CN105057692A - Green synthesis method of gold nanoparticles - Google Patents

Abstract

The invention discloses a green synthesis method of gold nanoparticles, which relates to a preparation method of gold nanoparticles. The invention aims to solve the technical problems that the existing chemical method for preparing gold nano particles is likely to cause environmental pollution due to too many chemical reagents and the cost of the physical method is high. The method of the present invention: 1. Wash and dry the citrus fruits, squeeze the juice, then centrifuge, take the supernatant, and dilute (1-10) times with deionized water to obtain a fruit juice solution; 2. Under room temperature, magnetic stirring After 2 minutes of the chloroauric acid solution, quickly add the fruit juice solution, wine red color appears and continue to stir for 20 minutes to end the reaction; the gold nanoparticle solution is obtained and stored in the refrigerator at 4°C. The invention adopts pure natural citrus fruit juice to greenly prepare gold nanoparticles (AuNPs), without adding any chemical reducing agent or chemical protecting agent. The synthesis method of the invention is simple and fast, has low cost and little environmental pollution.

Description

Green Synthesis of Gold Nanoparticles

technical field

The invention relates to a preparation method of gold nanoparticles.

Background technique

Gold nanomaterials have been widely valued because of their unique catalytic and optical properties, and their preparation and application have become a research hotspot in the field of nanotechnology. Compared with the traditional physical and chemical methods, the bioreduction method based on microorganisms or plants that has emerged in recent years has the advantages of low cost, environmental protection, and high stability of nanomaterials, and has become a novel preparation method for nano-gold materials with development prospects. At present, there are many methods for preparing gold nanomaterials. Due to different preparation processes, the particle size, purity and physical and chemical properties of the obtained particles are also different. The quality of the product obtained by the physical method is high, but the requirements for equipment are high, the production cost is expensive, and the ability to control the shape of gold nanoparticles is limited; the chemical method is flexible and diverse, and can be used to prepare gold nanoparticles with various shapes, but most The chemical method requires the introduction of more chemical reagents, which may cause certain environmental pollution problems.

Contents of the invention

The invention aims to solve the technical problems that the existing chemical method for preparing gold nanoparticles is likely to cause environmental pollution due to many chemical reagents and the technical problems of high cost of the physical method; the invention provides a green synthesis method of gold nanoparticles.

The green synthesis method of gold nanoparticles in the present invention is carried out according to the following steps: step 1, citrus fruits (i.e. Rutaceae citrus fruit) are washed and dried, squeezed the juice, then centrifuged, and the supernatant is taken, Dilute (1 to 10) times (by volume) with deionized water to obtain a fruit juice solution; step 2, under room temperature conditions, stir the chloroauric acid solution vigorously with a magnetic stirrer for 2 minutes and then quickly (quickly adding refers to In a short period of time, add chloroauric acid solution) into the fruit juice solution, wine red color appears and continue to stir for 20 minutes to end the reaction; the gold nanoparticle solution is obtained and stored in the refrigerator at 4°C.

Further defined, step 1 is carried out at a speed of 4000r/min. The citrus fruit described in step 1 is a kind of in orange, mandarin orange, lemon. The mass concentration of the chloroauric acid solution described in step two is 1% (w/v, ie weight/volume). The volume of the chloroauric acid solution described in step 2 is the same as the volume of the measured supernatant.

The invention adopts pure natural citrus fruit juice to greenly prepare gold nanoparticles (AuNPs), without adding any chemical reducing agent or chemical protecting agent. The synthesis method of the invention is simple and fast, has low cost and little environmental pollution.

The present invention utilizes orange, tangerine and lemon juice to successfully prepare gold nanoparticles (AuNPs). Among them, the absorption peaks of AuNPs prepared from 1×, 5× and 10× fruit juice were around 530 nm. The particle size distribution of AuNPs prepared from 1 × orange, orange and lemon juice was measured by laser particle size distribution analyzer, and the average particle size was 7.8±0.4nm, 11.8±0.5nm and 6.8±0.3nm, respectively.

At room temperature (25°C), the activities of AuNPs prepared from orange, tangerine and lemon juices were 76%, 47% and 69% after 120 hours respectively. Catalytic hydrogenation of p-nitrophenol was used as a probe reaction to evaluate the catalytic activity of AuNPs prepared from juices of different fruits. The study showed that the AuNPs prepared from the three fruit juices all had catalytic activity, and the catalytic activity of the AuNPs prepared from orange and lemon lasted longer than that prepared from orange juice.

Gold nanoparticles prepared from 1× orange juice were used to detect the pesticide residues of methion. With the increase of pesticide concentration, the color gradually changed from light wine red to purple to dark blue, indicating that the activity of acetylcholinesterase in the system was gradually inhibited, and the content of substrate gradually increased, which caused the particle size of gold nanoparticles to gradually increase. , with varying degrees of aggregation. The UV-Vis absorption spectrum basically presents similar results to the visual colorimetry. When the concentration of the pesticide gradually increased, the peak of the characteristic peak of the gold nanoparticles at the wavelength of 530nm began to decrease; at the same time, a new absorption peak appeared in the range of the wavelength of 650-780nm. The results show that different concentrations of pesticides can cause different degrees of aggregation of gold nanoparticles to form gold nanoparticles with different particle sizes, which in turn cause different wavelengths of the characteristic absorption peaks of gold nanoparticles. Selectivity experiments were carried out by adding 0.1 mg/mL glucose, sucrose, maltose, sodium chloride, potassium chloride, magnesium sulfate, calcium chloride and zinc acetate, and the color did not change after the reaction, indicating that gold nanoparticles synthesized based on orange juice The constructed pesticide detection system has high selectivity to common substances in general food.

Description of drawings

Fig. 1 is the transmission electron microscope picture () of gold nanoparticle; (1 * orange juice solution)

Fig. 2 is the ultraviolet-visible light absorption spectrogram of orange juice preparation gold nanoparticle , in Fig. 2, 1 represents the fruit juice solution that is diluted to 1 *, 2 represents the fruit juice solution that is diluted to 5 *, 3 represents the fruit juice solution that is diluted to 10 *, 4 represents the mother liquor, 5 represents the fruit juice solution diluted to 15×, and 6 represents the fruit juice solution diluted to 20×;

Fig. 3 is the particle size distribution figure that orange juice prepares gold nanoparticle;

Fig. 4 is the ultraviolet-visible light absorption spectrogram of orange juice preparation gold nanoparticle, in Fig. 4, 1 represents the fruit juice solution that is diluted to 1 *, 2 represents the fruit juice solution that is diluted to 5 *, 3 represents the fruit juice solution that is diluted to 10 *, 4 represents the mother liquor, 5 represents the fruit juice solution diluted to 15×, and 6 represents the fruit juice solution diluted to 20×;

Fig. 5 is the particle size distribution figure that orange juice prepares gold nanoparticle;

Fig. 6 is the ultraviolet-visible light absorption spectrogram of lemon juice preparation gold nanoparticle, in Fig. 6, 1 represents the fruit juice solution that is diluted to 1 *, 2 represents the fruit juice solution that is diluted to 5 *, 3 represents the fruit juice solution that is diluted to 10 *, 4 represents the mother liquor, 5 represents the fruit juice solution diluted to 15×, and 6 represents the fruit juice solution diluted to 20×;

Fig. 7 is the particle size distribution figure that lemon juice prepares gold nanoparticle;

Fig. 8 is the stability research figure of orange juice preparation gold nanoparticle;

Fig. 9 is the stability research figure of orange juice preparation gold nanoparticle;

Fig. 10 is the stability research figure of lemon juice preparation gold nanoparticle;

Fig. 11 is a graph showing the catalytic activity of gold nanoparticles prepared from orange juice;

Fig. 12 is the research diagram of the catalytic activity of gold nanoparticles prepared from orange juice;

Fig. 13 is a graph showing the catalytic activity of gold nanoparticles prepared from lemon juice;

Fig. 14 is the ultraviolet-visible light absorption spectrogram of 4 samples, in Fig. 14 a——gold nanoparticle system of the same volume, b—contrast 2, c—contrast 1, d—contrast 3 (the pesticide is methion) , e——contrast 4 (pesticide is phosphamide);

Figure 15 is the ultraviolet-visible light absorption spectrum of gold nanoparticles detecting different concentrations of pesticides. In Figure 15, 1 to 10 are in order: the concentration of methion is 0, 0.1×10 ^-4 , 0.5×10 ^-4 , 1.0× 10 ^-4 , 2.5×10 ^-4 , 5.0×10 ^-4 , 10×10 ^-4 , 25×10 ^-4 , 50×10 ^-4 and 100×10 ^-4 mg/mL, 1 is the control;

Figure 16 is a transmission electron microscope image of gold nanoparticles detecting pesticides containing methion at a concentration of 0.1×10 ^-4 mg/mL;

Figure 17 is a transmission electron microscope image of gold nanoparticles detecting pesticides containing methion at a concentration of 10×10 ^-4 mg/mL;

Fig. 18 is a transmission electron microscope image of gold nanoparticles detecting pesticides containing methion at a concentration of 100×10 ^-4 mg/mL.

Detailed ways

Specific embodiment 1: In this embodiment, oranges are used as raw materials to synthesize gold nanoparticles greenly. The specific method is carried out according to the following steps: Step 1. Take 1 orange, wash and dry it, extract the juice with a fruit juicer, and extract the juice Under the condition of 4000r/min, centrifuge for 3min; take the supernatant, collect the supernatant, take 10mL and dilute 1 time (1×) with deionized water to obtain a fruit juice solution.

Step 2, under room temperature conditions, 10mL chloroauric acid solution (1%, W/V) is placed in the reaction dish, after the magnetic stirrer stirs violently 2min, the fruit juice solution that step 1 obtains is added rapidly, the change of color occurs immediately, After the wine red color appeared, continue to stir for 20 minutes, then the reaction was terminated, and a gold nanoparticle (AuNPs) solution was obtained, which was stored in a refrigerator at 4°C.

The particle size distribution of the gold nanoparticles prepared by the method of this embodiment measured by a laser particle size distribution analyzer is shown in FIG. 3 . It can be seen from Figure 3 that the particle size of gold nanoparticles presents a normal distribution, mainly in the range of 7-9nm, accounting for 79% of the entire distribution system, and the average particle size reaches 7.8±0.4nm.

Embodiment 2: The difference between this embodiment and Embodiment 1 is that in step 1, the supernatant is diluted 5 times with deionized water. Other steps and parameters are the same as in the first embodiment.

Embodiment 3: This embodiment is different from Embodiment 1 in that: in step 1, the supernatant is diluted 10 times with deionized water. Other steps and parameters are the same as in the first embodiment.

The supernatant obtained in the method step 1 of the specific embodiment is diluted 1×, 5×, 10×, 15× and 20× respectively to prepare gold nanoparticles, and the ultraviolet-visible light absorption spectrum of the gold nanoparticles is shown in Figure 2 , it can be seen from Figure 2 that the gold nanoparticles prepared from orange juice with different concentrations have obvious absorption peaks between 520-570nm wavelength, and the peak heights are different. It can be seen that the gold nanoparticles prepared from fruit juices with different concentrations have obvious differences in particle size and content. The results show that the gold nanoparticles prepared by 1×, 5× and 10× fruit juice have absorption peaks around 530nm and have a smaller particle size; meanwhile, mother liquor (referring to supernatant), 15× and 20× The absorption peak of gold nanoparticles prepared from fruit juice gradually blue-shifted, appearing at a wavelength of 570nm, indicating that the particle size of gold particles gradually increased. Judging the content of gold nanoparticles by the peak value of the absorption peak, the content of gold nanoparticles prepared by 1× orange juice is the most in the system, and the peak value of the absorption peak reaches 0.46, which is much higher than that prepared by 15× and 20× orange juice. content of gold nanoparticles. Although the concentration of ascorbic acid contained in the mother liquor is the highest, the prepared gold nanoparticles are not dominant regardless of the particle size and content. (such as sugars, etc.), these biomacromolecules will cause the aggregation of newly generated gold species when the concentration is high, thereby increasing the particle size of the gold particles and delaying the generation of the gold particles.

Specific embodiment four: In this embodiment, oranges are used as raw materials for green synthesis of gold nanoparticles, and the specific method is carried out according to the following steps: Step 1, take 1 orange, wash and dry, squeeze the juice with a fruit juicer, and extract the juice Under the condition of 4000r/min, centrifuge for 3min; take the supernatant, collect the supernatant, take 10mL and dilute 1 times with deionized water to obtain a fruit juice solution.

Step 2, under room temperature conditions, 10mL chloroauric acid solution (1%, W/V) is placed in the reaction dish, after the magnetic stirrer stirs violently 2min, the fruit juice solution that step 1 obtains is added rapidly, the change of color occurs immediately, Wine red color appeared and continued to stir for 20 minutes to end the reaction to obtain a gold nanoparticle solution, which was stored in a refrigerator at 4°C.

The particle size distribution of the gold nanoparticles prepared by the method of this embodiment measured by a laser particle size distribution analyzer is shown in FIG. 5 . It can be seen from Figure 5 that the particle size distribution of gold nanoparticles in the preparation system of 1×orange juice also presents a normal distribution, with an average particle size of 11.8±0.5nm, mainly distributed in the range of 9-12nm. Compared with the same proportion of orange juice prepared gold nanoparticles particle size larger.

Embodiment 5: This embodiment is different from Embodiment 4 in that: in step 1, the supernatant is diluted 5 times with deionized water. Other steps and parameters are the same as in Embodiment 4.

Embodiment 6: This embodiment is different from Embodiment 4 in that: in step 1, the supernatant is diluted 10 times with deionized water. Other steps and parameters are the same as in Embodiment 4.

Dilute the supernatant obtained in step 4 of method 4 of specific embodiment 1×, 5×, 10×, 15× and 20× respectively to prepare gold nanoparticles, and the ultraviolet-visible light absorption spectrum of the gold nanoparticles is shown in Figure 4 , as can be seen from Figure 4, the ultraviolet-visible absorption spectrum of gold nanoparticles prepared from orange juice is basically consistent with that of gold nanoparticles prepared from orange juice. The absorption peaks of gold nanoparticles prepared from 1×, 5× and 10× fruit juices were around 540nm, which were larger than those prepared from orange juice. The content of gold nanoparticles in the preparation system of 1× fruit juice was lower than that prepared by the same concentration of orange juice, and the absorption peak was only about 0.32, indicating that the performance of orange juice to prepare gold nanoparticles was poorer than that of orange juice.

Specific embodiment seven: this embodiment uses lemon as raw material to synthesize gold nanoparticles greenly. The specific method is to carry out according to the following steps: step 1, take 1 lemon, wash and dry, squeeze the juice with a fruit juicer, and extract the juice Under the condition of 4000r/min, centrifuge for 3min; take the supernatant, collect the supernatant, take 10mL and dilute 1 times with deionized water to obtain a fruit juice solution.

Step 2, under room temperature conditions, 10mL chloroauric acid solution (1%, W/V) is placed in the reaction dish, after the magnetic stirrer stirs violently 2min, the fruit juice solution that step 1 obtains is added rapidly, the change of color occurs immediately, Wine red color appeared and continued to stir for 20 minutes to end the reaction to obtain a gold nanoparticle solution, which was stored in a refrigerator at 4°C.

Embodiment 8: This embodiment is different from Embodiment 7 in that: in step 1, the supernatant is diluted 5 times with deionized water. Other steps and parameters are the same as those in Embodiment 7.

Embodiment 9: This embodiment differs from Embodiment 7 in that: in step 1, the supernatant is diluted 10 times with deionized water. Other steps and parameters are the same as those in Embodiment 7.

Dilute the supernatant obtained in step 1 of the method of Embodiment 7 to 1×, 5×, 10×, 15× and 20× respectively to prepare gold nanoparticles, and the ultraviolet-visible light absorption spectrum of the gold nanoparticles is shown in Figure 6 . The results showed that the overall distribution of AuNPs prepared from lemon juice was almost the same as in oranges and tangerines. The absorption peaks of gold nanoparticles prepared from 1×, 5× and 10× fruit juices are around 530nm, and the content of gold nanoparticles in the preparation system of 1× fruit juice is the highest, and the absorption peak reaches 0.47, which is slightly higher than that of oranges and tangerines in the same proportion. preparation system. The preparation system of 1×lemon juice was selected for catalytic activity test. It can be seen from Figure 7 that in the preparation system of 1×lemon juice, the average particle size of gold nanoparticles is 6.8±0.3nm, and the particle size is mainly distributed in the range of 5-8nm.

Adopt following experiment to verify inventive effect:

1. Stability research

Carry out stability research on the gold nanoparticles prepared in specific embodiments 1, 4 and 7, respectively select the reaction solutions with time intervals of 0h, 12h, 24h, 48h and 120h, add them to cuvettes, and place them in ultraviolet-visible light spectroscopic In the photometer, the absorption spectrum of the gold nanoparticles is scanned, and the changes in the spectra are compared and analyzed to evaluate the stability; the results are shown in Figures 8-10.

It can be seen from Figure 8 that under normal temperature conditions (25°C), the absorption peak of gold nanoparticles prepared from orange juice gradually decreases with the prolongation of the time interval. The initial peak value is 0.63, and after 12 hours the peak value decreases to 0.57. After 120 hours, it decreased to 0.56, and after 120 hours, it decreased to 0.48; it shows that the gold nanoparticles maintain good activity after 5 days, and the activity is still maintained at about 76%, which can be applied to production practice.

The stability results of gold nanoparticles prepared from orange juice are shown in FIG. 9 . After standing for 12h and 24h, the decrease of the absorption peak value of the system is basically the same, indicating that the orange juice can maintain a good stability within a certain time interval. After 120h, the activity of the system was maintained at 47%, and the stability was much worse than that of gold nanoparticles prepared from orange juice.

It can be seen from FIG. 10 that the activity of gold nanoparticles prepared from lemon juice is maintained at 69% after 120 hours, which is relatively high.

In summary, the order of stability of gold nanoparticles prepared from three kinds of fruit juices from strong to weak is: orange>lemon>orange, and the stability of gold nanoparticles prepared from orange and lemon is the activity maintenance rate after 5 days Above 60%, it has a good application prospect.

2. Research on catalytic activity

To 25mL of p-nitrophenol solution (2.5× ^10-4 M), gold nanoparticles (0, 1, 2, 3, 4, 5mL) prepared by adding different doses of three kinds of fruit juices were sequentially added, and the system difference was used deionized water) and 1 mL of freshly prepared NaBH ₄ (0.25M). Place it in an ultraviolet-visible spectrophotometer, scan the absorption spectrum of the reaction system, and observe the change of the absorption peak at a wavelength of 400nm.

Catalytic hydrogenation of p-nitrophenol was used as a probe reaction to evaluate the catalytic activity of gold nanoparticles prepared from juices of different fruits. After gold nanoparticles are added to the normal reaction system, the hydrogenation reaction is obviously intensified, and the p-nitrophenol in the system is changed to p-aminophenol, which makes the peak of the original ultraviolet absorption peak at a wavelength of 400nm decrease. It can be seen from Figure 11 that with the increase in the number of gold nanoparticles prepared from orange juice, the process of the catalytic reaction changed significantly. When 1mL of gold nanoparticle sol was added, the degree of reaction was basically the same as the amount of p-nitrophenol left in the system without gold nanoparticles; when 2mL of gold sol was added, the catalytic reaction began to change significantly. When the gold sol added is 4mL, no obvious absorption peak can be seen at a wavelength of 400nm, indicating that the reaction has been completely completed under the catalysis of gold nanoparticles. It can be seen from Figure 12 that the gold nanoparticles prepared by orange juice also have obvious catalytic activity. When 1mL was added, the catalytic reaction changed significantly, and the absorption peak of p-nitrophenol dropped from 0.88 to 0.8; however, when gold When the sol was added to 5mL, there was still an absorption peak at a wavelength of 400nm, indicating that the reaction was not catalyzed completely, and the activity was still lower than that of gold nanoparticles prepared from orange juice. The catalytic activity of gold nanoparticles prepared from lemon juice is shown in Figure 13. When the gold sol added was 4mL, the absorption peak disappeared, indicating that the reaction was complete and the added gold nanoparticles had good catalytic activity. In summary, the catalytic activity of gold nanoparticles prepared from oranges and lemons is better than that prepared from orange juice, which may be related to the particle size.

3. Gold nanoparticles detection of organophosphorus pesticide residues

Acetylcholinesterase can specifically catalyze the hydrolysis of the substrate thioacetylcholine iodide (ATChI) into thioacetylcholine and acetic acid. The charge and S bond of thioacetylcholine can cause the aggregation of gold nanoparticles with negative charges on the surface, thereby Causes a change in the color of the entire system (theoretically from red to blue). However, when organophosphorus pesticides exist in the system, the activity of acetylcholinesterase is inhibited and cannot catalyze the hydrolysis reaction of the substrate, which hinders the aggregation degree of gold nanoparticles. Different concentrations of pesticides lead to different degrees of inhibition of acetylcholinesterase and different colors of the final system. According to this principle, a rapid detection system for pesticide residues based on gold nanoparticle colorimetric probes was constructed.

Firstly, thioacetylcholine iodide (ATChI) was selected as the reaction substrate, and 100 mL of 10 mM ATChI solution was prepared with deionized water. Add 18mg of silver nitrate powder to 5mL ATChI solution, shake fully for 10s, let it stand vertically for 1min, centrifuge to remove the precipitate, and take the supernatant (this step is used to remove I ions, because iodide ions can interfere with the determination of pesticides). Then add 1 mg of sodium chloride to the supernatant, shake fully for 10 s, stand vertically for 1 min, centrifuge to remove the precipitate, and take the supernatant (this step is used to remove excess Ag ions). At this time, the supernatant contains ATCh ⁺ , which can be used for the next colorimetric reaction.

Add 3mL sterile water, 0.1mL ATCh ⁺ solution, and 0.1mL enzyme-free buffer to 5mL centrifuge tubes, react fully for 10min, then add 0.5mL gold-solubilized collagen solution, and record the color change (sample 1).

Add 3mL of sterile water, 0.1mL of ATCh ⁺ solution and 0.1mL of acetylcholinesterase buffer (2U/mL) to a 5mL centrifuge tube and react fully for 10min, then add 0.5mL of gold-solubilized collagen solution and record the color change (sample 2).

Add 1mL of sterile water, 2mL of methion solution (0.01mg/mL) and 0.1mL of acetylcholinesterase buffer (2U/mL) to a 5mL centrifuge tube and react fully for 10min, then add 0.1mL of ATCh ⁺ solution. After 10 minutes, add 0.5mL gold-solubilized collagen solution, and record the color change (sample 3).

Add 1mL of sterile water, 2mL of ammonium phosphate working solution (0.01mg/mL) and 0.1mL of acetylcholinesterase buffer (2U/mL) to a 5mL centrifuge tube and react fully for 10min, then add 0.1mL of ATCh ⁺ solution. After 10 minutes, add 0.5mL gold-solubilized collagen solution, and record the color change (sample 4).

Add 2.5mL of sterile water and 5mL of phosphamide solutions of different concentrations (0, 0.1×10 ^-4 , 0.5×10 ^-4 , 1.0×10 ^-4 , 2.5×10 ^-4 , 5.0×10 ^-4 , 10×10 ^-4 , 25×10 ^-4 , 50×10 ^-4 and 100×10 ^-4 mg/mL) and 0.5mL acetylcholinesterase buffer (2U/mL) for 10 minutes after full reaction, then add 0.5mL ATCh ⁺ solution. After 10 minutes, add 2 mL of gold-solubilized collagen solution and record the color changes of the reaction solution at each concentration. The changes in the absorption spectra of different colorimetric systems were scanned by a UV-visible spectrophotometer.

In this experiment, acetylcholinesterase can specifically catalyze the hydrolysis of the substrate thioacetylcholine iodide (ATChI) into thioacetylcholine and acetic acid. When iodide ions are specifically removed from the system (some studies believe that iodide ions can combine with pesticides to interfere with the detection system), the abundant positive charges on the surface of the substrate can cause the aggregation of negatively charged gold nanoparticles on the surface, resulting in plasmon resonance phenomenon, which causes the color change of the whole system (theoretically from red to blue). However, when organophosphorus pesticides are present in the system, the activity of acetylcholinesterase is inhibited and cannot catalyze the hydrolysis reaction of the substrate. Subsequently, the substrate will combine with the gold nanoparticles to increase the aggregation degree of the gold nanoparticles. Different concentrations of pesticides lead to different degrees of inhibition of acetylcholinesterase and different colors of the final system. According to this principle, a rapid detection system for pesticide residues based on gold nanoparticle colorimetric probes was constructed.

In order to explore the principle of gold nanoparticles 90 organophosphorus pesticide residues, a colorimetric pre-experiment was set up according to the design, in which methion was used as a model of organophosphorus pesticides, and orange juice (1×) was used to prepare gold nanoparticles to participate in the reaction.

Design four experimental controls. Among them, the control 1 contained the substrate ATCh ⁺ and gold nanoparticles without acetylcholinesterase. This contrast is purple, and the original gold nanoparticles are wine red, indicating that some gold nanoparticles aggregate under this system, resulting in an increase in particle size and a change in color. In control 2, which contains substrate and enzyme solution, gold nanoparticles are added after reacting for a period of time, the color of the system basically remains unchanged, showing a light wine red. It shows that the particle size of gold nanoparticles does not become larger, because most of the substrates are decomposed by acetylcholinesterase, which fails to cause the adsorption and aggregation between charges. In control 3, the system contains methion and enzyme solution, after a period of reaction, the substrate is added, and after a period of reaction, gold nanoparticles are added. In control 4, the system is basically the same as that of control 3, except that methion is replaced by ammonium phosphate. From the colorimetric results of Controls 3 and 4, it can be seen that after adding pesticides, the color of the system changed from wine red to lavender, indicating that after adding pesticides, the activity of acetylcholinesterase was inhibited, and it could not normally catalyze the hydrolysis of the substrate ATCh ⁺ , so that the substrate remained A certain amount, resulting in the aggregation of gold nanoparticles.

In order to further accurately explore the colorimetric mechanism of gold nanoparticles to detect pesticides, the characteristic absorption peaks of gold nanoparticles in the four controls were characterized by a UV-visible spectrophotometer, and then the results of the colorimetric reaction construction were judged, as shown in Figure 14. The construction results of the colorimetric method showed that compared with the gold nanoparticle system with the same volume and the same concentration, the control 2 system was basically the same, indicating that when only the substrate and enzyme solution existed in the system, the product after enzymatic hydrolysis was not different. Affecting the particle size of gold nanoparticles did not cause obvious aggregation of gold nanoparticles. At the same time, the control 1, 3 and 4 can all cause the particle size of the gold nanoparticles to increase, causing the absorption peak to blue shift, and another obvious absorption peak appears at a wavelength of 630nm. Furthermore, the substrate can cause the aggregation of gold nanoparticles, and the particle size increases. The results showed that the higher the concentration of pesticide in the system, the more serious the aggregation of gold nanoparticles and the larger the particle size. According to the difference of color rendering degree and contrast in the pre-experiment, a detection system with reasonable peak detection sensitivity was designed for the detection of organophosphorus pesticide methion.

Detection of Pesticide Residues by Colorimetric Probe Method

According to the colorimetric principle preliminarily explored in the preliminary experiment, the redesigned colorimetric system was used to detect different concentrations of pesticides. The control (gold nanoparticles with the same concentration, no other reactive substances), and the concentration of methion-containing 100×10 ^-4 , 50×10 ^-4 , 25×10 ^-4 , 10×10 ^-4 , 5.0×10 ^-4 , 2.5×10 ^-4 , 1.0×10 ^-4 , 0.5×10 ^-4 , 0.1×10 ^-4 and 0mg /mL for color comparison, with the increase of pesticide concentration, the color gradually changed from light wine red to purple to dark blue, indicating that the activity of acetylcholinesterase in the system was gradually inhibited, and the content of the substrate gradually increased, resulting in the formation of gold nanoparticles. The particle size gradually increases, and different degrees of aggregation appear. In the colorimetric system where the concentration of methion pesticide was 100×10 ^-4 mg/mL, gold nanoparticles even precipitated. In the colorimetric system with methion pesticide concentrations of 0 and 0.1×10 ^-4 mg/mL, the overall color has almost no difference from the control. When the concentration of methion pesticide was 0.5×10 ^-4 mg/mL, the color of the system began to appear lavender, showing a clear colorimetric result.

The colorimetric detection results of different concentrations of pesticides were characterized by ultraviolet-visible light absorption spectroscopy, and the change process of the particle size of gold nanoparticles was further demonstrated. It can be seen from Figure 1 that the UV-Vis absorption spectrum basically presents similar results to the visual colorimetry. When the concentration of the pesticide gradually increased, the peak of the characteristic peak of the gold nanoparticles at the wavelength of 530nm began to decrease; at the same time, a new absorption peak appeared in the range of the wavelength of 650-780nm. The results show that different concentrations of pesticides can cause different degrees of aggregation of gold nanoparticles to form gold nanoparticles with different particle sizes, which in turn cause different wavelengths of the characteristic absorption peaks of gold nanoparticles. Among them, the absorption peaks of the systems with methion concentrations of 0 and 0.1×10 ^-4 mg/mL were significantly lower than those of the control, but the color change was not obvious in the colorimetric system. Combined with the analysis of Figures 3-8, it may be that the gold nanoparticles are slightly aggregated, resulting in a decrease in the peak value, but no new absorption peaks are generated, so there is no intuitive change in color.

Control in Figure 15 is the control; from 1 to 10 in order: the concentration of methion is 0, 0.1×10 ^-4 , 0.5×10 ^-4 , 1.0×10 ^-4 , 2.5×10 ^-4 , 5.0×10 ^{- 4} , 10×10 ^-4 , 25×10 ^-4 , 50×10 ^-4 and 100×10 ^-4 mg/mL.

Transmission electron microscopy was used to further characterize the gold in the colorimetric detection systems of different concentrations of pesticides (three systems were selected, containing methion concentrations of 0.1×10 ^-4 , 10×10 ^-4 and 100×10 ^-4 mg/mL respectively). The aggregation process of nanoparticles, the results are shown in Figures 16-18.

It can be seen from Figures 16-18 that three colorimetric systems containing different concentrations of methion were selected to participate in the morphology characterization, and the aggregation degree of gold nanoparticles showed obvious differences. When the system contained 0.1×10 ^-4 methion, the distance between gold nanoparticles was reduced compared to the system without pesticide (see Fig. 5), but no obvious aggregation was caused. When the system contained 10×10 ^-4 mg/mL of methion, the negatively charged gold nanoparticles on the surface were attracted together by the positively charged substrate ATCh ⁺ , and then aggregated. However, when the system contained 100×10 ^-4 mg/mL of methion, the aggregation of gold nanoparticles was intensified, and even a phenomenon of plate agglomeration appeared, forming large-size particles and precipitation. This result basically corresponds to the previous results of colorimetric reaction and UV-Vis absorption spectroscopy.

In summary, the colorimetric probe method based on gold nanoparticle aggregation to detect pesticide residues has good application value. At present, my country's maximum residue standard for the pesticide methion is 0.1mg/kg. According to the results in this paper, the detection limit of this experimental system is far lower than this standard, and it can be used to detect the residue of methion in food.

4. Selective detection research

Add 2.5mL sterile water, 5mL glucose, sucrose, citric acid, maltose, Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ and Zn ² solution (the system does not contain methion), then add 0.5mL acetylcholinesterase buffer (2U/mL) to the system and react for 10min, then add 0.5mLATh ⁺ solution; after 10min, add 2mL gold sol stock solution. Compared with the 0.01mg/mL methion system, record the color change caused by common substances in food, and then test the anti-interference ability of this detection system.

An important indicator of the colorimetric detection system is detection selectivity, that is, anti-interference. There are often other types of substances in real samples, and some substances will interfere with the detection system to a certain extent. The application object of this study is to detect pesticide residues in food, so this part is designed for common substances in food. The system containing 0.01mg/mL methion pesticide was selected as a reference, and 9 kinds of common substances in food were added for selectivity research, including sugars: glucose, sucrose, citric acid and maltose; metal ions: Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ and Zn ²⁺ ; the concentration of each interfering substance is 10 times the concentration of methion, that is, 0.1 mg/mL. The color of the system added with 0.01mg/mL methion was still dark blue with slight precipitation; meanwhile, after adding 0.1mg/mL glucose, sucrose, maltose, sodium chloride, potassium chloride, In the detection system of calcium and zinc acetate, the color did not change after the reaction, indicating that these substances failed to inhibit the activity of acetylcholinesterase to weaken the substrate concentration in the system. However, when citric acid was added to the reaction system, the color of the reaction changed from wine red to purple. The reason for this phenomenon may be that citric acid affects the activity of acetylcholinesterase, resulting in a high substrate concentration in the system, which leads to the aggregation of gold nanoparticles.

In summary, the organophosphorus pesticide detection system based on the gold nanoparticle colorimetric probe method has a high selectivity for common substances in general food.

Claims (5)

1. the green synthesis method of golden nanometer particle, is characterized in that the green synthesis method of golden nanometer particle carries out in the steps below:

Step one, citrus fruit cleaned and dries, squeeze the juice, then centrifugal, get supernatant, by volume dilute (1 ~ 10) doubly by deionized water, obtain juice solution;

Step 2, at ambient temperature, add juice solution rapidly with after magnetic stirring apparatus vigorous stirring chlorauric acid solution 2min, claret to appear continues to stir 20min and terminates reaction; Namely solution of gold nanoparticles is obtained.

2. the green synthesis method of golden nanometer particle according to claim 1, is characterized in that carrying out with 4000r/min rotating speed in step one.

3. the green synthesis method of golden nanometer particle according to claim 1, is characterized in that citrus fruit described in step one is orange, orange or lemon.

4. the green synthesis method of golden nanometer particle according to claim 2, is characterized in that the mass concentration of chlorauric acid solution described in step 2 is 1% (w/v).

5. the green synthesis method of golden nanometer particle according to claim 1, is characterized in that described in step 2, chlorauric acid solution consumption is identical with the volume of the supernatant measured.