A spectrum detection method for antiviral and antibacterial drug residues in water

Abstract

The present invention discloses a spectral detection method for antiviral and antibacterial drug residues in water. The detection method comprises the following steps: (1) adding a silver nanoparticle sol and a standard solution of an antiviral and antibacterial drug with a concentration of 1×10-4 to 1×10-10 mol / L into a centrifuge tube for mixing, and placing the centrifuge tube on a vortex mixer for vortex oscillation to obtain a standard test solution; (2) performing Raman spectroscopy on the standard test solution, recording the position and intensity of the characteristic peaks, and obtaining a linear equation for the antiviral and antibacterial drug to be detected; (3) adding the silver nanoparticle sol and the water sample to be detected into a centrifuge tube for mixing, placing the centrifuge tube on a vortex mixer for vortex oscillation to obtain a water sample test solution, and then performing Raman spectroscopy on the water sample test solution. The detection method proposed by the present invention is simple to operate, has high sensitivity, less pretreatment time, and strong signal stability.

Description

Technical Field

[0001] The invention relates to the technical field of Raman detection, and in particular to a spectrum detection method for antiviral and antibacterial drug residues in water. Background Art

[0002] At present, a considerable portion of antiviral and antibacterial drugs widely used in the medical, animal husbandry and other industries enter the environmental water bodies directly or through metabolites during use. Due to the anti-degradation characteristics and bioaccumulation of such drugs, they can be enriched through the food chain and eventually enter the human body, thus causing harm to human health. In addition, drug residues in the environment can also produce resistance genes, thereby destroying the ecological environment system.

[0003] Common methods for drug residue analysis in the environment include chromatography / mass spectrometry, microbial inhibition screening and immunoassay. In actual detection, these methods are time-consuming, require high sample pretreatment, have cumbersome processing, and have high requirements for operators. Raman spectroscopy, as a method that can be used for rapid on-site detection, has also been applied to the detection of such drug residues. However, in the actual detection process, due to the extremely low drug residue in the water body and the serious interference, the detection method based on Raman spectroscopy usually requires the use of enhanced reagents or enhanced substrates. At present, this method is limited by the preparation technology of the detection substrate or reagent, and its stability and sensitivity for drug residue detection always fail to meet the requirements of actual application. In summary, among the current methods for drug residue detection in water environments, chromatography-mass spectrometry can achieve very high sensitivity, but it cannot be detected on site due to operating conditions. The existing immunoassay and Raman spectroscopy detection methods can achieve rapid on-site detection, but the detection sensitivity and stability are not high.

[0004] At present, the Raman spectroscopy detection method of drug residues in water is generally affected by matrix interference and has the problems of low sensitivity and poor anti-interference. At the same time, the drug residues in the water environment are generally low, which further limits the application of this method. In order to achieve high-sensitivity detection, it is usually necessary to adopt the method of enhancing the Raman signal of the target object. At present, most of the methods for direct Raman spectroscopy detection of drug residues in water require a long adsorption time between drug molecules and metal nanocolloids (such as the use of Ag-TiO 2For base detection of aldosterone antibiotics, the Raman detection pretreatment time is 3 to 5 hours). The main method to shorten the pretreatment time is the salt agglomeration method. This method adds inorganic salts to the target sample to promote the agglomeration of silver (or gold) nanoparticles with Raman enhancement effects to generate hot spots, thereby increasing the Raman signal of the target. However, the Raman signal of the target in this method is unstable (for example, the document Anal. Chem. 2021, 93, 9373 uses the salt agglomeration method to detect fentanyl in urine. The signal of the target is usually greatly reduced after 2 to 3 minutes after enhancement). Summary of the invention

[0005] The present invention solves the above-mentioned problems existing in the prior art. The purpose of the present invention is to provide a spectrum detection method for antiviral and antibacterial drug residues in water. The present invention promotes the adsorption of detection targets on the surface of silver nanoparticles in a low-frequency vortex vibration manner and realizes the stable aggregation of silver nanoparticles so that the target generates a significant Raman enhancement signal, thereby realizing high-sensitivity detection of drug residues. The method has the advantages of simple operation, low cost, fast detection speed and high sensitivity, and can realize rapid detection of drug residues in water environment.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a spectrum detection method for antiviral and antibacterial drug residues in water, comprising the following steps:

[0007] (1) Silver nanoparticle sol was mixed with a concentration of 1×10 -4 ~1×10 -10 mol / L standard solution of antiviral and antibacterial drugs is added to a centrifuge tube and mixed, the volume ratio of silver nanoparticle sol to the standard solution of antiviral and antibacterial drugs is 1:3-3:1, and the centrifuge tube is placed on a vortex mixer for vortex oscillation for 1-20 minutes to obtain a standard test solution;

[0008] (2) performing Raman spectroscopy detection on the standard test solution obtained in step (1), recording the position and intensity of the characteristic peak, plotting a standard curve with the logarithm of the concentration of the standard solution of the antiviral or antibacterial drug as the abscissa and the intensity of the characteristic peak as the ordinate, and obtaining a linear equation of the antiviral or antibacterial drug to be tested;

[0009] (3) adding the silver nanoparticle sol and the water sample to be tested into a centrifuge tube at a volume ratio of 1:3 to 3:1 and mixing, and placing the centrifuge tube on a vortex mixer for vortex oscillation for 1 to 20 minutes to obtain a water sample solution to be tested, and then performing Raman spectroscopy detection on the water sample solution to be tested, and qualitatively identifying the types of antiviral and antibacterial drugs in the water sample to be tested according to step (2), substituting the obtained characteristic peak intensity into the linear equation obtained in step (2), and quantitatively determining the antiviral and antibacterial drugs in the water sample to be tested.

[0010] Preferably, the silver nanoparticle sol has a particle size of 50 to 80 nm.

[0011] Preferably, the silver nanoparticle sol is prepared by the following steps: dissolving silver nitrate in deionized water to form a silver nitrate aqueous solution with a concentration of 0.18 g / L, heating the silver nitrate aqueous solution to boiling, adding a sodium citrate solution with a mass fraction of 1%, keeping boiling for 30-60 minutes, and then cooling at room temperature to obtain the silver nanoparticle sol, wherein the volume ratio of the silver nitrate aqueous solution to the sodium citrate solution is 50:1.

[0012] Preferably, the total volume of the standard test solution described in step (1) is 3 to 4 mL, the pH is 4 to 9, and the temperature is 25°C to 90°C; the total volume of the water sample test solution described in step (3) is 3 to 4 mL, the pH is 4 to 9, and the temperature is 25°C to 90°C.

[0013] Further preferably, the volume ratio of the silver nanoparticle sol described in step (1) to the standard solution of antiviral and antibacterial drugs is 1:2.5-3:1, and the total volume of the standard test solution is 3.5-4.0 mL; the volume ratio of the silver nanoparticle sol described in step (3) to the water sample to be tested is 1:2.5-3:1, and the total volume of the water sample test solution is 3.5-4.0 mL.

[0014] Preferably, the rotation speed of the vortex mixer described in step (1) and step (3) is 100-2800 rpm, and the vortex oscillation is 10-20 min.

[0015] Preferably, the detection conditions of Raman spectroscopy detection in step (2) and step (3) are: laser wavelength 785nm, laser intensity 50-500mw, and sample collection time 4-20s.

[0016] Preferably, the antiviral and antibacterial drugs include cefazolin sodium and pefloxacin.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The detection method proposed in the present invention is simple to operate, has high sensitivity, requires little preprocessing time, and has strong signal stability.

[0019] 2. Compared with the existing methods for detecting drug residues in water environments, the method proposed in the present invention has the advantages of simple operation process, short pretreatment time, high sensitivity, on-site and portable detection: compared with the microbial inhibition screening method and the immunoassay method, the sensitivity and accuracy of the present invention are higher, and the pretreatment time is greatly shortened; compared with chromatography / mass spectrometry, the present invention eliminates the cumbersome operation process, has low detection cost, and can realize rapid on-site detection.

[0020] 3. In the method proposed by the present invention, controlling the vortex time to 1-20 min can promote the effective aggregation of silver nanoparticles, produce a resonance enhancement effect, form more and stronger local hot spots, and thus improve the detection sensitivity. Compared with the existing Raman detection method for drug residues in water environments, this method can significantly improve the adsorption stability between drug molecules and Raman enhancement reagents, so that the Raman enhancement signal can remain stable for more than 6 hours, and the Raman signal of the target treated by this method can remain stable for a long time.

[0021] 4. The method proposed in the present invention has high detection sensitivity for the detection of different types of drug residues in water and is a simple, fast, stable and universal detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a comparison diagram of the Raman spectra of cefazolin sodium before and after vortexing in Example 1;

[0023] Figure 2 is a graph showing the Raman enhanced detection working curve for cefazolin sodium in Example 1;

[0024] Figure 3 is the Raman peak spectrum of cefazolin sodium at different vortex times in Examples 1-5;

[0025] Figure 4 is a graph showing the relationship between the Raman peak intensity and the vortex time for cefazolin sodium in Example 5;

[0026] Figure 5 is a graph showing the working curve of Raman enhanced detection of pefloxacin in Example 6;

[0027] Figure 6 is a comparison diagram of the Raman spectra of pefloxacin at different vortex times in Example 7;

[0028] Figure 7 This is a graph showing the change in the highest peak intensity of pefloxacin in Example 7, which was vortexed for 6 minutes, left for 6 hours, and then vortexed again for 1 minute. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, but not to limit the present invention. Unless otherwise specified, the equipment used in the present invention is a conventional commercial product in the art. The water samples in the following examples were taken from a lake in the urban area of ​​Guangzhou.

[0030] The silver nanoparticle sol is prepared by the following steps: dissolving silver nitrate in deionized water to form a silver nitrate aqueous solution with a concentration of 0.18 g / L, heating the silver nitrate aqueous solution to boiling, adding a sodium citrate solution with a mass fraction of 1%, keeping boiling for 30-60 minutes, and then cooling at room temperature to obtain the silver nanoparticle sol, wherein the volume ratio of the silver nitrate aqueous solution to the sodium citrate solution is 50:1.

[0031] Example 1

[0032] The detection of cefazolin sodium in water specifically includes the following steps:

[0033] (1) Preparation of silver nanoparticle sol: 90 mg of silver nitrate was dissolved in 500 mL of deionized water and heated to boiling. 10 mL of 1% sodium citrate solution was added and kept boiling for 60 min. Then, the solution was cooled at room temperature to obtain a yellow-green silver nanoparticle sol with a diameter of 50 to 60 nm.

[0034] (2) Prepare 50 mL of 1×10 -4 mol / L、1×10 -5 mol / L、1×10 -6 mol / L、1×10 -7 mol / L and 1×10 -8 mol / L cefazolin sodium standard solution; 1mL of silver nanoparticle sol and 2.5mL of cefazolin sodium standard solution of different concentrations were mixed and added into a 10mL centrifuge tube, the pH value of the solution was adjusted to 6.3, the centrifuge tube was placed on a vortex mixer, the speed was set to 2800rpm, and the vortex oscillation was maintained for 20min to obtain a standard test solution.

[0035] At room temperature (25°C), the Raman spectrum of the standard solution of cefazolin sodium was collected using a fiber Raman detection system equipped with a 785nm laser. The Raman laser power was 100mW, and the Raman signal was collected twice, with each collection time of 15s. The characteristic peak 777cm -1 The position and intensity of the standard solution of cefazolin sodium are plotted with the logarithm of the concentration as the horizontal axis and the intensity of the characteristic peak as the vertical axis. Figure 2 As shown, the linear equation of cefazolin sodium was obtained.

[0036] In step (2), 1 mL of silver nanoparticle sol and 2.5 mL of 1×10 -4mol / L cefazolin sodium standard solution was mixed and added into a 10mL centrifuge tube, and the pH value of the solution was adjusted to 6.3. The centrifuge tube was placed on a vortex mixer, and the speed was set to 2800rpm. The vortex oscillation was maintained for 20min. The standard test solution without vortexing and the standard test solution after vortex oscillation for 20min were subjected to Raman detection respectively, and the detection conditions were the same as above.

[0037] Comparison of Raman spectra of cefazolin sodium before and after vortexing Figure 1 As shown in the figure, compared with the detection without vortexing process, the characteristic peak 777cm -1 The Raman signal is enhanced by 6.4 times in position calculation. The method proposed in the present invention can maintain the stability of the Raman signal for more than 6 hours after processing.

[0038] (3) 1 mL of silver nanoparticle sol and 2.5 mL of the water sample to be tested were added to a centrifuge tube and mixed, and the centrifuge tube was placed on a vortex mixer for vortex oscillation for 20 min to obtain a water sample solution to be tested.

[0039] At room temperature (25°C), the Raman spectrum of the standard solution of cefazolin sodium was collected using a fiber Raman detection system equipped with a 785nm laser. The Raman laser power was 100mW, and the Raman signal was collected twice, with each collection time of 15s. The characteristic peak 777cm -1 The position and intensity of the standard solution of cefazolin sodium were plotted with the logarithm of the concentration as the horizontal axis and the intensity of the characteristic peak as the vertical axis to draw a standard working curve (such as Figure 2 ), and the linear equation of cefazolin sodium was obtained. The detection limit of this method for cefazolin sodium in water was calculated to be 3.18×10 -9 mol / L, linear range is 1×10 -4 ~1×10 - 8 mol / L.

[0040] Comparing the method proposed in the present invention with the prior art, the detection conditions and results are shown in Table 1:

[0041] Table 1

[0042]

[0043] It can be seen from Table 1 that the method proposed in the present invention has the advantages of simple operation process, short pretreatment time, high sensitivity, on-site and portable detection, etc. compared with solid phase extraction-high performance liquid chromatography and enzyme-linked immunosorbent assay.

[0044] Example 2

[0045] 1 mL of silver nanoparticle sol and 2.5 mL of 1×10 -4mol / L cefazolin sodium standard solution was mixed and added into a 10 mL centrifuge tube, the solution pH was adjusted to 6.3, the centrifuge tube was placed on a vortex mixer, the speed was set to 2800 rpm, and the vortex oscillation was maintained for 15 min. The standard test solutions after vortex oscillation and mixing for 15 min were subjected to Raman detection respectively, and the detection conditions were the same as those in Example 1.

[0046] Example 3

[0047] 1 mL of silver nanoparticle sol and 2.5 mL of 1×10 -4 mol / L cefazolin sodium standard solution was mixed and added into a 10 mL centrifuge tube, the solution pH was adjusted to 6.3, the centrifuge tube was placed on a vortex mixer, the speed was set to 2800 rpm, and the vortex oscillation was maintained for 10 min. The standard test solutions after vortex oscillation and mixing for 10 min were subjected to Raman detection respectively, and the detection conditions were the same as those in Example 1.

[0048] Example 4

[0049] 1 mL of silver nanoparticle sol and 2.5 mL of 1×10 -4 mol / L cefazolin sodium standard solution was mixed and added into a 10 mL centrifuge tube, the solution pH was adjusted to 6.3, the centrifuge tube was placed on a vortex mixer, the speed was set to 2800 rpm, and the vortex oscillation was maintained for 5 min. The standard test solutions after vortex oscillation and mixing for 5 min were subjected to Raman detection respectively, and the detection conditions were the same as those in Example 1.

[0050] Example 5

[0051] 1 mL of silver nanoparticle sol and 2.5 mL of 1×10 -4 mol / L cefazolin sodium standard solution was mixed and added into a 10 mL centrifuge tube, the solution pH was adjusted to 6.3, the centrifuge tube was placed on a vortex mixer, the speed was set to 2800 rpm, and the vortex oscillation was maintained for 1 min. The standard test solutions after vortex oscillation and mixing for 1 min were subjected to Raman detection respectively, and the detection conditions were the same as those in Example 1.

[0052] The Raman peak spectra of cefazolin sodium at different vortex times in Examples 1-5 are shown in FIG. Figure 3 As shown in the figure, the Raman spectrum of cefazolin sodium was obtained by vortexing a 0.1 mmol cefazolin sodium solution for 1 min, 5 min, 10 min, 15 min and 20 min, respectively. -1 The Raman signal was enhanced by up to 6 times according to the position calculation.

[0053] The relationship between the Raman peak intensity and vortex time of cefazolin sodium is shown in the figure below: Figure 4The inventors vortexed a 0.1 mmol solution of cefazolin sodium for 25 min, and found that the Raman signal intensity of cefazolin sodium decreased compared to that of vortexing for 20 min.

[0054] Example 6

[0055] The method for detecting pefloxacin in water specifically comprises the following steps:

[0056] (1) Preparation of silver nanoparticle sol: 90 mg of silver nitrate was dissolved in 500 mL of deionized water and heated to boiling. 10 mL of 1% sodium citrate solution was added and kept boiling for 30 min, and then cooled at room temperature to obtain a yellow-green silver nanoparticle sol with a diameter of 50 to 80 nm.

[0057] (2) Prepare 50 mL of 1×10 -4 mol / L、1×10 -5 mol / L、1×10 -6 mol / L、1×10 -7 mol / L and 1×10 - 8 mol / L pefloxacin standard solution; 3mL of silver nanoparticle sol and 1mL of pefloxacin standard solution of different concentrations were mixed and added into a 10mL centrifuge tube, the solution pH was adjusted to 4, the centrifuge tube was placed on a vortex mixer, the speed was set to 100rpm, and the vortex oscillation was maintained for 1min.

[0058] Under 90℃ water bath conditions, the Raman spectrum of vortexed pefloxacin was collected using a fiber Raman detection system equipped with a 785nm laser. The Raman laser power was 50mW, and the Raman signal was collected twice, with each collection time of 20s. The characteristic peak 1391cm -1 The position and intensity of the standard solution of pefloxacin are plotted with the logarithm of the concentration of the standard solution of pefloxacin as the horizontal axis and the intensity of the characteristic peak as the vertical axis to draw a standard working curve (such as Figure 5 ), and the linear equation of pefloxacin was obtained.

[0059] (3) 3 mL of silver nanoparticle sol and 1 mL of the water sample to be tested were added to a centrifuge tube and mixed, and the centrifuge tube was placed on a vortex mixer for vortex oscillation for 1 min to obtain a water sample solution to be tested.

[0060] The concentration of pefloxacin in water was detected under the above experimental conditions. The detection limit of this method for pefloxacin in water was calculated to be 8.67×10 -7 mol / L, linear range is 1×10 -4 ~1×10 -6 mol / L.

[0061] Example 7

[0062] Referring to Example 6, pefloxacin was tested. The concentration was prepared to be 1×10 -4 mol / L of three pefloxacin standard solution samples, the samples were vortexed for 6 min, 20 s, and not vortexed, and their Raman spectra were detected and compared with the characteristic peak 1391 cm -1 The change of intensity, such as Figure 6 As shown in the figure, the peak intensity of the spectrum after vortexing for 6 minutes increased by 2 times compared with that without vortexing. In comparison, the peak intensity of the sample vortexed for only 20 seconds under the same conditions did not change significantly compared with that without vortexing.

[0063] After the sample was vortexed for 6 minutes, its Raman spectrum was measured, and then its Raman spectrum was measured after being left for 6 hours. After vortexing for 1 minute, its Raman spectrum was measured. The peak intensity changes were as follows: Figure 7 As shown in the above results, it can be seen that the vortex process in this method has an important influence on improving the detection performance of pefloxacin.

[0064] Example 8

[0065] With reference to Example 6, a detection method for pefloxacin is established, which specifically comprises the following steps:

[0066] (1) Prepare silver nanoparticle sol: dissolve 90 mg of silver nitrate in 500 mL of deionized water and heat to boiling. Add 10 mL of 1% sodium citrate solution and keep boiling for 30 min. Then cool at room temperature to obtain yellow-green silver nanosol with a diameter of 50 to 80 nm for silver nanoparticles.

[0067] (2) Prepare 50 mL of 1×10 -4 mol / L、1×10 -5 mol / L、1×10 -6 mol / L、1×10 -7 mol / L and 1×10 - 8 mol / L of pefloxacin standard solutions of different concentrations; 1 mL of silver nanoparticle sol and 3 mL of pefloxacin standard solutions of different concentrations were mixed and added into a 10 mL centrifuge tube, the solution pH was adjusted to 9, the centrifuge tube was placed on a vortex mixer, the speed was set to 2800 rpm, and the vortex oscillation was maintained for 1 min.

[0068] At room temperature (25°C), the Raman spectrum of vortexed pefloxacin was collected using a fiber Raman detection system equipped with a 785nm laser. The Raman laser power was 500mW, and the Raman signal was collected twice, with each collection time of 4s.

[0069] (3) 1 mL of silver nanoparticle sol and 3 mL of the water sample to be tested were added to a centrifuge tube and mixed, and the centrifuge tube was placed on a vortex mixer for vortex oscillation for 1 min to obtain a water sample solution to be tested.

[0070] The concentration of pefloxacin in water was detected under the above experimental conditions, and the working curve of the method was obtained. The detection limit of the method for pefloxacin in water was calculated to be 2.86×10 -6 mol / L.

[0071] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A spectral detection method for antiviral and antibacterial drug residues in water, It is characterized in that The steps include: (1) Silver nanoparticle sol was mixed with a concentration of 1×10 -4 ~ 1×10 -10 mol / L standard solution of antiviral and antibacterial drugs is added to a centrifuge tube and mixed, the particle size of the silver nanoparticle sol is 50-80 nm, the volume ratio of the silver nanoparticle sol to the standard solution of antiviral and antibacterial drugs is 1:3-3:1, and the centrifuge tube is placed on a vortex mixer for vortex oscillation for 10-20 min, the rotation speed of the vortex mixer is 100-2800 rpm, and a standard test solution is obtained; (2) Performing Raman spectroscopy on the standard test solution obtained in step (1), recording the position and intensity of the characteristic peak, plotting a standard curve with the logarithm of the concentration of the standard solution of the antiviral and antibacterial drug as the abscissa and the intensity of the characteristic peak as the ordinate, and obtaining a linear equation of the antiviral and antibacterial drug to be tested. The detection conditions of Raman spectroscopy detection are: laser wavelength 785 nm, laser intensity 50-500 mw, and sample collection time 4-20 s; (3) Adding the silver nanoparticle sol and the water sample to be tested into a centrifuge tube at a volume ratio of 1:3 to 3:1 and mixing, and placing the centrifuge tube on a vortex mixer for vortex oscillation for 10-20 min, wherein the rotation speed of the vortex mixer is 100-2800 rpm, to obtain a water sample solution to be tested, and then subjecting the water sample solution to Raman spectroscopy detection, qualitatively identifying the types of antiviral and antibacterial drugs in the water sample to be tested according to step (2), substituting the obtained characteristic peak intensity into the linear equation obtained in step (2), and quantitatively determining the antiviral and antibacterial drugs in the water sample to be tested, wherein the detection conditions of Raman spectroscopy detection are: laser wavelength 785 nm, laser intensity 50-500 mW, and sample collection time 4-20 s, and the antiviral and antibacterial drugs include cefazolin sodium and pefloxacin.

2. The method for spectral detection of antiviral and antibacterial drug residues in water according to claim 1, It is characterized in that The silver nanoparticle sol is prepared by the following steps: dissolving silver nitrate in deionized water to form a silver nitrate aqueous solution with a concentration of 0.18 g / L, heating the silver nitrate aqueous solution to boiling, adding a 1% sodium citrate solution by mass, and keeping boiling for 30-60 min, and then cooling at room temperature to obtain the silver nanoparticle sol, wherein the volume ratio of the silver nitrate aqueous solution to the sodium citrate solution is 50:

1.

3. The method for spectral detection of antiviral and antibacterial drug residues in water according to claim 1, It is characterized in that The total volume of the standard test solution described in step (1) is 3-4 mL, the pH is 4-9, and the temperature is 25°C-90°C.

4. The method for spectral detection of antiviral and antibacterial drug residues in water according to claim 1, It is characterized in that The total volume of the water sample solution to be tested in step (3) is 3-4 mL, the pH is 4-9, and the temperature is 25°C-90°C.

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