Electrochemiluminescence immunosensor for neuron-specific enolase detection

Abstract

This invention provides an electrochemiluminescent immunosensor for the detection of neuron-specific enolases (NSEs). Using a tetraphenylethylene metal-organic framework (La-TCBPE MOF) as the luminescent material, a large amount of secondary antibody Ab2 is loaded through carboxylation with EDC and NHS followed by amino group bonding, ultimately preparing a La-TCBPE / Ab2 signal probe solution. Combined with electrodeposited Au (dpAu) as the sensing interface, the constructed immunosensor achieves ultrasensitive detection of NSEs, providing a novel detection method for clinical NSEs.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection technology, specifically an electrochemiluminescent immunosensor for detecting neuron-specific enolases and its detection method. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine malignancy, accounting for approximately 13%-15% of all lung cancers. It has a rapid doubling time and metastasis rate, and a poor prognosis, with about 70% of patients diagnosed at an advanced stage. Neuron-specific enolase (NSE) is a cell-specific isotope of the glycolytic enzyme enolase, and its serum concentration is correlated with tumor progression, recurrence, and treatment efficacy in SCLC patients. Currently, NSE is the most reliable tumor marker for the diagnosis, prevention, and follow-up of small cell lung cancer (SCLC). The normal reference range for NSE is 0-16.3 ng / mL, and elevated NSE concentrations in body fluids may be associated with malignant proliferation. Therefore, early diagnosis and treatment are crucial for reducing lung cancer mortality.

[0003] Currently, the main methods for detecting NSE include liquid biopsy, high-performance liquid chromatography, PCR / FISH, colorimetry, spectral analysis, and fluorescence analysis. These methods face numerous disadvantages in clinical testing due to their high cost, lack of standardized testing criteria, cumbersome operation, expensive equipment, the need for professional training of operators, and long testing times. Summary of the Invention

[0004] To address the problems in existing technologies, this invention provides an electrochemiluminescent immunosensor for detecting neuron-specific enolases. The inventors used a tetraphenylethylene metal-organic framework (La-TCBPE MOF) as the ECL signal emitter. After activating the carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), the carboxyl groups were linked to a secondary antibody (Ab2) to prepare a La-TCBPE / Ab2 signal probe solution. The La-TCBPE luminescent material of this invention, as the ECL signal emitter, exhibits strong ECL intensity and stability, increases the light signal intensity of the biosensor, and, after carboxylation, becomes an effective binding site for the secondary antibody (Ab2), increasing the Ab2 loading. Simultaneously, deposition of Au (dpAu) improves conductivity and surface area, and allows for the binding of a large amount of primary antibody (Ab1) through Au-N bonds to generate the immunosensor. Therefore, the immunosensor constructed in this invention, using deposited Au (dpAu) as the sensor interface and La-TCBPE / Ab2 as the signal probe, achieves ultrasensitive detection of NSE, providing a novel detection method for clinical NSE.

[0005] Unless otherwise specified, all parts mentioned in this invention are parts by weight, and all percentages are mass percentages.

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

[0007] An electrochemiluminescent immunosensor for detecting neuron-specific enolase is characterized by the following method: A dry electrode is electrodeposited in HAuCl4 solution to form an Au electrode (deAu / GCE). Then, an Ab1 solution is added to the Au electrode and incubated at 4-6°C for 10-15 hours. Next, bovine serum albumin solution is added and incubated at 4-6°C for 30-50 minutes. Then, NSE solution is added and incubated at 4-6°C for 50-70 minutes. Finally, a signal probe solution is added and incubated at 4-6°C for 100-150 minutes, resulting in the electrochemiluminescent immunosensor for detecting neuron-specific enolase.

[0008] According to one embodiment of the present invention, the method for preparing the signal probe (tracer label) solution is as follows: EDC and NHS are dissolved in MES solution to obtain MES buffer. The MES buffer is slowly added to La-TCBPE dispersion under room temperature with a magnetic stirrer. After stirring for 20-40 min, Ab2 is added, and the mixture is stirred in an ice bath for 10-12 h. Then, the mixture is centrifuged, washed with ultrapure water, and the precipitate is redispersed in ultrapure water to obtain the signal probe (tracer label) solution.

[0009] According to one embodiment of the present invention, the preparation steps of the La-TCBPE are as follows: La(NO3)3·6H2O and H4TCBPE are dissolved in DMF, CH3COOH is added, ultrasonic treatment is performed for 2-5 min, and then the mixture is heated under reflux at 100-140℃ in a silicone oil bath for 5-8 h to separate and precipitate La-TCBPE.

[0010] Furthermore, the preparation steps of La-TCBPE according to the present invention are as follows: weigh 13.51 mg La(NO3)3·6H2O and 25.37 mg H4TCBPE and add them to a mixed solution of 3 mL DMF and 200 μL CH3COOH. Sonicate for 3 min, then heat under reflux at 120 °C in a silicone oil bath for 6 h. Finally, wash the obtained bright yellow precipitate with distilled water and anhydrous ethanol and dry it in a vacuum drying oven at 60 °C.

[0011] Specifically, the electrochemiluminescent immunosensor for detecting neuron-specific enolase is characterized by the following method for constructing the electrochemiluminescent immunosensor for detecting neuron-specific enolase:

[0012] (1) Preparation of signal probes:

[0013] 1) La-TCBPE:

[0014] Weigh 13.51 mg La(NO3)3·6H2O and 25.37 mg H4TCBPE and add them to a mixed solution of 3 mL DMF and 200 μL CH3COOH. Sonicate for 3 min, then heat under reflux at 120 °C in a silicone oil bath for 6 h. Finally, wash the resulting bright yellow precipitate with distilled water and anhydrous ethanol and dry it in a vacuum drying oven at 60 °C.

[0015] 2) Signal probe (tracer label) solution:

[0016] Weigh 153.36 mg EDC and 23 mg NHS and dissolve them in 1 mL MES solution to obtain MES buffer. Under room temperature, slowly add 1 mL MES buffer to 1 mL of 1 mg / mL La-TCBPE dispersion prepared in step 1). Stir for 30 minutes, then add 200 μL Ab2 (10 μg / mL), stir in an ice bath for 12 h, then centrifuge, wash with ultrapure water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the signal probe (Tracer label) solution.

[0017] (2) Au deposition process:

[0018] The glassy carbon electrode was polished for 3 min using 0.3 μm and 0.5 μm Al2O3, respectively, and then cleaned with ultrapure water and anhydrous ethanol to obtain a smooth interface. Then, an Au electrode (deAu / GCE) was formed by electrodeposition at -0.2 V for 30 s in HAuCl4 (1%) solution.

[0019] (3) Construction of electrochemiluminescence immunosensor

[0020] 1) Treat NSE-binding primary antibody (Ab1) and secondary antibody (Ab2) with 20mM Tris-HCl (pH=7.4) buffer at room temperature;

[0021] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 minutes, then rinse it with ultrapure water and set aside.

[0022] 3) Polish the electrodes obtained in step 2) to a mirror finish with Al2O3 powder of 0.3 μm and 0.05 μm respectively. Then, ultrasonically treat the electrodes in the order of ultrapure water and anhydrous ethanol, and dry them for subsequent use.

[0023] 4) The electrode obtained in step 3) was electrochemically activated in 0.5 M H2SO4, then rinsed with ultrapure water and dried;

[0024] 5) Electrodeposit the electrode from step 4) in HAuCl4 (1%) solution at -0.2V for 30s to form an Au electrode (deAu / GCE);

[0025] 6) Add 10 μL of Ab1 (10 μg / mL) solution dropwise to the electrode obtained in step 5) and incubate at 4°C for 12 h.

[0026] 7) Add 10 μL of 1% bovine serum albumin (BSA) to the electrode obtained in step 6) and incubate at 4°C for 40 min.

[0027] 8) Add 10 μL of NSE solution of different concentrations to the electrode obtained in step 7) and incubate at 4°C for 60 min.

[0028] 9) Add 10 μL of the tracel label solution described in claim 1 to the electrode prepared in step 8) and incubate at 4°C for 120 min to obtain an electrochemiluminescent immunosensor for NSE detection.

[0029] The present invention also provides a method for detecting NSE using the above-mentioned electrochemiluminescence immunoassay sensor.

[0030] According to one embodiment of the present invention, a method for detecting NSE using the above-described electrochemiluminescence immunoassay sensor is characterized by comprising the following steps:

[0031] 1) Different concentrations of the target substance NSE were dropped onto the electrodes of the sensor;

[0032] 2) The electrode in which NSE was added in step 1) was placed in a 0.1M PBS (pH=7.0) solution containing 100mM TEA for characterization, and its luminescence intensity was measured;

[0033] 3) Based on the linear relationship between luminescence intensity and the logarithm of NSE concentration obtained in step 2), plot the working curve;

[0034] 4) Detect the sample to be tested using the sensor described above, and calculate the NSE concentration of the sample to be tested by using the obtained current value through the working curve obtained in step 3).

[0035] Compared with existing technologies, this invention provides an electrochemiluminescent immunosensor for detecting NSE, with the outstanding advantage of using La-TCBPE as a novel ECL luminescent material and, for the first time, forming a binary ECL system with TEA as a co-reaction reagent, thereby achieving signal amplification and improving sensor sensitivity. This invention prepares a tetraphenylethylene metal-organic framework (La-TCBPE MOF) luminescent material, which is then carboxylated with EDC and NHS and bonded to amino groups to load a large amount of secondary antibody Ab2, ultimately preparing a La-TCBPE / Ab2 signal probe solution. Combined with electrodeposited Au (dpAu) as the sensing interface, a large amount of primary antibody (Ab1) is captured while improving the sensor's conductivity. The electrochemiluminescent immunosensor prepared by this invention has been successfully used for ultrasensitive detection of NSE. Compared with traditional NSE detection methods, this invention offers advantages such as high sensitivity, rapid detection, strong specificity, convenient operation, and inexpensive equipment and materials, providing a completely new analytical method for NSE detection.

[0036] The beneficial effects of this invention are:

[0037] 1) The use of tetraphenylethylene metal-organic framework (La-TCBPE MOF) luminescent material can greatly improve the low luminescence efficiency and poor luminescence stability of ligands, thereby improving sensor performance, achieving signal amplification, and increasing detection sensitivity.

[0038] 2) Using deposited Au as the substrate material has good conductivity and allows Ab1 to be fixed on the electrode.

[0039] 3) Antibodies have high specificity for the identification of targets, which can improve the selectivity of sensors, thus providing a new research direction and analytical method for the detection of trace NSE.

[0040] 5) All materials involved can be synthesized under laboratory conditions, which is simple to operate, the raw materials are inexpensive, and the amount used each time is very small, thus reducing the experimental cost.

[0041] 5) The entire detection and analysis method is clear and simple, highly sensitive, and has a rapid signal response.

[0042] 6) The electrochemiluminescence immunosensor prepared by this method can provide a new method for the detection of NSE; the electrochemiluminescence immunosensor prepared by this invention can also be applied to other applications such as food and drug, biological sample determination and environmental monitoring. Attached Figure Description

[0043] Figure 1 This is a comparison graph of the electrochemiluminescence signal intensity when using blank detectable material and when using NSE target material.

[0044] Figure 2 These are cyclic voltammetric graphs obtained with different modified electrodes in 5mM K3[Fe(CN)6] / K4[Fe(CN)6] solution at a scan rate of 100mV / s, covering a voltage range from -0.2 to 0.6V.

[0045] Figure 3 The results show the detection of different concentrations of NSE by the sensor of this invention. Figure A is a time-electrochemiluminescence intensity graph of NSE scanned by the sensor in 0.1M PBS (pH 7.0) with 100mM TEA, at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL, respectively. Figure B is a calibration curve of the sensor's electrochemiluminescence intensity versus the logarithmic value of different concentrations of NSE.

[0046] Figure 4 This is a time-electrochemiluminescence intensity graph obtained after 13 consecutive scans of a sensor incubated with 100 pg / mL NSE.

[0047] Figure 5 This is a graph showing the reproducibility of five sensors obtained from the same batch of NSE incubated with 100 pg / mL NSE under the same conditions.

[0048] Figure 6This is a specific detection diagram of the immunosensor. Among them, interfering agent 1 is glucose (Glu, 100 ng / mL), 2 is ascorbic acid (AA, 100 ng / mL), 3 is carcinoembryonic antigen (CEA, 100 ng / mL), 4 is dopamine (DA, 100 ng / mL), 5 is human serum albumin (HAS, 100 ng / mL), 6 is a control without interfering agents 1-5 and NSE, and 7 is a mixed sample containing interfering agents 1-5 and NSE (NSE, 1 ng / mL). The detection substance is neuron-specific enolase (NSE, 1 ng / mL). Detailed Implementation

[0049] The invention will now be described in further detail with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and the embodiments described herein should in no way be construed as limiting to these embodiments. Rather, the embodiments should be understood to cover any and all variations that become apparent from the teachings provided herein.

[0050] The main chemical reagents used in the embodiments of this invention are as follows: neuron-specific enolase (NSE) antigen and antibody (Ab1, Ab2) were purchased from Linc-Bio Science Co., Ltd. (Shanghai, China); lanthanum nitrate hexahydrate, tetrastyrene tetracarboxylic acid (H4TCBPE), chloroauric acid (HAuCl4), triethylamine (TEA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); N,N-dimethylformamide (DMF) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China); glacial acetic acid was purchased from Chongqing Chuandong Chemical (Group) Co., Ltd. (Chongqing, China); and bovine serum albumin (BSA) was purchased from Jekais Technology (Beijing) Co., Ltd. (Beijing, China).

[0051] Equipment used and technical parameters:

[0052] Instrumentation: Time / voltage-electrochemiluminescence intensity was measured using an MPI-E electrochemiluminescence workstation (Xi'an, China). Cyclic voltammetry (CV) was performed using a Metrohm Autolab BV electrochemiluminescence workstation (Modular, Switzerland). A three-electrode system was used for electrochemiluminescence detection: a modified glassy carbon electrode (4 mm in diameter) as the working electrode, a platinum wire as the counter electrode, and silver-silver chloride (saturated KCl) as the reference electrode.

[0053] Electrochemical detection employed a three-electrode system: a modified glassy carbon electrode (4 mm in 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 electrochemiluminescence three-electrode system was scanned at 100 mV / s in 0.1 M PBS (pH 7.0) containing 100 mM TEA. The electrochemical three-electrode system was also scanned at 100 mV / s in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution.

[0054] Example 1

[0055] (1) Preparation of signal probes

[0056] 1) La-TCBPE: Weigh 13.51 mg La(NO3)3·6H2O and 25.37 mg H4TCBPE and add them to a mixed solution of 3 mL LDM and 200 μL CH3COOH. Sonicate for 3 min. Then, transfer the solution to a 50 mL round flask and reflux in a silicone oil bath at 120 °C for 6 h. Finally, wash the resulting bright yellow precipitate several times with distilled water and anhydrous ethanol, and dry it in a vacuum drying oven at 60 °C.

[0057] 2) Tracer label: Weigh 153.36 mg EDC and 23 mg NHS and dissolve them in 1 mL MES solution to obtain MES buffer. Under room temperature, slowly add 1 mL of MES buffer to 1 mL of the 1 mg / mL La-TCBPE dispersion prepared in step 1). After stirring for 30 minutes, add 200 μL Ab2 (10 μg / mL), stir in an ice bath for 12 h, then centrifuge, wash with ultrapure water, and redisperse the precipitate in 1 mL of ultrapure water to obtain the tracer label solution.

[0058] (2) Operation of Au deposition substrate

[0059] 1) The glassy carbon electrode was polished for 3 min each using 0.3 μm and 0.5 μm Al2O3, and then washed with ultrapure water and anhydrous ethanol to obtain a smooth interface. Then, an Au electrode (deAu / GCE) was formed by electrodeposition at -0.2 V for 30 s in HAuCl4 (1%) solution.

[0060] (3) Construction of an electrochemiluminescence immunosensor for NSE detection

[0061] Follow these steps:

[0062] 1) Treat NSE-binding primary antibody (Ab1) and secondary antibody (Ab2) with 20mM Tris-HCl (pH=7.4) buffer at room temperature.

[0063] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 minutes, then rinse it with ultrapure water and set aside.

[0064] 3) Polish the electrodes obtained in step 2) to a mirror finish with Al2O3 powder of 0.3 μm and 0.05 μm respectively. Then, ultrasonically treat the electrodes in the order of ultrapure water and anhydrous ethanol, and dry them for subsequent use.

[0065] 4) The electrode obtained in step 3) was electrochemically activated in 0.5 M H2SO4, then rinsed with ultrapure water and dried;

[0066] 5) Electrodeposit the electrode from step 4) in HAuCl4 (1%) solution at -0.2V for 30s to form an Au electrode (deAu / GCE).

[0067] 6) Add 10 μL of Ab1 (10 μg / mL) solution dropwise to the electrode obtained in step 5) and incubate at 4°C for 12 h.

[0068] 7) Add 10 μL of 1% bovine serum albumin (BSA) to the electrode obtained in step 6) and incubate at 4°C for 40 min.

[0069] 8) Add 10 μL of NSE solution of different concentrations to the electrode obtained in step 7) and incubate at 4°C for 60 min.

[0070] 9) Add 10 μL of the tracel label solution described in claim 1 to the electrode prepared in step 8) and incubate at 4°C for 120 min to obtain an electrochemiluminescent immunosensor for NSE detection.

[0071] Example 2: Detection of NSE using an electrochemiluminescence immunosensor

[0072] The detection of NSE using the electrochemiluminescence immunosensor constructed in Example 1 was performed according to the following steps:

[0073] I. Drawing the working curve

[0074] 1) The modified electrode from Example 1 was used to detect the blank substance and NSE protein, and their electrochemiluminescence response signals were measured. The results are as follows: Figure 1 :

[0075] 2) The modified electrodes from steps 5) to 8) in the construction of the electrochemiluminescent immunosensor for NSE detection in Example 1 were placed in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV characterization. Their current response signals were measured, and the results are as follows: Figure 2 As shown: (a) bare glassy carbon electrode; (b) deposition of AuNPs; (c) addition of Ab1; (d) addition of BSA for sealing; (e) addition of NSE.

[0076] 3) 10 μL of the target compound NSE at different concentrations was added to the electrode of the immunosensor prepared in Example 1, and the luminescence intensity was measured respectively. For example... Figure 3 As shown in Figure A, the concentrations of a→g are 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL, respectively.

[0077] 4) Based on the linear relationship between the obtained luminescence intensity value and the logarithm of NSE concentration, plot the working curve (e.g., Figure 3 (As shown in B). The results showed that the luminescence intensity response value and the logarithm of NSE concentration had a good linear relationship in the range of 100 fg mL⁻¹ to 100 ng mL⁻¹, with a linear correlation coefficient of 0.9977 and a detection limit of 0.18 fg mL⁻¹; the results are as follows. Figure 3 As shown in B.

[0078] II. Sensor Stability Test: After performing 13 consecutive ECL measurements under optimal conditions, the sensor prepared in Example 1 showed no significant fluctuations in luminous intensity (e.g., ...). Figure 4 As shown in the figure, this indicates that the sensor has good stability.

[0079] III. Sensor Reproducibility Test: Sensors prepared in Example 1 using different glassy carbon electrodes incubated with the same concentration of NSE (100 pg / mL) were subjected to ECL measurements (e.g....). Figure 5 As shown in the figure, the relative standard deviation (RSD) of the batch difference was 1.67%, indicating that the sensor has good reproducibility.

[0080] IV. Sensor Specificity Test: To investigate the specificity of the proposed immunosensor, interfering substances that may exist in blood were used: glucose (Glu, 100 ng / mL), ascorbic acid (AA, 100 ng / mL), carcinoembryonic antigen (CEA, 100 ng / mL), dopamine (DA, 100 ng / mL), and human serum albumin (HSA, 100 ng / mL). The electrochemiluminescence intensity response values ​​of different interfering substances in 0.1 M PBS (pH = 7.0) containing 100 mM TEA were measured under the same concentration and conditions. The results (e.g.) Figure 6As shown in the figure, the proposed immunosensor based on the high specificity of NSE reaction has good specificity.

Claims

1. An electrochemiluminescent immunosensor for detecting neuron-specific enolases, characterized in that, The electrochemiluminescent immunosensor for detecting neuron-specific enolase is constructed as follows: A dried electrode is electrodeposited in HAuCl4 solution to form an Au electrode. Then, Ab1 solution is added dropwise to the Au electrode and incubated at 4-6℃ for 10-15 h. Next, bovine serum albumin solution is added dropwise and incubation continues at 4-6℃ for 30-50 min. Then, NSE solution is added dropwise and incubation continues at 4-6℃ for 50-70 min. Finally, a signal probe solution is added dropwise and incubation continues at 4-6℃ for 100-150 min, yielding the electrochemiluminescent immunosensor for detecting neuron-specific enolase. The signal probe solution is prepared as follows: EDC and NHS are dissolved in MES solution to obtain MES buffer. The MES buffer is slowly added to La-TCBPE dispersion at room temperature using a magnetic stirrer. After stirring for 20-40 min, Ab2 is added, and the mixture is stirred in an ice bath for 10-12 minutes. h, then centrifuged, washed with ultrapure water, and the precipitate was redispersed in ultrapure water to obtain the signal probe solution; the preparation steps of La-TCBPE are as follows: La(NO3)3·6H2O and H4TCBPE are dissolved in DMF, CH3COOH is added, ultrasonic treatment is performed for 2-5 min, and then heated under reflux at 100-140℃ in a silicone oil bath for 5-8 h to separate the precipitate La-TCBPE.

2. The electrochemiluminescence immunosensor as described in claim 1, characterized in that, The preparation steps of La-TCBPE are as follows: Weigh 13.51 mg La(NO3)3·6H2O and 25.37 mg H4TCBPE and add them to a mixed solution of 3 mL DMF and 200 µL CH3COOH. Sonicate for 3 min, then heat under reflux at 120 °C in a silicone oil bath for 6 h. Finally, wash the obtained bright yellow precipitate with distilled water and anhydrous ethanol and dry it in a vacuum drying oven at 60 °C.

3. An electrochemiluminescent immunosensor for detecting neuron-specific enolases, characterized in that, The method for constructing the electrochemiluminescent immunosensor for detecting neuron-specific enolases is as follows: (1) Preparation of signal probes: 1) La-TCBPE: Weigh 13.51 mg La(NO3)3·6H2O and 25.37 mg H4TCBPE and add them to a mixed solution of 3 mL DMF and 200 µL CH3COOH. Sonicate for 3 min, then heat under reflux in a silicone oil bath at 120 °C for 6 h. Finally, wash the resulting bright yellow precipitate with distilled water and anhydrous ethanol and dry it in a vacuum drying oven at 60 °C. 2) Signal probe solution: Weigh 153.36 mg EDC and 23 mg NHS and dissolve them in 1 mL MES solution to obtain MES buffer. Under room temperature, slowly add 1 mL MES buffer to 1 mL of the 1 mg / mL La-TCBPE dispersion prepared in step 1). After stirring for 30 minutes, add 200 µL of Ab2 solution with a concentration of 10 µg / mL. Stir in an ice bath for 12 h, then centrifuge and wash with ultrapure water. Redisperse the precipitate in 1 mL of ultrapure water to obtain the signal probe solution. (2) Construction of electrochemiluminescence immunosensor 1) Treat NSE with 20 mM Tris-HCl buffer (pH 7.4) at room temperature to bind primary antibody Ab1 and secondary antibody Ab2; 2) Soak the glassy carbon electrode in a piranha solution with a volume ratio of 98% H2SO4 / 30% H2O2 = 3:1 for 30 min, then rinse it with ultrapure water and set it aside. 3) Polish the electrodes obtained in step 2) to a mirror finish with Al2O3 powder of 0.3 μm and 0.05 μm respectively. Then, ultrasonically treat the electrodes in the order of ultrapure water and anhydrous ethanol, and dry them for subsequent use. 4) The electrode obtained in step 3) was electrochemically activated in 0.5 M H2SO4, then rinsed with ultrapure water and dried; 5) The electrode from step 4) is electrodeposited in a 1% HAuCl4 solution at -0.2 V for 30 s to form an Au electrode deAu / GCE; 6) Add 10 µL of Ab1 solution with a concentration of 10 µg / mL to the electrode obtained in step 5) and incubate at 4°C for 12 h. 7) Add 10 µL of 1% bovine serum albumin (BSA) to the electrode obtained in step 6) and incubate at 4°C for 40 min. 8) Add 10 µL of NSE solution of different concentrations to the electrode obtained in step 7) and incubate at 4°C for 60 min. 9) Add 10 µL of signal probe solution to the electrode prepared in step 8) and incubate it in a refrigerator at 4°C for 120 min to obtain an electrochemiluminescent immunosensor for NSE detection.

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