Method for rapidly detecting various antibiotics in water body

Abstract

The invention provides a method for rapidly detecting various antibiotics in a water body. The preparation method comprises the following steps: firstly, preparing an enhanced substrate for surface enhanced Raman spectroscopy analysis, and enabling the substrate to have high SERS activity by a method for re-dispersing large aggregates through salt regulation and ultrasonic treatment; secondly, preparing a'glass-PDMS (Polydimethylsiloxane)-glass' micro-fluidic chip; the enhanced substrate and a sample to be detected are premixed and then pumped into the micro-fluidic chip for SERS (Surface Enhanced Raman Scattering) detection. According to the invention, ultralow-concentration detection of multiple antibiotics in multiple water bodies can be realized; the method has the advantages of no need of marking, short detection time and high sensitivity, and can be used for rapid and high-precision detection of various antibiotics in a water body.

Description

Technical Field

[0001] The invention relates to the technical field of antibiotic detection, and in particular to a method for rapidly detecting multiple antibiotics in water. Background Art

[0002] China's aquaculture output has long ranked first in the world, accounting for more than 60% of the world's total output. With technological advances and increased demand, aquaculture has become an important part of China's food supply and rural economy. However, due to the high incidence of aquatic product diseases in aquaculture, the problem of some farmers using antibiotics has been repeatedly banned. The abuse of antibiotics seriously endangers the life and health of consumers and poses a huge risk to the sustainable development of the aquatic industry. Therefore, random inspections and long-term monitoring of banned drugs in aquaculture water quality are of great significance to ensuring the quality and safety of my country's aquatic products.

[0003] At present, conventional detection methods for antibiotics in water bodies mainly include chromatography, immunoassay, etc. These methods generally rely on large instruments, and the sample preparation is complicated and the detection time is long, which cannot meet the requirements of on-site real-time monitoring of antibiotics.

[0004] Surface-Enhanced Raman Scanning (SERS) is a highly sensitive analytical technique based on Raman spectroscopy that can significantly enhance the Raman signal of the analyte with an enhancement factor of up to 10. 6 to 10 14 times, and has received widespread attention in the fields of rapid detection of environment, food safety, chemistry and biomedicine. The key to high-intensity SERS signals lies in the preparation of high-performance SERS substrates. Compared with solid substrates, metal sol substrates have the characteristics of label-free, simple preparation, and high surface activity, and are more suitable for the measurement of analytes in aqueous solution systems. The method of aggregating and activating nanoparticles with coagulants or activators is commonly used to enhance SERS signals, but due to the uncontrollability of the aggregation state of nanoparticles, excessive aggregation may also lead to larger particle spacing, reduce the number of hot spots, and reduce the Raman signal intensity. Therefore, improving the aggregation state of nanoparticles and enhancing the stability of metal sol substrates are crucial to obtaining high-intensity SERS signals in solutions.

[0005] Microfluidics is a technology that manipulates the flow of liquids at the micrometer scale. By designing a sophisticated microchannel network, it can accurately control and process trace amounts of liquids at the nanoliter or microliter level. Combining microfluidics with surface-enhanced Raman spectroscopy can provide more possibilities for sample processing and analysis. Precise liquid manipulation can avoid the problem of uneven liquid distribution, and nanoparticles in a flowing state are less likely to agglomerate than in a static system. In addition, since microfluidic chips can perform experiments at the microliter to nanoliter level, the consumption of samples and reagents is greatly reduced. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for rapidly detecting multiple antibiotics in water by integrating surface enhanced Raman and microfluidic chips, which solves the problem of poor stability of metal sol substrate in SERS technology, and can realize label-free and highly sensitive SERS analysis of multiple antibiotics in water, meet the detection needs of on-site rapid detection and multi-batch sampling, and ensure water safety and aquatic product quality.

[0007] The present invention achieves the above technical objectives through the following technical solutions.

[0008] A method for rapidly detecting multiple antibiotics in water, the method is carried out according to the following steps:

[0009] S1: Preparation of silver sol reinforced substrate;

[0010] S2: Dispersing large aggregates using salt manipulation and ultrasound;

[0011] S3: Preparation of microfluidic chip;

[0012] S4: Detection of multiple antibiotics in water.

[0013] Wherein, the preparation of the silver sol enhanced substrate in step S1 is carried out according to the following steps:

[0014] Heat 200±50mL of 1±0.5mmol / L silver nitrate solution, stir and heat until boiling, quickly add 6±2mL of 1±0.5% trisodium citrate solution, heat and continue stirring for 1±0.5 hours to obtain a yellow-green silver nanoparticle solution.

[0015] Wherein, the step S2 utilizes salt regulation and ultrasonic dispersion of large aggregates, and is performed according to the following steps:

[0016] The silver sol enhanced substrate prepared in step S1 is mixed with the salt solution, wherein the volume ratio of the enhanced substrate to the salt solution is 10:3-10. After being mixed, the enhanced substrate is placed in an ultrasonic cleaning machine for ultrasonic dispersion for 3 to 10 minutes.

[0017] In a specific embodiment, the salt solution is a sodium chloride solution with a concentration of 1-2 mol / L.

[0018] In a specific embodiment, the volume ratio of the reinforcing substrate to the sodium chloride solution is 1:1.

[0019] Wherein, the preparation of the microfluidic chip in step S3 is carried out according to the following steps:

[0020] Pour the prepared PDMS onto the patterned silicon wafer mold, and cover it with a glass sheet with a PET film; after curing at 70±5°C for 30±10 minutes, peel off the PDMS together with the PET from the silicon wafer to obtain a PDMS film; drill holes at corresponding positions on the glass slide; put the PDMS film and the punched glass slide into a plasma cleaning device for surface treatment and then bond them; peel off the PET film as a transfer layer from the PDMS film, cover it with a glass slide, and make a "glass-PDMS film-glass" sandwich chip.

[0021] Wherein, the detection of multiple antibiotics in water in step S4 is carried out according to the following steps:

[0022] The enhanced substrate prepared in step S2 is mixed with the sample to be tested, and then a strong electrolyte solution with a concentration of 0.1-1 mol / L is added, mixed thoroughly, and pumped into the microfluidic chip prepared in step S3 for SERS detection, wherein the volume ratio of the enhanced substrate, the sample to be tested and the strong electrolyte solution is 1:5-9:0.1-0.17.

[0023] In a specific embodiment, the strong electrolyte solution is potassium bromide or hydrochloric acid.

[0024] In a specific embodiment, the detection conditions of SERS detection in the detection of antibiotics in water in step S4 are: laser wavelength 625nm, scanning time 10s, laser power 400mW.

[0025] The present invention adopts the above technical solution, and the beneficial effects are as follows:

[0026] 1. The present invention achieves moderate agglomeration of silver nanoparticles by precisely controlling the amount of sodium chloride added to form a uniform hot spot distribution, and redisperses large aggregates by ultrasonic treatment, thereby solving the problem of excessive agglomeration of nanoparticles and making the enhanced substrate highly stable.

[0027] 2. The microfluidic chip designed by the present invention can accurately control trace amounts of liquid and detect ultra-low concentrations of target drugs in complex water systems, while significantly saving costs and being more suitable for continuous detection of trace amounts of banned antibiotics in water. 3. The present invention can detect and distinguish different banned antibiotics in water.

[0028] 4. The present invention has good sensitivity in detecting antibiotics in water bodies, and successfully obtained SERS characteristic fingerprints of three common antibiotics with good signal-to-noise ratio, including malachite green, ofloxacin, and sulfathiazole.

[0029] 5. The present invention integrates microfluidics technology with surface enhanced Raman spectroscopy detection technology, which has the characteristics of label-free, high sensitivity, short detection time, simple operation, etc., and can realize real-time and rapid detection of multiple antibiotics in water. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention provides a flow chart of a method for rapidly detecting multiple antibiotics in water.

[0031] Figure 2 (A) TEM image of freshly prepared silver nanoparticles and after large aggregates were redispersed by salt regulation and ultrasound. (B) DLS image. The size distribution and agglomeration state of the nanoparticles are shown in the figure as density distribution (by volume) and scattered light intensity changes. (C) UV-visible spectrum.

[0032] Figure 3 It is a schematic diagram of the overall structure of the microfluidic chip in the present invention.

[0033] Figure 4 The surface enhanced Raman spectra and characteristic peaks (at 1080 cm) of sulfathiazole at different concentrations in (A) Tianyuan Lake water (B) giant salamander breeding water (C) breeding pond water 3 are shown in the present invention. -1 The linear relationship between the intensity of sulfathiazole and the concentration of sulfathiazole.

[0034] Figure 5 The surface enhanced Raman spectra and characteristic peaks (at 1175 cm) of malachite green of different concentrations in (A) Tianyuan Lake water (B) giant salamander breeding water (C) breeding pond water 3 are shown in FIG. -1 Linear relationship between the intensity of malachite green and the concentration of malachite green.

[0035] Figure 6 The surface enhanced Raman spectra and characteristic peaks (at 1623 cm) of ofloxacin at different concentrations in (A) Tianyuan Lake water (B) giant salamander breeding water (C) breeding pond water 3 are shown in the present invention. -1 The linear relationship between the intensity of ofloxacin and the concentration of ofloxacin.

[0036] Figure 7 It is a comparison diagram of Raman signals at characteristic peaks of (A) sulfathiazole (B) malachite green (C) ofloxacin single detection and mixed sample detection in the present invention. DETAILED DESCRIPTION

[0037] Below in conjunction with the accompanying drawings in the embodiments of the present invention, the technical scheme in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are some embodiments of the present invention, rather than all embodiments, and this does not constitute a limitation of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If the manufacturer is not indicated in the reagents or instruments and equipment used, they are deemed to be conventional products that can be purchased on the market.

[0038] Example 1 Improving SERS activity by salt regulation and ultrasonic dispersion of large aggregates

[0039] (I) Preparation of silver nanoparticles

[0040] Heat 200 mL of 1 mmol / L silver nitrate solution, stir and heat until boiling, quickly add 6 mL of 1% trisodium citrate solution, heat and continue stirring for 1 hour to obtain a yellow-green silver nanoparticle solution, which is stored in a 4°C refrigerator.

[0041] (ii) Redispersion of large aggregates

[0042] 10 uL of the silver nanoparticle solution prepared above was fully mixed with 10 uL of sodium chloride solution (1 mol / L), and then placed in an ultrasonic cleaner for ultrasonic treatment for 5 min to redisperse the large aggregates to obtain a reinforced substrate.

[0043] The surface morphology of silver nanoparticles was characterized. Figure 2 As shown in (A), the average size of freshly prepared nanoparticles is 60 nm, and the redispersed nanoparticles have an uneven distribution of particle sizes ranging from 100 to 1000 nm, with most of them around 120 nm, indicating the formation of dimers that mainly contribute to the SERS activity. Figure 2 (B) shows the change in the aggregation state of silver nanoparticles before and after salt regulation and redispersion of large aggregates. The scattered light intensity of silver nanoparticles increased after salt regulation and ultrasonic redispersion of large aggregates, indicating that the particles were moderately aggregated and did not undergo sedimentation due to excessive aggregation (manifested as a decrease in scattered light intensity). Figure 2 (C) The results show that the silver nanoparticles after salt regulation and ultrasonic redispersion of large aggregates have a new absorption peak at 560 nm, indicating the assembly of nanoparticles.

[0044] In summary, the present invention successfully improves the agglomeration state of nanoparticles by using salt regulation and ultrasonic dispersion of large aggregates. This method is label-free, simple and rapid, and can enhance the stability of the metal sol substrate, improve the coupling probability between particles in the solution, and increase the number of hot spots to enhance the Raman signal.

[0045] Example 2 Preparation of glass-PDMS film-glass sandwich microfluidic chip

[0046] (I) Microfluidic chip structure design

[0047] Microfluidic chip structure Figure 3 As shown in the figure, the chip is divided into four parts: filter, Tesla valve, detection area and liquid trap. First, due to the complex and diverse composition of water, a filter is designed to prevent impurities from clogging the pipeline. Secondly, the Tesla valve is used to conduct on one side to prevent liquid reflux, which plays a role in isolating the injection area and the detection area. Then, the size of the designed detection area is much larger than the size of the Raman detection spot, which is convenient for the alignment of the laser head. Finally, the liquid trap is set up using the surface tension effect, which can cause the fluid to break during evaporative flow and prevent the liquid in the detection area from evaporating quickly.

[0048] (II) Preparation of microfluidic chip

[0049] Mix the PDMS base material and the curing agent in a mass ratio of 10:1 and stir evenly. Pour the prepared PDMS onto the patterned silicon wafer mold and cover it with a glass sheet with a PET film. After curing at 70°C for 30 minutes, peel off the PDMS together with the PET from the silicon wafer to obtain a PDMS film. Drill holes at the corresponding positions on the glass slide. Put the PDMS film and the punched glass slide into a plasma cleaning device for surface treatment and then bond them. Peel off the PET film as a transfer layer from the PDMS film and cover it with a glass slide to make a "glass-PDMS film-glass" sandwich chip.

[0050] Example 3

[0051] A method for rapid detection of the antibiotic sulfathiazole in water includes four steps:

[0052] Step 1: Prepare an enhanced substrate for surface enhanced Raman spectroscopy analysis:

[0053] Heat 200 mL of 1 mmol / L silver nitrate solution, stir and heat until boiling, quickly add 6 mL of 1% trisodium citrate solution, heat and continue stirring for 1 hour to obtain a yellow-green silver nanoparticle solution with a size of about 60 nm.

[0054] Step 2: Use salt regulation and ultrasonic dispersion of large aggregates:

[0055] 10uL of silver nanoparticle solution was fully mixed with 10uL of sodium chloride solution (1mol / L), and then placed in an ultrasonic cleaner for ultrasonic treatment for 5min to redisperse the large aggregates to obtain a reinforced substrate.

[0056] Step 3: Preparation of microfluidic chip:

[0057] Mix the PDMS base material and the curing agent in a mass ratio of 10:1 and stir evenly. Pour the prepared PDMS onto the patterned silicon wafer mold and cover it with a glass sheet with a PET film. After curing at 70°C for 30 minutes, peel off the PDMS together with the PET from the silicon wafer to obtain a PDMS film. Drill holes at the corresponding positions on the glass slide. Put the PDMS film and the punched glass slide into a plasma cleaning device for surface treatment and then bond them. Peel off the PET film as the transfer layer from the PDMS film and cover it with a glass slide to make a "glass-PDMS film-glass" sandwich chip (such as Figure 3 shown).

[0058] Step 4: Detection of the antibiotic sulfathiazole in water:

[0059] 20uL of the enhanced substrate was fully mixed with 90uL of the sample to be tested, and then pumped into the microfluidic chip for SERS detection. The detection conditions were: laser wavelength 625nm, scanning time 10s, and laser power 400mW.

[0060] Preparation of samples to be tested:

[0061] Weigh 0.1277 g of sulfathiazole solid into a test tube, add 50 ml of ultrapure water, and prepare a concentration of 10 -3 mol / L sulfathiazole aqueous solution was used as a stock solution. -3 mol / L sulfathiazole was spiked into water and prepared into 4 different concentration gradients (10 -4 , 10 -5 , 10 -6 and 10 -7 mol / L) of the actual sample to be tested.

[0062] The aquaculture water samples were taken from Tianyuan Lake (Nanjing Medical University), a giant salamander breeding farm (Nanjing, Jiangsu Province) and aquaculture pond 3 (Nanjing, Jiangsu Province).

[0063] Sulfathiazole samples of different concentrations in different aquaculture water samples were mixed with the silver sol-enhanced substrate and injected into the inlet of the microfluidic chip at a flow rate of 0.02 m / s, and SERS detection was performed in the detection area.

[0064] Surface enhanced Raman spectra and characteristic peaks (at 1080 cm-1) of sulfathiazole at different concentrations in Tianyuan Lake water -1 The linear relationship between the intensity and the concentration of sulfathiazole is as follows Figure 4 (A) The results show that the Raman spectrum intensity of sulfathiazole is related to the concentration of 10 -4 -10 -7mol / L has a good linear relationship, the linear regression equation is: Y = 0.4857*X + 5.351, and the correlation coefficient is 0.9468. The detection limit of sulfathiazole in Tianyuan Lake water is 10 -7 mol / L(25.532ppb).

[0065] Surface enhanced Raman spectra and characteristic peaks (at 1080 cm) of sulfathiazole at different concentrations in giant salamander culture water -1 The linear relationship between the intensity and the concentration of sulfathiazole is as follows Figure 4 (B) The results show that the Raman spectrum intensity of sulfathiazole is related to the concentration of 10 -4 -10 -7 mol / L has a good linear relationship, the linear regression equation is: Y = 0.3049 * X + 4.859, and the correlation coefficient is 0.9927. The detection limit of sulfathiazole in the water of giant salamander breeding is 10 -7 mol / L(25.532ppb).

[0066] Surface enhanced Raman spectra and characteristic peaks (at 1080 cm-1) of sulfathiazole at different concentrations in pond 3 -1 The linear relationship between the intensity and the concentration of sulfathiazole is as follows Figure 4 (C) The results show that the Raman spectrum intensity of sulfathiazole is related to the concentration of 10 -4 -10 -7 mol / L has a good linear relationship, the linear regression equation is: Y = 0.2668 * X + 4.733, the correlation coefficient is 0.9072. The detection limit of sulfathiazole in the aquaculture pond 3 is 10 -7 mol / L(25.532ppb).

[0067] In summary, the antibiotic sulfathiazole can be detected in different types of water bodies with a detection limit as low as 10 -7 mol / L (25.532ppb), with good application value.

[0068] Example 4

[0069] A method for rapid detection of the antibiotic malachite green in water includes four steps:

[0070] Step 1: Prepare an enhanced substrate for surface enhanced Raman spectroscopy analysis:

[0071] Heat 200 mL of 1 mmol / L silver nitrate solution, stir and heat until boiling, quickly add 6 mL of 1% trisodium citrate solution, heat and continue stirring for 1 hour to obtain a yellow-green silver nanoparticle solution with a size of about 60 nm.

[0072] Step 2: Use salt regulation and ultrasonic dispersion of large aggregates:

[0073] 10uL of silver nanoparticle solution was fully mixed with 10uL of sodium chloride solution (1mol / L), and then placed in an ultrasonic cleaner for ultrasonic treatment for 5min to redisperse the large aggregates to obtain a reinforced substrate.

[0074] Step 3: Preparation of microfluidic chip:

[0075] Mix the PDMS base material and the curing agent in a mass ratio of 10:1 and stir evenly. Pour the prepared PDMS onto the patterned silicon wafer mold and cover it with a glass sheet with a PET film. After curing at 70°C for 30 minutes, peel off the PDMS together with the PET from the silicon wafer to obtain a PDMS film. Drill holes at the corresponding positions on the glass slide. Put the PDMS film and the punched glass slide into a plasma cleaning device for surface treatment and then bond them. Peel off the PET film as the transfer layer from the PDMS film and cover it with a glass slide to make a "glass-PDMS film-glass" sandwich chip (such as Figure 3 shown).

[0076] Step 4: Detection of the antibiotic malachite green in water:

[0077] 12uL of enhanced substrate and 60uL of sample to be tested were mixed with 20uL of potassium bromide (1mol / L) and pumped into the microfluidic chip for SERS detection. The detection conditions were: laser wavelength 625nm, scanning time 10s, laser power 400mW.

[0078] Preparation of samples to be tested:

[0079] Weigh 0.04635g of malachite green solid into a test tube, add 50ml of ultrapure water, and prepare a concentration of 10 -3 mol / L sulfathiazole aqueous solution was used as a stock solution. -3 mol / L malachite green was spiked into water and prepared into 8 different concentration gradients (10 -4 , 10 -5 , 10 -6 , 10 -7 , 0.5*10 -7 , 10 -8 , 0.5*10 -8 and 10 -9 mol / L) of the actual sample to be tested.

[0080] The aquaculture water samples were taken from Tianyuan Lake (Nanjing Medical University), a giant salamander breeding farm (Nanjing, Jiangsu Province) and aquaculture pond 3 (Nanjing, Jiangsu Province).

[0081] Malachite green samples of different concentrations from different aquaculture water samples were mixed with the silver sol-enhanced substrate and injected into the inlet of the microfluidic chip at a flow rate of 0.02 m / s, and SERS detection was performed in the detection area.

[0082] Surface enhanced Raman spectra and characteristic peaks (at 1175cm) of malachite green at different concentrations in Tianyuan Lake water -1 The linear relationship between the intensity and the concentration of malachite green is as follows Figure 5 (A) The results show that the Raman spectrum intensity of malachite green is related to the concentration of 10 -7 -10 -9 mol / L has a good linear relationship, the linear regression equation is: Y = 1.115*X + 12.84, the correlation coefficient is 0.9136. The detection limit of malachite green in Tianyuan Lake water is 10 -9 mol / L(0.93ppb).

[0083] Surface enhanced Raman spectra and characteristic peaks (at 1175cm) of malachite green at different concentrations in giant salamander culture water -1 The linear relationship between the intensity and the concentration of malachite green is as follows Figure 5 (B) The results show that the Raman spectrum intensity of malachite green is related to the concentration of 10 -7 -10 -9 mol / L has a good linear relationship, the linear regression equation is: Y = 0.8926 * X + 10.26, the correlation coefficient is 0.9665. The detection limit of malachite green in the water of giant salamander breeding is 10 -9 mol / L(0.93ppb).

[0084] Surface enhanced Raman spectra and characteristic peaks (at 1175 cm) of malachite green at different concentrations in pond 3 -1 The linear relationship between the intensity and the concentration of malachite green is as follows Figure 5 (C) The results show that the Raman spectrum intensity of malachite green is related to the concentration of 10 -7 -10 -9 mol / L has a good linear relationship, the linear regression equation is: Y = 0.9509 * X + 11.40, the correlation coefficient is 0.9392. The detection limit of malachite green in the aquaculture pond 3 is 10 -9 mol / L(0.93ppb).

[0085] In summary, the antibiotic malachite green can be detected in different types of water bodies with a detection limit as low as 10 -9 mol / L (0.93ppb), with good application value.

[0086] Example 5

[0087] A method for rapid detection of the antibiotic ofloxacin in water includes four steps:

[0088] Step 1: Prepare an enhanced substrate for surface enhanced Raman spectroscopy analysis:

[0089] Heat 200 mL of 1 mmol / L silver nitrate solution, stir and heat until boiling, quickly add 6 mL of 1% trisodium citrate solution, heat and continue stirring for 1 hour to obtain a yellow-green silver nanoparticle solution with a size of about 60 nm.

[0090] Step 2: Use salt regulation and ultrasonic dispersion of large aggregates:

[0091] 10uL of silver nanoparticle solution was fully mixed with 3uL of sodium chloride solution (2mol / L), and then placed in an ultrasonic cleaner for ultrasonic treatment for 5 minutes to redisperse the large aggregates to obtain a reinforced substrate.

[0092] Step 3: Preparation of microfluidic chip:

[0093] Mix the PDMS base material and the curing agent in a mass ratio of 10:1 and stir evenly. Pour the prepared PDMS onto the patterned silicon wafer mold and cover it with a glass sheet with a PET film. After curing at 70°C for 30 minutes, peel off the PDMS together with the PET from the silicon wafer to obtain a PDMS film. Drill holes at the corresponding positions on the glass slide. Put the PDMS film and the punched glass slide into a plasma cleaning device for surface treatment and then bond them. Peel off the PET film as the transfer layer from the PDMS film and cover it with a glass slide to make a "glass-PDMS film-glass" sandwich chip (such as Figure 3 shown).

[0094] Step 4: Detection of the antibiotic ofloxacin in water:

[0095] 13uL of enhanced substrate and 90uL of sample to be tested were added, and then 1uL of hydrochloric acid (0.1mol / L) was added and mixed thoroughly, and then pumped into the microfluidic chip for SERS detection. The detection conditions were: laser wavelength 625nm, scanning time 10s, and laser power 400mW.

[0096] Preparation of samples to be tested:

[0097] Weigh 0.1807g ofloxacin solid into a test tube, add 50ml of ultrapure water, and prepare a concentration of 10 -3 mol / L sulfathiazole aqueous solution was used as a stock solution. -3 mol / L malachite green was spiked into water and prepared into 5 different concentration gradients (10 -4, 10 -5 , 10 -6 , 10 -7 and 10 -8 mol / L) of the actual sample to be tested.

[0098] The aquaculture water samples were taken from Tianyuan Lake (Nanjing Medical University), a giant salamander breeding farm (Nanjing, Jiangsu Province) and aquaculture pond 3 (Nanjing, Jiangsu Province).

[0099] Ofloxacin samples of different concentrations from different aquaculture water samples were mixed with the silver sol enhanced substrate and injected into the inlet of the microfluidic chip at a flow rate of 0.02 m / s, and SERS detection was performed in the detection area.

[0100] Surface enhanced Raman spectra and characteristic peaks of ofloxacin at different concentrations in Tianyuan Lake water (at 1623 cm -1 The linear relationship between the intensity and ofloxacin concentration is as follows Figure 6 (A) The results show that the Raman spectrum intensity of ofloxacin is related to the concentration of 10 -4 -10 -8 mol / L has a good linear relationship, the linear regression equation is: Y = 0.3616 * X + 5.218, and the correlation coefficient is 0.9385. The detection limit of ofloxacin in Tianyuan Lake water is 10 -8 mol / L(3.6137ppb).

[0101] Surface enhanced Raman spectra and characteristic peaks (at 1623cm) of ofloxacin at different concentrations in water of giant salamander aquaculture -1 The linear relationship between the intensity and ofloxacin concentration is as follows Figure 6 (B) The results show that the Raman spectrum intensity of ofloxacin is related to the concentration of 10 -4 -10 -8 mol / L has a good linear relationship, the linear regression equation is: Y = 0.3840 * X + 5.143, the correlation coefficient is 0.9861. The detection limit of ofloxacin in the water of giant salamander breeding is 10 -8 mol / L(3.6137ppb).

[0102] Surface enhanced Raman spectra and characteristic peaks (at 1623 cm) of ofloxacin at different concentrations in pond 3 -1 The linear relationship between the intensity and ofloxacin concentration is as follows Figure 6 (C) The results show that the Raman spectrum intensity of ofloxacin is related to the concentration of 10 -4 -10 -8mol / L has a good linear relationship, the linear regression equation is: Y = 0.2672*X + 4.626, and the correlation coefficient is 0.9822. The detection limit of ofloxacin in the aquaculture pond 3 is 10 -8 mol / L(3.6137ppb).

[0103] In summary, the antibiotic ofloxacin can be detected in different types of water bodies with a detection limit as low as 10 -8 mol / L (3.6137ppb), with good application value.

[0104] Example 6

[0105] A method for rapid detection of mixed antibiotics in water

[0106] In order to expand the application scope of this method, a mixed antibiotic model in aquaculture water samples was designed for quantitative analysis.

[0107] Sulfathiazole, malachite green and ofloxacin were dissolved in crab culture water to prepare malachite green (10 -9 mol / L) and ofloxacin (10 -8 mol / L) concentration remained unchanged, sulfathiazole was subjected to five different concentration gradients (10 -4 , 10 -5 , 10 -6 and 10 -7 mol / L) of mixed drugs were used as the test samples, and SERS detection was performed using the method of Example 3. The characteristic peaks (1080 cm -1 The signal intensity at the characteristic peak (1080 cm -1 The comparison of signal strength at Figure 7 The results show that in the presence of multiple drugs, although there will be a certain degree of interference on the Raman signal intensity of sulfathiazole, the minimum detection limit of 10 -7 mol / L(25.532ppb), and the concentration is 10 -4 -10 -7 mol / L has a good linear relationship and does not affect the overall test results.

[0108] Sulfathiazole, malachite green and ofloxacin were dissolved in crab culture water to prepare sulfathiazole (10 -7 mol / L) and ofloxacin (10 -8 mol / L) concentration remained unchanged, malachite green 8 different concentration gradients (10 -4 , 10 -5 , 10 -6 , 10 -7, 0.5*10 -7 , 10 -8 , 0.5*10 -8 and 10 -9 mol / L) of mixed drugs were used as the test samples, and SERS detection was performed using the method of Example 4. The characteristic peaks (1175 cm -1 The signal intensity at the characteristic peak (1175 cm -1 The comparison of signal strength at Figure 7 (B) The results show that in the presence of multiple drugs, although there will be a certain degree of interference on the Raman signal intensity of malachite green, the minimum detection limit of 10 -9 mol / L(0.93ppb), and the concentration is 10 -7 -10 -9 mol / L has a good linear relationship and does not affect the overall test results.

[0109] Sulfathiazole, malachite green and ofloxacin were dissolved in crab culture water to prepare sulfathiazole (10 -7 mol / L) and malachite green (10 -9 mol / L) concentration remained unchanged, ofloxacin was subjected to 5 different concentration gradients (10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 - 8 mol / L) of mixed drugs were used as the test samples, and SERS detection was performed using the method of Example 5. The characteristic peaks (1623 cm -1 The signal intensity at the characteristic peak (1623 cm -1 The comparison of signal strength at Figure 7 (C) The results show that in the presence of multiple drugs, although there will be a certain degree of interference on the Raman signal intensity of ofloxacin, the minimum detection limit of 10 -8 mol / L(3.6137ppb), and the concentration is 10 -4 -10 -8 mol / L has a good linear relationship and does not affect the overall test results.

[0110] In summary, see Figure 1The microfluidic system designed in the present invention and the surface enhanced Raman spectroscopy technology combined technology have high sensitivity and stability for the determination of single drugs or mixed drugs of multiple antibiotics in water bodies, and can realize rapid and accurate drug quantitative analysis without labeling detection, which is of great significance for ensuring the quality and safety of aquatic products and the agricultural ecological environment.

[0111] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A method for rapid detection of multiple antibiotics in water, characterized in that: The method proceeds as follows: S1: Preparation of silver sol reinforced substrate; S2: Dispersing large aggregates using salt manipulation and ultrasound; S3: Preparation of microfluidic chip; S4: Detection of multiple antibiotics in water.

2. The method for rapid detection of multiple antibiotics in water according to claim 1, characterized in that: The preparation of the silver sol enhanced substrate in step S1 is carried out according to the following steps: Heat 200±50mL of 1±0.5mmol / L silver nitrate solution, stir and heat until boiling, quickly add 6±2mL of 1% trisodium citrate solution, heat and continue stirring for 1±0.5 hours to obtain a yellow-green silver nanoparticle solution.

3. The method for rapid detection of multiple antibiotics in water according to claim 1, characterized in that: The step S2 utilizes salt regulation and ultrasonic dispersion of large aggregates, and is performed according to the following steps: The enhanced substrate prepared in step S1 is mixed with the salt solution, wherein the volume ratio of the enhanced substrate to the salt solution is 10:3-10. After being mixed, the enhanced substrate is placed in an ultrasonic cleaning machine for ultrasonic dispersion for 5 minutes.

4. The method for rapid detection of multiple antibiotics in water according to claim 5, characterized in that: The salt solution is a sodium chloride solution with a concentration of 1-2 mol / L.

5. The method for rapid detection of multiple antibiotics in water according to claim 4, characterized in that: The volume ratio of the reinforcing substrate to the sodium chloride solution is 1:

1.

6. The method for rapid detection of multiple antibiotics in water according to claim 1, characterized in that: The preparation of the microfluidic chip in step S3 is carried out according to the following steps: Pour the prepared PDMS onto the patterned silicon wafer mold and cover it with a glass sheet with a PET film. After curing at 70±5℃, peel off the PDMS and PET from the silicon wafer to obtain a PDMS film. Drill holes at the corresponding positions on the glass slide. Put the PDMS film and the punched glass slide into a plasma cleaning device for surface treatment and then bond them. Peel off the PET film as a transfer layer from the PDMS film and cover it with a glass slide to make a "glass-PDMS film-glass" sandwich chip.

7. The method for rapid detection of multiple antibiotics in water according to claim 1, characterized in that: The detection of antibiotics in water in step S4 is carried out according to the following steps: The enhanced substrate prepared in step S2 is mixed with the sample to be tested, and then a strong electrolyte solution with a concentration of 0.1-1 mol / L is added, mixed thoroughly, and pumped into the microfluidic chip for SERS detection, wherein the volume ratio of the enhanced substrate, the sample to be tested and the strong electrolyte solution is 1:5-9:0.1-0.

17.

8. The method for rapid detection of multiple antibiotics in water according to claim 7, characterized in that: The strong electrolyte solution is potassium bromide or hydrochloric acid.

9. The method for rapid detection of multiple antibiotics in water according to claim 1, characterized in that: The detection conditions of SERS detection in the detection of antibiotics in water in step S4 are: laser wavelength 625nm, scanning time 10s, laser power 400mW.

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