode is obtained by applying a solution containing the covalent organic framework nanomaterial onto the surface of the electrode by dropping and drying.

6. The electrochemical-ELISA immunosensor of any one of claims 1-5 to , wherein the molar ratio of the thiol-containing aptamer nucleic acid strand to the thiol-containing primer in step (1) is (2-7): 1.

7. The electrochemical-ELISA immunosensor of any one of claims 1-6 to , wherein the primer-AuNPs-aptamer complex is prepared using Au-S bonds formed between AuNPs and thiol-modified aptamer nucleic acid chains.

8, electrochemical-ELISA detection method, characterized in that, the method is to use the electrochemical-ELISA immunosensor of any of claims 1-7 to to detect antigen targets.

9. The detection method according to claim 8, characterized in that it comprises the steps of:

(1) establishing a standard curve, namely constructing the electrochemical-ELISA immunosensor of any in claims 1-7, and measuring corresponding reduction peak current values, which are correspondingly marked as I, by using standard samples of target antigens with different concentrations_iThe reduction peak current value of the blank sample containing no target antigen was designated as I₀(ii) a With I_i/I₀Build a linear relationship with the concentration of the antigen of interest, i.e.Obtaining a standard curve;

(2) measuring a corresponding reduction peak current value by using a sample to be measured; and (3) calculating according to the standard curve obtained in the step (1) to obtain the content of the antigen target in the sample to be detected.

10, method for detecting the content of aflatoxin M1, which is characterized by comprising the following steps:

(1) constructing the electrochemical-ELISA immunosensor of any one of claims 1-7 and , measuring the corresponding reduction peak current values, respectively marked as I, by using aflatoxin M1 standard samples with different concentrations_i(ii) a The reduction peak current value of the blank sample without aflatoxin M1 is recorded as I₀(ii) a With I_i/I₀Constructing a linear relation with the concentration of the aflatoxin M1 to obtain a standard curve of the aflatoxin M1;

(2) and (3) measuring a corresponding reduction peak current value by using a sample to be measured with unknown aflatoxin M1 content, and obtaining the content of aflatoxin M1 in the sample to be measured according to the standard curve obtained in the step (1).

Technical Field

The invention relates to an electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic frameworks, and belongs to the technical field of electrochemical detection.

Background

Enzyme-linked immunosorbent assay (ELISA) relies on specific recognition of antigen and antibody, has the advantages of strong specificity, good stability, simple operation and the like, has been widely applied to the fields of food analysis, environment, medicine and the like, and becomes a gold standard for simultaneous detection of various samples, however, the traditional ELISA usually adopts biological enzyme (usually horseradish peroxidase or alkaline phosphatase) as a catalyst to catalyze the generation of colored substances, and uses a spectrophotometer to read signals, which limits the detection sensitivity and cannot meet the detection requirements for trace substances.

Therefore, there is an urgent need to develop methods that are accurate, sensitive, and suitable for trace species detection.

Disclosure of Invention

In order to solve the problems, the invention provides electrochemical-ELISA immunosensors based on rolling circle amplification DNA enzyme and covalent organic frameworks, which combines an electrochemical method with enzyme-linked immunosorbent assay, amplifies nucleic acid signals on an ELISA plate, modifies electrodes by functional nano materials, can avoid passivation of the electrodes in the traditional electrochemical immunoassay, can effectively amplify ELISA signals, reduces detection limit and realizes trace detection.

The th purpose of the invention is to provide electrochemical-ELISA immunosensor, which is characterized in that rolling circle amplification is carried out in an ELISA plate modified by a primer-gold nanoparticle-aptamer compound/antigen target/antibody sandwich structure, an amplification product is folded to form DNA enzyme, then the DNA enzyme is added into a solution containing o-aminophenol, and simultaneously an electrode modified by a covalent organic framework nano material is also immersed into the solution.

In embodiments of the invention, the covalent organic framework nanomaterial is COFs-TpBD.

In embodiments of the present invention, the preparation method of the primer-gold nanoparticles (AuNPs) -aptamer complex/antigen target/antibody sandwich structure modified elisa plate for rolling circle amplification comprises the following steps:

(1) preparation of primer-AuNPs-aptamer complex: preparing a primer-AuNPs-aptamer compound by using AuNPs, a sulfhydryl-containing aptamer and a sulfhydryl-containing primer;

(2) preparing a primer-AuNPs-aptamer/antigen target/antibody sandwich structure modified enzyme label plate: adding an antigen target substance on an ELISA plate containing a corresponding antibody, and then adding a primer-AuNPs-aptamer complex to form the primer-AuNPs-aptamer/antigen target substance/antibody sandwich structure modified ELISA plate.

In embodiments of the present invention, the primer-AuNPs-aptamer/antigen target/antibody sandwich modified ELISA plate was subjected to rolling circle amplification reaction.

In embodiments of the present invention, the rolling circle amplification process of the ELISA plate comprises adding a circular template, T4DNA ligase, T4DNA ligase buffer solution and ultrapure water into a primer-AuNPs-aptamer/antigen target/antibody modified ELISA plate, incubating at room temperature, washing the plate, adding phi29DNA polymerase buffer solution, dNTPs, BSA, ultrapure water and phi29DNA polymerase into the ELISA plate to obtain long single-chain DNA, and finally adding heme (hemin) and potassium ions into the ELISA plate, wherein the heme molecules are embedded into the long single-chain DNA and are folded into hemin/G-quadruplex structure, namely DNase.

In embodiments of the invention, the nucleotide sequence of the circular template is shown in SEQ ID NO. 1.

In embodiments of the present invention, the covalent organic framework nanomaterial modified electrode is obtained by dropping a solution containing the covalent organic framework nanomaterial on the surface of the electrode and drying the solution.

In embodiments of the present invention, the synthesis of covalent organic framework materials COFs-TpBD according to the solvothermal method specifically includes the steps of mixing 1,3, 5-trialdehyde phloroglucinol (Tp) (63mg, 0.3mmol), Benzidine (BD) (82.9mg, 0.45mmol), mesitylene, dioxane (1: 1, v: v, 6mL) and acetic acid (0.5mL, 3mol/L) into a polytetrafluoroethylene tube, sonicating the mixture for 10min to uniformly disperse the solution, sealing the tube, heating at 120 ℃ for 72h, collecting the resulting red precipitate by centrifugation, washing thoroughly with acetone, and vacuum drying at 60 ℃ for 12h to obtain a dark red powder.

In embodiments of the present invention, the molar ratio of the thiol-group-containing aptamer to the thiol-group-containing primer in step (1) is (2-7): 1, preferably 4: 1.

In embodiments of the present invention, AuNPs are prepared by Turkevich citric acid reduction, specifically comprising adding 2mL of chloroauric acid (1%) and 98mL of ultrapure water to a thoroughly washed 250mL round-bottomed flask, boiling at 120 ℃ and 1200rpm, rapidly adding 10mL of sodium citrate (1%, wt%), boiling for several minutes until the color changes from deep blue to wine red, stirring and boiling the mixture for 5 minutes to ensure uniform particle size, cooling the solution to room temperature, and storing at 4 ℃ for future use.

In embodiments of the invention, a primer-AuNPs-aptamer complex is prepared by utilizing an Au-S bond formed between AuNPs and a thiol-modified aptamer nucleic acid chain, and the specific preparation method comprises the steps of mixing the AuNPs with a thiol-modified aptamer, a thiol-modified primer and acetic acid (pH 5.2), incubating at room temperature for 12h, adding dNTP to seal redundant active sites on the surface of the AuNPs, adding NaCl, incubating at room temperature for 24h to increase the stability of the complex, centrifuging excess reagents at 10000rpm for 20min to remove, suspending the obtained precipitate in Tris-EDTA (TE, 0.1M, pH 7.4) buffer, and storing at 4 ℃ for later use.

In embodiments of the invention, the aptamer has the nucleotide sequence shown in SEQ ID NO. 2.

In embodiments of the invention, the nucleotide sequence of the primer is set forth in SEQ ID NO. 3.

In embodiments of the present invention, the sandwich modified elisa plate is prepared by adding an antibody corresponding to a target antigen to the elisa plate, incubating overnight at 4 ℃, adding BSA (1%) to the wells, incubating for 1h at 37 ℃ to block excess active sites, then injecting 0.1mL of standard target antigen solutions of different concentrations into each well, incubating for not less than 150min at 37 ℃, then adding primer-AuNPs-aptamer complexes to the wells, incubating for 2h at 37 ℃, forming a primer-AuNPs-aptamer/target antigen/antibody sandwich structure by specific recognition of the aptamers and the antibody to the target antigen, washing the plate twice with PBS-T (0.05% Tween-20, 0.1M PBS, pH 7.4) after each step, and washing times with PBS (0.1M PBS, pH 7.4).

In embodiments of the invention, the concentration of the T4DNA ligase is not less than 7U/mL and the concentration of the phi29DNA polymerase is not less than 0.2U/mL.

In embodiments of the invention, the concentration of heme is not less than 1 μ M.

In embodiments of the present invention, the time of the rolling circle amplification reaction is not less than 60 min.

In embodiments of the invention, o-aminophenol solution and H are added to the ELISA plate after rolling circle amplification₂O₂And reacting at room temperature. Said H₂O₂The concentration of (A) is not less than 25. mu.M. In the process, o-aminophenol is oxidized into a colored product 3-aminophenoxazine by using DNA enzyme catalysis, and the concentration of a target antigen can be indirectly obtained by measuring an electrochemical signal of the 3-aminophenoxazine; in addition, the color of the 3-aminophenoxazine changes with increasing concentration of the target antigen, and can be roughly quantitatively detected by naked eyes.

In embodiments of the invention, 3-AmmoniaThe phenylphenazine is theoretical molecular diametersThe benzene homologues of (a). Therefore, we chose to have a periodic columnar pi array and appropriate apertures

The two-dimensional porous COFs-TpBD of (1) as a candidate for sensing 3-aminophenoxazine. The enrichment amount of 3-aminophenoxazine is enhanced through pi-pi conjugated adsorption and the aperture effect of COFs-TpBD, thereby improving the response sensitivity and reducing the detection limit.

The second objective of the present invention is to provide electrochemical enzyme-linked immunoassay methods, which uses the electrochemical-ELISA immunosensor described above to detect the antigen target.

In embodiments of the invention, the method includes the steps of:

(1) establishment of a standard curve: constructing the electric sensor, and measuring the corresponding reduction peak current value by using target antigen standard samples with different concentrations to be recorded as I_iThe reduction peak current value of the blank sample containing no target antigen was designated as I₀(ii) a With I_i/I₀Constructing a linear relation with the concentration of the target antigen to obtain a standard curve;

(2) measuring a corresponding reduction peak current value by using a sample to be measured; and (3) calculating according to the standard curve obtained in the step (1) to obtain the content of the antigen target in the sample to be detected.

The third purpose of the invention is to provide methods for detecting the content of aflatoxin M1, which comprises the following steps:

(1) constructing the electric sensor for detecting the aflatoxin M1, and respectively measuring corresponding reduction peak current values by using standard samples of the aflatoxin M1 with different concentrations to serve as analysis signals I of the aflatoxin M1_iAnd recording the reduction peak current value of the blank sample without aflatoxin M1 as I₀(ii) a With I_i/I₀Constructing a linear relation with the concentration of the aflatoxin M1 to obtain the label of the aflatoxin M1A quasi-curve;

(2) and (3) measuring a corresponding reduction peak current value by using a sample to be measured with unknown aflatoxin M1 content, and obtaining the content of aflatoxin M1 in the sample to be measured according to the standard curve obtained in the step (1).

The invention has the beneficial effects that:

the invention combines the rolling circle amplification DNA enzyme nucleic acid signal amplification method and the covalent organic framework nano material signal amplification method for the first time, and opens up a new idea for constructing a high-throughput and high-sensitivity on-site immunosensor. Wherein, within the range of 0.5ng/mL to 80ng/mL, I_i/I₀(I_iIs a corresponding reduction peak current signal I after the aflatoxin M1 with the fixed concentration of is added₀The signal of the reduction peak current without adding aflatoxin M1) is in direct proportion to the concentration of aflatoxin M1, the correlation coefficient is 0.993, and the detection limit is 0.15ng/mL (S/N is 3). In addition, the color of 3-aminophenoxazine is directly correlated with the concentration of aflatoxin M1, so we can roughly quantify it with the naked eye.

The invention applies a rolling circle amplification nucleic acid signal amplification method to ELISA, uses the DNase obtained by amplification to catalyze o-aminophenol to oxidize and generate oxide 3-aminophenoxazine with electric activity, simultaneously prepares a modified electrode based on COFs-TpBD material, COF-TpBD is used as COFs material, has large specific surface area, porosity, large pi-pi conjugated system and thermal stability, can enrich 3-aminophenoxazine by intermolecular pi-pi aggregation, and in addition, the aperture of COF-TpBD

Greater than 3-aminophenoxazines

The molecular diameter of the protein can be further -step enhanced enrichment effect of 3-aminophenoxazine through aperture matching effect, the method can improve the detection sensitivity of aflatoxin M1 and can be better used for actual detection of aflatoxin M1, and a primer-AuNPs-aptamer is used as a recognition unit, rolling circle amplification is combined with COFs nano-materials, so that cascade signal amplification is realized, and the establishment of a cascade signal is realized sensitive, high-selectivity and high-flux electrochemical enzyme-linked immunoassay methods are established, meanwhile, rough quantitative analysis of aflatoxin M1 can be carried out through flesh eyes, and the method can also be used for measuring aflatoxin M1 in actual samples.

Drawings

FIG. 1 is a schematic diagram of the preparation and sensing of an electrochemical-ELISA immunosensor;

FIG. 2 is an agarose gel electrophoresis image of a rolling circle amplification reaction;

FIG. 3 is a scanning electron microscope atlas of COFs-TpBD modified electrode;

FIG. 4 shows the ratio of 1mmol/L [ Fe (CN) ] between GCE and COFs-TpBD/GCE₆]^3-/4-Cyclic voltammograms at different scanning speeds in solution (containing 0.1mol/L KCl);

FIG. 5 is the cyclic voltammetric behavior of COFs-TpBD/GCE in 3-aminophenoxazine solutions at different scan rates;

FIG. 6 is a graph illustrating the amplification of sensor signals; wherein a is naked GCE, b is an electrode which is amplified only by rolling circle and is not modified by COFs-TpBD; c is COFs-TpBD which is amplified by rolling circle and modified on the electrode;

FIG. 7 is a standard graph of aflatoxin M1 detection;

FIG. 8 is a graph of the optimization trend for experimental conditions, where A is the effect of primer/aptamer ratio on sensor signal; B. c, D is the effect of T4DNA ligase and phi29 polymerase concentration, reaction time on sensor electrical signal in Rolling Circle Amplification (RCA) reaction, respectively;

FIG. 9 is an optimization of experimental conditions, where A is the effect of AFM1 incubation time on sensor signal; B. c, D reaction time, hemoglobin concentration and H of o-aminophenol, respectively₂O₂The effect of concentration on sensor signal;

FIG. 10 is a selectivity test chart of the sensor.

Detailed Description

Obtaining experimental materials and reagents:

the aflatoxin M1 monoclonal antibody is available from Beijing Conno Biotech, Inc.; aflatoxin M1, AFTB1 and OTA standards are available from J&Limited science of KCompany, Beijing, China; the ring template (5 ' -p-TAG CAC GGA CATTTT CCC AAC CCG CCC TAC CCT TTTTTT TTT CCC AAC CCG CCC TAC CCT TTT GTA ACTGTT TCC TTC), the aflatoxin M1 aptamer (5 ' -SH-ACT GCT AGA GAT TTT CCACA) containing the sulfhydryl modification, the primer (5 ' -SH-GCT AGA AGG AAA CAG TTA C) containing the sulfhydryl modification and bovine serum albumin can be purchased from Shanghai worker, Shanghai in China; t4DNA ligase, phi29DNA polymerase, and deoxyribonucleic acid complex dNTP are available in New England Biolabs, Beijing, China; tris (hydroxymethyl) aminomethane, ethylenediaminetetraacetic acid, benzidine, 1,3, 5-trialdehyde phloroglucinol, and chloroauric acid (HAuCl)₄·4H₂O, 99.9%) is available from alatin, missouri, usa. Common chemical reagents such as ciprofloxacin and glucose can be purchased from the national medicine group, Shanghai, China. Ultrapure water (18.2M Ω cm) is available from Milli Q water purification system, USA.

The related experimental apparatus and equipment:

CHI 660C electrochemical workstation (Chenghua instruments, Inc., Shanghai, China), DYY-8C electrophoresis apparatus (six , Beijing, China), Bio-Rad GelDoc XR + imaging System (Bio-Red, USA), 1800 UV-visible spectrophotometer (Shimadzu, Japan), X-ray diffractometer (BRUKER AXS GMBH, Germany), Fourier Infrared spectrometer (Nicley, USA), scanning Electron microscope (Hitachi, Japan).

AuNPs preparation: 2mL of chloroauric acid (1%) and 98mL of ultrapure water were added to a 250mL round-bottom flask which had been thoroughly cleaned beforehand, and after boiling at 120 ℃ and 1200rpm, 10mL of sodium citrate (1%, wt%) was rapidly added and boiling was continued for several minutes until the color changed from deep blue to wine red; continuously stirring and boiling the mixture for 5min to ensure uniform particle size; after the solution was cooled to room temperature, it was stored at 4 ℃ for further use.

Preparing a COFs-TpBD modified electrode:

preparation of covalent organic framework material COFs-TpBD: 1,3, 5-trialdehyde phloroglucinol (Tp) (63mg, 0.3mmol), Benzidine (BD) (82.9mg, 0.45mmol), mesitylene: dioxane (1: 1, v: v, 6mL) and acetic acid (0.5mL, 3mol/L) were combined and added to a polytetrafluoroethylene tube; subjecting the mixture to ultrasonic treatment for 10min to uniformly disperse the solution; the tube was then sealed and heated at 120 ℃ for 72 h. Collecting the formed red precipitate by centrifugation, thoroughly washing with acetone, and vacuum drying at 60 deg.C for 12h to obtain dark red powder;

before electrode modification, polishing the electrode on a chamois leather by using 0.3 and 0.05 mu m aluminum oxide powder, and sequentially ultrasonically cleaning by using nitric acid (concentrated nitric acid: water, 1: 3, v/v), ethanol and ultrapure water; then, 5mg of COFs-TpBD was mixed with 5mL of ethanol, sonicated for 2h, and then a 1% Nafion ethanol solution was mixed with the COFs-TpBD dispersion 1: 1(v/v) mixing, and carrying out ultrasonic treatment until the dispersion is uniform; 5 mu.L of the COFs-TpBD-Nafion mixture was carefully applied dropwise to the electrode surface and dried at room temperature.