CN110819697A - Detection method of uranyl ions - Google Patents
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Abstract
The invention provides a simple DNA forceps probe which is used for amplifying and detecting uranyl ions based on a one-step method of DNA enzyme catalytic cracking. The two arms of the DNA tweezer probe are in close proximity in the original form, and therefore the fluorescent signal of the fluorophore at the end of the arm is significantly quenched. However, in the presence of uranyl ions, the structure of the DNA tweezer can change from "off" to "on" resulting in a strong fluorescent signal. The linear range of the obtained detection uranyl ions is 0.1nM to 60nM through the amplification of DNA enzyme catalytic cracking reaction, and the detection limit is 25 pM. Importantly, the whole detection process is very simple and only requires one operation step. In addition, the method has great potential and promising prospect for detecting uranyl ions in practical application.
Description
Technical Field
The invention relates to the field of detection of uranyl ions, in particular to the field of a method for detecting uranyl ions by amplification in one step based on a DNA forceps probe and DNA enzyme catalytic cracking.
Background
Enriched uranium can be used as nuclear fuel and nuclear weapons material. Worldwide uranium consumption may lead to uranium mining and nuclear waste releasing it into the environment, resulting in serious environmental pollution and human health problems. Uranium can be enriched into humans through the food chain, which can lead to severe childhood leukemia, lung cancer and other radiation-related diseases. Thus, the U.S. Environmental Protection Agency (EPA) sets a maximum contamination level of uranyl ions (130nM) in water.
To date, a number of techniques have been developed for uranium detection, including inductively coupled plasma mass spectrometry and atomic emission spectrometry, among others. However, they require expensive instruments and complicated operations. Recently, DNases that bind the enzyme chain (E-DNA) and the substrate chain (S-DNA) have been used to design biosensors for metal ions, such as uranyl ions, Mg2+, Cu2+, Pb2+, Zn2+ and Cd2 +. Various DNA enzyme-based methods for detecting uranyl ions have been reported, including colorimetric, fluorescent, and electrochemical methods. In addition, dnase-based probes have been used for fluorescence imaging of uranyl ions in living cells.
The DNA nano-machine is a DNA assembled nano-structure which can realize nano-mechanical motion at the nano-scale. DNA nanomachines are programmed and constructed from the general material "DNA" and have several unique advantages, such as ease of chemical synthesis, good thermal stability and functional modification. Moreover, DNA nanomachines are a promising platform for logical molecular computation with biological nanoscopic design, drug delivery, and with one-, two-, and three-dimensional nanostructures. DNA nanomachines such as DNA tweezers, DNA Walker, DNA motors, DNA gears and DNA nanocages have been designed with a serious degree of nanometer controllability and biocompatibility. DNA tweezers are typically nanomachines that respond to various external stimuli, including nucleic acids, metal ions, proteins, enzymes, and pH. To date, no DNA forceps have been used for metal ion detection in combination with DNase.
Disclosure of Invention
In order to solve the problems, the invention provides a method for amplifying and detecting uranyl ions based on a DNA forceps probe and a DNA enzyme catalytic cracking one-step method.
The invention comprises the following steps:
a method for detecting the concentration of uranyl ions in a solution comprises the following steps:
(1) preparing gold nanoparticles;
(2) the DNA sequence 4 is modified by the gold nanoparticles, one end of the DNA sequence 4 is thiolated, and the other end of the DNA sequence 4 is connected with a fluorescent group;
(3) preparing a DNA forceps probe by using the DNA sequence 4 obtained in the step (2) after the gold nanoparticles are modified;
(4) mixing the DNA forceps probe obtained in the step (3), a proper amount of uranyl ion specific DNA polymerase chain and a uranyl ion sample solution to be detected;
(5) and (4) detecting a fluorescence signal of the solution obtained in the step (4), and obtaining the concentration of the uranyl ions in the sample solution by using a standard curve.
Wherein, the DNA sequence 4 specifically comprises: HS-TACCCAAAAAACCT GGCTGCAACTCACTATrAGGAAGAGATGGACGTGACATACGGTACAAAAACCCTA-FAM.
Wherein, the DNA tweezers probe prepared together with the DNA sequence 4 in the step (3) also has DNA sequences 1-3, wherein the DNA sequence 1 is: TAGGCTTCGTAAGGTCCACATACATACATACACCAGCGAGAATGTTCCGT, DNA the sequence 2 is: TAGGGTTTTTGTACCGTACCGACGGAACATTCTCGCTGG, DNA SEQ ID NO: TGGACCTTACGAAGCCTAACTAGCCAGGTTTTTTGGGTA are provided.
Preferably, the uranyl ion-specific dnase chain is specifically: CACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGT are provided.
Preferably, the step (2) is specifically: thiolated DNA sequence 4 was mixed with gold nanoparticles in a 1: 1 for 12 hours to obtain the DNA sequence 4 modified by the gold nanoparticles.
Preferably, the step (3) is specifically: DNA tweezer probes were formed by mixing 100nM DNA sequences 1-4 in 100mM MES buffer (pH 5.5) and 300mM NaCl, then heating the mixture to 95 ℃ and slowly cooling.
Preferably, the step (4) is specifically: the 30nM uranyl ion-specific DNA polymerase chain and the solution of uranyl ions to be tested were mixed with DNA tweezers in 10mM MES buffer solution (pH 5.5) containing 300mM NaCl and incubated at 40 ℃ for 60 minutes.
Preferably, the fluorescent signal in step (5) is a fluorescent signal measured at 500nm to 600nm under excitation at 492 nm.
The invention constructs DNA tweezers for one-step amplification catalysis based on DNA enzyme, which is used for sensitive fluorescence detection of uranyl ions. DNA tweezers are formed by hybridization of DNA sequences. Fluorophores and gold nanoparticles (gold nanoparticles) are immobilized at the ends of the two arms of the DNA forceps, respectively. The two arms of the DNA tweezer are tightly connected by single-stranded DNA, resulting in quenching of the fluorescent signal. The linker sequence is then cleaved by the uranyl ion-specific dnase chain in the presence of the uranyl ion-specific dnase chain and uranyl, resulting in recovery of fluorescence intensity. The DNase can circularly cut other DNA tweezers to obviously improve the sensitivity.
The invention creatively combines DNA tweezers and DNA enzyme for metal ion detection, improves the sensitivity, also leads the detection process to be easy to operate and reduces the cost.
Drawings
Fig. 1 is a schematic diagram of the present invention.
FIG. 2 is a graph showing the intensity of fluorescence signals after changing the detection conditions.
FIG. 3(A) fluorescence spectra of DNA tweezers for different concentrations of uranyl ions: 0.1nM, 5nM, 10nM, 30nM, 60nM, 100nM, 150nM, 200 nM. (B) A relationship between fluorescence intensity and uranyl ion concentration. Illustration is shown: calibration plots of fluorescence intensity and uranyl ion between 0.1nM and 60 nM.
FIG. 4 is a graph showing the intensity of fluorescence signals generated by solutions containing the same concentration (60nM) of uranyl ions, Ca2+, Mg2+, Pb2+, Sn2+, Hg2+, Zn2+, Cu2+ and Co2 +.
Detailed Description
The present invention will be described in further detail with reference to embodiments.
As shown in fig. 1, the principle of the present invention is: the DNA tweezer structure is combined with sequences 1-4. The sequences 2 and 3 are each partially complementary to the end regions of sequence 1. They can hybridize to the ends of sequence 1, respectively, to form the two arms of the DNA tweezer. Then, sequence 4, modified with FAM and gold nanoparticles at both ends, can hybridize to a single portion of sequences 2 and 3, respectively, to form a complete DNA tweezer structure. The middle part of sequence 4 is tightly attached to both arms of the DNA tweezer, resulting in severe fluorescence quenching. The joining region has the same sequence as the substrate strand of the uranyl-specific dnase. It can be hybridized with uranyl ion specific DNA enzyme chain to form uranyl ion specific DNA enzyme. The linker region can be cleaved in the presence of uranyl ions to separate FAM and gold nanoparticles. The uranyl ion specific dnase chain can then be recombined with other DNA tweezers to form another dnase structure, and then catalytically cleave the linker of the DNA tweezers, so that the fluorescence signal is significantly restored. The concentration of uranyl can be quantitatively detected by fluorescence intensity.
The following experiments verify the feasibility of the detection method of the invention: and comparing the detection result of the optimal detection process with the detection result obtained after part of conditions of the optimal detection process are changed, and proving the feasibility of the method. The optimal detection process is as follows: preparing gold nanoparticles; DNA sequence 4 (HS-TACCCCAAAAACCTGGCAACTCACTATATrAGGAAGAGATGGACGTACAAACCCTTA-FAM) modified by the gold nanoparticles, wherein one end of the DNA sequence 4 is thiolated, and the other end is connected with a fluorescent group; preparing a DNA forceps probe by using the obtained DNA sequence 4 modified with the gold nanoparticles; mixing the obtained 100nM DNA forceps probe, 30nM uranyl ion specific DNA polymerase chain (CACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGT), and uranyl ion sample solution to be tested, and incubating at 40 deg.C for 60 min; the fluorescence signal of the resulting solution (signal of sample 6 in fig. 2) was measured. The sample 1 is a blank solution, i.e. the solution to be detected does not contain uranyl ions, the rest processes are the same as the optimal detection process, and under the condition that no uranyl ions exist, the DNA tweezers are still in an 'off' state, so that a weak fluorescence signal (the signal of the sample 1 in fig. 2) can be obtained. Sample 2 was a sample without the uranyl ion specific dnase chain and the rest of the process was identical to the optimal detection process (sample 2 signal in fig. 2), with similar fluorescence intensity as sample 1, indicating that no uranyl ion specific dnase chain could not form a uranyl ion specific dnase and that the linker of sequence 4 was still intact. Sample 3 is a method in which the specific DNA polymerase chain for uranyl ions in the optimal detection process is replaced with Pb2+Specific DNA polymerase chain: CATCTCTTCTCCGAGCCGGTCGAAATAGTGAGT, the rest of the process is the same as the optimal detection process (signal of sample 3 in fig. 2). The reason for the low fluorescence intensity of sample 3 is Pb2+The specific DNA polymerase chain can not form specific DNA enzyme of uranyl ion with sequence 4, so that the tweezers can not be opened when encountering uranyl ion. The sample 4 is half-reaction time, i.e. incubation is carried out at 40 ℃ for 30 minutes after mixing with the solution of uranyl ion sample to be detected, and the rest processes are the same as the optimal detection process (signal of sample 4 in fig. 2), fluorescenceThe intensity was significantly restored. This is because the cleavage reaction has proceeded to some extent in half the reaction time, and part of the tweezers has been opened. In sample 5, the molar ratio of the uranyl ion-specific dnase chain to the DNA tweezers was changed to 2:10, and the rest of the process was identical to the optimal detection process (signal of sample 5 in fig. 2), and the fluorescence intensity was reduced due to incomplete dnase cleavage reaction and a smaller amount of dnase.
To determine the fluorescent response of uranyl ions, various concentrations of uranyl ions were tested with the resulting DNA tweezer probe. As shown in fig. 3A, the fluorescence signal gradually increased with uranyl ions in the range of 0.1nM to 200 nM. In the range of 0.1nM to 60nM between the fluorescence intensity and the uranyl concentration, a good linear relationship can be obtained with a correlation coefficient of 0.993 (FIG. 3B). The detection limit of the sensitive DNA tweezers was evaluated to be 25pM according to the 3. sigma. blank standard. This limit of detection is comparable to other reported dnase-based methods, including fluorescence, colorimetric and electrochemical methods. RSD of 0.1nM uranyl ion measured in six replicates was 8.8%, indicating satisfactory reproducibility of the DNA tweezer probe.
In terms of specificity, the "sample solution containing uranyl ions" in the above-described optimal detection process was changed to a solution containing Ca2+, Mg2+, Pb2+, Sn2+, Hg2+, Zn2+, Cu2+, and Co2+ at the same concentration (60nM), and the fluorescence signal obtained in the remaining detection process was negligible (see fig. 4) as in the optimal detection process. It can be seen that the fluorescence intensity of other metal ions is much lower than that of uranyl ions. Meanwhile, experiments prove that even if the concentration of the interference ions is 100 times that of the uranyl ions, the interference generated by the interference ions can be ignored. The good selectivity of the method can be attributed to the strong specificity of the uranyl ion specific DNA polymerase chain.
Detecting uranyl ions in an actual sample:
the feasibility and applicability of the method for detecting uranyl ions are evaluated through different water samples (drinking water, tap water and river water). The water samples were purified by centrifugation and filtered through a 0.22 μm membrane. The pH of the above sample was adjusted to 5.5. The sample is then tested according to the optimal testing procedure. The uranyl concentration in the tap water sample determined in this way was 2.9nM and in the river was 4.7 nM. The recovery of the spiked samples was determined to be between 91.0% and 107.0%. In addition, the RSD is from 5.6% to 9.2%. The results show that the DNA forceps are feasible and can be used for practical water analysis.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (8)
1. A method for detecting the concentration of uranyl ions in a solution comprises the following steps:
(1) preparing gold nanoparticles;
(2) the DNA sequence 4 is modified by the gold nanoparticles, one end of the DNA sequence 4 is thiolated, and the other end of the DNA sequence 4 is connected with a fluorescent group;
(3) preparing a DNA forceps probe by using the DNA sequence 4 obtained in the step (2) after the gold nanoparticles are modified;
(4) mixing the DNA forceps probe obtained in the step (3), a proper amount of uranyl ion specific DNA polymerase chain and a uranyl ion sample solution to be detected;
(5) and (4) detecting a fluorescence signal of the solution obtained in the step (4), and obtaining the concentration of the uranyl ions in the sample solution by using a standard curve.
2. The method according to claim 1, characterized in that the DNA sequence 4 is in particular: HS-TACCAAAAACCTGGCAACTCACTATATrAGGAAGAGATGGACGGACGTGACGGACACACTACGGTACAAAACCCTA-FAM.
3. The method according to any of the preceding claims, characterized in that in step (3) DNA sequences 1 to 3 are also prepared together with DNA sequence 4 for the DNA tweezer probe, wherein DNA sequence 1 is: TAGGCTTCGTAAGGTCCACATACATACATACACCAGCGAGAATGTTCCGT, DNA the sequence 2 is: TAGGGTTTTTGTACCGTACCGACGGAACATTCTCGCTGG, DNA SEQ ID NO: TGGACCTTACGAAGCCTAACTAGCCAGGTTTTTTGGGTA are provided.
4. Method according to one of the preceding claims, characterized in that the uranyl ion-specific dnase chain is in particular: CACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGT are provided.
5. The method according to one of the preceding claims, characterized in that step (2) is embodied as: thiolated DNA sequence 4 was mixed with gold nanoparticles in a 1: 1 for 12 hours to obtain the DNA sequence 4 modified by the gold nanoparticles.
6. The method according to one of the preceding claims, characterized in that step (3) is embodied as: DNA tweezer probes were formed by mixing 100nM DNA sequences 1-4 in 100mM MES buffer (pH 5.5) and 300mM NaCl, then heating the mixture to 95 ℃ and slowly cooling.
7. The method according to one of the preceding claims, characterized in that step (4) is embodied as: the 30nM uranyl ion-specific DNA polymerase chain and the solution of uranyl ions to be tested were mixed with DNA tweezers in 10mM MES buffer solution (pH 5.5) containing 300mM NaCl and incubated at 40 ℃ for 60 minutes.
8. The method of any of the preceding claims, wherein the fluorescent signal in step (5) is a fluorescent signal measured at 500nm to 600nm under excitation at 492 nm.
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