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

Abstract

This invention belongs to the technical field of colorimetric chemical sensors, specifically relating to the preparation and application method of a smartphone-assisted surface molecularly imprinted colorimetric sensor for detecting norfloxacin. Using magnetic Fe3O4@KH540 nanoparticles as a matrix, magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) are prepared using surface molecular imprinting technology. Then, the MMIPs undergo a colorimetric reaction with H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB), and a colorimetric sensor for detecting norfloxacin is constructed in conjunction with a smartphone. This sensor exhibits specific recognition selectivity and binding affinity for NOR, is simple to operate, has a fast detection speed, low cost, requires no expensive instruments, and is suitable for on-site analysis.

Description

Technical Field

[0001] This invention belongs to the technical field of colorimetric chemical sensors, specifically relating to the preparation and application method of a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin. Background Technology

[0002] Norfloxacin is a broad-spectrum quinolone antibiotic widely used to treat urinary tract, respiratory, and gastrointestinal infections in humans and animals intended for consumption. However, overuse of norfloxacin can leave residues in animal products. These residues can cause drowsiness, loss of appetite, dizziness, and nausea in people who consume these products. Long-term use of residual norfloxacin can cause central nervous system disorders and induce antibiotic resistance (O. Bunkoed, P. Donkhampa, P. Nurerk. Microchemical Journal, 2020, 158:105127). Therefore, it is necessary to develop a reliable method for detecting norfloxacin.

[0003] Current methods for detecting NOR (non-reactive oxygen species) include chromatography, biosensors, and enzyme-linked immunosorbent assays (ELISA), which are typically expensive, time-consuming, and require complex sample pretreatment. Colorimetric sensors, on the other hand, offer advantages such as 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 possess advantages such as ease of preparation, low cost, and high chemical and mechanical stability. Surface-imprinted polymers are particularly favored by researchers due to their ease of preparation, large surface area, numerous recognition sites, and high stability, and are widely used to improve sensor selectivity. Therefore, surface-imprinted polymers, as recognition units in colorimetric sensors, provide 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 significant advantages, including low cost, high yield, highly 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 possess dual catalytic properties of peroxidase and catalase, as well as good stability and superparamagnetism. This invention uses Fe3O4 nanoparticles modified with the silane coupling agent 3-aminopropyltrimethoxysilane KH540 as a matrix, and prepares magnetic norfloxacin surface-imprinted nanozymes on their surface using a method of simultaneous graft polymerization and cross-linking imprinting to construct a colorimetric sensor capable of specifically recognizing and detecting norfloxacin. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing and applying a norfloxacin colorimetric sensor that is simple to operate and has a fast detection speed.

[0005] The technical solution adopted in this invention is: a method for preparing a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin, which is carried out according to the following steps:

[0006] Step 1: Preparation of modified Fe3O4@KH540 nanoparticles. The silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to the Fe3O4 dispersion and stirred in an alkaline environment for 24 h. After washing and drying, Fe3O4@KH540 nanoparticles were obtained.

[0007] Step 2: Preparation of prepolymer of functional monomer and template molecule. The template molecule norfloxacin (NOR) and the functional monomer methacrylic acid (MAA) are dissolved together in N,N'-dimethylformamide (DMF). After 8 h to 12 h, the prepolymer of functional monomer and template molecule is obtained.

[0008] Step 3: Preparation of magnetic norfloxacin surface molecularly imprinted nanoenzymes (MMIPs). The Fe3O4@KH540 nanoparticles prepared in Step 1 were added to N,N'-dimethylformamide (DMF) and fully swollen. Then, the prepolymer of the functional monomer and template molecule prepared in Step 2 was added, followed by the addition of a crosslinking agent. Nitrogen gas was purged to remove air, and an initiator was added. The reaction was carried out under nitrogen protection with continuous stirring to obtain magnetic norfloxacin surface molecularly imprinted nanoenzymes (MMIPs).

[0009] Step 4: Template molecule elution. The magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) are repeatedly eluted with elution buffer until no NOR is detected in the supernatant. The eluted magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) are the smartphone-assisted surface molecularly imprinted colorimetric sensors for detecting norfloxacin.

[0010] In step one, the Fe3O4 dispersion is an ethanol-water dispersion of Fe3O4, wherein the mass percentage concentration of Fe3O4 is 1.5-3% and the mass percentage concentration of ethanol is 80-90%. When the silane coupling agent 3-aminopropyltrimethoxysilane KH540 is added dropwise to the Fe3O4 dispersion, each 2 mL of KH540 corresponds to 1 g of Fe3O4. During washing, ethanol and purified water are used alternately for multiple washes.

[0011] In step two, each 0.5 g template molecule norfloxacin (NOR) corresponds to 0.6 mL to 1.0 mL of methacrylic acid and 35 mL of N,N'-dimethylformamide (DMF).

[0012] In step three, each 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 10 mL of N,N'-dimethylformamide (DMF) and 0.5 g of the template molecule norfloxacin (NOR) to prepare a prepolymer of the functional monomer and the template molecule. The crosslinking agent is N,N'-methylenebisacrylamide (MBA), and each 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 1.70 g to 2.10 g of N,N'-methylenebisacrylamide (MBA). The initiator is ammonium persulfate solution, and each 0.4 g of Fe3O4@kH540 nanoparticles corresponds to 0.014 to 0.018 g of ammonium persulfate. When preparing the ammonium persulfate solution, each 0.014 to 0.018 g of ammonium persulfate is dissolved in 5 mL of water to form the solution.

[0013] In step four, the eluent is a mixed solution of methanol and acetic acid, with a volume ratio of methanol to acetic acid of 9:1.

[0014] An application of a smartphone-assisted surface molecular imprinting colorimetric sensor is characterized by: adding a smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin to water, ultrasonically dispersing it to obtain a uniform MMIPs dispersion, adding norfloxacin NOR solution to the MMIPs dispersion, adsorbing it in a constant temperature incubator with a shaker, then removing it, sequentially adding buffer solution, hydrogen peroxide (H2O2) solution and 3,3',5,5'-tetramethylbenzidine (TMB) solution, mixing it evenly, reacting it in a 25 ℃ constant temperature incubator, using a magnet to adsorb the MMIPs, removing the supernatant and placing it in a 96-well plate of an ELISA reader, taking a picture with a smartphone to obtain RGB values, and obtaining grayscale values.

[0015] 0.02 g of magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) were dispersed in 5 mL of purified water and sonicated for 20 min to obtain an MMIPs dispersion. Then, 100 μL of the NOR solution to be tested was added to 300 µL of the MMIPs dispersion and incubated in a shaker for 20 min. After that, 300 μL of HAc-NaAc buffer (0.2 mol / L, pH=3.5), 250 μL of 0.06 mol / L H2O2 solution, and 50 μL of 0.008 mol / L TMB solution were added sequentially and mixed thoroughly. The mixture was then incubated at 25 °C for 4 min. The MMIPs were then magnetically adsorbed. The supernatant was then placed in a 96-well plate of a microplate reader, and the RGB values ​​were obtained by taking a picture with the Color Grab app on a smartphone. The grayscale value was also obtained. The concentration of the HAc-NaAc buffer was 0.2 mol / L and the pH was 3.5.

[0016] The solvents for the NOR and H2O2 solutions to be tested were both water, and the solvent for the 3,3',5,5'-tetramethylbenzidine TMB solution was dimethyl sulfoxide (DMSO).

[0017] The beneficial effects of this invention are:

[0018] (1) This invention uses Fe3O4 magnetic nanoparticles as biomimetic nanozymes to catalyze the oxidation of H2O2 into chromogenic substrates. It has both the catalytic activity of peroxidase and the rapid separation capability of magnetic separation. The surface molecularly imprinted polymers (MMIPs) constructed with Fe3O4 as the matrix have the catalytic activity of nanozymes and good magnetic separation capability. (2) The smartphone-assisted surface molecularly imprinted colorimetric sensor for detecting norfloxacin has the specific recognition selectivity of molecularly imprinted polymers, which can realize the specific detection of norfloxacin. (3) While constructing a colorimetric chemical sensor to detect norfloxacin, the detection results are processed by a smartphone, so that the sensor has the advantages of high sensitivity and specific adsorption capability, as well as simplicity, speed and on-site detection. (4) Through gray value analysis, it is found that 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. Attached Figure Description

[0019] Figure 1 It is a linear relationship between the grayscale value obtained from sensor testing and the concentration of NOR;

[0020] Figure 2 The sensor detects the color change of the solution when different concentrations of norfloxacin are used. Detailed Implementation

[0021] Example 1

[0022] 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to a dispersion of 1 g Fe3O4 nanoparticles in 45 mL of ethanol and 5 mL of purified water. The mixture was stirred for 24 h, and after washing and drying, Fe3O4@KH540 nanoparticles were obtained. 0.5 g of the template molecule norfloxacin (NOR) and 0.6 g of the functional monomer methacrylic acid (MAA) were dissolved together in 35 mL of N,N'-dimethylformamide (DMF). After 8 h, a prepolymer of the functional monomer and the template molecule was obtained. 0.4 g of the prepared Fe3O4@KH540 nanoparticles were added to 10 mL of N,N'-dimethylformamide (DMF) and allowed to swell completely. Then, the prepolymer of the functional monomer and template molecule prepared in step two was added, followed by 1.70 g of N,N'-methylenebisacrylamide (MBA) crosslinking agent. Nitrogen gas was purged for 30 min to remove air, and 0.014 g of the initiator ammonium persulfate was added. The mixture was stirred continuously for 6 h under nitrogen protection at 30 °C to obtain magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the magnetic norfloxacin surface molecularly imprinted nanozymes (MIPs) were removed and the template molecules were repeatedly eluted with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no NOR was detected in the supernatant, thus obtaining the eluted surface molecularly imprinted nanozymes (MMIPs).

[0023] Example 2

[0024] 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to a dispersion of Fe3O4 nanoparticles in 45 mL of ethanol and 5 mL of purified water. The mixture was stirred for 24 h, and then 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 together in 35 mL of N,N'-dimethylformamide (DMF). After 12 h, a prepolymer of the functional monomer and the template molecule was obtained. 0.4 g of the prepared Fe3O4@KH540 nanoparticles were added to 10 mL of N,N'-dimethylformamide (DMF) and allowed to swell completely. Then, the prepolymer of the functional monomer and template molecule prepared in step two was added, followed by 2.10 g of N,N'-methylenebisacrylamide (MBA) crosslinking agent. Nitrogen gas was purged for 30 min to remove air, and 0.018 g of the initiator ammonium persulfate was added. The mixture was stirred continuously for 10 h under nitrogen protection at 50 °C to obtain magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) were removed and the template molecules were repeatedly eluted with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no NOR was detected in the supernatant, thus obtaining the eluted surface molecularly imprinted nanozymes (MMIPs).

[0025] Example 3

[0026] 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to a dispersion of Fe3O4 nanoparticles in 45 mL of ethanol and 5 mL of purified water. The mixture was stirred for 24 h, and then washed and dried to obtain Fe3O4@KH540 nanoparticles. 0.5 g of the template molecule norfloxacin (NOR) and 0.8 g of the functional monomer methacrylic acid (MAA) were dissolved together in 35 mL of N,N'-dimethylformamide (DMF). After 12 h, a prepolymer of the functional monomer and the template molecule was obtained. 0.4 g of the prepared Fe3O4@KH540 nanoparticles were added to 10 mL of N,N'-dimethylformamide (DMF) and allowed to swell completely. Then, the prepolymer of the functional monomer and template molecule prepared in step two was added, followed by 1.93 g of N,N'-methylenebisacrylamide (MBA) crosslinking agent. Nitrogen gas was purged for 30 min to remove air, and 0.016 g of the initiator ammonium persulfate was added. The mixture was stirred continuously for 10 h under nitrogen protection at 40 °C to obtain magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the magnetic norfloxacin surface molecularly imprinted nanozymes (MIPs) were removed and the template molecules were repeatedly eluted with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no NOR was detected in the supernatant, thus obtaining the eluted surface molecularly imprinted nanozymes (MMIPs).

[0027] Example 4

[0028] 2 mL of silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to a dispersion of Fe3O4 nanoparticles in 45 mL of ethanol and 5 mL of purified water. The mixture was stirred for 24 h, and then washed and dried to obtain Fe3O4@KH540 nanoparticles. 0.5 g of the template molecule norfloxacin (NOR) and 0.8 g of the functional monomer methacrylic acid (MAA) were dissolved together in 35 mL of N,N'-dimethylformamide (DMF). After 10 h, a prepolymer of the functional monomer and the template molecule was obtained. 0.4 g of the prepared Fe3O4@KH540 nanoparticles were added to 10 mL of N,N'-dimethylformamide (DMF) and allowed to swell completely. Then, the prepolymer of the functional monomer and template molecule prepared in step two was added, followed by 1.93 g of N,N'-methylenebisacrylamide (MBA) crosslinking agent. Nitrogen gas was purged for 30 min to remove air, and 0.016 g of the initiator ammonium persulfate was added. The mixture was stirred continuously for 8 h under nitrogen protection at 40 °C to obtain magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs). After the reaction was completed, the magnetic norfloxacin surface molecularly imprinted nanozymes (MIPs) were removed and the template molecules were repeatedly eluted with a methanol / acetic acid (9:1, v / v) mixed elution buffer until no NOR was detected in the supernatant, thus obtaining the eluted surface molecularly imprinted nanozymes (MMIPs).

[0029] Example 5: An application method of a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin.

[0030] The process involved adding 0.02 g of MMIPs prepared in Example 4 to 5 mL of aqueous solution, sonicating for 20 min to obtain an MMIPs dispersion, adding 100 μL of the NOR solution to be tested to 300 μL of the MMIPs dispersion, incubating for 20 min in a constant-temperature shaker, then removing the sample and sequentially 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 μL of 0.008 mol / L 3,3',5,5'-tetramethylbenzidine (TMB) solution. The mixture was thoroughly mixed and incubated at 25°C. MMIPs were adsorbed using a magnet. The supernatant was then placed in a 96-well plate of a microplate reader, and the RGB values ​​and grayscale values ​​were obtained using a smartphone. Figure 1 As shown, the linear relationship between the grayscale value obtained from the sensor test and the NOR concentration is expressed by the linear equation y = 18.986x + 57.49545, with a linear correlation coefficient R. 2 =0.99141, and the calculated detection limit is 0.137 μM. Wherein Figure 2 The illustration shows the color changes of the solution when the sensor detects different concentrations of norfloxacin.

Claims

1. A method for using a smartphone-assisted surface molecular imprint colorimetric sensor to detect norfloxacin, characterized by the following steps: Step 1: Preparation of modified Fe3O4@KH540 nanoparticles. The silane coupling agent 3-aminopropyltrimethoxysilane KH540 was added dropwise to the Fe3O4 dispersion and stirred in an alkaline environment for 24 hours. After washing and drying, Fe3O4@KH540 nanoparticles were obtained. Step 2: Preparation of prepolymer of functional monomer and template molecule. The template molecule norfloxacin (NOR) and the functional monomer methacrylic acid (MAA) are dissolved together in N,N'-dimethylformamide (DMF). After 8-12 hours, the prepolymer of functional monomer and template molecule is obtained. Step 3: Preparation of magnetic norfloxacin surface molecularly imprinted nanoenzymes (MMIPs). The Fe3O4@KH540 nanoparticles prepared in Step 1 were added to N,N'-dimethylformamide (DMF) and fully swollen. Then, the prepolymer of the functional monomer and template molecule prepared in Step 2 was added, followed by the addition of a crosslinking agent. Nitrogen gas was purged to remove air, and an initiator was added. The reaction was carried out under nitrogen protection with continuous stirring to obtain magnetic norfloxacin surface molecularly imprinted nanoenzymes (MMIPs). Step 4: Template molecule elution. The magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) are repeatedly eluted with elution buffer until NOR is undetectable in the supernatant. The eluted magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) are the smartphone-assisted surface molecularly imprinted colorimetric sensor for detecting norfloxacin. Step 5: Add the smartphone-assisted surface molecular imprinting colorimetric sensor for detecting norfloxacin to water and ultrasonically disperse it to obtain a MMIPs dispersion. Add norfloxacin NOR solution to the MMIPs dispersion and incubate in a constant temperature shaker for adsorption. Then, remove the sensor and add buffer, hydrogen peroxide (H2O2) solution, and 3,3',5,5'-tetramethylbenzidine (TMB) solution in sequence. Mix well and incubate at 25°C. Use a magnet to adsorb the MMIPs. Remove the supernatant and place it in a 96-well plate of an ELISA reader. Take a picture with a smartphone to obtain the RGB values ​​and obtain the grayscale value.

2. The application method of a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin according to claim 1, characterized in that: in step one, the Fe3O4 dispersion is a 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, each 2 mL of KH540 corresponds to 1 g of Fe3O4; during washing, ethanol and purified water are used alternately for multiple washes.

3. The application method of a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin according to claim 1, characterized in that: in step two, each 0.5g template molecule norfloxacin (NOR) corresponds to 0.6mL~1.0mL of methacrylic acid and 35mL of N,N'-dimethylformamide (DMF).

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

5. The application method of a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin according to claim 1: In step four, the eluent is a mixed solution of methanol and acetic acid, with a volume ratio of methanol to acetic acid of 9:

1.

6. The application method of a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin according to claim 1, characterized in that: 0.02 g of magnetic norfloxacin surface molecularly imprinted nanozymes (MMIPs) were dispersed in 5 mL of purified water and ultrasonically dispersed for 20 min to obtain an MMIPs dispersion. Then, 100 μL of the NOR solution to be tested was added to 300 μL of the MMIPs dispersion and incubated in a shaker for 20 min at a constant temperature. After that, 300 μL of 0.2 mol / L HAc-NaAc buffer (pH 3.5), 250 μL of 0.06 mol / L H2O2 solution, and 50 μL of 0.008 mol / L TMB solution were added sequentially, mixed thoroughly, and reacted in a 25°C incubator for 4 min. The MMIPs were then magnetically adsorbed, and the supernatant was placed in a 96-well plate of a microplate reader. The RGB values ​​were obtained by taking a picture using the ColorGrab app on a smartphone, and the grayscale value was also obtained.

7. The application method of a smartphone-assisted surface molecular imprint colorimetric sensor for detecting norfloxacin according to claim 6, characterized in that: The solvents for the NOR and H2O2 solutions to be tested were both water, and the solvent for the 3,3',5,5'-tetramethylbenzidine TMB solution was dimethyl sulfoxide (DMSO).

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