CN105372307A - Preparation method and application of electrode for detecting transgenic CaMV35S promoter - Google Patents

Abstract

The invention provides a preparation method-level application for detecting transgenic CaMV35S promoter electrodes, including: the preparation steps of the reduced graphene oxide/gold nanocomposite material RGO/Au; the preparation steps of the silicon@cadmium telluride nanocomposite material Si@CdTe ; the step of preparing probe 1 (Probe1) on the ITO electrode; the step of modifying the target DNA (t-DNA) CaMV35S promoter to the probe 1 (Probe1); modifying the probe to the target DNA (t-DNA) CaMV35S promoter 2 (Probe2) steps. The present invention adopts rGO/Au? The dual signal amplification at both ends of NPs and Si@CdTe realized the sensitive detection of the transgenic CaMV35S promoter. In the concentration range of 0.05-100pM, the concentration of CaMV35S showed a good linear relationship with the photocurrent, and the detection limit could reach 0.017pM.

Description

Preparation method and application of an electrode for detecting transgenic CaMV35S promoter

technical field

The invention relates to the field of preparation of electrochemical electrodes, in particular to a preparation method and application of an electrode for detecting a transgenic CaMV35S promoter.

Background technique

Genetically Modified Crops (GMC for short) refers to the use of biotechnology to insert target genes identified and isolated from animals, plants or microorganisms into the plant genome to change its genetic composition and produce new agronomic traits. Since the first commercialization of genetically modified tomatoes in the United States in 1994, biotechnology has been widely used in modern agriculture. At present, there are more than 100 kinds of genetically modified organisms approved for commercial planting in the world, and 29 countries grow genetically modified crops. Many countries use some of the approved genetically modified crops as raw materials for food or feed production, and at the same time, the safety of genetically modified products has received widespread attention.

Currently, transgenic detection methods are mainly based on exogenous protein target detection and nucleic acid-based detection. Detection methods targeting exogenous proteins are based on the specific recognition of antigens by antibodies, mainly including enzyme-linked immunosorbent assay (ELISA), immunoassay strips and protein chips, but these methods have high background signals and difficult to maintain protein activity for a long time 1. The cost of developing specific antibodies is high, and the protein of processed transgenic products is denatured and cannot be detected. The most commonly used nucleic acid-based detection method is polymerase chain reaction (PCR), which is the most commonly used detection method for transgenics, but it requires a large amount of samples, cumbersome operations, time-consuming, and the accuracy of the results is easily affected by false positives and false negatives .

Graphene has higher mechanical strength, larger specific surface area and lower preparation cost. These characteristics make graphene a new excellent carrier, and the composite material formed by gold nanoparticles (AuNPs) and graphene not only has the original Excellent properties, such as large specific surface area, excellent electrical conductivity, catalytic performance, and excellent chemical stability, etc., and there is a synergistic effect between the two components, thus greatly improving the performance of the composite material and making graphene Au/AuNP composites exhibit many excellent properties and potential applications in the fields of catalysis, biosensors, spectroscopy, and energy storage.

Cadmium telluride quantum dots (CdTeQDs) are semiconductor nanomaterials. By adjusting the particle size, the band gap from the entire visible light band to the ultraviolet light band can be obtained. It has a large carrier mobility, high light absorption characteristics and Thermal stability, etc., can produce high-efficiency luminescence under ultraviolet light or electronic excitation. In terms of photoelectric properties, it not only has good luminescent performance, but also is a good electron acceptor material, so it has been widely studied and applied in electroluminescent devices and photovoltaic cell devices.

Silica nanospheres have the advantages of small size, good water solubility, large specific surface area, good biocompatibility, non-toxicity and the ability to couple with a variety of biomolecules, and are ideal carriers for quantum dots.

A photoelectrochemical sensor is a sensor that uses light energy as the excitation energy and an electrical signal as the detection signal.

In the invention, silicon balls (SiO ₂ @CdTe) coated with cadmium telluride are used as photoelectric signal markers. First, the surface of the ITO electrode is sequentially modified with graphene/gold (rGO/AuNPs), DNA probe 1 (Probe1), target DNA (T-DNA), DNA probe 2 (Probe2)-CdTe-coated silicon balls (SiO ₂ @CdTe), the constructed photoelectrochemical sensor greatly improves the sensitivity. The detection system uses ITO as the working electrode, the saturated calomel electrode as the reference electrode, the platinum electrode as the counter electrode, and the photocurrent as the detection signal. Through the detection of different concentrations of target DNA, a standard curve is established to achieve the accuracy of crops and products. purpose of quantitative testing.

Contents of the invention

The present invention aims at inventing a photoelectrochemical sensor integrating the advantages of label-free, high sensitivity, high selectivity, simple and easy operation, etc., and provides a quantitative detection containing The method of CaMV35S promoter transgenic crops and products solves the existing problems of high detection cost, complicated detection scheme, long detection time and low sensitivity.

The present invention is achieved through the following technical solutions:

Step 1, the preparation step of reduced graphene oxide/gold nanocomposite material RGO/Au;

Step 2. Preparation steps of silica nanospheres@cadmium telluride nanocomposite material SiO ₂ @CdTe;

Step 3. The step of preparing probe 1 (Probe1) on the ITO electrode: disperse RGO/Au in distilled water to obtain the RGO/Au dispersion liquid, then drop-coat it on the modified area on the surface of the ITO electrode, and dry it; take the dissolved probe 1 of tris-HCl buffer solution was drip-coated on the surface of the dried RGO/Au and dried; after rinsing the surface of the electrode, it was dried to obtain ITO-rGO/Au-Probe1;

Step 4. The step of modifying the target DNACaMV35S promoter to probe 1: take the tris-HCl buffer dissolved in the target DNACaMV35S promoter and apply it on the surface of the ITO-rGO/Au-Probe1, incubate at room temperature, rinse after drying Electrode, get ITO-rGO/Au-Probe1-t-DNA;

Step 5. The step of modifying the probe 2 to the target DNACaMV35S promoter: adding the distilled water dispersion of SiO ₂ @CdTe and the aqueous solution of N-hydroxysuccinimide to the ethyl-(3-dimethylaminopropyl) carbon The reaction is carried out in an aqueous solution of diimine hydrochloride. After the reaction, centrifuge and wash to obtain solid A. Add the obtained solid A to the tris-HCl buffer solution in which probe 2 is dissolved to obtain mixed solution A, and shake for a period of reaction. After a period of time, the solid product Probe2/SiO ₂ @CdTe was obtained; after Probe2/SiO ₂ @CdTe was dispersed in distilled water, a mixed solution B was obtained, and the mixed solution B was drop-coated on the ITO-rGO/Au-Probe1-t- Incubate on the DNA surface, rinse the electrode surface after drying, and obtain ITO-rGO/Au-Probe1-t-DNA-Probe2/SiO ₂ @CdTe.

In step 3, the concentration of the RGO/Au dispersion is 1-4 mg/mL, and the amount of the RGO/Au dispersion that is drop-coated on the ITO surface is 20 μL; The concentration is 4 μM.

In step 4, the amount of tris-HCl buffer dissolved with the target DNACaMV35S promoter is 20 μL, and the concentration of the target DNA is 0.05 pM-100 pM.

In step 5, the volume ratio of the distilled water dispersion of SiO ₂ @CdTe used, the aqueous solution of N-hydroxysuccinimide, and the aqueous solution of ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride 25:1:1; the concentration of the distilled water dispersion of SiO ₂ @CdTe used is 1mg/mL, the concentration of the aqueous solution of N-hydroxysuccinimide is 0.2M, ethyl-(3-dimethylaminopropyl ) The concentration of the aqueous solution of carbodiimide hydrochloride is 0.4M; In the mixed solution A, the concentration of the probe 2 in the tris-HCl buffer solution in which the probe 2 is dissolved is 10 μM, and in the mixed solution A, each 100 μL of tris-HCl buffer contains 1 mg of SiO ₂ @CdTe; in the mixture B, the concentration of Probe2/SiO ₂ @CdTe is 4 μM.

All electrode washing methods are to wash the electrodes with PBS buffer solution with pH 7.0 for 3 times, and all tris-HCl buffer solutions have a pH of 7.4 and a concentration of 4.5 mM.

Before modifying the probe 2 (Probe2) to the target DNA (t-DNA), take 20 μL of freshly prepared 0.01 mM 6-mercaptohexanol and drop it on the surface of ITO-rGO/Au-Probe1-t-DNA for blocking The excess active sites on rGO/Au were used after 2 hours, and the electrode was washed 3 times with PBS buffer at pH 7.0 to remove excess 6-mercaptohexanol.

The preparation method of SiO ₂ @CdTe is as follows: select silicon balls and add them to diethylene glycol diacrylate (PDDA), stir the reaction, after the stirring reaction is completed, centrifuge and wash, take the precipitate and add it to distilled water of CdTe to disperse solution, stirred and reacted, after the reaction was completed, centrifuged, washed, and dried to obtain the product, which was set aside.

The sequence of probe 1 (Probe1) used was 5'HS-GGCCATCGTTGAA3'; the sequence of CaMV35S (t-DNA) was 5'GGCAGAGGCATCTTCAACGATGGCC3'; the sequence of probe 2 (Probe2) was 5'GATGCCTCTGCC-NH23'.

The prepared electrode is used as a sensor for photoelectrochemical detection of the CaMV35S promoter, and the detection steps are as follows:

(1) Place the prepared ITO electrodes containing different concentrations of target DNA in PBS buffer at pH 7.0, and use a three-electrode system for electrochemical detection.

The three-electrode system: a saturated calomel electrode is a reference electrode, a platinum wire electrode is a counter electrode, and ITO is a working electrode;

(2) The scanning voltage ranges from -0.5V to 0V, the potential step is 4mV, the frequency is 25Hz, and the amplitude is 25mV;

(3) The photoelectric detection adopts the i-t test method, and the bias voltage is set to 0V; switch the lamp every 20s, record the change value of the current before and after the switch lamp, and according to the different photocurrent values generated by different concentrations of t-DNA, Draw a working standard curve;

(4) Replace the t-DNA solution with the sample solution to be tested, and perform detection according to the method for drawing the working curve.

The standard curve for CaMV35S detection refers to the standard curve prepared according to the photocurrent at different concentrations after the sensor reacts with different concentrations of CaMV35S and scans its photocurrent signal spectrum.

The beneficial effects of the present invention are:

(1) The present invention uses CaMV35S as the target DNA and the principle of specific complementary pairing of double probes to specifically recognize the CaMV35S transgene, and can detect transgenic crops and their products, greatly reducing the probability of false negatives and false positives.

(2) The present invention coats CdTe on the surface of silica nano-microspheres, so that N pieces of CdTe can be connected to a single Probe2, thereby performing signal amplification.

(3) The present invention realizes the sensitive detection of the transgenic CaMV35S promoter through dual signal amplification at both ends of rGO/AuNPs and SiO ₂ @CdTe. In the concentration range of 0.1-100pM, the CaMV35S concentration and photocurrent present a good linear relationship , the detection limit can reach 0.017pM.

Description of drawings

Fig. 1 is the photocurrent response diagram obtained by detecting different concentrations of CaMV35S in Example 4, wherein a is 0.05pM, b is 0.01pM, c is 0.5pM, d is 1pM, e is 10pM, f is 50pM, and g is 100pM ;

Fig. 2 is the experimental contrast figure in embodiment 3 and embodiment 7, wherein a is the photocurrent response of the blank electrode in embodiment 3, b is the photocurrent response of t-DNA, c is 2 base mismatched DNAs, d is 5 base mismatched DNA each, e is completely non-complementary DNA.

detailed description

The present invention will be further described below in conjunction with specific embodiment:

Both the silicon spheres and CdTe used in the present invention can be prepared by existing methods, and the silicon spheres used in the present invention refer to silicon dioxide nano-microspheres.

The preparation method of the graphene oxide used in the present invention is:

The preparation of GO adopts the modified Hummers method: under the conditions of ice-water bath and stirring, add 1g of natural flake graphite to 50mL concentrated H ₂ S ₂ O ₄ (98%), cool to zero; slowly add 0.5gKNO ₃ and 6gKMnO ₄ . React for 4 hours under the condition that the reaction temperature is controlled not to exceed 10°C. Then the system was transferred to a constant temperature water bath at 35° C. for stirring for 2 h, 300 mL of deionized water was added, and the reaction was continued for 2 h at ≤80° C. Reduce the remaining KMnO ₄ with excess 5% H ₂ O ₂ , wash with 5% HCl several times, and finally wash with enough deionized water until the solution no longer contains SO ₄ ^2- ions (BaCl ₂ detects no white precipitate) . The final product was transferred to a 65°C oven for drying and stored for future use.

The preparation method of rGO/Au is:

Weigh 25 mg of GO and ultrasonically disperse it in 50 mL of absolute ethanol solution, add 1 g of EDC and 0.5 g of NHS, and stir for 8 h at room temperature; then add 1.0 g of 2-ATP into the above reaction system, and continue stirring for 12 h at room temperature; Wash with water ethanol and double-distilled water, centrifuge for 3-5 times, and finally vacuum-dry at 60°C for 24 hours to obtain GO-SH; weigh 10 mg of the above-prepared GO-SH and ultrasonically disperse in 30 mL of double-distilled water, and transfer to In the three-hole flask, heat and reflux for 0.5h under stirring; then slowly add 10mL of freshly prepared sodium citrate solution (0.2gmL ^-1 ) into the three-hole flask, continue heating and reflux for 2.5h under stirring; then add 50μL HAuCl ₄ The solution (2wt%) was slowly added dropwise into the above reaction system, continued to heat and reflux for 0.5h, then stopped heating, and cooled to room temperature while stirring. Finally, the rGO/AuNPs were obtained by washing with twice distilled water, centrifuging for 3 to 5 times, and vacuum drying.

Example 1:

Preparation of SiO ₂ @CdTe

Take 1mg of 100nm silicon spheres, add them to 40mL of 0.4% diethylene glycol diacrylate (PDDA), stir for 1 hour, centrifuge at 7000 rpm for 5 minutes, wash with distilled water for 3 times, then take the precipitate and add it to 8 mL of distilled water containing 2.4 mg of CdTe was stirred and reacted for 1 hour, and the product was centrifuged, washed, and dried for later use.

Example 2:

Preparation of Probe2/SiO ₂ @CdTe:

Take 1 mL of 1 mg/mL SiO ₂ @CdTe dispersion in distilled water and 40 μL of 0.2M N-hydroxysuccinimide (NHS) into 40 μL of 0.4M ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), after reacting for 2 hours, centrifuge to remove excess EDC and NHS, add 100 μL of 10 μM Probe2tris-HCl buffer, and shake for 12 hours to obtain Probe2/SiO ₂ @CdTe.

Example 3:

(1) Pretreatment of ITO electrode: Cut the ITO conductive glass into 1cm*2cm size, clean it with detergent, ethanol, and ultrapure water for 30 minutes, then dry it with nitrogen, and fix it on a section of the ITO electrode with insulating tape An area of 1cm*0.5cm is used for modifying materials and testing;

(2) Take 20 μL of distilled water dispersion dispersed with 2mg/mLrGO/Au and evenly drop-coat it on the ITO conductive glass, and place it to dry;

(3) Take 10 μL of 4 μM Probe1 with sulfhydryl group drop-coated on the surface of the electrode in step (2), fix Probe1 on the surface of the electrode with the help of Au-S bonds, react for 12 hours, rinse the electrode with PBS buffer solution of pH 7.0 for 3 times, rinse Remove excess Probe1 and dry to obtain ITO-rGO/Au-Probe1;

(4) Take 20 μL of freshly prepared 0.01 mM 6-mercaptohexanol and add it dropwise on the surface of the electrode to seal the redundant active sites on rGO/Au. After 2 hours, rinse the electrode with PBS buffer solution with pH 7.0 for 3 times. To remove excess MCH;

(5) Take 20 μL of 4 μM Probe2/SiO ₂ @CdTe distilled water dispersion and apply it onto the surface of the electrode to form a control test of blank DNA. After incubation for 50 minutes, wash the electrode 3 times with PBS buffer at pH 7.0 to remove excess Probe2/SiO ₂ @CdTe to get ITO-rGO/Au-Probe1-Probe2/SiO ₂ @CdTe photoelectrochemical sensor.

Example 4:

(1) Pretreatment of ITO electrode: Cut the ITO conductive glass into 1cm*2cm size, clean it with detergent, ethanol, and ultrapure water for 30 minutes, then dry it with nitrogen, and fix it on a section of the ITO electrode with insulating tape An area of 1cm*0.5cm is used for modifying materials and testing;

(2) Take 20 μL of distilled water dispersion dispersed with 1 mg/mLrGO/Au and evenly drop-coat it on the ITO conductive glass, and let it dry;

(3) Take 10 μL of 4 μM Probe1 with sulfhydryl group drop-coated on the surface of the electrode in step (2), fix Probe1 on the surface of the electrode with the help of Au-S bonds, react for 12 hours, rinse the electrode with PBS buffer solution of pH 7.0 for 3 times, rinse Remove excess Probe1 and dry to obtain ITO-rGO/Au-Probe1;

(4) Take 20 μL of freshly prepared 0.01 mM 6-mercaptohexanol and add it dropwise on the surface of the electrode to seal the redundant active sites on rGO/Au. After 2 hours, rinse the electrode with PBS buffer solution with pH 7.0 for 3 times. To remove excess MCH;

(5) Take 20 μL of 0.05, 0.1, 0.5, 1, 10, 50, and 100 pM t-DNA respectively and drop them on the surface of 6 different ITO electrodes to make the target DNA and Probe1 complement each other, incubate at room temperature for 50 minutes, and use pH7 .0 PBS buffer solution to rinse the electrode 3 times to remove excess target DNA to obtain ITO-rGO/Au-Probe1-t-DNA;

(6) Take 20 μL of 4 μM Probe2/SiO ₂ @CdTe distilled water dispersion and apply it to the surface of the electrode, so that Probe2 and the target DNA are complementary paired to connect with the photoelectrochemical signal marker SiO ₂ @CdTe. After incubation for 50 minutes, use pH7.0 Rinse the electrode 3 times with PBS buffer to remove excess Probe2/SiO ₂ @CdTe, and obtain ITO-rGO/Au-Probe1-t-DNA-Probe2/SiO ₂ @CdTe photoelectrochemical sensor with corresponding concentration of t-DNA.

The ITO-rGO/Au-Probe1-t-DNA-Probe2/SiO ₂ @CdTe photoelectrochemical sensor prepared in this example was used to detect the CaMV35S promoter. The results are shown in Figure 1, and Figure 1 shows that it is in the range of 0.05 to 100pM. The detection of CaMV35S showed good linearity, and the detection limit reached 0.017pM, so the sensor can be used for the quantitative detection of CaMV35S.

Example 5:

(1) Pretreatment of ITO electrode: Cut the ITO conductive glass into 1cm*2cm size, clean it with detergent, ethanol, and ultrapure water for 30 minutes, then dry it with nitrogen, and fix it on a section of the ITO electrode with insulating tape An area of 1cm*0.5cm is used for modifying materials and testing;

(2) Take 20 μL of distilled water dispersion dispersed with 2mg/mLrGO/Au and evenly drop-coat it on the ITO conductive glass, and place it to dry;

(3) Take 10 μL of 4 μM Probe1 with sulfhydryl group drop-coated on the surface of the electrode in step (2), fix Probe1 on the surface of the electrode with the help of Au-S bonds, react for 12 hours, rinse the electrode with PBS buffer solution of pH 7.0 for 3 times, rinse Remove excess Probe1 and dry to obtain ITO-rGO/Au-Probe1;

(4) Take 20 μL of freshly prepared 0.01 mM 6-mercaptohexanol and add it dropwise on the surface of the electrode to seal the redundant active sites on rGO/Au. After 2 hours, rinse the electrode with PBS buffer solution with pH 7.0 for 3 times. To remove excess MCH;

(5) Take 20 μL of 0.05, 0.1, 0.5, 1, 10, 50, and 100 pM t-DNA respectively and drop them on the surface of 6 different ITO electrodes to make the target DNA and Probe1 complement each other, incubate at room temperature for 50 minutes, and use pH7 .0 PBS buffer solution to rinse the electrode 3 times to remove excess target DNA to obtain ITO-rGO/Au-Probe1-t-DNA;

(6) Take 1 mL of SiO ₂ @CdTe, add 40 μL of 400 mM ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 40 μL of 200 mM N-hydroxysuccinimide (NHS), and react for 2 hours , centrifuge to remove excess EDC and NHS, add 100 μL of tris-HCl buffer containing 10 μM Probe2, and shake for 12 hours to obtain Probe2/SiO ₂ @CdTe. Take 20 μL of 4 μM Probe2/SiO ₂ @CdTe distilled water dispersion drop-coated on the electrode surface, so that Probe2 and the target DNA are complementary paired to connect with the photoelectrochemical signal marker SiO ₂ @CdTe, after incubation for 50 minutes, buffer with PBS with pH 7.0 The electrode was rinsed three times with the solution to remove excess Probe2/SiO ₂ @CdTe, and the ITO-rGO/Au-Probe1-t-DNA-Probe2/SiO ₂ @CdTe photoelectrochemical sensor with the corresponding concentration of t-DNA was obtained.

Embodiment 6:

(1) Pretreatment of ITO electrode: Cut the ITO conductive glass into 1cm*2cm size, clean it with detergent, ethanol, and ultrapure water for 30 minutes, then dry it with nitrogen, and fix it on a section of the ITO electrode with insulating tape An area of 1cm*0.5cm is used for modifying materials and testing;

(2) Take 20 μL of distilled water dispersion dispersed with 4mg/mLrGO/Au and evenly drop-coat it on the ITO conductive glass, and let it dry;

(3) Take 10 μL of 4 μM Probe1 with sulfhydryl group drop-coated on the surface of the electrode in step (2), fix Probe1 on the surface of the electrode with the help of Au-S bonds, react for 12 hours, rinse the electrode with PBS buffer solution of pH 7.0 for 3 times, rinse Remove excess Probe1 and dry to obtain ITO-rGO/Au-Probe1;

(4) Take 20 μL of freshly prepared 0.01 mM 6-mercaptohexanol and add it dropwise on the surface of the electrode to seal the redundant active sites on rGO/Au. After 2 hours, rinse the electrode with PBS buffer solution with pH 7.0 for 3 times. To remove excess MCH;

(5) Take 20 μL of 0.05, 0.1, 0.5, 1, 10, 50, and 100 pM t-DNA respectively and drop them on the surface of 6 different ITO electrodes to make the target DNA and Probe1 complement each other, incubate at room temperature for 50 minutes, and use pH7 .0 PBS buffer solution to rinse the electrode 3 times to remove excess target DNA to obtain ITO-rGO/Au-Probe1-t-DNA;

(6) Take 1 mL of SiO ₂ @CdTe, add 40 μL of 400 mM ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 40 μL of 200 mM N-hydroxysuccinimide (NHS), and react for 2 hours , centrifuge to remove excess EDC and NHS, add 100 μL of tris-HCl buffer containing 10 μM Probe2, and shake for 12 hours to obtain Probe2/SiO ₂ @CdTe. Take 20 μL of 4 μM Probe2/SiO ₂ @CdTe distilled water dispersion drop-coated on the electrode surface, so that Probe2 and the target DNA are complementary paired to connect with the photoelectrochemical signal marker SiO ₂ @CdTe, after incubation for 50 minutes, buffer with PBS with pH 7.0 The electrode was rinsed three times with the solution to remove excess Probe2/SiO ₂ @CdTe, and the ITO-rGO/Au-Probe1-t-DNA-Probe2/SiO ₂ @CdTe photoelectrochemical sensor with the corresponding concentration of t-DNA was obtained.

Since the amount of modified rGO/Au in Examples 4 to 6 is different, the amount of the probe and the target DNA that are finally loaded are different, but the difference in the detection results is only limited to the magnitude of the photocurrent, so the three examples All electrodes can be used for photoelectrochemical detection of CaMV35S.

Embodiment 7:

(1) Pretreatment of ITO electrode: Cut the ITO conductive glass into 1cm*2cm size, clean it with detergent, ethanol, and ultrapure water for 30 minutes, then dry it with nitrogen, and fix it on a section of the ITO electrode with insulating tape An area of 1cm*0.5cm is used for modifying materials and testing;

(2) Take 20 μL of distilled water dispersion dispersed with 1 mg/mLrGO/Au and evenly drop-coat it on the ITO conductive glass, and let it dry;

(3) Take 10 μL of 4 μM Probe1 with sulfhydryl group drop-coated on the surface of the electrode in step (2), fix Probe1 on the surface of the electrode with the help of Au-S bonds, react for 12 hours, rinse the electrode with PBS buffer solution of pH 7.0 for 3 times, rinse Remove excess Probe1 and dry to obtain ITO-rGO/Au-Probe1;

(4) Take 20 μL of freshly prepared 0.01 mM 6-mercaptohexanol and add it dropwise on the surface of the electrode to seal the redundant active sites on rGO/Au. After 2 hours, rinse the electrode with PBS buffer solution with pH 7.0 for 3 times. To remove excess MCH;

(5) Take 20 μL of 200pM t-DNA, 2 base mismatched DNA, 5 base mismatched DNA and 20 μL of completely non-complementary DNA and drop them on the surface of 6 different ITO electrodes to make the DNA and Probe1 complement each other. Incubate at room temperature for 50 minutes, wash the electrode 3 times with PBS buffer solution of pH 7.0 to remove excess DNA, and obtain ITO-rGO/Au-Probe1-t-DNA;

(6) Take 20 μL of 4 μM Probe2/SiO ₂ @CdTe distilled water dispersion and apply it to the surface of the electrode, so that Probe2 and the target DNA are complementary paired to connect with the photoelectrochemical signal marker SiO ₂ @CdTe. After incubation for 50 minutes, use pH7.0 Rinse the electrode 3 times with PBS buffer solution to remove excess Probe2/SiO ₂ @CdTe to obtain a photoelectrochemical sensor corresponding to DNA.

Figure 2 is the comparative data of the sensors prepared in this example and Example 3. It can be found that the photoelectric corresponding signal of the sensor of this example decreases gradually with the increase of the number of base mismatches. When the electrode is modified with completely non-complementary DNA Afterwards, its photoelectric response value was basically the same as that of the blank control, indicating that the sensor had good selection specificity for CaMV35S.

In the above examples, the sequence of probe 1 used is 5'HS-GGCCATCGTTGAA3'; the sequence of CaMV35S (t-DNA) is 5'GGCAGAGGCATCTTCAACGATGGCC3'; the sequence of probe 2 is 5'GATGCCTCTGCC-NH23'; 2 base mismatches The sequence of DNA is: 5'GGCAGAGGCAACTTCAAGGATGGCC3'; the sequence of DNA with 5 base mismatches is: 5'GCCAGAGGCAACTTCAAGGATGCCG3'; the sequence of completely non-complementary DNA is: 5'CCTGCTCCGTAGCCTGCGTCATCTG3'.

SEQUENCELISTING

<110> Jiangsu University

<120> A preparation method and application of an electrode for detecting the transgenic CaMV35S promoter

<130> A preparation method and application of an electrode for detecting the transgenic CaMV35S promoter

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<170>PatentInversion3.5

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ggccatcgttgaa13

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ggcagaggcatcttcaacgatggcc25

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gatgcctctgcc12

Claims (9)

1. A preparation method for detecting a transgenic CaMV35S promoter electrode, characterized in that, comprising: Step 1, the preparation step of reduced graphene oxide/gold nanocomposite material RGO/Au; Step 2. Preparation steps of silica nanospheres@cadmium telluride nanocomposite material SiO ₂ @CdTe; Step 3, the step of preparing probe 1 on the ITO electrode: disperse RGO/Au in distilled water to obtain the RGO/Au dispersion liquid, then drop-coat it on the modified area on the surface of the ITO electrode, and dry it; take the tris solution with probe 1 - HCl buffer solution is drip-coated on the surface of the dried RGO/Au and dried; after rinsing the surface of the electrode, it is dried to obtain ITO-rGO/Au-Probe1; Step 4. The step of modifying the target DNACaMV35S promoter to probe 1: take the tris-HCl buffer dissolved in the target DNACaMV35S promoter and apply it on the surface of the ITO-rGO/Au-Probe1, incubate at room temperature, rinse after drying Electrode, get ITO-rGO/Au-Probe1-t-DNA; Step 5. The step of modifying the probe 2 to the target DNACaMV35S promoter: adding the distilled water dispersion of SiO ₂ @CdTe and the aqueous solution of N-hydroxysuccinimide to the ethyl-(3-dimethylaminopropyl) carbon The reaction is carried out in an aqueous solution of diimine hydrochloride. After the reaction, centrifuge and wash to obtain solid A. Add the obtained solid A to the tris-HCl buffer solution in which probe 2 is dissolved to obtain mixed solution A, and shake for a period of reaction. After a period of time, the solid product Probe2/SiO ₂ @CdTe was obtained; after Probe2/SiO ₂ @CdTe was dispersed in distilled water, a mixed solution B was obtained, and the mixed solution B was drop-coated on the ITO-rGO/Au-Probe1-t- Incubate on the DNA surface, rinse the electrode surface after drying, and obtain ITO-rGO/Au-Probe1-t-DNA-Probe2/SiO ₂ @CdTe. 2. by the method for claim 1, it is characterized in that, in step 3, the concentration of RGO/Au dispersion liquid is 1～4mg/mL, the amount of the RGO/Au dispersion liquid that is drip-coated on ITO surface is 20 μ L; The amount of tris-HCl buffer containing probe 1 was 10 μL, and the concentration of probe 1 was 4 μM. 3. The method according to claim 1, characterized in that in step 4, the amount of tris-HCl buffer dissolved with the target DNA is 20 μL, and the concentration of the target DNACaMV35S promoter is 0.05pM˜100pM. 4. The method according to claim 1, characterized in that, in step 5, the distilled water dispersion of SiO ₂ @CdTe, the aqueous solution of N-hydroxysuccinimide, ethyl-(3-dimethylammonia The volume ratio of the aqueous solution of propyl) carbodiimide hydrochloride is 25:1:1; the concentration of the distilled water dispersion of SiO ₂ @CdTe used is 1 mg/mL, and the concentration of the aqueous solution of N-hydroxysuccinimide is 0.2M, the concentration of the aqueous solution of ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 0.4M; The concentration of probe 2 was 10 μM, and in the mixture A, every 100 μL of tris-HCl buffer contained 1 mg SiO ₂ @CdTe; in the mixture B, the concentration of Probe2/SiO ₂ @CdTe was 4 μM. 5. The method according to any one of claims 1 to 4, characterized in that the preparation method of SiO ₂ @CdTe is: select silicon spheres and add them to diethylene glycol diacrylate phthalate, stir and react, After the stirring reaction is completed, centrifuged and washed, the precipitate is added to the distilled water dispersion of CdTe, stirred and reacted, and after the reaction is completed, centrifuged, washed, and dried to obtain the product for future use. 6. by the method described in claim 1～4 any one, it is characterized in that, used probe 1 sequence is 5'HS-GGCCATCGTTGAA3'; Target DNACaMV35S promoter sequence is 5'GGCAGAGGCATCTTCAACGATGGCC3'; Probe 2 sequence is 5'GATGCCTCTGCC-NH23'. 7. The method according to any one of claims 1 to 4, wherein, before modifying the probe 2 to the target DNA, 20 μL of 0.01 mM 6-mercaptohexanol that is freshly configured is added dropwise to the ITO-rGO/ The surface of Au-Probe1-t-DNA was used after 2 hours, and the electrode was washed 3 times with PBS buffer of pH 7.0. 8. by the method described in any one of claim 1～4, it is characterized in that, all electrode flushing methods are to flush electrode 3 times with the PBS damping fluid of pH7.0, and all tris-HCl buffering fluid pHs are 7.4, the concentration is 4.5mM. 9. The use of the electrode prepared by the method according to any one of claims 1 to 8, characterized in that the electrode is used for photoelectrochemical detection of CaMV35S promoter.