Electrochemical detection kit for highly sensitive detection of ERα based on AuNPs-SWCNT and HKUST-1

Abstract

The present invention discloses an electrochemical detection kit for highly sensitive detection of ERα based on AuNPs-SWCNT and HKUST-1, the kit comprising a glassy carbon electrode, PEI, HAuCl4·3H2O, SWCNT-COOH, H2SO4, H3BTC, hemin, Cu(NO3)2·3H2O, EDC, NHS, Apt, Ab, and MCH. The present invention successfully constructs an electrochemical detection kit for highly sensitive detection of ERα based on AuNPs-SWCNT composite material and hemin@HKUST-1. The present invention connects hemin@HKUST-1 to an antibody through an amide bond, and then the large specific surface area and stable porous structure enable HKUST-1 to amplify the signal after capturing heme and Ab. The hemin immobilized on HKUST-1 then undergoes an oxidation-reduction reaction with hydrogen peroxide to generate an electrochemical signal. The kit can achieve wide-range and highly sensitive detection of ERα, with a detection limit as low as 27.8 fg·mL ‑1 The kit has excellent selectivity, stability, reproducibility, and anti-interference properties and can be used for the detection of ERα in real samples.

Description

Technical Field

[0001] The present invention relates to an electrochemical detection kit for highly sensitive detection of ERα based on an AuNPs-SWCNT composite material and hemin@HKUST-1 and application thereof, belonging to the technical field of bioanalysis. Background Art

[0002] Breast cancer is the most common malignant tumor in women and one of the three most common cancers worldwide. Gene expression profiling can categorize breast cancer into three phenotypes: luminal (ERα-positive), human epidermal growth factor receptor 2 (HER-2)-overexpressing, and triple-negative. Approximately 75% of breast cancers are ERα-positive. Overexpression of estrogen receptor α (ERα) increases the expression of oncogenic proteins that promote breast cancer cell growth. Due to its crucial role in breast cancer development and progression, ERα is recognized as a biomarker and one of the most successful molecular targets. Therefore, ERα detection is crucial for the early diagnosis of breast cancer. Traditional methods for ERα detection include immunohistochemistry (IHC), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting (WB), and enzyme-linked immunosorbent assay (ELISA). However, these methods suffer from complex procedures and expensive instrumentation, limiting their clinical application. Therefore, there is a need to develop a simple, convenient, economical, sensitive, and efficient method for ERα detection.

[0003] With the continuous advancement of analytical technology, various promising methods for ERα analysis and detection have emerged. Among these methods, electrochemical biosensors have attracted considerable attention due to their advantages such as high sensitivity, strong specificity, and simple operation, as well as their excellent performance in detecting tumor biomarkers. Therefore, the present invention aims to provide a new electrochemical detection method with further improved detection performance. Summary of the Invention

[0004] 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 AuNPs-SWCNT composite material and hemin@HKUST-1 and its application. The kit has the advantages of good selectivity, strong stability, high sensitivity, and environmental friendliness.

[0005] In order to achieve the above object, one of the technical solutions of the present invention is:

[0006] An electrochemical detection kit for highly sensitive detection of ERα based on nanocomposites includes the following raw materials: a glassy carbon electrode, PEI, HAuCl4·3H2O, SWCNT-COOH, H2SO4, H3BTC, hemin, Cu(NO3)2·3H2O, EDC, NHS, Apt, Ab, MCH, PBS buffer, anhydrous ethanol, DMF, and H2O2.

[0007] Furthermore, some raw materials were prepared to the following concentrations: 1 mM for HAuCl4·3H2O solution and 1 mg·mL for SWCNT-COOH dispersion. -1 , the concentration of H2SO4 solution is 0.1N, the concentration of EDC solution is 0.1M, the concentration of NHS solution is 0.025M, the concentration of Apt solution is 1μM, and the concentration of Ab solution is 5μg·mL -1 , the concentration of MCH solution was 2 mM, the concentration of PBS buffer was 0.1 M, pH = 7.4, and the concentration of H2O2 solution was 0.1 M;

[0008] The sequence of Apt is 5′-SH-(CH 2 ) 6 -CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCATT G-3′.

[0009] One of the technical solutions of the present invention is: a method for detecting ERα, comprising the following steps:

[0010] (1) Add the AuNPs-SWCNT suspension dropwise onto the surface of the glassy carbon electrode for reaction;

[0011] (2) adding the Apt solution dropwise onto the electrode in step (1) to react;

[0012] (3) Immersing the electrode from step (2) in an MCH solution for reaction;

[0013] (4) adding the sample to be tested to the electrode in step (3) for reaction;

[0014] (5) Add the Ab-hemin@HKUST-1 suspension dropwise onto the electrode in step (4) for reaction;

[0015] (6) The electrode of step (5) is placed in a H2O2 solution, and the electrochemical signal is measured by square wave voltammetry.

[0016] Furthermore, the preparation method of AuNPs-SWCNT is as follows:

[0017] 2 mL of PEI and 20 mL of HAuCl4·3H2O solution were mixed evenly and heated to 80°C. When the mixed solution changed from light yellow to light red, it was cooled to room temperature with stirring to obtain an AuNPs solution. The synthesized AuNPs solution was added to an equal volume of SWCNT-COOH dispersion, 1 mL of H2SO4 solution was added, and the mixture was heated to 60°C for 1 h. The supernatant was removed by centrifugation and dried to obtain a solid product, AuNPs-SWCNT.

[0018] Furthermore, the preparation method of Ab-hemin@HKUST-1 suspension is as follows:

[0019] (1) Dissolve 0.5 g H3BTC and 120 mg hemin in 15 mL of a mixed solvent of anhydrous ethanol and DMF;

[0020] (2) Add 1.04 g of Cu(NO3)2·3H2O to 7.5 mL of ultrapure water;

[0021] (3) The two solutions of steps (1) and (2) were mixed evenly and reacted in a 60°C water bath for 7 h. After the reaction was completed, the reaction solution was cooled to room temperature. Then, the reaction solution was centrifuged, and the precipitate was washed with anhydrous ethanol until the supernatant became colorless and dried to obtain hemin@HKUST-1.

[0022] (4) hemin@HKUST-1 was dispersed in 5 mL of ultrapure water by ultrasonication, and equal volumes of EDC solution and NHS solution were added and reacted at 37 °C for 4 h to activate the carboxyl groups. The supernatant was removed by centrifugation and the precipitate was dried to obtain activated hemin@HKUST-1.

[0023] (5) 40 μL Ab solution and 5 mg activated hemin@HKUST-1 were added to PBS buffer and reacted for 4 h. After centrifugation to remove unbound antibody, the solid product Ab-hemin@HKUST-1 was resuspended in PBS buffer.

[0024] Furthermore, the mixed solvent of anhydrous ethanol and DMF is an equal volume mixture of anhydrous ethanol and DMF.

[0025] Furthermore, the glassy carbon electrode is pretreated first. The pretreatment method is: polishing the glassy carbon electrode to a mirror surface with aluminum oxide powder, cleaning, drying, and setting aside.

[0026] Furthermore, in step (1), the reaction temperature is 37°C, and the reaction time is 20 min; in step (2), the reaction temperature is 37°C, and the reaction time is 1 h; in step (3), the reaction temperature is 37°C, and the reaction time is 30 min; in step (4), the reaction temperature is 37°C, and the reaction time is 1 h; in step (5), the reaction temperature is 37°C, and the reaction time is 15 min to 90 min; in step (6), the scanning range of the square wave voltammetry is: -0.4 to 0.1 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.

[0027] One of the technical solutions of the present invention is: an application of the kit in preparing a reagent for detecting ERα.

[0028] The schematic diagram of the detection method of the present invention is as follows: Figure 1 shown.

[0029] This study uses an AuNPs-SWCNT composite and HKUST-1 as signal amplification materials, and hemin as a signal molecule to construct a highly sensitive kit for ERα detection. In this study, the AuNPs-SWCNT composite increases the conductivity and specific surface area of the electrode to achieve signal amplification, while also providing binding sites for Apt. Hemin@HKUST-1 can be linked to the antibody via an amide bond and serve as the signal molecule in the kit. The large specific surface area and stable porous structure of HKUST-1 enable signal amplification after capturing hemin and Ab. The Apt / ERα / Ab-hemin@HKUST-1 sandwich immune complex is formed through specific recognition between the antigen, antibody, and aptamer. Finally, the hemin immobilized on HKUST-1 undergoes a redox reaction with hydrogen peroxide, generating an electrochemical signal.

[0030] Beneficial effects of the present invention:

[0031] 1. The signal amplification effect of the AuNPs-SWCNT nanocomposite material and HKUST-1 in the present invention greatly improves the detection sensitivity.

[0032] 2. The present invention has the advantages of high efficiency, environmental protection, and easy operation.

[0033] 3. This study successfully constructed a highly sensitive electrochemical detection kit for ERα based on an AuNPs-SWCNT composite and hemin@HKUST-1. Hemin@HKUST-1 is linked to an antibody via an amide bond. The large surface area and stable porous structure of HKUST-1 enable signal amplification after capturing hemin and the antibody. The hemin immobilized on HKUST-1 then undergoes a redox reaction with hydrogen peroxide, generating an electrochemical signal.

[0034] 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 27.8 fg·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

[0035] Figure 1 Schematic diagram of the detection principle of the present invention.

[0036] Figure 2 (A) The UV spectrum of AuNPs and (B) the particle size distribution of AuNPs.

[0037] Figure 3 UV spectra of AuNPs, SWCNTs and AuNPs-SWCNT composites (A), zeta potential analysis of AuNPs, SWCNTs and AuNPs-SWCNT composites (B), AFM images of SWCNTs (C) and AuNPs-SWCNT composites (D).

[0038] Figure 4 SEM images of HKUST-1 (A) and hemin@HKUST-1 (B); XRD data of HKUST-1 and hemin@HKUST-1 (C); UV-visible spectra of HKUST-1 and hemin@HKUST-1 (D); and zeta potential analysis of HKUST-1 and hemin@HKUST-1 (E).

[0039] Figure 5SWV curves (A) of the AuNPs-SWCNT / Apt / MCH / ERα / Ab-hemin@HKUST-1 modified electrode (curve a) and electrodes without AuNPs-SWCNT (curve b), Apt (curve c), ERα (curve d), and Ab-hemin@HKUST-1 (curve e) modification. EIS (B) and CV (C) curves of bare GCE (curve a), GCE / AuNPs-SWCNT (curve b), GCE / AuNPs-SWCNT / Apt (curve c), GCE / AuNPs-SWCNT / Apt / MCH (curve d), GCE / AuNPs-SWCNT / Apt / MCH / ERα (curve e), and GCE / AuNPs-SWCNT / Apt / MCH / ERα / Ab-hemin@HKUST-1 (curve f).

[0040] Figure 6 AFM images of the modified electrode in the absence of ERα (A) and the presence of ERα (B); SEM images of the modified electrode in the absence of ERα (C) and the presence of ERα (D).

[0041] Figure 7 The relationship between the volume ratio of SWCNT to AuNPs and the current intensity (A); the relationship between the volume of AuNPs-SWCNT composite material (B) and the reaction time of Ab-hemin@HKUST-1 (C) and the current intensity.

[0042] Figure 8 For different concentrations of ERα (100 fg mL -1 to 100 ng·mL -1 ) SWV curve (A) and the relationship between current response value and ERα concentration (B) (the insert is the linear relationship curve).

[0043] Figure 9 is the current response value of the kit to different biomarkers.

[0044] Figure 10 The current response values of different concentrations of ERα in PBS buffer and 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

[0045] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0046] The Apt used was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and purified by HPLC-CE.

[0047] The sequence of Apt is 5′-SH-(CH 2 ) 6 -CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCA TTG-3′ (SEQ ID NO. 1).

[0048] ERα antibody (Ab) was purchased from Wuhan Cloud-Clone Technology Co., Ltd., and the product name is estrogen receptor α (ERa) polyclonal antibody (No.: PAB050Hu01).

[0049] Example 1: Kit

[0050] The electrochemical detection kit of the present invention comprises the following raw materials: glassy carbon electrode (GCE), polyethyleneimine (PEI), tetrachloroauric acid trihydrate (HAuCl4·3H2O), carboxyl carbon nanotubes (SWCNT-COOH), H2SO4, trimesic acid (H3BTC), hemin (hemin), copper nitrate trihydrate (Cu(NO3)2·3H2O), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), aptamer (Apt), ERα antibody (Ab), 6-mercapto-1-hexanol (MCH), PBS buffer, anhydrous ethanol, N,N-dimethylformamide (DMF), and H2O2.

[0051] Some raw materials need to be prepared into the following concentrations: HAuCl4·3H2O solution concentration is 1mM, SWCNT-COOH dispersion concentration is 1mg·mL -1 , the concentration of H2SO4 solution is 0.1N, the concentration of EDC solution is 0.1M, the concentration of NHS solution is 0.025M, the concentration of Apt solution is 1μM, and the concentration of Ab solution is 5μg·mL -1 The concentration of the MCH solution was 2 mM (the solvent was PBS buffer at a concentration of 0.1 M and a pH of 7.4), the concentration of the PBS buffer was 0.1 M and a pH of 7.4, and the concentration of the H2O2 solution was 0.1 M. Unless otherwise specified, all solutions were made with ultrapure water.

[0052] Example 2: Synthesis of AuNPs-SWCNT composites

[0053] 2 mL of PEI and 20 mL of HAuCl4·3H2O solution (1 mM) were mixed and heated to 80°C. When the mixed solution changed from light yellow to light red, the heating was stopped and the solution was cooled to room temperature under stirring to prepare the AuNPs solution. SWCNT-COOH was dispersed in ultrapure water by ultrasonication, and the synthesized AuNPs solution was added to an equal volume of SWCNT-COOH dispersion (1 mg·mL -1), 1 mL of H2SO4 solution (0.1 N) was added and heated to 60 °C for reaction for 1 h, the supernatant was removed by centrifugation, and the solid product AuNPs-SWCNT was obtained by drying.

[0054] Characterization of AuNPs:

[0055] (1) UV-visible absorption spectroscopy characterization

[0056] The synthesized AuNPs were characterized by UV-visible absorption spectroscopy. Figure 2 As shown in A, AuNPs showed a characteristic absorption peak of gold nanoparticles at 520 nm, indicating that AuNPs were successfully prepared.

[0057] (2) Particle size characterization

[0058] The particle size of AuNPs was measured by nanoparticle Zeta potential analyzer (DLS), and the results showed that the particle size distribution was around 20 nm ( Figure 2 B).

[0059] (3) Zeta potential characterization

[0060] The surface charge of AuNPs was measured by a nanoparticle Zeta potential instrument. Since PEI contains many amino functional groups, the surface of AuNPs should have a positive charge. The measurement results showed that the surface charge of AuNPs was +1.6 mV, indicating the successful synthesis of AuNPs.

[0061] Characterization of AuNPs-SWCNTs:

[0062] (1) UV-visible absorption spectroscopy characterization

[0063] The synthesized AuNPs-SWCNTs were characterized by UV-visible absorption spectroscopy. Figure 3 As shown in Figure A, compared with SWCNT, the AuNPs-SWCNT composite showed a weak characteristic absorption peak of nano-gold at 520 nm, indicating that AuNPs were successfully adsorbed on the SWCNT surface.

[0064] (2) Zeta potential characterization

[0065] The surface charge of AuNPs-SWCNT was measured by nanoparticle Zeta potential instrument. Figure 3 As shown in Figure B. SWCNT-COOH shows a negative potential due to the carboxyl groups on its surface. After binding with AuNPs, the surface is covered by AuNPs. Since the surface of AuNPs carries positive charges, the potential changes from -44.7 mV to +47.3 mV. This result indicates the successful synthesis of the complex.

[0066] (3) AFM characterization

[0067] The surface height of AuNPs-SWCNT was characterized by atomic force microscopy. Figure 3 C It can be seen that the surface height of SWCNT is 60.3 nm. After AuNPs are combined with SWCNT, the surface height of the AuNPs-SWCNT composite increases to 70.6 nm, indicating the successful synthesis of the composite.

[0068] Example 3: Synthesis of Ab-hemin@HKUST-1

[0069] (1) 0.5 g H3BTC and 120 mg hemin were dissolved in 15 mL of a mixed solvent of anhydrous ethanol and DMF (V 乙醇 :V DMF =1:1);

[0070] (2) Add 1.04 g of Cu(NO3)2·3H2O to 7.5 mL of water;

[0071] (3) The two solutions of steps (1) and (2) were mixed evenly and reacted in a 60°C water bath for 7 h. After the reaction was completed, the reaction solution was cooled to room temperature; the cooled reaction solution was transferred to a centrifuge tube, the upper solvent was removed by centrifugation, and the precipitate was washed several times with anhydrous ethanol until the supernatant became colorless; finally, the blue-green precipitate was placed in a 55°C vacuum oven and dried for 8 h to obtain hemin@HKUST-1; HKUST-1 was synthesized in the same manner, but hemin was not added during the reaction;

[0072] (4) The dried hemin@HKUST-1 was dispersed in 5 mL of ultrapure water by ultrasonication. Equal volumes of EDC solution (0.1 M) and NHS solution (0.025 M) were added and reacted on a shaker at 37 °C for 4 h to activate the carboxyl groups. The supernatant was removed by centrifugation and the solid product was dried in a vacuum oven at 55 °C for 8 h to obtain activated hemin@HKUST-1.

[0073] (5) 40 μL Ab solution (5 μg mL -1 ) and 5 mg of activated hemin@HKUST-1 were added to 1960 μL PBS buffer and reacted in a shaker at 37°C for 4 h. After removing unbound antibody by centrifugation, the solid product Ab-hemin@HKUST-1 was resuspended in 2 mL PBS buffer and set aside.

[0074] Characterization of HKUST-1 and hemin@HKUST-1:

[0075] (1) SEM characterization

[0076] The morphologies of the synthesized HKUST-1 and hemin@HKUST-1 were characterized by scanning electron microscopy. Figure 4 A and Figure 4 As shown in Figure B, both HKUST-1 and hemin@HKUST-1 have octahedral structures, indicating that encapsulating hemin does not change the structure of HKUST-1.

[0077] (2) XRD characterization

[0078] The crystal structures of HKUST-1 and hemin@HKUST-1 were characterized by X-ray diffractometer. Figure 4 As shown in Figure C, the XRD data of HKUST-1 and hemin@HKUST-1 are basically consistent, indicating that the encapsulation of hemin does not affect the crystal integrity of HKUST-1.

[0079] (3) UV-visible absorption spectroscopy characterization

[0080] HKUST-1 and hemin@HKUST-1 were characterized by UV-visible absorption spectroscopy. Figure 4 As shown in Figure 4, compared with HKUST-1, hemin@HKUST-1 has an absorption peak at 420 nm, and hemin also has a characteristic absorption peak at this wavelength, indicating that hemin is successfully encapsulated in HKUST-1.

[0081] (4) Zeta potential characterization

[0082] The surface potential of HKUST-1 and hemin@HKUST-1 was measured by a nanoparticle Zeta potential instrument. Figure 4 As shown in Figure E, the potential of the crystal changed from -5.98 mV to -13.1 mV after encapsulating hemin, indicating the successful synthesis of hemin@HKUST-1.

[0083] Example 4: Method for constructing a kit

[0084] (1) Electrode pretreatment

[0085] Polish the glassy carbon electrode to a mirror surface with alumina powder, then place it in an ultrasonic cleaner and ultrasonically clean it with ultrapure water, anhydrous ethanol, and ultrapure water in sequence, blow dry with nitrogen, and set aside;

[0086] (2) Electrode modification

[0087] ① Add 4 μL of AuNPs-SWCNT suspension (0.5 mg mL -1 ) was dropped on the electrode surface and reacted at 37°C for 20 min;

[0088] ② Drop 10 μL of Apt solution (1 μM) onto the modified electrode surface in step ① and react in a 37°C oven for 1 h;

[0089] ③ Immerse the modified electrode from step ② in 200 μL MCH solution (2 mM) and incubate at 37°C for 30 min to block unbound active sites;

[0090] ④ Drop 10 μL of the sample to be tested (containing ERα) onto the modified electrode surface in step ③ and react at 37°C for 1 h;

[0091] ⑤ Drop 10 μL of Ab-hemin@HKUST-1 suspension onto the modified electrode surface prepared in step ④ and incubate at 37°C for 1 h.

[0092] ⑥ Place the modified electrode from step ⑤ in a 0.1 M H₂O₂ solution and perform electrochemical measurements using square wave voltammetry (SWV; scan range: -0.4 to 0.1 V, rising potential: 4.0 mV, pulse amplitude: 25 mV, frequency: 15 Hz, rest time: 10 s). Calculate the ERα concentration in the sample based on the electrochemical signal.

[0093] During the preparation process, the electrode surface was rinsed with ultrapure water and dried with nitrogen gas after each reaction step.

[0094] Example 5: Feasibility test

[0095] In order to prove the feasibility of this method, SWV was used to record the current response of electrodes modified with different materials. Figure 5 As shown in Figure A, when AuNPs-SWCNT (curve b), Apt (curve c), ERα (curve d), and Ab-hemin@HKUST-1 (curve e) are missing during the construction process, no obvious peak current can be detected in the system. Only when the electrode is modified with AuNPs-SWCNT / Apt / MCH / ERα / Ab-hemin@HKUST-1 (curve a) can a clear current signal be observed. The reason for this phenomenon is that when any one material is missing, the subsequent material cannot be fixed on the electrode, and the electrical signal molecule hemin cannot react with H2O2 to generate redox current. This shows that in the absence of any component, the modified electrode cannot be successfully constructed, proving the feasibility of the method of the present invention.

[0096] Example 6: Characterization

[0097] The modified electrode was placed in 5 mM [Fe(CN)6] 3- / 4-In electrolyte solution, electrochemical impedance spectroscopy (EIS) is used to characterize the gradual modification process of the electrode surface. The semicircle diameter of the high-frequency region in the Nyquist plot represents the impedance value Rct of the modified electrode, which corresponds to the electrode surface [Fe(CN)6] 3- / 4- The electron transfer ability of the probe. Figure 5 As shown in B, after AuNPs-SWCNT was modified on the electrode, its Rct value (~122Ω, curve b) was significantly lower than that of the bare electrode (~210Ω, curve a). This is because the AuNPs-SWCNT composite material has a large specific surface area and outstanding conductive properties, which can effectively improve the conductivity of the electrode. This result shows that the AuNPs-SWCNT composite material was successfully immobilized on the electrode. After Apt modification, the phosphate group on the aptamer will react with [Fe(CN)6] 3- / 4- Strong electrostatic repulsion was generated, leading to an increase in Rct (~448Ω, curve c). Subsequently, the immobilization of MCH formed a denser film on the electrode surface, further hindering the electron transfer ability of the electrode surface, resulting in a further increase in Rct (~773Ω, curve d). Subsequently, due to the poor conductivity of biomolecules, after ERα was captured by the aptamer, Rct (~1006Ω, curve e) continued to increase. Finally, a large amount of Ab-hemin@HKUST-1 composite material was modified on the electrode, greatly increasing the electrode's steric hindrance and hindering electron transfer on the electrode surface, resulting in a significant increase in the impedance value (~1715Ω, curve f).

[0098] In addition, the electronic properties of the modified electrode surface were studied by cyclic voltammetry (CV) method. Figure 5 C. The current intensity of the AuNPs-SWCNT modified electrode (curve b) is higher than that of the bare electrode (curve a). This is because the modification of the AuNPs-SWCNT composite material reduces the Rct of the electrode surface, thereby increasing the current intensity. Thereafter, with the gradual modification of the electrode, the Rct of the electrode surface increases successively and the current intensity decreases successively. When Apt is successfully assembled onto the surface of the GCE / AuNPs-SWCNT electrode through the Au-S bond, its current intensity decreases significantly (curve c). After MCH self-assembles on the modified electrode, the current intensity decreases further (curve d). After ERα binds to Apt, the current intensity decreases again (curve e). When Ab-hemin@HKUST-1 is connected to ERα, the significantly increased steric hindrance seriously affects the charge transfer, and the conduction current intensity decreases accordingly (curve f). The CV results are consistent with the EIS results. The above results further demonstrate the successful preparation of the kit of the present invention.

[0099] The surface morphology of the modified electrode was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Figure 6 As shown in A, the surface height of the ERα blank group electrode (GCE / AuNPs-SWCNT / Apt / MCH) is 89.7nm. While the surface height of the experimental group electrode (GCE / AuNPs-SWCNT / Apt / MCH / ERα / Ab-hemin@HKUST-1) increases to 163.6nm ( Figure 6 B) SEM results of ERα blank group electrodes are shown in Figure 2. Figure 6 As shown in C, the electrode surface is relatively smooth, and AuNPs-SWCNT can be observed spreading on the electrode surface. When ERα exists, a large amount of Ab-hemin@HKUST-1 is introduced into the electrode surface, changing the morphology of the electrode surface ( Figure 6 D) AFM and SEM characterization results showed that the kit could be successfully constructed only when the target ERα was present, further demonstrating the feasibility of this method.

[0100] Example 7: Optimization of detection conditions

[0101] In order to achieve the best detection performance of the kit, the present invention studied the effects of the volume ratio of SWCNT to AuNPs, the modified volume of the AuNPs-SWCNT composite material, and the reaction time of ERα and Ab-hemin@HKUST-1 on the detection performance.

[0102] (1) Optimization of the volume ratio of SWCNT-COOH to AuNPs

[0103] The volume ratio of SWCNT-COOH to AuNPs has a certain influence on the current intensity of the test. The more AuNPs bound to the SWCNT surface, the more Apt is introduced, and the corresponding current intensity is stronger. Under the condition that the total volume of the reaction solution remains unchanged, the ratio of SWCNT to AuNPs is changed and the change in current intensity is observed. The results are shown in Figure 2. Figure 7 As shown in Figure A, when the volume ratio of the SWCNT-COOH dispersion to the AuNPs solution decreased from 4:1 to 1:1, the current intensity increased with the increase in the AuNPs ratio. However, further increases in the AuNPs ratio resulted in a decrease in the current. Therefore, in subsequent experiments, the volume ratio of the SWCNT-COOH dispersion to the AuNPs solution was set to 1:1.

[0104] (2) Modification volume optimization of AuNPs-SWCNT composites

[0105] The performance of the conductive film layer formed on the surface of the nanocomposite material is closely related to the signal amplification performance of the kit. Figure 7Figure B shows the relationship between the current intensity of the kit and the modified volume of the AuNPs-SWCNT composite. The current signal significantly increased as the composite volume varied from 1 to 4 μL. However, further increases in the composite volume did not result in a significant change in the current signal. Therefore, the optimal modification volume for the AuNPs-SWCNT composite was 4 μL.

[0106] (3) Optimization of the reaction time between ERα and Ab-hemin@HKUST-1

[0107] The amount of introduced signaling molecules is closely related to the reaction time between Ab-hemin@HKUST-1 and ERα. Therefore, it is necessary to determine the effect of reaction time on current intensity. Figure 7 As shown in Figure C, the current intensity increases with the reaction time of Ab-hemin@HKUST-1, reaching a maximum at 60 minutes and then remaining essentially constant. This result indicates that the reaction reaches equilibrium after 60 minutes, so the reaction time was set to 60 minutes in subsequent experiments.

[0108] Example 8: Analytical Performance

[0109] According to the optimal experimental conditions obtained in Example 7, the detection range and sensitivity of the kit were evaluated by changing the concentration of ERα. Figure 8 As shown in Figure 2, the current intensity increases with the increase of ERα concentration ( Figure 8 A). In addition, if Figure 8 As shown in B, at 100 fg·mL -1 to 100 ng·mL -1 Within the concentration range, there is a good linear relationship between the peak current intensity and the logarithm of ERα concentration. The corresponding linear equation is I = 29.81 + 5.41g C ERα (R 2 =0.998), where I represents the current (μA), C ERα represents ERα concentration (ng·mL -1 The detection limit (LOD) of the kit was 27.8 fg·mL obtained by linear equation. -1 (S / N=3). Compared with some other methods for detecting ERα, the kit based on AuNPs-SWCNT and hemin@HKUST-1 composite material has a wider detection range and lower detection limit.

[0110] Table 1 Comparison of analytical performance of this kit with other ERα detection methods

[0111]

[0112] Example 9: Selectivity, stability and reproducibility

[0113] To verify the selectivity of the kit, under the same experimental conditions, ERα was replaced with other biomarkers, including estrogen receptor β (ERβ), carcinoembryonic antigen (CEA), and carcinoembryonic antigen (CY21-1), and the current intensity was measured to verify the selectivity of the kit for ERα. A blank control (Blank) was also set up. The results are shown in Figure 2. Figure 9 As shown, ERβ, CEA and CY21-1 (100 ng·mL -1 ) and the corresponding current response values of ERα(10 ng·mL -1 ) current response values were 21.04%, 20.12% and 17.82%. This result indicates that the selectivity of the kit is satisfactory.

[0114] In addition, the reproducibility of the kit was evaluated by comparing the measured values within and between groups. The RSD of the inter-group determination was 3.22%, while the RSD of the intra-group determination was 2.30% (n=5). Afterwards, the stability of the kit was evaluated by comparing the current response signals of the newly constructed kit and the kit after storage for 2 weeks. It can be observed that the current signal of the kit after storage at 4°C for 2 weeks can still reach 93.4% of the original signal. The above results demonstrate that the kit has good reproducibility and stability.

[0115] Example 10: Anti-interference and actual sample detection

[0116] To verify the application of the kit in complex matrices, ERα was added to 10% (v / v) normal human serum and PBS buffer, respectively, to a concentration of 1 pg·mL -1 , 100 pg·mL -1 and 10 ng·mL -1 The current signal intensity of ERα in PBS buffer and 10% (v / v) human serum samples was measured and compared. Figure 10 As shown in A, the current intensity in 10% (v / v) normal human serum was 95.8% (1 pg·mL) in PBS buffer. -1 )、96.7%(100pg·mL -1 ) and 98.3% (10 ng·mL -1 ), which means that the kit has good anti-interference ability and is also applicable to matrices with complex components, indicating that the kit has great application prospects in actual sample detection.

[0117] In order to verify the accuracy of the kit in detecting ERα, it is necessary to apply the kit to breast cancer cells. Therefore, different amounts of nuclear protein of MCF-7 cells were extracted and the corresponding electrical signal intensity was measured using the kit. The measurement results are shown in Figure 2. Figure 10 As shown in B, the greater the number of cells, the stronger the corresponding electrical signal intensity, which indicates that this kit can be used to detect ERα in cells.

Claims

1. A highly sensitive electrochemical detection method for ERα for non-diagnostic and / or therapeutic purposes based on AuNPs-SWCNT and HKUST-1, characterized in that: The raw materials used include: glassy carbon electrode, PEI, HAuCl4·3H2O, SWCNT-COOH, H2SO4, H3BTC, hemin, Cu (NO3)2·3H2O, EDC, NHS, Apt, Ab, MCH; The sequence of Apt is: 5′-SH-(CH2)6-CCCGGCATGGTTGCGGAGCAGGAGTATAACACTACCATTG-3′; The detection method comprises the following steps: (1) Add the AuNPs-SWCNT suspension dropwise onto the surface of the glassy carbon electrode for reaction; (2) Add the Apt solution dropwise onto the electrode in step (1) to react; (3) Immerse the electrode in step (2) in MCH solution for reaction; (4) Add the sample to be tested onto the electrode in step (3) for reaction; (5) Add the Ab-hemin@HKUST-1 suspension dropwise onto the electrode in step (4) for reaction; (6) placing the electrode in step (5) in a H2O2 solution and measuring the electrochemical signal by square wave voltammetry; The preparation method of the Ab-hemin@HKUST-1 suspension is as follows: (1) Dissolve 0.5 g H3BTC and 120 mg hemin in 15 mL of a mixed solvent of anhydrous ethanol and DMF; (2) Add 1.04 g Cu (NO3)2·3H2O to 7.5 mL ultrapure water; (3) The two solutions of steps (1) and (2) were mixed evenly and reacted in a 60°C water bath for 7 h. After the reaction was completed, the reaction solution was cooled to room temperature. Then, the reaction solution was centrifuged, and the precipitate was washed with anhydrous ethanol until the supernatant became colorless and dried to obtain hemin@HKUST-1. (4) hemin@HKUST-1 was dispersed in 5 mL of ultrapure water by ultrasonication, and equal volumes of EDC solution and NHS solution were added and reacted at 37 °C for 4 h to activate the carboxyl groups. The supernatant was removed by centrifugation and the precipitate was dried to obtain activated hemin@HKUST-1. (5) 40 μL Ab solution and 5 mg activated hemin@HKUST-1 were added to PBS buffer and reacted for 4 h. After centrifugation to remove unbound antibody, the solid product Ab-hemin@HKUST-1 was resuspended in PBS buffer.

2. The electrochemical detection method according to claim 1, characterized in that The raw materials used also include PBS buffer, anhydrous ethanol, DMF, and H2O2.

3. The electrochemical detection method according to claim 1 or 2, characterized in that: Some raw materials need to be prepared into the following concentrations: HAuCl4·3H2O solution concentration is 1 mM, SWCNT-COOH dispersion concentration is 1 mg·mL -1 , the concentration of H2SO4 solution is 0.1 N, the concentration of EDC solution is 0.1 M, the concentration of NHS solution is 0.025 M, the concentration of Apt solution is 1 μM, and the concentration of Ab solution is 5 μg·mL -1 , the concentration of MCH solution was 2 mM, the concentration of PBS buffer was 0.1 M, pH = 7.4, and the concentration of H2O2 solution was 0.1 M.

4. The electrochemical detection method according to claim 1, characterized in that The preparation method of AuNPs-SWCNT is as follows: 2 mL of PEI and 20 mL of HAuCl4·3H2O solution were mixed evenly and heated to 80°C. When the mixed solution changed from light yellow to light red, it was cooled to room temperature with stirring to prepare an AuNPs solution. The synthesized AuNPs solution was added to an equal volume of SWCNT-COOH dispersion, and 1 mL of H2SO4 solution was added and heated to 60°C for 1 h. The supernatant was removed by centrifugation and dried to obtain a solid product, AuNPs-SWCNT.

5. The electrochemical detection method according to claim 1, characterized in that: The mixed solvent of anhydrous ethanol and DMF is a mixture of anhydrous ethanol and DMF in equal volumes.

6. The electrochemical detection method according to claim 1, characterized in that: Before use, the glassy carbon electrode is pretreated. The pretreatment method is: polish the glassy carbon electrode to a mirror surface with aluminum oxide powder, clean it, blow it dry, and set it aside.

7. The electrochemical detection method according to claim 1, characterized in that: In step (1) of the detection method, the reaction temperature is 37°C and the reaction time is 20 min; in step (2), the reaction temperature is 37°C and the reaction time is 1 h; in step (3), the reaction temperature is 37°C and the reaction time is 30 min; in step (4), the reaction temperature is 37°C and the reaction time is 1 h; in step (5), the reaction temperature is 37°C and the reaction time is 15 min~90 min; in step (6), the scanning range of the square wave voltammetry is: -0.4 to 0.1 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.

Images