Electrochemiluminescent aptamer sensor and detection method for HER2 detection in breast cancer

Abstract

This invention provides an electrochemiluminescent aptasensor for detecting HER2 in breast cancer. Using a nickel-based metal-organic framework (Ni-MOF) composite nanomaterial (PTCA@Ni-MOF) modified with the luminescent material 3,4,9,10-perylenetetracarboxylic acid (PTCA) as the signal material, the electrochemiluminescent sensor enables sensitive detection of HER2, providing a new diagnostic approach for early diagnosis of breast cancer patients. The detection method is clear and simple, with high sensitivity and rapid signal response.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence detection, and in particular to an electrochemiluminescence aptamer sensor and a detection method for detecting HER2 in breast cancer. Background Art

[0002] Malignant tumors pose a serious threat to human life and health. Among them, breast cancer is the leading cause of morbidity and mortality in women. HER2 is one of the most studied targets on the surface of tumor cells. It belongs to the EGF receptor family of transmembrane tyrosine kinases. It can regulate the processes of cell division, growth, differentiation, proliferation, migration and apoptosis. HER2 overexpression can be seen in gastric cancer, ovarian cancer, prostate cancer, lung cancer, bladder cancer, etc. It is most common in invasive breast cancer. Currently, the main diagnostic technologies available for detecting breast cancer are mammography, breast ultrasound and breast MRI. However, these methods require corresponding equipment, professional practitioners and expert analysis, and the detection cost is very high. Therefore, these methods are difficult to promote to most people, especially those with early breast cancer that has not yet been detected. Summary of the Invention

[0003] To address the challenges of the prior art, the present invention provides an electrochemiluminescent aptasensor for detecting HER2 in breast cancer. This sensor utilizes a nickel-based metal-organic framework (Ni-MOF) composite nanomaterial (PTCA@Ni-MOF) modified with the luminescent material 3,4,9,10-perylenetetracarboxylic acid (PTCA) as the signal material. This sensor provides sensitive detection of HER2, providing a new diagnostic approach for early diagnosis of breast cancer patients.

[0004] Unless otherwise specified, all parts described in the present invention are parts by weight and all percentages described are mass percentages.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] An electrochemiluminescent aptamer sensor for detecting HER2 in breast cancer is characterized in that the construction method of the electrochemiluminescent aptamer sensor for detecting HER2 in breast cancer is:

[0007] The electrode was immersed in a 1% (w / w) HAuCl4·4H2O solution and electrochemically deposited at a constant voltage of -0.2 V for 30-40 seconds to obtain a deposited gold nanoparticle (DpAu) film. The HER2-binding aptamer chain APT, the partially complementary chain cDNA of APT, and the partially complementary chain pDNA of cDNA were treated by immersion in Tris-HCl buffer (pH = 7.4) at room temperature and then dropwise added to the electrode with the deposited gold nanoparticle film and incubated at 4-5°C for 12-13 hours. A 1% (w / w) BSA solution was then dropwise added and incubated at room temperature for 60-80 minutes. The HER2-binding aptamer chain APT was then dropwise added by immersion in Tris-HCl buffer (pH = 7.4) at room temperature and incubated at room temperature for 2-3 hours. HER2 was then added dropwise and incubated at room temperature for 50-60 minutes. Finally, a signal probe (SP) solution was added dropwise and incubated at room temperature for 2-3 hours to obtain an electrochemiluminescent aptamer sensor for HER2 detection.

[0008] The preparation method of the signal probe (SP) solution is as follows:

[0009] pDNA was added to the PTCA@Ni-MOF dispersion and stirred in an ice bath for 10-12 h. The product was washed and centrifuged to obtain the signal probe. The synthesized signal probe was then centrifuged at 10,000-12,000 rpm, and the precipitate was redispersed in ultrapure water to obtain the signal probe (SP) solution.

[0010] The preparation method of the PTCA@Ni-MOF dispersion is as follows:

[0011] Ni-MOF was dispersed in ultrapure water and ultrasonically dispersed uniformly. A PTCA dispersion with a concentration of 1-3 mg / mL was added. After stirring at room temperature for 12-13 h, the mixture was centrifuged and washed three times with ultrapure water. The precipitate was dispersed in ultrapure water to obtain a PTCA@Ni-MOF dispersion.

[0012] The preparation method of the PTCA dispersion is:

[0013] Perylenetetracarboxylic acid dihydroanhydride (PTCDA) was dissolved in a 0.1 M NaOH solution and stirred at 80-90 °C for 2-3 h to obtain a yellow-green solution. 1.0 M HCl was then added dropwise to the solution until complete precipitation. The mixture was then centrifuged, washed with ultrapure water to a pH of 7.4, and dried at 60 °C to obtain a dark red powder, which was then dispersed with ultrapure water to obtain a PTCA dispersion.

[0014] The preparation method of Ni-MOF is:

[0015] Terephthalic acid (PTA) and nickel nitrate hexahydrate (Ni(NO3)2·6H2O) were stirred and dispersed in N,N-dimethylformamide (DMF). Then, a 0.4 M NaOH solution was added dropwise while stirring. Finally, the obtained mixed solution was transferred to a polytetrafluoroethylene-lined reactor and reacted at 100°C for 8 hours. After the reaction, it was cooled to room temperature and washed with DMF and ethanol. The obtained precipitate was dried at 60°C for 12 hours to obtain light green Ni-MOF nanosheets.

[0016] An electrochemiluminescent aptamer sensor for detecting HER2 in breast cancer, characterized by comprising the following steps:

[0017] (1) Preparation of substrate materials;

[0018] The electrode was immersed in 1% (w / w) HAuCl4·4H2O solution and electrochemically deposited at a constant voltage of -0.2 V for 30 s to obtain the deposited gold nanoparticle (DpAu) film;

[0019] (2) Preparation of signal probes;

[0020] 1) Ni-MOF:

[0021] 166 mg of terephthalic acid (PTA) and 96 mg of nickel nitrate hexahydrate (Ni(NO3)2·6H2O) were stirred and dispersed in 20 mL of N,N-dimethylformamide (DMF). 2 mL of 0.4 M NaOH solution was then added dropwise while stirring. The resulting mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined reactor and reacted at 100°C for 8 hours. After the reaction, the mixture was cooled to room temperature and washed several times with large amounts of DMF and ethanol. The resulting precipitate was dried at 60°C for 12 hours to obtain light green Ni-MOF nanosheets.

[0022] 2) PTCA:

[0023] 100 mg of perylenetetracarboxylic acid dihydroanhydride (PTCDA) was dissolved in 10 mL of 0.1 M NaOH solution and stirred at 80°C for 2 h to obtain a yellow-green solution. 1.0 M HCl was then added dropwise to the solution until complete precipitation. The mixture was then centrifuged, washed with ultrapure water to a pH of 7.4, and dried at 60°C to obtain a dark red powder.

[0024] 3) PTCA@Ni-MOF:

[0025] 1.5 mg of Ni-MOF was dispersed in 1.5 mL of ultrapure water and evenly dispersed by ultrasonication. 450 μL of 1 mg / mL PTCA dispersion was added thereto. After stirring at room temperature for 12 h, the mixture was centrifuged and washed three times with ultrapure water. The precipitate was dispersed in 1 mL of ultrapure water to obtain a PTCA@Ni-MOF dispersion.

[0026] 4) Signal Probe (SP):

[0027] 200 μL of pDNA was slowly added to the PTCA@Ni-MOF dispersion and stirred in an ice bath for 12 h. The product was washed and centrifuged to obtain the signal probe. The synthesized signal probe was then centrifuged at 10,000 rpm. The final precipitate was redispersed in 500 μL of ultrapure water and stored at 4 °C for future use.

[0028] (3) Construction of electrochemiluminescence (ECL) aptamer sensor for HER2 detection:

[0029] 1) Treat the HER2-binding aptamer chain (APT) and the partially complementary chain of APT (cDNA) with 20 mM Tris-HCl (pH = 7.4) buffer at room temperature and store at 4°C until use;

[0030] 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;

[0031] 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.

[0032] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0033] 5) immersing the electrode obtained in step 4) in a 1% (w / w) HAuCl4·4H2O solution and electrochemically depositing at a constant voltage of -0.2 V for 30 s to obtain a deposited gold nanoparticle (DpAu) film;

[0034] 6) Add 10 μL of the cDNA prepared in step 1) to the electrode prepared in step 5) and incubate at 4°C for 12 h.

[0035] 7) Add 10 μL of 1% (w / w) BSA solution dropwise to the electrode obtained in step 6) and incubate at room temperature for 60 min.

[0036] 8) Add 10 μL of the APT prepared in step 1) onto the electrode prepared in step 7) and incubate at room temperature for 2 h.

[0037] 9) Add 10 μL of target HER2 to the electrode prepared in step 8) and incubate at room temperature for 50 min;

[0038] 10) Add 10 μL of the signal probe (SP) dropwise onto the electrode prepared in step 9) and incubate at room temperature for 2 h to obtain an electrochemiluminescent aptamer sensor for HER2 detection.

[0039] The present invention also provides a method for detecting HER2 using the electrochemiluminescence (ECL) aptamer sensor.

[0040] A method for detecting HER2 using the electrochemiluminescence (ECL) aptamer sensor is characterized by comprising the following steps:

[0041] 1) adding different concentrations of the target human epidermal growth factor receptor 2 (HER2) to the electrode of the sensor;

[0042] 2) The electrode was placed in a 0.1 M PBS (pH = 7.4) solution containing 50 mM K2S2O8 for characterization, and its electrochemiluminescence intensity was measured;

[0043] 3) Based on the linear relationship between the luminescence intensity and the logarithm of the HER2 concentration obtained in step 2), a working curve is drawn;

[0044] 4) The sample to be tested is detected by the sensor, and the obtained current value is calculated using the working curve prepared in step 3) to obtain the HER2 concentration of the sample to be tested.

[0045] Compared with the prior art, the preparation method and application of the electrochemiluminescence (ECL) aptamer sensor for detecting HER2 of the present invention have the following outstanding features:

[0046] This invention prepares a nickel-based metal-organic framework (Ni-MOF) composite nanomaterial (PTCA@Ni-MOF) modified with the luminescent material 3,4,9,10-perylenetetracarboxylic acid (PTCA) as the signal material for an electrochemiluminescent sensor. A large amount of partially complementary DNA (pDNA) to cDNA is then loaded via the bonding between the metal particles and amino groups, ultimately creating a PTCA@Ni-MOF / pDNA signal probe solution. Regarding material selection, since efficient electron transfer can amplify the signal of the electrochemical sensor, the electrode surface is modified with deposited gold to enhance electron transfer efficiency. Therefore, using electrochemically deposited gold as the substrate and PTCA@Ni-MOF as the signal material, the present invention enables sensitive detection of HER2, providing a new diagnostic approach for early diagnosis of breast cancer patients. The prepared electrochemiluminescent aptamer sensor successfully demonstrated ultrasensitive HER2 detection. Compared to traditional HER2 detection methods, this invention offers advantages such as high sensitivity, strong specificity, rapid detection, convenient operation, low-cost equipment and materials, and no pollution, thus providing a novel analytical method for HER2 detection.

[0047] The beneficial effects of the present invention are:

[0048] 1) Nickel-based metal-organic framework (Ni-MOF) composite nanomaterials (PTCA@Ni-MOF) modified with the luminescent material 3,4,9,10-perylenetetracarboxylic acid (PTCA) can greatly improve the shortcomings of PTCA aqueous solution, such as low luminescence efficiency and easy electrode passivation, thereby improving sensor performance, achieving signal amplification, and increasing detection sensitivity.

[0049] 2) Aptamers are highly specific for target recognition and can improve the selectivity of sensors, thus providing new research directions and analytical methods for the detection of trace amounts of HER2.

[0050] 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.

[0051] 5) The entire detection and analysis method has clear and simple steps, high sensitivity, and rapid signal response.

[0052] 6) The electrochemical aptasensor prepared by this method can provide a new method for the detection of HER2. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1Cyclic voltammetry (A) and impedance voltammetry (B) of different modified electrodes were obtained in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a voltage range of -0.2 to 0.7 V at a scan rate of 100 mV / s.

[0054] Figure 2 Figure 2 is the result of detecting different concentrations of HER2 using the sensor of the present invention, wherein Figure (A) is a time-electrochemiluminescence intensity graph of the sensor scanning 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL of HER2 in 50 mM K2S2O8 in 0.1 M PBS (pH 7.4); Figure (B) is a calibration curve of the sensor electrochemiluminescence intensity and the logarithmic value of different HER2 concentrations.

[0055] Figure 3 Figure 2 is the sensor performance test result. Figure (A) shows the time-electrochemiluminescence intensity graph of the sensor incubated with 1 ng / mL and 10 pg / mL HER2 after 12 consecutive scans. Figure (B) shows the reproducibility results of the sensor obtained by simultaneously incubating 10 ng / mL HER2 with five different glassy carbon electrodes and scanning under the same conditions. Figure (C) is the specificity detection graph of the HER2 aptamer sensor, where the interfering substances are dopamine (DA, 10 ng / mL), prostate-specific antigen (PSA, 10 ng / mL), and human serum albumin (HSA, 10 ng / mL), as well as a mixture of the three. Implementation Method

[0056] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0057] The main chemical reagents used in the embodiments of the present invention are as follows:

[0058] Recombinant human HER-2 protein was purchased from Abcam (UK); p-terephthalic acid (PTA), nickel nitrate hexahydrate (Ni(NO₃)₂·6H₂O), NaOH, and HCl were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); N-dimethylformamide (DMF) and perylenetetracarboxylic dihydroanhydride (PTCDA) were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China); potassium persulfate (K₂S₂O₂) and bovine serum albumin (BSA) were purchased from J&K Scientific Co., Ltd. (Beijing, China); and ethanol was purchased from Chuandong Chemical Group Co., Ltd. All reagents were of analytical grade and used without further purification.

[0059] The aptamer involved was synthesized by Shanghai Bioengineering Co., Ltd. The specific sequence is as follows:

[0060] APT: 5'-TCTAAAAGGATTCTTCCCAAGGGGATCCAATTCAAACAGC-3'

[0061] cDNA:5'-GGAAGAATCCTTTTAGATTTTT-(CH2)6-SH-3'

[0062] pDNA:5'-TCTAAAAGGATTCTTCCTTTTT-(CH2)6-NH-3'

[0063] Equipment used and technical parameters:

[0064] Instrumentation: Time / voltage-based electrochemiluminescence (ECL) intensity measurements were performed using an MPI-EⅡ 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 used a three-electrode system: a modified glassy carbon electrode (4 mm diameter) as the working electrode, a platinum wire as the counter electrode, and silver-silver chloride (saturated with KCl) as the reference electrode. Electrochemical detection used 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). UV-visible absorption spectra were measured using a UV-2600 spectrophotometer (Shimadzu, Japan). Electrochemiluminescence (ECL) was performed at 200 mV / s using a three-electrode system in 50 mM K2S2O8 in 0.1 M PBS (pH 7.4). Electrochemical measurements were performed at 100 mV / s using a three-electrode system in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution.

[0065] Example 1 Preparation of an electrochemiluminescent aptamer sensor for detecting HER2 in breast cancer

[0066] Follow these steps:

[0067] (1) Preparation of substrate materials;

[0068] 1) Gold deposition: The electrode was immersed in a 1% (w / w) HAuCl4·4H2O solution and electrochemically deposited at a constant voltage of -0.2 V for 30 s to obtain a deposited gold nanoparticle (DpAu) film.

[0069] (2) Preparation of signal probes;

[0070] 1) Ni-MOF:

[0071] 166 mg of p-terephthalic acid (PTA) and 96 mg of nickel nitrate hexahydrate (Ni(NO₃)₂·6H₂O) were stirred and dispersed in 20 mL of N,N-dimethylformamide (DMF). Then, 2 mL of 0.4 M NaOH solution was slowly added dropwise to the solution while stirring. The resulting mixture was transferred to a 50 mL polytetrafluoroethylene-lined reactor and reacted at 100°C for 8 hours. After completion of the reaction, the mixture was cooled to room temperature and washed several times with large amounts of DMF and ethanol. The resulting precipitate was dried at 60°C for 12 hours, yielding pale green Ni-MOF nanosheets.

[0072] 2) PTCA:

[0073] Dissolve 100 mg of perylenetetracarboxylic dihydroanhydride (PTCDA) in 10 mL of 0.1 M NaOH solution and stir at 80°C for 2 h to obtain a yellow-green solution. Then, add 1.0 M HCl dropwise until complete precipitation. The mixture is then centrifuged, washed with ultrapure water to a pH of 7.4, and dried at 60°C to obtain a dark red powder.

[0074] 3) PTCA@Ni-MOF:

[0075] 1.5 mg of Ni-MOF was dispersed in 1.5 mL of ultrapure water and evenly dispersed by ultrasonication. 450 μL of 1 mg / mL PTCA dispersion was added and stirred at room temperature for 12 h. The mixture was centrifuged and washed three times with ultrapure water. The precipitate was dispersed in 1 mL of ultrapure water to obtain a PTCA@Ni-MOF dispersion.

[0076] 4) Signal Probe (SP):

[0077] 200 μL of amino-labeled pDNA was slowly added to the PTCA@Ni-MOF dispersion and stirred in an ice bath for 12 h. The product was washed and centrifuged to obtain the signal probe. Then, the synthesized signal probe was centrifuged at 10,000 rpm. The final precipitate was redispersed in 500 μL of ultrapure water and stored at 4°C for future use.

[0078] (3) Construction of an electrochemiluminescent aptamer sensor for HER2 detection:

[0079] 1) Treat the HER2-binding aptamer chain (APT) and the partially complementary chain of APT (cDNA) with 20 mM Tris-HCl (pH = 7.4) buffer at room temperature and store at 4°C until use;

[0080] 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.

[0081] 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.

[0082] 4) Electrochemically activate the electrode obtained in step 3) in 0.5 M H2SO4, then rinse with ultrapure water and dry;

[0083] 5) immersing the electrode obtained in step 4) in a 1% (w / w) HAuCl4·4H2O solution and electrochemically depositing at a constant voltage of -0.2 V for 30 s to obtain a deposited gold nanoparticle (DpAu) film;

[0084] 6) Add 10 μL of the cDNA prepared in step 1) to the electrode prepared in step 5) and incubate at 4°C for 12 h.

[0085] 7) Add 10 μL of 1% (w / w) BSA solution dropwise to the electrode obtained in step 6) and incubate at room temperature for 60 min.

[0086] 8) Add 10 μL of the APT prepared in step 1) onto the electrode prepared in step 7) and incubate at room temperature for 2 h.

[0087] 9) Add 10 μL of target HER2 to the electrode prepared in step 8) and incubate at room temperature for 50 min;

[0088] 10) Add 10 μL of the signal probe (SP) dropwise onto the electrode prepared in step 9) and incubate at room temperature for 2 h to obtain an electrochemiluminescent aptamer sensor for HER2 detection.

[0089] Example 2 Detection of HER2 using electrochemiluminescent aptamer sensor

[0090] The electrochemiluminescent aptamer sensor constructed in Example 1 was used to detect HER2 in the following steps:

[0091] 1. Draw a working curve

[0092] 1) The modified electrodes from steps 5) to 9) of the electrochemiluminescent aptasensor for HER2 detection in Example 1 were placed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV and EIS characterization. The current response signals were measured, and the results were as follows: Figure 1As shown in A: (a) bare electrode; (b) gold deposition; (c) cDNA addition; (d) BSA solution addition for blocking; (e) APT addition; (f) HER2 addition; (g) signal probe addition. The impedance response signal is measured, and the results are shown in Figure 1. Figure 1 As shown in B: (a) bare electrode; (b) gold deposition; (c) addition of cDNA; (d) addition of BSA solution for blocking; (e) addition of APT; (f) addition of HER2; (g) addition of signal probe.

[0093] 2) 10 μL of target HER2 at different concentrations was added to the electrode of the aptamer sensor prepared in Example 1, and the luminescence intensity was measured. Figure 2 As shown in A: The concentrations from bottom to top are: 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL.

[0094] 3) 10 μL of target HER2 at different concentrations was added to the electrode of the aptamer sensor prepared in Example 1, and the luminescence intensity was measured. Figure 2 As shown in A: The concentrations from bottom to top are: 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL.

[0095] 4) Based on the linear relationship between the obtained luminescence intensity value and the logarithmic value of HER2 concentration, draw a working curve (such as Figure 2 The results showed that the luminescence intensity response value and the logarithm of HER2 concentration showed a good linear relationship in the range of 1 pg / mL-100 ng / mL, with a linear correlation coefficient of 0.9988 and a detection limit of 0.15 pg / mL.

[0096] 2. Sensor stability test: The sensor prepared in Example 1 was subjected to 12 consecutive cycles of ECL measurement under optimal conditions on electrodes incubated with 1 ng / mL and 10 pg / mL HER2. The luminescence intensities were 98.67% and 100.2% of the original values, respectively (e.g. Figure 3 A), indicating that the sensor has good stability.

[0097] 3. Sensor reproducibility test: The sensor prepared in Example 1 by incubating the same concentration of HER-2 (10 ng / mL) with five different glassy carbon electrodes was subjected to ECL measurement (e.g. Figure 3 B), the relative standard deviation (RSD) was 1.87%, indicating that the sensor had good reproducibility.

[0098] IV. Sensor Specificity Test: To investigate the specificity of the proposed adaptive sensor, potential interfering substances present in serum were tested: dopamine (DA, 10 ng / mL), prostate-specific antigen (PSA, 10 ng / mL), and human serum albumin (HSA, 10 ng / mL), as well as a mixture of the three. The electrochemiluminescence intensity responses of the various interfering substances were measured under the same concentrations and conditions in 0.1 M PBS (pH = 7.4) containing 50 mM K₂S₂O₄. The results showed that (e.g. Figure 3 C), the proposed aptamer sensor based on the high-specific response of HER2 has good specificity.

Claims

1. An electrochemiluminescent aptamer sensor for detecting HER2 in breast cancer, characterized in that: The method for constructing the electrochemiluminescent aptamer sensor for detecting HER2 in breast cancer comprises the following steps: (1) Preparation of substrate materials; The electrode was immersed in 1% (w / w) HAuCl4·4H2O solution and electrochemically deposited at a constant voltage of -0.2 V for 30 s to obtain the deposited gold nanofilm; (2) Preparation of signal probes; 1) Ni-MOF: 166 mg of terephthalic acid and 96 mg of nickel nitrate hexahydrate were stirred and dispersed in 20 mL of N,N-dimethylformamide. 2 mL of 0.4 M NaOH solution was then added dropwise while stirring. The resulting mixed solution was transferred to a 50 mL polytetrafluoroethylene-lined reactor and reacted at 100 °C for 8 hours. After the reaction, it was cooled to room temperature and washed several times with large amounts of DMF and ethanol. The resulting precipitate was dried at 60 °C for 12 hours to obtain light green Ni-MOF nanosheets. 2) PTCA: 100 mg of perylenetetracarboxylic acid dihydroanhydride was dissolved in 10 mL of 0.1 M NaOH solution and stirred at 80°C for 2 h to obtain a yellow-green solution. 1.0 M HCl was then added dropwise to the solution until complete precipitation. The mixture was then centrifuged, washed with ultrapure water to a pH of 7.4, and dried at 60°C to obtain a dark red powder. 3) PTCA@Ni-MOF: 1.5 mg of Ni-MOF was dispersed in 1.5 mL of ultrapure water and evenly dispersed by ultrasonication. 450 μL of 1 mg / mL PTCA dispersion was added thereto. After stirring at room temperature for 12 h, the mixture was centrifuged and washed three times with ultrapure water. The precipitate was dispersed in 1 mL of ultrapure water to obtain a PTCA@Ni-MOF dispersion. 4) Signal probe: 200 μL of pDNA was slowly added to the PTCA@Ni-MOF dispersion and stirred in an ice bath for 12 h. The product was washed and centrifuged to obtain the signal probe. The synthesized signal probe was then centrifuged at 10,000 rpm. The final precipitate was redispersed in 500 μL of ultrapure water and stored at 4 °C for future use. (3) Construction of an electrochemiluminescent aptamer sensor for HER2 detection: 1) Treat the HER2-binding aptamer chain APT and the partially complementary chain cDNA of APT with 20 mM Tris-HCl buffer (pH 7.4) 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) immersing the electrode obtained in step 4) in a 1% (w / w) HAuCl4·4H2O solution and electrochemically depositing the gold nanofilm at a constant voltage of -0.2 V for 30 s; 6) Add 10 μL of the cDNA prepared in step 1) to the electrode prepared in step 5) and incubate at 4°C for 12 h. 7) Add 10 μL of 1% (w / w) BSA solution dropwise to the electrode obtained in step 6) and incubate at room temperature for 60 min. 8) Add 10 μL of the APT prepared in step 1) onto the electrode prepared in step 7) and incubate at room temperature for 2 h. 9) Add 10 μL of target HER2 to the electrode prepared in step 8) and incubate at room temperature for 50 min; 10) Add 10 μL of the signal probe dropwise onto the electrode prepared in step 9) and incubate at room temperature for 2 h to obtain an electrochemiluminescent aptamer sensor for HER2 detection. The specific sequences of APT, cDNA and pDNA are as follows: APT: 5'-TCTAAAAGGATTCTTCCCAAGGGGATCCAATTCAAACAGC-3' cDNA:5'-GGAAGAATCCTTTTAGATTTTT-(CH2)6-SH-3' pDNA:5'-TCTAAAAGGATTCTTCCTTTTT-(CH2)6-NH-3'.

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