A method for detecting anthracene in water based on nucleic acid aptamers

Abstract

The present invention provides a method for detecting anthracene in water based on nucleic acid aptamers, comprising the following steps: 1) preparing a detection reagent containing nucleic acid aptamers; 2) drawing a standard curve of anthracene standard solutions of different concentrations and corresponding Raman intensities; 3) adding the detection reagent to a water sample, measuring the Raman intensity after the reaction, and obtaining the content data of anthracene in the water sample using the standard curve. Compared with the prior art, the present invention uses polycyclic aromatic hydrocarbon-specific nucleic acid aptamers to control the synthesis of nanosilver chain structures with Raman effects to detect anthracene in water. The detection method has high sensitivity and good selectivity, with a minimum detection limit of 7.5 ppb, simple operation, simple pretreatment, no need for large instruments, low cost, and can be used for the detection of anthracene in water.

Description

Technical Field

[0001] The invention belongs to the field of chemical detection and relates to a method for detecting anthracene in water, specifically a method for controlling in-situ synthesis of nanosilver with Raman effect by using anthracene nucleic acid aptamers and detecting anthracene in water by using a Raman spectrometer. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs), also known as polycyclic aromatic hydrocarbons and condensed aromatic hydrocarbons, refer to aromatic hydrocarbons containing two or more benzene rings in the molecule. Some PAHs also contain nitrogen, sulfur and cyclopentane. PAHs occur in the combustion process of petroleum, coal, tar, etc. They are byproducts of incomplete combustion of these chemical fuels and are a common type of persistent organic pollutants in environmental and food pollutants. To date, more than 200 PAHs have been discovered, including naphthalene, anthracene, phenanthrene, anthracene, benzo(a)anthracene, benzo(b)fluoranthene, etc., most of which have varying degrees of carcinogenicity. The condensed ring structure of PAHs determines its lipophilicity, which means that they are more easily mixed with oil than with water. The larger the molecular weight of PAHs, the lower its water solubility and volatility. Based on these characteristics, PAHs in the environment are mainly found in soil, sediments and oily substances, and their content in water or air is relatively low. Despite this, they are still important pollutants in haze. Because of its high stability, it can migrate long distances with the flow of atmosphere and water, thereby polluting large areas of environmental media such as soil, water, and air, and accumulating in media and organisms for a long time, causing widespread and lasting serious harm.

[0003] In 1976, the U.S. Environmental Protection Agency listed 16 PAHs as priority pollutants, accounting for 12.4% of the total priority pollutants, and stipulated that the maximum acceptable total concentration of these 16 PAHs in water is 0.2 g / L. In 1989, my country's State Environmental Protection Agency also announced 17 priority PAHs pollutants, accounting for 25% of the first batch of priority pollutants in China's water. my country's "Urban Water Quality Standards" also stipulates that the total amount of these 6 PAHs shall not exceed 2 g / L.

[0004] Although the content of PAHs in the environment is trace, due to its universality and widespread existence, and its high structural stability, it can migrate, transform and accumulate over a long period of time and over a large range, and can enter the human body through the skin, mucous membranes, respiratory tract, digestive tract and other pathways to enrich to a high concentration, posing a serious threat to human life and health. With the increasingly serious risk of PAH pollution, timely, efficient and accurate detection of PAH pollutants in various environments is very important. Necessary detection helps to repair and protect the ecological environment and reduce the threat of PAH pollution to the general public. At present, the detection methods for PAHs include spectrophotometry, mass spectrometry, chromatography, enzyme-linked immunosorbent assay, surface enhanced Raman spectroscopy, etc.

[0005] In the existing technology, high performance liquid chromatography and other analytical methods are mostly used in the detection of polycyclic aromatic hydrocarbons anthracene in water samples. Although these analytical methods have certain detection effects, they have high requirements on instrument performance and the professional skills of detection personnel. In addition, the detection takes a long time and cannot achieve intuitive, efficient and rapid detection of the samples to be tested. Summary of the invention

[0006] In view of the above technical problems in the prior art, the present invention provides a method for detecting anthracene in water based on nucleic acid aptamers. The method for detecting anthracene in water based on nucleic acid aptamers is intended to solve the technical problem that the prior art method for detecting anthracene in water takes a long time to detect.

[0007] The present invention provides a method for detecting anthracene in water based on nucleic acid aptamers, comprising the following steps:

[0008] 1) preparing a detection reagent; the detection reagent is composed of a nucleic acid aptamer solution, a silver nitrate solution, a sodium borohydride solution and water, the nucleic acid aptamer sequence is 5'-TTTTTTCCGTTGGTGTGGTGG GCCTAGGGCCGGCGGCGCACAGCTGTTTTTTTT-3', the concentration of the nucleic acid aptamer solution is 200 nmol / L, the concentration of the silver nitrate solution is 0.10 mmol / L, and the concentration of the sodium borohydride solution is 0.10 mmol / L; in the detection reagent, the volume ratio of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water is 1:(8-10): (8-10):(0.8-1.2);

[0009] 2) Draw a standard curve of different concentrations of anthracene standard solution and the corresponding Raman intensity;

[0010] 3) Add the detection reagent to the water sample with a volume ratio of 1:1 between the detection reagent and water. Adjust the pH of the reaction solution to 5. Measure the Raman intensity after 10 minutes of reaction and use the standard curve to obtain the anthracene content data in the water.

[0011] Furthermore, in step 2), the process of drawing the standard curve is as follows:

[0012] 2-1) Add anthracene standard solutions of different concentrations into multiple containers;

[0013] 2-2) Adding nucleic acid aptamer solutions to the multiple containers in step 2-1) respectively, and incubating at 30° C. for 15 minutes;

[0014] 2-3) Add silver nitrate solution to the multiple containers in step 2-2) respectively, and incubate at 4°C for 5 minutes;

[0015] 2-4) respectively adding sodium borohydride solution to the multiple containers in step 2-3), reacting at 20° C. for 10 min, and adjusting the pH of the solution to 5;

[0016] 2-5) Determine the Raman intensity of the solutions in the multiple containers in step 2-4) and draw a standard curve of anthracene standard solutions of different concentrations and corresponding Raman intensities.

[0017] Furthermore, in step 2-5), the method for measuring the Raman intensity is: taking the solution in the container and placing it on a silicon wafer, and measuring the Raman intensity of the solution on the silicon wafer using a Raman spectrometer.

[0018] Furthermore, the excitation wavelength of the Raman spectrometer is 535 nm.

[0019] Further, in step 3), the process of adding the detection reagent to the water sample is:

[0020] 3-1) Adding the nucleic acid aptamer solution to the pretreated water sample and incubating at 30°C for 15 minutes to obtain a mixed solution A; the pretreatment refers to precipitating the water sample and filtering to remove impurity particles;

[0021] 3-2) Adding silver nitrate solution to the mixed solution A in step 3-1) and incubating at 4°C for 5 minutes to obtain a mixed solution B;

[0022] 3-3) Add sodium borohydride solution to the mixed solution B in step 3-2) and react at 20°C for 10 minutes, and adjust the pH of the solution to 5.

[0023] Furthermore, in step 3), the anthracene content in the water sample is 0-0.5 ppm.

[0024] As a preferred technical solution, the volume ratio of the nucleic acid aptamer solution, silver nitrate solution, sodium borohydride solution and water sample is preferably 1:9:9:1.

[0025] The method of the present invention can be used to determine the anthracene content in a water sample with an anthracene content of 0-0.5 ppm.

[0026] Aptamers are nucleic acid fragments with high specificity and high specificity for target substances that are screened from a large number of oligonucleotide libraries through the exponential enrichment ligand system in vitro evolution (SELEX) technology. Compared with protein antibodies and enzymes, aptamers have higher affinity, stability and specificity, and are easy to label and design sensors, which can be used for the detection of nucleic acids, proteins, inorganic metal ions, virus particles and cells. The present invention selects a nucleic acid aptamer that can specifically bind to anthracene and optimizes the sequence. Based on the nucleic acid aptamer, a Raman spectrometer is used to detect anthracene in water.

[0027] The detection principle of the present invention is: according to the structural characteristics of anthracene, a nucleic acid aptamer that can wrap the polycyclic structure of anthracene is designed and synthesized. When anthracene exists in water, an anthracene-specific nucleic acid aptamer is added, and the nucleic acid aptamer is tightly combined with anthracene. At this time, the anthracene sample is treated with in-situ reduction of nanosilver. Due to the electronegativity of nucleic acid and condition control, the nanosilver particles are tightly adsorbed on the surface of the aptamer, and a thick layer of nanosilver shell is formed on the outside thereof, achieving the effect of SERS (surface enhanced Raman spectroscopy) specific enhancement. Through the relationship between the concentration of anthracene and the Raman intensity, that is, the value of the Raman intensity is linearly related to the concentration of anthracene within a certain range, by analyzing the change of the Raman intensity, the detection of anthracene in water can be achieved.

[0028] Compared with the prior art, the present invention has the following characteristics:

[0029] 1) The present invention provides a method for detecting anthracene in water using a nucleic acid aptamer sensor based on surface enhanced Raman spectroscopy. When anthracene does not exist in the detection system, the Raman intensity of the in-situ reduced nanosilver is small. When anthracene is added to the system, anthracene and the nucleic acid aptamer specifically bind to each other, and then the nanosilver is in-situ reduced. The nanosilver will form a chain structure, and its corresponding Raman intensity will be enhanced. The quantitative detection of anthracene in water can be achieved by measuring the Raman intensity.

[0030] 2) The present invention further optimizes the process conditions such as the optimal reaction concentration, optimal reaction pH and optimal reaction time of the nucleic acid aptamer. The detection method has high sensitivity and good selectivity, with a minimum detection limit of 7.5 ppb, simple operation, simple pretreatment, no need for large instruments, low cost, and can be used for the detection of anthracene in water;

[0031] 3) The present invention uses anthracene-specific nucleic acid aptamers to control the synthesis of nanosilver with Raman effect for anthracene detection. The nucleic acid aptamers can not only specifically bind to anthracene, but also control the synthesis of nanosilver chain structures with Raman effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the principle of detecting anthracene in water based on nucleic acid aptamers in the present invention;

[0033] Figure 2 is a graph showing the relationship between the concentration of nucleic acid aptamers and the Raman intensity in the detection method of the present invention;

[0034] Figure 3 is a relationship diagram between pH value and Raman intensity in the detection method of the present invention;

[0035] Figure 4 is a graph showing the relationship between reaction time and Raman intensity in the detection method of the present invention;

[0036] Figure 5 It is a relationship diagram between anthracene concentration and Raman intensity in the detection method of the present invention (i.e., standard curve). DETAILED DESCRIPTION

[0037] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments. Example 1

[0038] like Figure 1 As shown, the method for rapid detection of anthracene in water using nucleic acid aptamer sensor based on surface enhanced Raman spectroscopy is as follows:

[0039] Preparation of detection reagents: Use distilled water to prepare a ssDNA nucleic acid aptamer solution with a concentration of 200 nmol / L. The nucleic acid aptamer sequence is 5'-TTT TTT CCG TTG GTG TGG TGG GCC TAG GGC CGG CGG CGC ACA GCT GTTTTT TTT-3'.

[0040] In 2012, M.Jo reported an aptamer with strong affinity for bicyclic compounds.

[0041] 5'-CCGCCGTTGGTGTGGTGGGCCTAGGGCCGGCGGCGCACAGCTGTTATAGAGTCTCCAGC-3', but it cannot bind to polycyclic compounds. We amplified a part of the original sequence by calculating the kd value, increased its length, cut off the non-binding sequence, and added thymine at both ends of the sequence. The ammonia nitrogen of thymine was used to enhance the affinity for the benzene ring through the hydrogen bond between the amino groups. After repeated experiments, it was found that this sequence has a strong specificity for anthracene; distilled water was used to prepare a silver nitrate solution with a concentration of 0.10mmol / L; distilled water was used to prepare a sodium borohydride solution with a concentration of 0.10mmol / L. The detection reagent is composed of a nucleic acid aptamer solution, a silver nitrate solution, a sodium borohydride solution and water. In the detection reagent, the volume ratio of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water is 1:(8-10): (8-10):(0.8-1.2).

[0042] Draw a standard curve of anthracene standard solutions of different concentrations and the corresponding Raman intensity: take 11 centrifuge tubes containing anthracene standard solutions of different concentrations, add detection reagents respectively, so that the anthracene content in the entire detection system is maintained at 0-0.5ppm, react at 25°C for 10 minutes, and synthesize nanosilver chain structures in situ; take the solutions in each centrifuge tube and place them on a silicon wafer, set the excitation wavelength to 535nm, and then use a Raman spectrometer to measure the corresponding Raman intensity, and then plot the different concentrations of anthracene and the corresponding Raman intensity to draw a standard curve, such as Figure 5 shown.

[0043] 4) Detection of anthracene in water samples: Add detection reagent to the water sample and react at 20°C for 10 minutes. Adjust the pH of the solution to 5, in situ reduce the nanosilver chain structure, and measure its Raman intensity. According to the Raman intensity obtained from the water sample, check the standard curve to obtain the anthracene content in the sample.

[0044] After multiple verifications, the method was used to determine water samples containing anthracene at concentrations of 0.1ppm, 0.25ppm, and 0.5ppm, and the recovery rates were 90-120%, proving the reliability of the method. The minimum detection limit of anthracene in water determined by this method is 7.5ppb. Example 2

[0045] The detection process in Example 1 was further optimized, and the Raman intensity of 0.5 ppm anthracene solution was measured under the conditions of aptamer concentration 0-200 nmol / L, pH value 4-11, and reaction time 0-30 min. The results are shown in Figure 2 , Figure 3 , Figure 4As shown, it can be seen that the optimal concentration of nucleic acid aptamers is above 70nmol / L, the optimal reaction pH is 5, and the optimal reaction time is 10min. Example 3

[0046] A method for detecting anthracene in water based on nucleic acid aptamers, wherein the anthracene content in the water sample is 0-0.5 ppm. The method comprises the following steps:

[0047] 1) Prepare a detection reagent including nucleic acid aptamers: including a nucleic acid aptamer solution with a concentration of 70nmol / L, a silver nitrate solution with a concentration of 0.10mmol / L, and a sodium borohydride solution with a concentration of 0.10mmol / L; in the nucleic acid aptamer solution, the nucleic acid aptamer sequence is 5'-TTT TTT CCG TTG GTG TGG TGG GCC TAG GGC CGG CGG CGC ACAGCT GTT TTT TTT-3'. The detection reagent is composed of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water. In the detection reagent, the volume ratio of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water is 1:(8-10): (8-10):(0.8-1.2).

[0048] 2) Draw a standard curve of different concentrations of anthracene standard solution and the corresponding Raman intensity:

[0049] 2-1) Add anthracene standard solutions of different concentrations into multiple containers;

[0050] 2-2) Adding nucleic acid aptamer solutions to the multiple containers in step 2-1) respectively, and incubating at 30° C. for 15 minutes;

[0051] 2-3) Add silver nitrate solution to the multiple containers in step 2-2) respectively, and incubate at 4°C for 5 minutes;

[0052] 2-4) respectively adding sodium borohydride solution to the multiple containers in step 2-3), reacting at 20° C. for 10 min, and adjusting the pH of the solution to 5;

[0053] 2-5) Determine the Raman intensity of the solutions in the multiple containers in step 2-4): Place the solutions in the containers on a silicon wafer, and measure the Raman intensity of the solutions on the silicon wafer using a Raman spectrometer with an excitation wavelength of 535 nm. Then draw a standard curve of different concentrations of anthracene standard solutions and corresponding Raman intensities.

[0054] 3) Add the detection reagent to the pretreated water sample:

[0055] 3-1) Adding the nucleic acid aptamer solution to the pretreated water sample and incubating at 30°C for 15 minutes to obtain a mixed solution A;

[0056] 3-2) Adding silver nitrate solution to the mixed solution A in step 3-1) and incubating at 4°C for 5 minutes to obtain a mixed solution B;

[0057] 3-3) Sodium borohydride solution was added to the mixed solution B in step 3-2) and reacted at 20°C for 10 minutes, and the pH of the solution was adjusted to 5. After the reaction, the Raman intensity was measured, and the content of anthracene in the water sample was obtained as 0.25 ppm using the standard curve. Example 4

[0058] A method for detecting anthracene in water based on nucleic acid aptamers, wherein the anthracene content in the water sample is 0-0.5 ppm. The method comprises the following steps:

[0059] 1) Prepare a detection reagent including nucleic acid aptamers: including a nucleic acid aptamer solution with a concentration of 150nmol / L, a silver nitrate solution with a concentration of 0.08mmol / L, and a sodium borohydride solution with a concentration of 0.12mmol / L; in the nucleic acid aptamer solution, the nucleic acid aptamer sequence is 5'-TTT TTT CCG TTG GTG TGG TGG GCC TAG GGC CGG CGG CGC ACAGCT GTT TTT TTT-3'. The detection reagent is composed of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water. In the detection reagent, the volume ratio of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water is 1:(8-10): (8-10):(0.8-1.2).

[0060] 2) Draw a standard curve of different concentrations of anthracene standard solution and the corresponding Raman intensity:

[0061] 2-1) Add anthracene standard solutions of different concentrations into multiple containers;

[0062] 2-2) Adding nucleic acid aptamer solutions to the multiple containers in step 2-1) respectively, and incubating at 30° C. for 15 minutes;

[0063] 2-3) Add silver nitrate solution to the multiple containers in step 2-2) respectively, and incubate at 10°C for 5 minutes;

[0064] 2-4) respectively adding sodium borohydride solution to the multiple containers in step 2-3) and reacting at 20° C. for 10 min;

[0065] 2-5) Determine the Raman intensity of the solutions in the multiple containers in step 2-4): Place the solutions in the containers on a silicon wafer, and measure the Raman intensity of the solutions on the silicon wafer using a Raman spectrometer with an excitation wavelength of 535 nm. Then draw a standard curve of different concentrations of anthracene standard solutions and corresponding Raman intensities.

[0066] 3) Add the detection reagent to the pretreated water sample:

[0067] 3-1) Adding the nucleic acid aptamer solution to the pretreated water sample and incubating at 30°C for 15 minutes to obtain a mixed solution A;

[0068] 3-2) Adding silver nitrate solution to the mixed solution A in step 3-1) and incubating at 4°C for 5 minutes to obtain a mixed solution B;

[0069] 3-3) Add sodium borohydride solution to the mixed solution B in step 3-2) and react at 20°C for 10 minutes, and adjust the pH of the solution to 5.

[0070] After the reaction, the Raman intensity was measured, and the content of anthracene in the water sample was obtained as 0.25 ppm using the standard curve. Example 5

[0071] A method for detecting anthracene in water based on nucleic acid aptamers, wherein the anthracene content in the water sample is 0-0.5 ppm. The method comprises the following steps:

[0072] 1) Prepare a detection reagent including nucleic acid aptamers: including a nucleic acid aptamer solution with a concentration of 100nmol / L, a silver nitrate solution with a concentration of 0.1mmol / L, and a sodium borohydride solution with a concentration of 0.1mmol / L; in the nucleic acid aptamer solution, the nucleic acid aptamer sequence is 5'-TTT TTT CCG TTG GTG TGG TGG GCC TAG GGC CGG CGG CGC ACA GCTGTT TTT TTT-3'. The detection reagent is composed of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water. In the detection reagent, the volume ratio of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water is 1:(8-10): (8-10):(0.8-1.2).

[0073] 2) Draw a standard curve of different concentrations of anthracene standard solution and the corresponding Raman intensity:

[0074] 2-1) Add anthracene standard solutions of different concentrations into multiple containers;

[0075] 2-2) Adding nucleic acid aptamer solutions to the multiple containers in step 2-1) respectively, and incubating at 30° C. for 15 minutes;

[0076] 2-3) Add silver nitrate solution to the multiple containers in step 2-2) respectively, and incubate at 4°C for 5 minutes;

[0077] 2-4) respectively adding sodium borohydride solution to the multiple containers in step 2-3), reacting at 20° C. for 10 min, and adjusting the pH of the solution to 5;

[0078] 2-5) Determine the Raman intensity of the solutions in the multiple containers in step 2-4): Place the solutions in the containers on a silicon wafer, and measure the Raman intensity of the solutions on the silicon wafer using a Raman spectrometer with an excitation wavelength of 532 nm. Then draw a standard curve of different concentrations of anthracene standard solutions and corresponding Raman intensities.

[0079] 3) Add the detection reagent to the pretreated water sample:

[0080] 3-1) Adding the nucleic acid aptamer solution to the pretreated water sample and incubating at 30°C for 15 minutes to obtain a mixed solution A;

[0081] 3-2) Adding silver nitrate solution to the mixed solution A in step 3-1) and incubating at 4°C for 5 minutes to obtain a mixed solution B;

[0082] 3-3) Add sodium borohydride solution to the mixed solution B in step 3-2) and react at 20°C for 10 minutes, and adjust the pH of the solution to 5.

[0083] After the reaction, the Raman intensity was measured, and the content of anthracene in the water sample was obtained as 0.25 ppm using the standard curve.

[0084] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for detecting anthracene in water based on nucleic acid aptamers, characterized in that The following steps are involved: 1) preparing a detection reagent; the detection reagent is composed of a nucleic acid aptamer solution, a silver nitrate solution, a sodium borohydride solution and water, the nucleic acid aptamer sequence is 5'-TTTTTTCCGTTGGTGTGGTGG GCCTAGGGCCGGCGGCGCACAGCTGTTTTTTTT-3', the concentration of the nucleic acid aptamer solution is 200 nmol / L, the concentration of the silver nitrate solution is 0.10 mmol / L, and the concentration of the sodium borohydride solution is 0.10 mmol / L; in the detection reagent, the volume ratio of the nucleic acid aptamer solution, the silver nitrate solution, the sodium borohydride solution and water is 1:(8-10): (8-10):(0.8-1.2); 2) Draw a standard curve of different concentrations of anthracene standard solution and the corresponding Raman intensity; 3) Add the detection reagent to the water sample, with the volume ratio of the detection reagent to the water sample being 1:1, adjust the pH of the reaction solution to 5, measure the Raman intensity after reacting for 10 minutes, and use the standard curve to obtain the anthracene content data in the water; In step 2), the process of drawing the standard curve is as follows: 2-1) Add anthracene standard solutions of different concentrations into multiple containers; 2-2) Adding nucleic acid aptamer solutions to the multiple containers in step 2-1) respectively, and incubating at 30° C. for 15 minutes; 2-3) Add silver nitrate solution to the multiple containers in step 2-2) respectively, and incubate at 4°C for 5 minutes; 2-4) respectively adding sodium borohydride solution to the multiple containers in step 2-3), reacting at 20° C. for 10 min, and adjusting the pH of the solution to 5; 2-5) measuring the Raman intensity of the solutions in the multiple containers in step 2-4), and drawing a standard curve of anthracene standard solutions with different concentrations and corresponding Raman intensities; In step 2-5), the method for measuring the Raman intensity is: taking the solution in the container and placing it on a silicon wafer, and measuring the Raman intensity of the solution on the silicon wafer using a Raman spectrometer; the excitation wavelength of the Raman spectrometer is 535nm; In step 3), the process of adding the detection reagent to the water sample is: 3-1) adding the nucleic acid aptamer solution to the pretreated water sample and incubating at 30° C. for 15 minutes to obtain a mixed solution A; the pretreatment refers to precipitating the water sample and filtering to remove impurity particles; 3-2) Adding silver nitrate solution to the mixed solution A in step 3-1) and incubating at 4°C for 5 minutes to obtain a mixed solution B; 3-3) Add sodium borohydride solution to the mixed solution B in step 3-2) and react at 20°C for 10 minutes, and adjust the pH of the solution to 5.

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