A method for rapidly detecting fleroxacin in water quality

Abstract

The application relates to the technical field of water quality detection, and discloses a method for rapidly detecting fluroxquinolone in water quality, which comprises the following steps: preparing a surface-enhanced Raman spectrum substrate, concentrating and pretreating a sample water sample, enriching and detecting fluroxquinolone. The fluroxquinolone in the water quality is detected by using the surface-enhanced Raman spectrum technology, the detection sensitivity is high, compared with traditional high-performance liquid chromatography and liquid chromatography-mass spectrometry, a complex sample pretreatment process is not needed, the detection time is greatly shortened, and rapid detection can be realized. The whole process does not need complex instrument equipment and strict reaction condition control, ordinary experiment personnel can master the method after simple training, and the method is favorable for large-scale popularization and application.

Description

Technical Field

[0001] This invention relates to the field of water quality testing technology, specifically a method for rapid detection of fluroxacin in water. Background Technology

[0002] Fleroxacin is a widely used quinolone antibiotic. Its extensive use in pharmaceuticals and aquaculture has led to its increasing detection in aquatic environments. Residues of fleroxacin in the aquatic environment not only pose a potential threat to aquatic ecosystems but may also enter the human body through the food chain, affecting human health. Therefore, accurate and rapid detection of fleroxacin in water is of great significance.

[0003] Currently, the main methods for detecting fleroxacin in water quality include high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). HPLC requires complex sample pretreatment, is time-consuming, and uses expensive equipment. While LC-MS offers high sensitivity, it also suffers from cumbersome sample pretreatment, high instrument costs, and requires highly skilled operators, making it unsuitable for rapid on-site detection. Therefore, there is an urgent need to develop a simple, rapid, and highly sensitive method for detecting fleroxacin in water quality. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a method for rapid detection of fluroxacin in water.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for rapid detection of fluroxacin in water, comprising the following steps:

[0008] S1. Preparation of surface-enhanced Raman spectroscopy substrate: A surface-enhanced Raman spectroscopy substrate was prepared by chemical deposition.

[0009] S2. Sample pretreatment: Take a certain volume of water sample and filter it through a 0.22μm filter membrane to remove suspended solids from the water sample; then concentrate the filtered water sample to 1 / 10 to 1 / 5 of the original volume using a rotary evaporator at 40 to 50℃ to obtain a concentrated water sample.

[0010] S3. Enrichment and Detection: 20–30 μL of concentrated water sample was dropped onto a surface-enhanced Raman spectroscopy (SERS) substrate and allowed to air dry at room temperature to enrich fleroxacin on the substrate surface. The substrate then was placed in a Raman spectrometer, and Raman spectroscopy was performed under the conditions of an excitation wavelength of 785 nm, a laser power of 10–50 mW, and an integration time of 1–10 s to obtain the Raman spectrum of the water sample. By comparing the spectrum with a standard Raman spectrum, the presence of fleroxacin in the water sample was determined, and a standard curve was established based on the characteristic peak intensity to achieve quantitative analysis of fleroxacin content.

[0011] Furthermore, the fabrication process of the surface-enhanced Raman spectroscopy substrate includes the following steps:

[0012] A1. Dissolve the template agent in a mixed solution of hydrochloric acid and ethanol, stir until completely dissolved, add tetrabutyl titanate, and continue stirring for 2-3 hours to form a sol; uniformly coat the above sol onto the surface of the pretreated titanium sheet, dry at 60-80℃ for 12-24 hours, and then calcine in a muffle furnace at 500-600℃ for 4-6 hours to remove the template agent, thereby obtaining a mesoporous titanium dioxide nanotube array;

[0013] A2. Add silver nitrate and sodium citrate to deionized water, heat to boiling and stir to obtain silver nano-seed solution; then, immerse the mesoporous titanium dioxide nanotube array in the silver nano-seed solution and let it stand at room temperature for 1-2 hours; next, dissolve silver nitrate and chloroauric acid in deionized water, store in the dark, add ascorbic acid and stir evenly, ascorbic acid should be prepared fresh for use, prepare a growth solution containing silver nitrate, chloroauric acid and ascorbic acid, immerse the substrate with adsorbed silver nano-seeds in the growth solution, react at room temperature for 30-60 minutes, and finally rinse with deionized water and dry to obtain silver-gold composite nanoparticle modified mesoporous titanium dioxide nanotube array substrate;

[0014] A3. Immerse the substrate in a Tris buffer solution containing 1–5 mg / mL polydopamine at pH 8.5 and react with shaking at room temperature for 6–12 h. Rinse the substrate with deionized water to remove unreacted polydopamine. Immerse the treated substrate in a solution of N,N-dimethylformamide containing 5–10 mM alkynyl-NHS ester and react at room temperature for 4–8 h. Then rinse the substrate sequentially with N,N-dimethylformamide and deionized water to remove residual reagents.

[0015] A4. Place the above substrate under ultraviolet light irradiation and treat it in a reaction tank containing 0.01-0.05 mol / L sodium hydroxide solution for 10-20 min;

[0016] A5. Dissolve the azide-modified fleroxacin aptamer in a phosphate buffer solution containing 0.1–0.5 mol / L sodium chloride, pH 7.2–7.6, to prepare an aptamer solution with a concentration of 10–30 μM. Immerse the photocatalytically oxidized substrate in the aptamer solution and let it stand at room temperature for 30–60 min. Rinse the substrate with phosphate buffer solution to remove unbound aptamers, rinse with deionized water, and air dry to obtain a surface-enhanced Raman spectroscopy substrate.

[0017] Furthermore, the template agent is one of triblock copolymer P123 or triblock copolymer F127.

[0018] Further, step A1 includes 0.5 to 1.5 parts template agent, 1 to 3 parts hydrochloric acid, 20 to 50 parts ethanol, and 2 to 5 parts tetrabutyl titanate.

[0019] Further, in step A2, the silver nanoseed solution comprises: 0.01–0.05 parts silver nitrate, 0.05–0.2 parts sodium citrate, and 100–200 parts water; the growth solution comprises: 0.005–0.02 parts silver nitrate, 0.005–0.02 parts chloroauric acid, 100–200 parts deionized water, and 0.01–0.05 parts ascorbic acid.

[0020] Furthermore, the ultraviolet light processing parameters in step A4 are a wavelength of 254 nm and a light intensity of 5–10 mW / cm². 2 .

[0021] Furthermore, the preparation method of the azide-modified fleroxacin aptamer includes the following steps:

[0022] B1. Using exponential enrichment ligand system evolution technology, with fleroxacin as the target molecule, aptamer sequences with high affinity and specificity for fleroxacin are screened in vitro from a random single-stranded oligonucleotide library. Bioinformatics analysis is performed on the screened aptamer sequences, and aptamer sequences with strong affinity, high specificity and structural stability are selected as target sequences. At the same time, appropriate modification sites are reserved at the 5' or 3' end of the aptamer sequence according to the subsequent modification requirements.

[0023] B2. The solid-phase phosphoramidite trimer method is adopted, using phosphoramidite monomers containing azido groups to replace conventional monomers. Azido groups are introduced into the aptamer at the preset modification sites. In specific operation, the synthesis instrument adds the corresponding phosphoramidite monomers in sequence according to the designed sequence. Through deprotection, coupling and oxidation chemical reactions, the oligonucleotide chain is gradually extended to complete the synthesis of azido-modified aptamers.

[0024] B3. The synthesized azide-modified aptamer is purified to remove unreacted monomers, byproducts, and impurities. Commonly used purification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and polyacrylamide gel electrophoresis (PAGE). RP-HPLC separates the aptamer and impurities based on the difference in partition coefficients between the stationary and mobile phases. PAGE, on the other hand, separates the aptamer based on differences in molecular size and charge. The purified azide-modified fleroxacin aptamer is recovered by cutting the gel bands.

[0025] Furthermore, the phosphoramid monomer containing an azide group is one of a 5'-azido-modified phosphoramid monomer or a 3'-azido-modified phosphoramid monomer.

[0026] (III) Beneficial Technical Effects

[0027] This invention uses surface-enhanced Raman spectroscopy to detect fleroxacin in water. Compared with traditional high-performance liquid chromatography and liquid chromatography-mass spectrometry, it eliminates the need for complex sample pretreatment, greatly shortens the detection time, and enables rapid detection.

[0028] The surface-enhanced Raman spectroscopy substrate prepared by this invention exhibits excellent enhancement effects for fleroxacin, and possesses high detection sensitivity, capable of detecting levels as low as 5 × 10⁻⁶ in water samples. -8 A mol / L solution of fleroxacin was prepared. The specific surface area and pore structure of the substrate were increased by fabricating a mesoporous titanium dioxide nanotube array, providing more adsorption sites for fleroxacin molecules. Simultaneously, the fleroxacin aptamer introduced by surface functionalization treatment could specifically bind to fleroxacin molecules, significantly improving the substrate's fleroxacin loading capacity. Using a polydopamine coating instead of a silane coupling agent provided better biocompatibility and chemical stability. Photocatalytic oxidation pretreatment generated hydroxyl groups on the substrate surface, enhancing surface activity and providing a favorable immobilization environment for the aptamer. The electrostatic adsorption immobilization process was mild, preserving the spatial structure and bioactivity of the aptamer and ensuring specific recognition of fleroxacin.

[0029] The entire process requires no complex instruments or equipment or strict control of reaction conditions. Ordinary laboratory personnel can master it with simple training, which is conducive to large-scale promotion and application. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, all components of the surface-enhanced Raman spectroscopy substrate formulation of this invention are commercially available.

[0032] All parts used in this invention are by weight.

[0033] Example 1

[0034] The template agent is a triblock copolymer P123.

[0035] Step A1 includes 0.5 parts template agent, 1 part hydrochloric acid, 20 parts ethanol, and 2 parts tetrabutyl titanate.

[0036] The silver nanoseed solution in step A2 includes: 0.01 parts silver nitrate, 0.05 parts sodium citrate, and 100 parts water; the growth solution includes: 0.005 parts silver nitrate, 0.005 parts chloroauric acid, 100 parts deionized water, and 0.01 parts ascorbic acid.

[0037] In step A4, the ultraviolet light processing parameters are: wavelength 254nm, light intensity 5mW / cm². 2 .

[0038] The phosphoramid monomer containing an azide group is a 5'-azide-modified phosphoramid monomer.

[0039] A method for rapid detection of fluroxacin in water includes the following steps:

[0040] S1. Preparation of surface-enhanced Raman spectroscopy substrate: A surface-enhanced Raman spectroscopy substrate was prepared by chemical deposition.

[0041] S2. Sample pretreatment: Take a certain volume of water sample and filter it through a 0.22μm filter membrane to remove suspended solids from the water sample; then concentrate the filtered water sample to 1 / 10 of its original volume using a rotary evaporator at 40℃ to obtain a concentrated water sample.

[0042] S3. Enrichment and Detection: 20 μL of concentrated water sample was dropped onto a surface-enhanced Raman spectroscopy (SERS) substrate and allowed to air dry at room temperature to enrich fleroxacin on the substrate surface. The substrate then was placed in a Raman spectrometer, and Raman spectroscopy was performed under the conditions of an excitation wavelength of 785 nm, a laser power of 10 mW, and an integration time of 1 s to obtain the Raman spectrum of the water sample. By comparing the spectrum with a standard Raman spectrum, the presence of fleroxacin in the water sample was determined, and a standard curve was established based on the characteristic peak intensity to achieve quantitative analysis of fleroxacin content.

[0043] The fabrication process of surface-enhanced Raman spectroscopy substrates includes the following steps:

[0044] A1. Dissolve the template agent in a mixed solution of hydrochloric acid and ethanol, stir until completely dissolved, add tetrabutyl titanate, and continue stirring for 2 hours to form a sol; uniformly coat the above sol onto the surface of the pretreated titanium sheet, dry at 60°C for 12 hours, and then calcine at 500°C for 4 hours in a muffle furnace to remove the template agent and obtain a mesoporous titanium dioxide nanotube array.

[0045] A2. Add silver nitrate and sodium citrate to deionized water, heat to boiling and stir to obtain silver nano-seed solution; then, immerse the mesoporous titanium dioxide nanotube array in the silver nano-seed solution and let it stand at room temperature for 1 hour; next, dissolve silver nitrate and chloroauric acid in deionized water, store in the dark, add ascorbic acid and stir evenly, and prepare the ascorbic acid fresh for use to prepare a growth solution containing silver nitrate, chloroauric acid and ascorbic acid; immerse the substrate with adsorbed silver nano-seeds in the growth solution and react at room temperature for 30 minutes; finally, rinse with deionized water and dry to obtain silver-gold composite nanoparticle modified mesoporous titanium dioxide nanotube array substrate;

[0046] A3. Immerse the substrate in a Tris buffer solution containing 1 mg / mL polydopamine at pH 8.5 and react with shaking at room temperature for 6 h. Rinse the substrate with deionized water to remove unreacted polydopamine. Immerse the treated substrate in a solution of N,N-dimethylformamide containing 5 mM alkynyl-NHS ester and react at room temperature for 4 h. Then rinse the substrate sequentially with N,N-dimethylformamide and deionized water to remove residual reagents.

[0047] A4. Place the above substrate under ultraviolet light irradiation and treat it in a reaction tank containing 0.01 mol / L sodium hydroxide solution for 10 min;

[0048] A5. The azide-modified fleroxacin aptamer was dissolved in a phosphate buffer solution containing 0.1 mol / L sodium chloride at pH 7.2 to prepare a 10 μM aptamer solution. The substrate that had undergone photocatalytic oxidation was immersed in the aptamer solution and allowed to stand at room temperature for 30 min. The substrate was then rinsed with phosphate buffer solution to remove unbound aptamers, rinsed with deionized water, and dried to obtain a surface-enhanced Raman spectroscopy substrate.

[0049] The preparation method of the azide-modified fleroxacin aptamer includes the following steps:

[0050] B1. Using exponential enrichment ligand system evolution technology, with fleroxacin as the target molecule, aptamer sequences with high affinity and specificity for fleroxacin were screened in vitro from a random single-stranded oligonucleotide library. Bioinformatics analysis was performed on the screened aptamer sequences, and aptamer sequences with strong affinity, high specificity and structural stability were selected as target sequences. At the same time, appropriate modification sites were reserved at the 5' end of the aptamer sequences according to subsequent modification requirements.

[0051] B2. The solid-phase phosphoramidite trimer method is adopted, using phosphoramidite monomers containing azido groups to replace conventional monomers. Azido groups are introduced into the aptamer at the preset modification sites. In specific operation, the synthesis instrument adds the corresponding phosphoramidite monomers in sequence according to the designed sequence. Through deprotection, coupling and oxidation chemical reactions, the oligonucleotide chain is gradually extended to complete the synthesis of azido-modified aptamers.

[0052] B3. The synthesized azide-modified aptamer is purified to remove unreacted monomers, byproducts, and impurities. Commonly used purification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and polyacrylamide gel electrophoresis (PAGE). RP-HPLC separates the aptamer and impurities based on the difference in partition coefficients between the stationary and mobile phases. PAGE, on the other hand, separates the aptamer based on differences in molecular size and charge. The purified azide-modified fleroxacin aptamer is recovered by cutting the gel bands.

[0053] Example 2

[0054] The template agent is triblock copolymer F127.

[0055] Step A1 includes 1 part template agent, 2 parts hydrochloric acid, 30 parts ethanol, and 4 parts tetrabutyl titanate.

[0056] The silver nanoseed solution in step A2 includes: 0.03 parts silver nitrate, 0.1 parts sodium citrate, and 150 parts water; the growth solution includes: 0.01 parts silver nitrate, 0.01 parts chloroauric acid, 150 parts deionized water, and 0.03 parts ascorbic acid.

[0057] In step A4, the ultraviolet light processing parameters are: wavelength 254nm, light intensity 8mW / cm². 2 .

[0058] The phosphoramid monomer containing an azide group is a 3'-azide-modified phosphoramid monomer.

[0059] A method for rapid detection of fluroxacin in water includes the following steps:

[0060] S1. Preparation of surface-enhanced Raman spectroscopy substrate: A surface-enhanced Raman spectroscopy substrate was prepared by chemical deposition.

[0061] S2. Sample pretreatment: Take a certain volume of water sample and filter it through a 0.22μm filter membrane to remove suspended solids from the water sample; then concentrate the filtered water sample to 1 / 8 of its original volume using a rotary evaporator at 45℃ to obtain a concentrated water sample.

[0062] S3. Enrichment and Detection: 25 μL of concentrated water sample was dropped onto a surface-enhanced Raman spectroscopy (SERS) substrate and allowed to air dry at room temperature to enrich fleroxacin on the substrate surface. The substrate then was placed in a Raman spectrometer, and Raman spectroscopy was performed at an excitation wavelength of 785 nm, a laser power of 30 mW, and an integration time of 5 s to obtain the Raman spectrum of the water sample. By comparing the spectrum with a standard Raman spectrum, the presence of fleroxacin in the water sample was determined. A standard curve was established based on the characteristic peak intensities to achieve quantitative analysis of fleroxacin content.

[0063] The fabrication process of surface-enhanced Raman spectroscopy substrates includes the following steps:

[0064] A1. Dissolve the template agent in a mixed solution of hydrochloric acid and ethanol, stir until completely dissolved, add tetrabutyl titanate, and continue stirring for 2.5 h to form a sol; uniformly coat the above sol onto the surface of the pretreated titanium sheet, dry at 70°C for 16 h, and then calcine at 550°C for 5 h in a muffle furnace to remove the template agent, thereby obtaining a mesoporous titanium dioxide nanotube array.

[0065] A2. Add silver nitrate and sodium citrate to deionized water, heat to boiling and stir to obtain silver nano-seed solution; then, immerse the mesoporous titanium dioxide nanotube array in the silver nano-seed solution and let it stand at room temperature for 1.5 h; next, dissolve silver nitrate and chloroauric acid in deionized water, store in the dark, add ascorbic acid and stir evenly, ascorbic acid should be prepared fresh for use, prepare a growth solution containing silver nitrate, chloroauric acid and ascorbic acid, immerse the substrate with adsorbed silver nano-seeds in the growth solution, react at room temperature for 45 min, finally rinse with deionized water and dry to obtain silver-gold composite nanoparticle modified mesoporous titanium dioxide nanotube array substrate;

[0066] A3. Immerse the substrate in a Tris buffer solution containing 3 mg / mL polydopamine at pH 8.5 and react with shaking at room temperature for 9 h. Rinse the substrate with deionized water to remove unreacted polydopamine. Immerse the treated substrate in a solution of N,N-dimethylformamide containing 6 mM alkynyl-NHS ester and react at room temperature for 6 h. Then rinse the substrate sequentially with N,N-dimethylformamide and deionized water to remove residual reagents.

[0067] A4. Place the above substrate under ultraviolet light irradiation and treat it in a reaction tank containing 0.03 mol / L sodium hydroxide solution for 15 min;

[0068] A5. The azide-modified fleroxacin aptamer was dissolved in a phosphate buffer solution containing 0.3 mol / L sodium chloride at pH 7.5 to prepare an aptamer solution with a concentration of 20 μM. The substrate that had undergone photocatalytic oxidation was immersed in the aptamer solution and allowed to stand at room temperature for 45 min. The substrate was then rinsed with phosphate buffer solution to remove unbound aptamers, rinsed with deionized water, and dried to obtain a surface-enhanced Raman spectroscopy substrate.

[0069] The preparation method of the azide-modified fleroxacin aptamer includes the following steps:

[0070] B1. Using exponential enrichment ligand system evolution technology, with fleroxacin as the target molecule, aptamer sequences with high affinity and specificity for fleroxacin were screened in vitro from a random single-stranded oligonucleotide library. Bioinformatics analysis was performed on the screened aptamer sequences, and aptamer sequences with strong affinity, high specificity and structural stability were selected as target sequences. At the same time, appropriate modification sites were reserved at the 3' end of the aptamer sequences according to subsequent modification requirements.

[0071] B2. The solid-phase phosphoramidite trimer method is adopted, using phosphoramidite monomers containing azido groups to replace conventional monomers. Azido groups are introduced into the aptamer at the preset modification sites. In specific operation, the synthesis instrument adds the corresponding phosphoramidite monomers in sequence according to the designed sequence. Through deprotection, coupling and oxidation chemical reactions, the oligonucleotide chain is gradually extended to complete the synthesis of azido-modified aptamers.

[0072] B3. The synthesized azide-modified aptamer is purified to remove unreacted monomers, byproducts, and impurities. Commonly used purification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and polyacrylamide gel electrophoresis (PAGE). RP-HPLC separates the aptamer and impurities based on the difference in partition coefficients between the stationary and mobile phases. PAGE, on the other hand, separates the aptamer based on differences in molecular size and charge. The purified azide-modified fleroxacin aptamer is recovered by cutting the gel bands.

[0073] Example 3

[0074] The template agent is a triblock copolymer P123.

[0075] Step A1 includes 1.5 parts template agent, 3 parts hydrochloric acid, 50 parts ethanol, and 5 parts tetrabutyl titanate.

[0076] The silver nanoseed solution in step A2 includes: 0.05 parts silver nitrate, 0.2 parts sodium citrate, and 200 parts water; the growth solution includes: 0.02 parts silver nitrate, 0.02 parts chloroauric acid, 200 parts deionized water, and 0.05 parts ascorbic acid.

[0077] In step A4, the ultraviolet light processing parameters are: wavelength 254nm, light intensity 10mW / cm². 2 .

[0078] The phosphoramid monomer containing an azide group is a 5'-azide-modified phosphoramid monomer.

[0079] A method for rapid detection of fluroxacin in water includes the following steps:

[0080] S1. Preparation of surface-enhanced Raman spectroscopy substrate: A surface-enhanced Raman spectroscopy substrate was prepared by chemical deposition.

[0081] S2. Sample pretreatment: Take a certain volume of water sample and filter it through a 0.22μm filter membrane to remove suspended solids from the water sample; then concentrate the filtered water sample to 1 / 5 of its original volume using a rotary evaporator at 50℃ to obtain a concentrated water sample.

[0082] S3. Enrichment and Detection: 30 μL of concentrated water sample was dropped onto a surface-enhanced Raman spectroscopy (SERS) substrate and allowed to air dry at room temperature to enrich fleroxacin on the substrate surface. The substrate then was placed in a Raman spectrometer, and Raman spectroscopy was performed at an excitation wavelength of 785 nm, a laser power of 50 mW, and an integration time of 10 s to obtain the Raman spectrum of the water sample. By comparing the spectrum with a standard Raman spectrum, the presence of fleroxacin in the water sample was determined, and a standard curve was established based on the characteristic peak intensity to achieve quantitative analysis of fleroxacin content.

[0083] The fabrication process of surface-enhanced Raman spectroscopy substrates includes the following steps:

[0084] A1. Dissolve the template agent in a mixed solution of hydrochloric acid and ethanol, stir until completely dissolved, add tetrabutyl titanate, and continue stirring for 3 hours to form a sol; uniformly coat the above sol onto the surface of the pretreated titanium sheet, dry at 80°C for 24 hours, and then calcine at 600°C for 6 hours in a muffle furnace to remove the template agent and obtain a mesoporous titanium dioxide nanotube array.

[0085] A2. Add silver nitrate and sodium citrate to deionized water, heat to boiling and stir to obtain silver nano-seed solution; then, immerse the mesoporous titanium dioxide nanotube array in the silver nano-seed solution and let it stand at room temperature for 2 hours; next, dissolve silver nitrate and chloroauric acid in deionized water, store in the dark, add ascorbic acid and stir evenly, ascorbic acid should be prepared fresh for use, prepare a growth solution containing silver nitrate, chloroauric acid and ascorbic acid, immerse the substrate with adsorbed silver nano-seeds in the growth solution, react at room temperature for 60 minutes, and finally rinse with deionized water and dry to obtain silver-gold composite nanoparticle modified mesoporous titanium dioxide nanotube array substrate;

[0086] A3. Immerse the substrate in a Tris buffer solution containing 5 mg / mL polydopamine at pH 8.5 and react with shaking at room temperature for 12 h. Rinse the substrate with deionized water to remove unreacted polydopamine. Immerse the treated substrate in a solution of N,N-dimethylformamide containing 10 mM alkynyl-NHS ester and react at room temperature for 8 h. Then rinse the substrate sequentially with N,N-dimethylformamide and deionized water to remove residual reagents.

[0087] A4. Place the above substrate under ultraviolet light irradiation and treat it in a reaction tank containing 0.05 mol / L sodium hydroxide solution for 20 min;

[0088] A5. The azide-modified fleroxacin aptamer was dissolved in a phosphate buffer solution containing 0.5 mol / L sodium chloride at pH 7.6 to prepare an aptamer solution with a concentration of 30 μM. The substrate that had undergone photocatalytic oxidation was immersed in the aptamer solution and allowed to stand at room temperature for 60 min. The substrate was then rinsed with phosphate buffer solution to remove unbound aptamers, rinsed with deionized water, and dried to obtain a surface-enhanced Raman spectroscopy substrate.

[0089] The preparation method of the azide-modified fleroxacin aptamer includes the following steps:

[0090] B1. Using exponential enrichment ligand system evolution technology, with fleroxacin as the target molecule, aptamer sequences with high affinity and specificity for fleroxacin were screened in vitro from a random single-stranded oligonucleotide library. Bioinformatics analysis was performed on the screened aptamer sequences, and aptamer sequences with strong affinity, high specificity and structural stability were selected as target sequences. At the same time, appropriate modification sites were reserved at the 5' end of the aptamer sequences according to subsequent modification requirements.

[0091] B2. The solid-phase phosphoramidite trimer method is adopted, using phosphoramidite monomers containing azido groups to replace conventional monomers. Azido groups are introduced into the aptamer at the preset modification sites. In specific operation, the synthesis instrument adds the corresponding phosphoramidite monomers in sequence according to the designed sequence. Through deprotection, coupling and oxidation chemical reactions, the oligonucleotide chain is gradually extended to complete the synthesis of azido-modified aptamers.

[0092] B3. The synthesized azide-modified aptamer is purified to remove unreacted monomers, byproducts, and impurities. Commonly used purification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and polyacrylamide gel electrophoresis (PAGE). RP-HPLC separates the aptamer and impurities based on the difference in partition coefficients between the stationary and mobile phases. PAGE, on the other hand, separates the aptamer based on differences in molecular size and charge. The purified azide-modified fleroxacin aptamer is recovered by cutting the gel bands.

[0093] Comparative Example 1: Step A1: The template agent was replaced with CTAB, the hydrochloric acid / ethanol solution was replaced with sodium hydroxide / ethanol solution, n-butanol was added as a co-solvent, the sol stirring time was 4 hours, the calcination temperature was 550℃, and the rest of the process was the same as in Example 1.

[0094] Comparative Example 2: Step A3 was removed, and the remaining processes were the same as in Example 1.

[0095] Comparative Example 3: Step S2 was modified to rotary evaporation concentration (40°C, 1 / 10 volume) instead of freeze-drying concentration, and the rest of the process was the same as in Example 1.

[0096] Experimental example:

[0097] 1. The recovery rates of fleroxacin in Examples 1-3 and Comparative Examples 1-3 were tested, and the results are shown in Table 1;

[0098] Table 1

[0099] Group Fluroxacin recovery rate (%) Example 1 95 Example 2 95 Example 3 96 Comparative Example 1 85 Comparative Example 2 72 Comparative Example 3 82

[0100] 2. The detection limits (mol / L) of the processes in Examples 1-3 and Comparative Examples 1-3 were tested, and the results are shown in Table 2;

[0101] Table 2

[0102] Group Detection limit (mol / L) Example 1 <![CDATA[5×10 -8 ]]> Example 2 <![CDATA[5×10 -8 <!-- 7 -->]]> Example 3 <![CDATA[5×10 -8 ]]> Comparative Example 1 <![CDATA[2×10 -7 ]]> Comparative Example 2 <![CDATA[1×10 -7 ]]> Comparative Example 3 <![CDATA[1×10 -6 ]]>

[0103] 3. The specific binding efficiency of fleroxacin was tested for the processes of Examples 1-3 and Comparative Examples 1-3, and the results are shown in Table 3;

[0104] Table 3

[0105] Group Specific binding efficiency (%) Example 1 95 Example 2 95 Example 3 95 Comparative Example 1 80 Comparative Example 2 60 Comparative Example 3 95

[0106] As shown in the table above, the process in this embodiment has a higher recovery rate of fleroxacin, a lower detection concentration of fleroxacin, and a higher specific binding efficiency with fleroxacin.

[0107] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapid detection of fluroxacin in water, characterized in that, Includes the following steps: S1. Preparation of surface-enhanced Raman spectroscopy substrate: A surface-enhanced Raman spectroscopy substrate was prepared by chemical deposition. S2. Sample pretreatment: Take a certain volume of water sample and filter it through a 0.22 μm filter membrane to remove suspended solids from the water sample; then concentrate the filtered water sample to 1 / 10 to 1 / 5 of the original volume using a rotary evaporator at 40~50℃ to obtain a concentrated water sample. S3. Enrichment and Detection: 20-30 μL of concentrated water sample was dropped onto a surface-enhanced Raman spectroscopy (SERS) substrate and allowed to air dry at room temperature to enrich fleroxacin on the substrate surface. The substrate then was placed in a Raman spectrometer, and Raman spectroscopy was performed under the conditions of an excitation wavelength of 785 nm, a laser power of 10-50 mW, and an integration time of 1-10 s to obtain the Raman spectrum of the water sample. By comparing the spectrum with a standard Raman spectrum, the presence of fleroxacin in the water sample was determined, and a standard curve was established based on the characteristic peak intensities to achieve quantitative analysis of fleroxacin content. The fabrication process of surface-enhanced Raman spectroscopy substrates includes the following steps: A1. Dissolve the template agent in a mixed solution of hydrochloric acid and ethanol, stir until completely dissolved, add tetrabutyl titanate, and continue stirring for 2-3 h to form a sol; uniformly coat the above sol onto the surface of the pretreated titanium sheet, dry at 60-80℃ for 12-24 h, and then calcine in a muffle furnace at 500-600℃ for 4-6 h to remove the template agent, thereby obtaining a mesoporous titanium dioxide nanotube array; A2. Add silver nitrate and sodium citrate to deionized water, heat to boiling and stir to obtain silver nano-seed solution; then, immerse the mesoporous titanium dioxide nanotube array in the silver nano-seed solution and let it stand at room temperature for 1-2 h; next, dissolve silver nitrate and chloroauric acid in deionized water, store in the dark, add ascorbic acid and stir evenly, ascorbic acid should be prepared fresh for use, prepare a growth solution containing silver nitrate, chloroauric acid and ascorbic acid, immerse the substrate with adsorbed silver nano-seeds in the growth solution, react at room temperature for 30-60 min, finally rinse with deionized water and dry to obtain silver-gold composite nanoparticle modified mesoporous titanium dioxide nanotube array substrate; A3. Immerse the substrate in a Tris buffer solution containing 1-5 mg / mL polydopamine at pH 8.5 and react with shaking at room temperature for 6-12 h. Rinse the substrate with deionized water to remove unreacted polydopamine. Immerse the treated substrate in a solution of N,N-dimethylformamide containing 5-10 mM alkynyl-NHS ester and react at room temperature for 4-8 h. Then rinse the substrate sequentially with N,N-dimethylformamide and deionized water to remove residual reagents. A4. Place the above substrate under ultraviolet light irradiation and treat it in a reaction tank containing 0.01~0.05 mol / L sodium hydroxide solution for 10~20 min; A5. Dissolve the azide-modified fleroxacin aptamer in a phosphate buffer solution containing 0.1–0.5 mol / L sodium chloride (pH 7.2–7.6) to prepare an aptamer solution with a concentration of 10–30 μM. Immerse the photocatalytically oxidized substrate in the aptamer solution and let it stand at room temperature for 30–60 min. Rinse the substrate with phosphate buffer solution to remove unbound aptamers, rinse with deionized water, and air dry to obtain a surface-enhanced Raman spectroscopy substrate.

2. The method for rapid detection of fluroxacin in water according to claim 1, characterized in that, The template agent is one of triblock copolymer P123 or triblock copolymer F127.

3. The method for rapid detection of fluroxacin in water according to claim 1, characterized in that, Step A1 includes 0.5 to 1.5 parts template agent, 1 to 3 parts hydrochloric acid, 20 to 50 parts ethanol, and 2 to 5 parts tetrabutyl titanate.

4. The method for rapid detection of fluroxacin in water according to claim 1, characterized in that, The silver nanoseed solution in step A2 includes: 0.01~0.05 parts silver nitrate, 0.05~0.2 parts sodium citrate, and 100~200 parts water; the growth solution includes: 0.005~0.02 parts silver nitrate, 0.005~0.02 parts chloroauric acid, 100~200 parts deionized water, and 0.01~0.05 parts ascorbic acid.

5. The method for rapid detection of fluroxacin in water according to claim 1, characterized in that, In step A4, the ultraviolet light processing parameters are a wavelength of 254 nm and a light intensity of 5~10 mW / cm2.

6. The method for rapid detection of fluroxacin in water according to claim 1, characterized in that, The preparation method of the azide-modified fleroxacin aptamer includes the following steps: B1. Using exponential enrichment ligand system evolution technology, with fleroxacin as the target molecule, aptamer sequences with high affinity and specificity for fleroxacin are screened in vitro from a random single-stranded oligonucleotide library. Bioinformatics analysis is performed on the screened aptamer sequences, and aptamer sequences with strong affinity, high specificity and structural stability are selected as target sequences. At the same time, appropriate modification sites are reserved at the 5' or 3' end of the aptamer sequence according to the subsequent modification requirements. B2. The solid-phase phosphoramidite trimer method is adopted, using phosphoramidite monomers containing azido groups to replace conventional monomers. Azido groups are introduced into the aptamer at the preset modification sites. In specific operation, the synthesis instrument adds the corresponding phosphoramidite monomers in sequence according to the designed sequence. Through deprotection, coupling and oxidation chemical reactions, the oligonucleotide chain is gradually extended to complete the synthesis of azido-modified aptamers. B3. The synthesized azide-modified aptamer is purified to remove unreacted monomers, byproducts, and impurities. Commonly used purification methods include reversed-phase high-performance liquid chromatography (RP-HPLC) and polyacrylamide gel electrophoresis (PAGE). RP-HPLC separates the aptamer and impurities based on the difference in partition coefficients between the stationary and mobile phases. PAGE, on the other hand, separates the aptamer based on differences in molecular size and charge. The purified azide-modified fleroxacin aptamer is recovered by cutting the gel bands.

7. The method for rapid detection of fluroxacin in water according to claim 6, characterized in that, The phosphoramid monomer containing an azide group is either a 5'-azido-modified phosphoramid monomer or a 3'-azido-modified phosphoramid monomer.