CN116087164A

CN116087164A - Method for simply and rapidly detecting aureomycin

Info

Publication number: CN116087164A
Application number: CN202211659940.2A
Authority: CN
Inventors: 李胎花; 钱美汝; 王雪; 王安琪; 戴晶; 韩建刚; 金龙; 李威
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2023-05-09
Anticipated expiration: 2042-12-22
Also published as: CN116087164B

Abstract

The invention discloses a simple and rapid aureomycin detection method, and belongs to the technical field of antibiotic detection. According to the method, a surfactant PEI, metal ions Na+ and a solution to be detected are added into an acidic buffer solution to form a system to be detected, a fluorescence photometer is adopted to detect the fluorescence intensity of the system to be detected, the fluorescence intensity at 425nm is enhanced, and the solution to be detected contains CTC. The fluorescence sensitization method for detecting CTC is simple and convenient to construct and operate, does not need complex pretreatment process, expensive instruments and professional technical operators, solves the problems of poor stability, difficult field detection and difficult visualization of the detection method in the prior art while retaining rapid detection, and has the advantages of high sensitivity, high accuracy and low detection cost.

Description

Method for simply and rapidly detecting aureomycin

Technical Field

The invention belongs to the technical field of antibiotic detection, and in particular relates to a method based on cationic surfactant PEI and metal ion Na under weak acid condition ⁺ A method for detecting aureomycin by synergistic fluorescence sensitization.

Background

Chlortetracycline (CTC) is a typical tetracycline antibiotic, contains 4 linearly fused tetracyclic nuclei, and has good antibacterial properties and therapeutic effects on various bacteria. CTCs, a candidate drug for bacterial infection, are widely used in animal husbandry for preventing intestinal infection and promoting growth rate, controlling reproductive cycle and breeding ability thereof, and the like, in addition to treating human diseases. At present, CTCs in the environment are mainly derived from livestock and poultry cultivation, pollution discharge in hospitals and pharmaceutical factories, and the like. The abuse of CTC causes a large amount of residues in animal foods, and the residues are further absorbed by human bodies through food chains or food nets, so that the human health is seriously threatened, adverse physiological reactions such as allergy, gastrointestinal disturbance, hepatotoxicity and the like are caused, and more serious, the environmental pollution is caused, the CTC with lower concentration is remained in livestock and poultry products, various pathogenic bacteria are easily induced to generate drug resistance, and the treatment of human and livestock and poultry diseases is not facilitated. The European Union and China prescribe a limit to the amount of CTC in animal-derived foods, wherein the maximum limit in muscle of CTC is 100 μg/kg (about 0.19 μmol/L); the highest limit in the liver is 300. Mu.g/kg (about 0.58. Mu. Mol/L); the highest limit in the kidney is 600 mug/kg (1.16 mu mol/L); the maximum limit in milk is 100. Mu.g/kg (0.19. Mu. Mol/L).

The traditional method for detecting CTC mainly comprises high performance liquid chromatography, enzyme-linked immunoassay, colloidal gold immunoassay, capillary electrophoresis, immunological method and the like. Although these methods have the advantages of higher sensitivity and reliability, simple operation, etc., they require higher detection cost, complex pretreatment process, expensive instruments, professional technical operators, etc., and at the same time, the experimental period time is long, the requirements for sample purity are higher, and some methods cannot meet the requirement of rapid screening of large-scale samples, so that the use is limited. Therefore, it is necessary to develop a simple, convenient and rapid method with high sensitivity, high accuracy and low detection cost.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a simple and rapid CTC detection method based on cationic surfactant PEI and metal ion Na under weak acid condition ⁺ Realizes the synergistic fluorescence sensitization detection of CTC. Another technical problem to be solved by the present invention is to provide a method for quantitatively detecting CTCs.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a simple and rapid method for detecting CTC comprises the following steps: adding a surfactant PEI and a metal ion Na into an acidic buffer solution ⁺ And forming a system to be detected by the solution to be detected, detecting the fluorescence intensity of the system to be detected by adopting a fluorescence photometer, and increasing the fluorescence intensity at 425nm to ensure that the solution to be detected contains CTC.

Further, the acidic buffer solution was an acetic acid buffer solution having a pH of 6, and the concentration of the acetic acid buffer solution was 20mmol/L.

Further, the PEI concentration in the system to be measured is 0.80%, na ⁺ The concentration was 0.5mmol/L.

A method for quantitatively detecting CTCs comprising the steps of:

1) Adding a surfactant PEI and a metal ion Na into an acidic buffer solution ⁺ And CTCs of different standard concentrations, wherein standard curves and standard curve equations are made by taking the CTC concentration as an abscissa and taking a relative fluorescence value as an ordinate;

2) Adding a surfactant PEI and a metal ion Na into an acidic buffer solution ⁺ And forming a system to be detected by the solution to be detected, detecting the fluorescence intensity of the system to be detected at 425nm by adopting a fluorescence photometer, and substituting the measured relative fluorescence value into a standard curve equation to obtain the CTC concentration in the solution to be detected.

Further, the relative fluorescence values are fluorescence intensities F and F ₀ Fluorescence value difference (. DELTA.F) ₄₂₅ )。

Further, the standard curve equation is: y= 47.879x-0.3854.

Further, the minimum detection limit of CTC is 2.7nmol/L.

Compared with the prior art, the invention has the beneficial effects that:

(1) The fluorescence sensitization method for detecting CTC is simple and convenient to construct and operate, and solves the problems of poor stability, difficult field detection and difficult visualization of the detection method in the prior art while retaining rapid detection.

(2) The fluorescence sensitization method for rapidly detecting CTC is suitable for naked eyes to observe the change of fluorescence color, has the advantages of high sensitivity, high accuracy, low detection cost and the like, and does not need complex pretreatment process, expensive instruments and professional technical operators.

(3) The fluorescence sensitization method constructed by the invention has good selectivity, other tetracycline antibiotics, fluoroquinolone antibiotics and macrolide antibiotics are little interfered, and when the method is used for detecting CTC, the interference of other antibiotics in a sample can be eliminated, and the result is more reliable.

(4) The method has good labeling recovery rate in the detection of the actual water sample, high accuracy and reliable detection result.

Drawings

FIG. 1 is a graph of a feasibility analysis of PEI-based fluorescence sensitization to detect CTC;

FIG. 2 is a graph of the effect of different pH values on CTC fluorescence intensity and PEI at pH 3-6; the buffer solution with the pH value of 3-6 in the diagram A is 20mmol/L acetic acid, the buffer solution with the pH value of 7-10 is 20mmol/L BR, and the buffer solution with the pH value of 3-6 in the diagram B is 20mmol/L acetic acid (containing 0.80% PEI);

FIG. 3 is a graph showing the effect of different surfactants on CTC fluorescence intensity; wherein the buffer solution is acetic acid (20 mmol/L, pH=6), the concentration of CTC is 10 mu mol/L, and the concentration of the surfactant is 0.8%;

FIG. 4 is a line graph of the effect of PEI at various concentrations on system fluorescence; wherein the buffer is acetic acid (20 mmol/L, pH=6), and the concentration of CTC is 10 μmol/L;

FIG. 5 is a graph of the effect of different metal ions on CTC fluorescence intensity; wherein the buffer is acetic acid (20 mmol/L, pH=6) containing 0.8% PEI, the concentration of CTC is 10. Mu. Mol/L, and the concentration of metal ions is 200. Mu. Mol/L;

FIG. 6 is a line graph of the effect of different concentrations of NaCl on the fluorescence of the system; wherein the buffer is acetic acid (20 mmol/L, pH=6) containing 0.8% PEI, and the concentration of CTC is 10. Mu. Mol/L;

FIG. 7 is a graph of fluorescence spectra of the effect of different concentrations of CTC on the system and the difference in CTC concentration and system fluorescence intensity (. DELTA.F) ₄₂₅ ) Is a linear relationship graph of (2);

FIG. 8 is a diagram showing a specificity analysis of the detection method of the present invention; in the figure, CTC concentration was 5. Mu. Mol/L and the concentration of the other different antibiotics was 10. Mu. Mol/L.

Detailed Description

The invention is further described below in connection with specific embodiments. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. In the following examples, unless otherwise indicated, all technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

The fluorescence sensitization method based on polyethyleneimine provided by the embodiment can realize simple and rapid quantitative detection of CTC, and the principle (figure 1) is that: under weak acidic condition (pH 6), adding weak fluorescent substance CTC into the system to be tested containing PEI, and generating strong fluorescence emission band at 425nm when excited at 345 nm. Under the weak acidic condition, a large amount of amine groups rich in the PEI structure promote the deprotonation of cations in the CTC zwitterionic state, so that the fluorescence effect of the CTC is enhanced. Halogen group (Cl) contained in CTC ^- ) It can also promote chelation of metal ions in the buffer solution. According to the fluorescence sensitization of PEI to CTC, an analysis method for rapidly detecting CTC is constructed.

1) Determination of the pH value

CTC was added to acetic acid buffer solution (20 mmol/L) at pH3-6, BR buffer solution (20 mmol/L) at pH7-10, and acetic acid buffer at pH3-6, respectivelyIn the solution (20 mmol/L,0.80% PEI), the final concentration of CTC was set to 20. Mu. Mol/L, and the fluorescence intensity of the system was measured. The ionization state of CTCs is controlled by both the pH of the solution and the acidic dissociation constants (i.e., pKa values), with dissociation constants pKa1, pKa2, pKa3 being about 3, 7, and 9. As shown in a in fig. 2, CTCs exist in a zwitterionic state with little fluorescence and are more stable when the pH is between pKa1 and pKa 2; CTC is a weak fluorescent substance when the pH is equal to pKa 2; when the pH value is higher than pKa2, CTC exists mainly in an anionic state, system F ₄₂₅ The fluorescence value of (2) is gradually increased, and the fluorescent material shows strong fluorescence at the pH value of 10. In a weak acid environment, PEI rich in amino groups is added to promote the deprotonation of cations in a CTC zwitterionic state, and as shown in B in FIG. 2, after PEI is added to pH3-6, the fluorescence enhancement effect of pH6 is obvious, so that the optimal pH value is selected to be pH6.

2) Determination of surfactant and concentration

CTC was added to acetic acid buffer (20 mmol/L, pH 6) containing 0.10% Tween 20, triton X-100, PEI and cetyltrimethylammonium bromide (CTAB), and the final concentration of CTC was set to 10. Mu. Mol/L, as shown in FIG. 3, and the effect of amine-enriched PEI on the fluorescence enhancement of CTC was most remarkable as compared with other surfactants, so PEI was selected as a sensitizer of the system.

The fluorescence intensity of the system was measured by setting PEI at 0%, 0.10%, 0.20%, 0.40%, 0.50%, 0.60%, 0.80%, 1% in acetic acid buffer (20 mmol/L, pH 6), and by setting control and experimental groups (0 and 10. Mu. Mol/L CTC), as shown in FIG. 4, when the percentage of PEI in acetic acid was more than 0.80%, system F ₄₂₅ The value of (2) is decreasing, so a PEI concentration of 0.80% is chosen.

3) Determination of metal ions and concentrations

200 mu mol/L of different metal ions, respectively Na, are added into an acetic acid buffer containing 0.80% PEI ⁺ 、K ⁺ 、Al ³⁺ 、Zn ²⁺ 、Mg ²⁺ 、Cr ³⁺ 、Cu ²⁺ 、Mn ²⁺ 、Co ²⁺ 、Ca ²⁺ 、Fe ³⁺ CTC (CTC)At a concentration of 10. Mu. Mol/L, as shown in FIG. 5, na is compared with other metal ions ⁺ And K ⁺ Has obvious fluorescence enhancement effect on PEI/CTC system, and takes into consideration of safety, na ⁺ Cheaper and often included in buffers, so Na is chosen ⁺ As sensitizer, the fluorescence value of the system is enhanced.

The fluorescence intensities of the systems were measured in 0.80% PEI-containing acetate buffer (20 mmol/L, pH 6) at concentrations of 0, 0.1, 0.2, 0.5, 0.8, 1.0, 2.0mmol/L NaCl, control and experimental groups (0 and 10. Mu. Mol/L CTC), respectively, as shown in FIG. 6, when Na ⁺ When the concentration is more than 0.5mmol/L, the system F ₄₂₅ The value of (2) is saturated, so that Na of 0.5mmol/L is selected ⁺ Concentration.

4) CTC standard concentration gradient curve drawing

Preparing CTC solutions with different concentrations, respectively adding into 20mmol/L acetic acid buffer solution with pH of 6 (containing 0.80% PEI, 0.5 mmol/LNa) ⁺ ) The CTC concentrations in the reaction solutions were set to 0, 0.005, 0.01, 0.1, 0.2, 0.5, 1, 2, 4, 5, 6, 8, and 10. Mu. Mol/L, respectively, and after 30 minutes of reaction at room temperature, the fluorescence intensities of the systems were measured using a fluorescence spectrometer. As shown in a in fig. 7, the fluorescence intensity of the system at 425nm gradually increased with increasing CTC concentration. By using fluorescence intensities F and F ₀ Is (DeltaF) ₄₂₅ ) A linear relationship is plotted as shown in B in FIG. 7, whereby it is found that ΔF is in the range of 0 to 10. Mu. Mol/L ₄₂₅ Has good linear relation with the concentration of CTC, and the linear regression equation is y= 47.879x-0.3854, and the detection limit is as low as 2.7nmol/L.

5) Specificity experiments with common antibiotics

To a solution containing 0.80% PEI, 0.5mmol/LNa ⁺ Adding aureomycin (CTC), tetracycline (TC), terramycin (OTC), doxycycline (DC), minocycline (MIN), ciprofloxacin (CIP), enrofloxacin (ENR), norfloxacin (NOR), streptozotocin (STZ), roxithromycin (ROX) and Erythromycin (ERY) respectively, reacting for 30MIN, and detecting fluorescence intensity. As shown in figure 8 of the drawings,the common antibiotics have little interference to the system, and only ΔF when CTC is contained ₄₂₅ The highest value of (2) indicates that the system has good selectivity to CTC.

Example 2 actual Water sample CTC labelling experiment

And performing standard recovery rate experiments on pretreated lakewater of the Xuanwu lake and the Hongze lake, mineral water of the farmer mountain spring, tap water of a laboratory and milk of a school supermarket respectively. Setting 0.5 mu mol/L, 1 mu mol/L and 2 mu mol/L as CTC concentration measured after final labeling of each water sample, measuring fluorescence intensity of each water sample by using a fluorescence spectrometer, and repeating three groups of experiments by using ultrapure water as a control group.

As shown in Table 1, the method has the standard adding recovery rate of CTC in 4 actual water samples such as mineral water, tap water, river water, lake water and the like and milk distributed between 88.7% and 99.7%, and shows that the system has accuracy and reliability for detecting CTC in the actual samples, and can be suitable for qualitative and quantitative detection of CTC in various environments.

TABLE 1 Source and recovery of different Water samples

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Claims

1. A method for simply and rapidly detecting aureomycin is characterized in that: adding a surfactant PEI and a metal ion Na into an acidic buffer solution ⁺ And forming a system to be detected by the solution to be detected, detecting the fluorescence intensity of the system to be detected by adopting a fluorescence photometer, and increasing the fluorescence intensity at 425nm to ensure that the solution to be detected contains CTC.

2. The method for simply and rapidly detecting aureomycin according to claim 1, wherein the acidic buffer solution is an acetic acid buffer solution with a pH of 6, and the concentration of the acetic acid buffer solution is 20mmol/L.

3. The method for simple and rapid detection of aureomycin according to claim 1, wherein the test bodyPEI concentration in the system was 0.80%, na ⁺ The concentration was 0.5mmol/L.

4. A method for quantitatively detecting aureomycin, which is characterized by comprising the following steps:

5. The method for quantitative detection of aureomycin according to claim 4, wherein the relative fluorescence values are fluorescence intensities F and F ₀ Fluorescence value difference Δf of (2) ₄₂₅ 。

6. The method for quantitatively detecting aureomycin according to claim 4, wherein CTCs are in a concentration range of 0 to 10 μmol/L, and the standard curve equation is: y= 47.879x-0.3854, the detection limit is as low as 2.7nmol/L.