CN115791726B - A method for ultrasensitive and highly selective detection of strontium ions based on G-quadruplex DNA - Google Patents
A method for ultrasensitive and highly selective detection of strontium ions based on G-quadruplex DNA Download PDFInfo
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Abstract
The invention discloses a method for detecting strontium ions with high sensitivity and high selectivity based on G-tetrad DNA, which comprises the steps of measuring a DNA aptamer and thioflavine T, and dissolving the DNA aptamer and the thioflavine T in a buffer solution to prepare a TiG-DNA sensor, wherein the nucleotide sequence of the DNA aptamer is 5'-AGGGTTAGGGTTAGGGTTAGGG-3'; adding strontium ion solution with known concentration, mixing uniformly, standing, detecting fluorescent signal, and constructing a standard curve according to fluorescence intensity attenuation; adding a sample to be detected into the TiG-DNA sensor, uniformly mixing, detecting a fluorescent signal, and substituting the fluorescent intensity detection value of the sample to be detected into calculation to obtain the Sr 2+ concentration of the sample to be detected. The detection method of the invention utilizes thioflavine T to trigger the DNA folding to form the G-tetrad biosensor, has high initial fluorescence intensity and specific identification of strontium ions, realizes the ultra-sensitive detection lower limit of 2.11nM and high selectivity, and provides a solution for detection and monitoring of strontium ions in the actual environment solution.
Description
Technical Field
The invention relates to the technical field of metal ion detection, in particular to a method for detecting strontium ions based on ultra-sensitivity and high selectivity of G-tetrad DNA.
Background
The use of nuclear energy inevitably creates a potential risk of nuclear accidents, posing a great threat to public health. 90 Sr is used as a fission product of 235 U, is a radionuclide, has a radioactive half-life as long as 29 years and can cause long-term damage. Although strontium has chemical properties similar to calcium, an element necessary for the human body can play an important role in various biological activities by substituting calcium, 90 Sr radiation also has a great influence on the health of the human body, and bone cancer and leukemia can be caused in serious cases. There is also evidence that: excessive strontium inhalation can lead to heart and nervous system diseases including severe dyspnea, anaphylaxis, and extreme tachycardia. At present, the radioactivity 90 Sr is detected in soil, water, food and biological entities, and the development of strontium detection technology has important significance due to the wide distribution of the radioactivity 90 Sr in natural environment and the harmful effect of the radioactivity on human health.
Various strontium detection techniques have been developed, including inductively coupled plasma atomic emission spectrometry (ICP-AES), atomic Absorption Spectrometry (AAS), X-ray fluorescence spectrometry (XRF), chemical Paper Sensors (CPS), and potentiometric titration. However, these techniques require complex sample pretreatment and large-scale instrumentation, and are not suitable for direct on-site detection and comprehensive monitoring of strontium ions. In addition, the number of the interference metal ions is huge, the Sr concentration in the environment is low, and the specificity and the sensitivity of the detection method can not meet the actual requirements.
With the development of sensing technology, the DNA biosensor provides a promising alternative method for rapidly, conveniently and cheaply detecting Sr by finding the specific recognition site of a specific substance. Wherein the G-quadruplex is a biological higher structure and can be formed by folding DNA or RNA rich in repetitive guanine (G). The 4 guanines are folded through hydrogen bonds to form a G-quadruplet, and form a basic structural unit of the quadruplet. Two or more G-quadrangles may be superimposed to form a G-tetrad structure. For example, chinese patent CN109799215B discloses a method for detecting Pb 2+ fluorescence based on G-tetrad DNA, which uses Pb 2+ to induce the single-stranded DNA rich in G base to form a change in G-tetrad configuration, thereby causing a change in fluorescence intensity, so as to realize detection of Pb 2+. However, in the prior art for detecting metal ions using G-quadruplex DNA, there are the following problems: the initial fluorescence intensity of the DNA sensor is not high because most fluorescent dyes have weak fluorescence due to the fact that the dyes themselves are slightly weak in the G-tetrad stacking process; and other technologies need proper fluorescent dye structures and Na + or K + ions and the like to assist in inducing specific DNA fragments to form G-quadruplets with specific configurations, so that the detection sensitivity of the G-quadruplet DNA is weakened. Due to the lack of a proper fluorescent signal molecule and the ambiguous action mechanism of the available signal molecule on the strontium ion detection of the G-tetrad DNA, a method for detecting the strontium ions based on the ultrasensitive high selectivity of the G-tetrad DNA is not discovered at present.
Disclosure of Invention
In view of the above, the invention provides a method for detecting strontium ions with ultra-sensitivity and high selectivity based on G-tetrad DNA, which solves the problems of low sensitivity and selectivity existing in the existing strontium ion chemical detection method.
The invention discloses a method for detecting strontium ions with ultra-sensitivity and high selectivity based on G-tetrad DNA, which comprises the following steps:
(1) Weighing a DNA aptamer and thioflavin T, and dissolving the DNA aptamer and the thioflavin T in Tris-HCl buffer solution to prepare TiG-DNA solution, wherein the nucleotide sequence of the DNA aptamer is 5'-AGGGTTAGGGTTAGGGTTAGGG-3', the concentration of the DNA aptamer in the TiG-DNA solution is 50nM, and the concentration of the thioflavin T is 7000nM;
(2) Adding TiG-DNA solution containing strontium ions with known concentration into the TiG-DNA solution in the step (1), uniformly mixing and standing, detecting fluorescence signals, and constructing a standard curve according to fluorescence intensity attenuation;
(3) Adding the TiG-DNA solution in the step (1) into the sample to be detected, uniformly mixing, detecting a fluorescent signal, and substituting the fluorescent intensity detection value of the sample to be detected into the calculation to obtain the Sr 2+ ion concentration of the sample to be detected.
Preferably, when the concentration of Sr 2+ ions is in the range of 10nM to 5. Mu.M, the concentration of Sr 2+ ions is in a linear relationship with the fluorescence intensity decay as in formula (1):
F＝70435.6-195.5CSr (1)
Wherein F is the fluorescence intensity of TiG-DNA solution after Sr 2+ ion is added, and C Sr (nM) is the concentration of Sr 2+ ion.
Preferably, the Tris-HCl buffer concentration is 10mM and the pH is 8.3.
The method for detecting strontium ions based on the ultra-sensitive and high-selectivity of the G-tetrad DNA provided by the application uses the G-tetrad TiG-DNA induced by fluorescent molecular thioflavin T as a biosensor, thereby realizing ultra-low detection limit of 2.11nM and higher detection selectivity. On the one hand, tiG-DNA has ultra-high initial fluorescence intensity due to electrostatic interaction and pi-pi accumulation between the G-tetrad and thioflavin T, so that the DNA has ultra-high detection sensitivity; on the other hand, thioflavin T induces DNA aptamer (5'-AGGGTTAGGGTTAGGGTTAGGG-3') to be converted into a G-tetrad structure in water environment to form TiG-DNA, and the coordination structure of TiG-DNA with specific recognition on Sr 2+ ions ensures that Sr 2+ ions have higher competitiveness on other coexisting metal ions and higher selectivity. The selection of the fluorescent dye and DNA aptamer sequences and their concentrations is of paramount importance in the present application.
Further, the detection method has extremely low detection limit, which is lower than the limit of the concentration of Sr 2+ ions in natural seawater (90.1 nM) and the concentration of Sr 2+ ions in drinking water regulated by the U.S. environmental protection agency (US EPA,3.4 nM). Therefore, the detection method provides an important solution for monitoring the concentration of Sr 2+ ions in seawater and drinking water caused by nuclear wastewater leakage.
Drawings
FIG. 1 shows the fluorescence test results of TiG-DNA, wherein a is the effect of different Sr 2+ ion concentrations on the fluorescence intensity of TiG-DNA, and b is the fitted curve of fluorescence intensity and Sr 2+ concentration;
FIG. 2 is an emission fluorescence plot of G-quadruplex DNA solutions prepared with different molar ratios of thioflavin to DNA aptamer;
FIG. 3 is a graph showing the fluorescence intensity decay after adding 100nM Sr 2+ ion solution to the G-quadruplex DNA solution of FIG. 2;
FIG. 4 is a graph showing the fluorescence decay trend of TiG-DNA solution when the concentration of interfering metal ions (such as Ca 2+、Ba2+、Na+ and K +) is 100 times higher than that of Sr 2+ ion, and E is a graph showing the fluorescence response of potential interfering ions to TiG-DNA solution, wherein c is the ability of single ion to trigger fluorescence decay;
FIG. 5 is a graph showing the results of comparison of the concentrations of Sr 2+ ions in mineral drinking water, natural seawater and simulated nuclear wastewater, respectively, by the method of example two and ICP-MS, in which TiG is a solution of 4-DNA for detecting Sr 2+ ions;
FIG. 6 is a schematic diagram of a method for detecting strontium ions with ultra-sensitivity and high selectivity by using a G-quadruplex DNA sensor according to the present invention.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
The DNA aptamer and Tris-HCl buffer referred to in the following examples were purchased from Biotechnology (Shanghai) Co., ltd, and the fluorescent dye thioflavin T, etc. was purchased from Shanghai Alasdine Biotechnology Co., ltd.
Embodiment one: a biosensor based on G-quadruplex DNA, comprising a DNA aptamer having a nucleotide sequence of 5'-AGGGTTAGGGTTAGGGTTAGGG-3' dissolved in Tris-HCl buffer at a concentration of 50nM and a TiG-DNA solution of thioflavin T at a concentration of 7000 nM.
Embodiment two: a method for detecting strontium ions with ultrasensitive high selectivity by using a G-tetrad DNA biosensor comprises the following steps:
(1) Measuring and dissolving the DNA aptamer and the fluorescent dye thioflavin T in Tris-HCl buffer solution with the concentration of 10mM and the pH of 8.3 respectively to obtain detection solution TiG-DNA, wherein the concentration of the DNA aptamer in the detection solution TiG-DNA is 50nM, the nucleotide sequence of the DNA aptamer is 5'-AGGGTTAGGGTTAGGGTTAGGG-3', the concentration of the thioflavin T is 7000nM, namely the molar ratio of the thioflavin T to the DNA is 140:1, a step of;
(2) Adding 2 mu L of Sr 2+ ion solution with known concentration into 2mL of TiG-DNA solution in the step (1), uniformly mixing, standing for 10min, detecting fluorescent signals, and constructing a standard curve according to fluorescence intensity attenuation;
Wherein the concentration of Sr 2+ ion is selected to be 10 nM-5 mu M, the fluorescence intensity gradually decreases with the increase of the concentration of Sr 2+ ion, and as a result, as shown in FIG. 1, the fluorescence intensity decays maximally when the concentration of Sr 2+ ion is 1mM, and the fluorescence intensity does not significantly decrease with the further increase of the concentration of Sr 2+ ion. In the concentration range of 0-100 nM, the linear relation between the concentration of Sr 2+ ions and the fluorescence intensity attenuation is shown as the formula (1):
F＝70435.6-195.5CSr (1)
Wherein F is fluorescence intensity after adding Sr 2+ ion, and C Sr (nM) is concentration of Sr 2+ ion. The result of calculating the LoD of TiG4-DNA on Sr 2+ ion by using 3 sigma method shows that the detection method realizes low LoD of 2.11nM, is the chemical detection method of Sr 2+ with highest sensitivity at present, and does not need large-scale instrument.
(3) Adding 2 mu L of sample to be detected into 2mL TiG-DNA solution in the step (1), uniformly mixing, detecting a fluorescent signal, substituting the fluorescent intensity detection value of the sample to be detected into calculation to obtain the concentration of Sr 2+ ions of the sample to be detected, and realizing quantitative detection of Sr 2+ ions.
Embodiment III: study of the Effect of thioflavin T and DNA aptamer in different molar ratios on fluorescence intensity of prepared G-tetrad DNA
As shown in FIGS. 2 and 3, thioflavin T was added to 50nM of DNA solution aptamer at various ratios (60:1-160:1), and when the molar concentration ratio of thioflavin T to DNA aptamer was less than 140:1, the fluorescence intensity increased with increasing amount of thioflavin T, whereas between 140:1-160:1, the fluorescence intensity was not significantly changed. And 100nM Sr 2+ ion solution is respectively added into G-tetrad DNA prepared by thioflavin T and DNA aptamer with different molar ratios, so that the fluorescence intensity is obviously attenuated, and the ratio of thioflavin T to DNA in the G-tetrad DNA with highest attenuation is 140:1.
Embodiment four: effect of different DNA aptamer sequences on strontium detection ability of prepared G-quadruplex DNA sensor
In contrast to the first example, the G-quadruplex DNA was prepared by selecting a different DNA aptamer and the fluorochrome thioflavin T, and the fluorescence intensity decay after adding 100nM Sr 2+ ion were tested, and as shown in the results of Table 1, when the DNA aptamer was single-stranded 5'-AGGGTTAGGGTTAGGGTTAGGG-3', the fluorescence intensity of the prepared G-quadruplex DNA was the strongest and the strontium ion detection ability thereof was the strongest. Wherein, the NO.1 and the NO.4 form double-stranded DNA, the NO.5 and the NO.6 form double-stranded DNA, and the CalfThymus DNA is calf thymus DNA.
TABLE 1 Effect of different DNA aptamers on the detection capability of prepared G-tetrad DNA on Sr 2+ ions
Fifth embodiment: detection of the selectivity of Sr 2+ ion Using the TiG-DNA solution of example one
Since a plurality of interference ions exist in the natural environment and coexist, the detection selectivity is important to the actual detection of Sr 2+ ions. Fluorescence decay analysis showed that 17 interfering ions with the same concentration as Sr 2+ ions, including the most abundant metal ions in the natural environment, had negligible effect on fluorescence intensity. Although Ca 2+ ion has very similar properties to Sr 2+ ion, the fluorescence decay caused by Sr 2+ ion is 3.39 times that caused by Ca 2+ ion (fig. 4 c). The effect of potentially interfering metal ions on the fluorescence decay caused by Sr 2+ ions was analyzed. The results show that even though the concentration of interfering metal ions (such as Ca 2+、Ba2+、Na+ and K +) is 100 times higher than the concentration of Sr 2+ ions, a negligible increase in fluorescence decay is observed (fig. 4 d). More importantly, all four interfering ions coexist at a concentration 100 times higher than the Sr 2+ ion, resulting in only a 5.01% increase in fluorescence decay (fig. 4 e). These results indicate that the TiG-DNA sensor of the first embodiment has significantly better selectivity for Sr 2+ ions than other metal ions, and is beneficial to the practical application of the detection method in complex natural environments.
Example six: test of Sr 2+ concentration in drinking mineral Water, natural seawater and Nuclear-simulated wastewater by the method of example two
The practicality of TiG-DNA detection of Sr 2+ ions was analyzed by testing the concentration of Sr 2+ ions in drinking mineral water, natural seawater and simulated nuclear wastewater, which represent samples of different pH values, different concentrations of Sr 2+ ions and different coexisting interfering ions, respectively. Seawater is obtained from the west coast of Hainan Haikou, and filtered with 0.22 μm filter membrane before use; simulated nuclear wastewater was prepared according to previous reports (CHEMICAL ENGINEERING Journal 2022,429,132265); the potable mineral water was purchased from the coconut palm group. To 1998. Mu.L of the above mixture was added 2. Mu.L of a water sample containing 50nM DNA aptamer, 7. Mu.M thioflavin T,10mM Tris (pH 8.3). Substituting the fluorescence intensity into a standard curve, and calculating the final concentration of Sr 2+ ions. The Sr 2+ concentration detected by this method in all samples is consistent with the detection value obtained by inductively coupled plasma mass spectrometry (ICP-MS), which is the most reliable method for detecting metal concentration, demonstrating the reliability and broad applicability of this detection method to Sr 2+ ion detection (fig. 5 a). Compared to the currently available chemical methods for detecting Sr 2+ ions, this detection method has a very low detection limit, below the limit for Sr 2+ ion concentration in drinking water prescribed by the united states environmental protection agency (US EPA,3.4 nM). Therefore, the detection method can monitor the change of the concentration of Sr 2+ ions in seawater and drinking water caused by nuclear wastewater leakage.
In conclusion, the method for detecting strontium ions by using the G-tetrad DNA sensor in an ultrasensitive and high-selectivity way is adopted. As shown in FIG. 6, on one hand, the fluorescent dye thioflavin T is selected to be mixed with DNA fragments with specific sequences, when thioflavin T is locked in G-tetrad DNA, electrostatic interaction and pi superposition between thioflavin T and the G-tetrad both enhance the fluorescence property of TiG-DNA, so that the fluorescent dye has extremely high fluorescence intensity; on the other hand, tiG-DNA has stronger binding affinity to Sr 2+ ions. The detection method has high initial fluorescence intensity of TiG-DNA and strong binding affinity of TiG-DNA to Sr 2+ ion, realizes ultra-sensitive detection limit and high selectivity of 2.11nM, and is one of the Sr 2+ ion detection methods with the best performance at present. The ultra-low detection limit greatly widens the applicability of the method to various environments containing trace Sr, and the detection reliability of the detection method is comparable with that of ICP-MS, and is considered as the most reliable technology in metal element detection. Therefore, the detection method has ultrahigh sensitivity, high specificity, wide applicability and high reliability, and provides a potential technology for protecting human beings from radioactive contamination and serving the sustainability of the nuclear energy industry.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather to enable any modification, equivalent replacement, improvement or the like to be made within the spirit and principles of the invention.
Claims (3)
1. A method for detecting strontium ions based on ultrasensitive high selectivity of G-tetrad DNA is characterized by comprising the following steps:
(1) Weighing a DNA aptamer and thioflavin T, and dissolving the DNA aptamer and the thioflavin T in Tris-HCl buffer solution to prepare TiG-DNA solution, wherein the nucleotide sequence of the DNA aptamer is 5'-AGGGTTAGGGTTAGGGTTAGGG-3', the concentration of the DNA aptamer in the TiG-DNA solution is 50nM, and the concentration of the thioflavin T is 7000nM;
(2) Adding TiG-DNA solution containing strontium ions with known concentration into the TiG-DNA solution in the step (1), uniformly mixing and standing, detecting fluorescence signals, and constructing a standard curve according to fluorescence intensity attenuation;
(3) Adding the TiG-DNA solution in the step (1) into the sample to be detected, uniformly mixing, detecting a fluorescent signal, and substituting the fluorescent intensity detection value of the sample to be detected into the calculation to obtain the Sr 2+ ion concentration of the sample to be detected.
2. The method for detecting strontium ions with high sensitivity and high selectivity based on G-tetrad DNA according to claim 1, wherein when the concentration of Sr 2+ ions is 10 nM-5 μM, the linear relation between the concentration of Sr 2+ ions and the fluorescence intensity attenuation is shown as formula (1):
F＝70435.6-195.5CSr (1)
Wherein F is the fluorescence intensity of TiG-DNA solution after Sr 2+ ion is added, C Sr is the concentration of Sr 2+ ion, and the unit is nM.
3. The method for detecting strontium ions with high sensitivity and high selectivity based on G-quadruplex DNA according to claim 1, wherein the concentration of the Tris-HCl buffer solution is 10mM, and the pH is 8.3.
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| CN109799215B (en) * | 2018-12-25 | 2021-09-28 | 北京农业质量标准与检测技术研究中心 | Pb based on G-quadruplex DNA2+Fluorescence sensing detection method |
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| CN110462039A (en) * | 2016-12-12 | 2019-11-15 | 科罗拉多大学董事会,法人团体 | Using biological RNA scaffolds selected in vitro to generate robust small molecule binding aptamers for genetically encoded biosensors |
| CN108458998A (en) * | 2018-01-29 | 2018-08-28 | 山西大学 | A method of the aptamers DNA silver nanoclusters based on label-free Fluorescence Increasing measure lead ion |
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