A visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots

Abstract

The invention relates to the field of fluorescent colorimetric sensor construction, and specifically discloses a visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots, which is used to solve the problems of complicated pre-treatment, high instrument cost, high detection site requirement and high operator specialization in existing antibiotic detection methods. The invention synthesizes iron-doped carbon quantum dots (Fe-CDs) by a hydrothermal method, characterizes the iron-doped carbon quantum dots (Fe-CDs), identifies the peroxidase activity of the iron-doped carbon quantum dots (Fe-CDs), performs dual-mode sensing detection of ampicillin and tetracycline based on an ultraviolet / fluorescence spectrophotometer, and finally develops a portable test strip, uses a smart phone sensing platform to automatically generate a linear fitting relationship between R, G, B values ​​and concentrations, realizes real-time, on-site, instrument-free and cost-effective instant detection, and will promote research on antibiotics in terms of environmental chemical behavior, temporal and spatial distribution and biological ecotoxicity.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent colorimetric sensor construction, and more specifically, to a visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots. Background Art

[0002] Currently, antibiotics have been widely used to prevent or treat bacterial infections in humans, animals and livestock. When released into the environment, antibiotics directly threaten human safety by disrupting normal metabolism and changing microbial communities. On the other hand, the continuous release of antibiotics into the environment can lead to chemical pollution and promote the generation and spread of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB). Antibiotic resistance (AR) reduces the therapeutic potential of antibiotics against human and animal pathogens, posing a serious threat to global public health.

[0003] Carbon quantum dots (CDs) are zero-dimensional nanomaterials smaller than 10 nm that have been widely used in different applications, including metal ion detection, photocatalysis, in vivo biological applications, and antibiotic detection, due to their excellent photoluminescence, catalytic properties, and water dispersibility. However, CDs often face challenges such as aggregation, aggregation-induced quenching (ACQ) effect, and excitation dependence in aqueous media, which limit their sensing and adsorption applications. Recent studies have shown that heteroatom doping can improve the catalytic properties of CDs, for example, Fe-doped carbon nanozymes have improved active site utilization during peroxidase catalysis, and their active site utilization is higher than that of carbon nanozymes doped with other metals (i.e., Cu, Co, Ni, and Mn). Antibiotic biosensors developed based on quantum dots have been booming, and such methods often have good potential for on-site applications and are becoming increasingly important in areas such as point-of-care testing (POCT), environmental monitoring, and agricultural breeding.

[0004] In recent years, people have developed a variety of antibiotic detection methods and widely used in various environmental media. At present, the main antibiotic detection methods are HPLC, LC-MS, etc., which have incomparable advantages in terms of throughput, sensitivity, precision, accuracy, etc. However, their pre-treatment is cumbersome, the instrument cost is high, the detection site requirements are high, and the operator specialization is high, which limits their application in the rapid detection of antibiotics on site. Therefore, it is of great significance to develop new technologies for rapid, convenient, sensitive and specific antibiotic detection. In order to solve the above problems, a technical solution is now provided. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots, performs dual-mode sensing detection of ampicillin and tetracycline based on colorimetry / fluorescence, and finally develops a portable test strip, which uses a smartphone sensing platform to automatically generate a linear fitting relationship between R, G, B values ​​and concentration, thereby realizing real-time, on-site, instrument-free and cost-effective instant detection to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots comprises the following steps:

[0008] Step 1, synthesizing iron-doped carbon quantum dots by a hydrothermal method;

[0009] Step 2, characterizing the iron-doped carbon quantum dots by transmission electron microscopy, UV-visible spectroscopy, fluorescence spectroscopy, and X-ray photoelectron spectroscopy;

[0010] Step 3, detecting the electron paramagnetic resonance signal by electron paramagnetic resonance spin capture technology, capturing free radicals and peroxidase activity kinetics test with 5,5-dimethyl-1-pyrroline-N-oxide and 2,2,6,6-tetramethylpiperidine capture agents at room temperature, and identifying the peroxidase activity of iron-doped carbon quantum dots;

[0011] Step 4, constructing a dual-mode sensing detection of ampicillin and tetracycline through the colorimetric signal of the Fe-CDs+TMB+H2O2 system at different ampicillin concentrations, the fluorescence signal of the system at different tetracycline concentrations, and the selectivity of the sensor under interference factors;

[0012] Step 5. Develop a portable test strip to achieve rapid visual detection of antibiotics through a smartphone sensing platform. Take a colorimetric photo with a smartphone, select an area in the photo, convert the photo color intensity into RGB values, and automatically generate a linear fitting relationship between R, G, B values ​​and concentration.

[0013] As a further embodiment of the present invention, in step 3, free radicals are captured by 5,5-dimethyl-1-pyrroline-N-oxide and 2,2,6,6-tetramethylpiperidine capture agents at room temperature and the peroxidase activity kinetics test is used to identify the peroxidase activity of iron-doped carbon quantum dots, and the specific steps are:

[0014] Step 31, catalyzing H2O2 to oxidize TMB to produce oxidized TMB through the catalysis of iron-doped carbon quantum dots, and the solution appears blue with a characteristic absorption peak at 652 nm;

[0015] Step 32: Draw a Michaelis-Menten curve by adjusting the concentrations of TMB and H2O2.

[0016] As a further embodiment of the present invention, in step 32, the concentration of TMB is in the range of 0.15-1.2 m·mol·L -1 , the concentration range of H2O2 is 0.8-3.8m·mol·L -1 .

[0017] As a further embodiment of the present invention, in step 32, the enzymatic reaction rate within the concentration range of TMB and the enzymatic reaction rate within the concentration range of H2O2 are described based on the Michaelis-Menten kinetic equation, wherein the formula for describing the enzymatic reaction rate within the concentration range of TMB is:

[0018]

[0019] Where: is the enzymatic reaction rate within the TMB concentration range, is the maximum initial velocity of TMB, is the Michaelis-Menten constant corresponding to TMB, specifically 0.256 m·mol·L -1 ,[S TMB ] is the concentration range of TMB.

[0020] As a further embodiment of the present invention, the formula for describing the enzymatic reaction rate within the concentration range of H2O2 is:

[0021]

[0022] Where: is the rate of the enzymatic reaction within the concentration range of H2O2, is the maximum initial velocity of H2O2, is the Michaelis-Menten constant corresponding to H2O2, specifically 0.772m·mol·L -1 , is the concentration range of H2O2.

[0023] As a further solution of the present invention, in step 4, a dual-mode sensing detection of ampicillin and tetracycline is constructed by using the colorimetric signal of the Fe-CDs+TMB+H2O2 system at different ampicillin concentrations, the fluorescence signal of the system at different tetracycline concentrations, and the selectivity of the sensor under interference factors. The specific steps are:

[0024] Step 41: Detect ampicillin by ultraviolet spectrophotometer. In the range of 0–300 μM, as the concentration of ampicillin increases, the absorbance at 652 nm gradually decreases, the blue color gradually fades, and the correlation coefficient R between the absorbance change at 652 nm and the ampicillin concentration is 2 is 0.998, and the fitted linear equation is:

[0025] y1=0.1006x1+0.2142;

[0026] Where: y1 is the absorbance change at 652nm, x1 is the concentration of ampicillin;

[0027] Step 42: Tetracycline was detected by fluorescence spectrophotometer. In the range of 0–600 μM, as the concentration of tetracycline increased, the fluorescence signal at 436 nm gradually weakened. The correlation coefficient R between the fluorescence change value at 436 nm and the tetracycline concentration was 2 is 0.995, and the fitted linear equation is:

[0028] y2=1431.53x2+1121.57;

[0029] Where: y2 is the fluorescence change value at 436nm, x2 is the tetracycline concentration.

[0030] As a further solution of the present invention, in step 5, a portable test strip is developed to realize rapid visual detection of antibiotics through a smartphone sensing platform, a colorimetric photo is taken by a smartphone, an area in the photo is selected, the color intensity of the photo is converted into RGB value, and a linear fitting relationship between R, G, B values ​​and concentration is automatically generated, and the specific steps are:

[0031] Step 51, printing iron-doped carbon quantum dots (Fe-CDs) on filter paper, developing a portable test strip, dropping the analyte, and using the complementary metal oxide semiconductor (CMOS) in the smartphone to capture the corresponding colorimetric / fluorescence image;

[0032] Step 52, extracting RGB information of the photo by using a color recognition application, and using G / R as a fitting parameter to achieve semi-quantitative determination;

[0033] Step 53: drip an ampicillin standard solution with a concentration range of 0 to 150 μg / ml onto the test paper and dry it naturally. As the concentration increases, the blue color of the test paper gradually fades. At this time, the correlation coefficient R between the gamma ratio change value and the concentration of the ampicillin standard solution is 2 is 0.999, and the fitted linear equation is:

[0034] y3=0.01246x3+0.5686;

[0035] Where: y3 is the gamma ratio change value, x3 is the concentration of ampicillin standard solution;

[0036] Step 54: drop a tetracycline standard solution with a concentration range of 0 to 200 μg / ml onto the test paper, and extract the RGB information of the photo. At this time, the correlation coefficient R between the gamma ratio change value and the concentration of the tetracycline standard solution is 2 is 0.991, and the fitted linear equation is:

[0037] y3=0.02402x4+1.1732;

[0038] Where: y3 is the gamma ratio change value, x4 is the concentration of tetracycline standard solution.

[0039] The present invention discloses a method for visually detecting ampicillin and tetracycline based on iron-doped carbon quantum dots: the present invention synthesizes iron-doped carbon quantum dots (Fe-CDs) by a hydrothermal method, characterizes the iron-doped carbon quantum dots (Fe-CDs), identifies the peroxidase activity of the iron-doped carbon quantum dots (Fe-CDs), performs dual-mode sensing detection of ampicillin and tetracycline based on colorimetric / fluorescence signals, and finally uses a smart phone sensing platform to automatically generate a linear fitting relationship between R, G, B values ​​and concentrations, thereby achieving real-time, on-site, instrument-free and cost-effective instant detection, and will promote research on environmental chemical behavior, temporal and spatial distribution and biological ecotoxicity of antibiotics; avoids the time and strict conditions required for enzyme culture, making it more suitable for real-time detection in various scenarios; the detection response is completed in a relatively short time, meeting the current growing demand for rapid detection; develops a portable test strip, and with the help of a color identifier, realizes a portable integrated device for smart phone sensing through 3D printing, which can realize quantitative detection result reading. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A schematic diagram of a process for visual detection of ampicillin and tetracycline based on iron-doped carbon quantum dots provided in Example 1 of the present invention;

[0041] Figure 2 This is a characterization image of the iron-doped carbon quantum dots (Fe-CDs) of the present invention;

[0042] Figure 3 This is an identification image of the peroxidase activity of the iron-doped carbon quantum dots (Fe-CDs) of the present invention;

[0043] Figure 4 This is a schematic diagram of the dual-mode sensing detection of ampicillin and tetracycline of the present invention;

[0044] Figure 5This is a schematic diagram of a portable paper sensor based on a smart phone according to the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the accompanying drawings in the present invention to clearly and completely describe the technical solution in the present invention. Obviously, the described technical solution is only a part of the present invention, not all of it. Based on the technical solution in the present invention, all other technical solutions obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0046] Example 1

[0047] Figure 1 The schematic diagram of the process of visual detection method of ampicillin and tetracycline based on iron-doped carbon quantum dots provided in the first embodiment of the present invention is shown. Figure 1 As shown, a visual detection method of ampicillin and tetracycline based on iron-doped carbon quantum dots in this embodiment includes the following steps:

[0048] Step 1, synthesizing iron-doped carbon quantum dots (Fe-CDs) by a hydrothermal method;

[0049] Step 2, characterizing the iron-doped carbon quantum dots (Fe-CDs) by transmission electron microscopy (TEM), UV-visible spectroscopy, fluorescence spectroscopy, and X-ray photoelectron spectroscopy (XPS) spectroscopy;

[0050] Step 3, detecting the electron paramagnetic resonance (EPR) signal by electron paramagnetic resonance spin trapping technology, capturing free radicals and peroxidase (POD) activity kinetics test with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) trapping agents at room temperature, and identifying the peroxidase activity of iron-doped carbon quantum dots (Fe-CDs);

[0051] Step 4, constructing a dual-mode sensing detection of ampicillin and tetracycline through the colorimetric signal of the Fe-CDs+TMB+H2O2 system at different ampicillin (Amp) concentrations, the fluorescence signal of the system at different tetracycline (TC) concentrations, and the selectivity of the sensor under interference factors;

[0052] Step 5: Develop a portable test strip to achieve rapid visual detection of antibiotics through a smartphone sensing platform. Take a colorimetric photo with a smartphone, select a specific area of ​​the photo, convert the photo color intensity into RGB values, and automatically generate a linear fitting relationship between R, G, B values ​​and concentration.

[0053] Among them, TMB refers to 3,3',5,5'-tetramethylbenzidine, which is a non-carcinogenic chromogenic substrate.

[0054] In step 1, the specific steps of synthesizing iron-doped carbon quantum dots (Fe-CDs) by hydrothermal method are as follows: 1.3 g of citric acid, 0.15 g of ferric chloride hexahydrate and 150 μL of ethylenediamine are dissolved in 20 mL of deionized water, the solution is transferred to a polytetrafluoroethylene-lined autoclave, and heated at 230° C. for 12 hours. After the mixture is cooled to room temperature, the brown-black suspension is filtered through a 0.22 μm filter membrane, and then dialyzed with deionized water for 12 hours and stored in a 4° C. refrigerator.

[0055] In step 2, the specific steps of characterizing the iron-doped carbon quantum dots (Fe-CDs) are as follows: the iron-doped carbon quantum dots (Fe-CDs) are synthesized and constructed by a hydrothermal method, and because of the fluorescent signal and POD enzyme activity, a unique colorimetric / fluorescence dual-mode detection is produced, and the iron-doped carbon quantum dots (Fe-CDs) are characterized by TEM images, fluorescence spectra, and XPS spectra;

[0056] like Figure 2 The characterization image of iron-doped carbon quantum dots (Fe-CDs) is shown in Figure 2. Figure 2 The transmission electron microscope (TEM) image of iron-doped carbon quantum dots (Fe-CDs) shown in Figure A shows that the material size distribution is uniform, with an average diameter of 3.1nm; Figure 2 Figure B shows the fluorescence spectrum display image of iron-doped carbon quantum dots (Fe-CDs). Iron-doped carbon quantum dots (Fe-CDs) exhibit an optimal emission wavelength at 436nm and appear blue under 365nm ultraviolet light, verifying that they have fluorescent properties; Figure 2 The X-ray photoelectron spectroscopy (XPS) spectrum of iron-doped carbon quantum dots (Fe-CDs) shown in C shows that Fe-CDs are mainly composed of four elements: C1s, O1s, N1s and Fe2p, proving the successful doping of Fe elements; Figure 2 D shows the X-ray photoelectron spectroscopy (XPS) spectrum display image of C1s, which is resolved into three peaks at 284.6, 285.9 and 287.9 ​​eV, which are attributed to three different types of carbon atoms.

[0057] In step 3, the peroxidase activity of iron-doped carbon quantum dots (Fe-CDs) is identified by capturing free radicals and peroxidase (POD) activity kinetics test using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP) capture agents at room temperature, and the specific steps are as follows:

[0058] Step 31, catalyzing H2O2 to oxidize TMB to produce oxidized TMB (ox-TMB) through the catalysis of iron-doped carbon quantum dots (Fe-CDs), and the solution appears blue with a characteristic absorption peak at 652 nm;

[0059] Step 32: Draw a Michaelis-Menten curve by adjusting the concentrations of TMB and H2O2.

[0060] In step 32, the concentration of TMB is in the range of 0.15-1.2 m·mol·L -1 , the concentration range of H2O2 is 0.8-3.8m·mol·L -1 .

[0061] In step 32, the Michaelis-Menten curve shows that the maximum initial velocity (Vmax) of TMB and H2O2 is 3.77×10 -8 and 3.54×10 -8 mol·L -1 ·s -1 , the corresponding Michaelis-Menten constant (K m ) are 0.256 and 0.772 m·mol·L -1 .

[0062] In step 32, the enzymatic reaction rate within the concentration range of TMB and the enzymatic reaction rate within the concentration range of H2O2 are described based on the Michaelis-Menten kinetic equation, wherein the formula for describing the enzymatic reaction rate within the concentration range of TMB is:

[0063]

[0064] Where: is the enzymatic reaction rate within the TMB concentration range, is the maximum initial velocity of TMB, is the Michaelis-Menten constant corresponding to TMB, specifically 0.256 m·mol·L -1 ,[S TMB ] is the concentration range of TMB.

[0065] The formula describing the rate of the enzymatic reaction within the concentration range of H2O2 is:

[0066]

[0067] Where: is the rate of the enzymatic reaction within the concentration range of H2O2, is the maximum initial velocity of H2O2, is the Michaelis-Menten constant corresponding to H2O2, specifically 0.772m·mol·L -1 , is the concentration range of H2O2.

[0068] like Figure 3 The identification image of the peroxidase activity of iron-doped carbon quantum dots (Fe-CDs) is shown in Figure 3 As shown in A, under the catalysis of iron-doped carbon quantum dots (Fe-CDs), H2O2 catalyzes the oxidation of TMB, and the solution appears blue due to the generation of ox-TMB; Figure 3 As shown in B, its characteristic absorption peak is at 652nm; Figure 3 The EPR spectrum shown in C further confirms the existence of free radical intermediates. Using DMPO / TEMP as a free radical scavenger, the signal peak of ·OH can be clearly observed. Figure 3 D and Figure 3 As shown in E, by adjusting TMB (0.15-1.2 m·mol·L -1 ) and H2O2 (0.8-3.8m·mol·L -1 ) concentration, while keeping other variables within the optimal concentration range, and plotting the Michaelis-Menten curve to clarify the kinetic mechanism of peroxidase activity. The maximum initial velocity (Vmax) of TMB and H2O2 was 3.77×10 -8 and 3.54×10 -8 mol·L -1 ·s -1 , the corresponding Michaelis-Menten constants (Km) are 0.256 and 0.772 m·mol·L -1 Compared with other enzymes reported in the literature, Fe-CDs not only exhibited a higher Vmax but also a significantly lower Km, showing a more pronounced affinity for TMB.

[0069] As a further solution of the present invention, in step 4, a dual-mode sensing detection of ampicillin and tetracycline is constructed by the colorimetric signal of the Fe-CDs+TMB+H2O2 system at different ampicillin (Amp) concentrations, the fluorescence signal of the system at different tetracycline (TC) concentrations, and the selectivity of the sensor under interference factors. The specific steps are:

[0070] Step 41: Detect ampicillin (Amp) by ultraviolet spectrophotometer. In the range of 0-300 μM, as the concentration of ampicillin (Amp) increases, the absorbance at 652 nm gradually decreases, the blue color gradually fades, and the correlation coefficient R between the absorbance change at 652 nm and the concentration of ampicillin (Amp) is 2 is 0.998, and the fitted linear equation is:

[0071] y1=0.1006x1+0.2142;

[0072] Where: y1 is the absorbance change at 652nm, x1 is the concentration of ampicillin (Amp);

[0073] Step 42: Tetracycline (TC) is detected by a fluorescence spectrophotometer. In the range of 0–600 μM, as the concentration of tetracycline (TC) increases, the fluorescence signal at 436 nm gradually weakens. The correlation coefficient R between the fluorescence change value at 436 nm and the tetracycline concentration fitting is 2 is 0.995, and the fitted linear equation is:

[0074] y2=1431.53x2+1121.57;

[0075] Where: y2 is the fluorescence change value at 436nm, x2 is the tetracycline concentration.

[0076] like Figure 4 The schematic diagram of the dual-mode sensing detection of ampicillin and tetracycline is shown in FIG. Figure 4 As shown in A, for the detection of ampicillin, by using a UV spectrophotometer, such as Figure 4 As shown in B, in the range of 0–300 μM, it was found that as the concentration of ampicillin increased, the absorbance at 652 nm gradually decreased, the blue color gradually faded, and the absorbance change at 652 nm showed a good linear relationship with the logarithm of the ampicillin concentration. In order to prove that this method has good specificity, Figure 4 As shown in C, various inorganic ions, representative amino acids, and common antibiotics were used as negative controls, and no significant interference with the detection of ampicillin was found. Figure 4 As shown in D, for the detection of tetracycline, Figure 4 As shown in Figure E, in the range of 0–600 μM, as the concentration of tetracycline increases, the fluorescence signal at 436 nm gradually weakens. The fluorescence change value at 436 nm shows a good linear relationship with the logarithm of the tetracycline concentration, as shown in Figure E. Figure 4 It was also found that potential interfering factors had little effect on the fluorescence intensity as shown in Figure F. Based on these findings, it can be concluded that the proposed sensor has excellent electrical polarity and anti-interference performance, further demonstrating that the sensor can become a high-precision and high-resolution tool for antibiotic detection.

[0077] In step 5, a portable test strip is developed to achieve rapid visual detection of antibiotics through a smartphone sensing platform. A colorimetric photo is taken by a smartphone, a specific area of ​​the photo is selected, the photo color intensity is converted to RGB value, and a linear fitting relationship between R, G, B values ​​and concentration is automatically generated. The specific steps are as follows:

[0078] Step 51, printing iron-doped carbon quantum dots (Fe-CDs) on filter paper, developing a portable test strip, dropping the analyte, and using the complementary metal oxide semiconductor (CMOS) in the smartphone to capture the corresponding colorimetric / fluorescence image;

[0079] Step 52, extracting RGB information of the photo by using a color recognition application, and using G / R as a fitting parameter to achieve semi-quantitative determination;

[0080] Step 53: drip an ampicillin standard solution with a concentration range of 0 to 150 μg / ml onto the test paper and dry it naturally. As the concentration increases, the blue color of the test paper gradually fades. At this time, the correlation coefficient R between the change value of the gamma ratio (G / R) and the concentration of the ampicillin standard solution is fitted. 2 is 0.999, and the fitted linear equation is:

[0081] y3=0.01246x3+0.5686;

[0082] Where: y3 is the change value of the gamma ratio (G / R), x3 is the concentration of the ampicillin standard solution;

[0083] Step 54: drop a tetracycline standard solution with a concentration range of 0 to 200 μg / ml onto the test paper, and extract the RGB information of the photo. At this time, the correlation coefficient R between the gamma ratio (G / R) change value and the concentration of the tetracycline standard solution is 2 is 0.991, and the fitted linear equation is:

[0084] y3=0.02402x4+1.1732;

[0085] Wherein: y3 is the change value of gamma ratio (G / R), and x4 is the concentration of tetracycline standard solution.

[0086] like Figure 5 The schematic diagram of the portable paper sensor based on smartphone is shown in Figure 4 As shown in A, iron-doped carbon quantum dots (Fe-CDs) are printed on filter paper to develop a portable test strip. The analyte is dropped into the test strip, and the corresponding colorimetric / fluorescence image is captured by the complementary metal oxide semiconductor (CMOS) in the smartphone. The RGB information of the photo is extracted using the Color Recognizer application, and G / R is used as the fitting parameter to conveniently achieve semi-quantitative determination. Figure 4 As shown in Figure B, a series of ampicillin standard solutions with concentrations ranging from 0 to 150 μg / ml were dropped onto the test paper and dried naturally. It was found that as the concentration increased, the blue color of the test paper gradually faded. Within this concentration range, the G / R change value showed a good linear relationship with the ampicillin concentration. Figure 4As shown in Figure C, the tetracycline standard solution with a concentration ranging from 0 to 200 μg / ml was dropped onto the test paper, and the RGB information of the photo was extracted. It was found that the G / R change value had a good linear relationship with the tetracycline concentration. The results showed that the portable smartphone detection platform can quickly realize the visual quantitative sensing of antibiotics with high sensitivity, which greatly expanded its application in the field of actual detection.

[0087] The embodiment of the present invention synthesizes iron-doped carbon quantum dots (Fe-CDs) by a hydrothermal method, characterizes the iron-doped carbon quantum dots (Fe-CDs), identifies the peroxidase activity of the iron-doped carbon quantum dots (Fe-CDs), performs dual-mode sensing detection of ampicillin and tetracycline based on an ultraviolet / fluorescence spectrophotometer, and finally develops a portable test strip, which uses a smartphone sensing platform to automatically generate a linear fitting relationship between R, G, B values ​​and concentrations, thereby achieving real-time, on-site, instrument-free and cost-effective instant detection, and will promote research on the environmental chemical behavior, temporal and spatial distribution and biological ecotoxicity of antibiotics; avoids the time and strict conditions required for enzyme culture, making it more suitable for real-time detection in various scenarios; the detection response is completed in a relatively short time, meeting the current growing demand for rapid detection; with the help of a color identifier, a portable integrated device for smartphone sensing is realized through 3D printing, which can realize quantitative detection result reading.

[0088] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

[0089] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots, characterized in that: The steps include: Step 1, synthesizing iron-doped carbon quantum dots by a hydrothermal method; Step 2, characterizing the iron-doped carbon quantum dots by transmission electron microscopy, UV-visible spectroscopy, fluorescence spectroscopy, and X-ray photoelectron spectroscopy; Step 3, detecting the electron paramagnetic resonance signal by electron paramagnetic resonance spin capture technology, capturing free radicals and peroxidase activity kinetics test with 5,5-dimethyl-1-pyrroline-N-oxide and 2,2,6,6-tetramethylpiperidine capture agents at room temperature, and identifying the peroxidase activity of iron-doped carbon quantum dots, the specific steps are as follows: Step 31, catalyzing H2O2 to oxidize TMB to produce oxidized TMB through the catalysis of iron-doped carbon quantum dots, and the solution appears blue with a characteristic absorption peak at 652 nm; Step 32, drawing a Michaelis-Menten curve by adjusting the concentrations of TMB and H2O2; Step 4: construct a dual-mode sensing detection of ampicillin and tetracycline through the colorimetric signal of the Fe-CDs+TMB+H2O2 system at different ampicillin concentrations, the fluorescence signal of the system at different tetracycline concentrations, and the selectivity of the sensor under interference factors. The specific steps are as follows: Step 41: Detect ampicillin by ultraviolet spectrophotometer. In the range of 0–300 μM, as the concentration of ampicillin increases, the absorbance at 652 nm gradually decreases, the blue color gradually fades, and the correlation coefficient R between the absorbance change at 652 nm and the ampicillin concentration is 2 is 0.998, and the fitted linear equation is: y1=0.1006x1+0.2142; Where: y1 is the absorbance change at 652nm, x1 is the concentration of ampicillin; Step 42: Tetracycline was detected by fluorescence spectrophotometer. In the range of 0–600 μM, as the concentration of tetracycline increased, the fluorescence signal at 436 nm gradually weakened. The correlation coefficient R between the fluorescence change value at 436 nm and the tetracycline concentration was 2 is 0.995, and the fitted linear equation is: y2=1431.53x2+1121.57; Where: y2 is the fluorescence change value at 436nm, x2 is the tetracycline concentration; Step 5. Develop a portable test strip to achieve rapid visual detection of antibiotics through a smartphone sensing platform. Take a colorimetric photo with a smartphone, select an area in the photo, convert the photo color intensity into RGB values, and automatically generate a linear fitting relationship between R, G, B values ​​and concentration.

2. A visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots according to claim 1, characterized in that: In step 32, the concentration of TMB is in the range of 0.15-1.2 mmol·L -1 , the concentration range of H2O2 is 0.8-3.8mmol·L -1 .

3. The visual detection method of ampicillin and tetracycline based on iron-doped carbon quantum dots according to claim 1, characterized in that: In step 32, the enzymatic reaction rate within the concentration range of TMB and the enzymatic reaction rate within the concentration range of H2O2 are described based on the Michaelis-Menten kinetic equation, wherein the formula for describing the enzymatic reaction rate within the concentration range of TMB is: Where: is the enzymatic reaction rate within the TMB concentration range, is the maximum initial velocity of TMB, is the Michaelis-Menten constant corresponding to TMB, specifically 0.256 mmol·L -1 ,[S TMB ] is the concentration range of TMB.

4. A visual detection method for ampicillin and tetracycline based on iron-doped carbon quantum dots according to claim 3, characterized in that: The formula describing the rate of the enzymatic reaction within the concentration range of H2O2 is: Where: is the rate of the enzymatic reaction within the concentration range of H2O2, is the maximum initial velocity of H2O2, is the Michaelis-Menten constant corresponding to H2O2, specifically 0.772mmol·L -1 , is the concentration range of H2O2.

5. The visual detection method of ampicillin and tetracycline based on iron-doped carbon quantum dots according to claim 1, characterized in that: In step 5, rapid visual detection of antibiotics is achieved through the smartphone sensing platform. A colorimetric photo is taken by the smartphone, an area in the photo is selected, the photo color intensity is converted into RGB values, and a linear fitting relationship between R, G, B values ​​and concentration is automatically generated. The specific steps are as follows: Step 51, printing iron-doped carbon quantum dots (Fe-CDs) on filter paper, developing a portable test strip, dropping the analyte, and using the complementary metal oxide semiconductor (CMOS) in the smartphone to capture the corresponding colorimetric / fluorescence image; Step 52, extracting RGB information of the photo by using a color recognition application, and using G / R as a fitting parameter to achieve semi-quantitative determination; Step 53: drip an ampicillin standard solution with a concentration range of 0 to 150 μg / ml onto the test paper and dry it naturally. As the concentration increases, the blue color of the test paper gradually fades. At this time, the correlation coefficient R between the gamma ratio change value and the concentration of the ampicillin standard solution is 2 is 0.999, and the fitted linear equation is: y3=0.01246x3+0.5686; Where: y3 is the gamma ratio change value, x3 is the concentration of ampicillin standard solution; Step 54: drop a tetracycline standard solution with a concentration range of 0 to 200 μg / ml onto the test paper, and extract the RGB information of the photo. At this time, the correlation coefficient R between the gamma ratio change value and the concentration of the tetracycline standard solution is 2 is 0.991, and the fitted linear equation is: y3=0.02402x4+1.1732; Where: y3 is the gamma ratio change value, x4 is the concentration of tetracycline standard solution.

Images