CN109142751B - Method for sensitively detecting tetrodotoxin TTX based on nucleic acid cleavage enzyme I immune marker - Google Patents
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Abstract
The invention belongs to the technical field of biological detection, and provides a method for sensitively detecting tetrodotoxin TTX based on a nucleic acid cleaving enzyme I label-free method, aiming at solving the problems of complex and tedious operation, long determination time, low reaction sensitivity, poor selectivity, high cost and the like of the existing method for determining tetrodotoxin. Adding nucleic acid aptamer into phosphate buffer solution, taking berberine as a fluorescent probe, adding target substance TTX to cause the configuration change of the nucleic acid aptamer so as to change the fluorescent signal of the system, adding nucleic acid shear enzyme I to reduce the background signal of the system, amplifying the fluorescent signal of the system, and detecting the fluorescence intensity under the excitation wavelength of 365nm and the emission wavelength of 530 nm. The sensitivity is obviously improved; the linear range for detecting the TTX content is wider than 0.03 nM-6000 nM, the detection limit is 0.015nM, and the detection can be completed only by one common fluorometer, so the detection cost is lower.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a method for sensitively detecting tetrodotoxin TTX based on a nucleic acid cleaving enzyme I label-free mode. The berberine is taken as a fluorescent probe, the configuration of the aptamer is changed by adding target substance TTX, so that the fluorescent signal of the system is changed, the background signal of the system is obviously reduced by the aid of the nucleic acid shear enzyme I, the fluorescent signal of the system is amplified, and the method for rapidly and sensitively detecting the TTX is established. The method has the advantages of simple operation, low cost, good selectivity, high sensitivity, high accuracy and wide linear range.
Background
Tetrodotoxin (TTX) is a kind of raw material contained in puffer fish and other living bodiesAnd (4) alkali. The molecular formula is C11H17O8N3The molecular weight is 319. Is a potent neurotoxin, currently there is no effective antidote that binds to the fast sodium ion channels on the cell membrane of nerve cells, blocking the action potential in the nerve. Its name comes from the globefish order, and most fishes in this order carry the toxin, including puffer fish, fugu bidentate, fugu tumblers, puffer fish, etc.
Tetrodotoxin is stable in chemical and thermal properties, cannot be destroyed by common cooking means such as salt curing or sun drying, and can be decomposed only by heating at high temperature for more than 30min or under alkaline conditions. The poisoning latency is short, the disease is acute, if the rescue is not timely, the death is realized within the fastest 10min after the poisoning, and the death is realized within 4-6h at the latest. However, clinical researches in recent years find that the pure toxin product has wide application in the aspect of medicine and has extremely high commercial value.
In the course of serious harm of tetrodotoxin and extensive application in medicine field, it is necessary to establish a quick, accurate and sensitive detection method.
An Aptamer (Aptamer) is a stretch of DNA (deoxyribonucleic acid), RNA (ribonucleic acid) sequence, XNA (nucleic acid analog) or peptide. Generally, oligonucleotide fragments are obtained from libraries of nucleic acid molecules using in vitro screening techniques, the exponential enrichment of ligands by exponential evolution, SELEX. It can be combined with various target substances with high specificity and high selectivity, so that it is widely used in the field of biosensors.
Nucleases are specific enzymes that are capable of cleaving phosphodiester bonds between nucleotides from the end or inside of a nucleic acid. Among exonucleases, Exo III and Exo I, in particular, show strong selectivity for aptamer cleavage in both folded and unfolded structures, and therefore, they are often used in biosensing technology for selective detection of target substances.
The TTX can be determined by bioassay, chromatography (TLC, HPLC, LC-MS, GC-MS), enzyme-linked immunosorbent assay (ELISA), etc. The mouse method in the bioassay method is a commonly used detection method and is a method for detecting TTX content by the Japanese law, but the method has the problems that the individual differences of mice are large, so that the reproducibility of the measurement result is poor, and the operation is complicated, time-consuming and labor-consuming; although TLC (thin layer chromatography) and TLC (high performance liquid chromatography) are simple to operate, the sensitivity is too low, quantitative detection and analysis cannot be carried out, and HPLC (high performance liquid chromatography), LC-MS (liquid chromatography-mass spectrometry) and GC-MS have the problems of complex sample pretreatment, more serious matrix influence, expensive instruments and higher detection cost; although the enzyme-linked immunosorbent assay has strong specificity, cross reaction may have great influence on the quantitative detection of tetrodotoxin due to the poor specificity of the monoclonal antibody, the enzyme-linked immunosorbent assay consumes long time, and the operation difficulty is large.
Disclosure of Invention
The invention provides a method for sensitively detecting tetrodotoxin TTX based on a nucleic acid cleaving enzyme I label-free method, aiming at solving the problems of complex operation, long determination time, low reaction sensitivity, poor selectivity, high cost and the like of the existing method for determining tetrodotoxin.
The invention is realized by the following technical scheme: a method for sensitively detecting tetrodotoxin TTX based on nucleic acid cleavage enzyme I label-free comprises the steps of adding nucleic acid aptamer and berberine into a phosphate buffer solution, taking the berberine as a fluorescent probe, adding target substance TTX to cause the configuration of the nucleic acid aptamer to change so as to change the fluorescent signal of a system, then adding nucleic acid cleavage enzyme I, reducing the background signal of the system by the nucleic acid cleavage enzyme I, amplifying the fluorescent signal of the system, and detecting the fluorescent intensity under the excitation wavelength of 365nm and the emission wavelength of 530 nm. The aptamer is: TTX-aptamer: 5'-ttt tta aag tgt gcc cac gga gcc gac agg-3' are provided.
The specific detection method comprises the following steps:
(1) establishing a standard curve: 2.0 to 6.0 mM Na at pH =7.5+，2.0～4.0 mM Mg2+Adding 3 mu L of aptamer with the concentration of 0.1-0.5 mu M and a plurality of target substances TTX with the concentration gradient into 1 mM phosphate buffer solution, adding 10 mu L of berberine solution with the concentration of 20-40 mu M as a fluorescent probe, and fixing the volume to 1 ml; standing at normal temperature for 5-10 min, adding nucleic acid cleaving enzyme I with concentration of 30-40U, incubating at 40-43 deg.C for 5-10 min, and determiningThe fluorescence intensity of the system is F, and a blank solution without adding target substance TTX is marked as F in the fluorescent aptamer sensing system by the same method0(ii) a With TTX concentration C as abscissa, (F-F)0)/F0Establishing a standard curve and a linear regression equation for the ordinate;
(2) detecting the TTX content in the sample to be detected and carrying out a recovery rate experiment: adding the collected sample to be detected into the phosphate buffer solution system in the step (1), and detecting the TTX content in the sample to be detected by taking berberine as a fluorescent probe; and (3) adding the TTX standard solution into a sample to be detected, detecting the content of TTX in the sample after the TTX standard solution is added according to the operation method in the step (1), and performing a recovery rate experiment.
The concentration gradient of the target substance TTX is at least 0.03nM, 3nM, 300 nM, 1000 nM, 2000 nM, 3000 nM, 5000 nM, 6000 nM. The reaction time of the blank solution of the fluorescent aptamer sensing system without adding target substance TTX in the nucleic acid cleavage enzyme I is 20-40 min.
The reaction time of the sample solution of the fluorescent aptamer sensing system in the addition of the nucleic acid cleavage enzyme I is preferably 10 min. The aptamer preferably stands for 10min after the target molecule TTX is added.
Preferably: aptamer concentration was 0.3 μ M; the berberine concentration is 20 μ M.
Compared with the prior method, the method has simple operation, and the sample does not need a complex pretreatment process; the method has high selectivity and is hardly influenced by the matrix; after the Exo I is added, the higher background interference of a system caused by the nonspecific binding of the fluorescent dye and the aptamer is obviously reduced, so that the sensitivity of the method is obviously improved; based on an Exo I label-free fluorescent aptamer sensing platform, the linear range of TTX content detection is wider than 0.03 nM-6000 nM, the detection limit is 0.015nM, the established method can be completed only by one common fluorometer, and the detection cost is low.
The method has better recovery rate of 99.6-101.8% when being applied to the detection of the TTX content in the human serum of the actual biological sample, and further proves the feasibility of the method applied to the TTX detection in the human serum of the biological sample.
The method establishes a sensing platform based on Exo I label-free fluorescent aptamer, uses Exo I as single-chain aptamer degrading enzyme, and detects the TTX content by observing the fluorescence intensity change of a system. The method has the characteristics of simple and rapid operation, low cost and the like, and has wide response range and high sensitivity when being used for TTX detection.
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FIG. 1 is a line graph of the system at different pH conditions; FIG. 2 shows Na in the system+A concentration investigation line graph; FIG. 3 is an aptamer concentration investigation line graph; FIG. 4 is a line diagram for examining berberine concentration; FIG. 5 is a line diagram for examining the temperature of the enzyme reaction; FIG. 6 is a graph showing the fluorescence intensity variation of the berberine/aptamer/Exo I complex system at three temperatures; FIG. 7 is a graph showing the fluorescence intensity change of the berberine/aptamer/Exo I complex system at three temperatures when TTX is added as a target; FIG. 8 is a graph showing the examination of the concentration of a nucleolytic enzyme; FIG. 9 is a TTX calibration curve and a linear regression equation; FIG. 10 is a TTX linear spectrum; FIG. 11 is a bar graph of a selective investigation of the sensing regime; fig. 12 is an experimental feasibility study.
Detailed Description
The method is further explained below with reference to the figures and examples.
Example 1: making a standard curve
Dissolving 3 μ L of aptamer solution with concentration of 0.1-0.5 μ M and 10 μ L of berberine solution with concentration of 20-40 μ M in PBS (1 mM, pH =7.5, containing 2.0-6.0 mM Na) containing different concentrations of TTX+，2.0～4.0 mM Mg2+) Setting the volume at middle constant, standing at room temperature for 5-10 min, adding Exo I with the concentration of 30-40U into the mixed solution, controlling the reaction temperature to be 40-43 ℃, reacting for 5-10 min, measuring the fluorescence intensity and marking as F, and preparing a blank solution of the fluorescent aptamer sensing system without adding target substance TTX by the same method; adding Exo I, reacting for 20-40min, and measuring fluorescence intensity and recording as F0(ii) a The excitation wavelength of the solution was 365nm and the emission wavelength was 530 nm.
The nucleic acid aptamers are: TTX-aptamer: 5'-ttt tta aag tgt gcc cac gga gcc gac agg-3' are provided.
TTX concentration is used as abscissa (F-F)0)/F0And establishing a standard curve and a linear regression equation for the ordinate.
Experimental example 1: the optimum pH of the system was examined. Selecting five buffer solutions with different pH solutions, respectively adding magnesium chloride, aptamer and TTX at pH = 6.5-8.5, finally adding a fluorescent probe, uniformly mixing and standing, adding Exo I for incubation, and determining the fluorescence intensity at the wavelength of 530nm by taking 365nm as the excitation wavelength. Preparing blank solution by the same method. The results are shown in FIG. 1. As is clear from FIG. 1, when the pH of the solution was 7.5, the change in the fluorescence intensity of the system was the largest, and the pH of the system was set to 7.5.
Experimental example 2: na in the system+And (5) concentration investigation. And (2) dissolving the TTX-aptamer, the berberine solution and sodium chloride solutions with different concentrations in a PBS (phosphate buffer solution) containing TTX, fixing the volume to be 1mL, standing at normal temperature, adding Exo I into the mixed solution, operating according to the step 1, and determining the fluorescence intensity at the wavelength of 530nm by taking 365nm as the excitation wavelength. Preparing blank solution by the same method. The results are shown in FIG. 2. As is clear from FIG. 2, the concentration of sodium chloride in the solution was 3.0 to 6.0 mM, since the change in fluorescence intensity of the system was the greatest at the concentration of sodium chloride in the solution of 3.0 to 6.0 mM.
Experimental example 3: and (5) examining the concentration of the aptamer. Experiments investigated the concentration and system of TTX (F-F) when different aptamer concentrations were added0)/F0The linear relationship of (c). The results are shown in FIG. 3. As can be seen from FIG. 3, the change in fluorescence intensity of the system was the greatest at an aptamer concentration of 0.3. mu.M. This concentration condition indicates a high sensitivity for detecting TTX, and therefore the concentration of aptamer was experimentally selected to be 0.3. mu.M.
Experimental example 4: and (5) inspecting the concentration of the berberine. The concentration and system of TTX (F-F) under the conditions of 10 muM, 20 muM and 30 muM are examined0)/F0The linear relationship of (c). The results are shown in FIG. 4. As can be seen from FIG. 4, the fluorescence intensity of the system changed most when the concentration of berberine was 20. mu.M. The high sensitivity of detecting TTX under the concentration condition is shown, so the berberine concentration selected by the experiment is 20 mu M.
Experimental example 5: and (5) observing the temperature of the enzyme reaction. The fluorescence intensity change value of the system under five temperature conditions is investigated in the experiment. The results are shown in FIG. 5. As can be seen from FIG. 5, the degradation of the enzyme is weak between 25 ℃ and 35 ℃ and is substantially complete at temperatures above 40 ℃, so the temperature of the enzyme reaction is selected experimentally to be above 40 ℃.
Experimental example 6: and (4) optimizing the temperature and time of the nuclease reaction. The experiment examines the fluorescence intensity change values of the berberine/aptamer/Exo I composite system at three temperatures, and the result is shown in figure 6. As can be seen from FIG. 6, the shorter the time required for the fluorescence value of the berberine/aptamer/Exo I complex system to reach the minimum value with the increase of the temperature. And (3) taking the temperature of 40-43 ℃ and the time of 20-40min as the reaction temperature and the reaction time of the berberine/aptamer/Exo I composite system according to the comprehensive experiment result.
The experiment further examines the change value of the fluorescence intensity of the system when the target substance TTX is added at three temperatures. The results are shown in FIG. 7. As can be seen from FIG. 7, the shorter the time required for the fluorescence value of the berberine/aptamer/TTX/Exo I complex system to reach the minimum value with the increase of the temperature. And (3) taking the temperature of 40-43 ℃ and the time of 5-10 min as the reaction temperature and the reaction time of the berberine/aptamer/TTX/Exo I composite system according to the comprehensive experiment result.
Experimental example 7: the concentration of the nucleic acid cleaving enzyme was examined. The results are shown in FIG. 8. The fluorescence intensity of the system is reduced along with the increase of the concentration of the Exo I, which shows that the degradation effect is enhanced along with the increase of the concentration of the Exo I, and when the concentration of the Exo I is more than a certain value, the fluorescence intensity of the system reaches the lowest value. In the experiment, 30-40U is selected as the optimal reaction concentration of Exo I.
Experimental example 8: TTX calibration curve and linear regression equation. TTX is in good linearity in the range of 0.03-6000 nM, and the detection limit is 0.015 nM. The results are shown in FIG. 9. The TTX linear spectrum is shown in FIG. 10.
Experimental example 9: selective investigation of the sensing system. Experiment investigates K+、Cl-、CO3 2-、PO4 3-Cysteine, ascorbic acid, glutathione, aspartic acid, lysine, serine, tryptophan, glucose and the like can interfere with the fluorescence response of the system, so as to test the selectivity of the sensing system. The results are shown in FIG. 11.
As can be seen from fig. 11, only TTX caused significant signal enhancement, while other substances caused little change in fluorescence even at a concentration 10 times or more higher than TTX. Because TTX aptamer has the capacity of being specifically combined with target molecules, experimental results show that the aptamer sensing system constructed by the invention has higher selectivity on TTX.
Experimental example 10: TTX detection in serum and recovery rate experiment. We used this method for the detection of TTX concentration in serum. The collected blood was centrifuged, and the supernatant was collected to obtain serum, which was then subjected to the procedure of example 1 to determine the TTX content of the sample, indicating that the serum contained no TTX. Meanwhile, TTX standard solutions with different concentrations are added into serum by adopting a standard addition method to carry out a recovery rate test, the data is the average value of six experimental results, the results are shown in table 1, the recovery rate is 99.63% -101.82%, and the results further prove the feasibility of the method applied to the accurate detection of the TTX concentration in the human serum.
TABLE 1 TTX detection and recovery Rate Table in serum
The results of this method compared to the reported method in the linear range and detection limits are shown in table 2.
TABLE 2 method comparison table for determining TTX content
Claims (6)
1. A method for sensitively detecting tetrodotoxin TTX based on a nucleic acid cleaving enzyme I immune label is characterized in that: adding nucleic acid aptamer and berberine into a phosphate buffer solution, taking the berberine as a fluorescent probe, adding target substance TTX to cause the configuration of the nucleic acid aptamer to change, so that the fluorescent signal of a system is changed, then adding nucleic acid shear enzyme I, reducing the background signal of the system by the nucleic acid shear enzyme I, amplifying the fluorescent signal of the system, and detecting the fluorescent intensity under the excitation wavelength of 365nm and the emission wavelength of 530 nm;
the aptamer is: TTX-aptamer: 5'-ttt tta aag tgt gcc cac gga gcc gac agg-3', respectively;
the specific detection method comprises the following steps:
(1) establishing a standard curve: 2.0 to 6.0 mM Na at pH =7.5+，2.0～4.0 mM Mg2+Adding 3 mu L of aptamer with the concentration of 0.1-0.5 mu M and a plurality of target substances TTX with the concentration gradient into 1 mM phosphate buffer solution, adding 10 mu L of berberine solution with the concentration of 20-40 mu M as a fluorescent probe, and fixing the volume to 1 ml; standing at normal temperature for 5-10 min, adding nucleic acid shear enzyme I with the concentration of 30-40U, incubating at 40-43 ℃ for 5-10 min, determining the fluorescence intensity F of the system, and marking the blank solution without target substance TTX in the fluorescent aptamer sensing system as F by the same method0(ii) a With TTX concentration C as abscissa, (F-F)0)/F0Establishing a standard curve and a linear regression equation for the ordinate;
(2) detecting the TTX content in the sample to be detected and carrying out a recovery rate experiment: adding the collected sample to be detected into the phosphate buffer solution system in the step (1), and detecting the TTX content in the sample to be detected by taking berberine as a fluorescent probe; and (3) adding the TTX standard solution into a sample to be detected, detecting the content of TTX in the sample after the TTX standard solution is added according to the operation method in the step (1), and performing a recovery rate experiment.
2. The method for detecting TTX based on the immune label sensitivity of nucleolytic enzyme I as claimed in claim 1, wherein the method comprises the following steps: the concentration gradient of the target substance TTX is at least 0.03nM, 3nM, 300 nM, 1000 nM, 2000 nM, 3000 nM, 5000 nM, 6000 nM.
3. The method for detecting TTX based on the immune label sensitivity of nucleolytic enzyme I as claimed in claim 1, wherein the method comprises the following steps: the reaction time of the blank solution of the fluorescent aptamer sensing system without adding target substance TTX in the nucleic acid cleavage enzyme I is 20-40 min.
4. The method for detecting TTX based on the immune label sensitivity of nucleolytic enzyme I as claimed in claim 1, wherein the method comprises the following steps: the reaction time of the sample solution of the fluorescent aptamer sensing system in the addition of the nucleic acid cleavage enzyme I is 10 min.
5. The method for detecting TTX based on the immune label sensitivity of nucleolytic enzyme I as claimed in claim 1, wherein the method comprises the following steps: the aptamer is kept standing for 10min after target molecule TTX is added.
6. The method for detecting TTX based on the immune label sensitivity of nucleolytic enzyme I as claimed in claim 1, wherein the method comprises the following steps: the aptamer concentration is 0.3 μ M; the berberine concentration is 20 μ M.
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