An electrochemiluminescent immunosensor for detecting CP4-EPSPS protein, its construction method, and its application.

Abstract

This invention provides an electrochemiluminescence immunosensor for detecting CP4-EPSPS protein, its construction method, and its applications, comprising the following steps: S1, antibody preparation; S2, preparation of GN-PAMAM-g-C3N4 composite material; S3, construction of the electrochemiluminescence immunosensor. According to this invention, a simple electrochemiluminescence immunosensor based on nitrogen-doped graphene, graphitic carbon nitride, and polyamide-amine composite material is proposed, which can be used to detect CP4-EPSPS protein in transgenic crops. When the RRS content in transgenic soybean is between 0.05% and 1.5%, the electrochemical reaction of the immunosensor is linear. The detection limit of this electrochemiluminescence immunosensor for transgenic soybean RRS is as low as 0.025%, and the immunosensor also exhibits high specificity and satisfactory stability, repeatability, and accuracy.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence immunosensors, and more specifically to an electrochemiluminescence immunosensor for detecting CP4-EPSPS protein, its construction method, and its applications. Background Technology

[0002] Genetically modified (GM) crops are crops whose DNA has been altered by introducing or inserting foreign genes into their genome. Compared to conventional crops, GM crops possess resistance to diseases and pests, or have increased nutritional content. Since 1996, GM crops have been commercially cultivated on a large scale. In 2019, the global area planted with GM crops was approximately 190.4 million hectares, an increase of more than 100 times. Soybeans, as one of the four major GM crops, account for 48% of the total GM crop planting area. Since the 1970s, glyphosate has been the most widely used herbicide in global agriculture due to its non-selective action against annual and perennial weeds. Since 1996, the glyphosate-resistant soybean variety RRS (Event GTS40-3-2) has been approved for commercial cultivation. It expresses the CP4-EPSPS protein from Agrobacterium rhizogenes, enabling the plant to develop tolerance to the glyphosate herbicide. However, public concerns remain regarding its environmental impact and safety as food and feed, making it necessary to test soybeans to determine if they are GM crops.

[0003] Methods for detecting genetically modified (GM) crops are typically based on detecting the inserted foreign gene or the specific protein expressed by the foreign gene. Based on these two principles, several main detection methods have emerged: PCR technology for gene detection, such as real-time quantitative PCR; and protein chip technology and immunoassay strip technology for detecting gene-expressed proteins. While PCR technology is sensitive, it requires sophisticated equipment, complex sample pretreatment (requiring crop genome extraction), and is time-consuming. Furthermore, PCR technology cannot detect protein levels in GM crops. Protein chip and immunoassay strip technologies primarily perform qualitative protein detection; although they have high specificity, they cannot achieve quantitative analysis and have relatively low sensitivity. Therefore, there is an urgent need to develop a new strategy that can detect GM crops with high sensitivity, speed, and quantification.

[0004] Electrochemiluminescence (ECL) immunosensors work by applying a voltage to electrodes, which induces high-energy electron transfer, exciting the ECL luminescent material. When the excited state returns to the ground state, luminescence occurs. During the specific binding of antigens and antibodies, the luminescence intensity changes, allowing the detection of protein concentration in the sample. Due to their high specificity, simplicity, speed, and low cost, ECL immunosensors have been widely used to detect malaria biomarkers, pathogens, pesticide residues, foodborne toxins, antibiotics, and other substances. However, an ECL sensor for detecting CP4-EPSPS has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide an electrochemiluminescence immunosensor for detecting CP4-EPSPS protein, its construction method, and its application, thereby solving the problem that existing detection methods for transgenic crops cannot simultaneously achieve high sensitivity, high stability, high repeatability, quantitative detection, and high accuracy.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] According to a first aspect of the present invention, a method for constructing an electrochemiluminescent immunosensor for detecting CP4-EPSPS protein is provided, comprising the following steps: S1, antibody preparation: diluting the antibody mAb to 0.3-0.8 mg / mL with 0.01 mol / L PBS at pH 7.4; S2, preparation of GN-PAMAM-g-C3N4 composite material: dispersing 1-2 mg of nitrogen-doped graphene (GN) solid sample in 1.7 mL of N,N-dimethylformamide, and adding 100-300 μL of graphitic carbon nitride (g-C3N4) solution after sonication for 1-1.5 hours, continuing sonication for 2-3 hours, and then adding 10-30 μL of [the composite material is missing here]. GN-PAMAM-g-C3N4 composite material was obtained by sonicating 20% ​​PAMAM for 1–1.5 hours. S3, Construction of the electrochemiluminescence immunosensor: The electrode surface was polished with alumina powder and washed with ultrapure water. 3–5 μL of the sonicated GN-PAMAM-g-C3N4 composite material was added to the cleaned electrode and dried under an infrared lamp. 3–5 μL of 5% glutaraldehyde solution was added to the composite material and reacted for 30 minutes to activate the amino groups in PAMAM. After the reaction, excess glutaraldehyde solution was washed away with ultrapure water, and 3 μL of antibody mAb was added. The reaction was carried out at room temperature in the dark for 30–40 minutes. The electrode surface was washed again, and 5 μL of PBS solution containing 5% BSA was added and reacted for 30–40 minutes to block non-specific binding sites. The surface was then washed with PBS to obtain an electrochemiluminescence immunosensor for detecting CP4-EPSPS protein.

[0008] Preferably, in step S2, the ratio of the amount of GN solid sample to g-C3N4 solution is 20 mg: 3 mL, and the amount of 20% PAMAM is 20 μL.

[0009] Preferably, in step S3, the electrode is selected from any one of glassy carbon electrode, gold electrode, and platinum electrode.

[0010] According to a second aspect of the present invention, an electrochemiluminescent immunosensor for detecting CP4-EPSPS protein is provided, which is constructed according to the above method.

[0011] According to a third aspect of the present invention, an electrochemiluminescence immunosensor is provided for the detection of transgenic crops, wherein the electrochemiluminescence immunosensor can be used for the detection of CP4-EPSPS protein in transgenic crops.

[0012] According to a preferred embodiment of the present invention, the application includes: 1) using an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as an auxiliary electrode, and the prepared electrochemiluminescence immunosensor as a working electrode, connecting them to a chemiluminescence detector, and connecting the electrochemical workstation and the chemiluminescence detector together; 2) measuring the electrochemiluminescence intensity in a mixed solution of 0.1M Na2S2O8 and 0.1M PBS, with a pH of 7.4, and the conditions for detecting CP4-EPSPS protein are: a voltage scan range from -1.5V to 0V, and scan rate, sampling interval, and settling time of 0.1V / S, 0.001V, and 2S, respectively; 3) performing all tests in triplicate, using the average signal intensity as the final result, and establishing a linear relationship between the luminescence intensity and the concentration of CP4-EPSPS protein by measuring the luminescence intensity corresponding to a series of different concentrations of transgenic crop water-extracted supernatant protein solutions. Based on this linear relationship, the concentration of CP4-EPSPS protein in unknown samples can be determined.

[0013] According to the electrochemiluminescence immunosensor provided by the present invention, the change in electrochemiluminescence intensity of RRS is proportional to the RRS concentration when the concentration of RRS standard containing CP4-EPSPS protein is in the range of 0.05% to 1.5%.

[0014] When the RRS content of genetically modified soybeans is between 0.05% and 1.5%, the electrochemical response of the immunosensor is linear (R... 2 =0.9954), the detection limit for RRS in genetically modified soybeans is as low as 0.025%. However, it should be understood that this invention is applicable to any genetically modified crop containing CP4-EPSPS protein, and is not limited to the detection of RRS in glyphosate-resistant soybean varieties.

[0015] According to a preferred embodiment of the present invention, step 3) includes: mixing standard protein powder of transgenic crop seeds with PBS solution of concentration of 0.01 mol / L, shaking vigorously for 3 to 5 minutes to ensure uniform mixing, then centrifuging at 8000 rpm for 5 minutes, collecting the supernatant, obtaining the concentration gradient required for detection by dilution, and during detection, dropping 5 μL of samples of different concentrations onto the electrochemiluminescence immunosensor, incubating at 37°C for 40 minutes, and then measuring the electrochemiluminescence intensity.

[0016] The inventiveness of this invention lies primarily in the first-ever construction of an electrochemiluminescence immunosensor for the qualitative and quantitative detection of CP4-EPSPS protein in genetically modified soybeans. This method uses a composite of nitrogen-doped graphene (GN) and graphitic carbon nitride (g-C3N4) as the substrate material and a CP4-EPSPS antibody as the recognition element. Application results in genetically modified crops demonstrate that this electrochemiluminescence immunosensor can detect genetically modified crops containing CP4-EPSPS protein. More importantly, the electrochemiluminescence immunosensor prepared according to this invention has a simple structure, provides quantitative detection results, and exhibits high detection sensitivity, demonstrating significant advantages.

[0017] In summary, this invention provides a simple electrochemiluminescence immunosensor based on a composite material of nitrogen-doped graphene, graphitic carbon nitride, and polyamide-amine (GN-PAMAM-g-C3N4) for detecting CP4-EPSPS protein in transgenic (GM) crops. In this immunosensor, signal amplification is achieved through carbon nitride. When the RRS content in transgenic soybeans is between 0.05% and 1.5%, the electrochemical response of the immunosensor is linear (R... 2 =0.9954). The detection limit for the RRS of transgenic soybeans was as low as 0.025%. This immunosensor also exhibited high specificity and satisfactory stability, repeatability, and accuracy. The results indicate that the constructed immunosensor provides a new method for highly sensitive and quantitative detection of CP4-EPSPS protein. Furthermore, by simply changing the antibody, this sensor can be applied to the detection of other proteins or pathogens, and can also be used for multi-component analysis. Therefore, the electrochemiluminescent immunosensor for detecting CP4-EPSPS protein provided by this invention has broad application prospects in the fields of transgenic detection or the detection of other proteins or pathogens. Attached Figure Description

[0018] Figure 1 The optimized conditions for the graphene and carbon nitride ratio are shown, with CV measurements (A) and ECL measurements (B).

[0019] Figure 2This is a schematic diagram illustrating the construction process of an electrochemiluminescence immunosensor based on GN-PAMAM-g-C3N4 composite material;

[0020] Figure 3 The CV measurements obtained at a scan rate of 50 mV / s using different electrodes in PBS (0.1 M, pH 7.4) are shown (A), and the ECL measurements obtained at a scan rate of 50 mV / s using different electrodes in Na2S2O8 (0.1 M, pH 7.4) are shown (B).

[0021] Figure 4 The performance analysis of the immunosensor is shown, including: standard curve for detecting soybean RRS (n=3) (A), specificity analysis of the sensor (B), stability analysis of the sensor: CV measurement (C), and ECL measurement (D). Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0023] Reagents and Instruments

[0024] Monoclonal antibodies (mAbs) against CP4-EPSPS protein were purchased from Shanghai Ronghui. PAMAM methanol solution (3rd generation, 20%) was purchased from Shanghai Maclean. GN and g-C3N4 were purchased from Xianfeng Nanomaterials Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma. Seed powder standards from transgenic crops used to test the performance of the immunosensors were purchased from ERM and AOCS, as shown in Table 1 below. All other chemicals and solvents were of analytical grade.

[0025] Table 1 Seed powder standards required for the experiment

[0026]

[0027] Electrochemical measurements were performed on a CHI 660E electrochemical workstation (Shanghai Chenhua), and electrochemical fluorescence luminescence measurements were performed on an MPI-E electrochemiluminescence detector (Xi'an Ruimai Company). A conventional three-electrode system was used: a glassy carbon electrode (GCE, d = 3 mm), a platinum electrode, and an Ag / AgCl electrode immersed in 3M saturated potassium chloride, serving as the working electrode, auxiliary electrode, and reference electrode, respectively. It should be understood that the working electrode is not limited to a glassy carbon electrode; it can also be a gold electrode, platinum electrode, etc.

[0028] Example 1: Preparation of GN-PAMAM-g-C3N4 composite material

[0029] 1.1 Preparation of GN-PAMAM-g-C3N4 composite material

[0030] The GN-PAMAM-g-C3N4 composite material was synthesized using a one-step ultrasonic composite method. 2 mg of nitrogen-doped graphene (GN) solid sample was dispersed in 1.7 mL of N,N-dimethylformamide (DMF), and after ultrasonic treatment for 1 hour, 300 μL of g-C3N4 solution was added, followed by another 3 hours of ultrasonic treatment. Then, 20 μL of 20% PAMAM was added to the composite material, and after another 1 hour of ultrasonic treatment, the synthesized composite material was obtained. This composite material is named GN-PAMAM-g-C3N4.

[0031] 1.2 Characterization of GN-PAMAM-g-C3N4 composite material

[0032] The GN-PAMAM-g-C3N4 composite material was prepared using a one-step ultrasonic method, which adsorbs PAMAM onto GN nanomaterials. SEM images were used to describe the micromorphology of the GN-PAMAM-g-C3N4 composite material coated on GCE.

[0033] Optimization of the ratio of 1.3GN to g-C3N4

[0034] The ratio of GN to g-C3N4 was determined by trying 20:1, 10:1, and 20:3 (mg:mL). At the optimal ratio, the current was approximately 220 μA, and the fluorescence ECL intensity was approximately 9000. Figure 1 As shown in A and B, the optimal ratio of GN to g-C3N4 is 20 mg: 3 mL, and the amount of PAMAM added is 20 μL.

[0035] Example 2: Construction of an electrochemiluminescence immunosensor based on GN-PAMAM-g-C3N4 composite material

[0036] 2.1 Antibody Preparation

[0037] Dilute the antibody mAb to 0.5 mg / mL with 0.01 mol / L PBS (pH 7.4), measure the protein concentration using NanoDrop, and store at 4°C protected from light.

[0038] 2.2 Preparation of GN-PAMAM-g-C3N4 composite material

[0039] The GN-PAMAM-g-C3N4 composite material was prepared using the method described in Example 1, wherein the ratio of GN to g-C3N4 was selected as 20:3. Specifically, 2 mg of nitrogen-doped graphene solid sample (GN) was dispersed in 1.7 mL of N,N-dimethylformamide (DMF), and after sonication for 1 hour, 300 μL of g-C3N4 solution was added, followed by sonication for another 3 hours. Then, 20 μL of 20% PAMAM was added to the composite material, and after sonication for another hour, the synthesized GN-PAMAM-g-C3N4 composite material was obtained.

[0040] 2.3 Construction of Electrochemiluminescence Immunosensor

[0041] The surface of the GCE electrode was successively polished with alumina powders of 1 μm, 0.3 μm, and 0.05 μm particle sizes, and then rinsed with ultrapure water. 5 μL of ultrasonically treated GN-PAMAM-g-C3N4 composite material was added dropwise to the cleaned GCE electrode and dried under an infrared lamp. After the surface was completely dry, 5 μL of 5% glutaraldehyde solution was added dropwise to the composite material and reacted for 30 minutes to activate the amino groups in PAMAM. After the reaction, excess glutaraldehyde solution was washed away with ultrapure water, and then 3 μL of mAb was added dropwise, reacting at room temperature in the dark for 40 minutes. The GCE electrode surface was cleaned again, and 5 μL of PBS solution containing 5% BSA was added dropwise and reacted for 40 minutes to block non-specific binding sites. After the reaction, the electrode was cleaned with PBS and stored at 4°C. A GCE / GN-PAMAM-g-C3N4 / mAb / BSA electrode was prepared, thus constructing an electrochemiluminescent immunosensor. The construction process is as follows: Figure 2 As shown.

[0042] 2.4 Electrochemical Measurement

[0043] Electrochemiluminescence intensity (ECL) measurements were performed in a mixed solution of 0.1 M Na2S2O8 and 0.1 M PBS (pH 7.4). The conditions for detecting CP4-EPSPS protein were: voltage scan range from -1.5 V to 0 V, scan rate, sampling interval, and settling time of 0.1 V / s, 0.001 V, and 2 s, respectively.

[0044] The signal strength change (ΔI) is calculated using the following formula:

[0045] ΔI=I-I0

[0046] Where I0 represents the peak ECL of the sample under test, and I represents the initial peak ECL of the electrode. All measurements were performed at room temperature.

[0047] To determine the characteristics of the assembly process, samples were taken from samples containing 5 mM [Fe(CN)6]. 3- / 4- CV measurements were performed using different electrodes in 0.1M KCl-containing PBS (0.1M, pH 7.4) at a scan rate of 50 mV / s and a scan range of -0.2V to 0.6V. Figure 3 ECL measurements were performed at a scan rate of 50 mV / s using different electrodes in PBS (0.1 M, pH 7.4) containing 0.1 M Na2S2O8 (as shown in A in the figure), and in PBS (0.1 M, pH 7.4) containing 0.1 M Na2S2O8. Figure 3 (As shown in B in the diagram).

[0048] The results are as follows Figure 3 As shown, the peak current of the bare GCE electrode is approximately 90 μA, while the peak current of the electrode modified with the GN-PAMAM-g-C3N4 composite material reaches 230 μA. Incubation with CP4-EPSPS further reduces the current. The changes on the electrode surface are significant, indicating that the electrochemiluminescence immunosensor was successfully assembled, and the GN-PAMAM-g-C3N4 composite material significantly enhances the current.

[0049] The electrode modified with the GN-PAMAM-g-C3N4 composite material achieved an ECL value of 9000. When the electrode was sealed with BSA and incubated with CP4-EPSPS, the signal value gradually weakened. The changes on the electrode surface were significant, indicating successful assembly of the electrochemiluminescence immunosensor, and the GN-PAMAM-g-C3N4 composite material significantly enhanced electrochemiluminescence.

[0050] Example 3: Sensitivity, specificity, and stability analysis of the electrochemiluminescence immunosensor

[0051] 3.1 Sensitivity Analysis

[0052] To evaluate the sensitivity of the electrochemiluminescence immunoassay sensor constructed in this invention for detecting CP4-EPSPS protein, this embodiment uses soybean, a crop with a large transgenic planting area, for detection. Under optimal conditions, different concentrations of soybean RRS were detected by ECL measurement. The ECL peak gradually decreased with increasing concentration because the increased amount of CP4-EPSPS protein on the electrode surface hindered electron transfer.

[0053] The results are as follows Figure 4 As shown in Figure A, within the range of 0.05% to 1.5%, the ECL intensity change (ΔI) of the RRS is directly proportional to the concentration. The linear regression equation for the RRS is as follows: ΔI = 4334.681x + 1139.879, showing a linear relationship (R... 2The sensitivity is very high, at 0.9954, and the detection limit for RRS samples is 0.025% (S / N = 3). Therefore, the sensitivity of the electrochemiluminescence immunosensor constructed according to the present invention is 0.025%.

[0054] 3.2 Specificity analysis

[0055] To evaluate the specificity of the electrochemiluminescence immunosensor for detecting CP4-EPSPS protein constructed in this invention, this embodiment also detected various transgenic crops containing 5% of different transgenic proteins, including: maize MIR162 (Bt-VIP3Aa), MIR604 (Bt-Cry3A), MON89034 (Bt-Cry1A105 / Cry2Ab), NK603 (CP4-EPSPS), TC1507 (Bt-Cry1F), 59122 (Cry34Ab1), rapeseed GT73 (CP4-EPSPS), soybean RRS (CP4-EPSPS), cotton MON88913 (CP4-EPSPS), and sugar beet H7-1 (CP4-EPSPS).

[0056] First, a 0.01 mol / L PBS solution was mixed with standard protein powders from different crop seeds at a mass ratio of 3:1, and the mixture was vigorously shaken for 3–5 minutes to ensure homogeneity. Then, the mixture was centrifuged at 8000 rpm for 5 minutes, and the supernatant was collected. The collected supernatant was diluted with 0.01 mol / L PBS, and the supernatant from the transgenic crop was diluted with the supernatant from the blank crop to obtain the concentration gradient required for detection. For detection, 5 μL of samples at different concentrations were dropped onto the prepared electrochemiluminescence immunoassay sensor and incubated at 37°C for 40 minutes.

[0057] All test results were performed in triplicate, with the average ECL intensity as the final result. Sensitivity and specificity data are expressed as mean ± standard deviation (SD) and calculated using Origin software.

[0058] The results are as follows Figure 4 As shown in B, this electrochemiluminescence immunosensor can detect corn, rapeseed, soybean, cotton and sugar beets containing CP4-EPSPS protein. The detection results are not affected by unknown matrix components or other proteins, such as BT-VIP3Aa, BT-Cry3A, Cry1A105, Cry2Ab, Cry1Ac or PAT.

[0059] 3.3 Stability Analysis

[0060] In this embodiment, the stability of the constructed electrochemiluminescence immunosensor was evaluated using GCE / GN-PAMAM-g-C3N4 / mAb / BSA as the working electrode. After storage at 4°C for 21 days, the operational stability of the sensor was studied by measuring the current values ​​under CV for 15 consecutive cycles and the current values ​​under ECL for 12 consecutive cycles. The results showed that the sensor retained 92.2% of the initial current and 81.9% of the initial ECL signal intensity, respectively.

[0061] The results are as follows Figure 4 As shown in C and D, stable current signal and ECL signal strengths were observed, with relative standard deviations (RSDs) of 0.60% and 2.13%, respectively.

[0062] In summary, the electrochemiluminescence immunosensor prepared according to the present invention exhibits satisfactory sensitivity, specificity, and stability.

[0063] Example 4: Actual Sample Analysis of the Electrochemiluminescence Immunosensor

[0064] To further verify the reliability of the electrochemiluminescence immunoassay sensor, recovery tests were conducted by adding different concentrations of RRS standards (0.05%, 0.5%, 1.0%, and 1.5%) to the corresponding blank samples, with the blank samples serving as controls. As shown in Table 2 below, the recoveries ranged from 98% to 114%, and the RSD was less than 10%, indicating that the sensor has good reliability.

[0065] Table 2 shows the recovery rate of the constructed electrochemiluminescence immunosensor for samples of known concentrations (n=3).

[0066]

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A method for constructing an electrochemiluminescence immunosensor for detecting CP4-EPSPS protein, characterized in that, Includes the following steps: S1, Antibody preparation: Dilute the antibody mAb to 0.3–0.8 mg / mL with 0.01 mol / L PBS at pH 7.4; S2, Preparation of GN-PAMAM-g-C3N4 composite material: 1-2 mg of GN solid sample was dispersed in 1.7 mL of N,N-dimethylformamide, and after sonication for 1-1.5 hours, 100-300 μL of g-C3N4 solution was added, and sonication was continued for 2-3 hours. Then, 10-30 μL of 20% PAMAM was added to the composite material and sonicated again for 1-1.5 hours to obtain GN-PAMAM-g-C3N4 composite material; S3, Construction of the electrochemiluminescence immunosensor: The electrode surface was polished with alumina powder and washed with ultrapure water. 3-5 μL of ultrasonically treated GN-PAMAM-g-C3N4 composite material was added to the cleaned electrode and dried under an infrared lamp. 3-5 μL of 5% glutaraldehyde solution was added to the composite material and reacted for 30 minutes to activate the amino groups in PAMAM. After the reaction, excess glutaraldehyde solution was washed away with ultrapure water, and 3 μL of antibody mAb was added. The reaction was carried out at room temperature in the dark for 30-40 minutes. The electrode surface was washed again, and 5 μL of PBS solution containing 5% BSA was added and reacted for 30-40 minutes to block non-specific binding sites. Finally, the electrode was washed with PBS to obtain an electrochemiluminescence immunosensor for detecting CP4-EPSPS protein.

2. The construction method according to claim 1, characterized in that, In step S2, the ratio of the amount of GN solid sample to g-C3N4 solution is 20 mg: 3 mL, and the amount of 20% PAMAM is 20 μL.

3. The construction method according to claim 1, characterized in that, In step S3, the electrode is selected from any one of glassy carbon electrode, gold electrode, and platinum electrode.

4. An electrochemiluminescent immunosensor for detecting CP4-EPSPS protein obtained by the construction method according to any one of claims 1 to 3.

5. An application of the electrochemiluminescence immunosensor according to claim 4 in the detection of transgenic crops, characterized in that, The electrochemiluminescence immunosensor can be used to detect CP4-EPSPS protein in transgenic crops.

6. The application according to claim 5, characterized in that, The applications include: 1) Using the Ag / AgCl electrode as the reference electrode, the platinum wire electrode as the auxiliary electrode, and the prepared electrochemiluminescence immunosensor as the working electrode, connect them to the chemiluminescence detector, and connect the electrochemical workstation and the chemiluminescence detector together. 2) Electrochemiluminescence intensity was measured in a mixed solution of 0.1M Na2S2O8 and 0.1M PBS. The pH of the mixed solution was 7.

4. The conditions for detecting CP4-EPSPS protein were: voltage scan range from -1.5V to 0V, scan rate, sampling interval and settling time of 0.1V / s, 0.001V and 2s, respectively. 3) All test results were performed in three parallel experiments, and the average signal intensity was used as the final result. The luminescence signal intensity corresponding to a series of transgenic crop standard protein solutions of different concentrations was measured to establish a linear relationship between luminescence intensity and CP4-EPSPS protein concentration. Based on this linear relationship, the concentration of CP4-EPSPS protein in unknown samples can be determined.

7. The application according to claim 6, characterized in that, Within the range of 0.05% to 1.5% concentration of RRS standards containing CP4-EPSPS protein, the change in electrochemiluminescence intensity of RRS is directly proportional to the RRS concentration.

8. The application according to claim 6, characterized in that, Step 3) includes: mixing standard protein powder from transgenic crop seeds with a 0.01 mol / L PBS solution, shaking vigorously for 3-5 minutes to ensure uniform mixing, then centrifuging at 8000 rpm for 5 minutes, collecting the supernatant, obtaining the concentration gradient required for detection by dilution, and during detection, dropping 5 μL of samples of different concentrations onto the electrochemiluminescence immunosensor, incubating at 37°C for 40 minutes, and then measuring the electrochemiluminescence intensity.

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