A nucleic acid aptamer of bisphenol a, nucleic acid aptamer derivatives and application thereof
By screening and modifying nucleic acid aptamers and their derivatives, the problems of high specificity and high affinity binding in the existing technology for bisphenol A detection have been solved, realizing a simple and economical bisphenol A detection method applicable to multiple detection platforms.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2026-02-25
- Publication Date
- 2026-08-04
AI Technical Summary
The lack of bisphenol A nucleic acid aptamers with high specificity and high affinity in existing technologies has led to bisphenol A detection methods relying on expensive equipment and complex operations, making it difficult to achieve simple, economical, and rapid detection.
We designed and screened nucleic acid aptamers and their derivatives with specific modifications, used PCR instruments and magnetic bead capture methods for screening, and combined multiple detection platforms to develop aptamer biosensors and rapid test strips.
It achieves highly specific recognition and high affinity binding of bisphenol A, significantly reducing detection costs and operational complexity, and is applicable to a variety of detection platforms.
Smart Images
Figure CN122012511B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a nucleic acid aptamer, a nucleic acid aptamer derivative, and their uses, specifically to a nucleic acid aptamer capable of binding to bisphenol A, a nucleic acid aptamer derivative, and their uses. Background Technology
[0002] Bisphenol A (BPA), also known as 2,2-bis(4-hydroxyphenylpropane), is a typical endocrine disrupting chemical (ECDS), primarily used in the production of polycarbonate, epoxy resins, and other polymeric materials. When these materials are heated, the unstable lipophilic compound BPA can migrate into water and food through food contact materials, and can also enter the environment through wastewater discharge from plastic manufacturing plants, seriously endangering human health. Studies have shown that BPA not only affects the central nervous system, immune system, and reproductive system, but also induces apoptosis in spermatogenic cells through extracellular molecular signaling patterns. Therefore, sensitive detection of BPA is crucial for the ecological environment and human health.
[0003] Currently, the detection of bisphenol A (BPA) mainly relies on instrumental analytical methods, including high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). While these methods offer high detection accuracy and specificity, their reliance on expensive equipment, cumbersome pretreatment procedures, and specialized technical personnel significantly limits their practical application. In contrast, biosensors, by converting biorecognition processes into detectable signals, enable real-time quantitative analysis of target analytes. They offer advantages such as high selectivity, high sensitivity, ease of operation, low cost, and continuous monitoring, attracting widespread attention in environmental monitoring, food safety, and medical diagnostics. Aptamers, as key recognition elements in biosensors, offer higher stability and lower preparation costs compared to traditional antibodies, and can achieve high batch-to-batch consistency through chemical synthesis. Furthermore, aptamers are easily modified, allowing for the introduction of specific functional groups through molecular design to construct high-performance sensors, playing a crucial role in disease diagnosis, drug screening, environmental pollutant monitoring, and food safety testing.
[0004] To date, research on screening methods for obtaining high-affinity and high-selectivity nucleic acid aptamers for bisphenol A (BPA) remains lacking. Successfully obtaining specific aptamers for BPA would drive the development of a series of rapid detection technologies, including aptamer biosensors and rapid test strips, thus providing simple and economical analytical tools for environmental pollutant detection. Therefore, research on screening methods for BPA-specific nucleic acid aptamers and their functionalized derivatives has significant practical value and application prospects. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a nucleic acid aptamer, nucleic acid aptamer derivative and its uses that can bind to bisphenol A with high specificity and high affinity, can be chemically synthesized, have good biocompatibility, small molecular weight, are stable and easy to store.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0007] A bisphenol A nucleic acid aptamer, characterized in that the nucleic acid aptamer has a DNA sequence as shown in SEQ ID NO:1.
[0008] Furthermore, a certain position on the nucleotide sequence of the nucleic acid aptamer is phosphorylated, oxymethylated, methylated, aminoized, thiolated, fluorinated, or isotopized.
[0009] Furthermore, the nucleotide sequence of the nucleic acid aptamer is bound with biotin, digoxigenin, fluorescent substances, nanomaterials, polyethylene glycol, peptides, proteins, enzymes, or folic acid labels.
[0010] Furthermore, the nucleic acid aptamer derivative is a thiophosphate backbone derived from the nucleotide sequence backbone of the nucleic acid aptamer, or a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer.
[0011] A method for screening the above-mentioned nucleic acid aptamers involves annealing a random library LibV1 and a primer mixture using a PCR instrument.
[0012] Furthermore, the nucleic acid aptamers or nucleic acid aptamer derivatives are screened using a magnetic bead capture method.
[0013] Compared with the prior art, the advantages of the present invention are:
[0014] (1) The nucleic acid aptamer provided by the present invention has a high specific recognition ability for bisphenol A molecules, strong binding affinity and good selectivity, which can significantly improve the reliability of detection results.
[0015] (2) The aptamers and their derivatives developed in this invention can be adapted to a variety of detection platforms (including surface plasmon resonance, microfluidic chips and portable sensors, etc.), which greatly reduces the detection cost and operational complexity compared with traditional instrument analysis methods. Attached Figure Description
[0016] Figure 1 This is the result of the affinity measurement between the nucleic acid aptamer probe and bisphenol A in the embodiments of the present invention.
[0017] Figure 2The results of fluorescence polarization detection of bisphenol A by the nucleic acid aptamer in this embodiment of the invention are shown. Detailed Implementation
[0018] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.
[0019] Example:
[0020] The bisphenol A aptamer provided in this embodiment has the DNA sequence shown in SEQ ID NO:1, and is capable of specifically binding to bisphenol A:
[0021] The nucleotide sequences of the above-mentioned nucleic acid aptamers are selected from naturally occurring or artificially synthesized sequences, or the same sequences from any other source.
[0022] It should be noted that the scope of protection of this invention covers all sequences containing this aptamer.
[0023] This invention covers sequences in which a certain position of the above-mentioned nucleic acid aptamer nucleotide sequence is phosphorylated, oxymethylated, methylated, fluorinated, aminated, thiolated, or isotopized.
[0024] This invention covers sequences on the nucleotide sequences of the aforementioned nucleic acid aptamers that bind biotin, digoxigenin, fluorescent substances, nanomaterials, polyethylene glycol, peptides, proteins, enzymes, or folic acid markers (or even radioactive substances).
[0025] The above-mentioned nucleic acid aptamers can be used to derive other nucleic acid aptamers. The nucleotide sequences of the derived nucleic acid aptamers can be any one of the following three sequences:
[0026] (1) The nucleotide sequence of the nucleic acid aptamer listed in this embodiment has more than 90% homology (for example, some complementary nucleotides can be deleted or added to the above nucleic acid aptamer sequence).
[0027] (2) The sequence that hybridizes with the nucleotide sequences of the nucleic acid aptamers listed in this embodiment;
[0028] (3) The RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer listed in this implementation.
[0029] The nucleotide sequence backbone of the nucleic acid aptamers listed in this embodiment can also be derived into a thiophosphate backbone, and the above nucleic acid aptamers can also be modified into corresponding locked nucleic acids or peptide nucleic acids.
[0030] The bisphenol A nucleic acid aptamer of this embodiment has the following uses: Applications in the detection of environmental, food, and biological hazards; Applications in separation and purification; Applications in drug design and development; Applications in the preparation of bisphenol A detection probes.
[0031] The CAS number of bisphenol A used in this embodiment is 80-05-7.
[0032] In this embodiment, the nucleic acid aptamers for bisphenol A are mainly screened using the magnetic bead capture method. The specific screening process includes the following steps:
[0033] (a) Initial Screening: Library Dissolution: Remove the libV1 library and 1ibV1-CS-biotin primer powder tubes and centrifuge at 12000 rpm for 10 min. Add 260 uL LPBS to the libV1-80nt library to dilute the 1ibV1 library to 5 μM. Add 50 uL of screening buffer to the 1ibV1-CS-biotin primer powder to dilute the 1ibV1-CS-biotin primer to 100 μM. Vortex the diluted library and libV1-CS-biotin separately to dissolve completely, and centrifuge at 12000 rpm for 1 min. Library and Primer Mixing: Add 26 uL of the dissolved 1ibV1-CS-biotin primer to the dissolved 1ibV1 library and mix well to a final concentration of approximately 10 uM. Centrifuge at 12000 rpm for 1 min. Library and primer complementarity pairing: The mixed libV1 library and 1ibV1-CS-biotin primers were aliquoted into eight-tube PCR instruments and subjected to slow annealing using a PCR instrument (slow annealing conditions: 95℃ for 10 min, slow cooling to 60℃ for 1 min, then slow cooling to 25℃, all at a cooling rate of 0.1℃ / s), and held at 25℃ for 1 min. The hybridization library was added to 1 mL of streptavidin-modified magnetic beads that had been washed five times and incubated on a shaker at room temperature for 1 hour. The magnetic beads were then adsorbed using a magnet, and the supernatant was removed. The beads were resuspended in 600 μL of DPBS buffer and washed six times. Bisphenol A target solution was then added, and the mixture was incubated on a shaker at room temperature for 1 hour. The magnetic beads were adsorbed using a magnet, and the supernatant was recovered to obtain the initial screening nucleic acid library containing a DNA-bisphenol A complex.
[0034] (b) Purification: The initial screening nucleic acid library obtained in step (a) was subjected to PCR amplification using the primers listed in step (a) with fluorescent primers at the 5' end and primers with spacers at the 3' end. The amplified products were separated into single strands using SDS-PAGE denaturing gel, followed by gel boiling, n-butanol concentration, and dialysis to form a secondary nucleic acid library. The PCR amplification conditions were: 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for N (optimal number of rounds) of amplification cycles; extension at 72℃ for 7 min. Gradient PCR was used, with the initial nucleic acid library as a template to optimize the annealing temperature, ultimately achieving an annealing temperature of 60℃. The optimal number of amplification rounds for each screening product was obtained through round optimization. After round optimization, the remaining libraries were amplified under the same conditions. The PCR-generated library was prepared into single strands for the next round of screening. The single strand preparation method was as follows: PCR product was mixed with n-butanol at a volume ratio of 1:5, vortexed for 30 s, and centrifuged at 6000 rpm for 2 min. The solution separated into layers; the upper n-butanol layer was removed, and the lower layer was retained, with a volume of approximately 50 μL. An equal volume of 2× TBE solution was added to the lower layer, and the mixture was heated at 95 ℃ for 10 min. While still hot, the sample was loaded onto a denaturing gel PAGE and run at 300 V for 20 min. Fluorescent bands were excised and collected under UV light, and the gel was fragmented. 1.2 mL of DPBS buffer was added to the fragmented gel, and the mixture was boiled at 95 ℃ for 15 min. After centrifugation, the supernatant was collected, and this process was repeated 1-2 times. A 1:5 volume ratio of n-butanol solution was added to the collected solution, vortexed for 30 s, and centrifuged at 6000 rpm for 2 min. The upper n-butanol layer was removed. Repeat 2-3 times to achieve a final sample volume of approximately 100 μL. Finally, dialyze the sample overnight in DPBS solution at 4 °C.
[0035] (c) Cycling: Replace the starting nucleic acid library with the secondary nucleic acid library obtained above and repeat steps (b) to (c) above. Use new streptavidinized magnetic beads in each cycle until a nucleic acid library containing nucleic acid aptamers that bind to bisphenol A with high affinity and high specificity is obtained.
[0036] (d) Screening efficiency evaluation: qPCR was used to determine the screening efficiency, i.e., the enrichment of DNA during the screening process. The detection signal is the C0 of the DNA library during the qPCR process. q The value was converted to the retention rate of the library by bisphenol A. First, a series of nucleic acid library solutions of different concentrations were prepared and measured using qPCR to obtain the concentration and C... qA linear equation related to the value was then used. Next, the concentration of the DNA library acquired through bisphenol A competition was measured, and the library retention rate was obtained based on the ratio of the measured amount to the input amount. Higher screening efficiency resulted in a higher retention rate. When the retention rate reached a plateau, the library was cloned and sequenced.
[0037] Examination 1: The affinity between bisphenol A and its nucleic acid aptamer in the above embodiment was mainly determined by qPCR. The specific determination process includes the following steps:
[0038] (1) Determination of nucleic acid concentration and qPCR determination of C q Standard curve between values
[0039] The relationship between nucleic acid aptamer probe concentration and C20 assay by qPCR q Linearity determination between values: Nucleic acid library Lib solutions of different concentrations (1000, 100, 10, 1, 0.1 pM) were prepared using DPBS as solvent; 10 μL of the prepared solution was transferred, and 2 μL of pre-prepared mixture solution (containing dNTPs, Evagreen enzyme, forward and reverse primers, and buffer) was added, and the mixture was vortexed and mixed. The linear relationship between the values was determined by the obtained C... q The values were linearly fitted to the logarithm of the concentration.
[0040] (2) Determination of the affinity of the probe for bisphenol A by qPCR:
[0041] First, 200 μL of streptavidin-encapsulated magnetic microsphere suspension was transferred, and then the magnetic beads were adsorbed with a magnet to remove the supernatant. The suspension was then washed four times with 200 μL of DPBS buffer.
[0042] 2 μL of 100 μM fixed probe was prepared using DPBS. It was first incubated with the aptamer probe for hybridization, and then incubated with the washed streptavidin magnetic microspheres for 35 min. The magnetic beads were then adsorbed with a magnet and the supernatant was removed. The microspheres were then washed twice with 200 μL of DPBS buffer, resuspended in 200 μL of DPBS, and divided into 5 equal parts.
[0043] Equal volumes of bisphenol A solutions at different concentrations (100, 400, 1600, 6400, and 25600 nM) were added to the magnetic bead suspensions containing the nucleic acid aptamer probes. The mixtures were reacted at room temperature for 15 min. The magnetic beads were then adsorbed using a magnet, and the supernatant was transferred and labeled 1, 2, 3, 4, and 5. 10 μL of the labeled solution was transferred to the corresponding numbered qPCR tubes, and 2 μL of pre-prepared mixture solution (containing dNTPs, Evagreen enzyme, forward and reverse primers, and buffer) was added to each tube. The mixtures were then vortexed and qPCR was used to quantify the nucleic acids in the solution. The obtained C... q Substituting the value into the linear equation established above, we obtain the number of aptamer molecules that have competed for different target concentrations, and then using Y=B max *X(K d +X) (Y is the retention rate of the nucleic acid molecule, X is the concentration of bisphenol A used, B is a constant, and max means maximum value) The formula is used to fit and calculate the K binding of the nucleic acid aptamer and the target. d Value. For example... Figure 1 As shown, the final fitted bisphenol A and its nucleic acid aptamer K d The value was 91 nmol / L.
[0044] Investigation 2: Bisphenol A was determined using fluorescence polarization technology. The specific determination process included the following steps:
[0045] (1) Hybrid sequence 1 is a sequence preceded by -SH- as shown in SEQ ID NO.2;
[0046] Hybrid sequence 2 consists of a -SH- prefixed to the sequence shown in SEQ ID NO. 3;
[0047] (2) The aptamer fluorescent probe 2 is a sequence as shown in SEQ ID NO.4 with -FAM- attached to it;
[0048] The DNA control fluorescent probe is a sequence as shown in SEQ ID NO.5 followed by -FAM-.
[0049] Preparation of nanosilver aptamer fluorescent probes and DNA control probes:
[0050] (a) Decahedral silver nanoparticles were selected as the nanomaterial. 1 mL of the decahedral silver nanoparticle solution was transferred, and 100 μL of 10 μM hybridization sequence 1 solution and 64 μL of 2 M sodium chloride solution were added. The mixture was shaken and incubated overnight at room temperature for 30 min. The prepared silver nanoparticle solution was centrifuged at 15000 rpm for 10 min to remove the supernatant. 1 mL of screening buffer was added, and the solution was centrifuged again at 15000 rpm for 10 min to remove the supernatant. This process was repeated three times. Next, 100 μL of 100 μM fluorescent probe 2 solution was added, and the solution was incubated at room temperature for 30 min. The solution was then centrifuged again to remove the supernatant, and 500 μL of screening buffer was added to resuspend the solution.
[0051] (b) Using decahedral silver nanoparticles as the nanomaterial, 1 mL of decahedral silver nanoparticle solution was transferred, and 100 μL of 10 μM hybridization sequence 2 solution and 64 μL of 2 M sodium chloride solution were added and vortexed to mix. The mixture was allowed to react at room temperature for 30 min and then incubated overnight. The prepared silver nanoparticle solution was centrifuged at 15000 rpm for 10 min, the supernatant was removed, and 1 mL of screening buffer was added. The mixture was then centrifuged at 15000 rpm for 10 min, and the supernatant was removed. This process was repeated three times. Next, 100 μL of 100 μM aptamer fluorescent probe 3 solution was added, and the mixture was allowed to react at room temperature for 30 min. Then, the mixture was centrifuged, the supernatant was removed, and the mixture was resuspended in 500 μL of screening buffer.
[0052] The above-mentioned nano-silver aptamer fluorescent probe and DNA control probe solutions were divided into five equal portions and placed into 96-well plates. 100 μL of bisphenol A solution with final concentrations of 40 nM, 160 nM, 640 nM, 2560 nM, and 10240 nM were added to each well, and the plates were reacted at room temperature for 10 min, followed by fluorescence polarization detection analysis. Figure 2 As can be seen, the fluorescence polarization value of the bisphenol A aptamer probe gradually decreases with increasing bisphenol A concentration, and there is a good linear relationship between the fluorescence polarization signal and the logarithm of the bisphenol A concentration. In contrast, the fluorescence polarization signal produced by the control tetrabromobisphenol A shows no correlation with bisphenol A, and the signal is weak. This demonstrates that the nucleic acid aptamer detection method provided by this invention can rapidly and effectively determine bisphenol A.
[0053] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.
Claims
1. A nucleic acid aptamer for bisphenol A, characterized in that, The DNA sequence of the nucleic acid aptamer is shown in SEQ ID NO:
1.
2. The nucleic acid aptamer according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid aptamer is bound with biotin, digoxigenin, fluorescent substances, nanomaterials, polyethylene glycol, protein, or folic acid labels.
3. A bisphenol A nucleic acid aptamer derivative, characterized in that, The nucleic acid aptamer derivative is a thiophosphate backbone derived from the nucleotide sequence backbone of the nucleic acid aptamer described in claim 1 or 2, or a corresponding locked nucleic acid or peptide nucleic acid modified from the nucleic acid aptamer described in claim 1 or 2.