An electrochemical detection kit for HER2 based on functionalized nanomaterials and ARGET ATRP and its application.

Abstract

This invention discloses an electrochemical detection kit for HER2 based on functionalized nanomaterials and ARGET ATRP, and its application. The kit includes GEC, PEDOT:PSS, HAuCl4, Apt, BSA, Ab-MWCNTs-BMP-Fc, DMSO, and LiClO4. DMSO-modified PEDOT:PSS is electrostatically adsorbed onto a GCE electrode. AuNPs are then electrodeposited onto the PEDOT:PSS / GCE electrode surface using a STEP method, serving as binding sites for Apt. Apt then connects to the deposited AuNPs via Au-S bonds, and BSA blocks unreacted binding sites. Subsequently, Apt specifically captures the target molecule HER2, which further specifically recognizes Abs on the Ab-MWCNTs-BMP-Fc, forming a sandwich structure. The modified electrode is immersed in lithium perchlorate electrolyte, and its detection performance is tested using SWV. This invention offers advantages such as high selectivity, high sensitivity, and time-saving, and has good applicability in human serum detection.

Description

Technical Field

[0001] This invention relates to a high-performance PEDOT:PSS platform HER2 electrochemical detection kit and its application based on carboxyl-functionalized MWCNTs and ARGET ATRP, belonging to the field of bioanalytical technology. Background Technology

[0002] Globally, breast cancer has become a significant factor seriously affecting women's health. Statistics show that in 2021, breast cancer, lung cancer, and colorectal cancer accounted for 50% of all new cancer cases in women. Breast cancer alone accounted for as much as 30%, surpassing lung cancer to become the most frequently diagnosed cancer in women. Therefore, establishing a rapid, accurate, and sensitive method for detecting breast cancer is of great importance.

[0003] Human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor family, is a common biomarker for breast cancer, overexpressed in 20%–30% of breast cancers. Elevated serum HER2 levels are associated with the occurrence of breast cancer and adverse reactions during treatment. Therefore, early detection of HER2 is crucial for timely treatment and prognosis of breast cancer. Currently, various methods have been reported for HER2 detection, including enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECL), DNA probe technology, electrochemiimmunoassay, immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH). Among these, IHC and FISH are the most common clinical methods, but they are highly invasive and time-consuming. In contrast, electrochemical methods have gained widespread attention due to their high selectivity, high sensitivity, and time-saving advantages, and body fluid detection methods are more convenient and less invasive for patients. Currently, methods using highly conductive materials to modify electrodes to improve sensor sensitivity are widely used. Poly(3,4-ethylenedioxythiophene) (PEDOT) has been widely used in electrochemical transistors, antistatic coatings, conductive coatings, and solar cells due to its excellent conductivity, biocompatibility, and environmental stability. Poly(4-styrene sulfonate) (PSS), as a dopant, is often mixed with PEDOT to alter the conformation of the PEDOT and PSS chains, thereby improving polymer stability. Furthermore, adding different additives to enhance the conductivity of the PEDOT:PSS mixture is also a common post-processing method.

[0004] Carbon nanotubes (CNTs) possess physical properties such as low density, high porosity, and good conductivity. Adding carbon nanomaterials to biosensors can increase current intensity, expand the dielectric coverage area, and improve biosensor performance. Multi-walled carbon nanotubes (MWCNTs) exhibit high conductivity, significantly enhancing electron transfer efficiency. This invention applies carboxyl-functionalized MWCNTs to the sensor. Carboxyl-functionalized MWCNTs have shorter walls, better dispersibility, and provide more reaction sites for the biosensor. Furthermore, atom transfer radical polymerization (ARGET ATRP), an electron transfer regeneration activator, is used as a signal amplification technique. This method offers stable and controllable reactions under mild conditions, improving sensitivity while also enhancing stability and reproducibility. This invention aims to develop a highly efficient electrochemical biological reagent kit based on PEDOT:PSS and functionalized MWCNTs for the detection of the breast cancer biomarker HER2. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a high-performance PEDOT:PSS platform HER2 electrochemical detection kit and its application based on carboxyl-functionalized MWCNTs and ARGETATRP. This kit overcomes the drawbacks of traditional HER2 detection methods, which require highly invasive in vivo detection and have long cycles. It offers advantages such as high selectivity, high sensitivity, and time-saving, and has good applicability in human serum detection.

[0006] To achieve the above objectives, one of the technical solutions of the present invention is as follows:

[0007] An electrochemical detection kit for HER2 based on functionalized nanomaterials and ARGET ATRP includes the following raw materials: GEC, PEDOT:PSS, HAuCl4, Apt, BSA, Ab-MWCNTs-BMP-Fc, DMSO, and LiClO4.

[0008] Furthermore, the Apt sequence is: 5'-SH-(CH2)6-GCAGCGGTGTGGGG-3'.

[0009] Furthermore, the preparation method of Ab-MWCNTs-BMP-Fc is as follows:

[0010] (1) Preparation of carboxyl-functionalized MWCNTs

[0011] 0.12 g MWCNTs were dispersed in 50 mL of a mixed solution of H2SO4 and HNO3 with a volume ratio of 3:1. After sonication, the solution was washed and centrifuged. The washing steps were repeated until the pH value reached neutral. The solution was then dried to obtain carboxyl-functionalized MWCNTs.

[0012] (2) Preparation of Ab-MWCNTs-BMP-Fc

[0013] First, equal volumes of 3 mM EDC solution, 3 mM NHS solution, and 3 mM BMP solution were mixed and incubated at 37 °C for 2 h to obtain a 1 mM aBMP solution. Then, 2.5 mg of carboxyl-functionalized MWCNTs were added to 10 mL of aBMP solution, and the mixture was sonicated for 2 h. After centrifugation, washing, and drying, MWCNTs-BMP was obtained. MWCNTs-BMP was then dispersed in 10 mL of freshly prepared ARGET ATRP solution, incubated at 37 °C for 2 h, washed, centrifuged, and dried to obtain MWCNTs-BMP-Fc. Subsequently, 10 mL of a mixed EDC and NHS solution was added to MWCNTs-BMP-Fc, and the mixture was reacted at 37 °C for 1 h. Finally, the reacted MWCNTs-BMP-Fc was dissolved in 5 mL of ultrapure water, and 100 μL of 1 μg·mL⁻¹ was added. -1 The Ab solution was reacted at 37°C for 1 hour; finally, after centrifugation, washing, and drying, Ab-MWCNTs-BMP-Fc powder was obtained; after completion, it was prepared into a 1 mg·mL solution with ultrapure water. -1 The Ab-MWCNTs-BMP-Fc solution was stored at 4°C for later use.

[0014] Furthermore, the preparation method of ARGET ATRP solution is as follows:

[0015] ① Dissolve CuBr2 and Me6TREN in DMSO to prepare a CuBr2 / Me6TREN solution with a concentration of 10 mM for both CuBr2 and Me6TREN;

[0016] ② Using PBS buffer as a solvent, prepare 2mM AA solution and 10mM FMMA solution respectively;

[0017] ③ Mix PBS buffer, CuBr2 / Me6TREN solution, AA solution and FMMA solution in a volume ratio of 7:1:1:1 to prepare ARGET ATRP solution.

[0018] Furthermore, during use, a portion of the raw materials is prepared as a solution: the PEDOT:PSS solution has a mass fraction of 1.5%, the HAuCl4 solution has a concentration of 1M, the Apt solution has a concentration of 1μM, the BSA solution has a mass fraction of 1%, and the Ab-MWCNTs-BMP-Fc solution has a concentration of 1 mg·mL⁻¹. -1 The concentration of the LiClO4 solution is 1M.

[0019] One of the technical solutions of the present invention is: a method for detecting HER2 using the kit, comprising the following steps:

[0020] (1) Preparation of highly conductive PEDOT:PSS solution

[0021] A 1.5% PEDOT:PSS solution by mass was mixed with DMSO at a volume ratio of 3:1 and stirred continuously at room temperature for 24 hours to obtain a DMSO-modified PEDOT:PSS solution, which was then stored at 4°C for later use.

[0022] (2) Electrode modification

[0023] ① The DMSO-modified PEDOT:PSS solution was dropped onto the pretreated GCE surface, reacted, washed, and dried;

[0024] ②Immerse the GCE electrode from step ① in HAuCl4 solution, electrodeposit gold nanoparticles using the STEP method, wash, and dry;

[0025] ③ Drop the Apt solution onto the GCE surface from step ②, react, wash, and dry;

[0026] ④ Immerse the GCE from step ③ in BSA solution, react, wash, and dry;

[0027] ⑤ Add the test solution to the GCE in step ④, react, wash, and dry;

[0028] ⑥ Add Ab-MWCNTs-BMP-Fc solution dropwise to the GCE from step ⑤, react, wash, and dry;

[0029] (3) Electrochemical determination

[0030] The modified GCE was immersed in LiClO4 solution and electrochemically detected using the SWV method.

[0031] Furthermore, the GCE electrode is first pretreated by polishing the surface of the GCE electrode with 0.3μm alumina powder. Then, the electrode is ultrasonically cleaned with ultrapure water, anhydrous ethanol and ultrapure water in sequence. The electrode is then detected by CV method in 0.2M KNO3 solution containing 1mM K3[Fe(CN)6] to ensure that the potential difference of the bare electrode is less than 80mV. Finally, the electrode is ultrasonically cleaned with anhydrous ethanol and ultrapure water.

[0032] Further, the reaction temperature of step ① is 35℃~40℃ and the time is 2.5~3.0h; the electrodeposition temperature of step ② is room temperature and the time is 180~240s; the reaction temperature of step ③ is 35℃~40℃ and the time is 1.0~1.5h; the reaction temperature of step ④ is 35℃~40℃ and the time is 0.5~1.0h; the reaction temperature of step ⑤ is 35℃~40℃ and the time is 1.0~2.0h; the reaction temperature of step ⑥ is 35℃~40℃ and the time is 1.0~2.0h; the SWV scanning range of step (2) is 0.1~0.6V, the scanning rate is 1.0V / s, and the potential increment is 4mV.

[0033] One of the technical solutions of the present invention is the application of the kit in the preparation of HER2 detection reagents.

[0034] A schematic diagram of the construction process of the reagent kit of the present invention is shown below. Figure 1 As shown.

[0035] First, DMSO-modified PEDOT:PSS was electrostatically adsorbed onto the GCE electrode. Then, AuNPs were electrodeposited onto the PEDOT:PSS / GCE electrode surface using the STEP method, serving as binding sites for Apt. Next, Apt linked to the deposited AuNPs via Au-S bonds, and unreacted binding sites were blocked with 1% BSA. Subsequently, Apt specifically captured the target analyte HER2, which further specifically recognized Abs on the Ab-MWCNTs-BMP-Fc layer, forming a sandwich structure. The modified electrode was then immersed in lithium perchlorate electrolyte, and its detection performance was tested using square wave voltammetry (SWV).

[0036] Beneficial effects of the present invention

[0037] 1. This invention uses materials with good conductivity, such as PEDOT:PSS and carboxyl-functionalized MWCNTs, which not only provide more connection sites for subsequent signal units, but also increase signal strength and significantly improve sensitivity.

[0038] 2. The ARGET ATRP aggregation signal amplification strategy is adopted. Its aggregation reaction is stable and controllable under mild conditions, and it can aggregate a large number of signal units, which greatly improves the detection sensitivity. At the same time, stability and reproducibility are also improved.

[0039] 3. This invention utilizes a DMSO-modified PEDOT:PSS coating to provide a substrate with high conductivity, and then electroplats an AuNPs layer onto its surface. Apt can autonomously attach to the electrode via Au-S bonds and specifically recognize and capture HER2. Subsequently, the Abs on the Ab-MWCNTs-BMP-Fc structure specifically recognize and connect to HER2, forming an Apt-HER2-Ab sandwich structure, thereby connecting a large number of signal monomers, ferrocene methyl methacrylate (FMMA), polymerized onto MWCNTs via ARGET ATRP, to the electrode. Finally, square wave voltammetry (SWV) is used to record the response value of the oxidation current, achieving highly sensitive detection of the target analyte. Under optimal conditions, the current signal exhibits a good linear relationship with the HER2 concentration, with the linear equation: I(μA)=8.23905lg[C HER2 / ng·mL -1 +25.3585(R) 2 =0.995), with a detection limit as low as 1.979 fg·mL. -1 Experimental results show that the present invention has good selectivity and stability, and is simple to operate and low in cost, and is expected to be extended to the detection of other targets and clinical applications. Attached Figure Description

[0040] Figure 1 A schematic diagram of the reagent kit construction process (A) and a schematic diagram of the ARGET ATRP principle (B).

[0041] Figure 2 SWV curves for Ab-MWCNTs-BMP-Fc / HER2 / Apt / AuNPs / PEDOT:PSS / GCE(a) and without PEDOT:PSS(b), AuNPs(c), Apt(d), HER2(e), and Ab-MWCNTs-BMP-Fc(f).

[0042] Figure 3 A represents the mixture containing 0.1 M KNO3 and 5 mM [Fe(CN)6]. 3- / 4- EIS curves of GCE at different stages in the solution. Figure 3 B represents the mixture containing 0.1 M KNO3 and 5 mM [Fe(CN)6]. 3- / 4- CV curves of GCE at different stages in the solution.

[0043] Figure 4 A is a 2D AFM image of PEDOT:PSS / GCE. 4B is a 2D AFM image of AuNPs / PEDOT:PSS / GCE. Figure 4C is a 2D AFM image of Ab-MWCNTs-BMP-Fc / HER2 / BSA / Apt / AuNPs / PEDOT:PSS / GCE.

[0044] Figure 5 A is a TEM image of unprocessed MWCNTs. Figure 5 B is a TEM image of MWCNTs treated with H2SO4 / HNO3. Figure 5 C is a TEM image of ARGET ATRP polymerized acidified MWCNTs.

[0045] Figure 6 A represents the optimization of the PEDOT:PSS volume conditions; Figure 6 B represents the optimization of the electrodeposition time conditions for the STEP method; Figure 6 C represents the optimization of incubation time conditions for Ab-MWCNTs-BMP-Fc.

[0046] Figure 7 A is a schematic diagram of the current signal intensity obtained by detecting different concentrations of HER2; Figure 7 B represents the linear relationship between the current signal intensity and different concentrations of HER2. In Figure A, curve af corresponds to concentrations of 0.01, 0.1, 1, 10, 100, and 1000 ng·mL, respectively. -1 HER2.

[0047] Figure 8 A represents the concentration of 100 ng / mL. -1 SWV reaction of HER2, CYFRA21-1, cTnI, ERα, ERβ, CEA and all antigen mixtures; Figure 8 B represents the change in the SWV reaction of the kit over time.

[0048] Figure 9 To prepare 100 pg / mL of PBS buffer, 5% (v / v) normal human serum, and 10% (v / v) normal human serum. -1 1 ng·mL -1 10 ng·mL -1 The SWV reaction of HER2. Detailed Implementation

[0049] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0050] HER2 antigen, HER2 antibody (Ab), and HER2 aptamer (Apt) were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. HER2 antigen is human epidermal growth factor receptor 2 antigen (product number: NO.D111419); HER2 antibody is human epidermal growth factor receptor 2 monoclonal coated antibody (product number: NO.D194027).

[0051] The Apt sequence is: 5'-SH-(CH2)6-GCAGCGGTGGGG-3' (SEQ ID NO.1).

[0052] Example 1: Material Preparation

[0053] (1) Preparation of carboxyl-functionalized MWCNTs

[0054] 0.12 g of multi-walled carbon nanotubes (MWCNTs) were dispersed in 50 mL of a 3:1 mixture of H2SO4 and HNO3. After sonication for 3 h, the mixture was washed with ultrapure water and centrifuged at 14000 rpm for 15 min. The washing process was repeated until the pH reached neutral. The mixture was then dried at 100 °C for 2 h to obtain carboxyl-functionalized MWCNTs.

[0055] (2) Preparation of Ab-MWCNTs-BMP-Fc

[0056] First, equal volumes of 3 mM carbodiimide (EDC) solution, 3 mM N-hydroxysuccinimide (NHS) solution, and 3 mM 2-bromoisobutyric acid (BMP) solution were mixed and incubated at 37 °C for 2 h to obtain a 1 mM activated BMP (aBMP) solution. Then, 2.5 mg of carboxyl-functionalized MWCNTs were added to 10 mL of aBMP solution and reacted under sonication for 2 h. The mixture was then centrifuged at 14000 rpm for 15 min and the precipitate was washed twice with ultrapure water. The precipitate was dried at 100 °C for 2 h to obtain MWCNTs-BMP. Next, MWCNTs-BMP was dispersed in 10 mL of freshly prepared ARGET ATRP solution and incubated at 37 °C for 2 h. After washing with ultrapure water, centrifuging at 14000 rpm for 15 min, and drying at 100 °C for 2 h, MWCNTs-BMP-Fc was obtained. Subsequently, 10 mL of a mixed solution of EDC (8 mM) and NHS (2 mM) was added to MWCNTs-BMP-Fc, and the mixture was reacted at 37 °C for 1 h to activate the carboxyl groups of MWCNTs. Then, the activated MWCNTs-BMP-Fc was dissolved in 5 mL of ultrapure water, and 100 μL of Ab solution (1 μg·mL⁻¹) was added. -1The reaction was carried out at 37°C for 1 hour. Finally, the precipitate was centrifuged at 14000 rpm for 15 minutes, washed twice with PBS buffer, and dried at 100°C for 2 hours to obtain Ab-MWCNTs-BMP-Fc powder. After completion, it was prepared into 1 mg / mL solutions using ultrapure water. -1 The Ab-MWCNTs-BMP-Fc solution was stored at 4°C for later use.

[0057] The preparation method of ARGET ATRP solution is as follows:

[0058] ① Dissolve CuBr2 and tris(2-dimethylaminoethyl)amine (Me6TREN) in dimethyl sulfoxide (DMSO) to prepare a CuBr2 / Me6TREN solution with a concentration of 10 mM for both CuBr2 and Me6TREN.

[0059] ② Using PBS buffer as a solvent, prepare 2mM ascorbic acid (AA) solution and 10mM FMMA solution respectively;

[0060] ③ Mix PBS buffer, CuBr2 / Me6TREN solution, AA solution and FMMA solution in a volume ratio of 7:1:1:1 to prepare ARGET ATRP solution.

[0061] Example 2: Reagent Kit

[0062] A high-performance PEDOT:PSS platform HER2 electrochemical detection kit based on carboxyl-functionalized MWCNTs and ARGET ATRP includes the following raw materials: glassy carbon electrode (GEC), poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonate) (PEDOT:PSS), HAuCl4, Apt, bovine serum albumin (BSA), Ab-MWCNTs-BMP-Fc, ultrapure water, anhydrous ethanol, KNO3, PBS buffer, dimethyl sulfoxide (DMSO), LiClO4, and H2SO4.

[0063] When using, prepare a portion of the raw materials into solutions: the PEDOT:PSS solution has a mass fraction of 1.5%, the HAuCl4 solution has a concentration of 1M, the Apt solution has a concentration of 1μM, the BSA solution has a mass fraction of 1%, and the Ab-MWCNTs-BMP-Fc solution has a concentration of 1 mg·mL⁻¹. -1 The concentration of the LiClO4 solution is 1M.

[0064] Example 3: Detection method of the kit

[0065] (1) Electrode pretreatment

[0066] First, the glassy carbon electrode (GEC) surface was polished with 0.3 μm alumina slurry. Then, the electrode was ultrasonically cleaned with ultrapure water, anhydrous ethanol and ultrapure water in sequence. Next, the electrode was detected by cyclic voltammetry (CV) in a 0.2 M KNO3 solution containing 1 mM K3[Fe(CN)6] to make the potential difference of the bare electrode less than 80 mV. Finally, the electrode was ultrasonically cleaned with anhydrous ethanol and ultrapure water.

[0067] (2) Preparation of highly conductive PEDOT:PSS solution

[0068] A 1.5% PEDOT:PSS solution by mass was mixed with DMSO at a volume ratio of 3:1 and stirred continuously at room temperature for 24 h to obtain a DMSO-modified PEDOT:PSS solution, which was then stored at 4 °C.

[0069] (3) Modified electrode

[0070] ① Drop 2.5 μL of DMSO-modified PEDOT:PSS solution onto the pretreated GCE surface, react at 37℃ for 3 h, wash, and dry;

[0071] ②Immerse the GCE electrode from step ① in 1M HAuCl4 solution, and use the STEP method to electrodeposit gold nanoparticles (AuNPs) in the solution onto the GCE from step ① as binding sites for Apt. The electrodeposition temperature is room temperature and the time is 180s. Wash and dry.

[0072] ③ Drop 10 μL of Apt solution (1 μM) onto the GCE surface from step ②, react at 37 °C for 1 h, wash, and dry;

[0073] ④ Immerse the GCE from step ③ in 300 μL of BSA solution (1% by mass), react at 37°C for 0.5 h, wash, and dry;

[0074] ⑤ Add 10 μL of the test solution (containing HER2 antigen) to the GCE in step ④, react at 37°C for 1 h, wash, and dry;

[0075] ⑥ Add 10 μL of Lab-MWCNTs-BMP-Fc solution (1 mg·mL) -1 The mixture was transferred to the GCE from step ⑤ and incubated at 37°C for 2 hours. The mixture was then washed and dried.

[0076] All the above steps involve washing with PBS buffer and drying with nitrogen gas.

[0077] (4) Electrochemical determination

[0078] The modified GCE was immersed in LiClO4 solution (1M) and electrochemically detected by square wave voltammetry (SWV) (scan range: 0.1–0.6 V, scan rate: 1.0 V / s, potential increment: 4 mV).

[0079] Example 4: Feasibility Verification

[0080] To evaluate the feasibility of using the kit of this invention to determine HER2, redox current signals of a series of modified electrodes were measured using SWV (1M LiClO4, potential range 0.1–0.6V) and compared. The results are as follows: Figure 2 As shown, a small current peak (curve b) appears when PEDOT:PSS is absent. This is because a small amount of AuNPs is directly electroplated onto the GCE, allowing a small number of signal units to successfully connect to the electrode. Without AuNPs (curve c), Apt (curve d), or HER2 (curve e) modification, only a tiny current peak occurs. This is because, without these substances, Ab-MWCNTs-BMP-Fc cannot be effectively connected to the electrode, and the weak signal is due to a small amount of Ab-MWCNTs-BMP-Fc attaching to the GCE via non-specific bonds. Simultaneously, no significant current signal is observed without Ab-MWCNTs-BMP-Fc (curve f). The electrode after the complete modification step exhibits a significant current peak at a potential of approximately 0.36V (curve a), indicating that the signal units are successfully connected to the electrode after the complete modification process. These experiments confirm the feasibility of this strategy for HER2 detection.

[0081] Example 5: Characterization

[0082] Electrochemical impedance spectroscopy (EIS) can sensitively monitor subtle changes in the impedance value of an electrode surface. Figure 3 The results showed that 5 mM [Fe(CN)6] contained 0.1 M KNO3 was used. 3- / 4- Characteristic changes at the modified electrode interface in electrolyte solution. Results are as follows: Figure 3 As shown in Figure A, the R of the bare electrode ct The Ω value is 197 Ω (curve a). However, when the highly conductive materials PEDOT:PSS (curve b) and AuNPs (curve c) are modified onto the electrode, the transfer of electrons between the electrolyte and electrode surface becomes faster due to the high conductivity of PEDOT:PSS and AuNPs. ct The rate of change continues to decrease. When Apt is modified onto the electrode, R... ct Increased to 110Ω (curve d). Similarly, BSA also has the ability to impede electron transfer, making R... ctIncreased to 198Ω (curve e). After the target HER2 was modified onto the electrode, R... ct Further increases (~377Ω, curve f). Finally, when Ab-MWCNTs-BMP-Fc are specifically recognized and attached to the electrode, R ct The Ω increased to 515 Ω (curve g). This is due to the poor conductivity and large steric hindrance of the polymer. Meanwhile, CV was used to characterize [Fe(CN)6]. 3- / 4- Electrode modification process ( Figure 3 B), whose trend of change is consistent with that of EIS. This indicates that the construction process of the present invention is successful.

[0083] The morphological changes of the modified glassy carbon electrode were observed using atomic force microscopy (AFM). The surface height changes of the two-dimensional AFM images of the electrodes after PEDOT:PSS, AuNPs / PEDOT:PSS, and all modification processes were compared. The results are as follows: Figure 4 As shown in Figure A, the electrode modified only with the PEDOT:PSS coating has a height of 25.2 nm. When AuNPs are electrodeposited onto the PEDOT:PSS coating, the height increases to 105.8 nm. Figure 4 B). Furthermore, the height of the two-dimensional AFM image of the electrode after the complete modification process increased to 170.1 nm ( Figure 4 C). The changes in AFM show that the height of the modified electrode continuously increases, indicating that the material was successfully modified onto the glassy carbon electrode.

[0084] Transmission electron microscopy (TEM) was used to observe detailed changes in MWCNTs at different treatment steps. The results are as follows: Figure 5 As shown in Figure A, the surface of untreated MWCNTs is smooth, and the inner walls are intact and undamaged. In contrast, the sidewalls of MWCNTs treated with H₂SO₄ / HNO₃ show obvious damage. Figure 5 B), the surface of MWCNTs polymerized by ARGET ATRP not only showed obvious fractures, but also had a rough surface with the presence of polymer. Figure 5 C). These studies show that the surface of MWCNTs is severely damaged after acid treatment, and the strength of carboxyl groups at the fracture sites increases. This indicates that the acidification process is simple and effective.

[0085] Example 6: Optimization of Detection Conditions

[0086] To optimize the performance of the kit, key conditions during electrode construction were optimized, including the volume of PEDOT:PSS, the electrodeposition time of the STEP method, and the incubation time of Ab-MWCNTs-BMP-Fc, in order to improve the analytical performance of the kit.

[0087] (1) PEDOT: PSS volume optimization

[0088] First, the effect of PEDOT:PSS volume on peak current was examined. Figure 6 A) The results show that within the range of 0.5 μL to 2.5 μL, the peak current increases with increasing PEDOT:PSS volume. However, when the amount of PEDOT:PSS exceeds 2.5 μL, the peak current decreases instead, because the excessively thick PEDOT:PSS coating hinders electron transfer. Therefore, the experimental results indicate that the optimal volume of PEDOT:PSS used on glassy carbon electrodes is 2.5 μL.

[0089] (2) Optimization of AuNPs electrodeposition time

[0090] like Figure 6 As shown in Figure B, the peak current increases with the electrodeposition time of the STEP method, reaching its maximum at 180 s. After this point, the peak current tends to stabilize. The results indicate that the optimal electrodeposition time for AuNPs is 180 s.

[0091] (3) Optimization of Ab-MWCNTs-BMP-Fc incubation time

[0092] like Figure 6 As shown in Figure C, the peak current increases continuously with the extension of the reaction time, reaching its maximum value at 120 min, and then no longer increases significantly. This indicates that Ab-MWCNTs-BMP-Fc reaches saturation at 120 min, therefore 120 min was selected as the optimal incubation time for Ab-MWCNTs-BMP-Fc.

[0093] Example 7: Performance Analysis

[0094] Under optimal conditions, the SWV reaction signal values ​​of the modified glassy carbon electrode were recorded in 1M LiClO4 solution to investigate the detection range and limit of detection of this strategy. The results are as follows: Figure 7 As shown, at 10 pg·mL -1 ~1μg·mL -1 Within the specified range, the peak SWV current exhibits a good linear relationship with the logarithm of HER2 concentration. The linear regression equation is: I(μA)=8.23905lg[C HER2 / ng·mL -1 +25.3585(R) 2 =0.995), the detection limit is: 1.979 fg·mL -1Compared with several other methods (Table 1), the method proposed in this invention for detecting HER2 has a wider detection range, a lower detection limit, and higher sensitivity. This indicates that the kit of this invention has potential application value in the early detection of cancer.

[0095] Table 1 Comparison of the kit of the present invention with several other HER2 detection methods

[0096]

[0097] Example 8: Selectivity, stability and reproducibility of the kit

[0098] To investigate the selectivity of this invention for different proteins, proteins from various sources were detected using this method, including a soluble fragment of cytokeratin 19 (CYFRA21-1), high-sensitivity troponin I (cTnI), estrogen receptor α (ERα), estrogen receptor β (ERβ), carcinoembryonic antigen (CEA), and a mixture of these proteins with HER2. The concentration of each protein was 100 ng / mL. -1 The result is as follows Figure 8 As shown in Figure A, the peak currents of proteins from other sources were significantly lower than those of HER2, while the mixed sample exhibited current intensities similar to those of pure HER2. This is because the sandwich structure significantly enhances the selectivity of the method through two specific recognition processes: aptamer-antigen and antigen-antibody. Therefore, this invention demonstrates high selectivity for HER2.

[0099] To demonstrate the stability of this invention, the modified electrode was stored at 4°C for different durations, and then tested using SWV. Figure 8 As shown in Figure B, the peak current decreases with prolonged storage time. Compared to the freshly prepared electrode, the signal intensity of the electrode stored for 14 days is 85.57% of that of the freshly prepared electrode. This indicates that the present invention has good stability. Meanwhile, under the same experimental conditions, reproducibility was evaluated by measuring the signal values ​​within and between groups (n=5). The RSDs within and between groups were 2.77% and 3.92%, respectively, indicating that the present invention has good reproducibility.

[0100] Example 9: Practical Application Value

[0101] To verify the detection performance of this invention in complex media, the signal values ​​of HER2 in 5% (v / v) and 10% (v / v) normal human serum (NHS) were measured. Different concentrations of HER2 (100 pg·mL⁻¹) were used. -1 1 ng·mL -1 10 ng·mL -1HER2 was added to 5% (v / v) and 10% (v / v) NHS and the signal values ​​were compared with those obtained from the same concentration of HER2 in 0.1M PBS buffer. The results are as follows: Figure 9 As shown, the peak current of the 5% (v / v) serum sample was 97.45% (100 pg·mL) of the PBS buffer. -1 ), 97.70% (1 ng·mL) -1 ), 97.56% (10 ng·mL) -1 The peak current of 10% (v / v) serum sample was 96.20% (100 pg / mL) of PBS buffer. -1 ), 95.34% (1 ng·mL) -1 ), 96.54% (10 ng·mL) -1 This indicates that the complex components in serum have only a minor interference effect on the measurement of signal values.

Claims

1. A HER2 electrochemical detection kit based on functionalized nanomaterials and ARGET ATRP, characterized in that, Including the following raw materials: GCE, PEDOT:PSS, HAuCl4, Apt, BSA, Ab-MWCNTs-BMP-Fc; The Apt sequence is: 5'-SH-(CH2)6-GCAGCGGTGTGGGG-3'; The preparation method of Ab-MWCNTs-BMP-Fc is as follows: (1) Preparation of carboxyl-functionalized MWCNTs 0.12 g MWCNTs were dispersed in 50 mL of a mixed solution of H2SO4 and HNO3 with a volume ratio of 3:

1. After sonication, the solution was washed and centrifuged. The washing steps were repeated until the pH value reached neutral. The solution was then dried to obtain carboxyl-functionalized MWCNTs. (2) Preparation of Ab-MWCNTs-BMP-Fc First, equal volumes of 3 mM EDC solution, 3 mM NHS solution, and 3 mM BMP solution were mixed and incubated at 37°C for 2 h to obtain a 1 mM aBMP solution. Then, 2.5 mg of carboxyl-functionalized MWCNTs were added to 10 mL of aBMP solution, and the mixture was sonicated for 2 h. After centrifugation, washing, and drying, MWCNTs-BMP was obtained. MWCNTs-BMP was then dispersed in 10 mL of freshly prepared ARGET ATRP solution, incubated at 37°C for 2 h, washed, centrifuged, and dried to obtain MWCNTs-BMP-Fc. Subsequently, 10 mL of a mixed EDC and NHS solution was added to MWCNTs-BMP-Fc, and the mixture was reacted at 37°C for 1 h. Finally, the reacted MWCNTs-BMP-Fc was dissolved in 5 mL of ultrapure water, and 100 µL of 1 µg·mL⁻¹ of methyl methacrylate (MCMA) was added. -1 The Ab solution was reacted at 37°C for 1 h; finally, after centrifugation, washing, and drying, Ab-MWCNTs-BMP-Fc powder was obtained; after completion, it was prepared into 1 mg·mL powder with ultrapure water. -1 The Ab-MWCNTs-BMP-Fc solution was stored at 4°C for later use. The preparation method of ARGET ATRP solution is as follows: ① Dissolve CuBr2 and Me6TREN in DMSO to prepare a CuBr2 / Me6TREN solution with a concentration of 10 mM for both CuBr2 and Me6TREN; ② Using PBS buffer as a solvent, prepare 2 mM AA solution and 10 mM FMMA solution respectively; ARGET ATRP solution was prepared by mixing PBS buffer, CuBr2 / Me6TREN solution, AA solution and FMMA solution in a volume ratio of 7:1:1:

1.

2. The reagent kit according to claim 1, characterized in that, It also includes DMSO and LiClO4.

3. The reagent kit according to claim 2, characterized in that, When using, prepare a portion of the raw materials into solutions: the PEDOT:PSS solution has a mass fraction of 1.5%, the HAuCl4 solution has a concentration of 1 M, the Apt solution has a concentration of 1 µM, the BSA solution has a mass fraction of 1%, and the Ab-MWCNTs-BMP-Fc solution has a concentration of 1 mg·mL⁻¹. -1 The concentration of the LiClO4 solution is 1 M.

4. A method for detecting HER2 using the kit described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of highly conductive PEDOT:PSS solution A 1.5% PEDOT:PSS solution by mass was mixed with DMSO at a volume ratio of 3:1 and stirred continuously at room temperature for 24 h to obtain a DMSO-modified PEDOT:PSS solution, which was then stored at 4 °C for later use. (2) Electrode modification ① The DMSO-modified PEDOT:PSS solution was dropped onto the pretreated GCE surface, reacted, washed, and dried; ②Immerse the GCE electrode from step ① in HAuCl4 solution, electrodeposit gold nanoparticles using the STEP method, wash, and dry; ③ Drop the Apt solution onto the GCE surface from step ②, react, wash, and dry; ④ Immerse the GCE from step ③ in BSA solution, react, wash, and dry; ⑤ Add the test solution to the GCE in step ④, react, wash, and dry; ⑥ Add Ab-MWCNTs-BMP-Fc solution dropwise to the GCE from step ⑤, react, wash, and dry; (3) Electrochemical determination The modified GCE was immersed in LiClO4 solution and electrochemically detected using the SWV method.

5. The method according to claim 4, characterized in that, The GCE electrode was first pretreated by polishing the surface with 0.3 μm alumina powder. Then, the electrode was ultrasonically cleaned with ultrapure water, anhydrous ethanol, and ultrapure water in sequence. The electrode was then tested by CV method in a 0.2 M KNO3 solution containing 1 mM K3[Fe(CN)6] to ensure that the potential difference of the bare electrode was less than 80 mV. Finally, the electrode was ultrasonically cleaned with anhydrous ethanol and ultrapure water.

6. The method according to claim 4, characterized in that, The reaction temperature of step ① is 35℃~40℃ and the time is 2.5~3.0 h; the electrodeposition temperature of step ② is room temperature and the time is 180~240 s; the reaction temperature of step ③ is 35℃~40℃ and the time is 1.0~1.5 h; the reaction temperature of step ④ is 35℃~40℃ and the time is 0.5~1.0 h; the reaction temperature of step ⑤ is 35℃~40℃ and the time is 1.0~2.0 h; the reaction temperature of step ⑥ is 35℃~40℃ and the time is 1.0~2.0 h; the SWV scanning range of step (2) is 0.1~0.6 V, the scanning rate is 1.0 V / s, and the potential increment is 4 mV.

7. The use of the kit as described in any one of claims 1-3 in the preparation of a reagent for detecting HER2.

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