CN102636643A - Immunosensor for detecting thiacloprid and preparation method of immunosensor - Google Patents

Abstract

The invention discloses an immunosensor for detecting thiacloprid and a preparation method thereof. The sensor is organically combined with graphene, nano-gold particles and chitosan, according to changes in current before and after detection, and different concentrations The thiacloprid will cause the detection current of the immunosensor to be different, and the thiacloprid in the solution can be detected accurately and efficiently, and the detection limit can reach nanogram level, and the detection method is simple, fast, stable and environmentally friendly. Friendly and economical at the same time.

Description

Immunosensor for detecting thiacloprid and preparation method thereof

technical field

The invention relates to a device and method in the technical field of chemical detection, in particular to an immune biosensor for measuring thiacloprid in a solution using graphene nanomaterials and a preparation method thereof.

Background technique

Thiacloprid is a neonicotinoid pesticide, which is a new broad-spectrum insecticide with high efficiency on piercing-sucking mouthparts pests. As one of the important sources of pollution to the environment and food, pesticides have attracted more and more attention from governments and the public. There are about 100 kinds of chemical pesticides that can destroy the balance of human hormones, and many chemical pesticides contain carcinogens; In addition to acute poisoning, the vast majority of chronic harm to the human body is mostly caused by contaminated food. China is a major exporter of agricultural products. The pesticide residues in China's exported agricultural products exceed international standards, which affects the export of agricultural products and their competitiveness in the international market, causing great economic losses. Therefore, it is still an urgent hope to find a fast and accurate determination technology for thiacloprid, and the research is of great significance.

Currently, the most widely used methods for the determination of thiacloprid mainly include liquid chromatography, liquid chromatography-mass chromatography, and high-performance liquid chromatography. However, these analytical methods require large-scale analytical instruments, and the process operation is cumbersome, so on-site detection cannot be realized.

Contents of the invention

The object of the present invention is to aim at above-mentioned deficiencies in the prior art, provide a kind of immunosensor for detecting thiacloprid and preparation method thereof, described sensor is through the organic combination between graphene, nano-gold particle and chitosan, Accurate and efficient detection of thiacloprid in the solution, the detection limit can reach nanogram level, and the detection method is simple, fast, environmentally friendly and economical.

To achieve the above object, the present invention adopts the following technical solutions:

An immunosensor for detecting thiacloprid (3-(6-chloro-3-pyridyl)methyl-1,3-thiazoline-2-ylidene) cyanamide, comprising electrodes, deposited on the The nano gold particle layer on the electrode, the graphene-chitosan layer coated on the described nano gold particle layer, the thiacloprid layer coated on the described graphene-chitosan layer, and coated on the The BSA layer on the thiacloprid layer.

Preferably, the electrode is a glassy carbon electrode, preferably a glassy carbon electrode that has been ground and polished by Al ₂ O ₃ powder liquid, and preferably the particle size of the Al ₂ O ₃ is 1.0 and 0.3 μm; the nano-gold particles are LHAuCL ₄ , preferably 100mg/LHAuCL ₄ solution; the graphene in the graphene-chitosan: the mass ratio of chitosan is 5:2, preferably the chitosan is 1mg/mL chitosan solution; The BSA is a BSA solution with a mass fraction of 5%.

The invention also discloses a method for preparing the immunosensor, comprising the following steps:

1, prepare the mixed solution of described graphene-chitosan:

a. Preparation of chitosan solution, heat 0.05M HCL to 80-90°C, add chitosan, stir the solution, add NaOH with a concentration of 0.1M after cooling, adjust pH to 7, and prepare chitosan solution , preferably at a concentration of 1 mg/mL;

B. graphene is dissolved in described chitosan solution, preferably graphene: the ratio of described chitosan solution is 5mg: 2mL, ultrasonic dispersion, preferably 1h, makes graphene-chitosan suspension;

2. After the electrode, preferably a glassy carbon electrode, is ground and polished by the Al ₂ O ₃ powder solution, ultrasonically in absolute ethanol and water for 3 minutes respectively, preferably 1.0 and 0.3 μm Al ₂ O ₃ powder solution;

3. Deposit nano-gold particles on the electrode first, then drop the graphene-chitosan suspension on the electrode, then drop the thiacloprid solution on the electrode, dry at 37°C, and finally place Soak in 5% BSA solution at 37°C for 30 min.

In a preferred embodiment, the method for depositing gold nanoparticles described in step 3 is: placing the electrode in a solution of gold nanoparticles, preferably _a 100mg/L HAuCL solution, and scanning at a constant potential for 60s at a potential of -0.2V .

In another preferred embodiment, the preferred concentration of the thiacloprid solution is 0.2 g/L.

The invention also discloses a method for detecting thiacloprid: immersing the immunosensor in a phosphate buffer solution containing free thiacloprid in different concentrations and a monoclonal antibody to thiacloprid with a titer concentration of 1:6400, Preferably, the pH of the phosphate buffer solution is 7.4, and the concentration is 0.1mol/L; and incubate at 37°C for 40 minutes; after washing with the phosphate buffer solution, perform a differential pulse voltammetry (DPV) scan in a 2mM K ₃ Fe(CN) ₆ solution , the scanning range is -0.2V to 0.5V, the amplitude is 0.05, the pulse width is 0.05s, the sampling width is 0.02s, the pulse period is 0.2s, and the relaxation time is 2s.

The experimental results showed that with the increase of the concentration of thiacloprid in the incubation solution, the peak current of DPV increased. Define the DPV peak current of the modified electrode incubated in the incubation solution containing only thiacloprid antibody as I ₀ , and the DPV peak current of the modified electrode incubated in the incubation solution containing free thiacloprid antigen as I _X , and calculate ΔI =I _X -I ₀ , a linear curve can be obtained by plotting ΔI against the concentration of thiacloprid (C). The concentration of thiacloprid in the range of 1-4000ng/mL is proportional to ΔI, the slope is 0.00202, and the linear correlation coefficient is 0.97728.

The invention has the following beneficial effects: because graphene has a large specific surface area and can well promote the electron transfer of bioelectrically active molecules, chitosan has the characteristics of ion exchange and adsorption for many ions, organic substances, and biomolecules. Therefore, the immunoelectrochemical sensor for immobilizing thiacloprid antibody of the present invention can stably and rapidly detect the concentration of residual thiacloprid at normal temperature.

Description of drawings

Figure 1 is a schematic diagram of the working principle of the competition mechanism of the immunosensor:

Fig. 2 is the cyclic voltage diagram of thiacloprid under different conditions.

Fig. 3 is a graph showing the DPV curves of different concentrations of thiacloprid detected by the immunosensor in the embodiment.

Fig. 4 is a linear graph of the change ΔI of the current in Fig. 3 and the concentration of thiacloprid.

Detailed ways

The following is a detailed description of the embodiments of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention and provides a detailed implementation plan and a specific operation process. But the scope of protection of the present invention is not limited to the following examples.

In the following examples, the preparation method of the immunosensor is as follows:

1. Preparation of chitosan solution: heat 0.05M HCL to 80-90°C, add weighed chitosan, stir the solution, add NaOH with a concentration of 0.1M after cooling, adjust the pH to 7, and prepare Concentration is the chitosan solution of 1mg/mL;

2. Weigh 5 mg of graphene and dissolve it in 2 mL of 1 mg/mL chitosan solution in step 1, and disperse it ultrasonically for 1 hour to obtain a graphene-chitosan suspension;

3. After grinding and polishing the glassy carbon electrode with a diameter of 3 mm by 1.0 and 0.3 μm Al ₂ O ₃ powder solution, ultrasonically in absolute ethanol and water respectively for 3 min;

4. Place the glassy carbon electrode finally obtained in step 3 in 100 mg/L HAuCL ₄ solution, scan at a potential of -0.2 V for 60 s, and then drop-coat 4 μL of graphene-chitosan suspension on the electrode surface, Then drop 2 μL of 0.2 g/L thiacloprid on the electrode, and dry it at 37°C; finally place it at 37°C and soak it in a BSA solution with a mass fraction of 5% for 30 minutes to prepare the immunosensor in this example .

In this embodiment, the pH of the phosphate buffer solution is 7.4, and the concentration is 0.1 mol/L.

In this embodiment, the scan range of the differential pulse voltammetry (DPV) scan is -0.2V to 0.5V, the amplitude is 0.05, the pulse width is 0.05s, the sampling width is 0.02s, the pulse period is 0.2s, and the relaxation time is 2s.

Figure 1 is a schematic diagram of the detection of thiacloprid in this embodiment. When the immunosensor enters the incubation solution containing the antibody containing thiacloprid, the free thiacloprid in the incubation solution competes with the solution with thiacloprid fixed on the electrode. Antibody responses to thiacloprid in The adsorption of the antibody on the electrode causes a change in the electrochemical signal of K ₃ [Fe(CN) ₆ ], thereby realizing the detection of thiacloprid; (a) the free thiacloprid in the solution and the thiacloprid immobilized on the electrode Clocloprid competes with the antibody in the solution to react, (b) the electrode is placed in 2mM K ₃ [Fe(CN) ₆ ] after cleaning, (c) electrochemical detection.

Example 1: Detection of Thiacloprid by Immunosensor

The immunosensor prepared above was immersed in a total volume of 50 μL of free thiacloprid solutions containing different concentrations, and a phosphate buffer solution of a monoclonal antibody to thiacloprid with a titer concentration of 1:6400, and incubated at 37°C For 40 min, after washing with phosphate buffer solution, differential pulse voltammetry (DPV) scanning was performed in 2mM K ₃ Fe(CN) ₆ solution. In this embodiment, the pH of the phosphate buffer solution is 7.4, and the concentration is 0.1 mol/L. In this embodiment, the scan range of the differential pulse voltammetry (DPV) scan is -0.2V to 0.5V, the amplitude is 0.05, the pulse width is 0.05, the sampling width is 0.02s, the pulse period is 0.2s, and the relaxation time is 2s. The experimental results showed that with the increase of the concentration of thiacloprid in the incubation solution, the peak current of DPV increased.

Figure 2 is the cyclic voltammetry results of thiacloprid. The cyclic voltammetry curve of the bare electrode (curve A) in 2mM K ₃ [Fe(CN) ₆ ] PBS solution shows a pair of obvious Fe(CN) ₆ ^3-/4- redox peak; when the electrode is modified with graphene/thiacloprid/chitosan (curve B), the peak current increases significantly, and graphene shows excellent electrochemical activity, which enhances the electronic Transmission; when the electrode is modified with nano-gold/graphene/thiacloprid/chitosan (curve C), the peak current intensity is greater, and the composite material of nano-gold and graphene shows more superior electrochemical performance; After the nano-gold/graphene/thiacloprid/chitosan modified electrode was incubated in the incubation solution containing thiacloprid antibody (curve D), the peak current decreased, which was due to the interaction between the thiacloprid antibody and the thiacloprid antibody on the electrode. The cloprid antigen reaction was caused by adsorption on the electrode, which hindered the transfer of electrons, which also indicated that the thiacloprid antigen had been modified on the electrode.

Figure 3 is the immunosensor in (a) containing only 8 μL of thiacloprid monoclonal antibody with a titer concentration of 1:6400, containing 8 μL of thiacloprid antibody with a titer concentration of 1:6400 and free thiacloprid at different concentrations (b) 4000ng/mL, (c) 3000ng/mL, (d) 2000ng/mL, (e) 500ng/mL, (f) 50ng/mL, (g) 10ng/mL, (h) 1ng/mL ( i) DPV curve in 2 mM K ₃ [Fe(CN) ₆ ] in PBS solution after incubation in 0 ng/mL incubation solution for 40 min. With the increase of thiacloprid concentration in the incubation solution, the DPV peak current increased. That is, the higher the concentration of free thiacloprid, the less antibody bound to the thiacloprid molecules immobilized on the electrode. Define the DPV peak current of the modified electrode incubated in the incubation solution containing only thiacloprid antibody as I ₀ , and the DPV peak current of the modified electrode after incubation as I _X , and calculate ΔI=I _X -I ₀ , and use ΔI for Plotting the thiacloprid concentration (C) yielded a linear curve (Figure 4). The concentration of thiacloprid in the range of 1-4000ng/mL is proportional to ΔI, the slope is 0.00202, and the linear correlation coefficient is 0.97728.

Embodiment 2: Determination of spiked thiacloprid in actual tomato samples

Step 1, tomato sample processing: Weigh 2±0.0050g tomato homogenate into 4 sample tubes of 10mL, add 200μL 0.2g/L thiacloprid standard solution to sample 4, add 100μL 0.2g/L thiacloprid to sample 3 Thiacloprid standard solution, add 25μL 0.2g/L thiacloprid standard solution to sample 2, do not add to sample 1, and add 3mL acetonitrile to the 4 sample tubes successively, the mixture is ultrasonically oscillated for 30min, and centrifuged at 2000r/m After 10 min, the supernatant was transferred to a nitrogen blowpipe. The extract was concentrated and evaporated at 50°C under nitrogen blowing, and the concentrate was dissolved in 1 mL of 0.1 mol/L phosphate buffer solution with a pH of 7.4 for electrochemical analysis.

Step 2, the determination of spiked thiacloprid in the actual tomato sample: take 5 μL of different tomato extract samples, and 8 μL of thiacloprid antibody solution with a titer concentration of 1:6400 and 0.1 mol/L antibody solution with a pH of 7.4 Phosphate buffer solution was mixed to make incubation solution, so that the total volume of each incubation solution was 50 μL, and the concentration of thiacloprid monoclonal antibody was the same, and the immunoelectrode was inserted into the incubation solution, incubated at 37°C for 40 min, rinsed with phosphate buffer solution and placed in 2 mM Differential pulse voltammetry (DPV) scans were performed in K ₃ Fe(CN) ₆ solution. Remove the peak current of the blank sample as I ₀ , the peak current of other samples I _X , and calculate ΔI=I _X -I ₀ , check the working curve (Fig. 4) to obtain the concentration of thiacloprid, the recovery results are shown in Table 1

Table 1 is the recovery rate of immunosensor detection of thiacloprid concentration in spiked tomatoes

Embodiment 3: Determination of spiked thiacloprid in actual apple sample

Step 1, Apple sample processing: Weigh 2±0.0050g apple homogenate into 4 sample tubes of 10mL, add 200 to sample 4, add 100μL 0.2g/L thiacloprid standard solution μL0.2g/L to sample 3 L thiacloprid standard solution, add 100μL 0.2g/L thiacloprid standard solution to sample 3, add 25μL 0.2g/L thiacloprid standard solution to sample 2, do not add to sample 1, and add in 4 sample tubes Add 3 mL of acetonitrile in sequence, the mixture is ultrasonically oscillated for 30 min, centrifuged at 2000 r/m for 10 min, and the supernatant is transferred to a nitrogen blowpipe. The extract was concentrated and evaporated at 50°C under nitrogen blowing, and the concentrate was dissolved in 1 mL of phosphate buffer solution with a pH of 7.4 for electrochemical analysis.

Step 2, Determination of spiked thiacloprid in actual apple samples: Take 5 μL of different apple extract samples, and 8 μL of thiacloprid antibody solution with a titer concentration of 1:6400 and 0.1 mol/L phosphoric acid with a pH of 7.4 The buffer solution was mixed to make the incubation solution, so that the total volume of each incubation solution was 50 μL, and the concentration of the thiacloprid monoclonal antibody was equal. The modified electrode was put into the incubation solution, incubated at 37°C for 40min, rinsed with phosphate buffer solution and placed in the incubation solution. Differential pulse voltammetry (DPV) scans were performed in 2mM K3Fe(CN)6 solution. Remove the peak current of the blank sample as I ₀ , the peak current of other samples I _X , and calculate ΔI=I _X -I ₀ , check the working curve (Fig. 4) to obtain the thiacloprid concentration, the recovery results are shown in Table 2

Table 2 is the recovery rate of immunosensor detection of thiacloprid concentration in spiked apples

Claims (10)

1. immunosensor that is used to detect thiophene worm quinoline; Comprise electrode, it is characterized in that also comprising: be deposited on nanogold particle layer on the said electrode, coat Graphene-shitosan layer on the said nanogold particle layer, coat the thiophene worm quinoline layer on said Graphene-shitosan layer and coat the BSA layer on the said thiophene worm quinoline layer.

2. according to the said described immunosensor of claim 1, wherein, said electrode is a glass-carbon electrode.

3. according to the said described immunosensor of claim 1, wherein, said electrode is through Al ₂O ₃Powder liquid sanding and polishing.

4. according to the said described immunosensor of claim 1, wherein, said nanogold particle is LHAuCL ₄

5. according to the said described immunosensor of claim 1, wherein, Graphene described in said Graphene-shitosan: the mass ratio of shitosan is 5: 2.

6. according to the said described immunosensor of claim 1, wherein, said BSA is that massfraction is 5% BSA solution.

7. method for preparing the arbitrary said immunosensor of claim 1-6 may further comprise the steps:

A, prepare the mixed solution of said Graphene-shitosan:

A. the preparation of chitosan solution is heated to 80-90 ℃ with the HCL of 0.05M, adds shitosan, agitating solution, and it is the NaOH of 0.1M that the cooling back adds concentration, regulates pH to 7, prepares chitosan solution;

B. Graphene is dissolved in the said chitosan solution, ultrasonic dispersion makes Graphene-shitosan suspending liquid;

B, with electrode through Al ₂O ₃Behind the powder liquid sanding and polishing, each ultrasonic 3min in absolute ethyl alcohol and water respectively;

C, with first depositing nano gold grain on the electrode after ultrasonic among the step B, then with the hanging drop of said Graphene-shitosan on electrode, again with thiophene worm quinoline drips of solution on electrode, 37 ℃ of oven dry down place at last and are immersed in BSA solution 30min under 37 ℃.

8. method according to claim 7, wherein, the method for depositing nano gold grain is described in the said step C: said electrode after ultrasonic is placed nanogold particle solution, constant potential scanning 60s under-0.2V electromotive force.

9. method according to claim 8, wherein, said nanogold particle solution is 100mg/LHAuCL ₄Solution.

10. method according to claim 7, wherein, the concentration of said thiophene worm quinoline solution is 0.2g/L.

Images