Method for detecting 17 beta-estradiol by using colorimetric-fluorescence dual-mode biosensor

Abstract

The invention discloses a method for detecting 17 beta-estradiol by using a colorimetric-fluorescent dual-mode biosensor, and belongs to the technical field of chemical analysis and detection. According to the method, a colorimetric-fluorescent dual-mode biosensor for detecting 17 beta-estradiol is established based on oxidase-like activity and fluorescence quenching characteristics of manganese dioxide nanosheets and catalytic characteristics of alkaline phosphatase by taking a nucleic acid aptamer as a recognition molecule. The synthesis cost of the nucleic acid aptamer is low, the method has universality, and other target objects can be detected by changing the nucleic acid aptamer; mBs-DNA-ALP is prepared by using a biotin-streptavidin reaction and a DNA hybridization reaction, and a structure conversion process from double-stranded DNA to an aptamer / target compound is realized by using targeted induction, so that a detection signal is amplified; according to the method for detecting 17 beta-estradiol through colorimetric-fluorescence signals, dual-mode output is achieved, results are verified and supplemented mutually, and the specificity and accuracy of the method are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection, and specifically relates to a method for detecting 17β-estradiol by a colorimetric-fluorescent dual-mode biosensor. Background Art

[0002] Endocrine disruptors may harm the endocrine functions of humans and aquatic organisms, and thus have become pollutants that have attracted much attention. Among endocrine disruptors, 17β-estradiol (E2) is a natural estrogen with high estrogenic activity and is a major female sex hormone. It is a key regulator of the growth, differentiation, and function of various tissues, including the reproductive system, mammary gland, skeletal system, and cardiovascular system. It can enter water bodies through the discharge of municipal sewage and industrial wastewater, animal feeding excretion, and other means. 17β-Estradiol may disrupt the endocrine system, further causing adverse effects on human reproduction, growth, and development, and endangering the health of future generations. In 2013, the Endocrine Society of the United States advocated for sensitive, accurate, and specific detection of 17β-estradiol.

[0003] Considering the adverse effects of 17β-estradiol on human health and the environment, researchers have developed various analytical methods for 17β-estradiol. Although traditional detection methods, such as HPLC and GC-MS, can achieve sensitive detection of 17β-estradiol, these methods require expensive equipment, complex operations, and long analysis times. ELISA (enzyme-linked immunosorbent assay)-based detection methods are relatively inexpensive and simple. However, due to the unsatisfactory specific binding of the 17β-estradiol receptor to the antibody, these methods lack selectivity and reproducibility. Therefore, it is extremely important to construct an effective 17β-estradiol detection method.

[0004] Aptamers are single-stranded oligonucleotides (DNA or RNA) obtained by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can specifically recognize and bind target molecules through conformational changes. Compared with antibodies, aptamers have the advantages of easy synthesis and modification, high affinity, strong specificity, low cost, and good stability, and have potential application value in the sensitive detection of biological macromolecules, organic small molecules, metal ions, etc. Compared with antibodies, aptamers exhibit more superior performance. For example, the in vitro screening process does not rely on animals or cells and is simple and easy to obtain. Secondly, the chemically synthesized aptamers have high purity and small differences between batches. The aptamer sequences obtained from the in vitro screening process are synthesized in large quantities and repeatedly by polymerase chain reaction (PCR) technology. Aptamers also have good chemical stability, are easy to store and use, are not easily inactivated, and can restore their native active conformation after denaturation. Finally, aptamers have a small molecular weight, good tissue penetration, and are easy to chemically modify, enabling multifunctionalization. Aptamers can be conveniently conjugated with various derivatives for chemical modification without loss of function, facilitating immobilization and method development, etc.

[0005] Manganese dioxide nanosheets (MnO2 NSs) exhibit many unique physicochemical properties due to their advantages such as a wide absorption light range, large specific surface area, high catalytic activity, and strong oxidation ability. Since the broad absorption spectrum of MnO2 NSs overlaps with the fluorescence excitation or emission spectra of most types of fluorescent dyes, MnO2 NSs have been widely studied as a reasonable quencher for fluorescence sensing systems. Therefore, MnO2 NSs are used as a very important nanomaterial in analytical detection.

[0006] Based on the peroxidase-like activity and fluorescence quenching characteristics of manganese dioxide nanosheets, the dual-mode biosensor combining colorimetric and fluorescence methods can largely eliminate instrumental errors and human errors, making the sensor have better reliability. Summary of the Invention

[0007] The object of the present invention is to provide a method for constructing a colorimetric-fluorescence dual-mode detection of 17β-estradiol by using aptamers as recognition molecules in combination with manganese dioxide nanosheets (MnO2 NSs) to achieve reliable and sensitive detection of 17β-estradiol (E2) in view of the problems existing in the existing detection technologies. The steps are as follows:

[0008] Prepare an MBs-DNA-ALP composite probe using magnetic beads (MB), aptamer (Aptamer) and its complementary strand (cDNA), and alkaline phosphatase (ALP), and prepare manganese dioxide nanosheets. Add an E2 buffer containing different concentrations of E2 to the prepared MBs-DNA-ALP composite probe and incubate it on a metal bath. After magnetic separation, take the supernatant into a new centrifuge tube, add ascorbic acid-2-phosphate (AAP) and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, and incubate it on a metal bath. Subsequently, add a manganese dioxide nanosheet solution to the above mixture and incubate it at room temperature for 10 min. Finally, in colorimetric detection, add sodium acetate-acetic acid (NaAc-HAc) buffer and 3,3',5,5'-tetramethylbenzidine (TMB) solution to the above mixture, incubate it in the dark at room temperature for 5 min, and then use a microplate reader to measure and record the absorbance and absorption spectrum of the solution; in fluorescence detection, add a DNA strand labeled with a fluorescent dye FAM (FAM-14G) and Tris-HCl buffer to the above mixture, incubate it in the dark at room temperature for 10 min, and use a fluorescence spectrophotometer to measure the fluorescence spectrum of the solution.

[0009] The Aptamer and cDNA are labeled with biotin at the 3' end of the nucleotide sequence, with a concentration of 10 μM each, and the magnetic beads and alkaline phosphatase are labeled with streptavidin.

[0010] The specific formula of the E2 buffer is 20 mM HEPES, pH = 7.4, 100 mM NaCl, 10 mM MgCl2, and the specific formula of the Tris-HCl buffer is 20 mM Tris-HCl, pH = 9, 5 mM MgCl2; the specific formula of the NaAc-HAc buffer is 200 mM, pH = 4.

[0011] The reaction temperature of E2 and the MBs-DNA-ALP composite probe is 25 °C, and the reaction time is 30 min.

[0012] The concentration of AAP is 500 μM, the concentration of manganese dioxide nanosheets is 0.13 mg / mL, the concentration of TMB is 4 mM, and the concentration of FAM-14G is 300 nM.

[0013] The mechanism of the present invention is as follows:

[0014] A composite probe (MBs-DNA-ALP) consisting of magnetic beads (MB), nucleic acid aptamer, its complementary strand (cDNA), and alkaline phosphatase (ALP) is used. The specific binding of E2 to its aptamer causes a significant conformational change in the aptamer, which in turn prompts the cDNA labeled with ALP (cDNA-ALP) to move away from the magnetic bead interface. After magnetic separation, cDNA-ALP catalyzes the dephosphorylation of ascorbic acid-2-phosphate (AAP), and the generated ascorbic acid (AA) reduces the nanozyme manganese dioxide nanosheets to Mn2+, thereby destroying the peroxidase-like catalytic activity and fluorescence quenching characteristics of the manganese dioxide nanosheets. Since 3,3',5,5'-tetramethylbenzidine cannot be oxidized by Mn2+ to form a blue oxidation product, the absorbance of the detection system at 652 nm decreases proportionally, achieving colorimetric signal output. Since the DNA strand labeled with the fluorescent dye FAM cannot be quenched by Mn2+ fluorescence, the fluorescence value of the detection system increases proportionally, achieving fluorescence signal output.

[0015] Advantages of the present invention:

[0016] (1) The nucleic acid aptamer used in the present invention has a low synthesis cost, and the method is universal. Detection of other targets can be achieved by changing the nucleic acid aptamer.

[0017] (2) By using the biotin-streptavidin reaction and DNA hybridization reaction, the present invention prepares MBs-DNA-ALP, and uses target-induced structural conversion from double-stranded DNA to aptamer / target complex, thereby amplifying the detection signal.

[0018] (3) The present invention constructs a colorimetric-fluorescent dual-mode nucleic acid aptamer sensor for detecting 17β-estradiol, with dual-mode output. The results are mutually verified and supplemented, improving the specificity and accuracy of the method. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the principle of the present invention.

[0020] Figure 2 It is a transmission electron microscope image of the MnO2 NSs prepared in Example 1.

[0021] Figure 3 It is an ultraviolet-visible absorption spectrogram of the AMB supernatant and the pure nucleic acid aptamer solution prepared in Example 2.

[0022] Figure 4 It is a feasibility verification of the colorimetric and fluorescent output signals of the method in Example 3.

[0023] Figure 5UV-Vis absorption spectra corresponding to adding different concentrations of E2 standard solutions (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20 μM) in the colorimetric detection of Example 3.

[0024] Figure 6 Linear relationship diagram between absorbance and E2 concentration in the colorimetric detection of Example 3.

[0025] Figure 7 Photos taken under sunlight corresponding to adding different concentrations of E2 standard solutions (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20 μM) in the colorimetric detection of Example 3.

[0026] Figure 8 Fluorescence spectra corresponding to adding different concentrations of E2 standard solutions (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 μM) in the fluorescence detection of Example 3.

[0027] Figure 9 Linear relationship diagram between relative fluorescence intensity and E2 concentration in the fluorescence detection of Example 3.

[0028] Figure 10 Specificity experiment of the E2 colorimetric-fluorescent dual-mode analysis detection method in Example 4. Detailed implementation mode

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1: Preparation of manganese dioxide nanosheets

[0031] First, 18 mL of 0.6 M tetramethylammonium hydroxide (TMA·OH) and 2 mL of 30 wt% H2O2 were mixed evenly in a 100 mL round-bottom flask. Subsequently, 10 mL of 0.3 M manganese chloride tetrahydrate (MnCl2·4H2O) solution was quickly added to the above solution, and the solution immediately turned dark brown. After continuously stirring vigorously at room temperature for 12 h, blocky manganese dioxide was collected through centrifugation, washing, drying and other steps. Subsequently, manganese dioxide nanosheets were obtained by ultrasonic exfoliation of the blocky manganese dioxide: 10 mg of blocky manganese dioxide solid was dissolved in 10 mL of ultrapure water, and the solution was ultrasonically exfoliated for 1 h using an ultrasonic cell disruptor to obtain a 1 mg / mL MnO2 material solution, which was placed at 4 °C for standby.

[0032] The transmission electron microscope image of the prepared MnO2 NSs is as Figure 2 shown.

[0033] Example 2: Preparation of MBs-DNA-ALP Composite Probe

[0034] (1) Take 100 μL of streptavidin-labeled magnetic beads (SA-MBs) (4 mg / mL), wash them 3 times with binding buffer (10 mM Tris-HCl, pH = 7.5, 1 mM EDTA, 1 M NaCl), and disperse them into 460 μL of binding buffer. Subsequently, add 40 μL of biotin-labeled E2 aptamer (10 μM) to the above SA-MBs, and incubate at room temperature on a vortex for 30 min. Wash the obtained E2 aptamer-modified MBs 3 times with binding buffer to remove the excess biotin-labeled aptamer, and resuspend them in 500 μL of binding buffer to obtain E2 aptamer-modified MBs (AMB).

[0035] (2) Add 50 μL of biotin-labeled cDNA (10 μM) and 5 μL of streptavidin-labeled alkaline phosphatase (SA-ALP) (1 mg / mL) to 445 μL of E2 buffer, and incubate at room temperature on a vortex for 30 min to obtain SA-ALP-modified cDNA (cDNA-ALP).

[0036] (3) After magnetically separating the supernatant of the obtained AMB, add 500 μL of cDNA-ALP prepared in (2), incubate, separate, and wash under the same conditions as in (1) to obtain MBs-DNA-ALP. Subsequently, resuspend the prepared MBs-DNA-ALP in 500 μL of E2 buffer and store it at 4 °C for later use.

[0037] To determine that the nucleic acid aptamer was successfully immobilized on the surface of the magnetic beads to form aptamer-functionalized magnetic beads, the supernatant obtained from the first separation was collected and its ultraviolet-visible absorption spectrum was scanned. As Figure 3 shown, according to the Lambert-Beer law, it can be seen that the concentration of the nucleic acid aptamer in the collected supernatant was significantly reduced. Thus, it can be seen that the nucleic acid aptamer has been successfully immobilized on the surface of the magnetic beads.

[0038] Example 3: Colorimetric-Fluorescent Detection and Analysis of E2

[0039] First, add 20 μL of E2 buffer containing different concentrations of E2 to 20 μL of the prepared MBs-DNA-ALP composite probe, and incubate it at 500 rpm on a metal bath for 30 min. After magnetic separation, take the supernatant into a new centrifuge tube, add 20 μL of AAP (500 μM) and Tris-HCl buffer, and react at 37 °C on a metal bath for 30 min. Subsequently, add 20 μL of manganese dioxide nanosheet solution (0.13 mg / mL) to the above mixture and incubate at room temperature for 10 min. Finally, in colorimetric detection, add 80 μL of NaAc-HAc buffer and 20 μL of TMB (4 mM) solution to the above mixture, incubate in the dark at room temperature for 5 min, and then use a microplate reader to measure and record the absorption spectrum of the solution and the absorbance at 652 nm; in fluorescence detection, add 20 μL of the DNA strand labeled with the fluorescent dye FAM (FAM-14G) and 80 μL of Tris-HCl buffer to the above mixture, incubate in the dark at room temperature for 10 min, and use a fluorescence spectrophotometer to measure the fluorescence spectrum of the solution.

[0040] The principle of this method is feasible ( Figure 4 ), the concentration of E2 is inversely proportional to the absorbance in the composite system, and the absorbance decreases with the increase of the concentration of E2 ( Figure 5 ), and the color of the system gradually fades ( Figure 6 ). The absorbance shows a good linear relationship in the range of E2 concentration from 0.1 to 2 μM (R2 = 0.9905) ( Figure 7 ), and the detection limit (LOD) is 0.07 μM. The concentration of E2 is proportional to the fluorescence intensity in the composite system, and the fluorescence intensity increases with the increase of the concentration of E2 ( Figure 8 ), and the relative fluorescence intensity shows a good linear relationship in the range of E2 concentration from 0.005 to 0.2 μM (R2 = 0.9915) ( Figure 9 ), and the detection limit (LOD) is 0.0036 μM, which can meet the detection of E2.

[0041] Example 4: Specificity evaluation of colorimetric-fluorescent detection of E2

[0042] To evaluate the specificity of the colorimetric-fluorescent dual-mode analysis and detection method for E2, structural analogues of 17β-estradiol were selected for specificity analysis, namely: estrone (E1), bisphenol A (BPA), progesterone (P), medroxyprogesterone acetate (MPA), deoxycholic acid (DA), diethylstilbestrol (DES), chloramphenicol (CAP), 2,4-D.

[0043] As Figure 10As shown, in colorimetric detection, the interaction between the aptamer and 17β-estradiol can cause an obvious absorbance change (ΔA) in the complex system. In fluorescence detection, the F0-F signal response in the presence of 17β-estradiol is much higher than that in the presence of structural analogs of 17β-estradiol and other substances. Thus, it can be seen that this dual-mode detection method has good selectivity for 17β-estradiol.

[0044] Example 5: Determination of E2 Content in Actual Samples

[0045] To evaluate its practicability in actual samples, we detected estradiol in tap water using the dual-mode method. Estradiol was diluted with tap water and quantitatively detected using the dual-mode method. As shown in Table 1, the spiked recovery rate of E2 in the actual sample was 98.30 - 108.95%, and the relative standard deviation (RSD) was less than 8.13%, indicating that this detection method can be applied to the detection of actual samples.

[0046] Table 1 Colorimetric-fluorescent Dual-Mode Detection of E2 in Actual Samples Developed Based on the Present Invention

[0047]

Claims

1. A method for detecting 17β-estradiol using a colorimetric-fluorescence dual-mode biosensor, characterized in that: The following steps are involved: MBs-DNA-ALP composite probes were prepared using magnetic beads, nucleic acid aptamers and their complementary chains, and alkaline phosphatase, and manganese dioxide nanosheets were prepared; E2 buffer containing different concentrations of E2 was added to the prepared MBs-DNA-ALP composite probe, and the mixture was incubated on a metal bath; after magnetic separation, the supernatant was taken into a new centrifuge tube, and ascorbic acid-2-phosphate and tris(hydroxymethylaminomethane) hydrochloride buffer were added, and the mixture was incubated on a metal bath; then, manganese dioxide nanosheet solution was added to the mixture, and the mixture was incubated at room temperature for 10 minutes; finally, in colorimetric detection, acetic acid-sodium acetate buffer and 3,3',5,5'-tetramethylbenzidine solution were added to the mixture, and the mixture was incubated at room temperature in the dark for 5 minutes, and the absorbance and absorption spectrum of the solution were measured and recorded using an enzyme marker; in fluorescence detection, DNA chains labeled with fluorescent dye FAM and Tris-HCl buffer were added to the mixture, and the mixture was incubated at room temperature in the dark for 10 minutes, and the fluorescence spectrum of the solution was measured using a fluorescence spectrophotometer.

2. The method for detecting 17β-estradiol using a colorimetric-fluorescence dual-mode biosensor as claimed in claim 1, characterized in that: The Aptamer and cDNA are labeled with biotin at the 3' end of the nucleotide sequence, with a concentration of 10 μM, and the magnetic beads and alkaline phosphatase are labeled with streptavidin.

3. The method for detecting 17β-estradiol using a colorimetric-fluorescence dual-mode biosensor as claimed in claim 1, characterized in that: The specific formula of the E2 buffer is 20mM HEPES, pH=4, 100mM NaCl, 10mM MgCl2; the specific formula of the Tris-HCl buffer is 20mM Tris-HCl, pH=9, 5mM MgCl2; the specific formula of the NaAc-HAc buffer is 200mM, pH=4.

4. The method for detecting 17β-estradiol using a colorimetric-fluorescence dual-mode biosensor as claimed in claim 1, characterized in that: The reaction temperature of E2 and MBs-DNA-ALP composite probe is 25° C., and the reaction time is 30 min.

5. The method for detecting 17β-estradiol using a colorimetric-fluorescence dual-mode biosensor as claimed in claim 1, characterized in that: The AAP concentration was 500 μM, the manganese dioxide nanosheet concentration was 0.13 mg / mL, the TMB concentration was 4 mM, and the FAM-14G concentration was 300 nM.

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