Preparation method and application of functionalized laser-induced graphene-based 17beta-estradiol electrochemical biosensor

Abstract

The invention belongs to the technical field of electrochemical biosensors, and particularly relates to a preparation method and application of a functionalized laser-induced graphene-based 17beta-estradiol electrochemical biosensor. According to the invention, a heat transfer printing laser-induced graphene electrode is firstly designed, and then the heat transfer printing laser-induced graphene electrode is functionalized based on the prepared MB (at) ZIF-8, so that integration and functionalization of a classical three-electrode system are realized, and meanwhile, an internal reference signal is provided for a ratio electrochemical biosensor. Besides, the two split aptamer fragments are connected through a T basic group, and an electrochemical probe label is combined to form an SS-Apt composite structure, so that precise recognition of E2 is realized, and an electrochemical probe signal can be enhanced; therefore, the ratio electrochemical biosensor with the functionalized heat transfer printing laser-induced graphene as the sensing substrate is constructed, sensitive and accurate detection of E2 is realized, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and particularly relates to a preparation method and application of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor. Background Art

[0002] Endocrine disruptors, also known as environmental estrogens, are a class of exogenous chemical substances that can interfere with the functions of the normal endocrine system in organisms and pose potential threats to human health and the ecosystem. Due to their wide environmental distribution and negative impacts, this substance is listed as one of the first 12 prohibited persistent organic pollutants.

[0003] 17β-Estradiol (E2) is one of the environmental estrogens with particularly strong effects. Its structure contains 18 carbon atoms and a four-ring framework, and has endocrine disrupting activity. E2 has a long residual time in the environment, is highly lipophilic, and is easily accumulated in aquatic organisms, posing a threat to human health, including changing the hormone levels of aquatic organisms, inducing sex reversal, and increasing the risk of cancer in humans. Therefore, it is crucial to sensitively and accurately detect E2 in environmental water bodies.

[0004] Currently, the national standard analytical method is liquid chromatography-tandem mass spectrometry (GB / T 21981-2008). This method requires strong professional operation and high cost, and is mainly applicable to laboratory detection. Electrochemical sensing methods have been widely used in the detection of E2 due to their advantages of rapid response, simple instrument, and high sensitivity. However, the traditional electrochemical three-electrode system has a relatively complex composition and is not suitable for on-site detection. At the same time, conventional electrochemical sensors measure samples through single-signal changes, which are affected by many external factors, such as electrode modification methods, test platforms, and environmental conditions. This will lead to slight differences in the background currents of different electrodes, thereby reducing the reliability of quantitative detection of samples.

[0005] Therefore, it is necessary to design an integrated sensing chip that can streamline the three-electrode system while ensuring good sensing performance to achieve sensitive and accurate on-site detection of E2. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention aims to design a sensing substrate of a functionalized laser-induced graphene (LIG) electrode to achieve the integrated integration of the classic three-electrode system and provide a reference signal for the sensor; construct an electrochemical probe-labeled DNA structure by connecting two split aptamers of E2 with a certain number of T bases. When E2 is present, it can accurately recognize E2 and generate a response signal; at the same time, combine the ratio strategy to construct a ratio electrochemical biosensor, and finally achieve highly sensitive and highly accurate detection of E2.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solutions;

[0008] A preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor is as follows:

[0009] (1) Pretreatment of polyimide (PI) film:

[0010] First, attach the PI film to the surface of the substrate material, and then successively clean the surface of the PI film with acetone and absolute ethanol (to remove surface impurities). After cleaning, a PI film substrate is obtained;

[0011] (2) Preparation of functionalized material MB@ZIF-8:

[0012] First, prepare a methylene blue (MB) solution, denoted as solution A; then dissolve 2-methylimidazole and zinc nitrate hexahydrate in ultrapure water respectively to obtain a 2-methylimidazole solution and a zinc nitrate hexahydrate solution, denoted as solution B and solution C respectively;

[0013] Add solution A to solution C for the first stirring, and then add solution B for the second stirring. After stirring, centrifuge and collect the product, and wash the product several times with ultrapure water to remove the reaction residual precursors; finally, vacuum dry the washed product to obtain the functionalized material MB@ZIF-8;

[0014] (3) Preparation of functionalized laser-induced graphene electrode:

[0015] S1. According to the designed planar three-electrode substrate pattern, control the laser to scan and directly write on the surface of the PI film substrate after the cleaning treatment in step (1) to obtain a laser-induced graphene electrode on the PI film, denoted as LIG; the laser-induced graphene electrode includes a working electrode, a reference electrode and a counter electrode, and conductive strips connected to the working electrode, the reference electrode and the counter electrode;

[0016] Then cover a layer of EVA film on the upper surface of the LIG, and then cover the PVC soft board on the upper part of the EVA film. After hot pressing treatment, the LIG is transferred to the PVC soft board. After annealing at room temperature, rinse with ethanol to separate it from the substrate and the PI film, realizing the thermal transfer of the LIG; subsequently, use the PI film to encapsulate the conductive strip area. The encapsulation is carried out along the direction connected to the electrode to the end of the conductive strip, and at the same time, the end of the conductive strip is exposed and does not completely cover the conductive strip; after this encapsulation, an integrated electrode (i.e., the thermally transferred LIG (Thermal Transfer LIG)) is obtained, denoted as TT-LIG;

[0017] S2. Dissolve the functionalized material MB@ZIF-8 obtained in step (2) in ultrapure water to obtain an MB@ZIF-8 solution; then take the MB@ZIF-8 solution and modify it on the working electrode surface of TT-LIG. After incubation, a functionalized TT-LIG electrode is obtained, denoted as MB@ZIF-8 / TT-LIG;

[0018] (4) Preparation of SS-Apt:

[0019] Connect split aptamer 1 and split aptamer 2 through the base sequence (T) to obtain SS-Apt (as the recognition element for E2); first, dissolve SS-Apt in Tris-HCl buffer to obtain an SS-Apt solution, then dilute it to obtain an SS-Apt dilution, and add TCEP to activate the thiol group. After standing, a mixture is obtained, denoted as solution H;

[0020] (5) Preparation of a functionalized laser-induced graphene-based E2 electrochemical biosensor:

[0021] First, modify the Au NPs solution in the working electrode area of MB@ZIF-8 / TT-LIG. After the first incubation, it is denoted as Au NPs / MB@ZIF-8 / TT-LIG;

[0022] Subsequently, modify the solution H prepared in (4) in the working electrode area of MB@ZIF-8 / TT-LIG and perform a second incubation (using the Au-S bond interaction to fix SS-Apt on the electrode surface). After incubating for a period of time, a functionalized laser-induced graphene-based E2 electrochemical biosensor is obtained, denoted as SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG.

[0023] Furthermore, in the step (1), the thickness of the PI film is 120 μm, and the substrate material is a silica glass plate with a thickness of 2 mm.

[0024] Furthermore, in the step (2), the concentration of solution A is 2.58×10-2M, the concentration of solution B is 1.67M, and the concentration of solution C is 0.25M; the dosage relationship of the mixture of solution A, solution B, and solution C is 4 mL: 90 mL: 10 mL; the first stirring time is 1 min; the second stirring time is 15 min; the centrifugation speed is 8000 rpm / min, and the time is 10 min; the vacuum drying temperature is 60 °C, and the time is 6 h; the number of times the product is washed with ultrapure water is 3 - 5 times.

[0025] Further, the planar three - electrode substrate pattern designed in S1 of step (3) includes a working electrode (WE), a reference electrode (RE), and a counter electrode (CE); where the working electrode is circular, and the arc - shaped reference electrode and counter electrode are on its outer side. The reference electrode and the counter electrode are arranged along the same circumference. The length of the reference electrode is less than that of the counter electrode, and one end of the reference electrode and the counter electrode has their openings facing each other and are not connected, while the other end is on the same horizontal line as the bottom of the working electrode;

[0026] Conductive strips are respectively connected to the lower ends of the working electrode, the reference electrode, and the counter electrode. The conductive strips have the same length and are parallel to each other; the working electrode, the reference electrode, and the counter electrode form a sensing area, and the three conductive strips form a wire area;

[0027] The laser is a 10.6 - μm carbon dioxide laser, and the laser power and induction rate are 3.6 W and 300 mm / s respectively. The laser scanning direction is parallel to the length direction of the conductive strip;

[0028] The length of the conductive strip is 9.5 mm and the width is 1.5 mm; the radius of the working electrode is 3 mm, the inner arc radius of the counter electrode is 4.5 mm, and the outer arc radius is 6 mm; the inner arc radius of the reference electrode is 4.5 mm, and the outer arc radius is 6 mm; the parameters of the hot - pressing treatment are 150 °C and 120 s; the annealing time is 5 - 8 min; the area where the PI film is used to encapsulate the conductive strip of the counter electrode extends downward from the top of the conductive strip (i.e., the connection with the electrode) to a length of 2 mm (used to isolate the electrolyte solution in the sensing area from entering the wire area).

[0029] Further, in S2 of step (3), the concentration of the MB@ZIF - 8 solution is 3 mg mL-1, and the modification dosage is 20 μL.

[0030] Further, in step (4), the DNA sequences (5'-3' end) of the SP1, SP2, and SS - Apt are as follows:

[0031] Split aptamer 1: AAGGGATGCCGTTTGGG

[0032] Split aptamer 2: CCCAAGTTCGGCATAGTG

[0033] SS - Apt:

[0034] HS - SH - AAGGGATGCCGTTTGGG - TTTTTTTTTTTT - CCCAAGTTCGGCATAGTG - Fc;

[0035] Further, in step (4), the pH of the Tris-HCl buffer solution is 7.4, the concentration is 10 mM, the concentration of the SS-Apt solution is 100 μM; the final concentration of TCEP in solution H is 5%; the standing time is 30 min; the concentration of solution H is 2.5 μM.

[0036] Further, in step (5), the concentration of the AuNPs solution is 4 μM, and the modification dosage is 20 μL; the temperature of the first incubation is 37 °C and the time is 30 min; the concentration of solution H is 2.5 μM, and the modification dosage is 20 μL, and the temperature of the second incubation is 4 °C and the time is 12 h.

[0037] The biosensor obtained by the present invention has a multi-layer structure. The bottom layer is a PVC board, the middle layer is an EVA film, and the upper layer is a porous graphene structure; this structure can provide a large specific surface area substrate for the assembly of the sensing interface to improve the detection sensitivity; the upper layer serves as the sensing interface and is the core layer for realizing the detection of the target substance.

[0038] The present invention also relates to the use of an electrochemical biosensor for detecting 17-β estradiol, and the steps are as follows:

[0039] (1) First, prepare E2 standard solutions with different concentrations; then take the electrochemical biosensor SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG, and modify the E2 standard solutions on its surface respectively. After incubation at room temperature, an electrochemical biosensor interface that has completed the recognition and detection is obtained; one concentration of the E2 standard solution corresponds to the modification of one electrochemical biosensor, and there is a one-to-one correspondence between the concentration and the electrochemical biosensor;

[0040] Then, wash the electrochemical biosensor interface that has completed the recognition and detection with phosphate buffer solution (PBS) to obtain a washed electrochemical biosensor, denoted as E2 / SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG;

[0041] (2) Test the response electrochemical signal of the sensor E2 / SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG by square wave voltammetry (SWV); the electrochemical signal generated by Fc is denoted as I Fc , and the electrochemical signal generated by MB is denoted as I MB ; perform a ratio process on the electrochemical signal generated by Fc and the electrochemical signal generated by MB to obtain I Fc / I MB ; its value is positively correlated with the concentration of the corresponding E2 solution, and each concentration of E2 will correspond to a I Fc / I MB value. According to I Fc / IMB Construct a standard linear curve by using the logarithm of the value and the concentration of E2;

[0042] (3) Detection of E2 in the sample: After processing the sample to obtain a sample solution, according to the operation in step (1), the difference is that the E2 standard solution is replaced with the sample solution; then continue to operate according to steps (2) and (3), and after electrochemical detection, obtain I Fc and I MB ; and perform ratio calculation; substitute the I Fc / I MB value into the standard curve constructed in step (3), and the detection of E2 in the unknown sample can be realized.

[0043] Furthermore, in step (1), the concentration of the E2 standard solution is 10 pg mL -1 -500 ng mL -1 , and the dosage of the modification is 20 μL for both; the time of incubation at room temperature is 50 min; the concentration of the PBS solution is 0.1 M and the pH is 7.4;

[0044] Furthermore, in step (2), the specific conditions for testing by cyclic voltammetry are as follows: the scanning voltage range is -0.5 to 0.5 V, the amplitude is 0.0025 V, and the frequency is 15 Hz.

[0045] In step (3), the electrochemical detection is performed by selecting square wave voltammetry (SWV) on a CHI1040C electrochemical workstation. The principle is as follows: The specific recognition of E2 by Apt will cause a conformational change of Apt at the electrode interface; the 3' end of Apt is labeled with an Fc signal probe; after recognizing E2, the Fc signal probe changes from a state far from the electrode interface to a state close to the electrode interface, and thus the Fc electrochemical signal increases, which is used as the response signal. At the same time, the functionalized TT-LIG electrode will generate the electrochemical signal of MB, which is used as the reference signal and forms a ratio signal I Fc / I MB .

[0046] Advantages of the present invention:

[0047] The present invention can realize the integrated integration of the classical three-electrode system, and at the same time can quickly prepare an electrode with a porous structure and excellent electrical conductivity, and realize its rapid and accurate transfer onto the EVA hot melt adhesive material; use the functionalized material MB@ZIF-8 to fill its porous structure, providing a reference signal for testing and more reactive sites at the same time. A ratiometric electrochemical biosensor with a functionalized laser-induced graphene electrode transferred by EVA hot melt adhesive as the sensing substrate, SS-Apt as the recognition element, Fc as the response signal, and MB as the reference signal is constructed, realizing the sensitive and accurate detection of E2. Description of the Drawings

[0048] Figure 1 Process diagram of constructing an electrochemical biosensor.

[0049] Figure 2 Design diagram of an integrated electrode (TT-LIG); where (A) is the designed integrated three-electrode pattern; (B) is the physical diagram of the LIG electrode; (C) is the physical diagram of the thermally transferred LIG electrode.

[0050] Figure 3 Characterization and exploration diagram of functionalized materials; where A is the scanning electron microscope (SEM) image of MB@ZIF-8; B is the X-ray energy dispersive spectroscopy (XRD) image of MB@ZIF-8 and ZIF-8; C is the physical diagram of the comparison before and after loading MB on ZIF-8.

[0051] Figure 4 Characterization diagram of the construction process of a functionalized laser-induced graphene-based E2 electrochemical biosensor: where A is the SEM image of the cross-section of the functionalized TT-LIG electrode; B is the SEM image of the surface of the functionalized TT-LIG electrode; C is the SEM image of the inside of the functionalized TT-LIG electrode.

[0052] Figure 5 Feasibility exploration and experimental condition optimization diagram of the preparation of a functionalized laser-induced graphene-based E2 electrochemical biosensor: where A is the CV curve of the sensor during the construction process; B is the EIS curve of the sensor during the construction process, and the inset is the equivalent circuit diagram; C is the SWV curve of the sensor in the presence of different concentrations of E2; D is the optimization of the aptamer concentration; E is the optimization of the number of T bases in SS-Apt; F is the optimization of the target binding time.

[0053] Figure 6 Selectivity experiment diagram (A), reproducibility experiment diagram (B), and stability experiment diagram (C) of a functionalized laser-induced graphene-based E2 electrochemical biosensor.

[0054] Figure 7 Effect diagram of the performance analysis of E2 using a functionalized laser-induced graphene-based E2 electrochemical biosensor: where A is the SWV curve of the sensor in the presence of different concentrations of E2; B is the standard curve for the determination of E2. Detailed implementation manners

[0055] The following will describe the embodiments of the present invention in detail with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0056] The pharmaceutical reagents mentioned in the present invention: The polyimide film is purchased from DuPont Company, USA; sodium citrate (Na3C6H5O7·2H2O), chloroauric acid (HAuCl4·3H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), ferric chloride hexahydrate (FeCl3·6H2O), zinc nitrate hexahydrate (Zn(NO3)2·6H2O), potassium chloride (KCl) are all purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.; chloroauric acid trihydrate, tris(2-carboxy)phosphine (TCEP), 2-methylimidazole are purchased from Aladdin Biotechnology Co., Ltd.; glacial acetic acid solution is purchased from China National Pharmaceutical Corporation; tris(hydroxymethyl)aminomethane (tris) is purchased from Alfa Aesar Chemical Co., Ltd.; methylene blue (MB), ethylene-vinyl acetate copolymer (EVA) are purchased from Macklin Biotechnology Co., Ltd. The reagent mentioned: single-stranded DNA (SS-Apt) is purchased from Sangon Biotech (Shanghai) Co., Ltd., and the corresponding sequences are as follows:

[0057] SP1: AAGGGATGCCGTTTGGG

[0058] SP2: CCCAAGTTCGGCATAGTG

[0059] SS-Apt: HS-SH-AAGGGATGCCGTTTGGG-TTTTTTTTTTTT-CCCAAGTTCGGCATAGTG-Fc; The present invention does not involve the invention of a sequence listing. The sequences mentioned are conventional primer sequences, so there is no need to provide a sequence listing.

[0060] The solutions used in the present invention: Use Tris-HCl (0.05 M Tris, 0.1 M NaCl, 0.2 M KCl, 5 mM MgCl2, pH = 7.4); Electrochemical detection and dilution of E2 are carried out with phosphate buffer (0.1 M PBS, pH = 7.4) and ultrapure water respectively. The solvents required for solution preparation and dissolution are ultrapure water.

[0061] Au NPs solution: Mix HAuCl4 and ultrapure water, heat in an oil bath until the solution boils completely, and then quickly add the Na3C6H5O7 solution; After completing the above steps, continue heating and stirring. When the color of the solution in the flask turns wine red, it is cooled to room temperature to obtain the Au NPs solution, which is stored at 4 °C for standby; The concentration of HAuCl4 is 0.1 M, and the concentration of the Na3C6H5O7 solution is 100 mg mL -1 , The dosage relationship of HAuCl4, ultrapure water and Na3C6H5O7 solution is 200 μL: 25 mL: 0.25 mL, and the time for continuous heating and stirring is 15 min.

[0062] The construction process of the electrochemical biosensor is as follows Figure 1 as shown.

[0063] The design diagram of the integrated electrode (TT-LIG) is as follows Figure 2 as shown, where (A) is the designed integrated three-electrode pattern; the integrated three-electrode pattern is designed in advance using Autodesk Computer Aided Design, the pattern resolution is adjusted to 350 dpi using Adobe Photoshop, and the electrode design pattern is arranged in an array through SeaCAD software at the upper computer control end. The actual LIG diagram is as shown in (B) in Figure 2 , and Figure 2 (C) in shows the actual diagram of the thermally transferred LIG. The specifically designed planar three-electrode substrate pattern includes a working electrode, a reference electrode, and a counter electrode; the working electrode is a circle with a radius of 3 mm, and the arc-shaped reference electrode and counter electrode are on its outer side. The reference electrode and the counter electrode are arranged along the same circumference. The length of the reference electrode is less than that of the counter electrode, and one end of the reference electrode and the counter electrode faces each other and is not connected, while the other end is on the same horizontal line as the bottom of the working electrode; the inner arc radius of the counter electrode is 4.5 mm, and the outer arc radius is 6 mm; the inner arc radius of the reference electrode is 4.5 mm, and the outer arc radius is 6 mm.

[0064] Conductive strips with a length of 9.5 mm and a width of 1.5 mm are respectively connected to the lower ends of the working electrode, the reference electrode, and the counter electrode, and the conductive strips are parallel to each other; the working electrode, the reference electrode, and the counter electrode form a sensing area, and the three conductive strips form a wire area.

[0065] The laser power and induction rate during processing are 3.6 W and 300 mm s -1 respectively; first, control the laser to scan and directly write on the surface of the PI film substrate in step (3) to obtain the LIG electrode; then, place the LIG, EVC film, and PVC board on the thermal transfer table in sequence, operate the hot embossing machine at 150 °C for 120 s, and anneal at room temperature for 5 min to obtain the thermally transferred LIG electrode; finally, encapsulate the electrode using polyimide tape, extending from the top of the conductive strip (i.e., the connection with the electrode) to the area with a length of 2 mm downward (used to isolate the electrolyte solution in the sensing area from entering the wire area) to obtain the thermally transferred LIG integrated electrode, denoted as TT-LIG.

[0066] (1) Characterization and exploration of the functionalized TT-LIG electrode prepared in the present invention

[0067] Characterization methods such as SEM and XRD were used to characterize the morphologies of the functionalized material MB@ZIF-8 and the functionalized TT-LIG electrode. As shown in the SEM images ( Figure 3 A and Figure 4) As shown, the edges and corners of ZIF-8 collapsed after loading MB; subsequently, XRD ( Figure 3 B) confirmed that the loading of MB did not damage the original framework of ZIF-8, and ZIF-8 changed from white to blue before and after loading MB ( Figure 3 C). The above characterizations proved that the functional material MB@ZIF-8 was successfully prepared. The functional material MB@ZIF-8 was modified on the surface of the TT-LIG working electrode to form a functional TT-LIG electrode. It was found from the SEM image that the functional material MB@ZIF-8 filled the pore structure of TT-LIG. The above characterizations proved that the functional TT-LIG electrode was successfully prepared.

[0068] (2) Exploration on the construction of the electrochemical biosensor prepared by the present invention

[0069] To verify the construction of the electrochemical biosensor, 6 groups of experiments were set up, and the test electrodes were TT-LIG; MB@ZIF-8 / TT-LIG; Au NPs / MB@ZIF-8 / TT-LIG; SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG; E2 / SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG, which were correspondingly recorded as test a, b, c, d, and e.

[0070] CV tests and EIS tests were carried out in a 5 mM K2[Fe(CN)6] 3- / 4- solution ( Figure 5 A and 5B); the CV curve on LIG showed the potentials of oxidation and reduction peaks; the charge transfer resistance (Rct) was determined by the diameter of the semicircle in the EIS Nyquist plot, which indicated the electron transfer kinetics at the electrode interface. The original TT-LIG electrode showed oxidation and reduction peaks at 0.15 V and -0.15 V respectively, accompanied by a large peak current, and its minimum Rct was 20 Ω, indicating high conductivity. After being functionalized with MB@ZIF-8, the Rct increased to 60 Ω, which was due to the poor conductivity of ZIF-8 itself, resulting in a decrease in conductivity and a decrease in the peak current value. After being modified with Au NPs, the Rct decreased to 50 Ω, improving the conductivity and increasing the peak current value, which was attributed to the high conductivity of Au NPs. After being modified with the aptamer, the Rct increased to 100 Ω, which was due to the steric hindrance hindering electron transfer, resulting in a decrease in conductivity and the smallest peak current value. When the target analyte was recognized, the electroactive substance Fc interacted with the electrode, promoting electron transfer, and the impedance decreased to 80 Ω, and the conductivity and peak current value increased. The above results confirmed the successful construction of the electrochemical biosensor.

[0071] (3) Feasibility exploration of the electrochemical biosensor (SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG) prepared by the present invention for detecting E2

[0072] To verify the feasibility of the sensor for E2 detection, three groups of experiments were set up to test the response of the sensor under different target states, namely no E2, addition of 1 ng / mL -1 of E2 and addition of 10 ng / mL -1 of E2, denoted as tests m, n, and k.

[0073] Square wave voltammetry (SWV) tests were performed on each group of experiments using 0.1 M PBS to evaluate the feasibility of the sensor for E2 detection. As Figure 5 shown in Figure C, the I Fc increases in the presence of E2 compared to when there is no E2. This is due to the labeled Fc signal probe in SS-Apt approaching the electrode interface; at the same time, I MB remains relatively unchanged, forming I Fc / I MB . In the absence of E2, I MB = 6.33 μA and I Fc = 1.80 μA were observed; after adding 1 ng / mL -1 of E2, I MB = 6.24 μA and I Fc = 2.03 μA were observed; when C E2 increased to 10 ng / mL -1 , I MB = 6.31 μA and I Fc = 2.19 μA; the increase in I Fc can be explained as: as C E2 increases, more Fc probes approach the electrode surface, resulting in an increase in the current response of Fc.

[0074] (4) Optimization of experimental conditions

[0075] S1. The split aptamer 1 and split aptamer 2 were connected by the base sequence (T) to obtain SS-Apt; SS-Apt was dissolved in Tris-HCl buffer to obtain an SS-Apt solution, which was then diluted to obtain an SS-Apt dilution. TCEP was added to activate the thiol groups to a final concentration of 5%. After standing, a mixture was obtained, denoted as solution H, with a concentration of 2.5 μM;

[0076] S2. A 20 μL solution of Au NPs with a concentration of 4 μM was modified on the surface of the functionalized TT-LIG working electrode and incubated at 37 °C for 30 min. Then, 20 μL of solution H was modified on the surface of the functionalized TT-LIG working electrode and incubated at 4 °C for 12 h; subsequently, 20 μL of 1 ng / mL -1The target E2 was incubated at room temperature for 50 min to generate an electrochemical signal. The SWV method of the CHI1040C electrochemical workstation was selected to test the current value of the sensor.

[0077] Since the detection range of the E2 electrochemical biosensor is affected by the solution concentration, the concentration of solution H was first optimized, and the set concentrations were 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, and 3.0 μM. As Figure 5 shown in D, with the increase in the concentration of solution H, I Fc increased significantly, but I Fc remained basically unchanged within 2.0 - 3.0 μM. Therefore, 2.5 μM was selected as the incubation concentration of solution H;

[0078] At the same time, the number of T bases in SS-Apt was optimized. By inserting 0, 6, 12, 18, and 24 T bases to construct variables, the influence of the length of SS-Apt on the Fc signal response sensitivity was explored. As Figure 5 shown in E, after inserting 6, 12, 18, and 24 T bases, the Fc signals increased by 6.58%, 15.71%, 14.04%, and 10.89% respectively. Therefore, inserting 12 T bases into SS-Apt was selected as the best response for the Fc signal.

[0079] In addition, regarding the influence of the binding time of E2 and the aptamer on the detection performance of the sensor, the incubation times at room temperature were set to 20 min, 30 min, 40 min, 50 min, and 60 min respectively; as Figure 5 shown in F, with the incubation time increasing from 20 min to 60 min, I Fc continued to increase. After 40 min, I Fc remained stable. Therefore, 50 min was selected as the best incubation time for E2.

[0080] (5) Exploration of the selectivity of the electrochemical biosensor prepared by the present invention for E2 detection

[0081] The specific method for exploration is as follows: Seven groups of experiments were set up, namely no target and interference substances, with 10 ng / mL -1 interference substance estriol (E3), with 10 ng / mL -1 interference substance bisphenol A (BPA), with 10 ng / mL -1 interference substance diethylstilbestrol (DES), and a mixture of interference substances with a concentration of 10 ng / mL -1 in all cases, with 1 ng / mL -1 target E2, and a mixture of interference substances and 1 ng / mL -1 interference substances and 1 ng / mL -1A mixture of the target E2. These seven groups of experiments are respectively denoted as the blank group, E3 group, BPA group, DES group, Mix group, E2 group, and Mix + E2 group.

[0082] The operations for the blank group are as follows: a) Modify 20 μL of the AuNPs solution onto the surface of the functionalized TT-LIG working electrode and incubate at 37 °C for 30 min. b) Modify 20 μL of solution H with a concentration of 2.5 μM onto the surface of the functionalized TT-LIG working electrode and incubate at 4 °C for 12 h; c) Wash the treated electrochemical biosensor with phosphate buffer (PBS). d) Subsequently, modify 20 μL of the blank sample and incubate at room temperature for 50 min. e) Select the SWV method on the CHI1040C electrochemical workstation to detect the current at the sensor interface and calculate the I Fc / I MB value.

[0083] The operations for the non-blank groups are as follows: a) Modify 20 μL of the AuNPs solution onto the surface of the functionalized TT-LIG working electrode and incubate at 37 °C for 30 min; b) Modify 20 μL of solution H with a concentration of 2.5 μM onto the surface of the functionalized TT-LIG working electrode and incubate at 4 °C for 12 h; c) Wash the treated electrochemical biosensor with phosphate buffer (PBS). d) Modify 20 μL of the interfering substance solution (E2, BPA, DES) or the mixture solution (Mix, E2, Mix + E2) and incubate at room temperature for 50 min; e) Select the SWV method on the CHI1040C electrochemical workstation to detect the current at the sensor interface and calculate the I Fc / I MB value.

[0084] From (A) in Figure 6 it can be seen that the signal changes significantly when the target is present, proving that the sensor has selectivity for E2 detection.

[0085] (6) Investigation of the reproducibility of the electrochemical biosensor prepared in the present invention for E2 detection

[0086] The specific operation for the investigation is as follows: Set up six groups of repeated experiments with the target E2 concentration of 1 ng mL -1 .

[0087] The operations for the six groups of repeated experiments are as follows: Modify 20 μL of the AuNPs solution onto the surface of the functionalized TT-LIG working electrode and incubate at 37 °C for 30 min. Then, modify 20 μL of solution H with a concentration of 2.5 μM onto the surface of the functionalized TT-LIG working electrode and incubate at 4 °C for 12 h; Subsequently, modify 20 μL, 1 ng mL -1Target E2 was incubated at room temperature for 50 min. The current at the sensor interface was detected by the SWV method on a CHI1040C electrochemical workstation, and I Fc / I MB value was calculated. This operation was repeated six times.

[0088] According to the results of the repeated experiments, it can be seen from (B) in Figure 6 that the signals of the six groups of repeated experiments were basically the same, and the relative standard deviation (RSD) was 1.7%, indicating that the sensor had good reproducibility for the detection of E2.

[0089] (7) Investigation of the stability of the electrochemical biosensor prepared in this invention for the detection of E2

[0090] The specific method for the investigation was as follows: Seven groups of experiments with the same conditions were set up, and one group was measured every day within seven days. The concentration of the target E2 was 1 ng mL -1 .

[0091] The experimental operation was as follows: On the first day, functionalized TT-LIG electrodes were fabricated in batches. A 20-μL Au NPs solution was modified onto the surface of the functionalized TT-LIG working electrode and incubated at 37 °C for 30 min. Then, a 20-μL solution H with a concentration of 2.5 μM was modified onto the surface of the functionalized TT-LIG working electrode and incubated at 4 °C for 12 h; subsequently, 20 μL of 1 ng mL -1 target E2 was modified onto some of the electrodes and incubated at room temperature for 50 min. The current at the sensor interface was detected by the SWV method on a CHI1040C electrochemical workstation, and I Fc / I MB value was calculated; the remaining electrodes without the modification of target E2 were placed in a 4 °C refrigerator for storage. In the following six days, a part of the electrodes was modified every day for the detection of target E2.

[0092] According to the results of the seven-day experiments, it can be seen from C in Figure 6 that the signals of the seven groups of repeated experiments were basically the same, and the relative standard deviation (RSD) was 0.8%, indicating that the sensor had good stability for the detection of E2.

[0093] Example 1:

[0094] (1) Pretreatment of the polyimide (PI) film:

[0095] First, a PI film with a thickness of 120 μm was affixed to the surface of the glass substrate material, and the surface of the PI film was sequentially cleaned with acetone and absolute ethanol (to remove surface impurities) to obtain the PI film after cleaning treatment;

[0096] (2) Preparation of the functionalized material MB@ZIF-8:

[0097] First, prepare a methylene blue (MB) solution with a concentration of 2.58×10 -2 M and reserve it for later use, denoted as Solution A. Dissolve 2-methylimidazole and zinc nitrate hexahydrate in ultrapure water respectively to obtain a 2-methylimidazole solution with a concentration of 1.67 M and a zinc nitrate hexahydrate solution with a concentration of 0.25 M, denoted as Solution B and Solution C respectively;

[0098] Subsequently, add Solution A to Solution C for the first stirring, and the resulting mixture is denoted as Solution D; then add Solution B to Solution D (where the volume ratio of Solution A, Solution B, and Solution C is 4 mL: 90 mL: 10 mL), conduct the second stirring. After vigorously stirring for 15 min, centrifuge at 8000 rpm for 10 min and collect the product. Wash the product 3 - 5 times with ultrapure water to remove the residual reaction precursors. Finally, vacuum-dry the washed product at 60 °C for 6 h to obtain the functionalized material MB@ZIF-8. (3) Preparation of the functionalized laser-induced graphene electrode:

[0099] The designed planar three-electrode substrate pattern includes a working electrode (WE), a reference electrode (RE), and a counter electrode (CE); among them, the working electrode is circular, and the arc-shaped reference electrode and counter electrode are on its outer side. The reference electrode and the counter electrode are arranged along the same circumference. The length of the reference electrode is less than that of the counter electrode, and one end of the reference electrode and the counter electrode faces each other and is not connected, while the other end is at the same horizontal line as the bottom of the working electrode; conductive strips are respectively connected to the lower ends of the working electrode, the reference electrode, and the counter electrode, and the conductive strips have the same length and are parallel to each other; the working electrode, the reference electrode, and the counter electrode form a sensing area, and the three conductive strips form a wire area;

[0100] First, control a 10.6 μm carbon dioxide laser, with a laser power of 3.6 W and an induction rate of 300 mm / s, to scan and directly write on the surface of the PI film substrate in step (1) to obtain a working electrode with a radius of 3 mm; a reference electrode and a counter electrode with an inner arc radius of 4.5 mm and an outer arc radius of 6 mm, and conductive strips with a length of 9.5 mm and a width of 1.5 mm connected to the working electrode, the reference electrode, and the counter electrode. Thus, the three electrodes and the conductive strips constitute a laser-induced graphene electrode, denoted as LIG;

[0101] Then transfer the LIG to the workbench of the hot stamping machine, cover a layer of hot melt adhesive film (EVA) on the upper part of the LIG, and then cover the PVC soft board on the upper part of the EVA. Set the heating temperature and heating time parameters of the hot stamping machine to 150 °C and 120 S. After the above preparations are completed, operate the extension rod to press the working module of the hot stamping machine onto the upper part of the PVC board, and anneal at room temperature for 5 min, then transfer the LIG to the PVC soft board. Then rinse with ethanol to separate it from the substrate and the polyimide film, realizing the thermal transfer of the LIG. Subsequently, encapsulate the conductive strip with a PI film, extending from the top of the conductive strip (i.e., the connection with the electrode) down to the area with a length of 2 mm (used to isolate the electrolyte solution in the sensing area from entering the wire area), obtaining a thermally transferred LIG integrated electrode, denoted as TT-LIG. Modify 20 μL of 3 mg mL -1 of the MB@ZIF-8 solution on the TT-LIG, and place it in an incubator at 37 °C for 30 min to obtain a functionalized TT-LIG electrode, denoted as MB@ZIF-8 / TT-LIG.

[0102] (4) Connect split aptamer 1 and split aptamer 2 through the base sequence (T) as the recognition element for E2, and denote it as SS-Apt. First, dissolve SS-Apt in Tris-HCl buffer with a pH of 7.4 and a concentration of 10 mM to obtain a SS-Apt solution with a concentration of 100 μM. Then dilute it with ultrapure water to 2.5 μM to obtain a SS-Apt dilution, and add 5% of TCEP to activate the thiol group, and let it stand for 30 min to obtain a mixed solution for standby.

[0103] (5) Preparation of a functionalized laser-induced graphene electrode electrochemical biosensor:

[0104] Modify 20 μL of a Au NPs solution with a concentration of 4 μM in the working electrode area of the MB@ZIF-8 / TT-LIG for the first incubation for 30 min. Obtain Au NPs / MB@ZIF-8 / TT-LIG. Subsequently, modify 20 μL of the mixed solution prepared in (4) in the working electrode area of the Au NPs / MB@ZIF-8 / TT-LIG and conduct the second incubation (using the Au-S bond interaction to fix the SS-Apt on the electrode surface); after the second incubation for 12 h, obtain a highly sensitive and highly selective electrochemical biosensor for detecting E2, denoted as SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG.

[0105] (6) First, prepare solutions with concentrations of 10 pg mL -1 , 50 pg mL -1 , 100 pg mL -1 , 500 pg mL -1 , 1 ng mL-1 , 5 ng / mL -1 , 10 ng / mL -1 , 50 ng / mL -1 , 100 ng / mL -1 , 500 ng / mL -1 E2 solution; Take 10 electrochemically biosensors constructed in step (5), and modify the surface of each with E2 solutions at concentrations of 10 pg / mL -1 , 50 pg / mL -1 , 100 pg / mL -1 , 500 pg / mL -1 , 1 ng / mL -1 , 5 ng / mL -1 , 10 ng / mL -1 , 50 ng / mL -1 , 100 ng / mL -1 , 500 ng / mL -1 E2 solution (one concentration of E2 solution corresponds to one electrochemically biosensor, and there is a one-to-one correspondence between the concentration and the electrochemically biosensor), incubate at room temperature for 50 min to obtain the electrochemically biosensor (E2 / SS-Apt / AuNPs / MB@ZIF-8 / TT-LIG) that has completed the recognition detection.

[0106] (7) Select the SWV method on the CHI1040C electrochemical workstation to detect the current of the electrochemically biosensor E2 / SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG in step (6). Since the Apt of E2 has specific recognition for E2, when E2 is present, the Fc signal probe labeled with SS-Apt approaches the electrode interface, and the electrochemical signal generated by Fc increases. The functionalized TT-LIG electrode generates an unchanged MB electrochemical signal.

[0107] The electrochemical signal generated by Fc is denoted as I Fc , and the electrochemical signal generated by MB is denoted as I MB ; Process the ratio of the electrochemical signal generated by Fc to the electrochemical signal generated by MB to obtain I Fc / I MB ; According to the linear relationship between the logarithm of the standard solution E2 concentration lgC E2 and I Fc / I MB , obtain the linear regression equation as I Fc / I MB = 0.18lgC E2 + 1.01.

[0108] According to Figure 7As can be seen from A-B in the figure, the linear range of the functionalized laser-induced graphene-based E2 electrochemical biosensor proposed by the present invention for E2 detection is 10 pg mL -1 ~500 ng mL -1 , spanning 4 orders of magnitude.

[0109] (8) Detection of pond water samples:

[0110] Collect 500 mL of pond water from a certain place in Zhenjiang, Jiangsu as the sample solution, filter it through a 500-mesh stainless steel sieve to remove most of the plankton and suspended matter in the water sample, and then filter and purify it with a 0.22-μm filter membrane. The filtered sample is stored in a brown reagent bottle in the dark as the sample solution to be tested for subsequent analysis. Add 0, 500, 1000, 5000 pg mL - 1 E2 standard solutions and mix well. Subsequently, modify 20 μL of the sample solution to be tested on the sensing interface, incubate at room temperature for 50 min, and then perform electrochemical testing by SWV method. Substitute the measured I Fc / I MB into the standard test curve to obtain its detection recovery rate. At the same time, the sample solution to be tested was tested by the national standard detection method of high performance liquid chromatography-tandem mass spectrometry, and the results are shown in Table 1.

[0111] Table 1: Detection recovery rate of E2 in pond water samples

[0112]

[0113] Note: "--" indicates not detected

[0114] As can be seen from Table 1, the electrochemical biosensor prepared by the present invention can sensitively, quantitatively and reliably detect E2 in the sample solution to be tested. It has a low preparation cost, a simple method, does not require professional training, is easy to operate, and has accurate detection results, and has high practical value.

[0115] Explanation: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above respective embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered within the scope of the claims of the present invention.

Claims

1. A preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor, characterized in that, It includes the following steps: (1) First, attach the PI film to the surface of the substrate material, and then sequentially clean the surface of the PI film with acetone and absolute ethanol. After cleaning, a PI film substrate is obtained; (2) Preparation of the functional material MB@ZIF-8: First, prepare a methylene blue solution, denoted as solution A; then dissolve 2-methylimidazole and zinc nitrate hexahydrate in ultrapure water respectively to obtain a 2-methylimidazole solution and a zinc nitrate hexahydrate solution, denoted as solution B and solution C respectively; Subsequently, add solution A to solution C for the first stirring, and the resulting mixture is denoted as solution D; Then add solution B to solution D for the second stirring. After stirring, centrifuge and collect the product, and wash the product several times with ultrapure water to remove the residual reaction precursors; finally, vacuum-dry the washed product to obtain the functional material MB@ZIF-8; (3) Preparation of the functionalized laser-induced graphene electrode: S1. According to the designed planar three-electrode substrate pattern, control the laser to scan and directly write on the surface of the PI film substrate after the cleaning treatment in step (1) to obtain a laser-induced graphene electrode on the PI film, denoted as LIG; the laser-induced graphene electrode includes a working electrode, a reference electrode, and a counter electrode, as well as conductive strips connected to the working electrode, the reference electrode, and the counter electrode; Then cover a layer of EVA film on the upper surface of the LIG, and then cover the PVC soft board on the upper part of the EVA film. After hot pressing treatment, the LIG is transferred to the PVC soft board. After annealing at room temperature, rinse with ethanol to separate it from the substrate and the PI film, realizing the thermal transfer of the LIG; subsequently, use the PI film to encapsulate the conductive strip area. The encapsulation is carried out along the direction connected to the electrode to the end of the conductive strip, and at the same time, the end of the conductive strip is exposed and does not completely cover the conductive strip; after this encapsulation, an integrated electrode is obtained, denoted as TT-LIG; S2. Dissolve the functional material MB@ZIF-8 obtained in step (2) in ultrapure water to obtain an MB@ZIF-8 solution; then take the MB@ZIF-8 solution and modify it on the surface of the working electrode of the TT-LIG. After incubation, a functionalized TT-LIG electrode is obtained, denoted as MB@ZIF-8 / TT-LIG; (4) Connect the split aptamer 1 and the split aptamer 2 through a base sequence to obtain SS-Apt; dissolve SS-Apt with Tris-HCl buffer solution to obtain an SS-Apt solution, and then dilute it to obtain an SS-Apt dilution solution, and add TCEP to activate the thiol group. After standing, a mixture is obtained, denoted as solution H; (5) Preparation of the functionalized laser-induced graphene-based E2 electrochemical biosensor: First, modify the Au NPs solution in the working electrode area of the MB@ZIF-8 / TT-LIG. After the first incubation, it is denoted as AuNPs / MB@ZIF-8 / TT-LIG; Subsequently, the solution H prepared in (4) was modified in the working electrode area of MB@ZIF-8 / TT-LIG and subjected to a second incubation. After incubating for a period of time, a functionalized laser-induced graphene-based E2 electrochemical biosensor was obtained, denoted as SS-Apt / AuNPs / MB@ZIF-8 / TT-LIG.

2. The preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor according to claim 1, wherein, In the step (1), the thickness of the PI film is 120 μm, and the substrate material is a silica glass plate with a thickness of 2 mm.

3. The preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor according to claim 1, characterized in that, In the step (2), the concentration of solution A is 2.58×10 -2 M, the concentration of solution B is 1.67 M, and the concentration of solution C is 0.25 M; the dosage relationship of the mixture of solution A, solution B, and solution C is 4 mL: 90 mL: 10 mL; the time of the first stirring is 1 min; the time of the second stirring is 15 min; the centrifugation speed is 8000 rpm / min, and the time is 10 min; the temperature of vacuum drying is 60 °C, and the time is 6 h; the number of times the product is washed with ultrapure water is 3 - 5 times.

4. The preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor according to claim 1, wherein, The planar three-electrode substrate pattern designed in S1 of step (3) includes a working electrode, a reference electrode, and a counter electrode; among them, the working electrode is circular, and the arc-shaped reference electrode and counter electrode are outside it. The reference electrode and the counter electrode are arranged along the same circumference. The length of the reference electrode is less than that of the counter electrode, and one end of the reference electrode and the counter electrode faces each other and is not connected, and the other end is on the same horizontal line as the bottom of the working electrode; Conductive strips are respectively connected to the lower ends of the working electrode, the reference electrode, and the counter electrode. The conductive strips have the same length and are parallel to each other; the working electrode, the reference electrode, and the counter electrode form a sensing area, and the three conductive strips form a wire area; The laser is a 10.6 μm carbon dioxide laser, and the laser power and induction rate are 3.6 W and 300 mm / s respectively. The laser scanning direction is parallel to the length direction of the conductive strip; The length of the conductive strip is 9.5 mm and the width is 1.5 mm; the radius of the working electrode is 3 mm, the inner arc radius of the counter electrode is 4.5 mm, and the outer arc radius is 6 mm; the inner arc radius of the reference electrode is 4.5 mm, and the outer arc radius is 6 mm; the parameters of the hot pressing treatment are 150 °C and 120 s; the annealing time is 5 - 8 min; among them, the area where the PI film is used to encapsulate the counter electrode conductive strip is the area extending downward from the top of the conductive strip to a length of 2 mm; In S2 of step (3), the concentration of the MB@ZIF-8 solution is 3 mg mL-1, and the modification dosage is 20 μL.

5. The preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor according to claim 1, wherein, In the step (4), the DNA sequences of split aptamer 1, split aptamer 2, and SS-Apt are as follows: Split aptamer 1: AAGGGATGCCGTTTGGG Split aptamer 2: CCCAAGTTCGGCATAGTG SS-Apt: HS-SH-AAGGGATGCCGTTTGGG-TTTTTTTTTTTT-CCCAAGTTCGGCATAGTG-Fc.

6. The preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor according to claim 1, characterized in that, In the step (4), the pH of the Tris-HCl buffer solution is 7.4, the concentration is 10 mM, and the concentration of the SS-Apt solution is 100 μM; among them, the final concentration of TCEP in solution H is 5%; the standing time is 30 min; the concentration of solution H is 2.5 μM.

7. The preparation method of a functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor according to claim 1, characterized in that, In step (5), the concentration of the AuNPs solution is 4 μM, and the modification dosage is 20 μL; the temperature of the first incubation is 37 °C and the time is 30 min; the concentration of solution H is 2.5 μM, and the modification dosage is 20 μL. The temperature of the second incubation is 4 °C and the time is 12 h.

8. The functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor prepared by the method according to any one of claims 1-7, characterized in that, The bottom layer of the functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor is a PVC plate, the middle layer is an EVA film, and the upper layer is a porous graphene structure.

9. Use of the functionalized laser-induced graphene-based 17β-estradiol electrochemical biosensor according to claim 8 for detecting 17β-estradiol, characterized in that, The steps are as follows: (1) First, prepare E2 standard solutions with different concentrations; then take the electrochemical biosensor SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG, and modify the E2 standard solutions on its surface respectively. After incubation at room temperature, an electrochemical biosensor interface that has completed the recognition detection is obtained; one concentration of the E2 standard solution corresponds to the modification of one electrochemical biosensor, and there is a one-to-one correspondence between the concentration and the electrochemical biosensor; Then, wash the electrochemical biosensor interface that has completed the recognition detection with a phosphate buffer solution to obtain the washed electrochemical biosensor, denoted as E2 / SS-Apt / Au NPs / MB@ZIF-8 / TT-LIG; (2) The response electrochemical signal of the sensor E2 / SS-Apt / AuNPs / MB@ZIF-8 / TT-LIG was tested by cyclic voltammetry; the electrochemical signal generated by Fc was denoted as I Fc , and the electrochemical signal generated by MB was denoted as I MB ; the ratio of the electrochemical signal generated by Fc to the electrochemical signal generated by MB was processed to obtain I Fc / I MB ; its value was positively correlated with the concentration of the corresponding E2 solution, and each concentration of E2 would correspond to an I Fc / I MB value. According to the I Fc / I MB value and the logarithm of the E2 concentration, a standard linear curve was constructed; (3) Detection of E2 in the sample: After processing the sample to obtain the sample solution, follow the operations in step (1), with the difference that the E2 standard solution is replaced by the sample solution; then continue to operate according to steps (2) and (3). After electrochemical detection, obtain I Fc and I MB ; and perform ratio calculation; Substitute the I Fc / I MB value into the standard curve constructed in step (3), and the detection of E2 in the unknown sample can be achieved.

10. The use according to claim 9, wherein, In step (1), the concentration of the E2 standard solution is 10 pg mL -1 -500 ng mL -1 , and the dosage of the modification is 20 μL for all; the time of incubation at room temperature is 50 min; the concentration of the PBS solution is 0.1 M and the pH is 7.4; in step (3), the specific conditions for cyclic voltammetry testing are as follows: the scanning voltage range is -0.5 to 0.5 V, the amplitude is 0.025 V, and the frequency is 15 Hz.

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