Transgenic crop detection method in one-step method signal enhancement mode

Abstract

The invention discloses a transgenic crop detection method in a one-step method signal enhancement mode, aminated CdSe / ZnS quantum dots (QDs) are coupled with an antigen to be detected, and an electrochemical luminescence detection signal can be enhanced by adopting a one-step incubation method, so that the detection time is remarkably shortened, and an antibody is saved.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay detection, and specifically relates to a transgenic detection method with a "one-step" signal enhancement mode. Background Art

[0002] With the continuous development and increasing maturity of bio-breeding technologies, their applications in the agricultural field have become increasingly widespread. As of 2023, 11 genetically modified crops are planted in 27 countries around the world, with a planting area of genetically modified crops reaching 206.3 million hectares, accounting for 23.7% of the total global crop planting area, which is 120 times higher than the 1.7 million hectares in 1996, with an average annual compound growth rate of 19.4%. Against the backdrop of ensuring food security, countries around the world have been paying increasing attention to crop variety innovation and genetic improvement using biotechnology. In China, only genetically modified insect-resistant cotton and virus-resistant papayas are allowed for commercial planting. Genetically modified organisms and their products are subject to a mandatory labeling system in the country, but the phenomenon of illegal genetically modified planting still occurs. Therefore, it is very necessary to monitor genetically modified crops.

[0003] Currently, the methods for transgenic detection mainly include nucleic acid-based PCR, Realtime-PCR, etc., and protein-based immunostrips, protein chips, etc. Nucleic acid target-based detection methods have advantages such as high sensitivity and strong accuracy, but due to the need for expensive thermal cyclers and skilled technicians, they can only be carried out in fully equipped laboratories. Protein target-based detection methods have advantages such as simple operation and strong specificity, but there are currently technical bottlenecks in improving sensitivity, and only qualitative detection results can be provided.

[0004] Electrochemiluminescence immunosensors have advantages such as high sensitivity, simple operation, strong specificity, and quantifiability. Traditional signal attenuation mode electrochemiluminescence immunosensors require one antibody, and the luminescent nanomaterials are directly modified on the electrode surface. Only one-step antigen incubation is required, with simple operation and short detection time. Traditional signal enhancement mode electrochemiluminescence immunosensors require two pairs of antibodies, and the antibody labeled with the latter nanomaterials is used as the electrochemiluminescence signal probe, which requires two-step incubation (antigen incubation + labeled antibody incubation). Its sensitivity is higher than that of the signal attenuation mode, but the detection time is long. Summary of the Invention

[0005] The purpose of the present invention is to provide a transgenic crop detection method with a "one-step" signal enhancement mode, which can achieve enhancement in the enhancement mode by using a one-step incubation method, significantly shorten the detection time, and save antibodies.

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

[0007] The method for detecting transgenic crops in the "one-step" signal enhancement mode includes the following steps:

[0008] 1) Coupling the antigen to be detected with amino-functionalized CdSe / ZnS quantum dots

[0009] Mix a PBS solution with a concentration of 0.01 - 0.02 M and crop seed powder in a volume-to-mass ratio of 5:1 - 3:1, shake vigorously for 2 - 3 min, and centrifuge at 6000 - 8000 rpm for 3 - 5 min to collect the crop supernatant; mix the supernatant with amino-functionalized CdSe / ZnS quantum dots with a concentration of 2.5 - 5 mM in a volume ratio of 2:1 - 1:1, add glutaraldehyde with a final concentration of 2.5 - 5%, perform a magnetic suspension reaction for 10 - 15 min, and after the reaction, add BSA with a final concentration of 1 - 3% to block for 30 - 45 min to obtain the solution to be detected;

[0010] 2) Detection

[0011] Drop the solution to be detected onto the surface of the GCE / GN-PAMAM / mAb / BSA immunosensor, incubate at 30 - 37 °C for 30 - 45 min, and then perform electrochemiluminescence analysis.

[0012] Preferably, in step 2), in the electrochemiluminescence analysis, the solution used contains 0.1 - 0.2 M of Na2S2O8, 0.1 - 0.2 M of KCl, and 0.1 - 0.2 M of PBS, with pH = 7 - 7.4.

[0013] Preferably, in step 2), in the electrochemiluminescence analysis, the voltage scanning range is -1.8 V to -0.2 V, the scanning rate is 0.2 - 0.3 V / s, the sampling interval time is 0.001 - 0.002 V, and the standing time is 2 - 3 s.

[0014] The specific construction steps of the GCE / GN-PAMAM / mAb / BSA immunosensor described in the present invention include:

[0015] 1) Preparation of GN-PAMAM composite material

[0016] Add 1 - 2 mg of solid powder of graphene nitride to 1 - 2 mL of N,N-dimethylformamide and ultrasonically treat for 3 - 3.5 h. Then, add 10 - 20 μL of 15 - 20 wt% PAMAM methanol solution to the dispersion, and ultrasonically treat at room temperature for 1 - 1.5 h to obtain the composite material GN-PAMAM;

[0017] 2) Construction of the electrochemiluminescence immunosensor

[0018] The electrode surface was polished with alumina powder, then washed successively with absolute ethanol and ultrapure water. 5 - 7.5 μL of the ultrasonically treated nano - composite material GN - PAMAM was dropped onto the electrode surface in two layers and dried under an infrared lamp. 5 - 7.5 μL of 2.5 - 5% glutaraldehyde solution was dropped onto the composite material and reacted at room temperature for 30 - 45 min to activate the amino groups in PAMAM. After the reaction, it was rinsed with 0.01 - 0.02 M PBS, then 1 - 2 μg of monoclonal antibody was added and incubated at 30 - 37 °C for 30 - 45 min. The electrode surface was washed again, and 4 - 5 μL of PBS solution containing 3 - 5% BSA was added and reacted for 30 - 45 min to block non - specific binding sites. Finally, it was washed with PBS to obtain the GCE / GN - PAMAM / mAb / BSA immunosensor.

[0019] Preferably, in step 3), the electrode is selected from any one of a glassy carbon electrode, a gold electrode, and a platinum electrode.

[0020] Advantages of the present invention:

[0021] 1. In the present invention, the amino - functionalized CdSe / ZnS quantum dots (QDs) are conjugated with the antigen to be detected. The electrochemical luminescence detection signal enhancement can be achieved by using a one - step incubation method, which significantly shortens the detection time and saves antibodies. In the traditional signal enhancement mode of conjugation, electrochemical luminescence immunosensors usually require two pairs of paired antibodies. The electrochemical luminescence signal is generated by the latter labeled antibody, and two - step incubation is required. After the antigen is captured by the sensor, the antibody labeled with the signal probe is incubated again.

[0022] 2. Compared with the detection of the conventional one - step signal - weakening mode sensor, the one - step signal - enhancement mode electrochemical immunosensor of the present invention has a wider linear range and can significantly improve the detection sensitivity. Brief description of the drawings

[0023] Figure 1 It is the SEM image of the GN - PAMAM composite material coated on the GCE in the embodiment of the present invention;

[0024] Figure 2 It is the CV measurement result obtained at a scanning rate of 50 mV / s in PBS (0.1 M, pH 7.4) using different electrodes during the sensor assembly process in the embodiment of the present invention;

[0025] Figure 3 It is the ECL measurement result obtained at a scanning rate of 50 mV / s in 0.1 M Na2S2O8 using different electrodes during the sensor assembly process in the embodiment of the present invention;

[0026] Figure 4Signal response and linear equation of the electrochemiluminescence immunosensor for detecting soybean RRS in the embodiments of the present invention;

[0027] Figure 5 Specificity comparison chart of the electrochemiluminescence immunosensor for different crops in the embodiments of the present invention;

[0028] Figure 6 Schematic diagram of the detection current signal and luminescence intensity of the electrochemiluminescence immunosensor in the embodiments of the present invention;

[0029] Figure 7 Comparison chart of the current and ECL intensity of the composite material GN-PAMAM-g-C3N4 prepared in the comparative example under different ratios of GN and g-C3N4;

[0030] Figure 8 Construction process of the electrochemiluminescence immunosensor in the comparative example;

[0031] Figure 9 Schematic diagram of the CV measurement results of the electrochemiluminescence immunosensor in the comparative example within different scanning ranges;

[0032] Figure 10 Schematic diagram of the ECL measurement results of the electrochemiluminescence immunosensor in the comparative example within different scanning ranges;

[0033] Figure 11 Comparison chart of the detection of different concentrations of soybean RRS by the electrochemiluminescence immunosensor in the comparative example through ECL measurement;

[0034] Figure 12 Specificity comparison chart of the electrochemiluminescence immunosensor for different crops in the comparative example;

[0035] Figure 13 Schematic diagram of the current signal and ECL signal intensity of the electrochemiluminescence immunosensor in the comparative example. Detailed implementation manners

[0036] The present invention will be further described below with reference to the embodiments and the drawings.

[0037] 1. Materials and equipment

[0038] The monoclonal antibody (mAb) against CP4-EPSPS protein was purchased from Ronghui (Shanghai, China).

[0039] Aminated CdSe / ZnS core-shell structured quantum dots (QDs) were purchased from Beida Jubang (Beijing, China).

[0040] Dendritic polymer PAMAM methanol solution (generation 3, 20%) was purchased from Chenyuan (Shandong, China).

[0041] Graphene nitride (GN) was purchased from Xianfeng Nano (Nanjing, China).

[0042] Bovine serum albumin (BSA) was purchased from Sigma (Louis, MO, USA).

[0043] The standard products of transgenic crop seed powder for testing the performance of immunosensors were purchased from ERM (Geel, Belgium, Europe) and AOCS (Urbana, Illinois, USA), as shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] Other chemicals and solvents were of analytical grade.

[0048] Electrochemical measurements were carried out on a CHI 660E electrochemical workstation (Chenhua, Shanghai, China), and electrochemiluminescence measurements were carried out on an MPI-E electrochemiluminescence detector (Ruimai, Xi'an, China). A conventional three-electrode system was used, and a glassy carbon electrode (GCE, d = 3 mm), a platinum electrode, and an Ag / AgCl electrode saturated with 3 M potassium chloride were used as the working electrode, counter electrode, and reference electrode, respectively.

[0049] Examples

[0050] 1. Coupling of the antigen to be detected with amino-functionalized CdSe / ZnS quantum dots

[0051] The crop protein was extracted by mixing a PBS solution with a concentration of 0.01 M and transgenic soybean seed powder at a volume-to-mass ratio of 3:1. After shaking vigorously for 5 min and centrifuging at 8000 rpm for 5 min, the supernatant of the crop was collected. Different concentrations of transgenic content supernatants were obtained by diluting with the blank crop supernatant. Then, 80 μL of amino-functionalized CdSe / ZnS quantum dots (5 mM) was mixed with 100 μL of supernatants with different concentrations of transgenic content, and magnetic suspension reaction was carried out for 10 min. After the reaction, BSA with a final concentration of 3% was added to block for 30 min to obtain the solution to be detected of quantum dot-coupled antigen.

[0052] 2. Preparation of the GCE / GN-PAMAM / mAb / BSA immunosensor

[0053] 1) Preparation of the GN-PAMAM composite

[0054] Add 2 mg of solid powder of GN to 2 mL of N,N-dimethylformamide and sonicate for 3 h. Then, add 20 μL of 20 wt% PAMAM methanol solution to the dispersion and sonicate for another 1 h at room temperature to obtain the composite material GN-PAMAM;

[0055] 2) Construction of electrochemiluminescence immunosensor

[0056] Polish the electrode surface with alumina powder, then wash it successively with absolute ethanol and ultrapure water. Drop 5 μL of the sonicated nanocomposite material GN-PAMAM onto the electrode surface in two layers and dry it under an infrared lamp. Drop 5 μL of 5% glutaraldehyde solution onto the composite material to react for 30 min to activate the amino groups in PAMAM. After the reaction, rinse it with 0.01 M PBS, add 2 μg of 0.5 mg / mL monoclonal antibody, and incubate at 37 °C for 30 min. Wash the electrode surface again and drop 5 μL of PBS solution containing 5% BSA to react for 30 - 40 min to block non-specific binding sites. Finally, wash it with PBS to obtain the GCE / GN-PAMAM / mAb / BSA sensor.

[0057] 3. Electrochemiluminescence detection

[0058] Drop the test solutions containing different concentrations of quantum dot-conjugated antigens onto the surface of the GCE / GN-PAMAM / mAb / BSA sensor respectively, and perform electrochemiluminescence (ECL) analysis after incubating at 37 °C for 30 min.

[0059] ECL measurement is carried out in 0.1 M PBS solution (pH 7.4) containing 0.1 M Na2S2O8 and 0.1 M KCl. The voltage scanning range is -1.8 V to -0.2 V, the scanning rate is 0.2 V / s, and the sampling interval and standing time are 0.001 V and 2 s respectively. The change in ECL signal (ΔI) is calculated according to the following formula: ΔI = I0 - I, where I0 represents the ECL signal value for detecting genetically modified crops, and I represents the ECL signal of the blank sample.

[0060] All measurements are carried out at room temperature.

[0061] To characterize the sensor assembly process, cyclic voltammetry (CV) measurements were performed using [Fe(CN)6] 3- / 4- as the redox probe. The scanning potential range of cyclic voltammetry is -0.2 V to 0.6 V, and the scanning rate is 50 mV / s.

[0062] Data statistical analysis: All samples were subjected to three parallel experiments, and the average value of the ECL signal was used as the final result. The sensitivity and specificity data were expressed as the mean ± standard deviation (SD) and calculated using Origin software.

[0063] 4. Conclusions

[0064] 4.1 Structural characterization of GN-PAMAM composite materials

[0065] The GN-PAMAM composite material was prepared by a one-step ultrasonic method. Figure 1 is the SEM image of the GN-PAMAM composite material coated on the GCE. As can be seen from Figure 1 it, the micromorphology of the GN-PAMAM composite material on the GCE shows that the composite material is evenly distributed on the electrode surface, forming a dense network structure.

[0066] 4.2 Assembly process of immunosensors

[0067] The characterization of the immunosensor assembly process was carried out by CV measurement in PBS (0.1 M, pH 7.4, containing 5 mM [Fe(CN)6]3- / 4- and 0.1 M KCl) and ECL measurement in PBS (0.1 M, pH 7.4, containing 0.1 M Na2S2O8 and 0.1 M KCl) at a scan rate of 50 mV / s.

[0068] Figure 2 is the CV measurement results obtained at a scan rate of 50 mV / s using different electrodes in PBS (0.1 M, pH 7.4) for the sensor assembly process. As can be seen from Figure 2 it, the peak current of the bare electrode is at a low level of about 90 μA, while the peak current of the electrode modified with GN-PAMAM composite material reaches about 195 μA. When mAb and BSA are modified on the electrode surface, due to the poor conductivity of the antibody and its attachment to the electrode surface, which hinders the electron transfer, the peak current gradually decreases. When the antibody captures CP4-EPSPS protein, the peak current continues to decline. The current value on the electrode surface changes continuously with the modification process, verifying the successful modification of each step of reagent materials and indicating the successful construction of the electrochemiluminescence immunosensor.

[0069] Figure 3 is the ECL measurement results obtained at a scan rate of 50 mV / s using different electrodes in 0.1 M Na2S2O8 for the sensor assembly process. As can be seen from Figure 3It can be seen that the ECL signal of the electrode modified with GN-PAMAM composite material is around 1500. After coupling antibodies and blocking with BSA, the ECL slightly increases. After the antibody captures the RRS supernatant protein labeled with 1% concentration of QDs, the luminescence intensity increases significantly, indicating the successful construction of the electrochemiluminescence immunosensor.

[0070] 4.3 Sensitivity

[0071] The test solutions containing different concentrations of quantum dot-conjugated transgenic soybean antigen (0.025%, 0.05%, 0.1%, 0.2%, 0.5%, 1.0%) were dropped onto the surface of the GCE / GN-PAMAM / mAb / BSA sensor, and electrochemiluminescence (ECL) analysis was performed after incubation at 37 °C for 30 min.

[0072] Linear regression analysis was performed on the obtained ECL change values to establish a standard curve. The detection limit calculation formula is LOD = 3SD / b (S / N = 3, SD is the standard deviation of the blank sample measured 10 times, and b is the slope of the standard curve of the ECL immunosensor luminescence change value versus concentration).

[0073] Figure 4 This is the signal response and linear equation of the electrochemiluminescence immunosensor for detecting soybean RRS. Among them, A1 is the calibration curve graph of RRS of the immunosensor, and the calibration curve for detecting soybean RRS (n = 3); A2 is the linear equation. In the range of 0.025% - 1%, the ECL intensity (ΔI) of RRS is proportional to the concentration, and the linear regression equation is as follows: ΔI = 7333.4x + 367.3, and the linear correlation coefficient R 2 is 0.9932, and the calculated detection limit (LOD) for the RRS sample is 0.01% (S / N = 3).

[0074] 4.4 Specificity

[0075] To detect the specificity of the constructed sensor for detecting CP4-EPSPS protein, transgenic crops with a transgenic protein content of 5% were detected, specifically including maize MIR162 (Bt-VIP3Aa), MIR604 (Bt-Cry3A), MON89034 (Bt-Cry1A105 / Cry2Ab), NK603 (CP4-EPSPS), TC1507 (Bt-Cry1F), 59122 (cry34Ab1). Soybean RRS (CP4-EPSPS). Figure 5For the specific comparison of the electrochemiluminescence immunosensor for different crops, it can be seen from the figure that the sensor can detect maize and soybean containing CP4-EPSPS protein. In addition, the detection results are not affected by the matrix or other transgenic proteins, such as BT-VIP3Aa, BT-Cry3A, Cry1A105, Cry2Ab or Cry1Ac.

[0076] 4.5 Stability

[0077] Using GCE / GN-PAMAM / mAb / BSA as the working electrode, the stability of the constructed immunosensing platform was evaluated. After storing the immunosensor at 4 °C for 14 days, it retained 94.9% of the initial current and 96.3% of the initial ECL signal, respectively. After 14 days of storage, the operating stability of the developed sensor was studied by measuring the CV current values for 15 consecutive cycles and the luminescence values under ECL for 12 consecutive cycles ( Figure 6 ). Stable current signals and luminescence intensities were observed, with relative standard deviations (RSDs) of 0.87% and 0.95%, respectively. Therefore, the constructed electrochemiluminescence sensor has good stability and reproducibility.

[0078] 4.6 Reliability

[0079] The applicability of the immunosensor was detected using the standard RRS soybean. Recovery verification tests were carried out by adding known concentrations of transgenic soybean RRS (0.05%, 0.2%, 0.5%, 1.0%) to blank samples. The samples were dropped onto the surface of the prepared working electrode, and the luminescence values of the immunosensor after capturing the samples were measured using ECL, and the recoveries were calculated. The recoveries (n = 3) of the immunosensor for detecting transgenic samples with known concentrations are shown in Table 2 in detail.

[0080] Table 2

[0081]

[0082]

[0083] As can be seen from Table 2, the recoveries are between 88% and 106.9%, and the RSD is less than 12%, indicating that the sensor has good reliability.

[0084] Comparative Example

[0085] 1. Construction of an electrochemiluminescence immunosensor based on GN-PAMAM-g-C3N4 composite

[0086] 1) Preparation of GN-PAMAM-g-C3N4 composite

[0087] The GN-PAMAM-g-C3N4 composite material was synthesized by a one-step ultrasonic composite method. 2 mg of the solid sample of nitrogen-doped graphene (GN) was dispersed in 1.7 mL of N, N-dimethylformamide (DMF), and 300 μL of g-C3N4 solution was added after 1 hour of ultrasonic treatment, followed by continuous ultrasonic treatment for 3 hours. Then, 20 μL of 20% PAMAM was added to the composite material and ultrasonicated for another 1 hour to obtain the synthesized composite material. This composite material is called GN-PAMAM-g-C3N4.

[0088] Optimization of the ratio of GN to g-C3N4 Figure 7 For the comparison chart of the current and ECL intensity of the composite material GN-PAMAM-g-C3N4 at different ratios of GN and g-C3N4, the ratios of GN and g-C3N4 were respectively tried as 20:1, 10:1, and 20:3 (mg:mL), as Figure 7 shown in A and B. The optimal ratio of GN and g-C3N4 is 20 mg:3 mL, the current is about 220 μA, and the fluorescence ECL intensity is about 9000. Therefore, the optimal ratio of GN and g-C3N4 is 20 mg:3 mL, and the addition amount of PAMAM is 20 μL.

[0089] 2) Construction of an electrochemiluminescence immunosensor

[0090] The surface of the GCE electrode was polished successively with alumina powders with particle sizes of 1 μm, 0.3 μm, and 0.05 μm, and the electrode surface was cleaned with ultrapure water after polishing. 5 μL of the ultrasonicated GN-PAMAM-g-C3N4 composite material was dropped onto the cleaned GCE electrode and baked dry under an infrared lamp. After the surface was completely dry, 5 μL of 5% glutaraldehyde solution was dropped onto the composite material to react for 30 minutes to activate the amino groups in PAMAM. After the reaction, the excess glutaraldehyde solution was washed away with ultrapure water, and then 3 μL of mAb was added and reacted for 40 minutes in the dark at room temperature. The surface of the GCE electrode was washed again, and 5 μL of PBS solution containing 5% BSA was added and reacted for 40 minutes to block the non-specific binding sites. The electrode after the reaction was washed with PBS and stored at a temperature of 4 °C. The GCE / GN-PAMAM-g-C3N4 / mAb / BSA electrode was prepared, that is, an electrochemiluminescence immunosensor was constructed.

[0091] Figure 8 The construction process of the electrochemiluminescence immunosensor for the comparative example.

[0092] 2. Electrochemical measurement

[0093] Electrochemiluminescence intensity (ECL) measurements were carried out 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 as follows: the voltage scanning range was from -1.5 V to 0 V, and the scanning rate, sampling interval, and standing time were 0.1 V / S, 0.001 V, and 2 S, respectively.

[0094] The change in signal intensity (ΔI) was calculated according to the following formula:

[0095] ΔI = I - I0

[0096] Where, I0 represents the ECL peak value of the sample to be measured, and I represents the initial ECL peak value of the electrode.

[0097] All measurements were carried out at room temperature.

[0098] To determine the characteristics of the assembly process, different electrodes were used in PBS (0.1 M, pH 7.4) containing 5 mM [Fe(CN)6] 3- / 4- and 0.1 M KCl, and CV measurements were carried out at a scanning rate of 50 mV / s in the scanning range of -0.2 V to 0.6 V (as Figure 9 shown), and ECL measurements were carried out at a scanning rate of 50 mV / s using different electrodes in PBS (0.1 M, pH 7.4) containing 0.1 M Na2S2O8 (as Figure 10 shown).

[0099] The results are as Figure 9 shown. It can be seen that the peak current value of the bare GCE electrode is about 90 μA, while the peak current of the electrode modified with GN-PAMAM-g-C3N4 composite material reaches 230 μA. After incubation with CP4-EPSPS, the current further decreases. The change on the electrode surface is obvious, indicating that the electrochemiluminescence immunosensor is successfully assembled, and the GN-PAMAM-g-C3N4 composite material significantly enhances the current.

[0100] The results are as Figure 10 shown. It can be seen that the ECL value of the electrode modified with GN-PAMAM-g-C3N4 composite material reaches 9000. When the electrode is blocked with BSA and incubated with CP4-EPSPS, the signal value gradually weakens. The change on the electrode surface is obvious, indicating that the electrochemiluminescence immunosensor is successfully assembled, and the GN-PAMAM-g-C3N4 composite material has a significant enhancing effect on electrochemiluminescence.

[0101] 3. Conclusions

[0102] 3.1 Sensitivity

[0103] To evaluate the sensitivity of the method for detecting CP4-EPSPS protein by the electrochemiluminescence immunosensor constructed in the comparative example, crops with a large transgenic planting area, soybeans, were detected. Under the optimal conditions, different concentrations of soybean RRS were detected by ECL measurement. The ECL peak gradually decreased with the increase in concentration because the increase in the amount of CP4-EPSPS protein on the electrode surface hindered electron transfer.

[0104] The results are as Figure 11 shown. In the range of 0.05% - 1.5%, the change in ECL intensity (ΔI) of RRS was proportional to the concentration. The linear regression equation of RRS was as follows: ΔI = 4334.681x + 1139.879, and the linear relationship (R 2 ) was very high, being 0.9954. The detection limit of the RRS sample was 0.025% (S / N = 3). Therefore, the sensitivity of the electrochemiluminescence immunosensor constructed according to the comparative example was 0.025%.

[0105] 3.2 Specificity

[0106] To evaluate the specificity of the method for detecting CP4-EPSPS protein by the electrochemiluminescence immunosensor constructed in the comparative example, transgenic crops with a transgenic protein content of 5% were also detected, 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).

[0107] First, a PBS solution with a concentration of 0.01 mol / L was mixed with the standard protein powder of different crop seeds at a mass ratio of 3:1, shaken vigorously for 3 - 5 minutes to ensure uniform mixing, and then centrifuged at 8000 rpm for 5 minutes using a centrifuge to collect the supernatant. The collected supernatant was diluted with 0.01 mol / L PBS, and the supernatant of transgenic crops was diluted with the supernatant of blank crops to obtain the concentration gradient required for detection. During detection, 5 μL of samples with different concentrations were dropped onto the prepared electrochemiluminescence immunosensor and incubated at 37 °C for 40 minutes.

[0108] All the test result data were subjected to three parallel experiments, and the average ECL intensity was used as the final result. The sensitivity and specificity data were expressed as the mean ± standard deviation (SD) and calculated using origin software.

[0109] The results are as Figure 12 shown. The electrochemiluminescence immunosensor can detect maize, rapeseed, soybean, cotton, and sugar beet containing CP4-EPSPS protein, and the detection results are not affected by unknown matrix components or other proteins, such as BT-VIP3Aa, BT-Cry3A, Cry1A105, Cry2Ab, Cry1Ac, or PAT.

[0110] 3.3 Stability

[0111] In this comparative example, GCE / GN-PAMAM-g-C3N4 / mAb / BSA was used as the working electrode to evaluate the stability of the constructed electrochemiluminescence immunosensor. After storing the electrochemiluminescence immunosensor 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.

[0112] The results are as Figure 13 shown in C and D below. Stable current signals and ECL signal intensities were observed, with relative standard deviations (RSDs) of 0.60% and 2.13%, respectively.

[0113] By comparing the examples and comparative examples of the present invention, it can be seen that the sensitivity of the present invention is higher than that of the comparative example. The sensitivity of the present invention is 0.01%, while the sensitivity of the comparative example is 0.025%, and it has good stability and reproducibility.

[0114] The present invention uses the exogenous protein CP4-EPSPS in genetically modified crops as the detection target. By directly labeling the antigen to be detected, a signal-enhanced electrochemiluminescence immunosensor can be constructed without the need to pre-label the antibody. High-sensitivity and quantitative detection of the target can be achieved with only one-step incubation. This method greatly simplifies the experimental steps and has the advantages of short detection time, high sensitivity, good stability, and reproducibility, providing a novel and simple method for the ultrasensitive detection of CP4-EPSPS protein in genetically modified crops. In addition, the new materials and new targets can be applied to immunoassays to improve the sensitivity of quantitative detection, showing good application prospects.

Claims

1. The transgenic crop detection method in the "one-step" signal enhancement mode is characterized in that It includes the following steps: 1) Coupling the antigen to be detected with amino-functionalized CdSe / ZnS quantum dots Mix a PBS solution with a concentration of 0.01 - 0.02 M and crop seed powder at a volume-to-mass ratio of 5:1 - 3:1, shake vigorously for 2 - 3 min, and centrifuge at 6000 - 8000 rpm for 3 - 5 min to collect the crop supernatant; mix the supernatant with amino-functionalized CdSe / ZnS quantum dots with a concentration of 2.5 - 5 mM at a volume ratio of 2:1 - 1:1, add glutaraldehyde with a final concentration of 2.5 - 5%, perform a magnetic suspension reaction for 10 - 15 min, and after the reaction, add BSA with a final concentration of 1 - 3% to block for 30 - 45 min to obtain the solution to be detected; 2) Detection Drop the solution to be detected on the surface of the GCE / GN-PAMAM / mAb / BSA immunosensor, and perform electrochemiluminescence analysis after incubation at 30 - 37 °C for 30 - 45 min.

2. The detection method according to claim 1, characterized in that, In step 2), in the electrochemiluminescence analysis, the solution used contains 0.1 - 0.2 M of Na2S2O8, 0.1 - 0.2 M of KCl, and 0.1 - 0.2 M of PBS, with pH = 7 - 7.

4.

3. The detection method according to claim 1 or 2, characterized in that In step 2), in the electrochemiluminescence analysis, the voltage scanning range is -1.8 V to -0.2 V, the scanning rate is 0.2 - 0.3 V / s, the sampling interval time is 0.001 - 0.002 V, and the standing time is 2 - 3 s.

4. The detection method according to claim 1, wherein The specific construction steps of the GCE / GN-PAMAM / mAb / BSA immunosensor include: 1) Preparation of the GN-PAMAM composite material Add 1 - 2 mg of solid powder of graphene nitride to 1 - 2 mL of N, N-dimethylformamide and ultrasonically treat for 3 - 3.5 h. Then, add 10 - 20 μL of 15 - 20 wt% PAMAM methanol solution to the dispersion and ultrasonically treat at room temperature for 1 - 1.5 h to obtain the composite material GN-PAMAM; 2) Construction of the electrochemiluminescence immunosensor Polish the electrode surface with alumina powder, then wash it successively with absolute ethanol and ultrapure water. Drop 5 - 7.5 μL of ultrasonically treated nano-composite material GN-PAMAM onto the electrode surface in two layers and dry it under an infrared lamp. Drop 5 - 7.5 μL of 2.5 - 5% glutaraldehyde solution onto the composite material and react at room temperature for 30 - 45 min to activate the amino groups in PAMAM. After the reaction, rinse with 0.01 - 0.02 M PBS, add 1 - 2 μg of monoclonal antibody, and incubate at 30 - 37 °C for 30 - 45 min. Wash the electrode surface again and drop 4 - 5 μL of PBS solution containing 3 - 5% BSA and react for 30 - 45 min to block non-specific binding sites. Finally, wash with PBS to obtain the GCE / GN-PAMAM / mAb / BSA immunosensor.

5. The detection method according to claim 4, wherein, In step 3), the electrode is selected from any one of a glassy carbon electrode, a gold electrode, and a platinum electrode.

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