CN109283147B - Homogeneous phase biosensing method for detecting kanamycin and application thereof - Google Patents
Homogeneous phase biosensing method for detecting kanamycin and application thereof Download PDFInfo
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Abstract
A homogeneous biosensing method for detecting kanamycin and its application, inhibit DNase activity through the double-stranded hybridization reaction of G-quadruplex DNA (S1) and kanamycin aptamer (S2), and kanamycin and its specificity bind release corresponding quantity S1 between aptamer, and bind hemin to form DNase to catalyze and develop signal transduction; furthermore, the hairpin structure formed between kanamycin and its aptamer can increase the sensitivity of the method by cycling the catalytic target analyte of exonuclease III after introduction of the complementary strand S3 hybridization; utilizing the aptamer specificity to identify the catalytic chromogenic reaction of the released DNA enzyme, and constructing the quantitative relation between the absorbance of a chromogenic product and the concentration of a kanamycin analyte; the method is convenient to operate, low in cost, high in sensitivity and good in stability, and has a good application value in rapid detection of kanamycin residues in complex media such as food, water and the like.
Description
Technical Field
The invention relates to the technical field of biological analysis, in particular to a homogeneous phase biosensing method for detecting kanamycin and application thereof.
Background
Kanamycin is an aminoglycoside high-efficiency broad-spectrum antibiotic, has good antibacterial effects on most enterobacteriaceae bacteria such as escherichia coli, klebsiella, enterobacter, proteus, shigella, salmonella, citrobacter, pulofilaria and yersinia, haemophilus influenzae, brucella, neisseria meningitidis, neisseria gonorrhoeae and the like, and is widely used for treating various gram-positive and gram-negative bacterial infectious diseases of human and animals. However, excessive use of kanamycin not only causes hearing loss, tinnitus, and even serious side effects such as nephrotoxic reaction and drug allergy, but also causes serious drug resistance. In recent years, the overproof antibiotic residues in food, water and soil caused by the abuse of antibiotics and the great threat to human health caused by the enrichment of food chains in human bodies have attracted general social attention. Therefore, many countries including China have strict restriction standards for antibiotic residues such as kanamycin in food.
At present, the national standard generally adopts a liquid chromatography-mass spectrometry combined method to detect kanamycin residues, but the method has expensive instruments and high detection cost, can be generally carried out only in a laboratory, and is difficult to meet the requirements of real-time and on-site detection in practical application. In order to solve the problem, people combine biological recognition effects such as antigens (antibodies), DNA and the like with analysis methods such as electrochemistry, colorimetry and the like in recent years, and develop a large number of biosensing methods which can be used for rapid and convenient detection of antibiotics. However, these methods are generally constructed based on heterogeneous analysis mode, and often require very complicated solid phase interface modification, and multiple steps of incubation, separation and washing operations, and even incorporate nano material signal amplification to improve the analysis sensitivity, so that not only the analysis time is long and the operation is complicated, but also the multiple steps of operation can affect the repeatability of the method to a certain extent, increase the analysis cost, and are very unfavorable for the wide use thereof. Therefore, it is of great significance to develop a new intelligent biosensing method which is simple to operate and has the excellent performances of high sensitivity, good accuracy, low cost and the like.
Disclosure of Invention
The invention aims to solve the problems of complex operation, higher cost, longer time, poor repeatability and the like of the existing method for detecting the clarithromycin, and provides a homogeneous phase biosensing method capable of simply and quickly detecting the content of the clarithromycin in a sample.
The invention relates to a homogeneous biosensing method for detecting kanamycin (Kana), which comprises the following steps:
(1) preparation of G-quadruplex signature sequence and kanamycin aptamer hybrid double strand
Adding 990 muL of tris (hydroxymethyl) aminomethane buffer solution with a concentration of 10mM pH =7.4 to the PV tube, the buffer solution contains 200mM NaCl, 10mM KCl, 1% DMSO by mass fraction and 0.05% Triton X-100 by mass fraction, and then 5 muL 10 muR M G-quadruplex DNA chain (S1) and 5 muL 10 muM kanamycin aptamer (S2) solution are added into the buffer solution, the G-quadruplex DNA strand (S1) has a base sequence of 5'-GGG TAG GGC GGG TTG GGA ACC TCA AGA CCA CTT GGA CAT TTT-3', the base sequence of the kanamycin aptamer (S2) is 5'-TGT CCA AGT GGT CTT GAG GTT TTTT-3', and an aptamer hybrid double-stranded complex solution is obtained for later use after shaking reaction for 45min at room temperature;
(2) homogeneous phase reaction determination of kanamycin (Kana) content in standard solution
6 parts of the aptamer hybridization double-stranded complex solution are taken, 100 mu L of each solution is added with 50 mu L of a 20mM Tris-HCl buffer solution containing 100mM NaCl and 2mM MgCl and the concentration gradient of the kalamycin (Kana) is 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL and 10ng/mL, and the pH value is =7.42And 5mM KCl, and then adding a 5 mu L2 mu M kanamycin aptamer partial complementary sequence (S3), a 5 mu L5U/mu L exonuclease III (Exo III) and 5 mu L1 mu M hemin (hemin) into each solution, wherein the alkali motif in the kanamycin aptamer partial complementary sequence (S3) is 5'-AAA AAA CCT GAC ACT AC-3'; vortex at 37 deg.C, mixing uniformly, reacting for 70min, heating to 65 deg.C, maintaining for 5min, and cooling to 25 deg.C; then, 70 μ L of a solution containing 0.4mM TMB and 0.4mM H was added to each solution2O2pH =5.0 citrate buffer solution containing 95.8mM Na2HPO452.1mM sodium citrate and 40mM KCl, and after 5min of the color reaction, 30 μ L2M H was continuously added thereto2SO4Terminating the color development reaction; measuring the absorbance value of the solution by using an ultraviolet-visible spectrophotometer to establish a quantitative relation between the absorbance and the kanamycin concentration;
(3) detection of kanamycin (Kana) content in sample
Taking a proper amount of sample and carrying out pretreatment, taking 50 mu L of the treated sample solution, adding 100 mu L of aptamer hybrid double-stranded complex solution, a 5 mu L2 mu M kanamycin aptamer partial complementary sequence (S3), 5 mu L5U/mu L exonuclease III (Exo III) and 5 mu L1 mu M heme chloride (hemin), wherein the alkali motif in the kanamycin aptamer partial complementary sequence (S3) is 5'-AAA AAA CCT GAC ACT AC-3'; vortex at 37 deg.C, mixing uniformly, reacting for 70min, heating to 65 deg.C, maintaining for 5min, and cooling to 25 deg.C; then, 70 μ L of a solution containing 0.4mM TMB and 0.4mM H was added to the sample solution2O2pH =5.0 citrate buffer solution containing 95.8mM Na2HPO452.1mM sodium citrate and 40mM KCl, and after 5min of the color reaction, 30 μ L2M was continuously added thereto H2SO4Terminating the color development reaction; and (3) measuring the absorbance value of the sample solution by using an ultraviolet-visible spectrophotometer, and calculating the content of kanamycin (Kana) according to the quantitative relation between the absorbance and the concentration of kanamycin.
The invention also provides application of the homogeneous phase biosensing method for detecting kanamycin in a detection sample.
The working principle of the invention is as follows: the G-quadruplex contained in S1 can be well inhibited from being combined with hemin (hemin) to form DNase through a DNA double-strand hybridization reaction between a G-quadruplex DNA chain (S1) and a kanamycin (Kana) aptamer (S2), and the specific combination between S2 serving as the kanamycin aptamer and a kanamycin (Kana) analyte can release a corresponding amount of S1, so that colorimetric signal transduction is carried out through a catalytic chromogenic reaction of the combination of S1 and the hemin (hemin) to form the DNase; in addition, after the hairpin-like structure formed by combining kanamycin (Kana) and the aptamer S2 of kanamycin (Kana) is introduced into the S2 partial complementary strand S3 for hybridization, kanamycin (Kana) and S3 are released through enzyme digestion reaction of exonuclease III (Exo III) to amplify a target analyte circulating signal, and therefore the sensitivity of an analysis method is effectively improved; the quantitative relation between the absorbance of a chromogenic product and the concentration of a kanamycin analyte can be constructed by utilizing the specific recognition of kanamycin and aptamer and the catalytic chromogenic reaction of free DNA enzyme released by the enzymatic reaction of exonuclease III.
Compared with the traditional antibody, the aptamer adopted by the invention has the advantages of better stability, lower cost and the like, and the better specificity recognition effect between the aptamer and a kanamycin analyte can ensure the excellent selectivity of the method in complex matrix analysis; the catalytic color reaction of DNA enzyme released by the reaction and the amplification of the enzymatic cycling reaction signal of exonuclease III enable the method to have high analysis sensitivity, and avoid the nonspecific adsorption influence possibly caused in the complex nano material preparation and signal amplification process in the traditional method; more importantly, all recognition reaction, circulation signal amplification and DNA enzyme formation are carried out in one step in homogeneous solution, so that the operation is very simple, and the repeatability of the method is well ensured, therefore, the method has outstanding superiority and practical value compared with the traditional method.
The biosensing method constructed by the invention can be conveniently and simply used for high-selectivity detection of the content of the kalamycin, has the excellent performances of high sensitivity, wide linear range, short detection time, low detection cost and the like, can realize quantitative response and accurate determination on the kalamycin within the concentration range of 0.1pg/mL-10ng/mL, and well overcomes the defects of complicated operation, high cost, long time, poor repeatability and the like of the traditional method.
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FIG. 1 is a schematic diagram of the detection principle of the present invention.
Detailed Description
Example 1
The homogeneous biosensing method for detecting kanamycin in the embodiment comprises the following steps:
(1) preparation of G-quadruplex signature sequence and kanamycin (Kana) aptamer hybrid double strand
Adding 990 muL of tris (hydroxymethyl) aminomethane buffer solution with a concentration of 10mM pH =7.4 to the PV tube, the buffer solution contains 200mM NaCl, 10mM KCl, 1% DMSO by mass fraction and 0.05% Triton X-100 by mass fraction, and then 5 muL 10 muR M G-quadruplex DNA chain (S1) and 5 muL 10 muM kanamycin (Kana) aptamer (S2) solution are added into the buffer solution, the base sequence of the G-quadruplex DNA strand (S1) is 5'-GGG TAG GGC GGG TTG GGA ACC TCA AGA CCA CTT GGA CAT TTT-3', the alkali sequence in the kanamycin aptamer (S2) is 5'-TGT CCA AGT GGT CTT GAG GTT TTTT-3', and an aptamer hybrid double-stranded complex solution is obtained for later use after shaking reaction for 45min at room temperature;
(2) homogeneous phase reaction determination of kanamycin content in standard solution
Taking 6 parts of the aptamer hybrid double-stranded complex solution, and adding the solutions with a concentration gradient of 0.0001ng/mL, 0.001ng/mL and 0.01ng/mL to the solutions respectively50 μ L of 20mM pH =7.4 Tris-HCl buffer solutions/mL, 0.1ng/mL, 1ng/mL and 10ng/mL containing 100mM NaCl, 2mM MgCl2And 5mM KCl, and then adding a 5 mu L2 mu M kanamycin aptamer partial complementary sequence (S3), a 5 mu L5U/mu L exonuclease III (Exo III) and 5 mu L1 mu M hemin (hemin) into each solution, wherein the alkali motif in the kanamycin aptamer partial complementary sequence (S3) is 5'-AAA AAA CCT GAC ACT AC-3'; vortex at 37 deg.C, mixing uniformly, reacting for 70min, heating to 65 deg.C, maintaining for 5min, and cooling to 25 deg.C; then, 70 μ L of a solution containing 0.4mM TMB and 0.4mM H was added to each solution2O2pH =5.0 citrate buffer solution containing 95.8mM Na2HPO452.1mM sodium citrate and 40mM KCl, and after 5min of the color reaction, 30 μ L2M H was continuously added thereto2SO4Terminating the color development reaction; the absorbance value of the solution was measured using an ultraviolet-visible spectrophotometer to establish a quantitative relationship between absorbance and kanamycin (Kana) concentration, and a working curve of the kanamycin standard solution was obtained, as shown in table 1 below.
TABLE 1 kanamycin Standard solution working curves
| Detection object | Linear range (ng/mL) | Coefficient of linear correlation | Detection limit (pg/mL) |
| Kanamycin | 0.0001-10 | 0.998 | 0.045 |
EXAMPLE 2 detection of kanamycin content in milk powder samples
According to the working curve obtained in example 1, the content of kanamycin in the milk powder sample is detected. 1g of commercially available milk powder was weighed and dissolved in 5 mL of 20mM Tris-HCl buffer solution with pH 7.4 and containing 100mM NaCl and 2mM MgCl2And 5mM KCl, taking 100 mu L of the dissolved milk powder solution, adding 20% by mass of acetic acid to adjust the solution to pH =4.6, centrifuging after 20min to remove coagulated protein and fat in the sample, filtering the sample solution by using a 0.22 mu m filter membrane, and adjusting the pH of the sample solution to =7.4 again; taking 50 muL of the treated sample solution, and adding 100 muL of the aptamer hybrid double-stranded complex solution, a 5 muL 2 muM kanamycin aptamer partial complementary sequence (S3), 5 muL 5U/muL exonuclease III (Exo III) and 5 muL 1 muM hemin (hemin), wherein the alkali motif in the kanamycin aptamer partial complementary sequence (S3) is 5'-AAA AAA CCT GAC ACT AC-3'; vortex at 37 deg.C, mixing uniformly, reacting for 70min, heating to 65 deg.C, maintaining for 5min, and cooling to 25 deg.C; then, 70 μ L of a solution containing 0.4mM TMB and 0.4mM H was added to the sample solution2O2pH =5.0 citrate buffer solution containing 95.8mM Na2HPO452.1mM sodium citrate and 40mM KCl, and after 5min of the color reaction, 30 μ L2M H was continuously added thereto2SO4Terminating the color development reaction; the absorbance value of the milk powder sample solution was measured by an ultraviolet-visible spectrophotometer, and the kanamycin (Kana) content was calculated based on the quantitative relationship between the absorbance and the kanamycin concentration in example 1.
The detection result shows that no kanamycin residue is detected in the milk sample. Kanamycin standard solutions with different concentrations are continuously added into the milk powder samples to carry out a standard adding recovery experiment, and the experimental results are shown in the following table 2.
TABLE 2 addition of recovery test results for milk powder sample solutions
| Serial number | Addition amount (μ M) | Average recovery (μ M) | RSD（%，n=5） | Average recovery (%) |
| 1 | 1 | 1.05 | 2.8 | 105.0 |
| 2 | 0.1 | 0.107 | 2.5 | 107.0 |
| 3 | 0.01 | 0.0096 | 3.3 | 96.0 |
As can be seen from Table 2 above, the Relative Standard Deviation (RSD) of the test results of this example 2 is 2.5-3.3%, and the recovery rate of spiking is 96.0-107.0%, which indicates that the analysis method of this example has higher accuracy and precision.
Example 3 detection of kanamycin content in Honey samples
The content of kanamycin in the honey samples was determined according to the working curve obtained in example 1. Weighing commercially available honey 2g, dissolving in 4mL 20mM Tris-HCl buffer solution with pH =7.4, containing 100mM NaCl, 2mM MgCl2And 5mM KCl; then filtering the sample solution by using a filter membrane of 0.22 mu m; taking 50 muL of the filtered sample solution, and adding 100 muL of the aptamer hybrid double-stranded complex solution, a 5 muL 2 muM kanamycin aptamer partial complementary sequence (S3), 5 muL 5U/muL exonuclease III (Exo III) and 5 muL 1 muM hemin (hemin), wherein the alkali motif in the kanamycin aptamer partial complementary sequence (S3) is 5'-AAA AAA CCT GAC ACT AC-3'; vortex at 37 deg.C, mixing uniformly, reacting for 70min, heating to 65 deg.C, maintaining for 5min, and cooling to 25 deg.C; then, 70 μ L of a solution containing 0.4mM TMB and 0.4mM H was added to the sample solution2O2pH =5.0 citrate buffer solution containing 95.8mM Na2HPO452.1mM sodium citrate and 40mM KCl, and after 5min of the color reaction, 30 μ L2M H was continuously added thereto2SO4Terminating the color development reaction; the absorbance value of the solution was measured by an ultraviolet-visible spectrophotometer, and the kanamycin (Kana) content was calculated from the quantitative relationship between the absorbance and the kanamycin concentration in example 1.
The detection result shows that no kanamycin residue is detected in the honey sample.
Claims (2)
1. A homogeneous biosensing method for detecting kanamycin is characterized by comprising the following steps:
(1) preparation of G-quadruplex signature sequence and kanamycin aptamer hybrid double strand
Adding 990 mu L of a tris (hydroxymethyl) aminomethane buffer solution with the concentration of 10mM, pH =7.4 into a PV tube, wherein the buffer solution contains 200mM NaCl, 10mM KCl, 1% DMSO by mass fraction and 0.05% Triton X-100 by mass fraction, adding 5 mu L of 10 mu M G-quadruplex DNA chain and 5 mu L of 10 mu M kanamycin nucleic acid aptamer solution, the base sequence of the G-quadruplex DNA chain is 5'-GGG TAG GGC GGG TTG GGA ACC TCA AGA CCA CTT GGA CAT TTT-3', the base sequence of the kanamycin nucleic acid aptamer is 5'-TGT CCA AGT GGT CTT GAG GTT TTTT-3', and performing oscillation reaction at room temperature for 45min to obtain an aptamer hybrid double-stranded complex solution for later use;
(2) homogeneous phase reaction determination of kanamycin content in standard solution
6 parts of the aptamer hybridization double-stranded complex solution are taken, 100 mu L of each solution is taken, 50 mu L of a 20mM pH =7.4 Tris-HCl buffer solution containing 100mM NaCl and 2mM MgCl and with the concentration gradient of the clarithromycin of 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL and 10ng/mL is added to the solution2And 5mM KCl, and then adding 5 mu L2 mu M kanamycin aptamer part complementary sequence, 5 mu L5U/mu L exonuclease III and 5 mu L1 mu M chlorhematin into each solution, wherein the base sequence in the kanamycin aptamer part complementary sequence is 5'-AAA AAA CCT GAC ACT AC-3'; vortex at 37 deg.C, mixing uniformly, reacting for 70min, heating to 65 deg.C, maintaining for 5min, and cooling to 25 deg.C; then, 70 μ L of a solution containing 0.4mM TMB and 0.4mM H was added to each solution2O2pH =5.0 citrate buffer solution containing 95.8mM Na2HPO452.1mM sodium citrate and 40mM KCl, and after 5min of the color reaction, 30 μ L2M H was continuously added thereto2SO4Terminating the color development reaction; measuring the absorbance value of the solution by using an ultraviolet-visible spectrophotometer to establish a quantitative relation between the absorbance and the kanamycin concentration;
(3) detection of kanamycin content in sample
Taking a proper amount of sample, carrying out pretreatment, taking 50 muL of the treated sample solution, adding 100 muL of aptamer hybrid double-stranded complex solution, 5 muL 2 muM kanamycin aptamer partial complementary sequence, 5 muL 5U/muL exonuclease III and 5 muL 1 muM chlorhematin into the sample solution, wherein the base sequence in the kanamycin aptamer partial complementary sequence is 5'-AAA AAA CCT GAC ACT AC-3'; vortex at 37 deg.C, mixing uniformly, reacting for 70min, heating to 65 deg.C, maintaining for 5min, and cooling to 25 deg.C;then 70 μ L of a solution containing 0.4mM TMB and 0.4mM H was added to the sample solution2O2pH =5.0 citrate buffer solution containing 95.8mM Na2HPO452.1mM sodium citrate and 40mM KCl, and after 5min of the color reaction, 30 μ L2M H was continuously added thereto2SO4Terminating the color development reaction; and (3) measuring the absorbance value of the sample solution by using an ultraviolet-visible spectrophotometer, and calculating the content of the kanamycin according to the quantitative relation between the absorbance and the concentration of the kanamycin.
2. The use of a homogeneous biosensing method for kanamycin detection according to claim 1 for the detection of kanamycin content in a sample.
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