Estradiol biosensor as well as construction method and application thereof

Abstract

The invention discloses an estradiol biosensor as well as a construction method and application thereof, and belongs to the technical field of biosensors. The estradiol biosensor comprises a sensing substrate and a DNA molecular machine, the sensing substrate is a gold electrode modified with IP-BP double chains; and the DNA molecular machine comprises an Aptamer-TP double strand and an FP-N3 probe. According to the biosensor, the estradiol aptamer probe is adopted for specific recognition of estradiol, a DNA molecular machine is constructed for signal cycle amplification, polymerization growth of an electric signal is carried out by means of an atom transfer radical polymerization (ATRP) reaction, efficient and amplified detection of estradiol can be achieved, the detection accuracy and sensitivity are improved, and the biosensor is suitable for mass production. The method can be applied to accurate detection of the E2 content in a clinical blood sample, and has important application value in the research fields of prediction of pregnancy outcome, endocrine diseases, precocious puberty and the like.

Description

Technical Field

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

[0002] Endogenous 17β-estradiol (E2) is a steroid estrogen secreted by ovarian granulosa cells. Its main function is to promote the development of female reproductive organs, maintain secondary sexual characteristics and reproductive functions, and is an important indicator in gonadal hormone detection. For example, when the E2 level exceeds the ovulation threshold, it proves that the placenta has taken over the function of the ovarian corpus luteum and maintains continued pregnancy. The level of E2 can reflect the quality of dominant follicles and ovarian corpus luteum function. Therefore, detecting the content of E2 has important value for predicting pregnancy outcomes, endocrine diseases, and precocious puberty.

[0003] Traditional detection methods mainly include chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Among them, chromatography and mass spectrometry techniques usually require sample pretreatment (such as sample derivatization), and the operations are complex and time-consuming. The antibodies in ELISA have the disadvantages of poor stability and easy inactivation. As a class of oligonucleotide fragments with specific recognition functions, nucleic acid aptamers are essentially nucleic acid molecules folded into specific three-dimensional structures, which specifically bind to target substances with high affinity. Nucleic acid aptamers have the characteristics of easy synthesis, easy modification, high stability, and no immunogenicity, and can be used to establish more efficient detection methods.

[0004] Toehold-mediated strand displacement reaction (TSDR) is a non-enzymatic process with characteristics such as strong controllability, predictable thermodynamics and kinetics, and has important value in constructing molecular machines, catalytic circuits, and logic gates. It should be noted that although the recycling of TSDR probes can achieve signal amplification, there are still problems of relatively high background signals, and the output of sensing signals often depends on the labeling of single signal molecules, which limits its application in substance detection and biosensors. Summary of the Invention

[0005] The object of the present invention is to provide an estradiol biosensor, a construction method thereof, and an application thereof to solve the problems existing in the above-mentioned prior art. The biosensor of the present invention uses estradiol aptamer probes to specifically recognize estradiol, constructs a DNA molecular machine for signal cycle amplification, and uses atom transfer radical polymerization (ATRP) reaction for the polymerization growth of electrical signals, which can achieve the efficient and amplified detection of estradiol, improve the detection accuracy and sensitivity, and can be applied to the accurate detection of E2 content in clinical blood samples, and has important 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 the regulation of electro-signal polymerization growth by a DNA molecular machine. The estradiol biosensor includes a sensing substrate and a DNA molecular machine;

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

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

[0010] The present invention also provides a construction method of the above estradiol biosensor, including the following steps:

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

[0012] S2. Anneal and hybridize the IP strand and the BP strand to obtain an IP-BP double strand; assemble the IP-BP double strand onto the AuE electrode to obtain a sensing substrate;

[0013] S3. Anneal and hybridize the Aptamer and the TP strand to obtain an Aptamer-TP double strand; modify an azide group on the FP strand to obtain an FP-N3 probe; obtain a DNA molecular machine.

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

[0015] Optionally, in step S2, the annealing and hybridization method is: mix the IP strand and the BP strand in an equimolar ratio, react at 90 °C - 100 °C for 8 - 12 min, and cool to 20 °C - 25 °C at a rate of 1 °C / min;

[0016] The assembly method is: activate the IP-BP double strand with tris(2-carboxyethyl)phosphine hydrochloride, then drop it onto the AuE electrode, react in the dark for 10 - 16 h, and then use mercaptohexanol to carry out a blocking reaction on the AuE electrode for 50 - 80 min;

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

[0018] Optionally, in step S3, the method of annealing and hybridization is as follows: Mix the Aptamer and the TP strand in an equimolar ratio, react at 90°C - 100°C for 8 - 12 min, and cool to 20°C - 25°C at a rate of 1 °C / min.

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

[0020] N1. After mixing the Aptamer-TP double strand and the FP-N3 probe, mix with the sample to be detected, dropwise add to the sensing substrate, and react for 80 - 150 min;

[0021] N2. Place the sensing substrate after the reaction in step N1 in a mixed solution containing 2-bromo-3-butynyl isobutyrate, ascorbic acid, and copper sulfate for click chemical reaction;

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

[0023] N4. Use the sensing substrate after the reaction in step N3 as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire as the auxiliary electrode to form a three-electrode system, and detect the current signal in a LiClO4 solution; calculate the estradiol content according to the standard curve.

[0024] Further, in step N1, the molar ratio of the Aptamer-TP double strand to the FP-N3 probe is 0.5 - 2:0.5 - 2; the concentration of the solution after mixing the Aptamer-TP double strand and the FP-N3 probe is 0.5 - 0.8 μM;

[0025] The volume ratio of the solution after mixing the Aptamer-TP double strand and the FP-N3 probe to the sample to be detected is 1:8 - 15; the amount added dropwise is 8 - 15 μL.

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

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

[0028] The present invention also provides the application of the above estradiol biosensor or the estradiol biosensor constructed by the above construction method in the preparation of estradiol detection products, and the detection products include detection kits.

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

[0030] Based on the electrochemically induced polymerization growth regulated by DNA molecular machines, the present invention prepares an estradiol biosensor and realizes the clinical detection of estradiol in blood samples. The present invention uses aptamers for specific recognition of target substances, improving the specificity of the biosensor; constructs DNA molecular machines based on strand displacement reactions for recycling of target substances, realizing the cyclic amplification of target substances; introduces alkyl halides and conducts electrochemically induced polymerization growth based on ATRP reactions, enabling efficient signal output of 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 the interference of interferents, obtain fully amplified signals, and improve the detection sensitivity and specificity.

[0031] The estradiol biosensor prepared by the present invention has the advantages of high accuracy, good selectivity, high sensitivity, etc., 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 technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following drawings are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings without creative efforts based on these drawings.

[0033] Figure 1 It is a schematic diagram of the detection principle of the estradiol biosensor of the present invention;

[0034] Figure 2CV response diagrams and EIS response diagrams of different sensing substrate interfaces in Example 2; among them, A are the CV response diagrams; B are the EIS response diagrams; curves a-d correspond to the AuE electrode, the electrode modified with IP-BP double strands, MCH / IP-BP / AuE after MCH blocking, and MCH / IP-BP / AuE after ATRP reaction in sequence;

[0035] Figure 3 Diagram of the experimental condition optimization results of the biosensor in Example 2; among them, A is the diagram of the change in the response signal of the biosensor at different IP-BP double strand concentrations; B is the diagram of the change in the response signal of the biosensor at different reaction times;

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

[0037] Figure 5 Diagram of the detection results of the E2 content in the blood samples of adult non-pregnant women (numbered a-c) and adult second-trimester pregnant women (numbered d-f). Detailed implementation manners

[0038] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0039] It should be understood that the terms described in the present invention are only used to describe specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0041] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the specification of the present invention, which will be obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention will be obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0042] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, that is, they are meant to include but not limited to.

[0043] The technical principle of the present invention is as follows:

[0044] The present invention constructs a biosensor for sensitive detection of estradiol (E2) based on the electrochemically mediated polymerization growth of DNA molecular machines. The detection principle of this biosensor is as Figure 1 shown. The DNA molecular machine consists of an Aptamer-TP double strand, an IP-BP double strand, and a fuel probe FP modified with an azide group (N3). The 3'-end of the IP strand contains a 6-nt toehold sequence (toehold 1), and the 5'-end contains a 7-nt toehold sequence (toehold 2), which can hybridize and complement with the 5'-end of the TP strand and the 3'-end of the FP strand respectively. When the target is absent, the IP strand hybridizes with the BP strand, and toehold 2 is blocked, inhibiting the TSDR reaction between the FP strand and the IP strand. When the target E2 is present, the Aptamer specifically binds to E2, releasing the TP strand. The TP strand undergoes a strand displacement reaction (TSDR1) with the help of toehold 1, displacing the BP strand from the double-stranded structure and exposing the toehold 2 of the IP strand. The FP strand hybridizes with the IP strand with the help of toehold 2, and then displaces the TP strand (TSDR2), enabling the TP strand to be recycled. After multiple TSDR cycles, a large number of IP-FP double strands are obtained on the electrode surface. The N3 at the end of the FP can undergo an azide-alkyne cycloaddition reaction to capture the initiator 2-bromoisobutyric acid 3-butynyl ester (BBriB). Subsequently, copper II / tris(2-dimethylaminoethyl)amine (Cu II / Me6TREN) is reduced to copper I / tris(2-dimethylaminoethyl)amine (Cu I / Me6TREN) under a constant voltage. Cu I / Me6TREN reacts with BBriB on the electrode surface to generate free radicals, which further polymerize ferrocene methanol methacrylate (FcMMA) onto the electrode surface, generating a significantly amplified current signal to achieve sensitive detection of the target E2.

[0045] Example 1

[0046] This embodiment provides an estradiol biosensor based on the regulation of electrical signal polymerization growth by DNA molecular machines, including a sensing substrate and DNA molecular machines. The construction method is as follows:

[0047] S1. Pretreatment of the gold electrode:

[0048] First, immerse the gold electrode (AuE) in a piranha solution (a mixed solution of 98% H2SO4 and 30% H2O2 with a volume ratio of 3:1) for 30 min, polish the gold electrode successively with 0.3 μm and 0.05 μm alumina powder, and perform cyclic voltammetry scanning (voltage: -0.3~1.5 V) detection on the electrode in a dilute sulfuric acid solution until a stable characteristic peak is obtained to obtain the AuE electrode.

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

[0050] Place equimolar amounts of the IP strand and the BP strand at 95 °C for annealing for 10 min, and then cool to 25 °C at a rate of 1 °C / min to form the IP-BP double strand. Subsequently, react 0.6 μM of the IP-BP double strand with tris(2-carboxyethyl)phosphine hydrochloride at room temperature for 1 h to open the disulfide bond of the IP, filter and purify to remove tris(2-carboxyethyl)phosphine hydrochloride to obtain the activated IP-BP double strand; take 10 μL of the activated IP-BP double strand, drop it on the surface of the AuE and react in the dark for 12 h to obtain the electrode modified with the IP-BP double strand. Finally, drop 10 μL of mercaptohexanol (MCH) on the electrode modified with the IP-BP double strand and react for 1 h to block the non-active sites to obtain MCH / IP-BP / AuE.

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

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

[0053] S3. Construction of the DNA molecular machine:

[0054] a. Place equimolar amounts of the Aptamer and the TP strand at 95 °C for annealing for 10 min, and then cool to 25 °C at a rate of 1 °C / min to obtain the Aptamer-TP double strand.

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

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

[0057] b. The FP strand 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 usage method of the estradiol biosensor constructed above is as follows:

[0060] N1. After mixing the Aptamer-TP double strand and the FP-N3 probe in 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 in a volume ratio of 1:10. Take 10 μL and drop it onto the sensing substrate MCH / IP-BP / AuE, and react for 100 min.

[0061] N2. Prepare a mixed solution containing 100 μM 3-butynyl 2-bromoisobutyrate (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 a click chemical reaction for 50 min to modify BBriB on the sensing substrate through a Cu(I)-catalyzed azide-alkyne cycloaddition reaction.

[0062] N3. Using dimethylformamide as a solvent, prepare a dimethylformamide solution containing 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); place the sensing substrate after the reaction in step N2 in the dimethylformamide solution, and apply a voltage of -0.56 V through an electrochemical workstation to react for 60 min to carry out an atom transfer radical polymerization reaction (ATRP) to generate polymeric 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, an Ag / AgCl electrode as the reference electrode, and a platinum wire as the auxiliary electrode to form a three-electrode system. Use 0.5 M LiClO4 as the electrolyte and perform square wave pulse SWV testing (potential: 0.1 - 0.3 V) to collect the current signal values.

[0064] When the sample to be detected is E2 solutions with different concentrations, a standard curve can be constructed based on the current signal values.

[0065] Example 2

[0066] In this example, the performance of the biosensor prepared in Example 1 was detected, including the following tests:

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

[0068] The AuE electrode, the electrode modified with IP-BP double strands, 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] containing 100 mM KCl 3− / 4− solution to observe the CV response and EIS response of different sensing substrate interfaces. The results are as Figure 2 shown.

[0069] From the CV response results ( Figure 2 in A), it can be seen that compared with the bare AuE electrode ( Figure 2 curve a in A), when the IP-BP double strands were modified onto the electrode surface, the current signal decreased ( Figure 2 curve b in A), which was due to the electrostatic repulsion between the negative charge of the DNA probe phosphate backbone and the [Fe(CN)6] 3- / 4- probe. After the electrode was blocked with MCH, the non-specifically adsorbed DNA probes were displaced, resulting in an increase in the current signal ( Figure 2 curve c in A). When the target E2 was added to initiate the ATRP reaction, a large amount of poly-FcMMA was generated on the electrode surface, and the current signal further increased ( Figure 2 curve d in A).

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

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

[0072] (2) Optimization of experimental conditions:

[0073] The changes in the response signals of the biosensor under different concentrations of IP-BP double strands (0.2 - 0.9 µM) and different reaction times (30 - 150 min) were investigated. The test method refers to Example 1, and the test sample is an 8 nM E2 solution. The results are as Figure 3 shown.

[0074] From the changes in the response signals of the biosensor under different concentrations of IP-BP double strands ( Figure 3 A), it can be seen that when the concentration of IP-BP double strands increases from 0.2 µM to 0.6 µM, the current signal gradually increases, and the sensor response signal is the largest at 0.6 µM. When the concentration of IP-BP double strands continues to increase, the impedance between the probes on the electrode surface increases, resulting in a decrease in the reaction efficiency and a gradual decrease in the current signal.

[0075] From the changes in the response signals of the biosensor under 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 flat after 100 min.

[0076] (3) Research on detection performance:

[0077] Using the biosensor of Example 1, different concentrations of E2 samples (0 fM, 100 fM, 1 pM, 10 pM, 80 pM, 800 pM, 8 nM, and 80 nM) were detected. Square wave pulse SWV tests were carried out, current signals were collected, and a standard curve was constructed to evaluate the sensitivity of the biosensor. The results are as Figure 4 shown.

[0078] From the SWV response curve ( Figure 4 A), it can be seen that the current peak increases with the increase in the 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. The linear equation is i = 11.06 + 0.8278 lgc (R 2(= 0.9928), and according to the three - fold relative standard deviation rule, the detection limit was calculated to be 35 fM. Compared with the detection limits (pM - nM) of traditional methods, this biosensor has higher sensitivity.

[0080] In addition, cortisol, estriol, and diethylstilbestrol were selected as interferents to detect the specificity of the biosensor. The results showed that compared with the response signal of 8 nM E2, the signal change obtained from interferents at 10 - fold concentration was less than 5.6%, demonstrating that the biosensor has excellent specificity.

[0081] (4) Verification of the detection performance of the biosensor:

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

[0083] The blood samples to be detected were diluted 10 - fold with phosphate buffer and detected according to the usage method in Example 1. The reacted sensing substrate was used as the working electrode, which formed a three - electrode system with an Ag / AgCl reference electrode and a platinum wire auxiliary electrode. The current signal of the three - electrode system was detected using a Chenhua CHI 660E electrochemical workstation. Using the above linear regression equation (i = 11.06 + 0.8278 lgc) for calculation, the concentration of E2 in the blood samples to be detected was obtained. The results are as Figure 5 shown, and there were significant differences in the concentration of E2 in different samples (a - f). Generally speaking, the concentration of E2 in blood samples from adult non - pregnant women (a, b, and c) was relatively low, with a concentration range of 124.9 pM to 886.8 pM. The concentration of E2 in blood samples from women in the second trimester of pregnancy (d, e, and f) increased significantly, with a concentration range of 2867 pM to 8762 pM. In addition, the same blood samples were detected using a commercial kit (Beckman 33540) commonly used in clinical diagnosis, and the detection results were consistent with those of the biosensor of the present invention, with a relative error between - 8.3% and 6.2%, indicating that the biosensor of the present invention has good detection ability and application even in complex blood samples.

[0084] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

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

5.

2. The construction method of the estradiol biosensor according to claim 1, characterized in that, It includes the following steps: S1. After soaking the gold electrode with piranha solution, it is polished to obtain an AuE electrode; S2. The IP strand and the BP strand are annealed and hybridized to obtain an IP-BP double strand; The IP-BP double strand is assembled onto the AuE electrode to obtain a sensing substrate; S3. The Aptamer and the TP strand are annealed and hybridized to obtain an Aptamer-TP double strand; an azide group is modified on the FP strand to obtain an FP-N3 probe; a DNA molecular machine is obtained.

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

4. The construction method according to claim 2, characterized in that In step S2, the annealing and hybridization method is: mixing the IP strand and the BP strand in an equimolar ratio, reacting at 90°C-100°C for 8-12 min, and cooling to 20°C-25°C at a rate of 1°C / min; The assembly method is: after activating the IP-BP double strand with tris(2-carboxyethyl)phosphine hydrochloride, dropping it onto the AuE electrode, reacting in the dark for 10-16 h, and then using mercaptohexanol to carry out a blocking reaction on the AuE electrode for 50-80 min; The concentration of the IP-BP double strand is 0.5-0.8 μM; the dropping amount is 8-15 μL.

5. The construction method according to claim 2, characterized in that, In step S3, the annealing and hybridization method is: mixing the Aptamer and the TP strand in an equimolar ratio, reacting at 90°C-100°C for 8-12 min, and cooling to 20°C-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 2-5 for detecting the content of estradiol for non-diagnostic purposes, characterized in that, The method for detecting the estradiol content includes the following steps: N1. After mixing the Aptamer-TP double strand and the FP-N3 probe, mixing with the sample to be detected, and dropping it onto the sensing substrate, reacting for 80-150 min; N2. The sensing substrate after the reaction in step N1 is placed in a mixed solution containing 2-bromo-2-methylpropionic acid 3-butynyl ester, ascorbic acid, and copper sulfate for a click chemical reaction; N3. The sensing substrate after the reaction in step N2 is placed in a dimethylformamide solution containing copper / tris(2-dimethylaminoethyl)amine, ferrocene methanol methacrylate, potassium bromide, and potassium hexafluorophosphate, and an ATRP reaction is carried out at a voltage of -0.56 V; N4. Using the sensing substrate after the reaction in step N3 as the working electrode, an Ag / AgCl electrode as the reference electrode, and a platinum wire as the auxiliary electrode, a three-electrode system is formed to detect the current signal in the LiClO4 solution; Calculate the estradiol content according to the standard curve.

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

8. The application according to claim 6, wherein In step N2, in the mixed solution, the final concentrations of 2-bromo-2-methylpropionic acid 3-butynyl ester, ascorbic acid, and copper sulfate are 80 - 120 μM, 15 - 25 μM, and 80 - 120 μM respectively; the time for the click chemical reaction is 40 - 60 min.

9. The application according to claim 6, characterized in that, In step N3, in the dimethylformamide solution, the final concentrations of copper / tris(2-dimethylaminoethyl)amine, ferrocenylmethanol methacrylate, potassium bromide, and potassium hexafluorophosphate are 80 - 120 μM, 80 - 120 μM, 80 - 120 mM, and 60 - 80 mM respectively; the time for the ATRP reaction 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 2-5 in the preparation of an estradiol detection product, characterized in that The detection product includes a detection kit.

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