Preparation method and application of a 17beta-estradiol photoelectro- electrochemical dual-ratio sensor based on split aptamer

Abstract

This invention belongs to the field of biosensor technology, specifically relating to a method for fabricating and applying a 17β-estradiol photoelectric-electrochemical dual ratio sensor based on a splitting aptamer. The invention designs an assembly of multiple probes at a single sensing interface. The splitting aptamer forms a sandwich structure to synergistically recognize 17β-estradiol and triggers HCR anchoring of CdTe QDs, further adsorbing MB; [Fe(CN)6]. 3‑ / 4‑ The probe outputs a redox current (denoted as I). [Fe(CN)6] 3‑ / 4‑ As a reference signal, MB acts as a dual-function probe to generate I. MB The redox current is denoted as I MB And sensitize and amplify the photocurrent (I) of CdTe QDs PEC ); through I MB / I [Fe(CN)6] 3‑ / 4‑ and I PEC / I [Fe(CN)6] 3‑ / 4‑ Dual ratio signals enable sensitive and accurate analysis of 17β-estradiol in natural water bodies.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensors, and particularly relates to a preparation method of a 17beta-estradiol photoelectric-electrochemical double-ratio sensor based on split aptamer and application thereof. BACKGROUND

[0002] Steroid estrogens are one of the 12 persistent organic pollutants prohibited by the Stockholm Convention on Persistent Organic Pollutants in 2002. As a kind of steroid estrogens, 17beta-estradiol (E2) can also interfere with the normal endocrine function of human beings at low concentrations, leading to endocrine dysfunction, male infertility and other diseases. Therefore, it is of great significance to develop and establish an easy, simple, high-sensitivity and high-precision 17beta-estradiol determination method for environmental quality and human health.

[0003] Aptamer-based biosensors have attracted a lot of attention due to their simplicity, rapidity and low cost. The two fragments of split aptamer can bind to specific sites of E2 in a synergistic manner, and this recognition mode makes the aptamer sensor almost free of false positives or non-specific signals. Nameghi et al. disclosed an electrochemical biosensor based on split aptamer for detecting E2, but the sensitivity was insufficient, and the output of single electrochemical signal made the detection result susceptible to environmental factors, resulting in insufficient accuracy. Therefore, on the basis of split aptamer biosensor, signal amplification and double-mode double-ratio strategy are introduced to solve the current technical problems. SUMMARY

[0004] The application aims to couple electrochemistry and electrochemical sensing technology, and develop a double-ratio aptamer sensor based on split aptamer. Multiple probes, split aptamer I and II (denoted as SPT I and SPT II), are assembled on a single sensing interface, which synergistically recognize 17beta-estradiol and trigger HCR anchoring CdTe quantum dots (QDs) to further adsorb methylene blue (MB). In terms of obtaining multiple signals, the redox current of [Fe(CN)6] 3- / 4- Probe output I [Fe(CN)6] 3- / 4- is used as a reference signal, and MB is used as a double-function probe: (1) generating the redox current of I MB ; (2) sensitizing CdTe QDs to amplify the photocurrent of I PEC as a response signal; through I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- Double-ratio signals, the sensitive and accurate analysis of 17beta-estradiol in natural water bodies is realized.

[0005] Split aptamer is a complete aptamer split into two fragments to recognize the target through the "sandwich" form. This recognition mode retains the original affinity and specificity, and can avoid false positive or non-specific signals. In addition, split aptamer and target recognition can trigger hybridization chain reaction (HCR), thereby improving the sensitivity and selectivity of the sensor. Aptamer sensor can combine various sensing methods, use the synergistic effect of different signal conversion modes and transmission mechanisms, explore the dual-mode sensing technology, and the two signal paths are relatively independent, allowing mutual cross-validation. The ratio strategy can quantitatively analyze the target by normalizing the two response signals, thereby eliminating the interference from environmental and experimental factors and improving the detection accuracy.

[0006] A preparation method of a 17β-estradiol photoelectric-electrochemical dual-ratio sensor based on split aptamer, comprising the following steps:

[0007] (1) Preparation of Au NPs solution:

[0008] Mix HAuCl4 solution and ultrapure water, after boiling, add Na3C6H5O7 solution, the color of the mixed solution gradually changes from light yellow to bright red, continue to react for a period of time, cool to room temperature, and the obtained solution is the Au NPs solution;

[0009] (2) Preparation of CdTe QDs-H1 and CdTe QDs-H2:

[0010] (a) Under magnetic stirring, mix ultrapure water, CdCl2·2.5H2O solution, trisodium citrate, Na2TeO3 solution, MPA solution and NaBH4 to obtain a brown solution, adjust the pH of the solution and continue to stir for a period of time to obtain a precursor solution; add the precursor solution into a reaction kettle for reaction, and obtain a reaction solution after reaction; mix the prepared reaction solution with ethanol, stand, centrifuge, wash, dry, disperse in ultrapure water again to obtain a CdTe QDs solution;

[0011] (b) Prepare a PBS solution containing 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) and N-hydroxysuccinimide (NHS), namely a mixed PBS solution; mix the CdTe QDs solution with the mixed PBS solution at room temperature to obtain an activated CdTe QDs solution;

[0012] The amplified chain of hybrid chain reaction (HCR) is two, recorded as H1 and H2; H1 is added into the activated CdTe QDs solution, then mixed for a period of time at room temperature, and reacted in dark, the product is collected by centrifugation after reaction, and then dispersed in PBS buffer, and the obtained mixture is recorded as CdTe QDs-H1;

[0013] (c) synchronizing the operation of step (b), the difference is that H1 is replaced by H2, and CdTe QDs-H2 is obtained;

[0014] (3) taking DNA single strand S1+SPT I, which is divided into SPT I and S1 two parts, wherein SPT I is split aptamer I; the one end of S1 is connected with thiol group for connecting with Au NPs; the DNA single strand complementary to S1 is recorded as S2;

[0015] After mixing equal volume of S1+SPT I and S2, the mixture is reacted in a metal bath to synthesize double-stranded DNA structure, recorded as dsDNA-1;

[0016] Taking the pretreated ITO electrode after cleaning treatment, the Au NPs solution prepared in step (1) is modified on the surface of the ITO electrode, and dried at room temperature to obtain Au NPs / ITO electrode; then dsDNA-1 is modified on the surface of the Au NPs / ITO electrode, and the sensor dsDNA-1 / Au NPs / ITO is obtained after incubation.

[0017] Preferably, the amount of the HAuCl4 solution, ultrapure water and Na3C6H5O7 solution in step (1) is 200 μL: 25 mL: 0.25 mL, wherein the concentration of the HAuCl4 solution is 0.1 mol·L -1 , and the concentration of the Na3C6H5O7 solution is 0.1 g·mL -1 ; the reaction is continued for 15-20 min.

[0018] Preferably, in step (a), the amount of the ultrapure water, CdCl2·2.5H2O solution, trisodium citrate, Na2TeO3 solution, MPA (3-mercaptopropionic acid) solution and NaBH4 mixed is 42 mL: 4 mL: 0.1 g: 4 mL: 99 μL: 50 mg, wherein the concentration of the CdCl2·2.5H2O solution is 0.04 mol·L -1 , the concentration of the Na2TeO3 solution is 0.01 mol·L -1 , and the concentration of the MPA solution is 0.23 mol·L -1 ;

[0019] The pH of the solution is adjusted by using 1 mol·L -1NaOH solution to 8.5, and stirring for 12 h; the reaction temperature in the reactor is 140℃, and the reaction time is 2 h; the volume ratio of the reaction solution to ethanol is 1:1; the centrifugal condition is 8000 rpm for 10 min; the concentration of the CdTe QDs solution is 30-60 μmol·L -1 .

[0020] Preferably, in step (b), the concentration of the PBS solution is 10 mmol·L -1 , the pH value is 7.4; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) in the mixed PBS solution is 10.4 g·L -1 , the concentration of N-hydroxysuccinimide (NHS) in the mixed PBS solution is 5.2 g·L -1 ; the amount of the CdTe QDs solution mixed with the mixed PBS solution is 100 μL:400 μL; the concentration of the CdTe QDs solution is 10-30 μmol·L -1 , and the reaction time is 30 min.

[0021] Preferably, in step (b), the concentration of H1 and H2 is both 100-150 μmol·L -1 ; the amount of H1 mixed with the activated CdTe QDs solution is 20 μL:500-550 μL, and the mixing time is 5-10 min at room temperature, and the light-free reaction is overnight at 4℃; the centrifugal condition is 11000 rpm for 30 min; the concentration of the CdTe QDs-H1 is 2-8 μmol·L -1 , wherein the concentration of the PBS buffer is 10 mmol·L -1 , and the pH value is 7.4.

[0022] Preferably, in step (3), the concentration of S1+SPT I and S2 is both 2-8 μmol·L -1 , the reaction temperature in the metal bath is 20-50℃, and the reaction time is 0.8-1.5 h.

[0023] The cleaning treatment of the ITO electrode is specifically as follows: sequentially cleaning with NaOH solution, ethanol and ultrapure water, wherein the concentration of the NaOH solution is 1 mol·L -1 ; the volume of the Au NPs solution modification is 15-25 μL, and the volume of the dsDNA-1 modification is 8-20 μL, and the concentration thereof is 1-4 μmol·L -1 ; the incubation temperature is 2-8℃, and the incubation time is 10-16 h.

[0024] The use of the prepared sensor for detecting 17β-estradiol is as follows:

[0025] S1, construction of standard curve:

[0026] (a) take DNA single strand SPT II+S3, which is divided into two parts of SPT II and S3, wherein SPT II is split aptamer II, and S3 is the trigger chain of HCR; after mixing SPT II+S3, CdTe QDs-H1 and CdTe QDs-H2, oscillation reaction is carried out, and dsDNA-2 is obtained after reaction;

[0027] (b) the sensor dsDNA-1 / Au NPs / ITO prepared in the above step (3) is used as a working electrode, a saturated Ag / AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; dsDNA-2 is mixed with 17β-estradiol standard solution to obtain a mixed solution; then the mixed solution is modified on the working surface, and after incubation at room temperature, the electrode is washed with PBS solution, and the washed electrode is recorded as E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO; finally, the MB solution is modified on the electrode surface, and after adsorption reaction at room temperature, it is washed to obtain MB / E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO; then photoelectrochemical test and electrochemical test are carried out to obtain photoelectrochemical signal and electrochemical signal; wherein the photocurrent is recorded as I PEC , the redox current of MB is recorded as I MB , and the redox current of [Fe(CN)6] 3- / 4- is recorded as I [Fe(CN)6] 3- / 4- ;

[0028] Two standard linear curves are established with the concentration of 17β-estradiol corresponding to the double ratio signals I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4-

[0029] S2, detection of actual sample 17β-estradiol:

[0030] First, the sample solution is prepared; according to the operation of step (b) of step S1, the difference is that the 17β-estradiol standard solution is replaced by the sample solution; after photoelectrochemical test and electrochemical test, photoelectrochemical signal and electrochemical signal are obtained, and the double ratio signals I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- are obtained, which are substituted into the standard linear curve of step S1 to realize the detection of the sample 17β-estradiol to be tested. ​

[0031] Preferably, the concentration of SPT II+S3 in (a) of step S1 is 8-15 μmol·L -1 Preferably, the concentration of CdTe QDs-H1 is 2-8 μmol·L -1 Preferably, the concentration of CdTe QDs-H2 is 2-8 μmol·L -1 Preferably, the volume ratio of SPT II+S3, CdTe QDs-H1 and CdTe QDs-H2 is 1:2:2; and the oscillation reaction time is 30-60 min.

[0032] Preferably, the volume ratio of dsDNA-2 to 17β-estradiol standard solution in (b) of step S1 is 1-5:1, and the concentration of 17β-estradiol standard solution is 0.1 pg·mL -1 -10000 pg·mL -1 Preferably, the volume of the mixed solution modification is 15-25 μL, the room temperature incubation time is 1.8-2.5 h, the volume of the MB solution modification is 15-25 μL, the concentration of the MB solution is 30-60 μmol·L -1 , and the adsorption reaction time is 2-10 min; the concentration of the PBS buffer is 10 mmol·L -1 , and the pH value is 7.4; the volume of the MB solution modification is 15-25 μL, the concentration of the MB solution is 30-60 μmol·L -1 , and the adsorption reaction time is 2-10 min.

[0033] Preferably, the photoelectrochemical test and the electrochemical test in steps S1 and S2 are measured and recorded by a type MC-TCX300 xenon lamp light source system and an Autolab PGSTAT 302N electrochemical workstation, respectively; wherein, the photoelectrochemical test is carried out in a 0.1 mol·L -1 PBS (pH=7.4) buffer solution, and an external bias voltage is 0 V; the electrochemical test is carried out in a mixed solution of 0.6 mL of 5 mmol·L -1 [Fe(CN)6] 3- / 4- , 5.4 mL of 0.1 mol·L -1 PBS (pH 7.4) buffer solution, and the potential range is-0.4 V-0.6 V.

[0034] Advantages of the present application:

[0035] (1) The 17beta-estradiol photoelectric-electrochemical dual-ratio sensor based on split aptamer is provided, a dual-ratio aptamer sensor coupled with photoelectrochemistry and electrochemistry is constructed by assembling multiple probes on a single sensing interface. The high specificity of the split aptamer is utilized to cooperatively recognize 17beta-estradiol and trigger HCR, thereby effectively improving the sensitivity of 17beta-estradiol detection; and a dual-ratio aptamer sensor is constructed by coupling photoelectrochemistry (PEC) and electrochemistry (EC) biosensing platforms through a dual-mode dual-ratio strategy, which not only retains the advantage of small background signal in PEC detection, but also eliminates the influence of the same factors on PEC and EC detection through the ratio strategy, thereby improving the accuracy of 17beta-estradiol detection.

[0036] (2) MB is selected as a bifunctional probe, which serves as a redox probe to generate an electrochemical signal and as a sensitizer to amplify the photoelectric signal of CdTe QDs, thereby realizing photoelectric-electrochemical synergy.

[0037] (3) The photoelectric-electrochemical dual-ratio sensor introduced in the application uses split aptamer as a specific recognition element, which can improve the selectivity of the 17beta-estradiol sensor, reduce the interference of other estrogens, trigger hybridization chain reaction for signal amplification, and realize sensitive and accurate analysis of 17beta-estradiol in natural water.

[0038] (4) The application uses the redox current signal of [Fe(CN)6] 3- / 4- as a reference, and uses the photoelectric signal of CdTe QDs and the electrical signal of MB as a dual ratio, and the 17beta-estradiol concentration is quantitatively detected by recording the dual ratio signal reaction. Multiple signals are obtained on the same electrode, which are used as references, thereby effectively reducing the interference of solution matrix and environmental factors on the electrode.

[0039] (5) The dual-ratio aptamer sensor constructed in the application has high sensitivity, good selectivity, high accuracy, and wide linear range, and the linear ranges are 0.1-5000 pg·mL -1 and 0.1-10000 pg·mL -1 , respectively. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a schematic diagram of the 17beta-estradiol photoelectric-electrochemical dual-ratio sensor based on split aptamer.

[0041] Figure 2 (A) is the photoelectric response of the sensor to 0.1 ng·mL -1 17beta-estradiol with and without MB, wherein

[0042] a is E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO;

[0043] b is MB / E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO;

[0044] Figure 2 (B) are Nyquist impedance plots for different electrodes, the inset is the equivalent circuit of Nyquist plot, where

[0045] a is ITO;

[0046] b is Au NPs / ITO;

[0047] c is dsDNA-1 / Au NPs / ITO;

[0048] d is E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO;

[0049] e is MB / E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO;

[0050] Figure 2 (C) are electrochemical responses with increasing concentration of 17β-estradiol, where

[0051] a is with 0 ng·mL -1 of 17β-estradiol added;

[0052] b is with 0.1 ng·mL -1 of 17β-estradiol added;

[0053] c is with 1 ng·mL -1 of 17β-estradiol added;

[0054] Figure 2 (D) are photoelectrochemical responses with increasing concentration of 17β-estradiol, where

[0055] a is with 0 ng·mL -1 of 17β-estradiol added;

[0056] b is with 0.1 ng·mL -1 of 17β-estradiol added;

[0057] c is with 1 ng·mL -1 of 17β-estradiol added;

[0058] Figure 3 (A) are electrochemical responses at different concentrations of 17β-estradiol (0.1 pg·mL -1 , 0.5 pg·mL -1 , 1 pg·mL-1 , 5 pg / mL -1 , 10 pg / mL -1 , 50 pg / mL -1 , 100 pg / mL -1 , 500 pg / mL -1 , 1000 pg / mL -1 , 5000 pg / mL -1 ); (B) photoelectrochemical response of (A) at different concentrations of 17β-estradiol (0.1 pg / mL -1 , 0.5 pg / mL -1 , 1 pg / mL -1 , 5 pg / mL -1 , 10 pg / mL -1 , 50 pg / mL -1 , 100 pg / mL -1 , 500 pg / mL -1 , 1000 pg / mL -1 , 5000 pg / mL -1 , 10000 pg / mL -1 ); (C) linear relationship plot of log of 17β-estradiol concentration vs. I MB / I [Fe(CN)6] 3- / 4- PEC / I [Fe(CN)6] 3- / 4- .

[0059] Figure 4 (A) and (B) are sensor selectivity tests; (C) and (D) are sensor stability tests. DETAILED DESCRIPTION

[0060] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. The detailed description set forth below in connection with the appended drawings is intended as a description of various embodiments of the present application and is not intended to represent the only embodiments in which the present application can be practiced. The detailed description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In some instances, well-known structures and functions have not been described in detail in order to avoid obscuring the concept of the present application.

[0061] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application.

[0062] ​Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict between the present specification and any document incorporated by reference, the present specification controls.

[0063] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the spirit or scope of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0064] The DNA sequences used in the present application are as follows:

[0065] S1+SPT I: 5'-SH-(CH2)6-AAA AAG GGG GTG AAG GGA TAC CCG ACC CTA AGC ATG CTT CCA GCT TAT TGA ATT ACA CGC AGA GGG TA-3'

[0066] S2: 5'-ATG CTT AGG GTC GGG TAT CCC TTC ACC CCC TTT TT-3'

[0067] SPT II+S3: 5'-GCG GCT CTG CGC ATT CAA TTG CTG CGC GCT GAA GCG CGG AAG CAC GGC GCC C-3'

[0068] H1: 5'-CTT AGG CGG GGG CGC CGT TTT-(CH2)6-NH2-3'

[0069] H2: 5'-NH2-(CH2)6-TTT CCG CCT AAG ACG GCG CCC-3'

[0070] The sequences of the split aptamer are shown in bold; S1 is connected to the electrode through an Au-S bond; S2 is complementary to S1; S3 is the trigger strand of HCR; H1 and H2 are the amplification strands of HCR.

[0071] The reagents S1+SPT I, SPT II+S3, S2, S3, H1 and H2 used in the present application are all purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0072] This application does not involve sequence listing applications. The above sequences are for illustrative purposes only and are all conventional reagent sequences, belonging to primer sequences.

[0073] Example 1:

[0074] (1) Preparation of Au NPs solution:

[0075] Add 200 μL of 0.1 mol·L⁻¹ solution to a three-hole flask. -1 Boil HAuCl4 solution and 25 mL of ultrapure water, then quickly add 0.25 mL of 0.1 g·mL⁻¹ solution. -1 The Na3C6H5O7 solution reacts, and the color of the solution gradually changes from light yellow to bright red. The above reaction solution continues to react for 15 minutes. After cooling to room temperature, the resulting solution is the Au NPs solution. The solution is stored at 4°C in the dark for later use.

[0076] (2) Preparation of CdTe QDs-H1 and CdTe QDs-H2 composite solutions:

[0077] (a) Preparation of CdTe QDs solution using microwave synthesis method.

[0078] Under magnetic stirring, 42 mL of ultrapure water and 4 mL of 0.04 mol·L⁻¹ were added sequentially to a 100 mL beaker. -1 CdCl2 solution, 0.1 g trisodium citrate solid, 4 mL 0.01 mol·L⁻¹ -1 Na₂TeO₃ solution, 99 μL 0.23 mol·L⁻¹ -1 MPA (3-mercaptopropionic acid) solution and 50 mg NaBH4 solid were reacted to produce a brown solution; then, 1 mol·L⁻¹ was used to react the solution with the solution of 3-mercaptopropionic acid (MPA) and 50 mg NaBH₄ solid to produce a brown solution. -1 The pH of the solution was slowly adjusted to 8.5 with NaOH solution and stirred continuously for 12 hours. The solution turned light brown and was recorded as the precursor solution. 15 mL of the precursor solution was added to a 30 mL reaction vessel and heated at 140 °C for 2 hours to obtain the reaction solution. The reaction solution was mixed with ethanol at a ratio of 1:1, allowed to stand for 10 minutes, centrifuged at 8000 rpm for 10 minutes, washed and dried, and then redispersed in ultrapure water to obtain the CdTe QDs solution.

[0079] (b) The amplification chains of the hybridization chain reaction (HCR) are denoted as H1 and H2;

[0080] 100 μL 26 μmol·L -1 CdTe QDs solution and 400 μL containing 10.4 g·L -1 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5.2 g·L -110 mmol·L⁻¹ N-hydroxysuccinimide (NHS) -1 PBS solution (pH 7.4) was mixed and reacted at room temperature for 30 min to obtain CdTe QDs with activated carboxyl groups; then 20 μL of 150 μmol·L⁻¹ PBS solution was added to the above solution. -1 H1 was mixed at room temperature for 5 min, then incubated overnight at 4°C in the dark; the product was then collected by centrifugation at 11000 rpm for 30 min, and then dispersed in 520 μL of 10 mmol·L⁻¹ solution. -1 The resulting solution, labeled CdTe QDs-H1, was prepared in PBS (pH 7.4) and stored at 4°C for later use.

[0081] (c) The same operation as step (b), except that H1 is replaced with H2 to obtain CdTe QDs-H2;

[0082] (3) Take a single strand of DNA S1+SPT I, which is divided into two parts, SPT I and S1. SPT I is the apposition aptamer I. One end of S1 is connected to a thiol group for connection with Au NPs. The single strand of DNA complementary to the bases of S1 is denoted as S2.

[0083] Equal volumes of 4 μmol·L -1 S1+SPT I and S2 were reacted in a metal bath at 37°C for 1 h to synthesize a double-stranded DNA structure. The solution after the reaction was denoted as dsDNA-1.

[0084] With 1 mol·L -1 The ITO electrode was sequentially cleaned with NaOH solution, ethanol, and ultrapure water to obtain a pretreated ITO electrode. 20 μL of the Au NPs solution prepared in step (1) was then applied to the surface of the ITO electrode. After the surface dried, 15 μL of 2 μmol·L⁻¹ NaOH solution was applied to the surface of the electrode. -1 dsDNA-1 was modified on the surface of Au NPs / ITO electrode and incubated at 4℃ for 12 h, denoted as dsDNA-1 / Au NPs / ITO.

[0085] Example 2:

[0086] Performance analysis of a 17β-estradiol photoelectric-electrochemical dual ratio sensor based on splitting aptamers:

[0087] (1) DNA single strand SPT II+S3 is divided into two parts, SPT II and S3, where SPT II is the apposition aptamer II and S3 is the trigger strand of HCR; after mixing SPT II+S3, CdTe QDs-H1 and CdTe QDs-H2, a shaking reaction is performed to obtain dsDNA-2.

[0088] 10 μmol·L-1 SPT II + S3, 5 μmol·L -1 CdTe QDs-H1 and 5 μmol·L -1 CdTe QDs-H2 were mixed in a volume ratio of 1:2:2, and HCR was carried out at 37°C with oscillation. After 30 min of oscillation reaction, dsDNA-2 was obtained;

[0089] (2) The sensor dsDNA-1 / Au NPs / ITO prepared in Example 1 was used as the working electrode, a saturated Ag / AgCl electrode was used as the reference electrode, and a platinum wire electrode was used as the counter electrode;

[0090] The dsDNA-2 was mixed with different concentrations of 17β-estradiol in a volume ratio of 3:1 (one concentration corresponds to one dsDNA-2, and they are in a one-to-one correspondence). Then, 20 μL of the mixed solution was modified on the surface of the electrode (dsDNA-1 / Au NPs / ITO), and after incubation at room temperature for 2 h, the electrode was washed with PBS solution (10 mmol·L -1 , pH 7.4), and was recorded as E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO. Finally, 20 μL of 50 μmol·L -1 MB solution was modified on the surface of the electrode, and after adsorption at room temperature for 5 min, the electrode was washed to obtain the sensor MB / E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO, which was subjected to photoelectrochemical and electrochemical tests;

[0091] The electrochemical and photoelectrochemical signals were measured and recorded by a model MC-TCX300 xenon lamp light source system and an Autolab PGSTAT 302N electrochemical workstation, respectively. The photoelectrochemical test was carried out in a 0.1 mol·L -1 PBS (pH=7.4) buffer solution, and the external bias voltage was 0 V. The electrochemical test was carried out in a mixture of 0.6 mL of 5 mmol·L -1 [Fe(CN)6] 3- / 4- and 5.4 mL of 0.1 mol·L -1 PBS (pH 7.4), and the potential range was -0.4 V to 0.6 V. The double-ratio signals I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- were measured, and the 17β-estradiol concentration corresponding to the double-ratio signal I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4-standard linear curve; wherein, I PEC is the photocurrent, I MB is the redox current of MB, I [Fe(CN)6] 3- / 4- is the redox current of [Fe(CN)6] 3- / 4- .

[0092] With the increase of the concentration of 17β-estradiol, I PEC and I MB gradually increase, while the concentration of [Fe(CN)6] 3- / 4- is constant, I [Fe(CN)6] 3- / 4- the signal remains unchanged, and linear relationships of I -1 / I -1 , I MB / I [Fe(CN)6] and I 3- / 4- / I PEC are obtained in the range of 0.1-5000 pg·mL [Fe(CN)6] and 0.1-10000 pg·mL 3- / 4- , respectively, and the linear regression equations are I MB / I [Fe(CN)6] 3- / 4- = 0.126 lgC E2 + 0.218 (R 2 = 0.998) and I PEC / I [Fe(CN)6] 3- / 4- = 0.036 lgC E2 + 0.147 (R 2 = 0.997) respectively. Figure 3 It is proved that the constructed sensor has a wide linear range and high sensitivity, and can realize accurate analysis of 17β-estradiol.

[0093] Figure 4 (A) and (B) are the selected performance test figures of the sensor: wherein blank refers to an aqueous solution, defined as a blank sample; the concentrations of estrone (E1), estriol (E3), bisphenol A (BPA) and diethylstilbestrol (DES) are 1 pg·mL -1 , which are interferents, and the signal response of 0.1 pg·mL -1 of 17β-estradiol (E2) is compared. It can be seen from the figure that the sensor will only produce obvious signal changes when the target 17β-estradiol is present, and the presence of other interfering ions has no obvious effect on the response signal, indicating that the sensor has good selectivity.

[0094] The prepared electrochemical biosensor was used to investigate the long-term stability of the dual-signal electrochemical biosensor. The electrode was stored in a refrigerator at 4°C after preparation, and then the prepared sensor was used to detect 0.1 pg·mL -1 of 17β-estradiol solution on the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th day, respectively; the signal changes of I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- were investigated.

[0095] Figure 4 The long-term stability of the sensor was (C) and (D). As can be seen from the figure, the relative standard deviations (RSD) of I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- were 2.5% and 2.7%, respectively, indicating that the prepared sensor had good long-term stability.

[0096] Example 3

[0097] Split aptamer-based 17β-estradiol photoelectrochemical-electrochemical dual-ratio sensor for detecting 17β-estradiol in natural water:

[0098] Based on this linear relationship, the inventors collected lake water samples from Jinshan Lake in Zhenjiang City, Jiangsu Province, filtered the samples with a 0.22 μm filter to remove solid impurities, and obtained sample liquids. Three filtered sample liquids were taken, and 17β-estradiol was added to each sample liquid to obtain three samples with different concentrations, with the final concentrations being 500, 1000, and 5000 pg·mL -1

[0099] The sensor dsDNA-1 / Au NPs / ITO prepared in Example 1 was used as the working electrode, a saturated Ag / AgCl electrode was used as the reference electrode, and a platinum wire electrode was used as the counter electrode. dsDNA-2 was mixed with the sample at a volume ratio of 3:1 to obtain a mixed solution, and then 20 μL of the mixed solution was modified on the surface of the working electrode. After incubation at room temperature for 2 h, the electrode was washed with PBS solution (10 mmol·L -1 , pH 7.4), and was recorded as E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO. Finally, 20 μL of 50 μmol·L -1 MB solution was modified on the surface of the electrode, and after adsorption at room temperature for 5 min, the electrode was washed for photoelectrochemical and electrochemical tests, and I MB / I [Fe(CN)6] was measured.3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- The signal change of 17β-estradiol is substituted into the constructed linear regression equation, so that the concentration of 17β-estradiol in the sample can be obtained, and the detection recovery rate is obtained, as shown in Table 1.

[0100] Table 1 Determination of 17β-estradiol in Jinshan Lake (n = 3)

[0101]

[0102] Note: "-" means not detected.

[0103] As can be seen from Table 1, I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- The recovery rates of E2 are 99.2% to 102.1% and 99.6% to 102.3%, respectively. In this embodiment, for the actual sample detection process, the constructed sensor can sensitively and accurately quantitatively detect 17β-estradiol in the sample to be detected, the overlapping part of the linear range can realize the verification of the double ratio, further improve the accuracy of the sensor detection, and the detection process does not need professional training and is simple to operate.

[0104] It is to be understood that the above description is only used to illustrate but not limit the technical solutions described in the present application; therefore, although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present application can still be modified or replaced equivalently; and all technical solutions and improvements without departing from the spirit and scope of the present application should be covered in the scope of the claims of the present application.

Claims

1. A method for the preparation of a split aptamer-based 17β-estradiol photoelectro- electrochemical dual-ratio sensor, characterized in that, The steps include: (1) Preparation of Au NPs solution: Mix HAuCl4 solution and ultrapure water, after boiling, add Na3C6H5O7 solution, the color of the mixed solution gradually changes from light yellow to bright red, continue to react for a period of time, and then cool to room temperature. The obtained solution is the Au NPs solution; (2) Preparation of CdTe QDs-H1 and CdTe QDs-H2: (a) Under magnetic stirring, mix ultrapure water, CdCl2·2.5H2O solution, trisodium citrate, Na2TeO3 solution, MPA solution and NaBH4 to obtain a brown solution. After adjusting the pH of the solution, continue to stir for a period of time to obtain a precursor solution; Put the precursor solution into a reaction kettle for reaction, and obtain a reaction solution after reaction. Mix the prepared reaction solution with ethanol, stand, centrifuge, wash, dry, and then disperse in ultrapure water to obtain a CdTe QDs solution; (b) Prepare a PBS solution containing 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxy succinimide, namely a mixed PBS solution. Mix the CdTe QDs solution with the mixed PBS solution at room temperature to obtain an activated CdTe QDs solution; The amplified chain of the hybridization chain reaction is 2, denoted as H1 and H2. Add H1 to the activated CdTe QDs solution, then mix at room temperature for a period of time, avoid light reaction, centrifuge to collect the product after reaction, and then disperse it in PBS buffer to obtain a mixed solution denoted as CdTe QDs-H1; (c) Perform the operation of step (b), with the only difference being that H1 is replaced by H2 to obtain CdTe QDs-H2; (3) Take DNA single strand S1+SPT I, which is divided into SPT I and S1 two parts, wherein SPT I is a split aptamer I; one end of S1 is connected with a thiol group for connection with Au NPs; and a DNA single strand complementary to S1 is denoted as S2; Mix equal volumes of S1+SPT I and S2, and then react in a metal bath to synthesize a double-stranded DNA structure denoted as dsDNA-1; Take an ITO electrode after cleaning treatment to obtain a pretreated ITO electrode, then modify the Au NPs solution prepared in step (1) on the surface of the ITO electrode, dry at room temperature, and obtain an Au NPs / ITO electrode; then modify dsDNA-1 on the surface of the Au NPs / ITO electrode, and obtain a sensor dsDNA-1 / Au NPs / ITO after incubation; (4) Take the DNA single strand SPT II+S3, which is divided into two parts of SPT II and S3, wherein SPT II is split aptamer II, and S3 is a trigger strand of HCR; mix SPT II+S3, CdTe QDs-H1 and CdTe QDs-H2, and then perform oscillation reaction, to obtain dsDNA-2 after reaction; mix dsDNA-2 with 17β-estradiol standard solution to obtain a mixed solution; then modify the mixed solution on the surface of dsDNA-1 / Au NPs / ITO prepared in step (3), incubate at room temperature, wash with PBS solution, and the electrode after washing is recorded as E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO; finally, modify MB solution on the surface of the electrode, adsorb at room temperature, and then wash to obtain MB / E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO.

2. The method for the preparation of a split aptamer-based 17p-estradiol photoelectro- electrochemical dual-ratiometric sensor according to claim 1, characterized in that, The amount of the HAuCl4 solution, ultrapure water and Na3C6H5O7 solution in step (1) is 200 μL: 25 mL: 0.25 mL, wherein the concentration of the HAuCl4 solution is 0.1 mol·L -1 , and the concentration of the Na3C6H5O7 solution is 0.1 g·mL -1 ; the reaction time is 15-20 min. The amount of the HAuCl4 solution, ultrapure water and Na3C6H5O7 solution in step (1) is 200 μL: 25 mL: 0.25 mL, wherein the concentration of the HAuCl4 solution is 0.1 mol·L -1 , and the concentration of the Na3C6H5O7 solution is 0.1 g·mL -1 ; the reaction time is 15-20 min.

3. The method for the preparation of split aptamer-based 17p-estradiol photoelectro- electrochemical dual-ratiometric sensor according to claim 1, characterized in that, In step (a), the amount of the ultrapure water, CdCl2·2.5H2O solution, trisodium citrate, Na2TeO3 solution, MPA solution and NaBH4 mixed is 42 mL:4 mL:0.1 g:4 mL:99 μL:50 mg, wherein the concentration of the CdCl2·2.5H2O solution is 0.04 mol·L -1 , the concentration of the Na2TeO3 solution is 0.01 mol·L -1 , and the concentration of the MPA solution is 0.23 mol·L -1 . The pH of the solution was adjusted slowly to 8.5 using a 1 mol L -1 NaOH solution, with stirring for a period of 12 h; the temperature of the reaction in the reactor was 140 °C, for a period of 2 h; the volume ratio of the reaction solution to ethanol was 1:1; the conditions for centrifugation were 8000 rpm for 10 min; the concentration of the CdTe QDs solution was 30-60 μmol L -1 .

4. The method for the preparation of split aptamer-based 17p-estradiol photoelectro- electrochemical dual-ratiometric sensor according to claim 1, characterized in that, The concentration of the PBS solution in step (b) is 10 mmol·L -1 , the pH value is 7.4; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the mixed PBS solution is 10.4 g·L -1 , the concentration of N-hydroxysuccinimide in the mixed PBS solution is 5.2 g·L -1 ; the amount of the CdTe QDs solution mixed with the mixed PBS solution is 100 µL:400 µL; the concentration of the CdTe QDs solution is 10~30 µmol·L -1 , and the reaction time is 30 min.

5. The method for the preparation of split aptamer-based 17p-estradiol photoelectro- electrochemical dual-ratiometric sensor according to claim 1, characterized in that, In step (b), the concentration of H1 and H2 is 100-150 µmol·L -1 ; the amount of H1 used in the activated CdTe QDs solution is 20 µL:500-550 µL, the mixing time at room temperature is 5-10 min, the dark reaction is at 4 ℃ overnight, the centrifugal condition is 11000 rpm for 30 min, and the concentration of CdTe QDs-H1 is 2-8 µmol·L -1 , wherein the concentration of PBS buffer is 10 mmol·L -1 , and the pH value is 7.

4.

6. The method for the preparation of a split aptamer-based 17p-estradiol photoelectro- electrochemical dual-ratiometric sensor according to claim 1, characterized in that, The concentration of S1+SPT I and S2 in step (3) is 2-8 µmol·L -1 The reaction temperature in the metal bath is 20-50 ℃, and the reaction time is 0.8-1.5 h. The ITO electrode is cleaned by using NaOH solution, ethanol and ultrapure water in sequence, wherein the concentration of the NaOH solution is 1 mol / L -1 ; the volume of the Au NPs solution is 15-25 μL, and the volume of the dsDNA-1 solution is 8-20 μL, and the concentration of the dsDNA-1 solution is 1-4 µmol / L -1 ; the incubation temperature is 2-8 ℃, and the incubation time is 10-16 h.

7. The method of preparation of split aptamer-based 17p-estradiol photoelectro- electrochemical dual-ratiometric sensor as claimed in claim 1, wherein, The concentration of SPT II+S3 in step (4) is 8-15 µmol·L -1 The concentration of CdTe QDs-H1 is 2-8 µmol·L -1 The concentration of CdTe QDs-H2 is 2-8 µmol·L -1 The volume ratio of SPT II+S3, CdTe QDs-H1 and CdTe QDs-H2 is 1:2:2; and the oscillation reaction time is 30-60 min.

8. The method of preparation of a split aptamer-based 17p-estradiol photoelectro- electrochemical dual-ratiometric sensor according to claim 1, characterized in that, The volume ratio of dsDNA-2 to 17β-estradiol standard solution in step (4) is 1-5:1, and the concentration of 17β-estradiol standard solution is 0.1 pg·mL -1 ~10000 pg·mL -1 , the volume of the mixed solution modification is 15-25 μL, the incubation time at room temperature is 1.8-2.5 h, the volume of the MB solution modification is 15-25 μL, and the concentration is 30-60 µmol·L -1 , the adsorption reaction time is 2-10 min; the concentration of PBS buffer is 10 mmol·L -1 , and the pH value is 7.

4.

9. Use of a split aptamer-based 17β-estradiol photoelectro- electrochemical dual-ratiometric sensor prepared according to the method of any one of claims 1-8 for the detection of 17β-estradiol, characterized in that, The steps are as follows: S1, construction of a standard curve: Based on the MB / E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO, photoelectrochemical test and electrochemical test are carried out to obtain photoelectrochemical signal and electrochemical signal; wherein the photocurrent is recorded as I PEC , the redox current of MB is recorded as I MB , the redox current of [Fe(CN)6] 3- / 4- is recorded as I [Fe(CN)6] 3- / 4- ; the concentration of 17β-estradiol is corresponded to the double ratio signal I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- Two standard linear curves are established; S2, detection of actual sample 17β-estradiol: First, prepare the sample liquid; according to the steps of step (4), the only difference is that the 17β-estradiol standard solution is replaced by the sample liquid, that is, the mixed solution is obtained by mixing dsDNA-2 with the sample liquid; then modify the mixed solution on the surface of dsDNA-1 / Au NPs / ITO prepared in step (3), incubate at room temperature, wash with PBS solution, and the electrode after washing is recorded as E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO; finally, modify MB solution on the surface of the electrode, adsorb at room temperature, and then wash to obtain the sample liquid treated MB / E2+dsDNA-2 / dsDNA-1 / Au NPs / ITO; The photoelectrochemical test and electrochemical test are carried out to obtain photoelectrochemical signals and electrochemical signals, and double-ratio signals I MB / I [Fe(CN)6] 3- / 4- and I PEC / I [Fe(CN)6] 3- / 4- The standard linear curve of step S1 is substituted to realize detection of the 17β-estradiol of the sample to be detected.

10. The use of claim 9, wherein the volume ratio of dsDNA-2 to sample solution in step S2 is 1-5:1, the volume of the mixed solution modification is 15-25 μL, the room temperature incubation time is 1.8-2.5 h, the volume of the MB solution modification is 15-25 μL, the concentration is 30-60 μmol·L -1 , and the adsorption reaction time is 2-10 min; the concentration of the PBS buffer is 10 mmol·L -1 , and the pH value is 7.

4. The photoelectrochemical test and the electrochemical test in steps S1 and S2 are measured and recorded by a model MC-TCX300 xenon lamp light source system and an Autolab PGSTAT 302N electrochemical workstation, respectively; wherein, Photoelectrochemical tests were carried out in 0.1 mol·L -1 PBS buffer solution with pH = 7.4, and the applied bias voltage was 0 V; electrochemical tests were carried out in a mixed solution of 0.6 mL of 5 mmol·L -1 [Fe(CN)6] 3- / 4- , 5.4 mL of 0.1 mol·L -1 PBS buffer solution with pH = 7.4, and the potential range was-0.4 V~0.6 V.

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