An estradiol biosensor and its construction method and application

Abstract

The present invention discloses an estradiol biosensor and its construction method and application, belonging to the field of biosensor technology. The estradiol biosensor of the present invention comprises a sensing substrate and a DNA molecular machine; the sensing substrate is a gold electrode modified with an IP-BP double chain; the DNA molecular machine comprises an Aptamer-TP double chain and an FP-N3 probe. The biosensor of the present invention uses an estradiol aptamer probe to specifically identify estradiol, constructs a DNA molecular machine for signal cyclic amplification, and uses an atom transfer radical polymerization (ATRP) reaction to perform polymerization growth of electrical signals, thereby achieving efficient and amplified detection of estradiol, improving detection accuracy and sensitivity, and can be applied to the accurate detection of E2 content in clinical blood samples. It has important application value in research fields such as predicting pregnancy outcomes, endocrine diseases, and precocious puberty.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and in particular to an estradiol biosensor and a construction method and application thereof. Background Art

[0002] Endogenous 17β-estradiol (E2) is a steroidal estrogen secreted by ovarian granulosa cells. Its primary function is to promote the development of female reproductive organs, maintain secondary sexual characteristics, and maintain reproductive function. It is a key indicator in gonadal hormone testing. For example, when E2 levels exceed the threshold for ovulation, it indicates that the placenta has taken over the function of the corpus luteum of the ovary, maintaining pregnancy. E2 levels can reflect the quality of the dominant follicle and the function of the corpus luteum. Therefore, measuring E2 levels is of great value in predicting pregnancy outcomes, endocrine diseases, and precocious puberty.

[0003] Traditional detection methods primarily include chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Chromatography and mass spectrometry techniques typically require sample pretreatment (such as sample derivatization) and are complex and time-consuming. Antibodies used in ELISAs suffer from poor stability and susceptibility to inactivation. Aptamers, a class of oligonucleotide fragments with specific recognition capabilities, are essentially nucleic acid molecules folded into a specific three-dimensional structure that binds specifically to targets with high affinity. Aptamers are easy to synthesize, modify, maintain high stability, and exhibit no immunogenicity, making them suitable for developing more efficient detection methods.

[0004] The Toehold-mediated strand displacement reaction (TSDR) is a non-enzymatic process characterized by highly controllable, predictable thermodynamics and kinetics, making it valuable in constructing molecular machines, catalytic circuits, and logic gates. It is worth noting that although the recycling of TSDR probes can achieve signal amplification, the problem of high background signal still exists, and the output of the sensing signal often depends on the labeling of a single signal molecule, which limits its application in substance detection and biosensors. Summary of the Invention

[0005] The present invention aims to provide an estradiol biosensor, its construction method, and its application to address the aforementioned problems of the prior art. The biosensor utilizes an estradiol aptamer probe for specific recognition of estradiol, constructs a DNA molecular machine for cyclic signal amplification, and utilizes atom transfer radical polymerization (ATRP) to polymerize and grow electrical signals. This enables efficient, amplified detection of estradiol, improving detection accuracy and sensitivity. The biosensor can be used to accurately detect E2 levels in clinical blood samples, and has significant application value in research fields such as predicting pregnancy outcomes, endocrine diseases, and precocious puberty.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an estradiol biosensor based on DNA molecular machines regulating electrical signal polymerization growth, the estradiol biosensor comprising a sensing substrate and a DNA molecular machine;

[0008] The sensing substrate is a gold electrode modified with an IP-BP double chain; the nucleotide sequence of the IP chain in the IP-BP double chain is shown in SEQ ID NO.1, and the nucleotide sequence of the BP chain is shown in SEQ ID NO.2;

[0009] The DNA molecular machine includes an Aptamer-TP double chain and an FP-N3 probe; the nucleotide sequence of Aptamer in the Aptamer-TP double chain is shown as SEQ ID NO.3, and the nucleotide sequence of the TP chain is shown as SEQ ID NO.4; the nucleotide sequence of the FP-N3 probe is shown as SEQ ID NO.5.

[0010] The present invention also provides a method for constructing the above-mentioned estradiol biosensor, comprising the following steps:

[0011] S1. After soaking the gold electrode in piranha solution, polishing it to obtain an AuE electrode;

[0012] S2, annealing and hybridizing the IP chain and the BP chain to obtain an IP-BP double chain; assembling the IP-BP double chain onto the AuE electrode to obtain a sensing substrate;

[0013] S3. The Aptamer and TP chains are annealed and hybridized to obtain an Aptamer-TP double chain; an azide group is modified on the FP chain to obtain an FP-N3 probe; and a DNA molecular machine is obtained.

[0014] Optionally, in step S1, the soaking treatment time is 20 to 40 minutes; the polishing method is: polishing with 0.3 μm and 0.05 μm alumina powder in sequence.

[0015] Optionally, in step S2, the annealing and hybridization method is: mixing the IP chain and the BP chain in equal molar ratios, reacting at 90°C to 100°C for 8 to 12 minutes, and cooling to 20°C to 25°C at a rate of 1°C / min;

[0016] The assembly method comprises: activating the IP-BP double chain with tris(2-carboxyethyl)phosphine hydrochloride, adding the double chain to the AuE electrode, reacting in the dark for 10 to 16 hours, and then blocking the reaction on the AuE electrode with mercaptohexanol for 50 to 80 minutes;

[0017] The concentration of the IP-BP double chain is 0.5-0.8 μM; the amount added is 8-15 μL.

[0018] Optionally, in step S3, the annealing and hybridization method is: mixing the Aptamer and the TP chain in equal molar ratios, reacting at 90°C to 100°C for 8 to 12 min, and cooling to 20°C to 25°C at a rate of 1°C / min.

[0019] The present invention also provides the use of the above-mentioned estradiol biosensor or the estradiol biosensor constructed by the above-mentioned construction method in detecting estradiol content for non-diagnostic purposes. The method for detecting estradiol content comprises the following steps:

[0020] N1. After mixing the Aptamer-TP double-stranded probe and the FP-N3 probe, mix with the sample to be tested, dropwise add to the sensor substrate, and react for 80-150 minutes;

[0021] N2. Placing the sensing substrate after the reaction in step N1 in a mixed solution containing 3-butynyl-2-bromoisobutyrate, ascorbic acid, and copper sulfate to perform a click chemistry reaction;

[0022] N3. Place the sensing substrate after the reaction in step N2 in a dimethylformamide solution containing copper / tris(2-dimethylaminoethyl)amine, ferrocenylmethanol methacrylate, potassium bromide, and potassium hexafluorophosphate, and perform ATRP reaction at a voltage of -0.56 V;

[0023] N4. Using the sensing substrate after the reaction in step N3 as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the auxiliary electrode to form a three-electrode system, the current signal is detected in the LiClO4 solution; and the estradiol content is calculated based on the standard curve.

[0024] Furthermore, in step N1, the molar ratio of the Aptamer-TP double-stranded and FP-N3 probe is 0.5-2:0.5-2; the concentration of the solution after the Aptamer-TP double-stranded and FP-N3 probe are mixed is 0.5-0.8 μM;

[0025] The volume ratio of the solution after the Aptamer-TP double-stranded and FP-N3 probes are mixed to the sample to be tested is 1:8-15; the amount of the dropwise addition is 8-15 μL.

[0026] Furthermore, in step N2, the final concentrations of 3-butynyl 2-bromoisobutyrate, ascorbic acid, and copper sulfate in the mixed solution are 80-120 μM, 15-25 μM, and 80-120 μM, respectively; and the click chemistry reaction time is 40-60 min;

[0027] Furthermore, in step N3, the final concentrations of copper / tris(2-dimethylaminoethyl)amine, ferrocenylmethanol methacrylate, potassium bromide, and potassium hexafluorophosphate in the dimethylformamide solution are 80-120 μM, 80-120 μM, 80-120 mM, and 60-80 mM, respectively; and the ATRP reaction time is 50-80 min.

[0028] The present invention also provides use of the above estradiol biosensor or the estradiol biosensor constructed by the above construction method in preparing an estradiol detection product, wherein the detection product includes a detection kit.

[0029] The present invention discloses the following technical effects:

[0030] The present invention prepares an estradiol biosensor based on the polymerization growth of electrical signals regulated by DNA molecular machines, enabling clinical detection of estradiol in blood samples. The present invention improves the specificity of the biosensor by using aptamers for specific recognition of targets; constructs DNA molecular machines based on chain displacement reactions for recycling of targets, achieving cyclic amplification of targets; introduces alkyl halides and performs polymerization growth of electrical signals based on ATRP reactions, enabling efficient signal output from the sensing substrate and improving the accuracy and feasibility of the biosensor. When detecting complex biological samples, the biosensor of the present invention can effectively resist interference from interfering substances, obtain fully amplified signals, and improve detection sensitivity and specificity.

[0031] The estradiol biosensor prepared by the present invention has the advantages of high accuracy, good selectivity, and high sensitivity. It can realize the quantitative detection of estradiol in clinical blood samples and has important application value in research fields such as predicting pregnancy outcomes, endocrine diseases and precocious puberty. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 Schematic diagram of the detection principle of the estradiol biosensor of the present invention;

[0034] Figure 2The CV response graphs and EIS response graphs of different sensor substrate interfaces in Example 2; wherein A is the CV response graph; B is the EIS response graph; curves a to d correspond to the AuE electrode, the electrode modified with double-chain IP-BP, the MCH / IP-BP / AuE after MCH blocking, and the MCH / IP-BP / AuE after ATRP reaction, respectively;

[0035] Figure 3 Figure 2 shows the experimental condition optimization results of the biosensor in Example 2; Figure A shows the response signal changes of the biosensor at different IP-BP double-strand concentrations; Figure B shows the response signal changes of the biosensor at different reaction times;

[0036] Figure 4 Figure 2 shows the detection results of the biosensor in Example 2 for E2 samples of different concentrations; wherein A is the SWV response curve of E2 samples of different concentrations; curves a to h are for E2 samples of 0, 100 fM, 1 pM, 10 pM, 80 pM, 800 pM, 8 nM, and 80 nM, respectively; B is the linear relationship between the SWV peak current signal value and the logarithm of the E2 concentration;

[0037] Figure 5 The graphs show the test results of E2 content in blood samples of non-pregnant adult women (numbered a to c) and blood samples of mid-pregnant adult women (numbered d to f). DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The technical principles of the present invention are as follows:

[0044] The present invention constructs a biosensor for sensitive detection of estradiol (E2) based on the electrochemical signal polymerization growth mediated by DNA molecular machines. The detection principle of the biosensor is as follows Figure 1 As shown, the DNA molecular machine consists of an aptamer-TP duplex, an IP-BP duplex, and a fuel probe (FP) modified with an azide group (N3). The IP strand contains a 6-nt toehold sequence (toehold 1) at its 3' end and a 7-nt toehold sequence (toehold 2) at its 5' end, which hybridize and complement with the 5' end of the TP strand and the 3' end of the FP strand, respectively. In the absence of the target, the IP strand hybridizes with the BP strand, blocking toehold 2 and inhibiting the TSDR reaction between the FP and IP strands. In the presence of the target E2, the aptamer specifically binds to E2, releasing the TP strand. The TP strand, with the help of toehold 1, undergoes a strand displacement reaction (TSDR1), displacing the BP strand from the duplex, exposing toehold 2 of the IP strand. The FP strand, with the help of toehold 2, hybridizes with the IP strand and subsequently displaces the TP strand (TSDR2), allowing the TP strand to be recycled. After multiple TSDR cycles, a large number of IP-FP double chains were obtained on the electrode surface. The N3 at the end of FP could undergo an azide-alkyne cycloaddition reaction to capture the 2-bromoisobutyric acid-3-butynyl ester (BBriB) initiator. Subsequently, copper II / tris(2-dimethylaminoethyl)amine (Cu II / Me6TREN) is reduced to copper I / tris(2-dimethylaminoethyl)amine (Cu I / Me6TREN), Cu I / Me6TREN reacts with BBriB on the electrode surface to generate free radicals that further polymerize ferrocenium methanol methacrylate (FcMMA) to the electrode surface, generating a significantly amplified current signal and achieving sensitive detection of the target E2.

[0045] Example 1

[0046] This embodiment provides an estradiol biosensor based on DNA molecular machines regulating electrical signal polymerization growth, comprising a sensing substrate and a DNA molecular machine. The construction method is as follows:

[0047] S1. Pretreatment of gold electrode:

[0048] First, a gold electrode (AuE) was immersed in piranha solution (a mixed solution of 98% H2SO4 and 30% H2O2 with a volume ratio of 3:1) for 30 min. The gold electrode was polished with 0.3 μm and 0.05 μm alumina powder respectively. The electrode was placed in a dilute sulfuric acid solution for cyclic voltammetry scanning (voltage: -0.3~1.5 V) until a stable characteristic peak was obtained to obtain an AuE electrode.

[0049] S2. Construction of sensing substrate MCH / IP-BP / AuE:

[0050] An equimolar ratio of IP and BP chains was annealed at 95°C for 10 minutes and then cooled to 25°C at a rate of 1°C / min to form an IP-BP duplex. Subsequently, 0.6 µM of the IP-BP duplex was reacted with tris(2-carboxyethyl)phosphine hydrochloride at room temperature for 1 hour to break the disulfide bonds of the IP. The activated IP-BP duplex was purified by filtration to remove the tris(2-carboxyethyl)phosphine hydrochloride, resulting in an activated IP-BP duplex. 10 µL of the activated IP-BP duplex was then dropwise applied to the AuE surface in the dark for 12 hours to produce an IP-BP duplex-modified electrode. Finally, 10 µL of mercaptohexanol (MCH) was added to the IP-BP duplex-modified electrode for 1 hour to block inactive sites, resulting in MCH / IP-BP / AuE.

[0051] IP chain: 5′-SH-(CH2)6-GATCATCGCTTCCAGCTTATTGAATTACACGC-3′ (SEQ ID NO. 1);

[0052] BP chain: 5'-TACTCATCAATTCAATAAGCTGGAAGCGATGAGTA-3' (SEQ ID NO. 2).

[0053] S3. Construction of DNA molecular machines:

[0054] a. Aptamer and TP chains in equal molar ratios were annealed at 95°C for 10 min and then cooled to 25°C at a rate of 1°C / min to obtain Aptamer-TP duplexes.

[0055] Aptamer chain: 5'-GCTCTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGAAGC-3' (SEQ ID NO. 3);

[0056] TP chain: 5'-GCGTGTAATTCAATAAGCTGGAAGC-3' (SEQ ID NO. 4).

[0057] b. The FP chain is modified with an azide group (N3) to obtain the FP-N3 probe.

[0058] FP-N3 probe: 5′-N3-AATTCAATAAGCTGGAAGCGATGATC-3′ (SEQ ID NO. 5).

[0059] The method for using the estradiol biosensor constructed above is as follows:

[0060] N1. Mix the Aptamer-TP double-stranded probe and the FP-N3 probe at a molar ratio of 1:1 to obtain a mixed solution with a concentration of 0.6 μM. Mix it with the sample to be tested at a volume ratio of 1:10. Take 10 μL and drop it onto the sensing substrate MCH / IP-BP / AuE and react for 100 minutes.

[0061] N2. Prepare a mixed solution containing 100 μM 2-bromoisobutyric acid-3-butynyl ester (BBriB), 20 μM ascorbic acid (AA), and 100 μM copper sulfate (CuSO4); place the sensing substrate after the reaction in step N1 in the mixed solution and perform click chemistry reaction for 50 minutes. BBriB is modified on the sensing substrate through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

[0062] N3, using dimethylformamide as solvent, prepare a 100 μM copper / tris (2-dimethylaminoethyl) amine (Cu II / Me6TREN), 100 μM ferrocenylmethanol methacrylate (FcMMA), 100 mM potassium bromide (KBr), and 70 mM potassium hexafluorophosphate (KPF6) in dimethylformamide solution; the sensing substrate after the step N2 reaction was placed in the dimethylformamide solution, and a voltage of -0.56 V was applied to the electrochemical workstation for 60 minutes to carry out atom transfer radical polymerization (ATRP) to generate polymerized poly-FcMMA (FcMMA) on the surface of the sensing substrate.

[0063] N4. Use the sensing substrate after the reaction in step N3 as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the auxiliary electrode to form a three-electrode system. Use 0.5 M LiClO4 as the electrolyte and perform a square wave pulse SWV test (potential: 0.1~0.3 V) to collect the current signal value.

[0064] When the samples to be tested are E2 solutions of different concentrations, a standard curve can be constructed according to the current signal values.

[0065] Example 2

[0066] This example tested the performance of the biosensor prepared in Example 1, including the following tests:

[0067] (1) Characterization of the sensing substrate interface:

[0068] The AuE electrode, the electrode modified with double-stranded IP-BP, the MCH / IP-BP / AuE after MCH blocking and the MCH / IP-BP / AuE after ATRP reaction were respectively placed in 1 mM [Fe(CN)6] 3− / 4− In the solution, the CV response and EIS response of different sensing substrate interfaces were observed. Figure 2 shown.

[0069] From the CV response results ( Figure 2 A) It can be seen that with the bare AuE electrode ( Figure 2 Compared with curve a), when IP-BP double chain is modified to the electrode surface, the current signal decreases ( Figure 2 Curve b of A) is due to the negative charge of the phosphate backbone of the DNA probe and the interaction between [Fe(CN)6] 3- / 4- The probe has electrostatic repulsion. After the MCH seals the electrode, the non-specifically adsorbed DNA probe is displaced, causing the current signal to increase ( Figure 2 When the target compound E2 is added to initiate the ATRP reaction, a large amount of poly-FcMMA is generated on the electrode surface, and the current signal further increases ( Figure 2 curve d of A).

[0070] From the EIS response results ( Figure 2 B) It can be seen that the semicircle diameter of the EIS curve represents the electron transfer impedance of the electrode. Figure 2 Compared with curve a), when IP-BP was modified onto the electrode surface, the impedance increased from 0.69 KΩ ( Figure 2 B of curve a) increases to 9.7 KΩ ( Figure 2 After MCH sealed the electrode, the impedance decreased to 8.6 KΩ ( Figure 2Curve c of B), the impedance after the target E2 triggers the ATRP reaction is 0.85 KΩ ( Figure 2 Curve d of B).

[0071] The CV and EIS detection results reveal the changes in the gradual construction process of the sensing substrate interface.

[0072] (2) Optimization of experimental conditions:

[0073] The response signal changes of the biosensor under different IP-BP double chain concentrations (0.2~0.9µM) and different reaction times (30~150min) were investigated. The experimental method was referred to Example 1, and the detection sample was 8 nM E2 solution. The results are shown in Figure 1. Figure 3 shown.

[0074] The response signal changes of the biosensor under different IP-BP double chain concentrations ( Figure 3 As shown in Figure A, the current signal gradually increases as the concentration of the IP-BP duplex increases from 0.2 µM to 0.6 µM, reaching a maximum sensor response at 0.6 µM. As the concentration of the IP-BP duplex continues to increase, the impedance between the probes on the electrode surface increases, reducing the reaction efficiency and causing the current signal to gradually decrease.

[0075] The response signal changes of the biosensor at different reaction times ( Figure 3 B) It can be seen that the response signal of the biosensor increases with the extension of the reaction time, and the current change tends to be gentle after 100 min.

[0076] (3) Detection performance research:

[0077] The biosensor of Example 1 was used to detect E2 samples of different concentrations (0 fM, 100 fM, 1 pM, 10 pM, 80 pM, 800 pM, 8 nM, and 80 nM). A square wave pulse SWV test was performed to collect current signals and construct a standard curve to evaluate the sensitivity of the biosensor. The results are shown in Figure 1. Figure 4 shown.

[0078] From the SWV response curve ( Figure 4 A) It can be seen that the current peak increases with the increase of E2 concentration.

[0079] From the standard curve ( Figure 4 B) It can be seen that there is a good linear relationship between the current signal value and the logarithm of the E2 concentration, and the linear equation is i = 11.06 + 0.8278 lgc (R 2= 0.9928), and the detection limit was calculated based on the 3-fold relative standard deviation rule to be 35 fM. Compared with the detection limit of traditional methods (pM~nM), this biosensor has higher sensitivity.

[0080] In addition, cortisol, estrogen triol, and diethylstilbestrol were selected as interfering substances to test the specificity of the biosensor. The results showed that compared with the response signal of 8 nM E2, the signal change obtained with a 10-fold concentration of interfering substances was less than 5.6%, demonstrating the excellent specificity of the biosensor.

[0081] (4) Biosensor detection performance verification:

[0082] Blood samples from non-pregnant adult women (numbered a, b, and c) and mid-pregnant women (numbered d, e, and f) were collected from Xuzhou Central Hospital on March 10, 2025.

[0083] The blood sample to be tested was diluted 10-fold with phosphate buffer and tested according to the method described in Example 1. The reacted sensing substrate was used as the working electrode, along with an Ag / AgCl reference electrode and a platinum auxiliary electrode to form a three-electrode system. The current signal of the three-electrode system was detected using a Chenhua CHI 660E electrochemical workstation. The linear regression equation (i = 11.06 + 0.8278 lgc) was used to calculate the concentration of E2 in the blood sample to be tested. The results are shown in Figure 1. Figure 5 As shown, E2 concentrations varied significantly between samples (af). Overall, E2 concentrations were lower in blood samples from non-pregnant adult women (a, b, and c), ranging from 124.9 pM to 886.8 pM. E2 concentrations were significantly elevated in blood samples from mid-pregnant women (d, e, and f), ranging from 2867 pM to 8762 pM. Furthermore, the same blood samples were tested using a commercially available kit (Beckman 33540), commonly used in clinical diagnostics. The results were consistent with those of the biosensor of the present invention, with relative errors ranging from -8.3% to 6.2%. This demonstrates that the biosensor of the present invention has good detection capabilities and applicability even in complex blood samples.

[0084] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An estradiol biosensor based on DNA molecular machinery regulating electrical signal polymerization growth, characterized in that: The estradiol biosensor comprises a sensing substrate and a DNA molecular machine; The sensing substrate is a gold electrode modified with an IP-BP double chain; the nucleotide sequence of the IP chain in the IP-BP double chain is shown in SEQ ID NO.1, and the nucleotide sequence of the BP chain is shown in SEQ ID NO.2; The DNA molecular machine includes an Aptamer-TP double chain and an FP-N3 probe; the nucleotide sequence of Aptamer in the Aptamer-TP double chain is shown as SEQ ID NO.3, and the nucleotide sequence of the TP chain is shown as SEQ ID NO.4; the nucleotide sequence of the FP-N3 probe is shown as SEQ ID NO.

5.

2. A method for constructing an estradiol biosensor according to claim 1, characterized in that: The steps include: S1. After soaking the gold electrode in piranha solution, polishing it to obtain an AuE electrode; S2, annealing and hybridizing the IP chain and BP chain to obtain IP-BP double chain; Assembling the IP-BP double chain onto the AuE electrode to obtain a sensing substrate; S3. The Aptamer and TP chains are annealed and hybridized to obtain an Aptamer-TP double chain; an azide group is modified on the FP chain to obtain an FP-N3 probe; and a DNA molecular machine is obtained.

3. The construction method according to claim 2, characterized in that In step S1, the soaking treatment time is 20 to 40 minutes; the polishing method is: polishing with 0.3 μm and 0.05 μm aluminum oxide powder in sequence.

4. The construction method according to claim 2, characterized in that In step S2, the annealing and hybridization method is as follows: mixing the IP chain and the BP chain in equal molar ratio, reacting at 90°C to 100°C for 8 to 12 minutes, and cooling to 20°C to 25°C at a rate of 1°C / min; The assembly method comprises: activating the IP-BP double chain with tris(2-carboxyethyl)phosphine hydrochloride, adding the double chain to the AuE electrode, reacting in the dark for 10 to 16 hours, and then blocking the reaction on the AuE electrode with mercaptohexanol for 50 to 80 minutes; The concentration of the IP-BP double chain is 0.5-0.8 μM; the amount added is 8-15 μL.

5. The construction method according to claim 2, characterized in that In step S3, the annealing and hybridization method is as follows: Aptamer and TP chain are mixed in equal molar ratios, reacted at 90°C to 100°C for 8 to 12 minutes, and cooled to 20°C to 25°C at a rate of 1°C / min.

6. Use of the estradiol biosensor according to claim 1 or the estradiol biosensor constructed by the construction method according to any one of claims 2 to 5 in detecting estradiol content for non-diagnostic purposes, characterized in that: The method for detecting estradiol content comprises the steps: N1. After mixing the Aptamer-TP double-stranded probe and the FP-N3 probe, mix with the sample to be tested, dropwise add to the sensor substrate, and react for 80-150 minutes; N2. Placing the sensing substrate after the reaction in step N1 in a mixed solution containing 3-butynyl-2-bromoisobutyrate, ascorbic acid, and copper sulfate to perform a click chemistry reaction; N3. Place the sensing substrate after the reaction in step N2 in a dimethylformamide solution containing copper / tris(2-dimethylaminoethyl)amine, ferrocenylmethanol methacrylate, potassium bromide, and potassium hexafluorophosphate, and perform ATRP reaction at a voltage of -0.56 V; N4. Using the sensing substrate after the reaction in step N3 as the working electrode, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the auxiliary electrode to form a three-electrode system, the current signal is detected in the LiClO4 solution; The estradiol content was calculated according to the standard curve.

7. The use according to claim 6, characterized in that In step N1, the molar ratio of the Aptamer-TP double-stranded and FP-N3 probe is 0.5-2:0.5-2; the concentration of the solution after the Aptamer-TP double-stranded and FP-N3 probe are mixed is 0.5-0.8 μM; The volume ratio of the solution after the Aptamer-TP double-stranded solution and the FP-N3 probe mixture to the sample to be tested is 1:8-15; the amount of the dropwise addition is 8-15 μL.

8. The use according to claim 6, characterized in that In step N2, the final concentrations of 3-butynyl 2-bromoisobutyrate, ascorbic acid, and copper sulfate in the mixed solution are 80-120 μM, 15-25 μM, and 80-120 μM, respectively; and the click chemistry reaction time is 40-60 min.

9. The use according to claim 6, characterized in that In step N3, the final concentrations of copper / tris(2-dimethylaminoethyl)amine, ferrocenylmethanol methacrylate, potassium bromide, and potassium hexafluorophosphate in the dimethylformamide solution are 80-120 μM, 80-120 μM, 80-120 mM, and 60-80 mM, respectively; and the ATRP reaction time is 50-80 min.

10. Use of the estradiol biosensor according to claim 1 or the estradiol biosensor constructed by the construction method according to any one of claims 2 to 5 in preparing an estradiol detection product, characterized in that: The detection product includes a detection kit.

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