CN110068561B - Bisphenol A fluorescence detection method based on atom transfer radical polymerization reaction and truncated aptamer - Google Patents
Bisphenol A fluorescence detection method based on atom transfer radical polymerization reaction and truncated aptamer Download PDFInfo
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Abstract
The invention discloses a bisphenol A fluorescence detection method based on atom transfer radical polymerization and truncated aptamer, which has the detection principle that hairpin DNA fixed on magnetic beads is unfolded in the presence of bisphenol A, the unfolded hairpin DNA can be connected with an ATRP reaction initiator PBIB through click reaction, a large number of fluorescent monomers are connected and polymerized on the unfolded hairpin DNA through ATRP reaction, and finally the fluorescence intensity measured by a fluorescence spectrometer is in positive correlation with the concentration of bisphenol A, so that the sensitive and accurate measurement of the concentration of bisphenol A is successfully realized. The result shows that the method has higher sensitivity, selectivity, anti-interference performance and accuracy for detecting the bisphenol A, and has huge practical application value in food safety and environmental monitoring.
Description
Technical Field
The invention relates to a bisphenol A fluorescence detection method based on atom transfer radical polymerization and a truncated aptamer, belonging to the technical field of biological analysis.
Background
Bisphenol A (BPA), which is called 2, 2-bis (4-hydroxyphenyl) propane, is an important synthetic raw material for polycarbonate and epoxy resin, and is also a manufacturing raw material for a plurality of common daily necessities such as food packaging materials, tableware, milk bottles and the like. BPA can permeate into food or beverages through food packaging containers or plastic films, and repeated use of these items or exposure to high heat environments can result in leaching of BPA, which can then be ingested by the human body. In addition, a large amount of bisphenol A is present in the environment due to landfill and disposal of household garbage in daily life and discharge of bisphenol A-containing wastewater in manufacturing plants. Many studies have shown that bisphenol a has strong endocrine disrupting activity and disrupts essential physiological processes. Bisphenol a is associated with a number of diseases such as thyroid function, metabolic diseases, neurological effects, cancer, etc. The traditional detection technology of bisphenol A comprises High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and the like, but the methods have the defects of complex sample pretreatment process, expensive instrument, professional operators and the like. Therefore, it is necessary to develop a new method for detecting bisphenol A with high selectivity and high sensitivity.
The aptamer is a single-stranded DNA or RNA which is obtained by screening through an exponential ligand enrichment evolution technology (SELEX) and has high affinity to specific substances (small molecular compounds, proteins, cells, ions and the like). Compared with the immune recognition detection technology, the aptamer is rapidly developed in the field of biosensing due to the advantages of easy synthesis, stable chemical property, easy modification, no immune prototype, low price and the like. The biosensing detection system established based on the aptamer has the advantages of high sensitivity, strong specificity and the like, and various biosensing detection methods for bisphenol A are emerging continuously since the bisphenol A aptamer is obtained by screening.
Meanwhile, in order to improve detection performance, various signal amplification techniques such as hybrid chain reaction, rolling circle amplification reaction, nanomaterial, polymerization reaction, and the like are applied to the field of biosensing. Among them, Atom Transfer Radical Polymerization (ATRP) has advantages of controllable reaction process, wide reaction monomer range, etc., and has been proposed in the 90 s to be widely used in the fields of materials, synthesis, etc., and its application in biosensing is also favored by researchers.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a bisphenol A fluorescence detection method based on atom transfer radical polymerization and truncated aptamers, which has the characteristics of high selectivity, high sensitivity and the like.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a bisphenol A fluorescence detection method based on atom transfer radical polymerization and truncated aptamer comprises the following steps:
(1) pretreatment of hairpin DNA:
heating hairpin DNA solution to 95 ℃ and keeping for 15min, and then cooling to room temperature;
② mixing the hairpin DNA solution (100 mu M) and the TCEP solution (10 mu M) according to the volume ratio of 1:1, and reacting the mixed solution in a constant temperature shaker at 37 ℃ for 3h in a dark place and preserving the mixed solution at-20 ℃ for later use; in use, the hairpin DNA solution is diluted to 1 μ M;
(2) amino Fe3O4Modification of magnetic beads
Firstly, a certain amount of amino Fe is measured3O4Magnetic bead stock solution (10mg/ml), carrying out magnetic separation, removing supernatant, washing with PBS buffer solution, carrying out magnetic separation, removing supernatant, adding PBS buffer solution, carrying out heavy suspension, and recovering to the volume of the measured magnetic bead stock solution;
measuring 20 mu L of magnetic bead resuspension, 20 mu L of Sulfo-SMCC solution (100 mu M) and 160 mu L of PBS buffer solution, mixing uniformly, and reacting for 2 hours in a constant temperature shaking table at 37 ℃ in a dark place;
thirdly, magnetically separating the mixed solution obtained in the second step, removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
adding 180 mu L of PBS buffer solution and 20 mu L of hairpin DNA solution (1 mu M), mixing uniformly, and reacting overnight in a constant temperature shaking table at 37 ℃ in a dark place;
(3) nucleic acid hybridization
Magnetically separating the reaction solution obtained in the step (2), removing supernatant, and washing with TE buffer solution;
adding 20 mu L of target solution and 180 mu L of TE buffer solution, mixing uniformly, and reacting for 2 hours in a constant temperature shaking table at 37 ℃ in a dark place;
(4) click reaction
Magnetically separating the reaction solution obtained in the step (3), removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
② adding 140 mul PBS buffer solution and 20 mul CuSO4Solution (1mM)20 mu L of PBIB solution (1mM) and 20 mu L of AA solution (2mM) are mixed evenly and reacted for 2 hours in a constant temperature shaking table at 37 ℃ in a dark place;
(5) atom Transfer Radical Polymerization (ATRP) reaction
Magnetically separating the reaction solution obtained in the step (4), removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
② 20 microliter of fluorescent monomer solution (1mM), 140 microliter of ultrapure water and 20 microliter of complex Cu are addedIIBr/Me6Mixing TREN solution (1mM) and 20 μ L AA solution (2mM), and reacting at 37 deg.C in a constant temperature shaking table in a dark place for 2 h;
(6) fluorescence detection
Magnetically separating the reaction liquid obtained in the step (5), removing supernatant, and washing with 30% (v/v) DMF solution and PBS buffer solution in sequence;
adding PBS buffer solution, mixing uniformly, and taking the mixed solution as a sample to perform fluorescence detection on a fluorescence spectrophotometer.
The sequence of the hairpin DNA is 5' -SH- (CH)2)6-CCACGCCGGTGGGTGGA ACGTGG-N3-3’。
The preparation method of the target solution comprises the following steps:
mixing ssDNA-A (100 mu M) and ssDNA-B (100 mu M) in a volume ratio of 1:1, heating the mixture to 95 ℃ and keeping the temperature for 15min, cooling to room temperature, and storing at-20 ℃ for later use;
② mixing 10 μ L of the mixed solution of the step I with a certain amount of sample to be tested, diluting to 500 μ L, reacting overnight in a constant temperature shaking table at 37 ℃ in the dark to obtain the target solution, and storing at-20 ℃ for later use.
The sequence of ssDNA-A is 5'-CCGGTGGGTGGA A-3'.
The sequence of ssDNA-B is 5'-TTCCACCCACCGG-3'.
Step (3) - ② in the TE buffer solution, MgCl with the concentration of 0.5mM is contained2。
The PBS buffer concentration was 0.1M and the pH was 7.4.
The excitation wavelength of the fluorescence detection in the step (6) is 489nm, and the slit width is 4 nm.
A schematic diagram of the detection method of the present invention is shown in FIG. 1.
The invention has the beneficial effects that:
1. compared with the traditional bisphenol A aptamer, the truncated bisphenol A aptamer (refer to the aptamer sequence reported in the high sensitive detection of bisphenol a by NanoAptamer assay with truncated aptamer. ACS Appl Mater Interfaces 9(17): the sequence is 5'-CCGGTGGGTGGAA-3') and the target have reduced binding steric hindrance, so that the two aptamers have higher affinity, and the selectivity and sensitivity of BPA detection are improved.
2. The method uses atom transfer radical polymerization as a signal amplification strategy, avoids the use of nano materials and biological enzymes (which are easily influenced by external environments such as pH and temperature) in the current common signal strategy, multiplies the signals, greatly improves the sensitivity, and has relatively higher stability and reproducibility.
3. In the presence of bisphenol A, the hairpin DNA fixed on the magnetic bead is unfolded, the unfolded hairpin DNA can be connected with an ATRP reaction initiator PBIB through a click reaction, a large number of fluorescent monomers are connected and polymerized on the unfolded hairpin DNA through an ATRP reaction, and finally, the fluorescence intensity measured by a fluorescence spectrometer is in positive correlation with the concentration of bisphenol A, so that the sensitive and accurate measurement of the concentration of bisphenol A is successfully realized. The results show that under the best experimental conditions, the logarithmic value of the concentration of bisphenol A and the fluorescence intensity are in a linear relation in the concentration range of 100fM to 100nM, and the linear equation is F3384 LogC +11153 (R)20.9985), detection limit was as low as 0.51 fM. When the method is used for detecting samples containing 100nM bisphenol A analogs BPB, BPC, BPF, DES and E3, the measured fluorescence intensities are respectively 11.90%, 30.10%, 21.90%, 19.20% and 15.80% of the signal intensity generated by 1nM BPA, and are basically equivalent to blank background signals, which shows that the method has high selectivity. In addition, the method provided by the invention is adopted to detect drinking water respectively containing 1pM, 100pM and 10000pM of BPA, and the recovery rates are respectively 102.0%, 91.1% and 122.5%. In conclusion, the method has higher sensitivity, selectivity, anti-interference performance and accuracy on the detection of the bisphenol A, and has the advantages of high sensitivity, high selectivity, high anti-interference performance and high accuracy in food safety and environmental monitoringHas great practical application value.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2 is a confocal fluorescence microscope photograph of magnetic beads modified with SSMCC/Hairpins/dsDNA-BPA/PBIB/P (FA) and magnetic beads modified with SSMCC/Hairpins/PBIB/P (FA).
FIG. 3 shows fluorescence spectra of magnetic beads in different modification states.
FIG. 4 shows the optimization of ATRP reaction time.
FIG. 5 is an optimization of fluorescent monomer concentration.
FIG. 6 is a graph (A) showing the fluorescence spectra of systems containing different concentrations of bisphenol A and a graph (B) showing the relationship between the concentration of bisphenol A and the corresponding fluorescence intensity. The concentration of bisphenol A in FIG. A was 100nM, 10nM, 1nM, 100pM, 10pM, 1pM, and 100fM in this order from top to bottom.
FIG. 7 is a graph showing a comparison of fluorescence intensity of 100nM bisphenol A analogs BPB, BPF, BPC, DES, E3 and 1nM bisphenol A under the same detection conditions.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Amino Fe3O4Magnetic beads were purchased from Border, McGr Biotech Inc.
The sequence of the hairpin DNA is: 5' -SH- (CH)2)6-CCACGCCGGTGGGTGGAACGTGG-N3-3’(SEQ ID NO.1)。
The sequence of ssDNA-A is: 5'-CCGGTGGGTGGAA-3' (SEQ ID NO. 2).
The sequence of ssDNA-B is: 5'-TTCCACCCACCGG-3' (SEQ ID NO. 3).
Example 1: construction of the detection method
A bisphenol A fluorescence detection method based on atom transfer radical polymerization and truncated aptamer comprises the following steps:
(1) pretreatment of hairpin DNA:
heating hairpin DNA solution to 95 ℃ and keeping for 15min, and then slowly cooling to room temperature;
② mixing the hairpin DNA solution (100 mu M) and the tris (2-carboxyethyl) phosphine hydrochloride (TCEP) solution (10 mu M) according to the volume ratio of 1:1, and keeping the mixed solution in a constant temperature shaking table at 37 ℃ in a dark place for reaction for 3h and at-20 ℃ for later use; when in use, ddH is added2O diluting the hairpin DNA solution to 1 μ M;
(2) amino Fe3O4Modification of magnetic beads
Measuring 50 mu L of amino Fe3O4Magnetic bead stock solution (10mg/ml), magnetic separation, supernatant removal, PBS buffer solution washing (0.1M, pH 7.4)3 times, magnetic separation, supernatant removal, PBS buffer solution adding heavy suspension and recovery to 50 u L;
measuring 20 mu L of magnetic bead resuspension, 20 mu L of Sulfo-SMCC solution (100 mu M) and 160 mu L of PBS buffer solution, uniformly mixing, and reacting for 2h in a constant temperature shaking table at 37 ℃ in the dark (the suspension state of the magnetic beads is kept in the process), so that the cross-linking agent Sulfo-SMCC is combined with the amino magnetic beads through amido bonds;
thirdly, magnetically separating the mixed solution obtained in the second step, removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
adding 180 mu L of PBS buffer solution and 20 mu L of hairpin DNA solution (1 mu M), uniformly mixing, and reacting overnight in a constant temperature shaking table at 37 ℃ in a dark place (the suspension state of magnetic beads is kept in the process), so that the hairpin DNA is bonded with the crosslinking agent Sulfo-SMCC;
(3) nucleic acid hybridization
Magnetically separating the reaction solution obtained in the step (2), removing supernatant, and washing with 200 mu L of TE buffer solution (pH 8) for 1 time;
② 20. mu.L of the objective solution and 180. mu.L of TE buffer (pH 8, containing 0.5mM MgCl)2) After uniform mixing, the mixture is reacted for 2 hours in a constant temperature shaking table at 37 ℃ in a dark place (the suspension state of the magnetic beads is kept in the process);
the target solution is a mixture of a sample to be detected and dsDNA, and the specific preparation method comprises the following steps:
mixing ssDNA-A (100. mu.M) and ssDNA-B (100. mu.M) in a volume ratio of 1:1, heating the mixture to 95 ℃ and keeping the temperature for 15min, slowly cooling to room temperature, and storing at-20 ℃ for later use (dsDNA);
secondly, mixing 10 mu L of the mixed solution obtained in the first step with 5 mu L of a sample to be detected, adding TE buffer solution to dilute the mixed solution to 500 mu L, and reacting the mixed solution overnight in a constant temperature shaking table at 37 ℃ in a dark place to obtain target solution, and storing the target solution for later use at-20 ℃;
(4) click reaction
Magnetically separating the reaction solution obtained in the step (3), removing supernatant, washing with 200 mu L PBS buffer solution, magnetically separating, and removing supernatant;
② adding 140 mul PBS buffer solution and 20 mul CuSO4Uniformly mixing the solution (1mM), 20 mu L of propargyl bromoisobutyrate (PBIB) solution (1mM) and 20 mu L of Ascorbic Acid (AA) solution (2mM), and reacting for 2h in a constant temperature shaking table at 37 ℃ in a dark place;
(5) atom Transfer Radical Polymerization (ATRP) reaction
Magnetically separating the reaction solution obtained in the step (4), removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
② 20 microliter of fluorescent monomer solution (1mM), 140 microliter of ultrapure water and 20 microliter of complex Cu are addedIIBr/Me6Mixing TREN solution (1mM) and 20 μ L AA solution (2mM), and reacting in a constant temperature shaking table at 37 deg.C in the dark for 2h (while maintaining the suspension state of the magnetic beads);
(6) fluorescence detection
Magnetically separating the reaction liquid obtained in the step (5), removing supernatant, and sequentially washing with 30% (v/v) DMF solution and PBS buffer solution for 2 times respectively;
② adding 3000 μ L PBS buffer solution, mixing uniformly, taking the mixture as a sample to perform fluorescence detection on a fluorescence spectrophotometer (excitation wavelength is 489nm, slit width is 4 nm).
The obtained fluorescence intensity is in positive correlation with the diluted bisphenol A concentration, the diluted bisphenol A concentration is obtained by calculation through a linear equation, and the concentration of the bisphenol A in the sample to be detected can be obtained through volume conversion.
Under the condition of a blue light excitation module, when BPA exists in a target solution, a great amount of punctate green fluorescent spots can be observed on the obtained final product SSMCC/Hairpins/dsDNA-BPA/PBIB/P (FA) modified magnetic beads under a confocal microscope; in contrast, when no BPA is present in the target solution, no significant fluorescence phenomenon is observed under a confocal microscope (FIG. 2) in the final product SSMCC/Hairpins/PBIB/P (FA) -modified magnetic beads, thus proving that the detection method of the invention is feasible.
Example 2: feasibility verification
Firstly, the fluorescence spectra of the magnetic beads in different modification states are respectively tested. As shown in FIG. 3, first, no fluorescence intensity was detected for the SSMCC/Hairpins/dsDNA-BPA/PBIB modified beads (curve b) without adding fluorescent monomer FA (fluorescent monomer-O-acrylate). In contrast, a distinct fluorescence emission peak was still observed after several washes after the addition of FA, since a large amount of FA was introduced onto the beads by ATRP, resulting in a very large fluorescence signal (curve a). This not only indicates that BPA binds to dsDNA and releases a single stranded DNA that can pair complementarily to the hairpin DNA loop sequence and unfold the fold structure, but also indicates that the assay has a higher signal-to-noise ratio (S/N) and sensitivity. Through comparative experiments, it is well demonstrated that the signal amplification strategy based on the truncated bisphenol a aptamer ATRP is feasible for the detection of BPA.
Example 3: optimization of detection conditions
(1) Optimization of reaction time
The relation between the ATRP reaction time and the fluorescence intensity is researched by detecting the fluorescence intensity of the magnetic beads with different ATRP reaction times. As a result, as shown in FIG. 4, the fluorescence intensity gradually increased with the lapse of the reaction time and stabilized at 120 min. Therefore, the ATRP reaction time was chosen to be 120 min.
(2) Optimization of fluorescent monomer concentration
The fluorescent monomer concentration can affect the length of the polymer chain and thus the detection performance. When the content of the fluorescent monomer in the system is insufficient, the fluorescence intensity does not reach the maximum. The fluorescence intensities of the magnetic beads with fluorescent monomer concentrations of 0.025mM, 0.05mM, 0.1mM, 0.2mM, and 0.3mM, and bisphenol A concentration of 100nM in the ATRP system were measured, respectively, and as shown in FIG. 5, the fluorescence intensity of the magnetic beads reached the maximum when the fluorescent monomer concentration was 0.1mM, and thereafter, the fluorescent monomer concentration was increased without significant increase, so that the fluorescent monomer concentration of 0.1mM was selected for the following experiment.
(3) Optimization of pH
pH value to CuIIBr/Me6TREN and CuI/Me6TREN (reduction of Cu from AA)IIBr/Me6 TREN) has a great influence. Under acidic conditions, protonation of various amine-containing ligands is favored, resulting in CuIIBr/Me6TREN and CuI/Me6Dissociation of TREN. From Cu in the absence of stabilizing ligandsI/Me6Cu dissociated in TREN1+Disproportionation is more likely to occur. Under alkaline conditions, CuIIBr/Me6TREN will convert to CuIIOH/Me6TREN, due to OH-Specific Br-Has stronger coordination ability. In general, both basic and acidic conditions are detrimental to the stability of the catalytic system and to ATRP. Therefore, pH 7 was chosen as the optimum parameter.
In summary, the optimal ATRP optimization conditions are: ATRP reaction time was 120min, fluorescent monomer concentration was 0.1mM, pH 7.
Example 4: sensitivity test
According to the optimal experimental conditions obtained by optimizing the conditions in example 3, the detection performance of the method of the invention on bisphenol A is studied by detecting the fluorescence intensity generated by bisphenol A (100fM, 1pM, 10pM, 100pM, 1nM, 10nM and 100nM) with different concentrations in the target solution. As shown in FIG. 6, the fluorescence intensity of the beads increased with increasing concentration of bisphenol A, because the higher the concentration of bisphenol A, the more free ssDNA-B is generated after binding reaction with dsDNA, which in turn leads to the larger number of hairpin DNA that is developed, and thus the more fluorescent monomers are introduced into the beads, the stronger the fluorescence intensity is generated. The logarithm value of the maximum fluorescence emission peak intensity and the concentration of bisphenol A shows good linear relation in the range of 100fM to 100nM, and the linear equation is F-3384 LogC +11153 (R)20.9985), the detection limit is 0.51fM, and the result shows that the method has higher detection sensitivity, lower detection limit and excellent performance.
Example 5: selectivity test
The fluorescence intensity of bisphenol A analogs 2, 2-bis (4-hydroxyphenyl) butane (BPB), 4' -dihydroxydiphenylmethane (BPF), 2-bis (4-hydroxy-3-methylphenyl) (BPC), Diethylstilbestrol (DES), estriol (E3) generated using the method of the present invention was investigated according to the optimal experimental conditions obtained by optimizing the conditions of example 3. Specifically, the difference in signal intensity produced by comparing 100nM of bisphenol A analog to 1nM of bisphenol A. As shown in fig. 7, the fluorescence intensities of BPB, BPC, BPF, DES, E3 are 11.90%, 30.10%, 21.90%, 19.20%, 15.80% of BPA, respectively, and the bisphenol a analog produces a smaller fluorescence signal relative to bisphenol a, substantially equivalent to the blank background signal, demonstrating the higher selectivity of the method of the invention due to the high affinity of the bisphenol a aptamer for bisphenol a, and the higher recognition ability for the bisphenol a analog.
Example 6: detection Performance in actual samples
According to the optimal experimental conditions obtained by optimizing the conditions in example 3, bisphenol A with quantitative concentration (final concentration of 1pM, 100pM and 10000pM) is dissolved in drinking water, the content of bisphenol A detected by using the method of the invention is researched, and the detection performance of the method of the invention in actual samples is researched. The results are shown in table 1, bisphenol A with final concentrations of 1pM, 100pM and 10000pM is respectively and quantitatively added into drinking water, and the recovery rates of the bisphenol A are 102.0%, 91.1% and 122.5% respectively when the method is used for detection, which shows that the method has better reliability and accuracy in actual samples, has stronger anti-interference capability and can be used for detection of the actual samples.
Table 1: recovery rate of bisphenol A at different concentrations
Sequence listing
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Claims (4)
1. A bisphenol A fluorescence detection method based on atom transfer radical polymerization and truncated aptamer is characterized by comprising the following steps:
(1) pretreatment of hairpin DNA:
heating hairpin DNA solution to 95 ℃ and keeping for 15min, and then cooling to room temperature;
secondly, mixing a hairpin DNA solution of 100 mu M and a TCEP solution of 10 mu M in a volume ratio of 1:1, and reacting the mixed solution in a constant temperature shaking table at 37 ℃ in a dark place for 3 hours and storing the mixed solution at-20 ℃ for later use; when in use, the mixed solution is diluted until the concentration of the hairpin DNA is 1 mu M;
(2) amino Fe3O4Modification of magnetic beads
Weighing a certain amount of 10mg/ml amino Fe3O4Magnetic bead stock solution, magnetic separation, supernatant removal, washing by PBS buffer solution, magnetic separation, supernatant removal, adding PBS buffer solution for resuspension and recovering to the volume of the measured magnetic bead stock solution;
measuring 20 mu L of magnetic bead resuspension, 20 mu L of 100 mu M sulfoc-SMCC solution and 160 mu L of PBS buffer solution, mixing uniformly, and reacting for 2 hours in a constant temperature shaking table at 37 ℃ in a dark place;
thirdly, magnetically separating the mixed solution obtained in the second step, removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
adding 180 mu L of PBS buffer solution and 20 mu L of 1 mu M hairpin DNA solution, uniformly mixing, and reacting overnight in a constant temperature shaking table at 37 ℃ in a dark place;
(3) nucleic acid hybridization
Magnetically separating the reaction solution obtained in the step (2), removing supernatant, and washing with TE buffer solution;
adding 20 mu L of target solution and 180 mu L of TE buffer solution, mixing uniformly, and reacting for 2 hours in a constant temperature shaking table at 37 ℃ in a dark place;
(4) click reaction
Magnetically separating the reaction solution obtained in the step (3), removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
② 140 mul PBS buffer solution and 20 mul 1mM CuSO are added4Uniformly mixing the solution, 20 mu L of 1mM PBIB solution and 20 mu L of 2mM AA solution, and reacting for 2 hours in a constant temperature shaking table at 37 ℃ in a dark place;
(5) atom Transfer Radical Polymerization (ATRP)
Magnetically separating the reaction solution obtained in the step (4), removing supernatant, washing with PBS buffer solution, magnetically separating, and removing supernatant;
② 20 microliter of 1mM fluorescent monomer solution, 140 microliter of ultrapure water and 20 microliter of 1mM complex Cu are addedIIBr/Me6Mixing TREN solution and 20 μ L2 mM AA solution, and reacting in constant temperature shaking table at 37 deg.C in dark for 2 hr; the fluorescent monomer is fluorescein-O-acrylate;
(6) fluorescence detection
Magnetically separating the reaction liquid obtained in the step (5), removing supernatant, and washing with 30% (v/v) DMF solution and PBS buffer solution in sequence;
adding PBS buffer solution, uniformly mixing, and performing fluorescence detection on the mixed solution serving as a sample on a fluorescence spectrophotometer;
the hairpin DNA has the sequence of 5' -SH- (CH)2)6-CCACGCCGGTGGGTGGA ACGTGG-N3-3’；
The preparation method of the target solution comprises the following steps:
mixing 100 mu M ssDNA-A and 100 mu M ssDNA-B in a volume ratio of 1:1, heating the mixed solution to 95 ℃, keeping the temperature for 15min, cooling to room temperature, and storing at-20 ℃ for later use;
secondly, mixing 10 mu L of the mixed solution obtained in the first step with a certain amount of a sample to be detected, diluting to 500 mu L, and reacting overnight in a constant temperature shaking table at 37 ℃ in a dark place to obtain a target solution, and storing at-20 ℃ for later use;
the sequence of ssDNA-A is 5'-CCGGTGGGTGGA A-3'; the sequence of ssDNA-B is 5'-TTCCACCCACCGG-3'.
2. The method for detecting fluorescence of bisphenol A based on atom transfer radical polymerization and truncated aptamer according to claim 1, wherein the TE buffer solution in step (3) -step (2) contains MgCl with a concentration of 0.5mM2。
3. The method for detecting fluorescence of bisphenol A based on atom transfer radical polymerization and truncated aptamer according to claim 1, wherein the concentration of PBS buffer is 0.1M and the pH is 7.4.
4. The method for detecting bisphenol A fluorescence based on atom transfer radical polymerization and truncated aptamer according to claim 1, wherein the excitation wavelength of fluorescence detection in step (6) is 489nm and the slit width is 4 nm.
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