阅读说明：本技术 检测转基因蛋白的无标记型AuNPs-Thi电化学免疫传感器及其制备方法 (Unmarked AuNPs-Thi electrochemical immunosensor for detecting transgenic protein and preparation method thereof ) 是由 曾海娟 王金斌 杨倩雯 于 2021-08-30 设计创作，主要内容包括：检测转基因蛋白的无标记型AuNPs-Thi电化学免疫传感器及其制备方法,在玻碳电极表面电沉积金纳米粒子AuNPs,硫堇Thi稳定吸附在AuNPs表面形成AuNPs-Thi膜；转基因蛋白-mAb抗体共价结合于AuNPs-Thi膜上,再以BSA进行封闭。本发明在电极表面电沉积AuNPs后吸附适量Thi,显著增强电化学信号,再通过氨基交联作用将转基因蛋白的抗体连接到AuNPs-Thi膜上,利于传感器的长期稳定性和灵敏度,该方法简单、快速,所需抗体量少,具有较高的灵敏度、良好的特异性和重现性,为电化学免疫检测转基因作物中的转基因蛋白提供了一种新方法。(A non-marking AuNPs-Thi electrochemical immunosensor for detecting transgenic protein and a preparation method thereof are disclosed, wherein gold nano particles AuNPs are electrodeposited on the surface of a glassy carbon electrode, and thionine Thi is stably adsorbed on the surface of the AuNPs to form an AuNPs-Thi film; the transgenic protein-mAb antibodies were covalently bound to AuNPs-Thi membranes and blocked with BSA. According to the invention, a proper amount of Thi is adsorbed after AuNPs are electrodeposited on the surface of an electrode, an electrochemical signal is obviously enhanced, and then the antibody of the transgenic protein is connected to the AuNPs-Thi membrane through an amino crosslinking effect, so that the long-term stability and sensitivity of the sensor are facilitated.)

1. A working electrode of the unmarked AuNPs-Thi electrochemical immunosensor is a glassy carbon electrode, gold nanoparticles AuNPs are electrodeposited on the surface of the glassy carbon electrode, and thionine Thi is stably adsorbed on the surface of the AuNPs through electrostatic adsorption to form an AuNPs-Thi membrane; the transgenic protein-mAb antibody is covalently bound to AuNPs-Thi membrane with 3-5% BSA as blocking agent.

2. The unlabeled AuNPs-Thi electrochemical immunosensor according to claim 1, wherein the gold nanoparticles AuNPs are used in amounts of: with 1-1.5% HAuCl₄Solution deposition for 3-4 cyclesThe potential range is as follows: 0V to 1.1V and 50mV/s, and the dosage of the thionine Thi is 5 to 10 mu g.

3. The unlabeled AuNPs-Thi electrochemical immunosensor according to claim 1, wherein the amount of the transgenic protein-mAb antibody is 2.5-3 μ g and the amount of the blocking agent BSA is 5-10 μ L.

4. The label-free AuNPs-Thi electrochemical immunosensor according to any one of claims 1-3, wherein the transgenic protein is a PAT protein and the transgenic protein-mAb antibody is a PAT-mAb antibody.

5. The method for preparing the unlabeled AuNPs-Thi electrochemical immunosensor for detecting transgenic proteins according to claim 1, comprising the following steps:

1) soaking the cleaned glassy carbon electrode in HAuCl₄In the solution, gold nano particles are electrodeposited on the surface of an electrode by a CV method;

2) then modifying 5-10 mu L of Thi on the surface of the glassy carbon electrode deposited with the gold nanoparticles at room temperature by adopting a dripping method, stably adsorbing the Thi on the surface of AuNPs by electrostatic adsorption to form an AuNPs-Thi membrane, and activating for 30-45min at room temperature by using 0.25-0.5% glutaraldehyde;

3) then, dropwise adding the transgenic protein-mAb antibody to the activated electrode surface obtained in the step 2) for modification, and incubating for 40-60min at 30-37 ℃;

4) and (3) dropwise adding 3-5% BSA (bovine serum albumin) on the surface of the electrode, standing at 30-37 ℃ for 40-60min, blocking the non-specific binding sites, washing, and drying at room temperature to obtain the unmarked AuNPs-Thi electrochemical immunosensor for detecting the transgenic protein.

6. The method for preparing the unlabeled AuNPs-Thi electrochemical immunosensor for detecting transgenic proteins according to claim 5, wherein in the step 1), HAuCl is added₄The concentration of the solution is 1-1.5%, and 3-4 circles of electrodeposition are carried out; in the step 2), 0.75-1.0mg/mL thionine solution of Thi and 30mi of room-temperature activation time of 0.5% glutaraldehyde are usedn; in step 3), the amount of transgenic protein-mAb antibody was 2.5. mu.g, and in step 4), 5% BSA was 5. mu.L.

7. The method for preparing the unlabeled AuNPs-Thi electrochemical immunosensor for detecting transgenic proteins of claim 6, wherein the preparation of the electrodes is characterized by cyclic voltammetry, and [ Fe (CN) ]₆]^3-/^4-As a redox probe, the CV scan ranged from-0.2V to 0.6V, and the scan rate was 50 mV/s.

8. The use of the unlabeled AuNPs-Thi electrochemical immunosensor of claim 1 to detect transgenic proteins.

9. The application of the unlabeled AuNPs-Thi electrochemical immunosensor according to claim 8, wherein in the application, the platinum wire electrode is a counter electrode, the Ag-AgCl electrode is a reference electrode, and the PAT protein in the transgenic crops is determined by using a differential pulse voltammetry method.

10. The application of the unlabeled AuNPs-Thi electrochemical immunosensor of claim 9 to detection of transgenic proteins, wherein the PAT protein in transgenic crops is determined by differential pulse voltammetry, and the parameters are set as follows: potential range: 0-0.4V; potential increment: 4 mV; amplitude: 0.05V; pulse width: 0.06 s; sampling width: 0.02 s; standing for 2 s; incubation time: 30 min; all measurements were performed at room temperature.

Technical Field

The invention belongs to the technical field of food safety immunodetection, and particularly relates to a marker-free AuNPs-Thi electrochemical immunosensor for detecting transgenic protein and a preparation method thereof.

Background

With the continuous development of genetic engineering technology, the planting area of transgenic crops is enlarged year by year, and the safety of the transgenic crops becomes the focus of attention of people. Although the mandatory marking regulations of transgenic crops and products have been implemented in China, many cases of illegal planting and selling of transgenic crops still occur. Therefore, the development of a simple, rapid and highly sensitive transgenic crop identification method has important significance for the supervision of the transgenic crops and products thereof.

In the detection of transgenic crops, the detection based on the nucleic acid level has high sensitivity, but depends on instruments and has certain technical requirements on operators; the detection method based on the protein level has the advantages of simple operation, short detection time and low requirement on the pretreatment of the sample, and is most suitable for the on-site rapid detection of the target. Among them, the immunosensor has the advantages of being quantifiable, strong in specificity, easy to operate and the like, and has become a research hotspot of a rapid detection method at present.

In the detection of the PAT protein, a rapid detection method mostly depends on an immunological technology, and the immunological rapid detection technology, such as an immunochromatographic test strip and the like, can only provide a semi-quantitative detection result. A rapid, sensitive and quantitative detection method is urgently needed for trace detection of crops in the links of fields, storage and the like.

The detection method based on the PCR technology has high sensitivity, but the experimental process is complicated and takes long time. An immunochromatographic strip (ICS) can rapidly detect a target, but has low sensitivity and cannot quantify the target. Electrochemical immunosensor (ECL) has higher sensitivity, but the traditional ECL reagent has some disadvantages, such as high toxicity and long time consumption, and the performance of different detection methods of the existing transgenic protein is compared in table 1.

Table 1 comparison of the performance of different detection methods for transgenic proteins.

Electrochemical immunosensors can be classified into label-free immunosensors and label-free immunosensors, and are of great interest due to their high sensitivity and simplicity of operation. Although the labeled immunosensor has high sensitivity, it requires the antibody or antigen to be labeled with other electroactive substances during the analysis process. The label-free immunosensor does not need to label an antibody or an antigen, and the target sample can be quickly detected through simple antigen-antibody specific reaction.

Compared with a labeled immunosensor, the unlabeled immunosensor has the advantages of simple preparation process, low cost, easiness in miniaturization, rapidness, simplicity and convenience and the like, is widely applied to immunoassay in various fields, and is a quantitative detection method with a higher application prospect.

Wang Z et al (A novel oriented immunosensor based on AuNPs-thionine-CMWCNTs and staphylococcus protein A for interacting in-6 analysis in complex biological samples [ J ]. analytical Chimica acta 2020,1140:145-152) developed an immunosensor based on AuNPs/Thi/CMWCNTs (carboxylated multiwalled carbon nanotubes) for highly sensitive detection of interleukin-6 (IL-6), and the scheme selects a composite material modified electrode formed by 3 materials to achieve the purpose of highly sensitive detection of a target object, but the multiwalled carbon nanotube modified electrode is easy to generate aggregation phenomenon, and the constructed immunosensor may have poor parallelism.

Disclosure of Invention

The invention aims to provide a marker-free AuNPs-Thi electrochemical immunosensor for detecting transgenic protein and a preparation method thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

a working electrode of the unmarked AuNPs-Thi electrochemical immunosensor is a glassy carbon electrode, gold nanoparticles AuNPs are electrodeposited on the surface of the glassy carbon electrode, and thionine Thi is stably adsorbed on the surface of the AuNPs through electrostatic adsorption to form an AuNPs-Thi membrane; the transgenic protein-mAb antibody is covalently bound to AuNPs-Thi membrane with 3-5% BSA as blocking agent.

Further, the dosage of the gold nanoparticles AuNPs is as follows: with 1-1.5% HAuCl₄The solution is deposited for 3-4 circles, and the potential range is as follows: 0V to 1.1V and 50mV/s, and the dosage of the thionine Thi is 5 to 10 mu g.

The dosage of the transgenic protein-mAb antibody is 2.5-3 mug, and the dosage of the blocking agent BSA is 5-10 muL.

Preferably, the transgenic protein is a PAT protein and the transgenic protein-mAb antibody is a PAT-mAb antibody.

The invention provides a preparation method of an unmarked AuNPs-Thi electrochemical immunosensor for detecting transgenic proteins, which comprises the following steps:

1) soaking the cleaned glassy carbon electrode in HAuCl₄In the solution, gold nanoparticles are electrodeposited on the surface of an electrode by Cyclic Voltammetry (CV);

2) then modifying 5-10 mu L of Thi on the surface of the glassy carbon electrode deposited with the gold nanoparticles at room temperature by adopting a dripping method, stably adsorbing the Thi on the surface of AuNPs by electrostatic adsorption to form an AuNPs-Thi membrane, and activating for 30-45min at room temperature by using 0.25-0.5% glutaraldehyde;

3) then, dropwise adding the transgenic protein-mAb antibody to the activated electrode surface obtained in the step 2) for modification, and incubating for 40-60min at 30-37 ℃;

4) and (3) dropwise adding 3-5% BSA (bovine serum albumin) on the surface of the electrode, standing at 30-37 ℃ for 40-60min, blocking the non-specific binding sites, washing, and drying at room temperature to obtain the unmarked AuNPs-Thi electrochemical immunosensor for detecting the transgenic protein.

Further, in step 1), HAuCl₄The concentration of the solution is 1-1.5%, and 3-4 circles of electrodeposition are carried out; in the step 2), the used Thi is 0.75-1.0mg/mL of thionine solution, and the room-temperature activation time of 0.5% glutaraldehyde is 30 min; in step 3), the amount of transgenic protein-mAb antibody was 2.5. mu.g, and in step 4), 5% BSA was 5. mu.L.

The preparation process of the electrode is characterized by adopting cyclic voltammetry, and the preparation process is represented by [ Fe (CN)₆]^3-/^4-As a redox probe, the CV scan ranged from-0.2V to 0.6V, and the scan rate was 50 mV/s.

Preferably, the whole construction process is washed with double distilled water and dried at room temperature.

The invention provides an application of the unmarked AuNPs-Thi electrochemical immunosensor in detection of transgenic proteins.

Further, in the application, the platinum wire electrode is a counter electrode, the Ag-AgCl electrode is a reference electrode, and Differential Pulse Voltammetry (DPV) is adopted to measure the PAT protein in the transgenic crops.

In the above application, PAT protein in transgenic crops was measured by Differential Pulse Voltammetry (DPV), and the parameters were set as follows: potential range: 0-0.4V; potential increment: 4 mV; amplitude: 0.05V; pulse width: 0.06 s; sampling width: 0.02 s; standing for 2 s; incubation time: 30 min; all measurements were performed at room temperature.

The invention takes exogenous transgenic protein (such as PAT protein) in transgenic crops as a detection target, and utilizes AuNPs and Thi to construct a simple label-free electrochemical immunosensor. Thionine (Thi) is a cationic dye with positive charges, has a symmetrical structure, can be used as an active substance for accelerating electron transfer, is used for enhancing current signals due to the specific electrochemical activity, and is also used as a connecting agent to be connected with an antibody through an amino group.

The gold nanoparticles are electrodeposited on the surface of the electrode by a CV method to form a uniform film structure, the immobilization capacity of the surface of the original electrode is greatly improved, and then the thionine Thi is modified on the surface of the electrode on which the gold nanoparticles are deposited by a dripping method and activated. AuNPs electrodeposited on the surface of the electrode promote electron transmission, signal amplification of the sensor is realized, Thi is stably adsorbed on the surface of the AuNPs layer through electrostatic adsorption, the surface area and the biological solid carrying capacity of the electrode are increased, electrochemical signals in the immunosensor can be remarkably enhanced, and after the Thi is modified on the surface of the electrode, the peak current value is further increased and can reach 150 muA.

Therefore, comparisons were made with different concentrations and adsorption times for the Thi modifications.

According to the invention, the dosage of thionine Thi is 5-10 mu g, the adsorption effect of Thi on the AuNPs surface directly influences the sensitivity of the immunosensor, when the concentration of thionine is 0.75-1.0mg/mL, the current change value is increased along with the increase of the concentration of Thi, when the concentration of Thi is 1.0mg/mL, the current change value is 40 mu A, and when the concentration of Thi is more than 1.0mg/mL, the current change value begins to decrease and tends to be stable, and excessive Thi can be attached to the surface of an electrode to block electron transmission, so that the thionine solution with the concentration of 0.75-1.0mg/mL is selected.

In the invention, the current value of the electrode surface is changed along with the modification process, the peak current is increased after Thi modification, and when the mAb antibody and BSA are modified on the electrode surface sequentially, the mAb antibody and BSA have poor conductivity, so that the transfer of electrons is blocked, and the peak current is gradually reduced. After the mAb antibody captures the transgenic protein, the peak current at the electrode surface continues to decrease. And (3) substituting the current value in the sample to be detected into the standard curve to obtain the transgene content in the sample by measuring the standard substances with different concentrations and drawing the standard curve.

Compared with the prior art, the invention has the following beneficial effects:

the invention constructs a novel unmarked electrochemical immunosensor by utilizing AuNPs and Thi, and the novel unmarked strategy causes the current change on the surface of an electrode by simple immunoreaction, makes quick and accurate analysis on a target biological target and realizes high-sensitivity detection on transgenic crops.

According to the invention, the process of electrodepositing AuNPs on the surface of the electrode is simple, the complex experimental process of the traditional electrode modification method is simplified, and the AuNPs as a low-dimensional functional nano material can increase the surface area of the sensor, improve the solid loading capacity, promote the charge transfer between the oxidation compound and the sensor, promote the electron transmission and realize the signal amplification of the sensor.

According to the invention, a proper amount of Thi is adsorbed after AuNPs is electrodeposited on the surface of the electrode, so that the conductivity of the electrode is improved, the surface area of the electrode is enlarged, more binding sites are provided, and a simple and rapid unmarked immunosensor is constructed, wherein the AuNPs have good conductivity, the electron transfer rate is increased, and the sensitivity of the immunosensor is further improved; the Thi is stably adsorbed on the surface of the AuNPs layer through electrostatic adsorption, so that the surface area and the biological solid loading capacity of the electrode are increased, and the electrochemical signal in the immunosensor can be obviously enhanced.

According to the invention, the transgenic protein is captured on the immunosensor through immune reaction, quantitative detection can be realized only within 30min on the premise of ensuring high sensitivity, the test time is greatly shortened, the experimental steps are greatly simplified, the detection method is simple and rapid, the amount of the required antibody is small, and the method has high sensitivity, good specificity and reproducibility, and provides a new method for electrochemical immunodetection of the PAT protein in the transgenic crops.

The immunosensor constructed by the invention effectively improves the detection sensitivity of PAT protein, the detection limits of the immunosensor to PAT protein of soybean A2704-12 and corn BT-176 are 0.02% and 0.03%, respectively, after the immunosensor is stored for 15d and 33d at 4 ℃, the sensor can still keep 87.5% and 82.5% of the initial signal value, and the RSD is 0.92%.

Drawings

FIG. 1 is a schematic diagram of the construction of an electrochemical immunosensor for detecting PAT proteins in example 1 of the present invention.

Fig. 2 is an SEM image of AuNPs deposited on the surface of an electrode in example 1 of the present invention.

FIG. 3 is a step-by-step assembly process of a CV-characterized immunosensor in example 1 of the present invention.

FIG. 4 is a graph showing the peak current of DPV of soybean A2704-12 at various concentrations in example 2 of the present invention.

FIG. 5 is a standard curve of the immunosensor in example 2 of the present invention for detecting soybean A2704-12.

FIG. 6 is a graph showing the peak current of DPV for maize BT-176 at various concentrations in example 2 of the present invention.

FIG. 7 is a standard curve of the immunosensor detecting corn BT-176 in example 2 of the present invention.

FIG. 8 shows the results of specific detection by the immunosensor in example 2 of the present invention.

FIG. 9 shows the stability of the CV measurement immunosensor in example 2 of the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples.

Monoclonal antibodies against the PAT protein (PAT-mAb) were purchased from Shanghai Holly Biometrics; glutaraldehyde, Bovine Serum Albumin (BSA), and Thionine (Thionine, Thii) are all available from Sigma (St. Louis, Mo., USA); all transgenic crop seed powder standard lines with 100% and 5% protein content were purchased from ERM (gel, Belgium, europe) and AOCS (Urbana, Illinois, USA), and all transgenic crop seed powder with 1% protein content was purchased from rural areas of agriculture; all chemicals and solvents were analytical reagents.

The electrochemical measurements are all carried out at CHI660E electrochemical workstation (Shanghai Chenghua), and the three-electrode system is adopted for detection at room temperature; the working electrode is a glassy carbon electrode (GCE, d is 3mm), the platinum electrode is a counter electrode, and the Ag/AgCl electrode is a reference electrode. Uv-vis absorption spectroscopy was performed using a NanoDrop2000c spectrophotometer (Thermo Scientific, MA, USA); the microscopic appearance of the decorated material was characterized by Scanning electron microscopy (SEM, japan).

Example 1 construction of a Label-free AuNPs-Thi electrochemical immunosensor for detection of PAT proteins

The working electrode of the unmarked electrochemical immunosensor is a glassy carbon electrode, gold nano particles AuNPs are electrodeposited on the surface of the glassy carbon electrode, and thionine Thi is stably adsorbed on the surface of the AuNPs through electrostatic adsorption to form an AuNPs-Thi film; the PAT-mAb antibody was covalently bound to AuNPs-Thi membrane with BSA as blocking agent, see FIG. 1, and was constructed as follows:

1) polishing and cleaning the working electrode according to steps, and soaking the cleaned glassy carbon electrode in 1% HAuCl₄In the solution, gold nanoparticles are electrodeposited on the surface of an electrode by a CV method, and the potential range is as follows: 0V to 1.1V, and the scanning rate is 50 mV/s;

2) thi (5 mu L) is modified on the surface of a glassy carbon electrode GCE by adopting a dropping method, and after the Thi is dropped on the glassy carbon electrode, the glassy carbon electrode is activated for 30min at room temperature by 0.5% glutaraldehyde.

Wherein, because the adsorption effect of Thi on the AuNPs surface directly influences the sensitivity of immunosensor, consequently, adopt different concentrations and adsorption time to compare when modifing Thi:

thi with the concentration of 0.5mg/mL, 1mg/mL and 2mg/mL is respectively selected to be modified on the surfaces of different glassy carbon electrodes, after 2 hours of room temperature incubation, a current signal is measured by a DPV method, and the result shows that the current change value is less than 25 muA when 0.5mg/mL and 2mg/mL, and reaches 40 muA when 1 mg/mL.

After modifying the electrode by using Thi with proper concentration, respectively comparing the peak currents under the conditions of room temperature treatment for 40min, 60min, 90min, 120min and overnight at 4 ℃, determining the peak current signals by using a DPV method, selecting the time with large peak current change as proper incubation time, and displaying the results: the signal intensity of the current gradually increased with increasing reaction time, and the maximum current response was obtained after overnight treatment at 4 ℃.

Through multiple experimental screening, when the glass carbon electrode is dripped to a Thi 4 ℃ overnight under the selected treatment condition of 1.0mg/mL, a better signal amplification effect can be obtained, and the high-sensitivity detection of the sensor is realized.

In order to more intuitively see the change of the electrode surface in the modification process, the microscopic appearance of the modified electrode surface is observed through a scanning electron microscope, as shown in fig. 2, the electrodeposited AuNPs form a uniform film structure on the electrode surface, which proves that the AuNPs are successfully modified, and the immobilization capacity of the original electrode surface is greatly improved.

3) Adding PAT-mAb antibody dropwise onto electrode surface activated by glutaraldehyde, and incubating at 37 deg.C for 40 min;

4) 5% BSA (5 mu L) is dripped on the surface of the electrode, the electrode is placed for 40min at 37 ℃, the rest active sites are blocked, double distilled water is used for washing in the whole modification process, the drying is carried out at room temperature, the unmarked AuNPs-Thi electrochemical immunosensor for detecting PAT protein is obtained, and the sensor is stored for standby at 4 ℃.

The preparation process of the electrode was characterized by cyclic voltammetry, [ Fe (CN) ]₆]^3-/^4-As a redox probe, a probe containing 5mM [ Fe (CN)₆]^3-/4-And 0.1M KCl in PBS (0.1M, pH 7.4), with a CV scan range of-0.2V to 0.6V, the construction of the immunosensor was characterized one by a CV at a scan rate of 50mV/s, see FIG. 3.

As can be seen from FIG. 3, the bare GCE has lower peak current, and after a layer of AuNPs is electrodeposited on the surface, the peak current can reach 120 muA, and after Thi is modified on the surface of the electrode, the peak current value is further increased to reach 150 muA; when mAb and BSA were successively modified on the electrode surface, the transfer of electrons was hindered due to poor conductivity of mAb and BSA, and the peak current gradually decreased. When the antibody captures the PAT protein, the peak current continues to decrease.

The current value of the electrode surface is continuously changed along with the modification process, the successful modification of the reagent material in each step and the successful construction of the electrochemical immunosensor are verified, and meanwhile, the AuNPs/Thi can greatly enhance the current signal.

Example 2 analysis of detection Performance of the constructed immunosensor

1. Sample processing and measuring method

Transgenic crop seed standards to be tested (see table 2) were mixed with 0.01M PBS buffer (pH 7.4) at a ratio of 1: 3 mass to volume ratio.

TABLE 2 list of transgenic crop seed meal standards used

And adding the seed powder into PBS, violently shaking for 3-5 min, centrifuging at 6000rpm for 5min, collecting supernatants in a layered manner, diluting the supernatants into transgenic protein solutions with different concentrations for detection, and taking the supernatants of corresponding non-transgenic crops as experimental blank controls. The actual seed samples were ground to a powder by a food grinder or mortar, with a ratio of 1: and 5, adding double distilled water according to a proportion of 5, shaking and uniformly mixing, centrifuging at 8000rpm for 5min, and taking the supernatant for detection.

PAT protein was measured in transgenic plants by Differential Pulse Voltammetry (DPV) in the presence of 5mM [ Fe (CN)₆]^3-/^4-And 0.1M KCl in PBS buffer (0.1M, pH 7.4), with the parameters set to: the potential range is 0-0.4V; potential increment, 4 mV; amplitude, 0.05V; pulse width, 0.06 s; sampling width, 0.02 s; standing for 2 s; incubation time, 30 min; the current difference Δ I is calculated according to equation (1):

ΔI＝I₀-I formula (1)

Wherein, I₀The measured current values for the blank crop supernatant and for the transgenic crop supernatant I were obtained, all measurements being carried out at room temperature.

2. Establishment of sensor standard curve and sensitivity analysis

Under the condition screened out in the example 1, a marker-free immunosensor based on AuNPs/Thi is constructed, a transgenic soybean standard A2704-12 and a transgenic corn standard BT-176 with the concentrations of 0.05%, 0.1%, 0.2%, 0.5%, 1.0% and 1.5% are respectively dripped on the surface of a prepared working electrode for DPV measurement, the obtained current change value is analyzed to establish a standard curve, and the detection limit is calculated.

The sensitivity of the constructed immunosensor is detected, the peak current changes along with the change of the concentration of the detected protein, and a good linear relation is presented in a certain concentration range. The detection results of soybean A2704-12 and corn BT-176 standard products containing protein with the concentration range of 0.05% -1.5% are shown in figures 4 and 6, and the peak current is continuously reduced along with the increase of the concentration of PAT protein, because the increase of the PAT protein on the surface of an electrode hinders the electron transfer.

The corresponding standard curve linear regression equation is respectively that delta I is 22.424x +10.708 (R)²0.9936) and Δ I13.873 x +5.7094 (R)²0.9933) (see fig. 5 and 7), where Δ I is the current signal change (μ a) and x is the concentration of the protein. Finally, the detection limit of the soybean A2704-12 is 0.02%, and the detection limit of the corn BT-176 is 0.03% (S/N is 3).

3. Specificity and stability detection of immunosensor

Specificity is an important index of the immunosensor, and whether the specific adsorption of PAT protein can be realized or not influences the accuracy and sensitivity of the sensor.

To evaluate the specificity of the immunosensor developed, 11 interfering proteins ubiquitous in transgenic crops were tested, including 5% corn BT-176(BT-Cry1Ac/PAT), corn MIR604(BT-Cry3A), corn MON89034(BT-Cry1a105/Cry2Ab), corn MON88017(CP4-EPSPS/Cry3Bb1), soybean RRS (CP4-EPSPS), cotton MON88913(CP4-EPSPS), and beet H7-1(CP4-EPSPS), 1% corn MON810(BT-Cry1Ab), corn BT-11 (BT-1 Ab/PAT), soybean a2704-12(PAT), and rape T45(PAT), and the supernatant of 11 transgenic crops was used as a blank control to evaluate the specificity of the immunosensor constructed and to verify its stability

As shown in FIG. 8, the peak current change of the PAT protein is obviously higher than that of other proteins, the sensor can successfully detect soybean and corn standard products containing the PAT protein and is not interfered by proteins such as CP4-EPSPS, BT-Cry1Ab, BT-Cry3A, BT-Cry1A105/Cry2Ab and the like, and the developed immunosensor is proved to have good specific recognition capability.

The constructed GCE/AuNPs/Thi/mAb/BSA still maintains 87.5% and 82.5% of the original signal value after being stored at 4 ℃ for 15d and 33 d. After storage for 33d, the CV scans continuously for 15 cycles to obtain a stable current signal (fig. 9), with an RSD of 0.92%.

Therefore, the immunosensor constructed by the embodiment has good stability and repeatability, and can be applied to analysis of transgenic crop proteins.

3. Recovery rate test of immunosensor

The standard addition recovery experiments were performed with 0.05%, 0.1%, 0.5% and 1% transgenic maize BT-176 and transgenic soybean a2704-12 seed standards, corresponding blank samples were blank controls, and samples at each concentration were repeated three times to evaluate the applicability and accuracy of the constructed immunosensor, with the results shown in table 3.

Table 3 recovery of PAT protein from immunosensor spiked samples (n ═ 3)

As can be seen from Table 3, the relative standard deviation RSD is less than 15.0%, the recovery rate of the corn sample is 98-113%, and the recovery rate of the soybean sample is 85-108%, which indicates that the constructed electrochemical immunosensor has good accuracy for detecting the PAT protein, and can be applied to the actual sample detection of transgenic crops.