CN117990895A

CN117990895A - Fluorescence analysis method for detecting kanamycin content by using malachite green

Info

Publication number: CN117990895A
Application number: CN202311813848.1A
Authority: CN
Inventors: 郭丽敏; 高仕超; 靳雅柯; 任蕾; 庞维荣
Original assignee: Shanxi University of Chinese Mediciine
Current assignee: Shanxi University of Chinese Mediciine
Priority date: 2023-12-26
Filing date: 2023-12-26
Publication date: 2024-05-07

Abstract

The invention belongs to the field of antibiotic detection, and particularly relates to a fluorescence analysis method for detecting kanamycin content by using malachite green. Aiming at the technical problems of expensive reagent and low sensitivity in the prior art, the invention uses cheaper malachite green as a fluorescent probe and uses a nucleic acid aptamer as a recognition element, and establishes a fluorescent analysis method for detecting kanamycin content. The method has the advantages of good specificity, simple operation, low cost, extremely high sensitivity and low toxicity, and can be applied to detection of kanamycin content in actual samples.

Description

Fluorescence analysis method for detecting kanamycin content by using malachite green

Technical Field

The invention belongs to the field of antibiotic detection, and particularly relates to a fluorescence analysis method for detecting kanamycin content by using malachite green.

Background

Kanamycin sulfate (KANAMYCIN SULFATE) belongs to aminoglycoside anti-infective drugs, has the advantages of good water solubility, wide antibacterial spectrum, low bacterial resistance, low plasma protein binding rate, convenient use and low price, and is widely applied to livestock and poultry farming industry. As kanamycin usage increases year by year, the hazards created by it are also becoming of increasing concern. Abuse of kanamycin can cause serious harm to human body, such as cytotoxicity, nephrotoxicity, drug resistance and the like, and is considered to be one of the most toxic antibiotic pollution. The maximum allowable residual amount of kanamycin in animal-derived foods such as honey is regulated in countries around the world. Therefore, it is particularly necessary to establish a simple, sensitive and highly specific kanamycin detection method. Currently, the detection methods of kanamycin residue mainly include High Performance Liquid Chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS/MS) and ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).

Although the detection methods of the instruments are high in sensitivity and good in selectivity, the detection cost is high, the operation is complex, and special detection is needed; in addition, the pretreatment steps of the actual samples are tedious and time-consuming, which also has a certain limitation on the application of the instrumental analysis method. Another common method is an immunization method, which has the advantages of simple operation and high sensitivity, but the preparation of the antibody has the problems of large technical difficulty, poor stability and the like, and the detection result is easy to generate false positive.

The nucleic acid aptamer is a single-stranded oligonucleotide capable of specifically recognizing a target, and an analysis method based on the nucleic acid aptamer attracts attention of researchers because the nucleic acid aptamer meets the detection requirements of simplicity, rapidness, high sensitivity and high specificity. The optical biosensor detects by using the light signal initiated by the reaction of the detected substance and the detection reagent as the basis, has the advantages of simplicity, high sensitivity and quick response, and is widely applied to the fields of biomedicine and food detection. The gold nano ion colorimetric method has the problems of expensive reagent and low sensitivity; novel aptamer and aptamer structure switch rule are not optimal detection methods because of long time consumption.

Disclosure of Invention

Aiming at the technical problems, the invention establishes a fluorescence analysis method for detecting kanamycin content by using cheaper malachite green as a fluorescent probe and using a nucleic acid aptamer as an identification element. The method has the advantages of good specificity, low cost, extremely high sensitivity and low toxicity, and can be applied to detection of kanamycin content in actual samples.

In order to achieve the above purpose, the present invention is realized by the following technical scheme:

A fluorescence analysis method for detecting kanamycin content by using malachite green is established by taking malachite green as a fluorescent probe and taking kanamycin nucleic acid aptamer as an identification element.

Further, the kanamycin aptamer has a sequence 5'-TGGGGGTTGAGGCTAAGCCGA-3'.

A fluorescence assay for detecting kanamycin content as described above, comprising the steps of:

(1) Adding malachite green solution into kanamycin aptamer solution for suspension reaction;

(2) Then adding a sample to be tested, and carrying out suspension reaction;

(3) After the reaction was completed, the fluorescence value of the solution was measured.

Further, the concentration of the kanamycin nucleic acid aptamer solution in the step (1) ranges from 2.0 to 15.0 mu mol/L; the concentration range of the malachite green solution is 2.0-20 mu mol/L.

Further, the molar ratio of kanamycin aptamer solution to malachite green solution in step (1) is 1:2.

Further, the suspension reaction time in the step (1) was 40min, and the reaction conditions were 25℃and 650rpm.

Further, the suspension reaction time in the step (2) was 20min, and the reaction conditions were 25℃and 650rpm.

Further, the basic parameters for determining the fluorescence value of the solution in the step (3) are as follows: the 600nm is the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high.

Use of a fluorescence assay as described above for detecting kanamycin content.

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

1. The malachite green selected by the invention can emit weak fluorescence, and after kanamycin aptamer is added into the malachite green solution, the malachite green is protected by the aptamer, so that the fluorescence signal is enhanced to a certain extent.

2. After kanamycin is added, kanamycin can cause the selective combination of an aptamer and kanamycin, so that a hairpin structure is formed, and the aptamer can protect malachite green, so that a fluorescence signal is greatly enhanced.

3. The invention can be used for detecting the content of kanamycin and also can be used for quantitative analysis of kanamycin.

4. The method has good specificity, low cost, extremely high sensitivity, good selectivity and low toxicity.

Drawings

FIG. 1 is a schematic diagram of the detection of kanamycin content according to the present invention.

FIG. 2 is a chart showing the fluorescence spectrum of kanamycin for detection in the present invention.

FIG. 3 (1) is a graph showing fluorescence spectra at different K+ concentrations according to the present invention; (2) is a fluorescence spectrum chart under different Na+ concentrations.

FIG. 4 is a graph showing fluorescence spectra at different pH values according to the present invention.

FIG. 5 is a graph showing fluorescence spectra of malachite green at different concentrations according to the present invention.

FIG. 6 is a graph showing fluorescence spectra at different kanamycin aptamer concentrations according to the present invention.

FIG. 7 (1) is a graph showing the spectrum of the kanamycin/aptamer/malachite green system of the invention at various concentrations of kanamycin; (2) A linear plot of ΔF/F ₀ of the present invention as a function of kanamycin concentration is shown.

FIG. 8 is a graph showing fluorescence spectra of different antibiotics detected by the method of the present invention.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1

Solution preparation

1. Preparation of kanamycin solution

Kanamycin solutions at concentrations of 0.00, 0.06, 0.12, 0.30, 0.60, 1.20, 2.25, 4.50, 9.00, 15.00, 24.00 and 30.00. Mu. Mol/L were prepared with buffer solutions (10 mmol/L Tris-HCl+30mmol/L K ⁺+10mmol/L Na⁺, pH 7.5), respectively.

2. Preparation of kanamycin aptamer solution

Preparing kanamycin nucleic acid aptamer solutions with concentrations of 3, 6, 9, 24, 30 and 45 mu mol/L respectively by using a buffer solution (10.0 mmol/L Tris-HCl+30.0mmol/L K ⁺+10.0mmol/L Na⁺, pH value 7.5);

3. Preparation of malachite green solution

Malachite green solutions at concentrations of 6, 15, 24, 30, 45 and 60. Mu. Mol/L were prepared with buffer solutions (10.0 mmol/L Tris-HCl+30.0mmol/L K ⁺+10.0mmol/L Na⁺, pH 7.5).

4. Preparation of honey solution

(1) Taking 3mL of jujube flower honey, diluting with pure water for 20 times, mixing uniformly, and centrifuging at 10000rpm/min for 10min;

(2) Taking supernatant, and filtering with qualitative filter paper;

(3) Filtering with 0.22 μm filter membrane to obtain Mel solution.

5. Preparation of kanamycin Honey solution

Using the honey solution obtained in 4, kanamycin honey solutions with concentrations of 0.12, 0.24, 0.60, 2.40 and 6.00. Mu. Mol/L were prepared, respectively.

Example 2

Feasibility experiment

(1) Sample preparation of malachite green solution: the malachite green solution obtained in example 1 was diluted to 10. Mu. Mol/L with a buffer solution;

(2) Sample preparation of malachite green/aptamer solution: adding 40 mu L of 30umol/L malachite green solution into 40 mu L of 15 mu mol/L kanamycin aptamer solution, placing into a suspension instrument for reaction for 40min, wherein the reaction condition is 25 ℃ and 650rpm;

(3) Malachite green/aptamer/kanamycin solution sample preparation: mixing 40 mu L of malachite green solution with 30 mu mol/L and 40 mu L of kanamycin aptamer solution with 15 mu mol/L, placing into a suspension instrument for reaction for 40min, wherein the reaction condition is 25 ℃ and 650rpm; then 40. Mu.L of kanamycin solution with the concentration of 24.00. Mu. Mol/L obtained in the example 1 is added, the reaction is carried out for 20min in a suspension instrument under the reaction condition of 25 ℃ and 650rpm, and the final concentration of kanamycin in the solution is 8. Mu. Mol/L;

(4) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; the reacted solution was poured into a cuvette to measure its fluorescence value, and the fluorescence intensity was recorded.

As shown in FIG. 2, the line a is malachite green fluorescence, and the signal is weak; the line b is malachite green/nucleic acid aptamer, and the malachite green is protected by the nucleic acid aptamer, so that the fluorescence signal is enhanced to a certain extent; the c line is malachite green/nucleic acid aptamer/kanamycin, so that the aptamer and kanamycin can be combined selectively, a hairpin structure is formed, and the aptamer can protect malachite green, so that a fluorescence signal is greatly enhanced.

The fluorescence signal of the fluorescence analysis method for detecting the kanamycin content, which is established by taking malachite green as a fluorescent probe and taking kanamycin aptamer as an identification element, is strong and has feasibility.

Example 3

Experimental condition optimization

Effect of K ⁺ concentration

(1) K ⁺ solution was added to 10mmol/L Tris-HCl+10mmol/L Na ⁺ (pH 7.5) to prepare buffer solutions having K ⁺ concentrations of 0, 15, 30, 60, 90, 150mmol/L, respectively, and all of the reagents were prepared from these buffer solutions, namely 15. Mu. Mol/L kanamycin aptamer solution, 30. Mu. Mol/L malachite green solution, and 5.00. Mu. Mol/L kanamycin solution;

(2) Adding 40 mu L of 30 mu mol/L malachite green solution into 40 mu L of 15 mu mol/L kanamycin aptamer solution, placing into a suspension instrument for reaction for 40min, wherein the reaction condition is 25 ℃ and 650rpm;

(3) Adding 40 mu L of 4.50 mu mol/L kanamycin solution into the solution (2), and reacting for 20min in a suspension instrument under the reaction condition of 25 ℃ and 650rpm;

(4) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; and (3) injecting the reacted solution into a cuvette to measure the fluorescence value, and recording the fluorescence intensity, wherein the final concentration of K ⁺ in the solution to be measured is 0, 5, 10, 20, 30 and 50mmol/L.

As a result, as shown in FIG. 3 (1), in the range of 0 to 50mmol/L, as the concentration of K ⁺ increases, ΔF/F ₀ increases and decreases; when the concentration of K ⁺ is 30mmol/L, the fluorescence signal change value delta F/F ₀ reaches the highest point. Therefore, the K+ concentration is 30mmol/L, which is the optimal condition for experiments.

Effect of na ⁺ concentration

(1) Na ⁺ solution was added to 10mmol/L Tris-HCl+30mmol/L K ⁺ solution (pH 7.5) to prepare buffer solutions having Na ⁺ concentrations of 0, 3, 15, 30, 60, 150mmol/L, respectively, and all of the reagents, namely 15. Mu. Mol/L kanamycin aptamer solution, 30. Mu. Mol/L malachite green solution and 5.00. Mu. Mol/L kanamycin solution were prepared from these buffer solutions;

(3) Adding 40 mu L of 4.50 mu mol/L kanamycin solution into the solution in the step (2), and reacting for 20min in a suspension instrument under the reaction condition of 25 ℃ and 650rpm;

(4) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; the reacted solution is injected into a cuvette to measure the fluorescence value, the fluorescence intensity is recorded, and the final concentration of Na ⁺ in the solution to be measured is 0, 1, 5, 10, 20 and 50mmol/L.

As shown in FIG. 3 (2), in the range of 0 to 50mmol/L, as the Na+ concentration increases, deltaF/F ₀ rises first and then becomes stable; when Na+ reaches 10mmol/L, the fluorescence signal change value DeltaF/F ₀ reaches the highest point. Therefore, the Na+ concentration of 10mmol/L is the optimal condition for the experiment.

Influence of pH

(1) All solution reagents, namely 15. Mu. Mol/L kanamycin aptamer solution, 30. Mu. Mol/L malachite green solution and 4.50. Mu. Mol/L kanamycin solution were prepared with buffer solutions (10 mmol/LTris-HCl+30mmol/L K ⁺+10mmol/L Na⁺) having pH of 6.8, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, respectively;

As shown in FIG. 4, when the pH of Tris-HCl is in the range of 6.8-10.0, the DeltaF/F ₀ rises first and then becomes stable as the pH value increases; the fluorescence signal change value DeltaF/F ₀ reached the highest point when the pH of Tris-HCl was 7.5, so that the optimal condition for the experiment was the case when the pH of Tris-HCl was 7.5.

4. Influence of malachite green concentration

(1) To 6 parts of kanamycin aptamer solution containing 40. Mu.L of 15. Mu. Mol/L, 40. Mu.L of malachite green solution obtained in example 1 (the concentrations are 6, 15, 24, 30, 45 and 60. Mu. Mol/L, respectively) were added, and the mixture was put into a suspension apparatus to react for 40min at 25℃and 650rpm;

(2) Adding 40 mu L of 4.50 mu mol/L kanamycin solution into 6 parts of the solution (1) respectively, and reacting for 20min in a suspension instrument under the reaction condition of 25 ℃ and 650rpm;

(3) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; the 6 parts of the solution after the reaction were poured into a cuvette to measure the fluorescence value, and the fluorescence intensity was recorded. The final concentrations of malachite green in the test solutions were 2, 5, 8, 10, 15 and 20. Mu. Mol/L.

As a result, as shown in FIG. 5, the effect of malachite green at various concentrations on the detection system was examined in the range of 2 to 20. Mu. Mol/L, and as the concentration of malachite green increased, deltaF/F ₀ showed a tendency to rise and fall after that (F ₀ was an aptamer/malachite green fluorescent signal, F was a kanamycin/aptamer/malachite green fluorescent signal), and at a malachite green content of 10. Mu. Mol/L, the DeltaF/F ₀ value was the largest, and as a result, it was shown that the concentration of malachite green solution was 10. Mu. Mol/L as an experimental optimum condition.

5. Effects of kanamycin aptamer concentration

(1) 40. Mu.L of 30. Mu. Mol/L malachite green solution was added to 40. Mu.L of the kanamycin aptamer solution obtained in example 1 (concentrations of 3, 6, 15, 24, 30 and 45. Mu. Mol/L, respectively), and the mixture was placed in a suspension apparatus to react for 40min at 25℃and 650rpm;

(2) Adding 40 mu L of 4.50 mu mol/L kanamycin solution into the solution (1) respectively, and reacting for 20min in a suspension instrument under the reaction condition of 25 ℃ and 650rpm;

(3) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; and 6 parts of the reacted solution are injected into a cuvette to measure the fluorescence value, the fluorescence intensity is recorded, and the final concentration of the aptamer in the solution to be measured is 1,2, 5, 8, 10 and 15 mu mol/L.

As a result, as shown in FIG. 6, in the range of 1.0 to 15.0. Mu. Mol/L, the fluorescence signal variation value DeltaF/F ₀ shows a tendency to rise sharply and then fall finally and smoothly; at a nucleic acid aptamer concentration of 5. Mu. Mol/L, ΔF/F ₀ reached a maximum, so that 5. Mu. Mol// L kanamycin aptamer was the optimal condition for the experiment.

Example 4

(1) Adding 40 mu L of 10umol/L malachite green solution into 40 mu L of 5 mu mol/L kanamycin aptamer solution, placing into a suspension instrument for reaction for 40min, wherein the reaction condition is 25 ℃ and 650rpm;

(2) Then, 40. Mu.L of kanamycin solution (0, 0.06, 0.12, 0.30, 0.60, 1.20, 2.25, 4.50, 9.00, 15.00, 24.00 and 30.00. Mu. Mol/L) of the concentration obtained in example 1 was added, and the mixture was reacted in a suspension apparatus at 25℃and 650rpm for 20 minutes;

(3) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; the resultant solution was poured into a cuvette to measure its fluorescence value, and the fluorescence intensity was recorded, and the final concentration of kanamycin in the solution to be measured was 0.00, 0.02, 0.04, 0.10, 0.20, 0.40, 0.75, 1.50, 3.00, 5.00, 8.00 and 10.00. Mu. Mol/L.

The kanamycin concentration was measured under the optimized conditions and the results are shown in FIG. 7. Along with the continuous increase of the kanamycin concentration, the fluorescence signal value of the system is obviously increased, which indicates that the nucleic acid aptamer is specifically combined with kanamycin to form a hairpin structure for protecting malachite green, and the fluorescence signal is further enhanced.

In the range of 0.02-3.00. Mu. Mol/L, kanamycin concentration (x) is linearly related to ΔF/F ₀ (y), and the linear relation is as follows: y=0.817x+0.144, (r2=0.996), and the detection limit LOD is 7.4nmol/L.

Example 5

Specificity detection assay

(1) Adding 40 mu L of 30umol/L malachite green solution into 40 mu L of 15 mu mol/L kanamycin aptamer solution, placing into a suspension instrument for reaction for 40min, wherein the reaction condition is 25 ℃ and 650rpm;

(2) Five antibiotics of ciprofloxacin hydrochloride (Ciprofloxacin hydrochloride), oxytetracycline hydrochloride (Oxytetracycline Hydrochloride), tetracycline hydrochloride (TETRACYCLINE HYDROCHLORIDE), chloramphenicol (chloramphenicol) and aureomycin hydrochloride (Chlortetracycline Hydrochloride) are selected as interfering agents to prepare interfering agent solutions with the concentration of 15 mu mol/L;

(3) Adding 40 mu L of the single interfering reagent solution obtained in the step (2) and five coexisting solutions of interfering reagents into 6 parts of the solution in the step (1) respectively, and reacting for 20min in a suspension instrument under the reaction condition of 25 ℃ and 650rpm;

(4) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; and (3) injecting the reacted solution into a cuvette to measure the fluorescence value, and recording the fluorescence intensity, wherein the final concentration of each interfering reagent in the solution to be measured is 5 mu mol/L.

As a result, as shown in FIG. 8, the fluorescence signal change value DeltaF/F ₀ of these 5 antibiotics was very low; 5 antibiotics are mixed together for investigation, and the fluorescence signal change value delta F/F ₀ is very low, which indicates that ciprofloxacin hydrochloride, oxytetracycline hydrochloride, tetracycline hydrochloride, chloramphenicol and aureomycin hydrochloride can not replace kanamycin; kanamycin was mixed with 5 antibiotics and the signal of the mixture was detected to be similar to that generated by kanamycin alone.

The results demonstrate the high selectivity of the aptamer for kanamycin, the use of other antibiotics alone does not generate fluorescent signals, and the mixing of various antibiotics with kanamycin does not affect kanamycin detection.

Example 6

Detection of kanamycin in Honey samples

(2) Then, 40. Mu.L of the kanamycin honey solution obtained in example 1 (kanamycin concentration of 0.12, 0.36, 0.60, 2.40 and 6.00. Mu. Mol/L, respectively) was added, and the mixture was reacted in a suspension apparatus at 25℃and 650rpm for 20 minutes;

(3) The 600nm is set as the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high; the resultant solution was poured into a cuvette to measure its fluorescence value, and the fluorescence intensity was recorded, and the final concentrations of kanamycin in the solutions to be measured were 0.04, 0.12, 0.20, 0.80 and 2.00. Mu. Mol/L.

As shown in Table 1, the amount of kanamycin detected in honey was not significantly different from that detected in the buffer solution, and the recovery rate was between 94.2% and 106.2%.

This demonstrates that the detection of kanamycin under the influence of honey is still sensitive and accurate, which further demonstrates the utility of the invention. The invention has low cost, extremely high sensitivity, good selectivity and low toxicity, and can be applied to detection of kanamycin in actual samples.

TABLE 1 recovery of kanamycin at various concentrations in Honey

The above-described embodiments represent only specific embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A fluorescence analysis method for detecting kanamycin content by using malachite green is characterized in that the analysis method is a fluorescence analysis method for detecting kanamycin content, which is established by taking malachite green as a fluorescent probe and taking kanamycin nucleic acid aptamer as an identification element.

2. The fluorescence assay for detecting kanamycin content according to claim 1, wherein the sequence of the kanamycin aptamer is 5'-TGGGGGTTGAGGCTAAGCCGA-3'.

3. A fluorescence assay for detecting kanamycin content according to claim 1, comprising the steps of:

(2) Then adding a sample to be tested, and carrying out suspension reaction;

4. The fluorescence assay for detecting kanamycin content according to claim 3, wherein the concentration of the kanamycin aptamer solution in step (1) is in the range of 2.0 to 15.0 μmol/L; the concentration range of the malachite green solution is 2.0-20 mu mol/L.

5. The fluorescence assay for detecting kanamycin content according to claim 3, wherein the molar ratio of kanamycin aptamer solution to malachite green solution in step (1) is 1:2.

6. A fluorescence assay for detecting kanamycin content according to claim 3, wherein the suspension reaction time in step (1) is 40min, the reaction conditions are 25 ℃,650rpm.

7. A fluorescence assay for detecting kanamycin content according to claim 3, wherein the suspension reaction time in step (2) is 20min, the reaction conditions are 25 ℃,650rpm.

8. A fluorescence assay for detecting kanamycin content according to claim 3, wherein the basic parameters for determining the fluorescence value of the solution in step (3) are: the 600nm is the excitation wavelength, the 619-850 nm range is the emission wavelength, the excitation and emission broadband are 10nm, and the sensitivity is high.

9. Use of a fluorescence assay according to claim 1 for detecting kanamycin content.