Electrochemical detection kit for highly sensitive detection of ERα based on the competition between ERα and GO for aptamers and its application

Abstract

The present invention discloses an electrochemical detection kit for highly sensitive detection of ERα based on the competitive interaction between ERα and GO for aptamers, and its application. The kit comprises the following raw materials: a gold electrode, Apt, MCH, GO-Br, Me6TREN, FMMA, CuBr2, AA, and LiClO4. In this study, aptamers with thiol groups attached to their ends first self-assemble onto the gold electrode surface via gold-sulfur bonds. Next, MCH is used to block excess binding sites. GO-Br then adsorbs the aptamers via π-π stacking, attaching them to the electrode surface. The BIBB on the GO-Br surface acts as an initiator for the ATRP reaction, initiating polymerization and introducing a large number of electrical signal molecules onto the GO surface, generating electrical signals. Finally, ERα competes with GO-Br for the aptamer. Due to the stronger binding ability of ERα with the aptamer, GO falls off the electrode surface, along with the electrical signal molecules, resulting in quenching of the electrical signal. The kit offers advantages such as good selectivity, strong stability, high sensitivity, and environmental friendliness.

Description

Technical Field

[0001] The present invention relates to an electrochemical detection kit for highly sensitively detecting ERα based on the competitive effect of ERα and GO on an aptamer and an application thereof, belonging to the technical field of bioanalysis. Background Art

[0002] Breast cancer is the most common malignant tumor in women and the leading cause of cancer-related death in women. Breast cancer is an estrogen-dependent tumor, and the level of estrogen receptors is closely related to its occurrence and development. Estrogen receptors are ligand-dependent transcription factors that stimulate the overexpression of cell growth-related genes by binding to estrogen, causing the growth and proliferation of tumor cells. Studies have shown that the 5-year survival rate of patients with early breast cancer can exceed 90% after treatment, which means that early diagnosis can effectively improve the patient's survival rate. Estrogen receptor α (ERα) is positive in more than 75% of breast cancer cases and plays an important role in the early diagnosis of breast cancer. Therefore, achieving rapid and accurate detection of ERα is of great significance to improving the survival rate of breast cancer patients.

[0003] Current methods for detecting ERα primarily include immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and western blotting. Most of these methods require expensive equipment and specialized personnel, and their application is limited by their lack of sensitivity for detecting trace levels of ERα in the serum of patients with early-stage breast cancer. Therefore, there is an urgent need for a simple, convenient, economical, sensitive, and efficient method for detecting trace levels of ERα.

[0004] Electrochemical biosensors can identify specific substances and convert their concentrations into measurable electrical signals, thereby enabling the detection of target objects. With the development of fields such as molecular biology and nanomaterials, electrochemical biosensors have been widely used to detect various biomolecules. In addition, the introduction of polymerization reactions and nanomaterials into traditional electrochemical sensing systems can increase the loading capacity of electrochemical signal units and achieve highly sensitive detection of targets. The purpose of the present invention is to provide an electrochemical detection kit for highly sensitive detection of ERα based on the competitive effect of ERα and GO on aptamers, combined with ARGET ATRP technology. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an electrochemical detection kit for highly sensitive detection of ERα based on the competition between ERα and GO on aptamers and its application. The kit has the advantages of good selectivity, strong stability, high sensitivity, and green environmental protection.

[0006] In order to achieve the above object, one of the technical solutions of the present invention is:

[0007] An electrochemical detection kit for highly sensitive detection of ERα based on the competitive effect of ERα and GO on the aptamer includes the following raw materials: gold electrode, Apt, MCH, GO-Br, Me6TREN, FMMA, CuBr2, AA, LiClO4, ultrapure water, and anhydrous ethanol.

[0008] Furthermore, the preparation method of GO-Br is:

[0009] (1) 220 μL of DETA was ultrasonically dispersed in 1780 μL of 95% (v / v) ethanol solution. 20 mg of GO was weighed and added to the solution under ultrasonication. Ultrasonication was continued to completely disperse the GO. The reaction was stirred at room temperature.

[0010] (2) After the reaction is completed, the supernatant in step (1) is removed by centrifugation, and the precipitate is washed and dried at room temperature to obtain the product GO-NH2;

[0011] (3) Ultrasonic dispersion of the product GO-NH2 from step (2) in DMSO, adding 300 μL BIBB, stirring at room temperature for reaction, and after the reaction is completed, centrifuging and removing the supernatant to obtain the product;

[0012] (4) The product of step (3) is washed and dried at room temperature to obtain the product GO-Br.

[0013] Furthermore, some raw materials need to be prepared into solutions when used: the concentration of Apt solution is 1 μM, the concentration of MCH solution is 2 mM, the concentration of GO-Br solution is 0.1 mM to 0.6 mM, the concentration of AA solution is 2 mM, the concentration of FMMA solution is 10 mM, and the concentration of LiClO4 solution is 1 M.

[0014] Furthermore, the sequence of Apt is: 5'-SH-(CH2)6-CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCATTG-3'.

[0015] One of the technical solutions of the present invention is: a method for detecting ERα using the kit, comprising the following steps:

[0016] (1) Add Apt solution dropwise to the surface of the gold electrode to react;

[0017] (2) Immersing the electrode from step (1) in an MCH solution for reaction;

[0018] (3) adding the GO-Br solution dropwise onto the electrode in step (2) to react;

[0019] (4) Immersing the electrode from step (3) in an ATRP reaction solution for reaction;

[0020] (5) placing the electrode of step (4) in a LiClO4 solution and measuring the electrochemical signal by square wave voltammetry;

[0021] (6) adding the sample to be tested to the electrode in step (5) for reaction;

[0022] (7) The electrode of step (6) is placed in a LiClO4 solution, and the electrochemical signal is measured by square wave voltammetry.

[0023] Furthermore, the ATRP reaction solution was prepared by mixing 140 μL ultrapure water, 20 μL FMMA solution, 20 μL AA solution and 20 μL CuBr2 / ME6TREN solution before use.

[0024] Furthermore, the preparation method of the CuBr2 / Me6TREN solution is as follows: before use, CuBr2 and Me6TREN are dissolved in DMSO to prepare a CuBr2 / Me6TREN solution with a CuBr2 and Me6TREN concentration of 10 mM and 12 mM, respectively.

[0025] Furthermore, in step (1), the reaction temperature is 37° C., and the reaction time is 1 h; in step (2), the reaction temperature is 37° C., and the reaction time is 30 min; in step (3), the reaction temperature is 37° C., and the reaction time is 30 min to 180 min; in step (4), the reaction temperature is 37° C., and the reaction time is 20 min to 120 min; the scanning range of the square wave voltammetry in steps (5) and (7) is: 0.2 V to 0.6 V, the rising potential is 4.0 mV, the pulse amplitude is 25 mV, the frequency is 15 Hz, and the rest time is 10 s; in step (6), the reaction temperature is 37° C., and the reaction time is 1 h.

[0026] One of the technical solutions of the present invention is: an application of the kit in preparing a reagent for detecting ERα.

[0027] The schematic diagram of the detection method of the present invention is as follows Figure 1 shown.

[0028] This study constructs a highly sensitive kit for detecting ERα based on signal quenching through competition between ERα and GO for aptamers. In this study, aptamers with thiol groups attached to their termini self-assemble onto the surface of a gold electrode via gold-sulfur bonds. MCH is then used to block excess binding sites. GO-Br is then attached to the electrode surface via π-π stacking, adsorbing the aptamers. The BIBB on the GO-Br surface acts as an initiator for ATRP, initiating polymerization and introducing a large number of electrical signal molecules onto the GO surface, generating an electrical signal. Finally, ERα competes with GO-Br for the aptamer. Due to the stronger binding affinity between ERα and the aptamer, GO is detached from the electrode surface, along with the electrical signal molecules, resulting in signal quenching.

[0029] Beneficial effects of the present invention:

[0030] 1. The present invention is based on the ATRP polymerization signal amplification strategy, which improves the detection sensitivity of the kit.

[0031] 2. This invention utilizes the competitive interaction between GO and ERα for ERα aptamers to construct a quenching kit, effectively reducing the impact of nonspecific adsorption on the results. GO-Br acts as an initiator for the ATRP reaction, triggering the reaction in the presence of a catalyst and a monomer, generating an electrical signal. When ERα is added, ERα competes with GO for the aptamer, causing GO and the signal molecule to fall off, resulting in observed signal quenching.

[0032] 3. The present invention has the advantages of high efficiency, environmental protection, and easy operation.

[0033] 4. Under the optimal experimental conditions, the detection sensitivity of the kit was studied and it was found that the kit can achieve wide range and high sensitivity detection of ERα. -1 to 100 ng·mL -1 A good linear relationship exists between the peak current intensity and the logarithm of ERα concentration within a wide concentration range, and the detection limit is as low as 0.17 pg·mL -1 Furthermore, the kit exhibits excellent selectivity, stability, reproducibility, and anti-interference properties. More importantly, it can detect ERα in real samples. These experimental results demonstrate the value of this kit in biomarker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the detection method of the present invention.

[0035] Figure 2 XPS spectra (A) and Raman spectra (B) of GO and GO-Br.

[0036] Figure 3SWV curves (A) of the Apt / MCH / GO-Br / FMMA / ERα modified electrode (curve e) and electrodes without Apt (curve b), GO-Br (curve c), FMMA (curve d), and ERα (curve a) modification. EIS (B) and CV (C) curves of a bare gold electrode (curve a), and electrodes modified with Apt (curve b), Apt / MCH (curve c), Apt / MCH / GO-Br (curve d), Apt / MCH / GO-Br / FMMA (curve e), and Apt / MCH / GO-Br / FMMA / ERα (curve f).

[0037] Figure 4 The height of the electrode before polymerization (A); the height of the electrode after polymerization (B) and the height of the electrode after adding ERα (C).

[0038] Figure 5 The relationship between GO-Br concentration (A) and reaction time (B) and the current intensity of the reagent kit; the relationship between ATRP reaction time and the current intensity of the reagent kit (C).

[0039] Figure 6 SWV curves (A) and current response values of different ERα concentrations and ERα concentration (1 pg·mL -1 to 100 ng·mL -1 ) (B) (the inserted figure is a linear relationship curve).

[0040] Figure 7 is the current response value of the kit to different biomarkers (ERα, ERβ, CEA and CY21-1).

[0041] Figure 8 The current response values of different concentrations of ERα in PBS buffer and PBS buffer containing 10% (v / v) normal human serum (A) and the SWV signals generated by the kit when detecting different amounts of MCF-7 cell nuclear proteins (B). DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0043] The aptamer (Apt) used was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and purified by HPLC-CE.

[0044] The sequence of Apt is 5′-SH-(CH 2 ) 6 -CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCA TTG-3′ (SEQ ID NO. 1).

[0045] Example 1: Synthesis of GO-Br

[0046] (1) 220 μL of diethylenetriamine (DETA) was ultrasonically dispersed in 1780 μL of 95% (v / v) ethanol solution. 20 mg of graphene oxide (GO) was slowly added to the solution under ultrasonication. Ultrasonication was continued for 30 min to completely disperse the GO. The mixture was stirred at room temperature for 24 h.

[0047] (2) After the reaction is completed, the supernatant in step (1) is removed by centrifugation, and the mixture is washed three times with anhydrous ethanol, methanol, and acetone, respectively, and the supernatant is removed by centrifugation. The precipitate is placed in a vacuum drying oven and dried at room temperature for 8 hours to obtain the product GO-NH2;

[0048] (3) ultrasonically dispersing the product of step (2) into 10 mL of dimethyl sulfoxide (DMSO), adding 300 μL of 2-bromoisobutyryl bromide (BIBB), and stirring at room temperature for 8 h. After the reaction is completed, centrifugation is performed to remove the supernatant to obtain the product;

[0049] (4) The product of step (3) was washed with anhydrous ethanol and ultrapure water, and dried in a vacuum drying oven at room temperature for 8 h to obtain the product GO-Br.

[0050] Characterization of GO-Br

[0051] (1) Wide-area XPS spectrum characterization

[0052] The elemental compositions of GO and functionalized GO materials were analyzed by X-ray photoelectron spectroscopy (XPS). Figure 2 ). Figure 2 Figure A shows the wide-area XPS spectra of GO and GO-Br. Unmodified GO lacks nitrogen and bromine, while the GO-Br spectrum contains peaks for these elements. Furthermore, the C / O ratio of GO is 0.52, while after the reaction, the C / O ratio of GO-Br decreases to 0.67. This reduction in O content indicates that the oxygen-containing groups in GO have been successfully reduced.

[0053] (2) Raman spectroscopy characterization

[0054] The materials before and after modification were characterized by Raman spectroscopy. In the Raman spectrum, the peak at 1600 cm -1 The peak near 1350cm is the characteristic signal peak of GO, which is called "G" band. -1 The peak near the graphene oxide is caused by the disordered structure, which is called the "D" band. Modification of the GO surface will lead to changes in the disordered structure, thereby causing changes in the intensity of the "D" band. Figure 2As shown in Figure B, when GO is modified to GO-Br, the ID / IG value changes from 1.00 to 1.11. This phenomenon occurs because the modification of DETA and BIBB affects the lattice of GO. XPS and Raman spectroscopy results indicate that the synthesis of GO-Br is successful.

[0055] Example 2: Kit

[0056] The electrochemical detection kit of the present invention comprises the following raw materials: a gold electrode, an aptamer (Apt), 6-mercaptohexanol (MCH), GO-Br, tris[2-(dimethylamino)ethyl]amine (Me6TREN), ferrocenylmethyl methacrylate (FMMA), copper bromide (CuBr2), ascorbic acid (AA), lithium perchlorate (LiClO4), ultrapure water, and anhydrous ethanol.

[0057] Some raw materials need to be prepared as solutions: the concentration of the Apt solution is 1 μM, the concentration of the MCH solution is 2 mM, the concentration of the GO-Br solution is 0.1 mM to 0.6 mM, the concentration of the AA solution is 2 mM, the concentration of the FMMA solution is 10 mM, and the concentration of the LiClO4 solution is 1 M. Unless otherwise specified, the solvent for all solutions is ultrapure water.

[0058] Example 3: Method for constructing a kit

[0059] (1) Electrode pretreatment

[0060] First, the gold electrode was polished to a mirror finish using aluminum oxide powder. Then, the electrode was ultrasonically cleaned with ultrapure water and anhydrous ethanol and then immersed in piranha acid (V 98％H2SO4 :V H2O2 =3:1) for 15 minutes. Then, ultrasonically clean the electrode with ultrapure water and anhydrous ethanol and scan the CV curves in 0.5 M sulfuric acid solution until the images overlap. Finally, ultrasonically clean the electrode surface with ultrapure water and blow dry it with nitrogen before use.

[0061] (2) Modified electrode

[0062] ① Apt modification: 10 mL of Apt solution (1 μM) was added to the pretreated gold electrode and reacted at 37°C for 1 h;

[0063] ②MCH modification: The modified electrode from step ① was immersed in 200 μL MCH solution (2 mM) and reacted at 37°C for 0.5 h to block the unbound active sites;

[0064] 3. GO-Br modification: add 10 μL GO-Br solution (0.4 mM) to the electrode surface modified in step 2 and react at 37°C for 120 min.

[0065] ④FMMA modification: Immerse the electrode modified in step ③ in 200 μL ATRP reaction solution and react at 37°C for 80 min;

[0066] ⑤ Immerse the modified electrode prepared in step ④ in a LiClO4 solution (1 M) and perform electrochemical measurements using square wave voltammetry (SWV, scan range: 0.2 V–0.6 V, rising potential: 4.0 mV, pulse amplitude: 25 mV, frequency: 15 Hz, rest time: 10 s);

[0067] ⑥ERα modification: add 10 μL of the sample solution to be tested (containing 1 pg mL -1 ~100 ng·mL -1 ERα), react at 37°C for 1 h;

[0068] ⑦ Immerse the modified electrode from step ⑥ in a 1 M LiClO₄ solution and perform electrochemical measurements using square wave voltammetry (SWV; scan range: 0.2 V–0.6 V, rising potential: 4.0 mV, pulse amplitude: 25 mV, frequency: 15 Hz, rest time: 10 s). Calculate the ERα concentration in the sample solution based on the difference in signal intensity between the two electrochemical measurements.

[0069] The ATRP reaction solution was prepared immediately before use by mixing 140 μL of ultrapure water, 20 μL of FMMA solution (10 mM), 20 μL of AA solution (2 mM), and 20 μL of CuBr2 / Me6TREN solution. The CuBr2 / Me6TREN solution was prepared immediately before use by dissolving CuBr2 and Me6TREN in DMSO to a concentration of 10 mM CuBr2 and 12 mM Me6TREN, respectively.

[0070] During the preparation process, the electrode surface was rinsed with ultrapure water after each reaction step in step (2) and dried with nitrogen.

[0071] Example 4: Feasibility Verification

[0072] In order to prove the feasibility of this method, square wave voltammetry (SWV) was used to record the current response of electrodes modified with different materials. Figure 3As shown in Figure A, the electrode modified with Apt / MCH / GO-Br / FMMA produces the strongest electrical signal (curve a). Further modification of ERα causes ERα to compete with GO for the aptamer, resulting in the shedding of the electrical signal molecule from the electrode surface and a significant decrease in the electrical signal (curve e). Electrodes lacking modification with Apt (curve b), GO (curve c), or FMMA (curve d) exhibit only weak signal peaks, indicating that the kit cannot be successfully constructed without any of these modifications, demonstrating the feasibility of the method of the present invention.

[0073] Afterwards, the stepwise modification process of the electrode was investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Figure 3 B shows the change in the impedance value of the electrode after each modification step. The diameter of the semicircle in the Nyquist plot represents the magnitude of the impedance value. As can be seen from the figure, the impedance value of the polished bare gold electrode is low (269.2Ω, curve a). When the aptamer is fixed on the electrode surface, the impedance value increases significantly (842.8Ω, curve b). This is because the aptamer has many phosphate groups, which react with the [Fe(CN)6] 3- / 4- Electrostatic repulsion is generated, leading to an increase in impedance. Subsequently, MCH is used to block unbound active sites, further increasing the impedance (1178.2Ω, curve c). Subsequently, GO-Br adsorbs the aptamer through π-π stacking. Due to the poor conductivity of GO, the impedance of the electrode surface continues to increase (1540.3Ω, curve d). Subsequently, the ATRP reaction occurs on the GO surface, and a large amount of polymer greatly increases the steric hindrance of the electrode surface, resulting in a sharp increase in surface resistance (2765.8Ω, curve e). Finally, ERα competes with GO for the aptamer. Because the binding ability between ERα and the aptamer is stronger than that between GO and ERα, GO falls off the electrode surface, and the signal molecules attached to GO also fall off, thereby reducing the impedance of the electrode surface (1987.2Ω, curve f). EIS results indicate that the fully modified electrode was successfully constructed through step-by-step modification. Figure 3 C shows the CV curves of the electrode surface after each modification step. With the gradual modification of Apt, GO-Br, and FMMA, the peak current gradually decreases. Finally, modification with ERα causes the GO-Br and FMMA to fall off the electrode surface, resulting in a slight increase in the peak current. This result corresponds to the EIS results. These EIS and CV results demonstrate the successful construction of a fully modified electrode.

[0074] Example 5: Characterization

[0075] The surface height of the electrodes before and after polymerization was characterized by atomic force microscopy (AFM). Figure 4In A, the height of the electrode surface before polymerization is 20.0 nm. After polymerization, due to the aggregation of a large number of signal molecules on the electrode surface, the surface height of the electrode should increase, and the experimental results show that this is indeed the case. Figure 4 As can be seen in B, the electrode surface height becomes 35.7nm after polymerization. This result indicates that the polymerization reaction has successfully occurred on the electrode. After that, ERα was added and the electrode surface height was measured again. The results are as follows Figure 4 As shown in C, the height decreases to 21.3 nm, indicating that some GO and signal molecules are detached from the electrode surface.

[0076] The water contact angle (WCA) can effectively reveal changes in groups on a material's surface. The results showed that due to the hydrophobic nature of the bare gold electrode, the surface WCA was 95.7°. After the aptamer was immobilized on the electrode, the WCA decreased to 84.0° due to the numerous hydrophilic groups on the single-stranded DNA. MCH then blocked unbound sites on the electrode surface, slightly increasing the WCA to 85.4°. This is because MCH possesses both hydrophilic groups and hydrophobic carbon chains, resulting in no significant change in the WCA. After GO adsorption, the WCA decreased to 74.5°, due to GO being a hydrophilic nanomaterial. ATRP then occurred on the electrode surface, introducing a large amount of hydrophobic ferrocene onto the electrode surface, causing the WCA to increase dramatically to 91.9°. Finally, when ERα competed with GO for the aptamer, some GO and ferrocene fell off the electrode surface, causing the WCA to decrease slightly to 87.0°. These results further demonstrate the feasibility of the proposed method.

[0077] Example 6: Optimization of detection conditions

[0078] In order to achieve the best detection performance, the present invention studied the effects of the concentration and reaction time of GO-Br and the reaction time of ATRP on the detection performance.

[0079] (1) Optimization of GO-Br concentration

[0080] The concentration of GO-Br has a certain influence on the current intensity of the detection. Figure 5 As shown in Figure A, as the GO-Br concentration increases between 0.1 and 0.4 mM, the signal intensity increases. After reaching 0.4 mM, the signal intensity stabilizes and no longer increases. This is because BIBB is an initiator for the ATRP reaction. The more GO-Br adsorbed by the aptamer, the more polymerized products are formed in the ATRP reaction, and the signal intensity of the kit increases accordingly. When the GO-Br concentration reaches a certain value, the aptamer reaches saturation with GO-Br, and the signal intensity no longer changes. Experimental results indicate that the optimal GO-Br concentration for the reaction is 0.4 mM.

[0081] (2) Optimization of GO-Br reaction time

[0082] The reaction time between GO-Br and aptamer is also related to the polymerization reaction of signal molecules. Figure 5 As shown in Figure B, the signal intensity gradually increases within the first 120 minutes of the reaction. Thereafter, as the reaction time increases, the signal intensity no longer changes significantly. This indicates that the reaction is complete within 120 minutes. Therefore, the reaction time between GO-Br and the aptamer was set to 120 minutes in subsequent experiments.

[0083] (3) Optimization of ATRP reaction time

[0084] The reaction time of ATRP is also an important factor affecting the formation of polymers. Figure 5 As shown in Figure C, the electrical signal continuously increases from 20 to 80 minutes. As the reaction time increases, the electrical signal no longer changes over time. This result indicates that the ATRP polymerization reaction is complete within 80 minutes. Based on these experimental results, the ATRP reaction time was set to 80 minutes.

[0085] Example 7: Analytical Performance

[0086] According to the optimal experimental conditions obtained in Example 6, the detection range and sensitivity of the kit were evaluated by changing the concentration of ERα. Figure 6 As shown in Figure 2, the current intensity decreases with the increase of ERα concentration ( Figure 6 A). In addition, if Figure 6 As shown in B, at 1 pg·mL -1 to 100 ng·mL -1 Within the concentration range, there is a good linear relationship between the SWV signal quenched by the addition of ERα and the logarithm of the ERα concentration, and the corresponding linear equation is ΔI = 1.1984 + 0.303logC ERα (R 2 =0.998). Based on the linear equation, the detection limit of the kit was 0.17 pg·mL. -1 (S / N=3). Compared with the detection methods reported in the literature, this method has a lower detection limit and a wider detection range.

[0087] Table 1 Comparison of analytical performance of this kit with other ERα detection methods

[0088]

[0089] Example 8: Selectivity, stability and reproducibility

[0090] In order to verify the selectivity of the kit, ERα (1 ng·mL -1) were replaced by estrogen receptor β (ERβ), carcinoembryonic antigen (CEA), and cytokeratin 19 fragment (CY21-1) (10 ng mL -1 ) and compared the SWV signals generated by the kit to verify the selectivity of the kit. Figure 7 As shown, the electrical signals generated by the kits modified with ERβ, CEA, and CY21-1 were essentially the same as those in the blank group, while the electrical signal generated by the kit modified with ERα was significantly decreased. This is because only ERα is able to specifically bind to the aptamer, causing the signal molecule to fall off the electrode surface and resulting in signal quenching. Other proteins cannot compete with GO for the aptamer and therefore do not attenuate the signal. This result demonstrates that the kit is capable of specifically detecting ERα.

[0091] Furthermore, the stability and reproducibility of the kit were also crucial. After storing the kit at 4°C for two weeks, the SWV signal was measured again. The results showed that the current intensity maintained at 92.3% of its initial value. The reproducibility of the kit was then verified by comparing the variability of the measured data within and between groups. The results showed that the RSD within the group was 2.69% and the RSD between groups was 6.03%. Therefore, the stability and reproducibility of the kit were satisfactory.

[0092] Example 9: Anti-interference and actual sample detection

[0093] To verify the anti-interference ability of the kit in complex matrices, a 0.1 M PBS buffer solution containing 10% (v / v) normal human serum was used to prepare a 1 pg mL -1 , 100pg mL -1 and 10 ng mL -1 The electrical signal intensity of the kit was measured and compared with the electrical signal intensity of the ERα sample prepared with 0.1M PBS buffer at the same concentration. Figure 8 As shown in Figure A, the electrical signal intensities of ERα in PBS buffer containing 10% normal human serum were 98.3%, 96.9%, and 104.3% of those in PBS buffer, respectively. The results indicate that the detection performance of this kit is not affected by complex matrices and has strong anti-interference capabilities.

[0094] In order to further verify the accuracy of the test results of the kit, ERα in the nuclear protein extract of breast cancer MCF-7 cells was measured. The measurement results are as follows Figure 8 As shown in Figure B, the greater the number of MCF-7 cells, the greater the concentration of ERα in the nuclear extract, and the stronger the electrical signal quenched by the kit, which is consistent with the experimental results. Therefore, this kit can be applied to the detection of ERα in real samples.

Claims

1. A highly sensitive electrochemical detection method for non-diagnostic and / or therapeutic purposes of ERα based on the competitive effect between ERα and GO on aptamers, characterized in that: The raw materials used include: gold electrode, Apt, MCH, GO-Br, Me6TREN, FMMA, CuBr2, AA, LiClO4; The preparation method of GO-Br is: ① DETA was ultrasonically dispersed in 95% (v / v) ethanol solution. GO was weighed and added to the solution under ultrasonication. Ultrasonication was continued to completely disperse GO. The reaction was stirred at room temperature. ② After the reaction is completed, the supernatant in step ① is removed by centrifugation, the precipitate is washed, and dried at room temperature to obtain the product GO-NH2; ③ Ultrasonic dispersion of the product GO-NH2 from step ② in DMSO, adding BIBB, stirring at room temperature for reaction, and after the reaction is completed, centrifugation to remove the supernatant to obtain the product; ④ Wash the product of step ③ and dry it at room temperature to obtain the product GO-Br; The detection method comprises the following steps: (1) Add Apt solution to the surface of the gold electrode for reaction; (2) Immerse the electrode in step (1) in MCH solution for reaction; (3) Add the GO-Br solution dropwise onto the electrode in step (2) to react; (4) Immerse the electrode in step (3) in the ATRP reaction solution and react; (5) placing the electrode from step (4) in a LiClO4 solution and measuring the electrochemical signal by square wave voltammetry; (6) Add the sample to be tested onto the electrode in step (5) for reaction; (7) placing the electrode from step (6) in a LiClO4 solution and measuring the electrochemical signal by square wave voltammetry; The ATRP reaction solution was prepared by mixing 140 μL ultrapure water, 20 μL FMMA solution, 20 μL AA solution, and 20 μL CuBr2 / ME6TREN solution before use.

2. The electrochemical detection method according to claim 1, characterized in that The raw materials used also include ultrapure water and anhydrous ethanol.

3. The electrochemical detection method according to claim 1, characterized in that: Some raw materials need to be prepared into solutions when used: the concentration of Apt solution is 1 μM, the concentration of MCH solution is 2 mM, the concentration of GO-Br solution is 0.1 mM~0.6 mM, the concentration of AA solution is 2 mM, the concentration of FMMA solution is 10 mM, and the concentration of LiClO4 solution is 1 M.

4. The electrochemical detection method according to claim 1, characterized in that The sequence of Apt is: 5'-SH-(CH2)6-CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCATTG-3'.

5. The electrochemical detection method according to claim 1, characterized in that: The preparation method of CuBr2 / Me6TREN solution is as follows: before use, CuBr2 and Me6TREN are dissolved in DMSO to prepare a CuBr2 / Me6TREN solution with CuBr2 and Me6TREN concentrations of 10 mM and 12 mM, respectively.

6. The electrochemical detection method according to claim 1, characterized in that: In step (1), the reaction temperature is 37°C, and the reaction time is 1 h; in step (2), the reaction temperature is 37°C, and the reaction time is 30 min; in step (3), the reaction temperature is 37°C, and the reaction time is 30 min~180 min; in step (4), the reaction temperature is 37°C, and the reaction time is 20 min~120 min; in steps (5) and (7), the scanning range of square wave voltammetry is: 0.2 V~0.6 V, the rising potential is 4.0 mV, the pulse amplitude is 25 mV, the frequency is 15 Hz, and the rest time is 10 s; in step (6), the reaction temperature is 37°C, and the reaction time is 1 h.

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