Carbon dot nano-enzyme for detecting ciprofloxacin, preparation method of carbon dot nano-enzyme and sensor
By using iron-doped carbon quantum doped Fe-CDs nanoenzyme materials, the problems of cumbersome detection steps, strong instrument dependence and high cost in the prior art are solved, and efficient, sensitive and accurate ciprofloxacin detection is achieved, with low detection cost and simple operation.
Patent Information
- Application Number
- CN202411763180.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-03
- Publication Date
- 2025-03-11
AI Technical Summary
The prior art has problems such as cumbersome steps, strong instrument dependence and high cost when detecting ciprofloxacin, making it difficult to achieve efficient, sensitive and accurate detection.
Iron-doped carbon quantum dot Fe-CDs were used as nanoenzyme materials, and β-cyclodextrin and ferric chloride hexahydrate were used as carbon sources and iron dopants. Fe-CDs nanoenzymes were prepared by one-step hydrothermal method, and carbon dot nanoenzymes were obtained for detection of ciprofloxacin by dialysis purification.
It realizes efficient, sensitive and accurate detection of ciprofloxacin in water, avoids the use of complex instruments, has low detection cost, is easy to operate, and has good reproducibility and sensitivity.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of nanozymes, and in particular relates to a carbon dot nanozyme for detecting ciprofloxacin, a preparation method thereof and a sensor. Background Art
[0002] Ciprofloxacin is a broad-spectrum fluoroquinolone drug that is widely used to treat bacterial infections. However, with the increase in medical needs, the pollution of ciprofloxacin and its metabolites in the environment has gradually attracted widespread attention. Ciprofloxacin and its metabolites can enter the environment with the excrement of livestock and poultry and remain in the soil, water and other environments. At present, antibiotics such as ciprofloxacin have been detected in rivers, lakes and groundwater, which has greatly destroyed the balance of aquatic ecosystems. Therefore, it is urgent to develop an efficient and sensitive method for detecting ciprofloxacin.
[0003] At present, the methods for detecting ciprofloxacin mainly include fluorescence spectroscopy, high performance liquid chromatography, electrochemical method, chemiluminescence, etc. These methods have their unique advantages, but also have the following defects: cumbersome steps, strong instrument dependence, high cost, etc. The colorimetric method has the advantages of low cost, high sensitivity, and simple operation, which has attracted great attention from researchers in the field of antibiotic drug detection.
[0004] Nanozyme is a kind of nanomaterial with enzymatic catalytic activity. It can imitate the catalytic function of natural enzymes, has the advantages of high catalytic activity, stability, and multifunctionality, and is widely used in biosensor construction and catalytic reaction research. Among them, carbon quantum dots are one of the important members of nanozyme materials. Due to their good biocompatibility and multifunctional catalytic properties, they are widely used in biosensing, medical diagnosis, and environmental monitoring. Although the large conjugated structure gives it excellent electron transfer ability, its catalytic performance still needs to be further improved. The heteroatom doping strategy can significantly improve its electronic properties and introduce new reactive active sites. Metal ion doping can adjust the electron density inside the carbon dots, thereby changing its physical and chemical properties to increase oxidation activity. Summary of the invention
[0005] The object of the present invention is to provide a carbon dot nanozyme for detecting ciprofloxacin and a preparation method and a sensor thereof, which can efficiently, sensitively and accurately detect ciprofloxacin in water;
[0006] In order to achieve the above object, the technical solution adopted by the present invention is:
[0007] A method for preparing a carbon dot nanozyme for detecting ciprofloxacin comprises the following steps:
[0008] The ferric chloride and β-cyclodextrin are dissolved in deionized water, and heated to react to obtain a reaction solution. The reaction solution is filtered and dialyzed to obtain a Fe-CDs carbon dot nanozyme solution.
[0009] Preferably, the mass volumes of the ferric chloride, β-cyclodextrin and deionized water are 0.04 g:0.3 g:20 mL.
[0010] Preferably, the heating reaction temperature is 160° C., and the reaction time is 4 h-10 h.
[0011] In another aspect of the present invention, a carbon dot nanozyme for detecting ciprofloxacin prepared according to the above-mentioned preparation method is provided.
[0012] In another aspect, the present invention provides a method for detecting ciprofloxacin using the carbon dot nanozyme described above, comprising the following steps: mixing Fe-CDs carbon dot nanozyme solution, 3,3′,5,5′-tetramethylbenzidine solution, H 2 O 2 The solution and acetic acid-sodium acetate buffer solution are added to the sample to be tested to obtain a first mixed solution, and the reaction is carried out for 5 minutes. The absorption value of the first mixed solution at 652nm is measured and substituted into the first linear equation to obtain the content of ciprofloxacin in the sample.
[0013] Preferably, the concentration of the Fe-CDs carbon dot nanozyme solution is 0.1 mg / mL, the concentration of the 3,3′,5,5′-tetramethylbenzidine solution is 3.3 mM, and the concentration of H 2 O 2 The concentration of the solution is 10 mM, and the concentration of the acetic acid-sodium acetate buffer is 0.1 M; the Fe-CDs carbon dot nanozyme solution, 3,3′,5,5′-tetramethylbenzidine solution, H 2 O 2 The volume ratio of solution and acetic acid-sodium acetate buffer was 50:10:10:500.
[0014] Preferably, the first linear equation is: ΔA 652 =4.1104×c CIP -0.083, R 2 =0.996, where c CIP is the concentration of ciprofloxacin solution, △A 652 It is the absorbance change at 652 nm before and after adding ciprofloxacin to the second mixed solution.
[0015] In another aspect, the present invention provides a sensor for detecting ciprofloxacin, comprising the carbon dot nanozyme for detecting ciprofloxacin as described above.
[0016] In another aspect, the present invention provides a method for detecting ciprofloxacin using the above-mentioned sensor, comprising the following steps:
[0017] (1) Add Fe-CDs carbon dot nanozyme solution and acetic acid-sodium acetate buffer solution to the test paper, let it stand and dry, then add H 2 O 2 solution and 3,3′,5,5′-tetramethylbenzidine solution;
[0018] (2) Add the sample to be tested onto the test paper and let it stand;
[0019] (3) The color of the test paper is photographed by a terminal device, and the color of the test paper is converted into a corresponding RGB value. The RGB value is substituted into the second linear equation to obtain the content of ciprofloxacin in the sample, where the second linear equation is R(R+G+B)=0.1328C CIP +0.1366, R 2 =0.99.
[0020] Preferably, the concentration of the Fe-CDs carbon dot nanozyme solution is 0.1 mg / mL, the concentration of the 3,3′,5,5′-tetramethylbenzidine solution is 0.6 mM, and the concentration of H 2 O 2 The concentration of the solution is 2mM, and the concentration of the acetic acid-sodium acetate buffer is 0.1M; the Fe-CDs carbon dot nanozyme solution, 3,3′,5,5′-tetramethylbenzidine solution, H 2 O 2 The volume ratio of solution and acetic acid-sodium acetate buffer is 50:50:50:500.
[0021] The carbon dot nanozyme of the present invention is iron-doped carbon quantum dot Fe-CDs, using β-cyclodextrin as a carbon source and ferric chloride hexahydrate as an iron dopant, preparing iron-doped carbon quantum dots by a one-step hydrothermal method, and obtaining a light yellow Fe-CDs solution after dialysis purification. β-cyclodextrin can significantly improve the affinity of the carbon dot nanozyme with the catalytic substrate, and the doping of iron ions can further improve the catalytic oxidation ability of the carbon dot nanozyme.
[0022] The Fe-CDs used in the present invention are easy to synthesize, have strong stability, and have high response sensitivity to ciprofloxacin. A portable sensor constructed based on Fe-CDs can avoid the use of complex and expensive instruments, and the quantitative detection of ciprofloxacin of different concentrations and the evaluation test of ciprofloxacin in actual water samples can be achieved by combining a test strip with an image acquisition device.
[0023] Compared with other methods for detecting ciprofloxacin, this method has the advantages of being rapid and effective, stable performance, low background interference, simple operation, low detection cost and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The transmission electron microscope image and particle size distribution diagram of Fe-CDs prepared in the present invention;
[0025] Figure 2 The X-ray diffraction pattern of Fe-CDs prepared in the present invention;
[0026] Figure 3 The X-ray photoelectron spectrum of Fe-CDs prepared by the present invention;
[0027] Figure 4 This is the infrared spectrum of Fe-CDs prepared by the present invention;
[0028] Figure 5 The ultraviolet spectrum of Fe-CDs prepared in the present invention catalyzes 3,3′,5,5′-tetramethylbenzidine substrate;
[0029] Figure 6 The UV-visible spectrum of ciprofloxacin added dropwise to the Fe-CDs solution prepared by the present invention;
[0030] Figure 7 It is a linear graph between ciprofloxacin concentration and absorbance change value;
[0031] Figure 8 This is a diagram showing the color change of the test strip after adding ciprofloxacin;
[0032] Fig. 9 It is a linear graph between ciprofloxacin concentration and RGB value of the test strip; DETAILED DESCRIPTION
[0033] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.
[0034] 1. Preparation and characterization of Fe-CDs
[0035] A method for preparing a carbon dot nanozyme for detecting ciprofloxacin comprises the following steps:
[0036] Step 1: Weigh 0.04 g of ferric chloride hexahydrate and 0.3 g of β-cyclodextrin, add them to 20 mL of deionized water, stir for half an hour until completely dissolved, transfer the mixed solution to a tetrafluoroethylene high-pressure reactor and place it in an oven, and heat the reaction at 160°C for 6 hours to obtain a light yellow liquid.
[0037] Step 2: Purify the light yellow solution with a 0.22 μm filter membrane, and then dialyze it with a 1000 Da dialysis bag for 6 hours, replacing ultrapure water every 2 hours. After the dialysis is completed, prepare a 0.1 mg / mL Fe-CDs stock solution and store it at 4°C.
[0038] Characterization of properties Figure 1 , Figure 2 , Figure 3 and Figure 4 .
[0039] Figure 1 Transmission electron microscopy image and particle size distribution diagram of the prepared Fe-CDs, showing that the average particle size of Fe-CDs is 8.21±0.91nm, and they are evenly distributed on the copper mesh covered with ultra-thin carbon film.
[0040] Figure 2 The X-ray diffraction pattern of the prepared Fe-CDs shows that Fe-CDs present two characteristic peaks at 28° and 41°, corresponding to the (002) and (100) crystal planes of carbon, respectively.
[0041] Figure 3 This is the X-ray photoelectron spectrum of the prepared Fe-CDs, which shows that Fe-CDs are mainly composed of Fe(2p), C(1s) and O(1s).
[0042] Figure 4 This is the infrared spectrum of the prepared Fe-CDs, which shows that there are functional groups such as OH, CH, OCO, CO and Fe-O on the surface of Fe-CDs.
[0043] 2. Characterization of Fe-CDs catalytic oxidation performance
[0044] The evaluation method of the catalytic performance of Fe-CDs nanozymes is as follows:
[0045] Step 1: Accurately weigh 7.9 mg of 3,3′,5,5′-tetramethylbenzidine powder and dissolve it in 10 mL of anhydrous ethanol. Ultrasonicate for 10 min to fully dissolve it to obtain a stock solution with a concentration of 3.3 mM.
[0046] Step 2: Take 50 μL of Fe-CDs stock solution (0.1 mg / mL), add 0.5 mL of acetic acid-sodium acetate buffer (0.1 M, pH = 4.5), 10 μL of H 2 O 2 solution (2 mM) and 50 μL 3,3′,5,5′-tetramethylbenzidine solution (3.3 mM), incubated at 40°C for 5 min, observed the color change of the reaction system and measured the absorbance change of the oxidation product at 652 nm using an ultraviolet spectrophotometer.
[0047] Carbon dot nanozymes in H 2 O 2In the presence of ciprofloxacin, it can catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine solution to generate a blue product. The catalytic performance of the nanozyme can be evaluated by observing the ultraviolet absorption of the blue product at 652nm. The catalytic reaction is carried out under acidic conditions, and the pH value of the reaction solution is adjusted with acetic acid-sodium acetate buffer. Ciprofloxacin can inhibit the catalytic performance of the nanozyme, thereby realizing the detection of ciprofloxacin.
[0048] Among them, Figure 5 As shown, Fe-CDs can significantly catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine substrate to generate a blue product, which exhibits strong absorption at 652 nm.
[0049] 3. Quantitative detection of ciprofloxacin based on Fe-CDs
[0050] The method for quantitative detection of ciprofloxacin by Fe-CDs nanozyme comprises the following specific steps:
[0051] Step 1: Add 50 μL of Fe-CDs stock solution (0.1 mg / mL), 10 μL of 3,3′,5,5′-tetramethylbenzidine solution (3.3 mM) and 10 μL of H 2 O 2 Solution (10 mM). Measure the absorbance at 652 nm and record it as A Blank .
[0052] Step 2: gradually add ciprofloxacin standard solution to the solution in step 1, and measure the absorbance of the mixed solution at 652 nm after reacting for 5 min, which is recorded as A. 652 The absorbance change of the solution and the corresponding color change during the entire titration process are shown in Figure 6 .
[0053] Step 3: Use Origin software to calculate the absorbance change △A of the mixed solution at 652nm 652 (A Blank -A 652 ) as the ordinate and ciprofloxacin concentration as the abscissa, and the linear relationship between the two was plotted and fitted. The results are shown in Figure 7 .
[0054] in, Figure 6 It shows that with the addition of ciprofloxacin standard solution, the ability of Fe-CDs to catalyze TMB oxidation decreases, and the green color gradually weakens, which is manifested in the decrease of absorbance at 652nm. Figure 7 This is the linear relationship between the absorbance change of the Fe-CDs-catalyzed 3,3′,5,5′-tetramethylbenzidine oxidation product at 652nm and the concentration of ciprofloxacin. The linear equation is △A 652=4.1104×c CIP -0.083, correlation coefficient R 2 =0.996, the linear range is 0.03-0.1mM, and the minimum detection limit is 1.46μM.
[0055] 4. Detection of ciprofloxacin content in actual water based on Fe-CDs
[0056] In this embodiment, the samples to be tested are Lingde Lake water and tap water.
[0057] Step 1: Select water from Lingde Lake and tap water, filter through a 0.22 μm microporous membrane to remove particle suspension, dilute by half and store in a 4°C refrigerator for later use;
[0058] Step 2: Take 200 μL of water sample, add 50 μL of Fe-CDs stock solution (0.1 mg / mL), 10 μL of 3,3′,5,5′-tetramethylbenzidine solution (3.3 mM), 10 μL of H 2 O 2 Solution (10mM), 500μL acetic acid-sodium acetate buffer (0.1M, pH=4.5), react for 5min and record the absorbance value A of the solution at 652nm Blank .
[0059] Step 3: Add different concentrations of ciprofloxacin solution to the solution to make the final concentration of ciprofloxacin in the solution 0.03, 0.05, and 0.08 mM, and measure the absorbance value A of the mixed solution at 652 nm. 652nm , and calculate the absorbance change △A after adding ciprofloxacin 652 =A Blank -A 652nm
[0060] Step 4: 652 Substitute into the linear equation △A 652 =4.1104×c CIP -0.083, the corresponding spiked concentration of ciprofloxacin was calculated and the spiked recovery of ciprofloxacin in the actual sample was measured. All experiments were repeated six times, and the results are shown in Table 1.
[0061] Table 1 shows that the Fe-CDs-based ciprofloxacin detection method can be used for the quantitative detection of ciprofloxacin in actual samples, with spiked recoveries ranging from 96.3% to 104%, and relative standard deviations less than 3.8%, thus having good reproducibility.
[0062] Table 1: Detection of ciprofloxacin content in actual water samples using Fe-CDs
[0063]
[0064] 5. Portable sensors based on Fe-CDs
[0065] Step 1: Select appropriate filter paper and cut it into 1×2 cm strips for later use;
[0066] Step 2: Add 500 μL of acetic acid-sodium acetate buffer (0.1 M, pH = 4.5) and 50 μL of Fe-CDs solution (0.1 mg / mL) on the surface of the test strip and let it stand for 20 min to dry;
[0067] Step 3: Add 50 μL of H 2 O 2 solution (2 mM) and 50 μL of 3,3′,5,5′-tetramethylbenzidine solution (0.6 mM), the color of the test strip immediately turned blue, and it was dried in air for 10 min before use;
[0068] Step 4: Add ciprofloxacin solutions of different concentrations to the surface of the test strip, place it at room temperature for 20 minutes, collect the test strip image, and convert the color of the test strip into the corresponding RGB value. Figure 8 The results showed that the blue color of the test strip gradually faded as the concentration of ciprofloxacin increased.
[0069] Step 5: According to the given RGB value, with R (R + G + B) as the ordinate and the ciprofloxacin concentration as the abscissa, a linear relationship with the ciprofloxacin concentration is fitted. The results are shown in Fig. 9 . Fig. 9 It is a linear relationship between the R(R+G+B) value of the test strip color and the ciprofloxacin concentration. The linear equation is R(R+G+B)=0.1328c CIP +0.1366, correlation coefficient R 2 =0.99, and the minimum detection limit is 15.1μM.
[0070] Specifically, the test strip image is captured by an image capture device, wherein the image capture device may be a camera, a smart phone or a PDA. After the image capture device captures the test strip image, the color of the test strip is converted into a corresponding RGB value through an image processing module.
Claims
1. A method for preparing a carbon dot nanozyme for detecting ciprofloxacin, characterized in that: The following steps are involved: The ferric chloride and β-cyclodextrin are dissolved in deionized water, and heated to react to obtain a reaction solution. The reaction solution is filtered and dialyzed to obtain a Fe-CDs carbon dot nanozyme solution.
2. The preparation method according to claim 1, characterized in that: The mass volumes of the ferric chloride, β-cyclodextrin and deionized water are 0.04 g:0.3 g:20 mL.
3. The preparation method according to claim 1, characterized in that: The heating reaction temperature is 160° C., and the reaction time is 4 h to 10 h.
4. The carbon dot nanozyme for detecting ciprofloxacin prepared according to the preparation method according to any one of claims 1 to 3.
5. The method for detecting ciprofloxacin using the carbon dot nanozyme according to claim 4, characterized in that: The method comprises the following steps: adding Fe-CDs carbon dot nanozyme solution, 3,3′,5,5′-tetramethylbenzidine solution, H2O2 solution and acetic acid-sodium acetate buffer solution to a sample to be tested to obtain a mixed solution, and measuring the absorption value of the mixed solution at 652 nm and substituting it into a first linear equation to obtain the content of ciprofloxacin in the sample.
6. The method according to claim 5, characterized in that The concentration of the Fe-CDs carbon dot nanozyme solution is 0.1 mg / mL, the concentration of the 3,3′,5,5′-tetramethylbenzidine solution is 3.3 mM, the concentration of the H2O2 solution is 10 mM, and the concentration of the acetic acid-sodium acetate buffer solution is 0.1 M; the volume ratio of the Fe-CDs carbon dot nanozyme solution, 3,3′,5,5′-tetramethylbenzidine solution, H2O2 solution and acetic acid-sodium acetate buffer solution is 50:10:10:
500.
7. The method according to claim 5, characterized in that The first linear equation is: △A 652 =4.1104×c CIP -0.083, R 2 =0.996, where c CIP is the concentration of ciprofloxacin solution, △A 652 It is the absorbance change of the mixed solution at 652nm before and after adding ciprofloxacin.
8. A sensor for detecting ciprofloxacin, characterized in that: It comprises the carbon dot nanozyme for detecting ciprofloxacin as claimed in any one of claims 1 to 3.
9. The method for detecting ciprofloxacin using the sensor according to claim 8, characterized in that: The steps include: (1) Fe-CDs carbon dot nanozyme solution and acetic acid-sodium acetate buffer were added dropwise onto the test paper, and after standing to dry, H2O2 solution and 3,3′,5,5′-tetramethylbenzidine solution were added dropwise; (2) Add the sample to be tested onto the test paper and let it stand; (3) Collect the color of the test paper, convert the color of the test paper into the corresponding RGB value, and substitute the RGB value into the second linear equation to obtain the content of ciprofloxacin in the sample, where the second linear equation is R(R+G+B)=0.1328 C CIP +0.1366,R 2 =0.99。 10. The method according to claim 9, characterized in that The concentration of the Fe-CDs carbon dot nanozyme solution is 0.1 mg / mL, the concentration of the 3,3′,5,5′-tetramethylbenzidine solution is 0.6 mM, the concentration of the H2O2 solution is 2 mM, and the concentration of the acetic acid-sodium acetate buffer solution is 0.1 M; the volume ratio of the Fe-CDs carbon dot nanozyme solution, 3,3′,5,5′-tetramethylbenzidine solution, H2O2 solution and acetic acid-sodium acetate buffer solution is 50:50:50:500.