CN112304914B - Method for detecting tetracycline antibiotics based on fluorescence spectrometry - Google Patents
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Abstract
The invention provides a method for detecting tetracycline antibiotics based on fluorescence spectroscopy, which comprises the following steps: mixing the raw materials, namely firstly mixing the nano Fe 3 O 4 Mixing with tetracycline antibiotics and incubating, then uniformly mixing with rhodamine, hydrogen peroxide and potassium iodide, and adding a buffer solution to adjust the pH value to 3-5 to obtain a raw material mixed solution; forming a fluorescent solution, and carrying out water bath heating reaction on the raw material mixed solution at the temperature of 30-70 ℃ to form a chemical fluorescent solution; and (2) detecting the fluorescence intensity F of the chemical fluorescence solution by using a fluorescence spectrometer, and establishing a regression equation for detecting the tetracycline antibiotic according to the relation between the detected fluorescence intensity F and the concentration c of the tetracycline antibiotic. The method provided by the invention has the advantages of simple and convenient operation, higher sensitivity and good accuracy, and provides possibility for further analysis and application.
Description
Technical Field
The invention relates to a quantitative detection method of a fluorescence spectrometry, in particular to a method for detecting tetracycline antibiotics based on the fluorescence spectrometry.
Background
Antibiotics are widely known in human and veterinary medicine as pharmaceutical and food additives. According to the data of the food and drug administration of China, the annual yield of antibiotic materials in China in 2012 reaches 210,000 tons. Antibiotics can be released into the environment through various ways, and the residual excess antibiotics in the environment can cause environmental pollution and increase the drug resistance of bacteria. The use of antibiotics in large quantities can also lead to their accumulation in everyday food products, such as meat, eggs and dairy products, which accumulate in the human body through the food chain and impair human health. Therefore, it is very important to detect and control antibiotics in food.
The current detection methods of antibiotics mainly include chromatography, mass spectrometry, immunoassay, electrochemical method, fluorescence spectrometry, and the like. Among them, fluorescence spectroscopy is widely used because of its convenience and sensitivity. For example, aptamer-modified gold nanoparticles (AuNPs) are used to detect sulfadimidine; kanamycin is detected by utilizing aptamer-modified silver nanoparticles (AgNPs); the detection of chloramphenicol using aptamer sensors based on AuNPs, biotin and streptavidin, and the like. However, these methods usually require aptamers as recognition units, which increase the complexity of the operation and limit the scope of application. Therefore, a simple and sensitive method for antibiotic detection is urgently needed.
Tetracycline antibiotics (TCs) are one of the most widely used antibiotics, and mainly include the three antibiotics Oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC), and in addition, doxycycline (DC). A paper published in 2019 by Huang et al discloses a novel method for simultaneously extracting and determining TC, CTC and DC from eggs, which adopts a radiation type multi-componentMolecular Imprinting Solid Phase Extraction (MISPE) -PDMS/glass array chip coating magnetic graphene oxide (Fe) 3 O 4 /GO)(Huang,et al.,Chip-based multi-molecularly imprinted monolithic capillary array columns coated Fe 3 O 4 GO for selective extraction and mutation determination of tetracycline, chlorotetracycline and deoxytetracycline in genes, microchemical Journal 150 (2019), 104097. Although the method provided by Huang et al can identify the tetracycline antibiotics TC, CTC and DC from the eggs, the method has the problems of complicated modification process, complicated operation, need of complicated large-scale instruments such as HPLC, etc., and is not suitable for on-site analysis.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting tetracycline antibiotics based on fluorescence spectroscopy, so as to solve the above problems.
Further, it is an object of the present invention to provide a method for detecting tetracycline antibiotics based on fluorescence spectroscopy, which enables the quantitative detection of OTC, TC and CTC from food simply and conveniently.
Specifically, the invention provides a method for detecting tetracycline antibiotics based on fluorescence spectrometry, which comprises the following steps:
mixing the raw materials, namely firstly mixing the nano Fe 3 O 4 Mixing with tetracycline antibiotics, incubating, uniformly mixing with rhodamine, hydrogen peroxide and potassium iodide, and adding buffer solution to adjust pH value to 3-5 to obtain raw material mixed solution;
forming a fluorescent solution, and carrying out water bath heating reaction on the raw material mixed solution at the temperature of 30-70 ℃ to form a chemical fluorescent solution;
and (2) detecting the fluorescence intensity F of the chemical fluorescence solution by using a fluorescence spectrometer, and establishing a regression equation for detecting the tetracycline antibiotic according to the relation between the detected fluorescence intensity F and the concentration c of the tetracycline antibiotic.
Based on the above, the nano Fe 3 O 4 The preparation method comprises the following steps: feCl 3 Dissolving NaAc and the NaAc in the ethylene glycol, and uniformly mixing to obtain a mixed solution;reacting the mixture at 180-210 ℃ for 6-9 h, and cooling to room temperature to obtain an iron oxide mixture; collecting a black product from the iron oxide mixture with an external magnet; washing and drying the black product to obtain the nano Fe 3 O 4 。
Based on the above, the step of mixing the raw materials comprises: 10 mu L of 0.2-1.0 mg/mL nano Fe 3 O 4 Adding 10 mu L of tetracycline antibiotics into a 1.5mL centrifuge tube, incubating for 4-9 min, and adding 10 mu L of 0.2-1.2 mM Rh6G and 10 mu L of 10-120 mM H 2 O 2 10 mu L of 350-550 mM KI is added into the centrifuge tube and diluted to 1mL by HAc-NaAc buffer solution, so as to obtain the raw material mixed solution.
Based on the above, the step of forming a fluorescent solution includes: and uniformly mixing the raw material mixed solution, and heating in water bath at 30-70 ℃ for 20-60 min to form the chemical fluorescent solution.
Based on the above, the steps of detecting the luminescence and establishing the regression equation include: setting the width of a slit of the fluorescence spectrometer to be 0.5nm, detecting the fluorescence intensity F of the chemiluminescence solution at the position of 530-600 nm, and establishing a regression equation for detecting tetracycline antibiotics according to the relation between the detected fluorescence intensity F and the concentration c of the tetracycline antibiotics.
Based on the above, the tetracycline antibiotic is OTC, TC, CTC or any combination thereof.
Based on the above, when the tetracycline antibiotic is OTC with the concentration of 40-2400 nM, the regression equation is established as F =9.40049-0.00308 × c, wherein the correlation coefficient R 2 =0.99672, the lower detection limit was 20nM.
Based on the above, when the tetracycline antibiotic is TC with the concentration of 30-1800 nM, the regression equation is F =9.26289-0.00422 × c, wherein the correlation coefficient R 2 =0.99244, with a detection lower limit of 10nM.
Based on the above, when the tetracycline antibiotic is CTC with the concentration of 50-1500 nM, the regression equation established is F =9.91375-0.00555 xc, wherein the correlation coefficient R 2 ＝0.99453 at a lower detection limit of 40nM.
Compared with the prior art, the method for detecting the tetracycline antibiotics based on the fluorescence spectrometry provided by the invention firstly comprises the steps of detecting TCs and nano Fe 3 O 4 (abbreviated as "Fe 3 O 4 NPs ") in the presence of dissolved oxygen, fe in the complex 3 O 4 NPs can catalyze H 2 O 2 Formation of hydroxyl radicals (. OH),. OH and I - Reaction to form I 3 - ，I 3 - Forms an association with rhodamine to quench fluorescence. Because the fluorescence intensity change of rhodamine has good linear relation with the concentration of TCs, the detection lower limit is low, and the selectivity is good, the method for detecting TCs based on fluorescence spectrometry provided by the invention has the advantages of simple and convenient operation, higher sensitivity and good accuracy, and provides possibility for further analysis and application.
In addition, the detection method provided by the invention does not need recognition units such as antibodies and aptamers, does not need complex chemical synthesis operation or complex modification process, is simpler and quicker in operation, does not need strict storage conditions, and has low cost; the detection method uses ferroferric oxide as peroxidase, is more stable and is not easily influenced by environmental conditions; does not need complex large-scale instruments, and can be used for detecting the content of tetracycline antibiotics in actual foods such as milk, eggs, honey and the like.
Drawings
FIG. 1 is a schematic diagram of the detection principle of tetracycline antibiotics provided by the present invention.
FIG. 2 is a diagram of the UV-Vis spectrum of 2, 3-dihydroxybenzoic acid with or without OTC provided by the present invention.
FIG. 3 shows Fe provided in the examples of the present invention 3 O 4 Concentration versus fluorescence intensity variation factor Δ F.
FIG. 4 is a graph showing the relationship between Rh6G concentration and the change factor Δ F of fluorescence intensity according to the example of the present invention.
FIG. 5 is a graph of KI concentration versus the fluorescence intensity variation factor Δ F provided by an embodiment of the present invention.
FIG. 6 is a drawing H provided by an embodiment of the present invention 2 O 2 Concentration versus fluorescence intensity change factor Δ F.
FIG. 7 is a graph showing the relationship between the pH of the buffer solution and the change factor Δ F of fluorescence intensity according to an embodiment of the present invention.
FIG. 8 is a graph showing the relationship between the reaction temperature and the fluorescence intensity variation factor Δ F according to the example of the present invention.
FIG. 9 is a graph showing the relationship between the reaction time and the fluorescence intensity variation factor Δ F according to the example of the present invention.
FIG. 10 is a graph showing the change in fluorescence intensity of rhodamine 6G at various CTC concentrations provided in the examples of the present invention.
FIG. 11 is a graph showing the change in fluorescence intensity of rhodamine 6G at different TC concentrations provided by embodiments of the present invention.
FIG. 12 is a graph showing the change of fluorescence intensity of rhodamine 6G at different OTC concentrations provided by the embodiment of the invention.
FIG. 13 is a schematic diagram of a CTC concentration detection standard curve provided in an embodiment of the present invention.
FIG. 14 is a diagram illustrating a standard curve for detecting TC concentration according to an embodiment of the present invention.
FIG. 15 is a schematic diagram of a standard curve for detecting the concentration of OTC provided by the embodiment of the present invention.
FIG. 16 is a histogram of the specificity study provided by the example of the invention.
Detailed Description
The technical solution of the present invention is further described in detail by the following embodiments.
The reagent related to the embodiment of the invention is as follows: feCl 3 NaAC and H 2 O 2 Purchased from national drug group chemical agents limited (china); rhodamine 6G, KI and salicylic acid were obtained from alatin (shanghai); oxytetracycline, chlortetracycline, and tetracycline were purchased from mecoline (shanghai); penicillin, streptomycin, kanamycin, and naringin are all from solibao (beijing); milk, eggs and honey were obtained from supermarkets near zheng zhou university.
The embodiment of the invention relates to the following instruments: x-ray photoelectron spectroscopy (Thermo ESCAL AB250 XI), transmission electron microscope (Jepan, JEM-2100), fluorescence spectrometer (Edinburgh Spectrofluorometer FS 5), particle size and Zeta potentiometer (Micromeritics Nanopus-3), microplate reader (Molecular Devices), infrared spectrometer (Thermo Nicolet NEXUS 470).
Fe used in the examples of the present invention 3 O 4 NPs are self-made and are prepared by a process comprising: 0.52g FeCl was weighed 3 Adding the mixture into 25mL of ethylene glycol, stirring the mixture to dissolve the mixture, adding 2.25g of NaAc, and violently shaking the mixture for 30 minutes. Then the mixed solution is transferred to a reaction kettle, reacted for 8 hours at 200 ℃, naturally cooled to room temperature, and then a black product is collected by an external magnet. The black product was washed 3 times with ethanol and distilled water, respectively, and then dried in a lyophilizer.
The embodiment of the invention provides a method for detecting TCs based on fluorescence spectroscopy, which comprises the following specific steps:
mixing the raw materials, namely mixing 10 mu L of 0.2-1.0 mg/mL nano Fe 3 O 4 Adding 10 mu L of tetracycline antibiotics into a 1.5mL centrifuge tube for incubation for 4-9 min, then adding 10 mu L of 0.2-1.2 mM Rh6G and 10 mu L of 10-120 mM H 2 O 2 Adding 10 mu L of 350-550 mM KI into the centrifuge tube, and diluting the KI to 1mL by using HAc-NaAc buffer solution to obtain a raw material mixed solution; wherein, the TCs are OTC, TC and CTC;
forming a fluorescent solution, uniformly mixing the raw material mixed solution, and heating in a water bath at the temperature of between 30 and 70 ℃ for reaction for 20 to 60min to form a chemical fluorescent solution;
and (2) setting the width of a slit of the fluorescence spectrometer to be 0.5nm by luminescence detection and establishment of a regression equation, detecting the fluorescence intensity F of the chemofluorescence solution at 557nm, detecting the fluorescence intensity F of the chemofluorescence solution by using the fluorescence spectrometer, and establishing the regression equation for detecting the tetracycline antibiotic according to the relation between the detected fluorescence intensity F and the concentration c of the tetracycline antibiotic.
Therefore, the method for detecting TCs based on fluorescence spectroscopy provided by the invention mainly relates to Fe 3 O 4 Concentration of NPs, concentration of rhodamine, concentration of KI、H 2 O 2 The concentration of the raw material mixture, the pH value of the raw material mixture, the reaction temperature, the reaction time and the like, and these factors have important influences on the detection result. Before analyzing the influence of the above factors on the detection result, the feasibility of the detection method needs to be verified.
1. Feasibility test of detection method
The detection principle corresponding to the method for detecting TCs based on fluorescence spectrometry is shown in figure 1, and TCs and Fe 3 O 4 The NPs are complexed to form complexes in the presence of dissolved oxygen, fe therein 3 O 4 NPs catalyze H 2 O 2 Formation of OH, OH and I - Reaction to form I 3 - ，I 3 - Association with Rh6G to form
A polymer, whose fluorescence is quenched; meanwhile, the change of the fluorescence intensity of Rh6G has a good linear relation with the concentration of TCs. Therefore, the feasibility of the method for detecting TCs based on fluorescence spectrometry provided by the invention is tested by comparing the concentration of OH in the reaction system under the condition of existence of OTC.
The conditions of feasibility experiments (hereinafter referred to as feasibility experiments) of the detection method provided by the invention are tested: 20 μ L of 0.8mg/L Fe 3 O 4 NPs, 30. Mu.L of 1. Mu.M OTC, 50. Mu.L of 6mM salicylic acid, 50. Mu.L of 6mM H 2 O 2 Adding the mixture into a 96-well polystyrene white plate, then incubating for 30min at 37 ℃, and then detecting the UV-Vis spectrum in a 460-600 nm range by using a microplate reader. The parallel experiments were repeated 7 times in this condition.
Blank set of test conditions: basically the same as the feasibility experiment conditions, the main difference is that: the OTC solution in the above feasibility experimental conditions was replaced with an equal amount of purified water. In this condition, 7 replicates were performed.
Through experiments, the following results are found: when the wavelength of the excitation light is 527nm, rh6G has a strong fluorescence emission signal at 557nm, and TCs and Fe 3 O 4 In the presence of NPs, RThe fluorescence intensity of h6G was significantly reduced.
When salicylic acid is added to the reaction system constructed under the above feasibility test conditions, OH attacks salicylic acid and generates 2, 3-dihydroxybenzoic acid, which has a maximum absorption at 535nm, so that the OD value of 2, 3-dihydroxybenzoic acid reflects the concentration of OH. As shown in FIG. 2, the OD of 2, 3-dihydroxybenzoic acid was significantly higher in the presence of OTC.
The OD values of both the present and blank groups were analyzed by using paired T-test. The p values of the two groups were less than 0.01, which means that there was a significant difference between the two groups, i.e., fe after addition of OTC 3 O 4 NPs can catalyze H 2 O 2 More OH is produced. Thus, based on Fe 3 O 4 NPs detection of TCs is feasible.
2. Optimized analysis test for influence factors of detection system
The method for detecting TCs based on fluorescence spectroscopy provided by the present invention is further illustrated below with respect to the influence of these factors on the detection result.
Wherein the emission fluorescence intensity at 557nm was recorded as F, while the fluorescence intensity of the blank solution without OTC was recorded as F in the following optimization analysis test of each influencing factor 0 Δ F = (F) 0 -F)/F 0 Defined as the factor of change in fluorescence intensity. In order to obtain better detection results, the following reaction conditions were subjected to an optimization analysis using OTC with the fluorescence intensity variation factor as a dependent variable.
2.1Fe 3 O 4 Optimization analysis test of NPs concentration
The test parameter conditions are as follows: fe 3 O 4 NPs concentration of 0.2, 0.4, 0.6, 0.8, 1.0mg/mL, rh6G concentration of 1.0mM, KI concentration of 450mM, H 2 O 2 The concentration was 90mM, the pH of HAc-NaAc buffer was 3.5, the reaction temperature was 50 ℃ and the reaction time was 50min.
Fe 3 O 4 The optimized detection result of NPs concentration is shown in fig. 3: with Fe 3 O 4 The value of the fluorescence intensity change factor is gradually increased when the concentration of the NPs is increased and is in Fe 3 O 4 The value of the fluorescence intensity change factor reaches the maximum at the concentration of NPs of 0.8mg/mL and then decreases. Therefore, 0.8mg/mL of Fe was selected 3 O 4 Optimum concentration of NPs.
2.2 optimized assay for rhodamine concentration
The test parameter conditions are as follows: fe 3 O 4 NPs concentration of 0.8mg/mL, rh6G concentration of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2mM, KI concentration of 450mM 2 O 2 The concentration was 90mM, the pH of HAc-NaAc buffer was 3.5, the reaction temperature was 50 ℃ and the reaction time was 50min.
The optimized detection result of Rh6G concentration is shown in fig. 4: the fluorescence intensity change factor value gradually increased with the increase of Rh6G concentration, and the optimized detection result at Rh6G concentration is shown in fig. 3: the value of the fluorescence intensity change factor gradually increased with the increase in Rh6G concentration, and reached a maximum at an Rh6G concentration of 1.0mM and then decreased. Therefore, the optimum concentration of Rh6G was selected to be 1.0 mM.
2.3 optimization of KI concentration analysis test
The test parameter conditions are as follows: fe 3 O 4 NPs concentration is 0.8mg/mL, rh6G concentration is 1.0mM, KI concentration is 350, 400, 450, 500, 550mM respectively, H 2 O 2 The concentration was 90mM, the pH of HAc-NaAc buffer was 3.5, the reaction temperature was 50 ℃ and the reaction time was 50min.
The optimized detection result of the KI concentration is shown in FIG. 5: the value of the factor of change in fluorescence intensity increased first and then decreased gradually as the concentration of KI increased, and reached a maximum at a KI concentration of 450 mM. Therefore, an optimal concentration of 450mM for KI was chosen.
2.4H 2 O 2 Optimized assay for concentration
The test parameter conditions are as follows: fe 3 O 4 NPs concentration of 0.8mg/mL, rh6G concentration of 1.0mM, KI concentration of 450mM, H 2 O 2 The concentrations were 10, 30, 60, 90 and 120mM, respectively, the pH of the HAc-NaAc buffer was 3.5, the reaction temperature was 50 ℃ and the reaction time was 50min.
H 2 O 2 Optimized detection of concentrationThe measurement results are shown in FIG. 6: with H 2 O 2 The fluorescence intensity change factor value increases and then decreases with increasing concentration, and is at H 2 O 2 The value of the fluorescence intensity change factor reached the maximum at a concentration of 90 mM. Therefore, 90mM was chosen as H 2 O 2 The optimum concentration of (c).
2.5 optimization of the pH value of HAc-NaAc buffer
The test parameter conditions are as follows: fe 3 O 4 NPs concentration was 0.8mg/mL, rh6G concentration was 1.0mM, KI concentration was 450mM 2 O 2 The concentrations were 10, 30, 60, 90 and 120mM, respectively, the pH values of HAc-NaAc buffers were 3.0, 3.5, 4.0, 4.5 and 5.0, respectively, the reaction temperature was 50 ℃ and the reaction time was 50min.
The optimized detection result of the HAc-NaAc buffer solution pH value is shown in FIG. 7: the fluorescence intensity change factor value is zigzag with the increase of the pH value of the HAc-NaAc buffer solution, and reaches the maximum at the pH value of 3.5 of the HAc-NaAc buffer solution. Therefore, the optimal pH for HAc-NaAc buffer was chosen to be 3.5.
2.6 optimized assay of reaction temperature
The test parameter conditions are as follows: fe 3 O 4 NPs concentration of 0.8mg/mL, rh6G concentration of 1.0mM, KI concentration of 450mM, H 2 O 2 The concentration is 90mM, the pH value of HAc-NaAc buffer solution is 3.5, the reaction temperature is 30, 40, 50, 60 and 70 ℃ respectively, and the reaction time is 50min.
The optimized detection results of the reaction temperature are shown in fig. 8: the value of the fluorescence intensity variation factor varied in a zigzag manner with the increase of the reaction temperature, and reached the maximum at a reaction temperature of 50 ℃. Therefore, 50 ℃ was selected as the optimum reaction temperature.
2.7 optimization of reaction time analysis
The test parameter conditions are as follows: fe 3 O 4 NPs concentration was 0.8mg/mL, rh6G concentration was 1.0mM, KI concentration was 450mM 2 O 2 The concentration is 90mM, the pH value of HAc-NaAc buffer solution is 3.5, the reaction temperature is 50 ℃, and the reaction time is 20, 30, 40, 50 and 60min.
The results of the optimized detection of the reaction time are shown in fig. 9: the value of the fluorescence intensity change factor varied in a zigzag manner with the increase of the reaction time, and reached the maximum at a reaction time of 50min. Therefore, 50min was chosen as the optimal reaction time.
3. Establishment of a Standard Curve
Under the condition of the same other conditions, CTC, TC and OTC are respectively taken as detection targets, and the fluorescence change of rhodamine 6G is detected by changing the corresponding concentration.
Specifically, the test parameter conditions are respectively as follows: the concentration of CTC is 0, 50, 300, 500, 700, 1000, 1200, 1500nM respectively;
the concentration of TC is 0, 30, 300, 600, 900, 1200, 1500 and 1800nM respectively;
concentrations of OTC were 0, 40, 400, 800, 1200, 1600, 2000, 2400nM, respectively;
Fe 3 O 4 NPs concentration was 0.8mg/mL, rh6G concentration was 1.0mM, KI concentration was 450mM 2 O 2 The concentrations are 90mM, the pH value of HAc-NaAc buffer solution is 3.5, the reaction temperatures are 50 ℃ respectively, and the reaction time is 50min respectively.
Three replicates were performed for each concentration of each TCs and the results are shown in figures 10-12, respectively. Based on the detection results shown in fig. 10 to 12, respectively, and using the concentration of each TCs as the abscissa and the fluorescence intensity F as the ordinate, linear fitting was performed to obtain linear equations for each TCs, as shown in fig. 13 to 15, respectively.
The method for determining the lower detection limit of the linear equation established by the method provided by the invention comprises the following steps:
CTC samples in 5 low concentration regions at 5nM, 10nM, 20nM, 30nM and 40nM concentrations were measured for their mean fluorescence intensity according to the above-described detection method. The fluorescence intensity of three blanks was measured simultaneously in triplicate for each concentration, and the mean value of the fluorescence intensity minus the three standard deviations was calculated as F Blank -3SD, detected below F Blank The concentration corresponding to the fluorescence intensity of-3 SD was the lower detection limit. Linear Range and detection of Linear equations for fitted CTCsThe lower line is shown in table 1 below.
TC samples of 5 low concentration regions at 5nM, 10nM, 15nM, 20nM, and 25nM concentrations were measured for their mean fluorescence intensity according to the above-described detection method. The fluorescence intensity of three blanks was measured simultaneously in triplicate for each concentration, and the mean value of the fluorescence intensity minus the three standard deviations was calculated as F Blank -3SD, detected below F Blank The concentration corresponding to the fluorescence intensity of-3 SD was the lower detection limit. The linear range and the lower detection line of the fitted linear equation of TC are shown in table 1 below.
OTC samples in 4 low concentration regions with concentrations of 5nM, 10nM, 20nM and 30nM, respectively, were measured for their mean fluorescence intensity according to the above-described detection method. The fluorescence intensity of three blanks was measured simultaneously in triplicate for each concentration, and the mean value of the fluorescence intensity minus the three standard deviations was calculated as F Blank -3SD, detected below F Blank The concentration corresponding to the fluorescence intensity of-3 SD was the lower detection limit. The linear range and the lower detection line of the fitted linear equation of OTC are shown in table 1 below.
TABLE 1 comparison of Linear Range and lower detection limits for the method of the present invention with existing detection methods
Wherein the "electrochemical" related data in Table 1 are derived from "Zheng, D.D., et al, an electrochemical biosensor for the direct detection of the aerobic in mouse blood and urine, analysis, 2013.138 (6): p.1886-1890.", "high performance liquid chromatography" related data are derived from "Perez-Si, I.2015., specification of the aerobic in milk by polymer, specification of the aerobic in capillary blood, 718.42-46", "colorimetric" related data are derived from mineral, H.I.A.S.M.and G.F.development, analysis chip acta, 2012.42-46 ".
As can be seen from table 1: the TCs detection method provided by the embodiment of the invention has better sensing performance, is simple, and does not need special modification, thereby greatly reducing the complexity of operation and expanding the application range of the TCs detection method.
4. Verification test of TCs detection method provided by the embodiment of the invention
4.1 specificity
In order to evaluate the selectivity of the method for detecting TCs provided by the embodiment of the present invention for TCs, the signal response of the method to certain interfering substances was investigated. As shown in FIG. 16, TCs can significantly cause Rh6G fluorescence quenching compared with other substances, resulting in a significant change in Δ F, indicating that the method for detecting TCs based on fluorescence spectroscopy provided by the present invention has higher selectivity for TCs.
Wherein the specifically added interfering substances are: 0.5238g of casein was weighed and dissolved in 4.365mL of water at a concentration of 1.2X 10 -4 g/L. 0.4253g of milk protein is weighed out and dissolved in 5.316mL of purified water with the concentration of 8.5X 10 -5 g/L; 6.7mg of potassium chloride was weighed, and 8.98mL of purified water was added thereto and diluted 10-fold to a concentration of 1mM. Weighing 10.5mg of calcium chloride, dissolving in 9.45mL of purified water, and diluting by 10 times to obtain 1mM calcium chloride; 5.2mg of alpha-lactose was weighed out and dissolved in 7.6mL of purified water at a concentration of 2mM. Weighing 7.1mg of sucrose, dissolving in 10.38mL of purified water, and dissolving at the concentration of 2mM; weighing 4.4mg of naringin, dissolving in 3.79mL of purified water, diluting by 20 times, and adjusting the concentration to 100 mu M; weighing 9.8mg of penicillin, dissolving in 5.5mL of purified water, diluting by 50 times, and adjusting the concentration to 100 mu M; weighing 4.9mg kanamycin, dissolving in 4.22mL purified water, diluting 20 times, and the concentration is 100 MuM; 7.6mg of streptomycin was weighed, dissolved in 5.2mL of pure water, and diluted 10-fold to a concentration of 100. Mu.M.
4.2 recovery of food samples from the spiked samples
In order to evaluate the utility of the method for detecting TCs provided in the embodiments of the present invention, antibiotics were detected in actual food samples. The embodiment of the invention detects the concentration of TCs in milk, egg and honey samples under the optimal condition of each parameter.
4.2.1 recovery of milk samples with Standard addition
The detection method of TCs in the milk sample comprises the following steps: adding a 2M trichloroacetic acid solution to milk and carrying out ultrasonic treatment for 15 minutes, and then centrifuging the mixed solution at 3000rpm for 15 minutes to form a milk supernatant; taking the milk supernatant, adjusting the pH value to be neutral by using NaOH solution, filtering the solution by using a 0.22 mu m filter membrane, storing the solution at 4 ℃, and then respectively adding TCs with different concentrations into the milk supernatant to form a milk sample to be detected containing antibiotics; by adopting the method for detecting TCs provided by the embodiment of the invention, the fluorescence intensity of the milk sample to be detected is detected under the optimal condition of each parameter obtained by the part 2 of the influence factor optimization analysis test; and (3) calculating the concentration of TCs in the milk sample to be detected according to the detected fluorescence intensity and a linear equation fitted by combining the part of '3 and the establishment of the standard curve'. The results of the measurements are shown in tables 2 to 4.
TABLE 2 results of OTC detection and recovery in milk samples
| Adding amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 200 | 224.09±45.02 | 112.04 | 20.09 |
| 1200 | 1173.31±121.03 | 97.77 | 10.31 |
| 2400 | 2551.77±50.34 | 106.32 | 1.97 |
TABLE 3 CTC detection results and recovery in milk samples
| Adding amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 300 | 366.57±29.33 | 122.19 | 8.00 |
| 900 | 981.42±61.14 | 109.04 | 6.22 |
| 1500 | 1555.53±16.18 | 103.7 | 1.04 |
TABLE 4 results of TC detection and recovery in milk samples
| Adding amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 200 | 251.01±17.45 | 125.50 | 6.95 |
| 1100 | 1225.8±34.02 | 111.43 | 2.77 |
| 1800 | 1778.28±26.44 | 98.79 | 1.48 |
As can be seen from tables 2 to 4: the method for detecting TCs provided by the embodiment of the invention has the advantages that the detected value and the added value are basically consistent, and the recovery rate is between 97.77% and 125.50%, thus the method provided by the embodiment of the invention can be effectively applied to milk sample detection.
4.2.2 recovery of egg samples from the feed
The detection method of TCs in the egg sample comprises the following steps: adding 20mL of PBS buffer solution with pH value of 7.2 into 5g of homogenized eggs, vortexing for 1min, and then shaking for 10min; taking 0.25mL of supernatant, adding 0.25mL of PBS buffer solution, mixing uniformly, and centrifuging at 5000r/min for 1.5min to form egg supernatant; taking the egg supernatant, adjusting the pH value to be neutral by using NaOH solution, filtering by using a 0.22-micron filter membrane, storing at 4 ℃, and then adding TCs with different concentrations into the egg supernatant respectively to form an egg sample to be detected, wherein the egg sample contains antibiotics; by adopting the method for detecting TCs provided by the embodiment of the invention, the fluorescence intensity of the egg sample to be detected is detected under the optimal condition of each parameter obtained by the part 2 of the influence factor optimization analysis test; and (3) calculating the concentration of TCs in the egg sample to be detected according to the detected fluorescence intensity and a linear equation fitted by combining the part of '3 and the establishment of the standard curve'. The results of the measurements are shown in tables 5 to 7.
TABLE 5 results and recovery of OTC in egg samples
| Adding amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 300 | 361.95±66.78 | 120.65 | 18.45 |
| 1500 | 1647.36±77.02 | 109.82 | 4.67 |
| 2400 | 2431.21±32.49 | 101.3 | 1.33 |
TABLE 6 CTC detection results and recovery in egg samples
| Addition amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 300 | 320.45±13.06 | 106.81 | 16.3 |
| 1000 | 973.55±58.41 | 97.35 | 5.99 |
| 1500 | 1366.97±58.70 | 91.13 | 4.29 |
TABLE 7 TC assay results and recovery in egg samples
| Addition amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 200 | 177.38±30.42 | 88.69 | 17.1 |
| 1000 | 1109.17±38.66 | 110.91 | 3.48 |
| 1800 | 1665.35±24.63 | 92.51 | 1.47 |
As can be seen from tables 5 to 7: the method for detecting TCs provided by the embodiment of the invention has the advantages that the detected value and the added value are basically consistent, and the recovery rate is between 88.69% and 120.65%, so that the method provided by the embodiment of the invention can be effectively applied to egg sample detection.
4.2.3 recovery of Honey sample from the Standard
The method for detecting TCs in the honey sample comprises the following steps: weighing 2.5g of honey into a 10mL centrifugal bottle, adding 5mL of phosphate buffer solution with pH =6.0, shaking for dissolution, then adding 30mL of extracting solution, performing vortex extraction for 5min, and centrifuging at the rotating speed of 8000r/min for 5min to form honey supernatant; taking the honey supernatant, adjusting the pH value to be neutral by using NaOH solution, filtering by using a 0.22 mu m filter membrane, storing at 4 ℃, and then respectively adding TCs with different concentrations into the honey supernatant to form a honey sample to be detected containing antibiotics; by adopting the method for detecting TCs provided by the embodiment of the invention, the fluorescence intensity of the honey sample to be detected is detected under the optimal condition of each parameter obtained by the part 2 of the influence factor optimization analysis test; and (3) calculating the concentration of TCs in the honey sample to be detected according to the detected fluorescence intensity and a linear equation fitted by combining the part of '3 and the establishment of the standard curve'. The results of the measurements are shown in tables 8 to 10.
TABLE 8 OTC test results and recovery in honey samples
| Adding amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 300 | 268.41±48.34 | 89.47 | 18.45 |
| 1500 | 1495.76±95.37 | 99.71 | 6.37 |
| 2400 | 2352.48±31.66 | 98.02 | 1.34 |
TABLE 9 CTC test results and recovery in Honey samples
| Addition amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 300 | 268.08±50.88 | 89.36 | 18.9 |
| 1000 | 903.88±55.73 | 90.38 | 6.16 |
| 1500 | 1384.42±17.73 | 92.29 | 1.28 |
TABLE 10 TC assay results and recovery in Honey samples
| Adding amount (nM) | Measured value (nM) | Recovery (%) | RSD(％) |
| 200 | 172.67±39.92 | 86.33 | 23.1 |
| 1000 | 1016.22±81.92 | 101.62 | 8.06 |
| 1800 | 1576.02±42.23 | 87.55 | 2.67 |
As can be seen from tables 8 to 10: the method for detecting TCs provided by the embodiment of the invention has the advantages that the detected value and the added value are basically consistent, and the recovery rate is between 86.33% and 101.62%, thus the method provided by the embodiment of the invention can be effectively applied to honey sample detection.
Therefore, the method for detecting TCs based on fluorescence spectroscopy provided by the embodiment of the invention uses Fe 3 O 4 NPs are fluorescent probes and can detect TCs in actual samples. Fe 3 O 4 NPs have enzyme-like activity and can catalyze H 2 O 2 Generation of OH, TCs and Fe 3 O 4 When NPs form a complex, OH and I are also produced in the presence of dissolved oxygen - Reaction to form I 3 - ，I 3 - Binding to Rh6G forms an association, which quenches the fluorescence. Therefore, the method for detecting TCs by fluorescence spectrometry provided by the embodiment of the invention has higher sensitivity and good accuracy in complex actual sample detection, and provides possibility for further analysis application.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention and not to limit it; although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: modifications of the embodiments of the invention or equivalent substitutions for parts of the technical features are possible; without departing from the spirit of the present invention, it is intended to cover all aspects of the invention as defined by the appended claims.
Claims (8)
1. A method for detecting tetracycline antibiotics based on fluorescence spectroscopy, comprising:
firstly, mixing the raw materials to obtain nano Fe 3 O 4 Mixing and incubating with tetracycline antibiotics, then uniformly mixing with rhodamine, hydrogen peroxide and potassium iodide, and adding a buffer solution to adjust the pH value to 3-5 to obtain a raw material mixed solution, wherein the tetracycline antibiotics are OTC, TC, CTC or any combination thereof;
forming a fluorescent solution, and carrying out water bath heating reaction on the raw material mixed solution at the temperature of 30-70 ℃ to form a chemical fluorescent solution;
and (2) detecting the fluorescence intensity F of the chemical fluorescence solution by using a fluorescence spectrometer, and establishing a regression equation for detecting the tetracycline antibiotic according to the relation between the detected fluorescence intensity F and the concentration c of the tetracycline antibiotic.
2. The method for detecting tetracycline antibiotics of claim 1, based on fluorescence spectroscopy, characterized in that the nano-Fe 3 O 4 The preparation method comprises the following steps: feCl 3 Dissolving NaAc and the NaAc in the ethylene glycol, and uniformly mixing to obtain a mixed solution; reacting the mixture at 180-210 ℃ for 6-9 h, and cooling to room temperature to obtain an iron oxide mixture; collecting a black product from the iron oxide mixture using an external magnet; washing and drying the black product to obtain the nano Fe 3 O 4 。
3. The method for detecting tetracycline antibiotics based on fluorescence spectroscopy of claim 1 or 2, wherein the step of mixing raw materials comprises: 10 mu L of 0.2-1.0 mg/mL nano Fe 3 O 4 Adding 10 mu L of tetracycline antibiotics into a 1.5mL centrifuge tube for incubation for 4-9 min, then adding 10 mu L of 0.2-1.2 mM Rh6G and 10 mu L of 10-120 mM H 2 O 2 10 mu L of 350-550 mM KI is added into the centrifuge tube and diluted to 1mL by HAc-NaAc buffer solution, so as to obtain the raw material mixed solution.
4. The method for detecting tetracycline antibiotics based on fluorescence spectroscopy of claim 3, wherein the step of forming a fluorescent solution comprises: and uniformly mixing the raw material mixed solution, and heating in water bath at 30-70 ℃ for 20-60 min to form the chemical fluorescent solution.
5. The method for detecting tetracycline antibiotics based on fluorescence spectroscopy of claim 4, wherein the steps of luminescence detection and establishing regression equations comprise: setting the width of a slit of the fluorescence spectrometer to be 0.5nm, detecting the fluorescence intensity F of the chemiluminescence solution at the 530-600 nm position, and establishing a regression equation for detecting tetracycline antibiotics according to the relation between the detected fluorescence intensity F and the concentration c of the tetracycline antibiotics.
6. The method for detecting tetracycline antibiotics of claim 5, wherein when the tetracycline antibiotics is OTC at a concentration of 40-2400 nM, the regression equation established is F =9.40049-0.00308 xc, where the correlation coefficient R is 2 =0.99672, detection lower limit is 20nM.
7. The method for detecting tetracycline antibiotics of claim 5, wherein when the concentration of the tetracycline antibiotics is TC of 30-1800 nM, the regression equation is established as F =9.26289-0.00422 xc, wherein the correlation coefficient R is 2 =0.99244, with a detection lower limit of 10nM.
8. The method for detecting tetracycline antibiotics of claim 5, wherein when the tetracycline antibiotics are CTCs at a concentration of 50-1500 nM, the regression equation established is F =9.91375-0.00555 xc, where the correlation coefficient R is 2 = 0.99453, lower detection limit 40nM.
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