CN113049562A - Fluorescence method for rapidly and quantitatively detecting BPA - Google Patents

Abstract

The invention discloses a method for rapidly and quantitatively detecting BPA, which uses the specificity of a BPA aptamer to realize rapid and quantitative detection of BPA in a sample; specifically, the method comprises the steps of synthesizing an aptamer sequence which is rich in C and G and has FAM marks at the 5' end aiming at the structure of BPA, and detecting the fluorescence intensity of a specific peak value after mixing the aptamer sequence with BPA solutions with different concentrations in the presence of graphene oxide and polyvinylpyrrolidone so as to draw a standard curve; quantitatively detecting the concentration of BPA in the sample by comparing the fluorescence of the sample to be detected with a standard curve; the method can be widely applied to the fields of food, agriculture, medicine and the like.

Description

Fluorescence method for rapidly and quantitatively detecting BPA

Technical Field

The invention belongs to the field of bisphenol A substance residue detection, and particularly discloses a fluorescence method for rapidly and quantitatively detecting BPA.

Background

BPA, bisphenol A, is an organic compound having the formula C₁₅H₁₆O₂Is an important technical raw material for synthesizing materials such as polycarbonate, epoxy resin and the like, and is widely applied to plastic materials of daily life. Has been used for manufacturing plastic (milk) bottles for 60 yearsA drinking cup for infants, and an inner coating of a food and beverage (milk powder) can. BPA is ubiquitous and has its silhouette from mineral water bottles, medical devices, and the interior of food packaging. BPA is difficult to degrade and can leach from the container into foods and beverages. In recent years, studies have shown that BPA has an estrogen-like activity and in severe cases can lead to precocious puberty, metabolic disorders and even cancer. Therefore, the determination of the BPA content is of positive significance. At present, methods for detecting BPA mainly include chromatography, mass spectrometry, chemiluminescence, electrochemical methods and the like. However, the above detection methods either require expensive large-scale instruments and require high operator requirements, such as chromatography and mass spectrometry; or low detection specificity, complex detection method, high labor and time consumption, toxic chemical reagents and the like. Therefore, there is a need to develop a method with good sensitivity and simple operation.

Disclosure of Invention

In view of the above situation, the invention aims to provide a rapid, accurate and highly sensitive detection method for BPA, and the invention designs an aptamer labeled by a fluorescent probe and detects BPA through a fluorescent signal.

The technical scheme of the invention is as follows:

a fluorescence method for rapidly and quantitatively detecting BPA comprises the following steps

S1, synthesizing an aptamer sequence which is rich in C and G and has FAM marks at the 5' end aiming at the structure of BPA; the length of the sequence is between 50 and 70 bp; the aptamer sequence is stored in a Tris solution, namely a solution A;

s2, adding BPA with different masses into the solution A, and standing for 10-60min to form solutions B with different concentrations;

s3, preparing a graphene oxide solution, uniformly mixing the graphene oxide solution with the equivalent accumulated vinyl pyrrolidone solution, and standing for 10-60min to obtain a solution C;

s4, mixing the solutions B and C, standing for 20-80min to obtain a solution D, and starting to detect the fluorescence signals of the solutions D containing BPA with different concentrations;

and S5, drawing a standard curve according to the detection result, then carrying out concentration detection on the unknown sample, and calculating the concentration of the unknown sample from the standard curve according to the fluorescence signal value.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the aptamer sequence in the step S1 is SEQ ID No.1= -FAM-5 'CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the solution A in the step S1 is a Tris solution with the aptamer sequence concentration of 1mM and the pH value of 7.4.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the volume of the solution A in the step S2 is in the range of 50-200 ul.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the concentration of the graphene oxide solution in the step S3 is 50-200 ug/ml.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the concentration of the polyvinylpyrrolidone in the step S3 is 10-40 ug/ml.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the volume of the solution B in the step S4 is 800ul, and the volume of the solution C is 200 ul.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the regression equation of the standard curve in the step S5 is Y =2.640+ 1.156X.

Further, in the fluorescence method for rapidly and quantitatively detecting BPA, the peak wavelength of the fluorescence signal in the steps S4 and S5 is 515 nm.

Further, the fluorescence method for rapidly and quantitatively detecting BPA specifically comprises the following steps:

s1, synthesizing an aptamer of BPA, wherein the sequence = SEQ ID NO.1= FAM-5 'CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'; preparing a 1mM Tris solution with pH 7.4 containing 1 μ M of the aptamer;

s2, taking 100 mul of the aptamer solution, mixing the aptamer solution with BPA with different concentrations, and standing for 30 min;

s3, preparing graphene oxide, preparing 50ug/mL with distilled water before use, uniformly mixing with an isometric 20 mug/mL polyvinylpyrrolidone solution, and standing for 30 min;

s4, adding 90 mu L of 10mM Tris solution with pH of 7.4 into the solution prepared in S2, and adding a proper amount of distilled water until the total volume is 800 mu L; adding 200 muL of the solution prepared in the step S3, and standing for 50min to start detection;

and S5, drawing a standard curve according to the detection result, then carrying out concentration detection on the unknown sample, and calculating the concentration of the unknown sample from the standard curve according to the fluorescence signal value.

According to the technical scheme, the invention has the following beneficial effects

According to the invention, the BPA is specifically identified by the aptamer, so that the rapid detection of the BPA is realized, the method has the advantages of high specificity, small required sample amount, high sensitivity, simplicity and convenience in operation, low requirements on instruments and operators, short detection time and high applicability.

Drawings

FIG. 1 is a graph of the fluorescence spectra of BPA at different concentrations in example 3 of the present invention;

FIG. 2 is a standard curve for BPA in example 3 of the present invention.

Detailed Description

The invention will be further elucidated by means of several specific examples, which are intended to be illustrative only and not limiting.

Example 1

A fluorescence method for rapidly and quantitatively detecting BPA comprises the following steps:

s1, synthesizing an aptamer of BPA, wherein the sequence = SEQ ID NO.1= FAM-5 'CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'; preparing a 1mM Tris solution with pH 7.4 containing 1 μ M of the aptamer;

s2, mixing 50 mul of the aptamer solution with BPA with different concentrations, and standing for 30 min;

s3, preparing graphene oxide, preparing 100ug/mL with distilled water before use, uniformly mixing with an isometric 10 mug/mL polyvinylpyrrolidone solution, and standing for 10 min;

s4, adding 90 into the solution prepared in S2Mu.l 10mM Tris solution pH 7.4 (containing 0mM CaCL)₂) Adding a proper amount of distilled water until the total volume is 800 mu L; adding 200 muL of the solution prepared in the step S3, and standing for 20min to start detection;

and S5, drawing a standard curve according to the detection result, then carrying out concentration detection on the unknown sample, and calculating the concentration of the unknown sample from the standard curve according to the fluorescence signal value.

Example 2

A fluorescence method for rapidly and quantitatively detecting BPA comprises the following steps:

s1, synthesizing an aptamer of BPA, wherein the sequence = SEQ ID NO.1= FAM-5 'CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'; preparing a 1mM Tris solution with pH 7.4 containing 1 μ M of the aptamer;

s2, taking 100 mul of the aptamer solution, mixing the aptamer solution with BPA with different concentrations, and standing for 60 min;

s3, preparing graphene oxide, preparing 200ug/mL with distilled water before use, uniformly mixing with an isometric polyvinylpyrrolidone solution of 40 mug/mL, and standing for 60 min;

s4. to the solution prepared in S2, 90ul of 10mM Tris solution (containing 200mM CaCL) of pH 7.4 was added₂) Adding a proper amount of distilled water until the total volume is 800 mu L; adding 200 muL of the solution prepared in the step S3, and standing for 80min to start detection;

and S5, drawing a standard curve according to the detection result, then carrying out concentration detection on the unknown sample, and calculating the concentration of the unknown sample from the standard curve according to the fluorescence signal value.

Example 3

A fluorescence method for rapidly and quantitatively detecting BPA comprises the following steps:

s1, synthesizing an aptamer of BPA, wherein the sequence = SEQ ID NO.1= FAM-5 'CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'; preparing a 1mM Tris solution with pH 7.4 containing 1 μ M of the aptamer;

s2, taking 100 mul of the aptamer solution, mixing the aptamer solution with BPA with different concentrations, and standing for 30 min;

s3, preparing graphene oxide, preparing 50ug/mL with distilled water before use, uniformly mixing with an isometric 20 mug/mL polyvinylpyrrolidone solution, and standing for 30 min;

s4, adding 90 mu l of 10mM Tris solution (containing 0mM CaCL and pH 7.4) into the solution prepared in S2₂) Adding a proper amount of distilled water until the total volume is 800 mu l; and adding 200 mu L of the solution prepared in the step S3, standing for 50min, and then starting detection, wherein fluorescence spectrum curves at different BPA concentrations are shown in the attached figure 1, the BPA concentrations are 10ug/mL, 20ug/mL and 50ug/mL sequentially from bottom to top, the corresponding fluorescence intensities a.u are 16.84, 29.52 and 57.55 respectively, and the peak wavelength is found to be near 515 nm.

S5, drawing a standard curve according to the detection result, wherein the curve is shown in figure 2, the concentration of BPA is 0ug/mL, 5ug/mL,10ug/mL, 20ug/mL and 50ug/mL in sequence, the corresponding fluorescence intensity a.u is 2.38, 8.38, 16.84, 29.52 and 57.55 respectively, the regression equation of the curve is Y =2.640+1.156X, and R is²Greater than 0.95, good fitting effect; and then, detecting the concentration of the unknown sample, and comparing the concentration of the unknown sample with a standard curve according to the fluorescence signal value to obtain the concentration of the unknown sample.

According to the embodiment, the rapid detection of the BPA is realized by the specific recognition of the aptamer to the BPA, the method has the advantages of high specificity, small required sample amount, high sensitivity, simplicity and convenience in operation, low requirements on instruments and operators, short detection time and high applicability.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

SEQUENCE LISTING

<110> Suzhou Jianxiong professional technical institute

<120> fluorescence method for rapidly and quantitatively detecting BPA

<130> 2021

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 63

<212> DNA

<213> artificial

<400> 1

ccggtgggtg gtcaggtggg atagcgttcc gcgtatggcc cagcgcatca cgggttcgca 60

cca 63

Claims (10)

1. A fluorescence method for rapidly and quantitatively detecting BPA is characterized by comprising the following steps

S1, synthesizing an aptamer sequence which is rich in C and G and has FAM marks at the 5' end aiming at the structure of BPA; the length of the sequence is between 50 and 70 bp; the aptamer sequence is stored in a Tris solution, namely a solution A;

s2, adding BPA with different masses into the solution A, and standing for 10-60min to form solutions B with different concentrations;

s3, preparing a graphene oxide solution, uniformly mixing the graphene oxide solution with the equivalent accumulated vinyl pyrrolidone solution, and standing for 10-60min to obtain a solution C;

s4, mixing the solutions B and C, standing for 20-80min to obtain a solution D, and starting to detect the fluorescence signals of the solutions D containing BPA with different concentrations;

and S5, drawing a standard curve according to the detection result, then carrying out concentration detection on the unknown sample, and calculating the concentration of the unknown sample from the standard curve according to the fluorescence signal value.

2. The fluorescence method for rapid quantitative detection of BPA according to claim 1, wherein the aptamer sequence in step S1 is SEQ ID No.1= FAM-5 'CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'.

3. The fluorescence method for rapid and quantitative detection of BPA according to claim 1, wherein the solution A in step S1 is Tris solution with aptamer sequence concentration of 1mM and pH 7.4.

4. The fluorescence method for rapid quantitative detection of BPA according to claim 1, wherein the volume of solution A in step S2 is in the range of 50-200 ul.

5. The fluorescence method for rapidly and quantitatively detecting BPA according to claim 1, wherein the concentration of the graphene oxide solution in the step S3 is 50-200 ug/ml.

6. The fluorescence method for rapid quantitative detection of BPA according to claim 1, wherein the concentration of polyvinylpyrrolidone in step S3 is 10-40 ug/ml.

7. The fluorescence method for rapid quantitative detection of BPA according to claim 1, wherein the volume of solution B in step S4 is 800ul and the volume of solution C is 200 ul.

8. The fluorescence method for rapid quantitative detection of BPA according to claim 1, wherein the regression equation of the standard curve in step S5 is Y =2.640+ 1.156X.

9. The fluorescence method for rapid and quantitative detection of BPA according to claim 1, wherein the peak wavelength of the fluorescence signal in steps S4 and S5 is 515 nm.

10. The fluorescence method for rapidly and quantitatively detecting BPA according to claim 1, which comprises the following steps:

s1, synthesizing an aptamer of BPA, wherein the sequence = SEQ ID NO.1= FAM-5 'CCG GTG GGT GGT CAG GTG GGA TAG CGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3'; preparing a 1mM Tris solution with pH 7.4 containing 1 μ M of the aptamer;

s2, taking 100 mul of the aptamer solution, mixing the aptamer solution with BPA with different concentrations, and standing for 30 min;

s3, preparing graphene oxide, preparing 50ug/mL with distilled water before use, uniformly mixing with an isometric 20 mug/mL polyvinylpyrrolidone solution, and standing for 30 min;

s4, adding 90 mu L of 10mM Tris solution with pH of 7.4 into the solution prepared in S2, and adding a proper amount of distilled water until the total volume is 800 mu L; adding 200 muL of the solution prepared in the step S3, and standing for 50min to start detection;

and S5, drawing a standard curve according to the detection result, then carrying out concentration detection on the unknown sample, and calculating the concentration of the unknown sample from the standard curve according to the fluorescence signal value.

Images