A preparation method of a flexible Raman detection substrate and its application in rapid detection of drug residues in water

Abstract

The present invention discloses a preparation method of a flexible Raman detection substrate and its application in rapid detection of drug residues in water. By using a vortex evaporation method to assemble a flexible SERS substrate with a silver ring array on a sealing film, the low-cost and large-scale preparation of a flexible substrate with high sensitivity and high reproducibility is realized, which can achieve the rapid detection of drug residues in the water environment, solve the problem of poor uniformity in the preparation of flexible SERS substrates, and also solve the problem that drug residues in water cannot be rapidly detected.

Description

Technical Field:

[0001] The present invention relates to a flexible surface enhanced Raman scattering substrate material, and particularly to a method for preparing a flexible Raman detection substrate and its application in rapid detection of drug residues in water. Background Art:

[0002] Raman spectroscopy is a non-destructive and rapid detection technology, which can obtain the fingerprint information of target molecules and has the characteristics of specific recognition, and has been applied in the fields of chemical analysis, biomedical detection, etc. Compared with the traditional Raman spectroscopy detection technology, the surface enhanced Raman spectroscopy (SERS) technology can significantly enhance the Raman signals of target molecules adsorbed on the surface by means of the plasma resonance enhancement effect of noble metal nanomaterials, thereby improving its detection sensitivity, and thus has been widely studied. Currently, the core problem that plagues the wide application of SERS technology lies in the lack of detection substrates with high sensitivity and low cost. Among the existing SERS detection substrates, flexible SERS substrates have the characteristics of high porosity, low preparation cost, flexibility, foldability and bendability, and have incomparable advantages over rigid SERS substrates in applications such as extraction, filtration, and concentration of probe molecules in complex sample systems. Flexible substrates are mainly prepared by methods such as in-situ reduction, physical deposition, electrospinning, and inkjet printing, and flexible material substrates mainly focus on three types: bio-based materials, polymers, and carbon materials. The above methods can prepare SERS substrates with strong stability, high reproducibility, and good enhancement effect, but they have the disadvantages of complex production processes and high instrument costs.

[0003] The method of self-assembly of prefabricated metal nanoparticles to prepare SERS substrates is an efficient and low-cost method for preparing detection substrates, which includes solvent evaporation assembly method and gravity self-assembly method. These methods have lower costs and can be prepared on a large scale, but the uniformity of the substrates is poor, and the stability and reproducibility of the prepared substrates need to be further improved.

[0004] In terms of detecting target substances, antibiotic drugs have been widely used in industries such as medical treatment and livestock breeding. However, during the use process, a considerable part of these drugs will directly or enter the environmental water body in the form of metabolites. These drugs have the characteristics of anti-degradation and bioaccumulation, resulting in their enrichment in the ecosystem through the food chain and entering the human body, causing harm to human health. In addition, the residues of these drugs in the environment may lead to the generation of resistance genes, causing damage to the ecological environment system. At present, the methods for detecting drugs in water are mainly laboratory instrument detections such as high performance liquid chromatography and liquid chromatography mass spectrometry, and there is a huge demand for portable and rapid detection technologies such as Raman spectroscopy. Summary of the Invention:

[0005] The object of the present invention is to provide a method for preparing a flexible Raman detection substrate and its application in rapid detection of drug residues in water. By means of vortex evaporation, a flexible SERS substrate with a silver ring array is formed on a sealing film through the deposition and assembly of silver nanoparticle aerosol, realizing the low-cost and large-scale preparation of a flexible substrate with high sensitivity and high reproducibility. The obtained detection substrate can be used for the rapid detection of drug residues in water environment, solving the problems of poor uniformity in the preparation of flexible SERS substrates and the inability to rapidly detect drug residues in water.

[0006] The present invention is realized through the following technical solutions:

[0007] A method for preparing a flexible Raman detection substrate, the method comprising the following steps: adding silver nanoparticle sol and deionized water into a centrifuge tube respectively and mixing them, placing the centrifuge tube on the sample plate of a vortex oscillator, covering the tube orifice of the centrifuge tube with a piece of flexible sealing film, and fixing a flat plate above for vortex oscillation, so that the silver nanoparticles in the solution are deposited and assembled on the sealing film in the form of aerosol. After vortexing for a certain time, take out the sealing film covering the centrifuge tube (the silver nanoparticles are deposited on the sealing film to form a silver ring), rinse it with deionized water, and dry it with nitrogen to obtain a flexible surface-enhanced Raman spectroscopy detection substrate with a silver ring.

[0008] The preparation method of the silver nanoparticle sol is as follows: dissolve silver nitrate in deionized water, heat it to boiling, add sodium citrate solution, keep boiling for 30 - 60 min, and then cool it at room temperature to obtain a yellow-green silver nanoparticle sol. The prepared silver nanoparticles (AgNPs) have a particle size of 50 - 60 nm. Centrifuge the prepared AgNPs at a speed of 10000 rpm for 10 minutes. Extract the precipitate, and then redisperse it with deionized water.

[0009] The volume ratio of the silver nanoparticle sol to the deionized water is (1 - 1.5):(1 - 2.5).

[0010] The rotation speed of the vortex oscillator is 1400 - 2800 rpm, and the oscillation time is controlled to be 5 - 15 min.

[0011] The centrifuge tube is one or more than two.

[0012] When there are more than two centrifuge tubes, cover the tube orifices of all centrifuge tubes with a piece of flexible sealing film and fix a flat plate above for vortex oscillation to obtain an array of flexible surface-enhanced Raman spectroscopy detection substrates with silver rings. Prepare an array of flexible SERS substrates with silver rings, which can be cut into single detection substrates for use during detection, or multiple samples can be added one by one to form a detection sample array for use.

[0013] The flexible sealing film is a dense bag PE film, a fresh-keeping PE film or a sealing Parafilm film.

[0014] The present invention also protects the application of the flexible Raman detection substrate in the rapid detection of drug residues in water, including the following steps: Prepare drug solutions with different concentrations of 1×10 -4 ~1×10 -10 mol / L respectively. Drop 5 - 20 μL of the drug solutions with different concentrations onto the prepared detection substrate, and detect them with a Raman spectrometer at room temperature to obtain a working curve of the relationship between the target substance at different concentrations and the intensity of its Raman spectral characteristic peaks or peak area intensities, thereby establishing a Raman enhanced detection method for the target substance.

[0015] The laser wavelength of the used Raman spectrometer is 785 nm, the laser power is 50 - 500 mW, and the sample acquisition time is 2 - 10 s.

[0016] The drug is selected from any one of drugs such as sulfapyridine, sulfachloropyridazine, pefloxacin, fleroxacin, and ferulic acid.

[0017] This method can be applied to the highly sensitive detection of the above-mentioned drugs in water, but is not limited to the above-mentioned drug types.

[0018] The lowest detection concentration of the method is 3.69 - 28.4 μg / L, and the linear range is 3 orders of magnitude.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1) The present invention has the advantages of simple preparation process, short assembly time, high sensitivity, high reproducibility, and portability: Compared with the lithography method with relatively high reproducibility, the operation process of the present invention is simpler and the cost is lower. It can achieve batch preparation and on-site rapid detection, and has sensitivity and reproducibility similar to those of the lithography method (the relative standard deviation (RSD) of the SERS substrate prepared by the present invention is between 3.61 - 14.38%. For the two-dimensional silver nanogrid SERS substrate prepared by Chen et al. through the lithography method, the RSD corresponding to the Raman intensity of 10 - 5M Rh6G for repeated tests is 5.8 - 13.4%); Compared with the direct evaporation method, the substrate prepared by the present invention has higher uniformity, higher detection reproducibility and stability, and the SERS signal intensity obtained from the prepared substrate is 5 times higher than that of the substrate prepared by the direct evaporation method.

[0021] 2) The sealing film used for the flexible substrate is a high-polymer material such as a dense bag PE film, a fresh-keeping PE film, or a sealing Parafilm film, which has a low Raman background signal, no fluorescence interference, excellent elasticity and stability, and can adapt to complex detection environments. The polymer material is inexpensive, easy to process, and has the advantages of stability, uniformity, economy and high efficiency, which is of great significance for the practical application of the SERS substrate.

[0022] 3) The method of the present patent technology has high detection sensitivity for the detection of sulfonamide drug residues in water, and is a simple, rapid, stable and highly reproducible method. Description of the Drawings:

[0023] Figure 1 It is a Raman spectrum comparison diagram of sulfapyridine by the flexible detection substrate prepared in Example 1;

[0024] Figure 2 It is a working curve of the change of the Raman characteristic peak intensity of sulfapyridine with concentration;

[0025] Figure 3 It is a working curve of the change of the Raman characteristic peak intensity of sulfachloropyridazine with concentration;

[0026] Figure 4 It is the Raman peak spectrum of sulfapyridine detected by the SR SRES substrate assembled at different vortex rotation speeds in Example 3;

[0027] Figure 5 It is the working curve of the intensity at 744 cm -1 of the characteristic peak of pefloxacin with the change of concentration;

[0028] Figure 6 It is the working curve of the intensity at 1111 cm -1 of the Raman spectrum characteristic peak of ferulic acid with the change of concentration. Detailed Embodiments:

[0029] The following is a further description of the present invention, rather than a limitation of the present invention.

[0030] Example 1: Preparation of a flexible Raman detection substrate and rapid detection of sulfapyridine residues in water

[0031] First, 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 wt% sodium citrate solution and keep boiling for 60 min, then cool at room temperature to obtain yellow-green silver nano sol. The diameter of silver nanoparticles is 50 - 60 nm. Centrifuge the prepared AgNPs at a speed of 10000 rpm for 10 min. Extract the precipitate and then redisperse it with deionized water to remove the excess sodium citrate in the solution. Add 1 mL of silver nanoparticle sol and 1.5 mL of deionized water into several centrifuge tubes (without lids) and place them on the sample plate of a vortex oscillator. Cover the mouths of all centrifuge tubes on the sample plate with a piece of cut flexible Parafilm membrane, and then fix it with a flat plate above and perform vortex oscillation. During the oscillation process, control the rotation speed of the vortex mixer at 2800 rpm and the oscillation time at 5 min to prepare a flexible SERS substrate with a silver ring array.

[0032] Prepare 50 ml of sulfapyridine standard solutions with different concentrations of 1×10 -8 ~1×10 -4 mol / L (2.49 μg / L to 24.9 mg / L); during the detection process, 10 μL of target solutions with different concentrations were dropped onto the prepared SERS substrate.

[0033] At room temperature (25 °C), the Raman spectrum of sulfapyridine was collected using a fiber Raman detection system equipped with a 785 nm laser. The Raman laser power was 300 mW, and the Raman signal was collected cumulatively 2 times, with each collection time being 5 s.

[0034] After assembling silver rings on the sealing film using this method, the Raman signal was calculated to be enhanced by 126 times at the characteristic peak position of 1600 cm -1 . Detect the concentration of sulfapyridine in water under the above experimental conditions, and the working curve of this method is as shown in Figure 2 . Calculate that the lowest detection concentration of sulfapyridine in water by this method is 24.9 μg / L, and the linear range is 24.9 μg / L to 24.9 mg / L.

[0035] Detect sulfapyridine in actual water samples using the detection method established in Example 1. The recovery rate of this method is greater than 88.1%, and the RSD is less than 13.9%.

[0036] Example 2: Detect residual sulfachloropyridazine in water

[0037] Refer to Example 1. First, prepare silver nanoparticle sol. Dissolve 90 mg of silver nitrate in 500 mL of deionized water and heat it to boiling. Add 10 mL of 1 wt% sodium citrate solution and keep boiling for 60 min, then cool it at room temperature to obtain yellow-green silver nano sol. Centrifuge the prepared AgNPs at a speed of 10000 rpm for 10 min. Extract the precipitate and then redisperse it with deionized water to remove the excess sodium citrate in the solution. Add 1.5 mL of silver nanoparticle sol and 1 mL of deionized water into several centrifuge tubes (without lids) and place them on the sample plate of a vortex oscillator. Cover the mouths of all centrifuge tubes on the sample plate with a piece of cut flexible sealing Parafilm, and then fix a flat plate above to perform vortex oscillation. During the oscillation process, control the rotation speed of the vortex mixer to be 2500 rpm and the oscillation time to be 12 min to prepare a flexible SERS substrate with a silver ring array.

[0038] Prepare 50 ml of -8 ~1×10 -4Standard solutions of sulfachloropyridazine at different concentrations of mol / L (2.84 μg / L to 28.47 mg / L); during the detection process, 5 μL of target solutions at different concentrations were dropped onto the prepared SERS substrate.

[0039] At room temperature (25 °C), the Raman spectrum of sulfachloropyridazine was collected using a fiber Raman detection system equipped with a 785 nm laser. The Raman laser power was 300 mW, the Raman signal was collected cumulatively 2 times, and the collection time for each time was 5 s.

[0040] The concentration of sulfachloropyridazine in water was detected under the above experimental conditions, and the working curve of this method was obtained as Figure 3 shown. The lowest detection concentration of sulfachloropyridazine in water by this method was calculated to be 28.4 μg / L, and the linear range was 28.47 μg / L to 28.47 mg / L.

[0041] The sulfachloropyridazine in the actual water sample was detected by the detection method established in Example 2. The recovery rate of this method was greater than 82.9%, and the RSD was less than 6.4%.

[0042] Example 3: Detection of residual sulfapyridine in water

[0043] Referring to the experimental conditions of Example 1, silver ring SERS substrates were obtained at vortex oscillation speeds of 0 rpm, 1120 rpm, 1400 rpm, 1680 rpm, 1960 rpm, 2240 rpm, 2520 rpm, and 2800 rpm respectively. The substrates were used to detect sulfapyridine and obtain Raman spectrum detection maps, and the Raman signal was enhanced by 126 times calculated based on the characteristic peak at 1600 cm -1 position.

[0044] Example 4: Detection of residual pefloxacin in water

[0045] Referring to Example 1, the method for detecting pefloxacin is established in the following way. The process is as follows: First, prepare silver nanoparticle sol. Dissolve 90 mg of silver nitrate in 500 mL of deionized water and heat it to boiling. Add 10 mL of 1 wt% sodium citrate solution and keep boiling for 30 min, then cool it at room temperature to obtain yellow-green silver nano sol. Centrifuge the prepared AgNPs at a speed of 10000 rpm for 10 min. Extract the precipitate and then redisperse it with deionized water to remove the excess sodium citrate in the solution. Add 1 mL of silver nanoparticle sol and 2.5 mL of deionized water into several centrifuge tubes (without lids) and place them on the sample plate of a vortex oscillator. Use a piece of cut flexible fresh-keeping PE film to uniformly cover the mouths of all centrifuge tubes on the sample plate, and then fix a flat plate above to perform vortex oscillation. During the oscillation process, control the rotation speed of the vortex mixer to be 1400 rpm and the vortex time to be 5 min to prepare a flexible SERS substrate with a silver ring array.

[0046] Prepare 10 ml of pefloxacin standard solution with a concentration range of 10 μg / L - 10 mg / L; during the detection process, drop 20 μL of target solution with different concentrations onto the prepared SERS substrate. Detect pefloxacin with a Raman spectrometer equipped with a 785 nm laser. The Raman laser power is 500 mW, and the Raman signal is collected cumulatively 2 times, with each collection time being 2 s.

[0047] Detect the concentration of pefloxacin in water under the above experimental conditions. According to the change of the intensity at the characteristic peak of 744 cm -1 with the concentration, the working curve of this method is obtained as Figure 5 shown. Calculate that the lowest detection concentration of pefloxacin in water by this method is 10 μg / L, and the linear range is 10 μg / L - 10 mg / L.

[0048] Example 5: Detect the residual ferulic acid in water

[0049] Referring to Example 1, a detection method for ferulic acid was established. The process is as follows: First, prepare silver nanoparticle sol. Dissolve 90 mg of silver nitrate in 500 mL of deionized water and heat it to boiling. Add 10 mL of 1 wt% sodium citrate solution and keep boiling for 60 min, then cool it at room temperature to obtain yellow-green silver nano sol. Centrifuge the prepared AgNPs at a speed of 10,000 rpm for 10 min. Extract the precipitate and then redisperse it with deionized water to remove the excess sodium citrate in the solution. Add 1 mL of silver nanoparticle sol and 1.5 mL of deionized water into several centrifuge tubes (without lids) and place them on the sample plate of a vortex oscillator. Use a piece of cut flexible sealing Parafilm to uniformly cover the mouths of all centrifuge tubes on the sample plate, and then fix a flat plate above to perform vortex oscillation. During the vortex process, control the rotation speed of the vortex mixer at 2800 rpm and the vortex time at 15 min to prepare a flexible SERS substrate with a silver ring array.

[0050] Prepare 10 ml of ferulic acid standard solutions with different concentrations from 10 μg / L to 10 mg / L; during the detection process, drop 5 μL of target solutions with different concentrations onto the prepared SERS substrate. Detect ferulic acid with a Raman spectrometer equipped with a 785 nm laser. The Raman laser power is 50 mW, and the Raman signal is collected 2 times in total, with each collection time being 10 s.

[0051] Detect the concentration of ferulic acid in water under the above experimental conditions. According to the change of its intensity at the characteristic peak of 1111 cm -1 with the concentration, obtain the working curve of this method as Figure 6 shown, and calculate that the detection limit of this method for ferulic acid in water is 10 μg / L, and the linear range is 10 μg / L - 10 mg / L.

[0052] Example 6: Detect the residual fleroxacin in water

[0053] Referring to Example 1, use a dense bag PE film as the material for preparing the flexible detection substrate, and detect fleroxacin in water with the prepared silver ring array detection substrate. The detection limit of this substrate for fleroxacin is 3.69 μg / L.

Claims

1. A preparation method of a flexible Raman detection substrate, characterized in that, the method comprises the following steps: Silver nanoparticle sol and deionized water are respectively added into a centrifuge tube and mixed, and placed on the sample plate of a vortex oscillator. The tube mouth of the centrifuge tube is covered with a piece of flexible sealing film, and a flat plate is fixed above for vortex oscillation, so that the silver nanoparticles in the solution are deposited and assembled on the sealing film in the form of aerosol. After vortexing for a certain time, the sealing film covering the centrifuge tube is taken out, rinsed thoroughly with deionized water, and dried with nitrogen to obtain a flexible surface-enhanced Raman spectroscopy detection substrate with a silver ring.

2. The preparation method according to claim 1, characterized in that, the preparation method of the silver nanoparticle sol is as follows: Dissolve silver nitrate in deionized water, heat to boiling, add sodium citrate solution, keep boiling for 30 - 60 min, then cool at room temperature to obtain a yellow-green silver nanoparticle sol, centrifuge at a speed of 10000 rpm for 10 minutes, extract the precipitate, and then redisperse it with deionized water.

3. The preparation method according to claim 1, characterized in that, the volume ratio of the silver nanoparticle sol to the deionized water is (1 - 1.5):(1 - 2.5).

4. The preparation method according to claim 1, characterized in that, the rotation speed of the vortex oscillator is 1400 - 2800 rpm, and the oscillation time is controlled to be 5 - 15 min.

5. The preparation method according to claim 1, characterized in that, the centrifuge tube is one or more than two.

6. The preparation method according to claim 5, characterized in that, when there are more than two centrifuge tubes, the tube mouths of all centrifuge tubes are covered with a piece of flexible sealing film and a flat plate is fixed above for vortex oscillation to obtain an array of flexible surface-enhanced Raman spectroscopy detection substrates with silver rings.

7. The preparation method according to claim 1, characterized in that, the flexible sealing film is a dense bag PE film, a fresh-keeping PE film or a sealing Parafilm film.

8. Application of the flexible Raman detection substrate obtained by the preparation method according to claim 1 in the rapid detection of drug residues in water, characterized in that, it comprises the following steps: Prepare drug solutions with different concentrations of 1×10 -4 ~1×10 -10 mol / L respectively. Drop 5 - 20 μL of the drug solutions with different concentrations onto the prepared detection substrate, and detect them with a Raman spectrometer under room temperature conditions to obtain a working curve showing the relationship between the target substance and the intensity of its Raman spectral characteristic peaks or peak area intensities at different concentrations, thereby establishing a Raman enhanced detection method for the target substance.

9. The application according to claim 8, characterized in that, the laser wavelength of the Raman spectrometer used is 785 nm, the laser power is 50 - 500 mW, and the sample collection time is 2 - 10 s.

10. The application according to claim 8, characterized in that, the drug is selected from any one of sulfapyridine, sulfachloropyridazine, pefloxacin, fleroxacin and ferulic acid.

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