A Biosensor for Detecting 17β-Estradiol and Its Application

Abstract

The present invention discloses a biosensor for detecting 17β-estradiol and its application. The biosensor of the present invention includes split probes STWS-a, STWS-b, and AuNPs@track hairpin. In the presence of 17β-estradiol, 17β-estradiol specifically induces the assembly of STWS-a and STWS-b into STWS, bringing the E6-a and E6-b at the tails of the two probes close together to form a complete E6-DNAzyme sequence, which hybridizes with AuNPs@track hairpin. With the assistance of Mg<supgt;2+< / supgt>, track hairpin is catalytically cleaved by E6-DNAzyme, releasing the FAM-labeled short chain. STWS is released and autonomously moves to the adjacent track hairpin, and the FAM-labeled short chain is released, achieving fluorescence signal accumulation, and finally realizing the specific detection of 17β-estradiol.

Description

Technical Field

[0001] The present invention relates to the technical field of detection and analysis, and in particular to a biosensor for detecting 17β-estradiol and its application. Background Art

[0002] 17β-Estradiol (E2) is a natural endocrine disrupting chemical, mainly produced in the follicles of vertebrates, and is considered the most biologically active estrogen. In view of its significant effects on promoting the growth of animal skeletal muscle and increasing milk production, 17β-estradiol has been wantonly abused in the livestock and poultry industry in recent years. However, the illegal use of 17β-estradiol easily leads to food residues, which accumulate in the human body through the food chain, threatening human health. Relevant toxicological studies have shown that even low concentrations of E2 can seriously disrupt the balance of human hormone metabolism, affect the reproductive system function, and increase the risk of cancer.

[0003] Traditional detection methods mainly include high performance liquid chromatography (HPLC), gas chromatography (GC), and liquid / gas chromatography-mass spectrometry (HPLC / GC-MS), etc., but they require large instruments and complex sample pretreatment, so they are not suitable for rapid food safety detection. In addition, although the commonly used immunological methods at present have the advantages of strong specificity, high sensitivity and convenient use, the antibody as the recognition molecule limits the stability of the method and is extremely vulnerable to interference from the external environment. To overcome the above defects, there is an urgent need to develop a detection method for 17β-estradiol with high stability, specificity and sensitivity to ensure food safety. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a biosensor for detecting 17β-estradiol and its application.

[0005] The first object of the present invention is to provide a biosensor for detecting 17β-estradiol based on a target self-service 3D-DNA walker, including split probes STWS-a, STWS-b and AuNPs@track hairpin, and the AuNPs@track hairpin is dispersed in a mixed solution of MgCl2, NaCl and Tween-20; wherein, the nucleotide sequence of the split probe STWS-a is as shown in SEQ ID NO.1, and the nucleotide sequence of the split probe STWS-b is as shown in SEQ ID NO.2.

[0006] SEQ ID NO.1:

[0007] 5’-TCTGTCAGCGATGGGACGACATGGA-3’;

[0008] SEQ ID NO.2:

[0009] 5’-TCCATCAACGAAGTGCGTCCGTCCCCACCCATGTAAGCTT-3’.

[0010] In one embodiment of the present invention, the MgCl2 concentration in the dispersion mixing solution is 10 - 20 mM, the NaCl concentration is 200 - 600 mM, and the Tween-20 concentration is 0.02 - 0.08% (v / v).

[0011] In one embodiment of the present invention, the AuNPs@track hairpin is prepared by the following method:

[0012] Mix the track hairpin with TCEP and heat, then cool to obtain a stable hairpin structure. Add AuNPs and mix evenly to obtain a mixed solution. Add buffer to make the NaCl concentration in the mixed solution reach 0.02 - 0.07 M, and then add NaCl to increase the NaCl concentration in the mixed solution to 0.0 - 0.6 M in increments of 0.1 M NaCl, with an interval of 8 hours between each addition of NaCl. At the same time, maintain a Tween-20 concentration of 0.03 - 0.08% (v / v) during the "aging" process to obtain the AuNPs@track hairpin; the nucleotide sequence of the track hairpin is as shown in SEQ ID NO.3. (Because one end of the track hairpin is labeled with SH (mercapto group), disulfide bonds will form between SH groups, so the track hairpins in the solution will be connected by disulfide bonds to form dimers. Therefore, TCEP needs to be added for reaction to form a stable hairpin structure.)

[0013] SEQ ID NO.3:

[0014] 5’-SH-[T] 31 -CGACCTGTCTATATCAAGCTTTrAGGACAGATTTTTTTTTCAGGTCG-FAM-3’ (synthesized by Sangon Biotech (Shanghai) Co., Ltd.).

[0015] In one embodiment of the present invention, a functional group or molecule is connected to the 5' end or 3' end of the nucleotide sequence of the track hairpin, and a ribonucleotide rA is modified in the middle.

[0016] In one embodiment of the present invention, the functional group or molecule is selected from fluorescein, biotin, amino group, mercapto group, digoxin, radioisotope, enzyme label, or nano-luminescent material.

[0017] The second object of the present invention is to provide a kit, and the kit includes the biosensor described above.

[0018] The third object of the present invention is to provide the application of the biosensor and the kit in detecting 17β-estradiol.

[0019] In an embodiment of the present invention, the specific application method includes the following steps: adding the splitting probe STWS-a, the splitting probe STWS-b, and AuNPs@track hairpin to the 17β-estradiol test solution to be measured, stirring and mixing for reaction, and detecting the fluorescence spectrum value of the obtained reaction solution to achieve qualitative or quantitative detection of 17β-estradiol.

[0020] In an embodiment of the present invention, the molar ratio of the splitting probe STWS-a to STWS-b is 1:0.3 - 4; the total molar amount of the splitting probe STWS-a and STWS-b to AuNPs@track hairpin is 200 - 10:1.

[0021] In an embodiment of the present invention, the reaction conditions are as follows: the reaction temperature is 25 - 37°C, and the reaction time is 1 - 3 h.

[0022] In an embodiment of the present invention, in the detection of the fluorescence spectrum value, the excitation wavelength is 475 - 495 nm, and the emission wavelength is 510 - 700 nm.

[0023] The detection principle of the present invention is as Figure 1 shown. By fusing the split aptamer (Split-a or Split-b) of 17β-estradiol and a partial sequence (E6-a or E6-b) of the E6-DNAzyme sequence, two splitting probes (STWS-a and STWS-b) with specific bifunctions are rationally designed. In the presence of 17β-estradiol, 17β-estradiol specifically induces the assembly of STWS-a and STWS-b into STWS, enabling the E6-a and E6-b at the tails of the two probes to be closely approximated to form a complete E6-DNAzyme sequence. Then, the activated E6-DNAzyme can hybridize with the track hairpin on AuNPs@track hairpin. With the assistance of Mg 2+ the track hairpin is catalytically cleaved by E6-DNAzyme, releasing the FAM-labeled short chain. Subsequently, STWS is released and autonomously moves to the adjacent track hairpin. Therefore, more FAM-labeled short chains are released, achieving a large signal accumulation, and finally realizing the specific detection of 17β-estradiol.

[0024] The said E6-a (5’-3’): TCTGTCAGCGAT;

[0025] The said E6-b (5’-3’): CACCCATGTAAGCTT.

[0026] The said E6-DNAzyme sequence (5’-3’): TCTGTCAGCGATCCGGAACGGCACCCATGTAAGCTT.

[0027] The above technical solution of the present invention has the following advantages compared with the prior art:

[0028] (1) The present invention ingeniously designs a cleavage probe that can specifically and highly affinity recognize 17β-estradiol by using the cleavage aptamer of 17β-estradiol. The constructed cleavage probe integrates the recognition element and the driving element, and serves as a walking strand to initiate DNA walker amplification, greatly simplifying the operation process of DNA walker and improving the detection sensitivity. The detection limit can reach 0.28 pM.

[0029] (2) The present invention uses the cleavage aptamer as the recognition element. Compared with directly using the complete aptamer as the recognition element, the cleavage aptamer only undergoes inducible structural recombination and forms a specific functional structure when the target exists. Therefore, only extremely low background signals will be generated, greatly improving the detection accuracy.

[0030] (3) The present invention uses nano-gold as the anchoring substrate for the DNA walker track to construct a 3D-DNA walker. Compared with the traditional 1D-DNA walker and 2D-DNA walker, the reaction rate of DNA walker is significantly improved and the detection time is shortened.

[0031] (4) The DNA walker in the present invention is driven by E6-DNAzyme and does not require the participation of protease, greatly improving the stability of DNA walker. Compared with protease, DNAzyme has the advantages of low cost, not easy to denature and easy to prepare. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention and in combination with the drawings, wherein

[0033] Figure 1 is the schematic diagram (SEM) of the self-service 3D-DNA walker detection of 17β-estradiol based on the target of the present invention.

[0034] Figure 2It is the transmission electron microscopy characterization diagram (TEM) of gold nanoparticles (AuNPs) in Example 1 of the present invention.

[0035] Figure 3 It is the ultraviolet-visible spectrum of the preparation of AuNPs@trackhairpin in Example 2 of the present invention.

[0036] Figure 4 It is the fluorescence spectrum (A) of the target self-service 3D-DNA walker under different concentrations of 17β-estradiol in Example 4 of the present invention; the corresponding standard curve (B).

[0037] Figure 5 It is the specificity analysis of detecting 17β-estradiol based on the target self-service 3D-DNA walker in Example 5 of the present invention. Detailed implementation manners

[0038] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0039] Example 1: Preparation of gold nanoparticles

[0040] First, add 1 mL of 1% chloroauric acid aqueous solution to a three-necked round-bottom flask containing 98 mL of ultrapure water, stir vigorously and heat to boiling in an oil bath. Then quickly add 1 mL of 5% trisodium citrate aqueous solution. After reacting for 30 min, the color of the solution gradually changes from light yellow to dark wine red. Finally, transfer the round-bottom flask to room temperature, stir and cool to room temperature, and then store the prepared AuNPs solution in the dark at 4 °C for later use. The morphology and size of the gold nanoparticles are measured by transmission electron microscopy (TEM) and dynamic light scattering instrument, as Figure 2 shown. The prepared gold nanoparticles have uniform size and an average particle size of 15 nm.

[0041] Example 2: Preparation of AuNPs@trackhairpin

[0042] In the 17β-estradiol detection method based on the target self-service 3D-DNA walker, it includes a DNA-functionalized gold nanoparticle (AuNPs@trackhairpin), and its specific preparation steps: Add 100 μL of track hairpin modified with FAW with a concentration of 4 μM (the sequence is 5’-SH-[T] 31-CGACCTGTCTATATCAAGCTTTrAGGACAGATTTTTTTTTCAGGTCG-FAM-3’) was mixed with 10 μL of 20 mM TCEP at 25 °C for 2 h to cleave disulfide bonds. Then, the trackhairpin solution was heated at 95 °C for 10 minutes and then slowly cooled on ice to obtain a stable hairpin structure. Next, 500 μL of AuNPs (1.79 nM) was added and shaken well. Then, PBS buffer (10×) was added dropwise to the above solution until a concentration of 0.05 M NaCl was reached. Then, 2 M NaCl was used to increase the NaCl concentration to 0.5 M in 0.1 M NaCl increments, with an 8-hour interval, while maintaining a 0.05% tween-20 concentration during the "aging" process. Finally, the excess trackhairpin was removed by centrifugation at 11,000 rpm for 20 minutes. Finally, AuNPs@track hairpin was resuspended in Tris-HCl buffer (10 mM, pH 8.3) and stored at 4 °C for further use. Characterized by ultraviolet-visible spectroscopy, as Figure 3 shown, the functionalized gold nanoparticles presented a characteristic peak of nucleic acid at 260 nm, indicating the successful functionalization of trackhairpin on the surface of gold nanoparticles.

[0043] Example 3: Operating steps of target self-service 3D-DNA walker

[0044] For a typical operating step of target self-service 3D-DNA walker, 50 μL of 17β-estradiol at different concentrations was mixed with an equal volume of split probes (STSW-a and STSW-b, 200 nM respectively). Subsequently, 100 μL of 2 nM AuNPs@trackhairpin dispersion solution containing 20 mM MgCl2, 500 mM NaCl, 0.05% (v / v) Tween-20 was mixed with the above solution. The mixed solution was incubated at 37 °C for 2 h. After incubation, fluorescence data was collected in a 96-well clear-bottom black polystyrene microplate by a Synergy H1 multimode microplate reader.

[0045] Example 4: Performance characterization of target self-service 3D-DNA walker for detecting 17β-estradiol

[0046] According to the operating steps in Example 3, the target self-service 3D-DNA walker for detecting 17β-estradiol was studied by detecting 17β-estradiol at different concentrations (1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50 nM). As Figure 4As shown in Figure A, as the target concentration increased from 1 pM to 50 nM, the fluorescence signal response gradually increased and showed a good linear relationship with the logarithm of the 17β-estradiol concentration ( Figure 4 B). The regression equation was fitted as ΔF = 7906.8 LgC E2 + 25463.6, and the correlation coefficient value R 2 was 0.9982, where F is the fluorescence signal intensity and C E2 is the 17β-estradiol concentration. According to the 3-fold signal-to-noise ratio principle (LOD = 3δ / κ, where δ is the standard deviation of blank parallel determination and κ is the slope of the calibration curve), the limit of detection (LOD) was calculated to be 0.28 pM. The relative standard deviation of 11 repeated determinations of 5 ng / mL lysozyme was 2.7%.

[0047] Example 5: Specificity analysis of detecting 17β-estradiol based on target self-help 3D-DNA walker

[0048] To evaluate the specificity of detecting 17β-estradiol based on target self-help 3D-DNA walker, several other hormone structural analogs that may exist in food samples were determined. As Figure 5 shown, compared with the single determination of 17β-estradiol, in the presence of sodium deoxycholate (DCA), sodium dehydroepiandrosterone sulfate (DIS), estrone (E1), ethinyl estradiol (EE), and dihydrotestosterone (DHT), the signal of the determined 17β-estradiol did not change significantly. This result confirmed that the detection of 17β-estradiol based on target self-help 3D-DNA walker has excellent specificity.

[0049] Example 6: Spiked recovery of actual samples

[0050] To evaluate the practical application performance of the target self-help 3D-DNA walker for detecting 17β-estradiol in complex environments, the method proposed in the present invention was used to analyze spiked actual samples (water, milk, yogurt, infant milk powder, beef, and shrimp) added with different concentrations of 17β-estradiol (0, 0.2, 0.5, and 2 μg / kg). 5 g of milk was dispersed in 10 mL of ethyl acetate and sonicated continuously for 10 minutes. Subsequently, the mixed solution was centrifuged at 8000 r / min for 5 min, and the supernatant was collected. The obtained supernatant was dispersed in Tris-HCl buffer containing 10% DMSO after rotary evaporation and diluted to 10 mL for standby. The pretreatment processes of water samples, yogurt, and infant milk powder samples were the same as those of milk samples. For beef and shrimp samples, 5 g of minced beef or shrimp was added to 10 mL of sodium acetate buffer, vortexed for 2 min, then 20 μL of glucuronidase and arylsulfatase were added, and enzymolysis was carried out at 50 °C for 2 h. Then 10 mL of ethyl acetate was added, and finally, it was processed according to the treatment steps of milk samples. As shown in Table 1, the spiked recovery rate of 17β-estradiol ranged from 95.6% to 106.4%, and the relative standard deviation (RSD) was from 1.8% to 6.3%. This result indicates that the target self-help 3D-DNA walker detection strategy constructed in the present invention can effectively resist the interference of complex matrices in actual samples and has good potential for practical analysis.

[0051] Table 1. Analytical results of spiked 17β-estradiol in actual samples

[0052]

[0053]

[0054] a ND: Not detected

[0055] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention. SEQUENCE LISTING <110> Jiangnan University <120> A biosensor for detecting 17β-estradiol and its application <130> 3 <160> 3 <170> PatentIn version 3.3 <210> 1 <211> 25 <212> DNA <213> (Synthetic) <400> 1 tctgtcagcg atgggacgac atgga 25 <210> 2 <211> 40 <212> DNA <213> (Synthetic) <400> 2 tccatcaacg aagtgcgtcc gtccccaccc atgtaagctt 40 <210> 3 <211> 78 <212> DNA <213> (Synthetic) <400> 3 tttttttttt tttttttttt tttttttttt tcgacctgtc tatatcaagc tttraggaca 60 gatttttttt tcaggtcg 78

Claims

1. A biosensor for detecting 17β-estradiol, characterized in that, Including splitting probes STWS-a, STWS-b, and AuNPs@track hairpin, where the AuNPs@track hairpin is dispersed in a mixed solution of MgCl2, NaCl, and Tween-20; among them, the nucleotide sequence of the splitting probe STWS-a is as shown in SEQ ID NO.1, and the nucleotide sequence of the splitting probe STWS-b is as shown in SEQ ID NO.2; The AuNPs@track hairpin is prepared by the following method: Mix the track hairpin with TCEP and heat, cool to obtain a stable hairpin structure, add AuNPs and mix evenly to obtain a mixed solution, add buffer to make the NaCl concentration in the mixed solution reach 0.02 - 0.07M, and then add NaCl to increase the NaCl concentration in the mixed solution to 0.2 - 0.6M while keeping the Tween-20 concentration constant, and finally obtain the AuNPs@track hairpin; the nucleotide sequence of the track hairpin is as shown in SEQ ID NO.

3.

2. The biosensor according to claim 1, characterized in that, The concentration of MgCl2 in the dispersion mixed solution is 10 - 20 mM, the concentration of NaCl is 200 - 600 mM, and the concentration of Tween-20 is 0.02 - 0.08% (v / v).

3. The biosensor according to claim 1, characterized in that, A functional group or molecule is connected to the 5' end or 3' end of the nucleotide sequence of the track hairpin.

4. The biosensor according to claim 3, characterized in that, The functional group or molecule is selected from labeled fluorescein, biotin, amino group, mercapto group, digoxin, radioisotope, enzyme label, or nano-luminescent material.

5. A kit, characterized in that, The kit includes the biosensor according to any one of claims 1 - 4.

6. Use of the biosensor according to any one of claims 1 - 4 and the kit according to claim 5 in detecting 17β-estradiol.

7. The application according to claim 6, characterized in that The specific application method includes the following steps: Add the splitting probe STWS-a, splitting probe STWS-b, and AuNPs@track hairpin to the 17β-estradiol test solution to be detected, stir and mix for reaction, and detect the fluorescence spectrum value of the obtained reaction solution to achieve qualitative or quantitative detection of 17β-estradiol.

8. The application according to claim 7, wherein The reaction conditions: The reaction temperature is 25 - 37°C, and the reaction time is 1 - 3 h.

9. The application according to claim 7, wherein In the detection of the fluorescence spectrum value, the excitation wavelength is 475 - 495 nm, and the emission wavelength is 510 - 700 nm.

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