An electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection and its detection method

Abstract

The present invention belongs to the technical field of HER-2 detection in breast cancer, and specifically relates to an electrochemiluminescence resonance energy transfer (ECL-RET) aptamer sensor for HER-2 detection and a detection method thereof. The method for constructing the ECL-RET aptamer sensor for HER-2 detection comprises the following steps: dropping a dispersion of a luminescent material CS@HOF-102 onto the surface of a glassy carbon electrode and drying at room temperature; then dropping a capture probe solution onto the electrode and incubating; then dropping a 6-mercaptohexanol solution onto the electrode and incubating; and finally dropping a quenching probe Au@Ag2S / HBA solution onto the electrode and incubating, thereby obtaining the ECL-RET aptamer sensor for HER-2 detection. The ECL-RET aptamer sensor uses CS@HOF-102 as the luminescent material and Au@Ag2S / HBA as the quenching probe, utilizes the electrochemiluminescence resonance energy transfer (ECL-RET) effect, and achieves ultrasensitive detection of HER-2 through an "on-off-on" detection mode, thereby providing a new diagnostic approach for early diagnosis of breast cancer patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of breast cancer HER-2 detection, and particularly relates to an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection and a detection method thereof. Background Art

[0002] Breast cancer (BC) is a malignant tumor caused by the uncontrolled proliferation and subsequent malignant transformation of epithelial cells in breast tissue. Compared to other cancers, breast cancer ranks first in both incidence and mortality among female malignancies, and both rates are on the rise. Accurate diagnosis and timely treatment of breast cancer in its early stages not only effectively prevents metastasis to vital organs but also significantly improves treatment success rates and patient survival, while minimizing recurrence rates. Therefore, there is a need for the development of cost-effective, sensitive, and rapid detection methods for the early detection of breast cancer. Human epidermal growth factor receptor 2 (HER-2) is a transmembrane glycoprotein involved in regulating cell growth and differentiation. Its overexpression is associated with poor cell differentiation, shortened survival, early recurrence and metastasis, aggressiveness, and poor clinical prognosis. Therefore, HER-2 protein, as a biomarker for breast cancer, is crucial for prognostic diagnosis and targeted treatment.

[0003] Currently, commonly used HER-2 detection methods include histological and serological methods. Histological methods include immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). However, histological methods are highly invasive for patients, subject to significant subjective influences, and cannot achieve continuous testing and monitoring. Serological methods offer the advantages of simplicity, quantification, objectivity, and real-time detection, and can be used as a supplement to histological testing. Methods for detecting serum HER-2 include enzyme-linked immunosorbent assay (EIA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay. However, existing detection technologies still suffer from low sensitivity and specificity. Summary of the Invention

[0004] To address the problems of the prior art, the present invention provides an electrochemiluminescence resonance energy transfer (ECL-RET) aptasensor for HER-2 detection. This aptasensor, constructed from luminescent and quenching materials, uses CS@HOF-102 as the luminescent material and Au@Ag2S / HBA as the quenching probe. By utilizing the electrochemiluminescence resonance energy transfer (ECL-RET) effect and an "on-off-on" detection mode, it achieves ultrasensitive detection of HER-2, providing a new diagnostic approach for early diagnosis of breast cancer patients.

[0005] The present invention solves the technical problem by adopting the following technical solutions:

[0006] Electrochemiluminescence (ECL) is a combination of chemiluminescence and electrochemistry, with the advantages of both technologies, such as low background signal, wide dynamic range, high sensitivity, easy control, and simple equipment.

[0007] Aptamers are small single-stranded oligonucleotides (DNA or RNA) that can recognize and bind to target substances with high specificity and affinity. They are screened through the systematic evolution of ligands by exponential enrichment (SELEX) technique, and their sequence lengths mostly range from 15 to 60 bases. Compared with antibodies, aptamers have advantages such as fast synthesis, low manufacturing cost, good stability, and greater modifiability. They can also mimic natural biological interfaces containing biomarkers, giving them higher detection sensitivity.

[0008] The present invention aims to provide an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection. The method for constructing the electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection is as follows:

[0009] The luminescent material CS@HOF-102 dispersion was dropped onto the surface of a glassy carbon electrode and dried at room temperature; then the capture probe solution was dropped onto the electrode and incubated; then the 6-mercaptohexanol solution was dropped onto the electrode and incubated; finally, the quenching probe Au@Ag2S / HBA solution was dropped onto the electrode and incubated to obtain an electrochemiluminescent aptamer sensor for HER-2 detection.

[0010] Furthermore, the preparation method of the luminescent material CS@HOF-102 dispersion is as follows;

[0011] 100 mg of chitosan (CS) was weighed and dissolved in 10 mL of glacial acetic acid solution (1 wt%) and dispersed evenly. 1 mg of HOF-102 was weighed and dispersed in 1 mL of ultrapure water. The mixture was added to 0.5 mL of the chitosan solution and magnetically stirred for 12 h to obtain a uniformly dispersed CS@HOF-102 solution. After centrifugation and washing with ultrapure water, the precipitate was dispersed in 1 mL of ultrapure water to obtain a CS@HOF-102 dispersion.

[0012] Furthermore, the preparation method of HOF-102 nanomaterial is as follows:

[0013] 40 mg of H4TNAPy was dissolved in 12 mL of N,N-dimethylformamide (DMF) solution and then heated at 120°C in a silicone oil bath for 1 h to obtain a clear yellow solution. After cooling to room temperature, the solution was poured into 32 mL of acetone and stirred (400 rpm) for 2 min. The suspension was stirred for 12 h and then centrifuged at 8000 rpm for 5 min. The yellow precipitate was further washed with acetone and then dried at room temperature to obtain the yellow HOF-102 material.

[0014] Furthermore, the preparation method of the Au@Ag2S / HBA quenching probe solution is as follows: add 250 μL of 2 μM HER-2 binding aptamer (HBA) to 750 μL of Au@Ag2S NPs solution, stir in an ice bath for 12 h, centrifuge, wash with water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the Au@Ag2S / HBA quenching probe solution.

[0015] Furthermore, the preparation method of Au@Ag2S NPs solution is as follows: 1 mL of 1% HAuCl4 solution is added to 100 mL of ultrapure water and boiled, and then 2.5 mL of 1% trisodium citrate solution is quickly added and continued to boil for 15 min. After cooling, the volume is restored to the original volume with ultrapure water to obtain a transparent wine-red solution, which is gold nanoparticles (AuNPs), which is stored at 4°C; under stirring, 1 mL of silver nitrate (4 mg / mL) is added to 50 mL of Au NPs boiling solution; then 1 mL of 1% trisodium citrate is added dropwise to the reaction solution, and heated to reflux at 135°C for 1 h; the color of the solution changes from wine red to orange, and Au@Ag NPs are obtained; after cooling to room temperature, the Au@Ag NPs solution is stored in the dark at 4°C; 0.3 mL of 10 mM sodium sulfide is added to 10 mL of Au@Ag NPs solution and stirred at room temperature for 12 h; NPs solution was stored at 4°C.

[0016] Furthermore, the method for constructing an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection is as follows:

[0017] 1) Treat the HER-2 binding aptamer (HBA) and capture probe chain (CP) with 20 mM Tris-HCl (pH = 7.4) buffer at room temperature and store at 4°C until use;

[0018] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 minutes, then rinse with ultrapure water for later use.

[0019] 3) The electrodes obtained in step 2) were polished to a mirror surface using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The electrodes were then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dried for later use.

[0020] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0021] 5) Add 10 μL of the luminescent material CS@HOF-102 dispersion onto the cleaned glassy carbon electrode surface in step 4) and dry at room temperature;

[0022] 6) Add 10 μL of the capture probe strand prepared in step 1) onto the electrode prepared in step 5) and incubate at 4-5°C for 12 h.

[0023] 7) Add 10 μL of 1% 6-mercaptohexanol (MCH) solution dropwise to the electrode obtained in step 6) and incubate at room temperature for 30 minutes.

[0024] 8) 10 μL of the Au@Ag2S / HBA quenched probe solution was added dropwise to the electrode prepared in step 7) and incubated at room temperature for 2 h to obtain an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection.

[0025] The present invention also aims to provide a method for detecting an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection, comprising the following steps:

[0026] 1) adding different concentrations of the target human epidermal growth factor receptor HER-2 to the electrode of the sensor;

[0027] 2) The electrode was placed in a 0.01 M PBS (pH = 7.0) solution containing 20 mM TEA for characterization, and its luminescence intensity was measured;

[0028] 3) Based on the linear relationship between the luminescence intensity and the logarithmic value of the HER-2 concentration obtained in step 2), a working curve is drawn.

[0029] An electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection is prepared mainly by the following method:

[0030] (1) Preparation of luminescent materials;

[0031] 1) HOF-102: 40 mg of H4TNAPy was dissolved in 12 mL of N,N-dimethylformamide and heated at 120°C in a silicone oil bath for 1 h to yield a clear yellow solution. After cooling to room temperature, the solution was poured into 32 mL of acetone and stirred at 400 rpm for 2 min. The suspension was stirred for 12 h and then centrifuged at 8000 rpm for 5 min. The resulting yellow precipitate was further washed with acetone and dried at room temperature to yield the yellow HOF-102 material.

[0032] 2) CS@HOF-102 dispersion: Weigh 100 mg of chitosan and dissolve it in 10 mL of glacial acetic acid solution (1 wt%) until uniformly dispersed. Weigh 1 mg of HOF-102 prepared in step 1) and disperse it in 1 mL of ultrapure water. Add it to 0.5 mL of CS solution and magnetically stir for 12 h to obtain a uniformly dispersed CS@HOF-102 solution. After centrifugation and washing with ultrapure water, the precipitate was dispersed in 1 mL of ultrapure water to obtain a CS@HOF-102 dispersion.

[0033] (2) Preparation of quenching probes;

[0034] 1) Au@Ag2S: Add 1 mL of 1% HAuCl4 solution to 100 mL of ultrapure water and boil. Then, quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, bring the volume back to the original volume with ultrapure water to obtain a transparent wine-red solution, which is the gold nanoparticles. Store at 4°C. While stirring, add 1 mL of silver nitrate (4 mg / mL) to 50 mL of the boiling AuNPs solution. Then, add 1 mL of 1% trisodium citrate dropwise to the reaction solution, and heat under reflux at 135°C for 1 hour. The color of the solution changes from wine-red to orange, yielding Au@Ag nanoparticles. After cooling to room temperature, store the Au@Ag NPs solution at 4°C in the dark. Add 0.3 mL of 10 mM sodium sulfide to 10 mL of the Au@Ag NPs solution and stir at room temperature for 12 hours. This yields the Au@Ag2S nanoparticle solution, which is stored at 4°C.

[0035] 2) Au@Ag2S / HBA: Add 250 μL of 2 μM HER-2 binding aptamer to 750 μL of the Au@Ag2S NPs solution prepared in step 1). Stir on ice for 12 h, centrifuge, and wash with water. Redisperse the precipitate in 1 mL of ultrapure water to obtain the Au@Ag2S / HBA quenching probe solution.

[0036] (3) Construction of an electrochemiluminescent aptamer sensor for HER-2 detection:

[0037] 1) Treat the HER-2 binding aptamer and capture probe strands with 20 mM Tris-HCl (pH 7.4) buffer at room temperature and store at 4°C until use.

[0038] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 minutes, then rinse with ultrapure water for later use.

[0039] 3) The electrodes obtained in step 2) were polished to a mirror surface using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The electrodes were then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dried for later use.

[0040] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0041] 5) Add 10 μL of the luminescent material CS@HOF-102 dispersion onto the cleaned glassy carbon electrode surface in step 4) and dry at room temperature;

[0042] 6) Add 10 μL of the CP prepared in step 1) onto the electrode prepared in step 5) and incubate at 4°C for 12 h.

[0043] 7) Add 10 μL of 1% 6-mercaptohexanol solution to the electrode obtained in step 6) and incubate at room temperature for 30 min;

[0044] 8) 10 μL of the Au@Ag2S / HBA quenched probe solution was added dropwise to the electrode prepared in step 7) and incubated at room temperature for 2 h to obtain an electrochemiluminescent aptamer sensor for HER-2 detection.

[0045] Compared with the prior art, the beneficial technical effects of the present invention are:

[0046] 1) This invention utilizes electrochemiluminescence resonance energy transfer (ECL-RET) technology, employing a chitosan (CS)-modified hydrogen-bonded organic framework (HOF-102) as the luminescent material and Au@Ag2S / HBA as the quenching probe to fabricate an electrochemiluminescent aptamer sensor. This sensor achieves ultrasensitive detection of HER-2 using an on-off-on detection mode. Compared to traditional HER-2 detection methods, this method offers advantages such as high sensitivity, strong specificity, rapid detection, and ease of use, providing a novel approach for HER-2 detection.

[0047] 2) The chitosan (CS)-modified hydrogen-bonded organic framework material (HOF-102) can greatly improve the low luminescence efficiency and poor luminescence stability of the ligand H4TNAPy, thereby improving the sensor performance and achieving signal amplification while increasing the detection sensitivity.

[0048] 3) The electrochemiluminescence resonance energy transfer (ECL-RET) aptamer sensor prepared in the present invention has high specificity for target recognition and can improve the selectivity of the sensor, thereby providing a new research direction and analytical method for the detection of trace HER-2.

[0049] 4) All the materials involved can be synthesized under laboratory conditions, which are simple to operate, the raw materials are inexpensive, low in toxicity, and environmentally friendly. In addition, the amount used each time is extremely small, which reduces the experimental cost.

[0050] 5) The electrochemical aptamer sensor prepared in this invention provides a new method for detecting HER-2; the entire detection and analysis method is clear and simple, with high sensitivity and rapid signal response. The electrochemiluminescent aptamer sensor prepared in this invention can also be used for other biological sample analysis, food, drug, and environmental monitoring.

[0051] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a comparison chart of the electrochemiluminescence signal intensities when the sensor of the present invention is used with a blank test object and with a HER-2 target object for detection.

[0053] Figure 2 These are cyclic voltammetry characterization graphs of different modified electrodes in the present invention obtained in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a voltage range from -0.2 to 0.6 V at a scan rate of 100 mV / s.

[0054] Figure 3 The UV spectrum of Au@Ag2S NPs of the present invention (a) and the ECL spectrum of HOF-102 (b) are shown.

[0055] Figure 4Figure 2 is the result of detecting different concentrations of HER-2 by the sensor of the present invention, wherein Figure A is a time-electrochemiluminescence intensity graph of the sensor scanning 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL of HER-2 in 20 mM TEA in 0.01 M PBS (pH 7.0); Figure B is a calibration curve of the sensor electrochemiluminescence intensity and the logarithmic value of different HER-2 concentrations.

[0056] Figure 5 Figure 3 is the stability test result of the sensor of the present invention, which is a time-electrochemiluminescence intensity graph obtained after the sensor was incubated with 100 ng / mL HER-2 and scanned continuously for 15 cycles.

[0057] Figure 6 The intra- and inter-batch reproducibility results are obtained by simultaneously incubating 100 ng / mL HER-2 on different glassy carbon electrodes and scanning them under the same conditions.

[0058] Figure 7 This is a specificity test graph for the aptasensor of the present invention, where the test substance is epidermal growth factor 2 (HER-2, 1 ng / mL), a blank sample without interfering substances and HER-2, and a mixed sample containing interfering substances and HER-2 (1 ng / mL). The interfering substances are epidermal growth factor receptor 1 (HER-1, 100 ng / mL), epidermal growth factor receptor 3 (HER-3, 100 ng / mL), glucose (Glu, 100 ng / mL), dopamine (DA, 100 ng / mL), and ascorbic acid (AA, 100 ng / mL). DETAILED DESCRIPTION

[0059] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope that the present invention is intended to protect.

[0060] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0061] The main chemical reagents used in the embodiments of the present invention are as follows:

[0062] Human epidermal growth factor receptor 2 (HER-2) standard was purchased from Abcam (Cambridge, UK); chitosan (CS), silver nitrate (AgNO3), and trisodium citrate (C6H5Na3O7·2H2O) were purchased from Kron Reagent (Chengdu, China); H4TNAPy [6,6',6'',6'''-(pyrene-1,3,6,8-tetramethylphenyl)tetrakis(2-naphthoic acid)] was purchased from Biter Pharmaceuticals Co., Ltd. (Shanghai, China); sodium sulfide (Na2S) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); chloroauric acid (HAuCl4) was purchased from Sigma (USA); 6-mercaptohexanol (MCH) and potassium persulfate (K2S2O8) were purchased from J&K Scientific Ltd (Beijing, China); and N,N-dimethylformamide (DMF) was purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0063] The aptamer involved was synthesized by Shanghai Bioengineering Co., Ltd. The specific sequence is as follows:

[0064] Sequence of HER-2 binding aptamer chain (HBA): 5'-NH2-(CH2)6-GGGCCGTCGAACACGAGCATGGTGCGTGGACCTAGGATGACCTGAGTACTGTCC-3'

[0065] Sequence of capture probe (CP): 5'-NH2-(CH2)6-TTTTTGGACAGTACTCAGGTCATCCTAGG-3'

[0066] Equipment used and technical parameters:

[0067] Instrumentation: Time / voltage-based electrochemiluminescence (ECL) intensity measurements were performed using an MPI-E electrochemiluminescence workstation (Xi'an, China). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed using a Metrohm Autolab BV electrochemical workstation (Modular Instruments, Switzerland). Electrochemiluminescence detection was performed using a three-electrode system: a modified glassy carbon electrode (4 mm diameter) as the working electrode, a platinum wire as the counter electrode, and a silver-silver chloride (saturated with KCl) reference electrode. Electrochemical detection was performed using a three-electrode system: a modified glassy carbon electrode (4 mm diameter) as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference electrode. pH was monitored using a pH meter (S210 SevenCompact, Mettler-Toledo, Shanghai, China). The three-electrode system was scanned at 100 mV / s in 0.01 M PBS (pH 7.0) containing 20 mM TEA. The electrochemical three-electrode system was scanned at 100 mV / s in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution. Example 1

[0068] Prepare the CS@HOF-102 dispersion luminescent material by following the steps below:

[0069] 1) HOF-102: 40 mg of H4TNAPy was dissolved in 12 mL of N,N-dimethylformamide (DMF) and heated at 120°C in a silicone oil bath for 1 h to yield a clear yellow solution. After cooling to room temperature, the solution was poured into 32 mL of acetone and stirred at 400 rpm for 2 min. The suspension was stirred for 12 h and then centrifuged at 8000 rpm for 5 min. The resulting yellow precipitate was further washed with acetone and dried at room temperature to yield the yellow HOF-102 material.

[0070] 2) CS@HOF-102 dispersion: Weigh 100 mg of chitosan (CS) and dissolve it in 10 mL of glacial acetic acid solution (1 wt%) until uniformly dispersed. Weigh 1 mg of HOF-102 prepared in step 1) and disperse it in 1 mL of ultrapure water. Add 0.5 mL of CS solution and magnetically stir for 12 h to obtain a uniformly dispersed CS@HOF-102 solution. After centrifugation and washing with ultrapure water, the precipitate was dispersed in 1 mL of ultrapure water to obtain a CS@HOF-102 dispersion. Example 2

[0071] Prepare the Au@Ag2S / HBA quenching probe by following the steps below:

[0072] 1) Au@Ag2S: Add 1 mL of 1% HAuCl4 solution to 100 mL of ultrapure water and boil. Then, quickly add 2.5 mL of 1% trisodium citrate solution and continue boiling for 15 minutes. After cooling, bring the volume back to the original volume with ultrapure water to obtain a transparent wine-red solution, which is gold nanoparticles (AuNPs). Store at 4°C. While stirring, add 1 mL of silver nitrate (4 mg / mL) to 50 mL of the boiling AuNPs solution. Then, add 1 mL of 1% trisodium citrate dropwise to the reaction solution, and heat under reflux at 135°C for 1 hour. The color of the solution changes from wine-red to orange, yielding Au@Ag nanoparticles (Au@Ag NPs). After cooling to room temperature, store the Au@AgNPs solution at 4°C in the dark. Add 0.3 mL of 10 mM sodium sulfide to 10 mL of the Au@AgNPs solution and stir at room temperature for 12 hours. The Au@Ag2S nanoparticle (Au@Ag2S NPs) solution was obtained and stored at 4°C.

[0073] 2) Au@Ag2S / HBA: Add 250 μL of 2 μM HER-2 binding aptamer (HBA) to 750 μL of the Au@Ag2S NPs solution prepared in step 1). Stir on ice for 12 h, centrifuge, and wash with water. Re-disperse the precipitate in 1 mL of ultrapure water to obtain the Au@Ag2S / HBA quenching probe solution. Example 3

[0074] To construct an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection, follow the steps below:

[0075] 1) Treat the HER-2 binding aptamer (HBA) and capture probe (CP) with 20 mM Tris-HCl (pH = 7.4) buffer at room temperature and store until use;

[0076] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 minutes, then rinse with ultrapure water for later use.

[0077] 3) The electrodes obtained in step 2) were polished to a mirror surface using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The electrodes were then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dried for later use.

[0078] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0079] 5) 10 μL of the luminescent material CS@HOF-102 dispersion prepared in Example 1 was dropped onto the surface of the cleaned glassy carbon electrode in step 4) and dried at room temperature;

[0080] 6) Add 10 μL of the CP prepared in step 1) onto the electrode prepared in step 5) and incubate at 4°C for 12 h.

[0081] 7) Add 10 μL of 1% 6-mercaptohexanol (MCH) solution dropwise to the electrode obtained in step 6) and incubate at room temperature for 30 minutes.

[0082] 8) 10 μL of the Au@Ag2S / HBA quenched probe solution prepared in Example 2 was added dropwise to the electrode prepared in step 7) and incubated at room temperature for 2 h to obtain an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection. Example 4

[0083] The electrochemiluminescence resonance energy transfer aptamer sensor constructed in Example 3 was used to detect HER-2 in the following steps:

[0084] Draw a working curve

[0085] 1) The modified electrode of Example 3 was used to detect blank substance and HER-2 protein, and the electrochemiluminescence response signals were measured. The results were as follows: Figure 1 .

[0086] 2) The modified electrodes from steps 5) to 8) of the electrochemiluminescent aptasensor for HER-2 detection in Example 3 were placed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV characterization. The current response signals were measured, and the results were as follows: Figure 2 Shown: (a) bare glassy carbon electrode; (b) dropwise addition of CS@HOF-102; (c) dropwise addition of CP; (d) dropwise addition of MCH for sealing; (e) dropwise addition of Au@Ag2S / HBA; (f) dropwise addition of HER-2.

[0087] 3) The modified electrodes were placed in 0.01 M PBS (pH = 7.0) containing 20 mM TEA for characterization and their electrochemiluminescence response signals were measured. The ECL spectrum of HOF-102 and the UV spectrum of Au@Ag2S / HBA were measured respectively. The results are as follows: Figure 3 Shown: (a) UV spectrum of Au@Ag2S / HBA; (b) ECL spectrum of HOF-102.

[0088] 4) 10 μL of target HER-2 at different concentrations was added to the electrode of the immunosensor prepared in Example 3, and the luminescence intensity was measured. Figure 4 As shown in A: The concentrations from left to right are: 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL.

[0089] 5) Based on the linear relationship between the obtained luminescence intensity value and the logarithmic value of HER-2 concentration, draw a working curve (such as Figure 4 The results showed that the luminescence intensity response value and the logarithm of HER-2 concentration showed a good linear relationship in the range of 100 fg / mL to 100 ng / mL, with a linear correlation coefficient of 0.9991 and a detection limit of 0.39 fg / mL. Figure 4 As shown in B.

[0090] 2. Sensor stability test: The sensor prepared in Example 3 was subjected to 15 consecutive cycles of ECL measurement under optimal conditions (e.g. Figure 5 There is no obvious fluctuation in the luminescence intensity, indicating that the sensor has good stability.

[0091] 3. Sensor reproducibility test: The sensor prepared in Example 3 by incubating the same concentration of HER-2 (100 ng / mL) with five different glassy carbon electrodes was subjected to ECL measurement (e.g. Figure 6 The intra-batch relative standard deviation (RSD) was 3.38%, and the inter-batch relative standard deviation (RSD) was 2.87%, indicating that the sensor had good reproducibility.

[0092] IV. Sensor Specificity Test: To investigate the specificity of the proposed adaptive sensor, the following interfering substances, possibly present in serum, were tested: epidermal growth factor receptor 1 (HER-1, 100 ng / mL), epidermal growth factor receptor 3 (HER-3, 100 ng / mL), glucose (Glu, 100 ng / mL), dopamine (DA, 100 ng / mL), and ascorbic acid (AA, 100 ng / mL). The electrochemiluminescence intensity responses of the different interfering substances were measured in 0.01 M PBS (pH = 7.0) containing 20 mM TEA at the same concentration and under the same conditions. The results showed that (e.g. Figure 7 As shown in Figure 3 ), the proposed aptamer sensor based on the high specific reaction of HER-2 has good specificity.

[0093] The present invention utilizes a chitosan (CS)-modified hydrogen-bonding organic framework (HOF-102) as the luminescent material and Au@Ag2S nanoparticles as the signal quenching material. A large number of HER-2 aptamer chains (HBAs) are then loaded via the bonding between the metal particles and amino groups, ultimately creating an Au@Ag2S / HBA quenching probe solution. The chitosan (CS)-modified hydrogen-bonding organic framework (HOF-102) prepared in this invention exhibits strong and stable anodic ECL activity. Furthermore, the core-shell Au@Ag2S nanoparticles have a broad UV absorption range, and the UV spectrum of the signal quenching material closely overlaps with the ECL spectrum of HOF-102, enabling efficient resonance energy transfer and quenching of the ECL signal. Through this approach, the prepared electrochemiluminescence resonance energy transfer aptamer sensor was successfully used for ultrasensitive HER-2 detection. Compared to traditional HER-2 detection methods, this invention offers advantages such as high sensitivity, strong specificity, rapid detection, convenient operation, and no pollution, thus providing a new analytical method for HER-2 detection.

[0094] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0095] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. An electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection, characterized in that: The method for constructing an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection includes: The luminescent material CS@HOF-102 dispersion was added dropwise onto the surface of a glassy carbon electrode and dried. A capture probe solution was then added dropwise onto the electrode and incubated. A 6-mercaptohexanol solution was then added dropwise onto the electrode and incubated. Finally, a quenching probe Au@Ag2S / HBA solution was added dropwise onto the electrode and incubated. This yielded an electrochemiluminescent aptamer sensor for HER-2 detection. The preparation method of the luminescent material CS@HOF-102 dispersion is as follows; HOF-102 nanomaterial powder was weighed and dispersed in ultrapure water to obtain a HOF-102 dispersion. Chitosan was dissolved in glacial acetic acid solution and added to the HOF-102 dispersion. The mixture was stirred for 12 h, centrifuged, washed, and the precipitate was dispersed in ultrapure water to obtain a uniformly dispersed CS@HOF-102 dispersion. The preparation method of the Au@Ag2S / HBA quenching probe solution is as follows: add the HER-2 binding aptamer to the Au@Ag2S NPs solution, stir in an ice bath for 12 h, centrifuge, wash with water, and redisperse the precipitate in ultrapure water to obtain the Au@Ag2S / HBA quenching probe solution.

2. The electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection according to claim 1, wherein: The preparation method of HOF-102 nanomaterial is as follows: H4TNAPy was dissolved in N,N-dimethylformamide solution and heated at 120°C for 1 hour to obtain a clear yellow solution. After cooling to room temperature, the solution was poured into acetone and stirred for 2 minutes. The suspension was stirred for 12 hours and then centrifuged at 8000 rpm for 5 minutes. The yellow precipitate was further washed with acetone and then dried at room temperature to obtain yellow HOF-102 nanomaterials.

3. The electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection according to claim 1, characterized in that: The preparation method of Au@Ag2S NPs solution is as follows: 1% HAuCl4 solution is added to ultrapure water and boiled, and then 1% trisodium citrate solution is quickly added and boiled for 15 minutes. After cooling, the volume is restored to the original volume with ultrapure water to obtain a transparent wine-red solution, which is gold nanoparticles, and stored at 4°C; silver nitrate is added to the boiling Au NPs solution under stirring; then trisodium citrate is added dropwise to the reaction solution and heated under reflux at 135°C for 1 hour; the color of the solution changes from wine-red to orange, and Au@Ag NPs are obtained; after cooling to room temperature, the Au@Ag NPs solution is stored in the dark at 4°C; sodium sulfide is added to the Au@AgNPs solution and stirred at room temperature for 12 hours to obtain the Au@Ag2S NPs solution.

4. The electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection according to claim 1, wherein: The construction method of the electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection is as follows: 1) Treat the HER-2 binding aptamer and capture probe strands with Tris-HCl buffer at room temperature and store at 4°C until use; 2) Soak the glassy carbon electrode in piranha solution for 30 minutes, then rinse with ultrapure water for later use; 3) The electrodes obtained in step 2) were polished to a mirror surface using 0.3 μm and 0.05 μm Al2O3 powders, respectively. The electrodes were then ultrasonically treated with ultrapure water, anhydrous ethanol, and ultrapure water in that order, and dried for later use. 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry; 5) Add the luminescent material CS@HOF-102 dispersion dropwise onto the cleaned glassy carbon electrode surface in step 4) and dry at room temperature; 6) Add the capture probe chain prepared in step 1) dropwise onto the electrode prepared in step 5) and incubate at 4-5°C for 12 h; 7) Add 1% 6-mercaptohexanol solution dropwise to the electrode obtained in step 6) and incubate at room temperature for 30 min; 8) The Au@Ag2S / HBA quenched probe solution was added dropwise to the electrode prepared in step 7) and incubated at room temperature for 2 h to obtain an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection.

5. The method for detecting an electrochemiluminescence resonance energy transfer aptamer sensor for HER-2 detection according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) adding different concentrations of the target human epidermal growth factor receptor HER-2 to the electrode of the sensor; 2) The electrode was placed in a 0.01 M PBS solution containing 20 mM TEA for characterization, and its luminescence intensity was measured; 3) Based on the linear relationship between the luminescence intensity and the logarithmic value of the HER-2 concentration obtained in step 2), a working curve is drawn.

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