A norfloxacin sensor and its preparation and detection methods
The norfloxacin sensor, which combines CTS/Ag-NiWO4 composite material with multi-walled carbon nanotubes, solves the problems of insufficient sensitivity and detection limit in the existing technology, and realizes efficient quantitative detection of norfloxacin with the advantages of high sensitivity and low detection limit.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHENGDU NORMAL UNIV
- Filing Date
- 2023-03-17
- Publication Date
- 2026-05-26
AI Technical Summary
The lack of novel electrode catalytic materials with high sensitivity, low detection limit, and wide linear range in the existing technology leads to poor performance of electrochemical catalytic sensors in norfloxacin detection.
Norfloxacin sensor was prepared by combining CTS/Ag-NiWO4 composite material with multi-walled carbon nanotubes. Detection was performed by cyclic voltammetry. The Ag-NiWO4 catalyzes the reaction of norfloxacin and generates a current signal, which is then combined with the adsorption of chitosan to achieve quantitative detection.
This method achieves highly sensitive detection of norfloxacin concentration, with low detection limit, wide linear range, simple detection method, high average recovery rate, good linearity, and higher sensitivity than existing technologies.
Smart Images
Figure CN116448860B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of electrochemical sensor technology, and in particular to a norfloxacin sensor and its preparation and detection methods. Background Technology
[0002] Norfloxacin, also known as norfloxacin, is a third-generation quinolone antibiotic. It exhibits high antibacterial activity against aerobic Gram-negative bacilli and shows good in vitro antibacterial activity against most Enterobacteriaceae, including Enterobacter spp., Klebsiella spp., Salmonella spp., and Yersinia spp. Norfloxacin kills bacteria by inhibiting DNA synthesis and replication by acting on the A subunit of bacterial DNA gyrase. Therefore, it is used in livestock and poultry farming to prevent disease. However, excessively high levels of norfloxacin residues in livestock and poultry can lead to dizziness, nausea, loss of appetite, and delayed bone formation in children, affecting their development. Therefore, national standards limit the amount of norfloxacin residues in livestock and poultry farming; for example, the maximum residue limit for pork is 0.02 mg / kg. Therefore, detecting the norfloxacin content in meat is of great significance.
[0003] Currently, high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) are the most commonly used methods for detecting norfloxacin, but they are costly and cumbersome to operate. Electrochemical catalytic sensors for norfloxacin detection offer advantages such as simple operation, low manufacturing cost, high sensitivity, short response time, good selectivity, and stability. However, existing research lacks studies on novel electrode catalytic materials with high sensitivity, low detection limits, and wide linear ranges. Therefore, the application of electrochemical catalytic sensors in the detection of antibiotics in food has attracted widespread attention. Summary of the Invention
[0004] To address the lack of novel electrode catalytic materials with high sensitivity, low detection limit, and wide linear range in existing technologies, this invention provides a norfloxacin sensor and its preparation and detection methods.
[0005] The present invention provides the following technical solution: a norfloxacin sensor, comprising a base electrode, a first solid film and a second solid film, wherein the first solid film is a multi-walled carbon nanotube that has been acidified and washed to a neutral pH, and the second solid film is a CTS / Ag-NiWO4 composite material, wherein the first solid film and the second solid film are sequentially stacked on the outer surface of the base electrode.
[0006] A method for preparing a norfloxacin sensor includes the following steps:
[0007] S1, silver-doped nickel tungstate, chitosan and ultrapure water were mixed and dispersed to obtain CTS / Ag-NiWO4 composite material;
[0008] S2, to prepare a multi-walled carbon nanotube suspension;
[0009] S3, polishing and activating the surface of the base electrode;
[0010] S4, the multi-walled carbon nanotube solution is drop-coated onto the activated surface of the base electrode and then refrigerated to form a first solid film;
[0011] S5, the CTS / Ag-NiWO4 composite material is attached to the surface of the first solid film and then refrigerated to form a second solid film.
[0012] Preferably, in S1, the silver-doped nickel tungstate and chitosan are mixed and then ultrapure water is added for ultrasonic dispersion to obtain the CTS / Ag-NiWO4 composite material.
[0013] Preferably, the mass ratio of the nickel tungstate doped with silver to chitosan is 1:1.
[0014] Preferably, step S2 includes the following steps:
[0015] S21, use a glass rod to dip multi-walled carbon nanotubes and place them in nitric acid for ultrasonic dispersion and acidification to form a solution;
[0016] S22, the solution is centrifuged to remove the liquid, then ultrapure water is added and the solution is repeatedly centrifuged and washed until the pH value of the solution is neutral, and then it is placed in an oven to dry.
[0017] S23, Weigh a certain amount of dried multi-walled carbon nanotubes, add ultrapure water, and disperse them ultrasonically to form the multi-walled carbon nanotube solution.
[0018] Preferably, in step S4, the multi-walled carbon nanotube solution is drop-coated onto the surface of the base electrode and placed in a refrigerator at 4°C to form a uniform first solid film on the surface of the base electrode.
[0019] Preferably, in step S5, the CTS / Ag-NiWO4 composite material is drop-coated onto the surface of the first solid film and placed in a refrigerator at 4°C to form a uniform second solid film on the surface of the first solid film.
[0020] A norfloxacin sensor detection method includes an electrolytic device for cyclic voltammetry, the electrolytic device comprising a three-electrode system, the three-electrode system including a working electrode, and the detection method comprising the following steps.
[0021] T1, prepare a series of norfloxacin solutions with decreasing concentrations, and use cyclic voltammetry and the electrolysis device to detect the series of norfloxacin solutions, and plot a standard curve or reference table of norfloxacin concentration versus current.
[0022] T2, after processing the meat sample to be tested to obtain a sample solution, the sample solution is tested using cyclic voltammetry and the electrolysis device.
[0023] T3, based on the current value obtained in step T2, refer to the standard curve or comparison table to determine the concentration of norfloxacin residue in the meat sample to be tested;
[0024] The working electrode is a norfloxacin sensor or a sensor prepared according to a norfloxacin sensor preparation method.
[0025] Preferably, step T1 includes the following steps:
[0026] T11, prepare PBS buffer;
[0027] T12, prepare a norfloxacin solution with a basic concentration, and use ultrapure water to gradient dilute the norfloxacin solution with the basic concentration to obtain a series of norfloxacin solutions with decreasing concentrations;
[0028] T13, transfer PBS buffer into the electrolytic cell of the electrolysis device as the base solution, and take a series of norfloxacin solutions with increasing concentrations starting from the lowest concentration. Take multiple volumes of each concentration and add them to the base solution and mix well. Use cyclic voltammetry and the electrolysis device for detection. Plot a standard curve or comparison table of norfloxacin concentration versus current value based on the detection results.
[0029] Preferably, step T2 includes the following steps:
[0030] T21, prepare acetonitrile-sodium hydroxide solution and PBS buffer;
[0031] T22. After grinding the meat sample to be tested, add the acetonitrile-sodium hydroxide solution, disperse and centrifuge, take the supernatant, add the PBS buffer and dichloromethane, disperse and centrifuge, take the lower organic phase, add n-hexane, centrifuge, and take the lower solution as the sample solution for later use.
[0032] T23, the PBS buffer solution is transferred to the electrolytic cell of the electrolysis device as the base solution, the sample solution is added to the base solution and mixed, and the cyclic voltammetry and the electrolysis device are used for detection under the same working conditions as in step T1.
[0033] The beneficial effects of this invention are: Ag-NiWO4 catalyzes the reaction of norfloxacin, which in turn causes electron transfer and generates a current signal. Multi-walled carbon nanotubes, Ag-NiWO4, and chitosan are used to construct the sensor interface, enabling quantitative detection of norfloxacin concentration. This invention not only has strong adsorption, catalytic, and conductivity capabilities, but also has simple and easy preparation and detection methods, high sensitivity, and low detection limits. Attached Figure Description
[0034] Figure 1 This is a schematic diagram illustrating the operation of one embodiment of the preparation method of the present invention.
[0035] Figure 2 This is a schematic diagram of one embodiment of the electrolysis apparatus of the present invention.
[0036] Figure 3 The standard curve of norfloxacin concentration versus current value was plotted for this invention. Implementation
[0037] The embodiments of the present invention will be described in more detail below with reference to the accompanying drawings and reference numerals, so that those skilled in the art can implement them after reading this specification. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Example
[0038] The present invention provides a norfloxacin sensor, comprising a base electrode, a first solid film and a second solid film, wherein the first solid film is a multi-walled carbon nanotube and the second solid film is a CTS / Ag-NiWO4 composite material, and the first solid film and the second solid film are sequentially stacked on the outer surface of the base electrode. Example
[0039] This invention provides a method such as Figure 1 The method for preparing the norfloxacin sensor shown includes the following steps:
[0040] S1. Take 0.5 mg of silver-doped nickel tungstate (Ag-NiWO4) and 0.5 mg of chitosan (CTS), mix them, add 500 μL of ultrapure water and disperse them using an ultrasonic cleaner to obtain the CTS / Ag-NiWO4 composite material.
[0041] S2, to prepare a suspension of multi-walled carbon nanotubes (MWCNTs).
[0042] S21. A small amount of multi-walled carbon nanotubes was dipped into a glass rod and acidified in nitric acid at a volume ratio of 1:1, while being ultrasonically dispersed into a suspension for 6 hours.
[0043] S22, the acidified suspension is centrifuged to remove the liquid, and the multi-walled carbon nanotubes obtained by centrifugation are washed with ultrapure water and then centrifuged to remove water. This process is repeated multiple times until the pH is neutral. The prepared multi-walled carbon nanotubes are then placed in an oven to dry.
[0044] S23, weigh 1 mg of dried multi-walled carbon nanotubes, add 1 mL of ultrapure water, and ultrasonically disperse to form a multi-walled carbon nanotube suspension of 1 mg / mL.
[0045] S3 uses a glassy carbon electrode as the base electrode, and the surface of the base electrode is polished and activated. The polishing and activation methods both employ commonly used methods in existing electrochemistry, and the specific steps are as follows:
[0046] S31. Add small amounts of 0.3μm diameter alumina powder to the polishing disc multiple times, add appropriate amount of ultrapure water and polish the glassy carbon electrode for 15 minutes until the electrode surface is polished to a mirror finish; then rinse the polished glassy carbon electrode with ultrapure water, place it in ultrapure water for ultrasonic cleaning for 5 minutes, then place it in anhydrous ethanol for ultrasonic cleaning for 5 minutes, and finally place it in ultrapure water for ultrasonic cleaning for 5 minutes.
[0047] S32. Place the cleaned glassy carbon electrode in 5 mL of 0.1 mol / mL sulfuric acid solution and activate the glassy carbon electrode by cyclic voltammetry in a potential range of -1.0 to 1.8 V for 50 cycles. After activation, rinse the glassy carbon electrode with ultrapure water and place it in ultrapure water for later use.
[0048] S4. Take 3 μL of the 1 mg / mL multi-walled carbon nanotube solution and drop it onto the surface of the glassy carbon electrode. Place it in a refrigerator at 4°C and refrigerate it upside down for 4 hours to allow it to solidify naturally and form a uniform first solid film.
[0049] S5, then drop-coat 3μL of the CTS / Ag-NiWO4 composite material onto the surface of the first solid film, place it in a refrigerator at 4℃ and refrigerate it upside down for 4 hours to allow it to cure naturally and form a uniform second solid film. The preparation is complete and the CTS / Ag-NiWO4 / MWCNT sensor is obtained.
[0050] All reagents used in the above steps were of analytical grade. In other embodiments, carbon paste electrodes, metal electrodes, etc., can also be used as the base electrodes. The applicant's research found that Ag-NiWO4 can effectively catalyze the reaction of norfloxacin, thereby causing electron transfer and generating a current signal, which is presented as a cyclic voltammetry image in an electrochemical workstation. Carbon nanotubes have a one-dimensional hollow tubular structure, with the tube wall composed of a single or multiple layers of graphene sheets. The tube diameter is on the nanometer scale, the tube length is on the micrometer scale, and the aspect ratio is huge, exhibiting metallic or semiconductor properties. As an electrode modification material, it has good conductivity when used in chemical reactions and has wide applications in the field of modified electrodes. Chitosan (CTS) is a cationic polyamine with a high charge density at pH < 6.5, thus it can adsorb the -COO of norfloxacin. - Anions, possessing the properties of polyelectrolytes, exhibit a double-helix structure containing amino and hydroxyl groups. They can form intermolecular hydrogen bonds with norfloxacin, effectively allowing norfloxacin to adhere to the electrode surface and accelerate the catalytic reaction. Chitosan and Ni in Ag-NiWO4... 2+Chelation occurs, allowing chitosan to combine with Ag-NiWO4, thus achieving the effect of Ag-NiWO4 being stably fixed on the electrode surface and not easily falling off.
[0051] This invention utilizes multi-walled carbon nanotubes and CTS / Ag-NiWO4 composite materials to construct the sensor interface, thereby improving sensor sensitivity. Furthermore, leveraging the principle of Ag-NiWO4 catalyzing the reaction of norfloxacin, quantitative detection of norfloxacin concentration can be achieved. The prepared sensor not only possesses strong catalytic, conductive, and adsorption capabilities but also boasts advantages such as simple detection method, convenient operation, high sensitivity, and low detection limit. Its lowest detection limit for norfloxacin concentration reaches 2.399 ng / mL, while the lowest detection limit achievable by commonly used high-performance liquid chromatography (HPLC) is only 2.5 ng / mL, and that by reversed-phase HPLC is 50 ng / mL. The average recovery rate is 97.9%, and the lowest detection limit achievable by liquid chromatography-mass spectrometry (LC-MS) is 0.0564-0.8653 μg / kg. Moreover, this invention achieves a detection limit of 3.998 × 10⁻⁶ μg / kg. -2 ~3.998×10 -6 The current values obtained within the concentration range of μg / L showed a good linear relationship with the negative logarithm of norfloxacin concentration, such as... Figure 3 As shown, its linear regression equation is I = 0.1008(-lgC) + 8.8079, and the correlation coefficient R0 is... 2 =0.9767, where I is the current value and -lgC is the negative logarithm of the norfloxacin concentration. Example
[0052] This invention provides a detection method for norfloxacin using a sensor, comprising an electrolytic device for cyclic voltammetry. The electrolytic device includes an electrolytic cell and a three-electrode system disposed within the electrolytic cell. The three-electrode system includes a working electrode, a counter electrode, and a reference electrode. The working electrode is a CTS / Ag-NiWO4 / MWCNT sensor prepared in Example 1, the counter electrode is a platinum electrode, and the reference electrode is a saturated calomel electrode. A schematic diagram of the electrolytic device is shown below. Figure 2 As shown.
[0053] The detection method includes the following steps:
[0054] T1. A series of norfloxacin solutions with increasing concentrations were prepared. The series of norfloxacin solutions were tested using cyclic voltammetry and the electrolysis device to plot a standard curve of norfloxacin concentration versus current.
[0055] The specific steps are as follows:
[0056] T11. Weigh a certain amount of disodium hydrogen phosphate into a beaker, add ultrapure water to the beaker to dissolve and bring the volume to a final volume to prepare a 0.05 mol / L PBS buffer solution A. Weigh a certain amount of sodium dihydrogen phosphate into another beaker, add ultrapure water to the beaker to dissolve and bring the volume to a final volume to prepare a 0.05 mol / L PBS buffer solution B. Mix solutions A and B in a certain ratio to prepare a PBS buffer solution with pH=5.00.
[0057] T12: A certain amount of norfloxacin was dissolved in ultrapure water to prepare a norfloxacin solution with a basic concentration of 0.1 mg / mL. This norfloxacin solution with the basic concentration was then serially diluted with ultrapure water to prepare a series of norfloxacin solutions with decreasing concentrations, which were used as standard solutions for detection. The concentrations of norfloxacin in these solutions were 3.998 × 10⁻⁶. -2 3.998×10 -3 3.998×10 -4 3.998×10 -5 3.998×10 -6 μg / L.
[0058] T13. Set the operating conditions of the electrolysis device, including a CV detection settling time of 2 s, an initial voltage of -1.0 V, a peak voltage of 1.0 V, a sampling interval of 0.001 V, a scan rate of 0.05 V / s, and a sensitivity of 10⁻⁵ A / V. Transfer 5 mL of PBS buffer to the electrolysis cell as the base solution. Starting from the lowest concentration, take a series of norfloxacin solutions with decreasing concentrations for detection. Take 2 μL, 4 μL, 6 μL, and 8 μL of each concentration and add them to the base solution, mix well, and perform detection using cyclic voltammetry and the electrolysis device. Based on the detection results, plot a standard curve of norfloxacin concentration versus current value with the negative logarithm of the norfloxacin concentration on the x-axis and the detected current value on the y-axis. Figure 3 As shown. The plotting method adopted is a commonly used plotting method in electrochemistry.
[0059] T2, after processing the meat sample to be tested to obtain a sample solution, the sample solution is tested using cyclic voltammetry and the electrolysis device.
[0060] The specific steps are as follows:
[0061] T21. Measure 84 mL of anhydrous acetonitrile and add it to 16 mL of 0.1 mol / L sodium hydroxide solution. Mix well to obtain an acetonitrile-0.1 mol / L sodium hydroxide solution with a volume ratio of 84:16. Weigh a certain amount of disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium chloride, mix them, dissolve them in a beaker, and make up to volume with ultrapure water to prepare a 0.02 mol / L PBS buffer.
[0062] T22, using pork as the meat sample to be tested, after grinding the pork sample, weigh 3.0178g, add 9mL of the acetonitrile-0.1mol / L sodium hydroxide solution, sonicate for 10min, centrifuge for 10min, take 4mL of supernatant, add 4mL of 0.02mol / L PBS buffer, then add 8mL of dichloromethane, sonicate for 10min, centrifuge for 10min, take the lower organic phase, add 2mL of n-hexane, centrifuge for 5min, and take the lower solution as the sample solution for later use.
[0063] T23. Set the operating conditions of the electrolysis device to be the same as in step T13. Transfer 5 mL of PBS buffer into the electrolysis cell as the base solution. Add 5 μL of the sample solution to the base solution and mix well. Use cyclic voltammetry and the electrolysis device for detection.
[0064] T3, based on the current value obtained in step T2, the concentration of norfloxacin residue in the meat sample to be tested is determined by referring to the standard curve.
[0065] In other embodiments, the linear relationship between norfloxacin concentration and current value can also be represented by a reference table.
[0066] The above describes one or more embodiments of the present invention in a relatively specific and detailed manner, but it should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.
Claims
1. A norfloxacin sensor characterized by: It includes a base electrode, a first solid film, and a second solid film. The first solid film is a multi-walled carbon nanotube, and the second solid film is a CTS / Ag-NiWO4 composite material. The first solid film and the second solid film are sequentially stacked on the outer surface of the base electrode.
2. A method of preparing a norfloxacin sensor, characterized by: Includes the following steps, S1, silver-doped nickel tungstate, chitosan and ultrapure water were mixed and dispersed to obtain CTS / Ag-NiWO4 composite material; S2, to prepare a multi-walled carbon nanotube suspension; S3, polishing and activating the surface of the base electrode; S4, the multi-walled carbon nanotube solution is drop-coated onto the activated surface of the base electrode and then refrigerated to form a first solid film; S5, the CTS / Ag-NiWO4 composite material is attached to the surface of the first solid film and then refrigerated to form a second solid film.
3. The method for preparing a norfloxacin sensor according to claim 2, characterized in that: In step S1, the silver-doped nickel tungstate is mixed with chitosan and then ultrapure water is added for ultrasonic dispersion to obtain the CTS / Ag-NiWO4 composite material.
4. The method for preparing a norfloxacin sensor according to claim 3, characterized in that: The mass ratio of silver-doped nickel tungstate to chitosan is 1:
1.
5. The method for preparing a norfloxacin sensor according to claim 2, wherein: Step S2 includes the following steps: S21, use a glass rod to dip multi-walled carbon nanotubes and place them in nitric acid for ultrasonic dispersion and acidification to form a solution; S22, the solution is centrifuged to remove the liquid, then ultrapure water is added and the solution is repeatedly centrifuged and washed until the pH value of the solution is neutral, and then it is placed in an oven to dry. S23, Weigh a certain amount of dried multi-walled carbon nanotubes, add ultrapure water, and disperse them ultrasonically to form the multi-walled carbon nanotube solution.
6. The method for preparing a norfloxacin sensor according to claim 2, wherein: In step S4, the multi-walled carbon nanotube solution is drop-coated onto the surface of the base electrode and placed in a refrigerator at 4°C to form a uniform first solid film on the surface of the base electrode.
7. The method for preparing a norfloxacin sensor according to claim 2, wherein: In step S5, the CTS / Ag-NiWO4 composite material is drop-coated onto the surface of the first solid film and placed in a refrigerator at 4°C to form a uniform second solid film on the surface of the first solid film.
8. A method for detecting norfloxacin sensor comprising an electrolytic device applied to cyclic voltammetry, said electrolytic device comprising a three-electrode system, said three-electrode system comprising a working electrode, characterized by: The detection method Includes the following steps, T1, prepare a series of norfloxacin solutions with decreasing concentrations, and use cyclic voltammetry and the electrolysis device to detect the series of norfloxacin solutions, and plot a standard curve or reference table of norfloxacin concentration versus current. T2, after processing the meat sample to be tested to obtain a sample solution, the sample solution is tested using cyclic voltammetry and the electrolysis device. T3, based on the current value obtained in step T2, refers to the standard curve or comparison table to determine the concentration of norfloxacin residue in the meat sample to be tested; the working electrode is a norfloxacin sensor according to claim 1 or a sensor prepared by a norfloxacin sensor preparation method according to any one of claims 2 to 7.
9. The method of claim 8, wherein the method is for detecting norfloxacin. Step T1 includes the following steps: T11, prepare PBS buffer; T12, prepare a norfloxacin solution with a basic concentration, and use ultrapure water to gradient dilute the norfloxacin solution with the basic concentration to obtain a series of norfloxacin solutions with decreasing concentrations; T13, transfer PBS buffer into the electrolytic cell of the electrolysis device as the base solution, and take a series of norfloxacin solutions with increasing concentrations starting from the lowest concentration. Take multiple volumes of each concentration and add them to the base solution and mix well. Use cyclic voltammetry and the electrolysis device for detection. Plot a standard curve or comparison table of norfloxacin concentration versus current value based on the detection results.
10. The method of claim 8, wherein the method is for detecting norfloxacin. Step T2 includes the following steps: T21, prepare acetonitrile-sodium hydroxide solution and PBS buffer; T22. After grinding the meat sample to be tested, add the acetonitrile-sodium hydroxide solution, disperse and centrifuge thoroughly, take the supernatant, add the PBS buffer and dichloromethane, disperse and centrifuge thoroughly, take the lower organic phase, add n-hexane, centrifuge, and take the lower solution as the sample solution for later use. T23, the PBS buffer solution is transferred to the electrolytic cell of the electrolysis device as the base solution, the sample solution is added to the base solution and mixed, and the cyclic voltammetry and the electrolysis device are used for detection under the same working conditions as in step T1.