Preparation and application method of smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin

Abstract

The invention belongs to the technical field of colorimetric chemical sensors, and particularly relates to a preparation and application method of a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin, magnetic Fe3O4 (at) KH540 nanoparticles are taken as a matrix, magnetic norfloxacin surface molecular imprinting nano-enzyme MMIPs is prepared by adopting a surface molecular imprinting technology, then the MMIPs, H2O2 and 3, 3 ', 5, 6, 7-tetramethyl-3, 5, 6-tetramethyl-3, 5, 6-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3-tetramethyl-3 The colorimetric sensor for detecting norfloxacin is constructed by carrying out a chromogenic reaction on 3, 3 ', 5, 5'-tetramethyl benzidine TMB and cooperating with a smartphone. The sensor has specific recognition selectivity and binding affinity for NOR, is simple to operate, high in detection speed and low in cost, does not need expensive instruments, and can be suitable for field analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of colorimetric chemical sensors, and in particular relates to a preparation and application method of a smart phone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin. Background Art

[0002] Norfloxacin is one of the broad-spectrum quinolone antibiotics and is widely used to treat urinary tract, respiratory tract and gastrointestinal tract infections in humans and food animals. However, excessive use of norfloxacin may leave its residues in livestock products. These residues can cause drowsiness, anorexia, dizziness and nausea in people who consume these products. Long-term use of residual norfloxacin can cause central nervous system diseases and induce antibiotic resistance (O. Bunkoed, P. Donkhampa, P.Nurerk. Microchemical Journal, 2020, 158: 105127). Therefore, it is necessary to develop a reliable method to detect norfloxacin.

[0003] Current methods for detecting NOR include chromatography, biosensors, and enzyme-linked immunosorbent assays, which are usually expensive, time-consuming, and require complex sample pretreatment. Colorimetric sensors have the advantages of simple operation, fast detection speed, low cost, no need for expensive instruments, and suitability for on-site analysis. Molecularly imprinted polymers exhibit strong specific recognition of template molecules, and have the advantages of easy preparation, low cost, high chemical and mechanical stability. Surface molecularly imprinted polymers are particularly favored by researchers due to their easy preparation, large surface area, multiple recognition sites, and high stability, and are widely used to improve the selectivity of sensors. Therefore, surface molecularly imprinted polymers as recognition units of colorimetric sensors provide colorimetric sensors with advantages such as high sensitivity and specific recognition (C. Dong, H. Shi, Y. Han, Y. Yang, R. Wang, J. Men. Journal of Membrane Science, 2021, 145: 110231). Compared with natural enzymes, nanozymes have many obvious advantages, including low cost, high yield, adaptable enzyme-like activity and excellent stability (Z. Zhang, X. Zhang, B. Liu. Journal of the American Chemical Society, 2017, 139: 5412–5419). Fe3O4 magnetic nanoparticles have the dual catalytic properties of peroxidase and catalase, as well as good stability and superparamagnetism. The present invention uses Fe3O4 nanoparticles modified with silane coupling agent 3-aminopropyltrimethoxysilane KH540 as a matrix, and uses a method of simultaneous graft polymerization and cross-linking imprinting on its surface to prepare a magnetic norfloxacin surface molecular imprinting nanozyme to construct a colorimetric sensor that can specifically recognize and detect norfloxacin. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a preparation and application method of a norfloxacin colorimetric sensor with simple operation and fast detection speed.

[0005] The technical solution adopted by the present invention is: a method for preparing a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin, which is carried out according to the following steps: Step 1: Prepare modified Fe3O4@KH540 nanoparticles, add silane coupling agent 3-aminopropyltrimethoxysilane KH540 dropwise into Fe3O4 dispersion, stir in alkaline environment for 24 h, wash and dry to obtain Fe3O4@KH540 nanoparticles; Step 2: prepare a prepolymer of a functional monomer and a template molecule, dissolve the template molecule norfloxacin NOR and the functional monomer methacrylic acid MAA in N,N'-dimethylformamide DMF, and obtain a prepolymer of the functional monomer and the template molecule after 8 to 12 hours; Step 3, preparing the magnetic norfloxacin surface molecularly imprinted nanozyme MMIPs, adding the Fe3O4@KH540 nanoparticles prepared in step 1 to N,N'-dimethylformamide DMF to fully swell, adding the prepolymer of the functional monomer and template molecule prepared in step 2, and then adding the cross-linking agent, passing nitrogen to remove the air, adding the initiator, and continuously stirring the reaction under nitrogen protection to obtain the magnetic norfloxacin surface molecularly imprinted nanozyme MMIPs; Step 4: Elution of the template molecule: repeatedly elute the magnetic norfloxacin surface molecular imprinting nanozyme MMIPs with an elution solution until no NOR is detected in the supernatant, thereby obtaining the eluted magnetic norfloxacin surface molecular imprinting nanozyme MMIPs, which is a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin.

[0006] In step 1, the Fe3O4 dispersion is an Fe3O4 ethanol-water dispersion, wherein the mass percentage concentration of Fe3O4 is 1.5-3%, the mass percentage concentration of ethanol is 80-90%, and when the silane coupling agent 3-aminopropyltrimethoxysilane KH540 is added dropwise to the Fe3O4 dispersion, every 2 mL of KH540 corresponds to 1 g of Fe3O4; during washing, ethanol and purified water are used alternately for multiple washings.

[0007] In step 2, every 0.5 g of the template molecule norfloxacin NOR corresponds to 0.6 mL~1.0 mL of methacrylic acid and 35 mL of N,N'-dimethylformamide DMF.

[0008] In step three, every 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 10 mL of N,N'-dimethylformamide DMF, 0.5 g of the template molecule norfloxacin NOR prepared as a prepolymer of functional monomer and template molecule, the cross-linking agent is N,N'-methylenebisacrylamide MBA, every 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 1.70 g~2.10 g of N,N'-methylenebisacrylamide MBA, the initiator is ammonium persulfate solution, every 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 0.014~0.018 g of ammonium persulfate. When preparing the ammonium persulfate solution, every 0.014~0.018 g of ammonium persulfate is dissolved in 5 mL of water to form.

[0009] In step 4, the eluent is a mixed solution of methanol / acetic acid, and the volume ratio of methanol to acetic acid is 9:1.

[0010] An application of a smartphone-assisted surface molecular imprinting colorimetric sensor is characterized in that: a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin is added into water, ultrasonically dispersed to obtain a MMIPs dispersion, a norfloxacin NOR solution is added into the MMIPs dispersion, adsorbed in a constant temperature incubation oscillator, then taken out, a buffer solution, a hydrogen peroxide H2O2 solution and a 3,3',5,5'-tetramethylbenzidine TMB solution are added in sequence, mixed evenly, reacted in a 25°C constant temperature box, MMIPs are adsorbed by a magnet, the supernatant is taken out and placed in a 96-well plate of an ELISA instrument, a smartphone is used to take a photo to obtain RGB values, and a grayscale value is obtained.

[0011] Each 0.02 g of magnetic norfloxacin surface molecularly imprinted nanozyme MMIPs was dispersed in 5 mL of purified water and ultrasonically dispersed for 20 min to obtain MMIPs dispersion. Then, 100 uL of the NOR solution to be tested was added to 300 µL of MMIPs dispersion, adsorbed in a constant temperature incubator for 20 min, then taken out, and 300 uL of HAc-NaAc buffer (0.2 mol / L, pH=3.5), 250 uL of 0.06 mol / L H2O2 solution and 50 uL of 0.008 mol / L TMB solution were added in sequence, mixed evenly, reacted in a constant temperature incubator at 25 °C for 4 min, MMIPs were adsorbed by a magnet, the supernatant was taken out and placed in a 96-well plate of a microplate reader, and the APP Color Grab in the smartphone was used to take pictures to obtain RGB values ​​and grayscale values. The concentration of HAc-NaAc buffer was 0.2 mol / L, and pH=3.5.

[0012] The solvents of the NOR solution and H2O2 solution to be tested are both water, and the solvent of the 3,3',5,5'-tetramethylbenzidine TMB solution is dimethyl sulfoxide DMSO.

[0013] The beneficial effects of the present invention are: (1) The present invention uses Fe3O4 magnetic nanoparticles as bionic nanozymes to catalyze H2O2 oxidation of chromogenic substrates, which have both the catalytic activity of peroxidase and the rapid separation ability of magnetic separation. The surface molecular imprinting polymer MMIPs constructed with Fe3O4 as the matrix has the catalytic activity of nanozymes and good magnetic separation ability; (2) The smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin has the specific recognition selectivity of molecular imprinting polymers and can achieve specific detection of norfloxacin; (3) While constructing a colorimetric chemical sensor to detect norfloxacin, a smartphone is used to process the detection results, so that the sensor has high sensitivity and specific adsorption capacity, and has the advantages of simple, rapid and on-site detection; (4) Through gray value analysis, the colorimetric sensor has a wide linear range (0.16 μmol / L ~ 2.50 μmol / L) for norfloxacin, and its detection limit is 0.137 μmol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the linear relationship between the gray value obtained by the sensor test and the concentration of NOR; Figure 2 It is the color change of the solution when the sensor detects different concentrations of norfloxacin. DETAILED DESCRIPTION

[0015] Example 1 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to a dispersion of 1 g of Fe3O4 nanoparticles dispersed in 45 mL of ethanol and 5 mL of purified water, stirred for 24 h, and washed and dried to obtain Fe3O4@KH540 nanoparticles. 0.5 g template molecule norfloxacin NOR and 0.6 g functional monomer methacrylic acid MAA were dissolved in 35 mL N,N'-dimethylformamide DMF, and a prepolymer of the functional monomer and the template molecule was obtained after 8 h; 0.4 g of the prepared Fe3O4@KH540 nanoparticles was added to 10 mL N,N'-dimethylformamide DMF to fully swell, and then the prepolymer of the functional monomer and the template molecule prepared in step 2 was added, and then 1.70 g N,N'-methylenebisacrylamide MBA cross-linking agent was added, and nitrogen was passed for 30 min to remove the air, and 0.014 g initiator ammonium persulfate was added. Under nitrogen protection at 30 °C, stirring was continued for 6 h to obtain magnetic norfloxacin surface molecularly imprinted nanozymes MMIPs. After the reaction, the magnetic norfloxacin surface molecular imprinting nanozyme MIPs was taken out, and the template molecules were repeatedly eluted with a mixed eluent of methanol / acetic acid (9:1, v / v) until NOR was no longer detected in the supernatant, thereby obtaining the eluted surface molecular imprinting nanozyme MMIPs.

[0016] Example 2 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to the dispersion of Fe3O4 nanoparticles in 45 mL of ethanol and 5 mL of purified water, stirred for 24 h, and washed and dried to obtain Fe3O4@KH540 nanoparticles. 0.5 g of the template molecule norfloxacin NOR and 1.0 g of the functional monomer methacrylic acid MAA were dissolved in 35 mL N,N'-dimethylformamide DMF, and a prepolymer of the functional monomer and the template molecule was obtained after 12 h; 0.4 g of the prepared Fe3O4@KH540 nanoparticles was added to 10 mL N,N'-dimethylformamide DMF to fully swell, and then the prepolymer of the functional monomer and the template molecule prepared in step 2 was added, and then 2.10 g of N,N'-methylenebisacrylamide MBA cross-linking agent was added, and nitrogen was passed for 30 min to remove the air, and 0.018 g of initiator ammonium persulfate was added. Under nitrogen protection at 50 °C, stirring was continued for 10 h to obtain magnetic norfloxacin surface molecularly imprinted nanozymes MMIPs. After the reaction, the magnetic norfloxacin surface molecular imprinting nanozyme MMIPs was taken out, and the template molecules were repeatedly eluted with a mixed eluent of methanol / acetic acid (9:1, v / v) until NOR was no longer detected in the supernatant, thereby obtaining the eluted surface molecular imprinting nanozyme MMIPs.

[0017] Example 3 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to the dispersion of Fe3O4 nanoparticles in 45 mL of ethanol and 5 mL of purified water, stirred for 24 h, and washed and dried to obtain Fe3O4@KH540 nanoparticles. 0.5 g of template molecule norfloxacin NOR and 0.8 g of functional monomer methacrylic acid MAA were dissolved in 35 mL N,N'-dimethylformamide DMF, and a prepolymer of the functional monomer and the template molecule was obtained after 12 h; 0.4 g of the prepared Fe3O4@KH540 nanoparticles was added to 10 mL N,N'-dimethylformamide DMF to fully swell, and then the prepolymer of the functional monomer and the template molecule prepared in step 2 was added, and then 1.93 g of N,N'-methylenebisacrylamide MBA cross-linking agent was added, and nitrogen was passed for 30 min to remove the air, and 0.016 g of initiator ammonium persulfate was added. Under nitrogen protection at 40 °C, stirring was continued for 10 h to obtain magnetic norfloxacin surface molecularly imprinted nanozymes MMIPs. After the reaction, the magnetic norfloxacin surface molecular imprinting nanozyme MIPs was taken out, and the template molecules were repeatedly eluted with a mixed eluent of methanol / acetic acid (9:1, v / v) until NOR was no longer detected in the supernatant, thereby obtaining the eluted surface molecular imprinting nanozyme MMIPs.

[0018] Example 4 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to the dispersion of Fe3O4 nanoparticles in 45 mL of ethanol and 5 mL of purified water, stirred for 24 h, and washed and dried to obtain Fe3O4@KH540 nanoparticles. 0.5 g of template molecule norfloxacin NOR and 0.8 g of functional monomer methacrylic acid MAA were dissolved in 35 mL N,N'-dimethylformamide DMF, and a prepolymer of the functional monomer and the template molecule was obtained after 10 h. 0.4 g of the prepared Fe3O4@KH540 nanoparticles was added to 10 mL N,N'-dimethylformamide DMF to fully swell, and then the prepolymer of the functional monomer and the template molecule prepared in step 2 was added, and then 1.93 g of N,N'-methylenebisacrylamide MBA cross-linking agent was added, and nitrogen was passed for 30 min to remove the air. 0.016 g of initiator ammonium persulfate was added, and the mixture was stirred continuously for 8 h at 40 °C under nitrogen protection to obtain magnetic norfloxacin surface molecularly imprinted nanozymes MMIPs. After the reaction, the magnetic norfloxacin surface molecular imprinting nanozyme MIPs was taken out, and the template molecules were repeatedly eluted with a mixed eluent of methanol / acetic acid (9:1, v / v) until NOR was no longer detected in the supernatant, thereby obtaining the eluted surface molecular imprinting nanozyme MMIPs.

[0019] Example 5 An application method of a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin.

[0020] The method includes adding 0.02 g of MMIPs prepared in Example 4 to 5 mL of aqueous solution, ultrasonicating for 20 min to obtain MMIPs dispersion, adding 100 μL of the NOR solution to be tested to 300 μL of MMIPs dispersion, adsorbing in a constant temperature incubator for 20 min, then taking out, adding 300 μL of HAc-NaAc (0.2 mol / L, pH=3.5) buffer, 250 μL of 0.06 mol / L H2O2 solution and 50 uL of 0.008 mol / L 3,3',5,5'-tetramethylbenzidine TMB solution in sequence, mixing evenly, reacting in a constant temperature incubator at 25°C, adsorbing MMIPs with a magnet, taking out the supernatant and placing it in a 96-well plate of an ELISA reader, acquiring RGB values ​​with a smart phone, and obtaining grayscale values. Figure 1 As shown in the figure, the linear relationship between the gray value obtained by the sensor test and the concentration of NOR is as follows: the linear equation is y=18.986x+57.49545, and the linear correlation coefficient is R 2 =0.99141, and the calculated detection limit is 0.137μM. Figure 2 The inset shows the color change of the solution when the sensor detects different concentrations of norfloxacin.

Claims

1. A method for preparing a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin, characterized in that: Follow the steps below: Step 1: Prepare modified Fe3O4@KH540 nanoparticles, add silane coupling agent 3-aminopropyltrimethoxysilane KH540 dropwise into Fe3O4 dispersion, stir in alkaline environment for 24 h, wash and dry to obtain Fe3O4@KH540 nanoparticles; Step 2: prepare a prepolymer of a functional monomer and a template molecule, dissolve the template molecule norfloxacin NOR and the functional monomer methacrylic acid MAA in N,N'-dimethylformamide DMF, and obtain a prepolymer of the functional monomer and the template molecule after 8 to 12 hours; Step 3, preparing the magnetic norfloxacin surface molecularly imprinted nanozyme MMIPs, adding the Fe3O4@KH540 nanoparticles prepared in step 1 to N,N'-dimethylformamide DMF to fully swell, adding the prepolymer of the functional monomer and template molecule prepared in step 2, and then adding the cross-linking agent, passing nitrogen to remove the air, adding the initiator, and continuously stirring the reaction under nitrogen protection to obtain the magnetic norfloxacin surface molecularly imprinted nanozyme MMIPs; Step 4: Elution of the template molecule: repeatedly elute the magnetic norfloxacin surface molecular imprinting nanozyme MMIPs with an elution solution until no NOR is detected in the supernatant, thereby obtaining the eluted magnetic norfloxacin surface molecular imprinting nanozyme MMIPs, which is a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin.

2. The method for preparing a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin according to claim 1, characterized in that: In step 1, the Fe3O4 dispersion is an Fe3O4 ethanol-water dispersion, wherein the mass percentage concentration of Fe3O4 is 1.5-3%, the mass percentage concentration of ethanol is 80-90%, and when the silane coupling agent 3-aminopropyltrimethoxysilane KH540 is added dropwise to the Fe3O4 dispersion, every 2 mL of KH540 corresponds to 1 g of Fe3O4; during washing, ethanol and purified water are used alternately for multiple washings.

3. The method for preparing a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin according to claim 1, characterized in that: In step 2, every 0.5 g of the template molecule norfloxacin NOR corresponds to 0.6 mL~1.0 mL of methacrylic acid and 35 mL of N,N'-dimethylformamide DMF.

4. The method for preparing a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin according to claim 1, characterized in that: In step three, every 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 10 mL of N,N'-dimethylformamide DMF, 0.5 g of the template molecule norfloxacin NOR prepared as a prepolymer of functional monomer and template molecule, the cross-linking agent is N,N'-methylenebisacrylamide MBA, every 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 1.70 g~2.10 g of N,N'-methylenebisacrylamide MBA, the initiator is ammonium persulfate solution, every 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 0.014~0.018 g of ammonium persulfate. When preparing the ammonium persulfate solution, every 0.014~0.018 g of ammonium persulfate is dissolved in 5 mL of water to form.

5. The method for preparing a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin according to claim 1, characterized in that: In step 4, the eluent is a mixed solution of methanol / acetic acid, and the volume ratio of methanol to acetic acid is 9:

1.

6. An application of a smartphone-assisted surface molecular imprinting colorimetric sensor prepared by the method for preparing a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin in claim 1, characterized in that: The smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin was added to water and ultrasonically dispersed to obtain a MMIPs dispersion. The MMIPs dispersion was then taken out and a buffer solution, a hydrogen peroxide H2O2 solution and a 3,3',5,5'-tetramethylbenzidine TMB solution were added in sequence. The mixture was mixed evenly and reacted in a constant temperature incubator at 25 ℃. The MMIPs were adsorbed by a magnet. The supernatant was taken out and placed in a 96-well plate of a microplate reader. The RGB value was obtained by taking a photo with a smartphone, and the grayscale value was obtained.

7. The method for using a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin according to claim 6, characterized in that: Each 0.02 g of magnetic norfloxacin surface molecularly imprinted nanozyme MMIPs was dispersed in 5 mL of purified water and ultrasonically dispersed for 20 min to obtain MMIPs dispersion. Then, 100 uL of the NOR solution to be tested was added to 300 µL of MMIPs dispersion, adsorbed in a constant temperature incubator for 20 min, then taken out, and 300 uL of HAc-NaAc buffer (0.2 mol / L, pH=3.5), 250 uL of 0.06 mol / L H2O2 solution and 50 uL of 0.008 mol / L TMB solution were added in sequence, mixed evenly, reacted in a constant temperature incubator at 25 °C for 4 min, MMIPs were adsorbed by a magnet, the supernatant was taken out and placed in a 96-well plate of a microplate reader, and the APP Color Grab in the smartphone was used to take pictures to obtain RGB values ​​and grayscale values. The concentration of HAc-NaAc buffer was 0.2 mol / L, and pH=3.

5.

8. The method for using a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin according to claim 6, characterized in that: The solvents of the NOR solution and H2O2 solution to be tested are both water, and the solvent of the 3,3',5,5'-tetramethylbenzidine TMB solution is dimethyl sulfoxide DMSO.

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