CN110819697B - Detection method of uranyl ions - Google Patents

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 uranyl ions for detection is 0.1nM to 60nM by the amplification of DNA enzyme catalytic cracking reaction, and the detection limit is 25pM. 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 the uranyl ions in practical application.

Description

A kind of detection method of uranyl ion

technical field

The invention relates to the field of detection of uranyl ions, in particular to the field of a method for one-step amplification and detection of uranyl ions based on DNA tweezers probes and DNase catalyzed cleavage.

Background technique

Enriched uranium can be used as nuclear energy fuel and nuclear weapons material. The worldwide consumption of uranium may lead to the release of uranium mining and nuclear waste into the environment, which can lead to serious environmental pollution and human health problems. Uranium can be enriched into the human body through the food chain, which can lead to severe childhood leukemia, lung cancer, and other radiation-related diseases. Therefore, the U.S. Environmental Protection Agency (EPA) has set a maximum contamination level (130nM) for uranyl ions in water.

So far, many 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, DNA enzymes that combine enzyme strands (E-DNA) and substrate strands (S-DNA) have been used to design biosensors for metal ions such as uranyl ions, Mg2+, Cu2+, Pb2+, Zn2+, and Cd2+. A variety of DNase-based methods for the detection of uranyl ions have been reported, including colorimetric, fluorescent, and electrochemical methods. In addition, DNase-based probes have been used for fluorescent imaging of uranyl ions in living cells.

A DNA nanomachine is a DNA-assembled nanostructure that can realize nanomechanical motion at the nanoscale. DNA nanomachines are programmed and constructed from the universal material "DNA", which has some unique advantages such as easy chemical synthesis, good thermal stability and functional modification. Moreover, DNA nanomachines are promising platforms for bionanodesign, drug delivery, and logical molecular computing with one-, two-, and three-dimensional nanostructures. Serious DNA nanomachines with nanoscale controllability and biocompatibility have been designed, such as DNA tweezers, DNA walkers, DNA motors, DNA gears, and DNA nanocages. DNA tweezers are typical nanomachines that can respond to different external stimuli, including nucleic acids, metal ions, proteins, enzymes, and pH. So far, there is no DNA tweezers combined with DNase for metal ion detection.

Contents of the invention

In order to solve the above problems, the present invention provides a method for the detection of uranyl ions based on one-step amplification based on DNA tweezers probe and DNase-catalyzed cleavage.

The present invention comprises the steps:

A method for detecting the concentration of uranyl ions in a solution, comprising the steps of:

(1) preparing gold nanoparticles;

(2) the DNA sequence 4 modified with the gold nanoparticles, one end of the DNA sequence 4 is thiolated, and the other end is connected to a fluorescent group;

(3) prepare DNA tweezers probes with the DNA sequence 4 obtained in step (2) after modifying the gold nanoparticles;

(4) mixing the DNA tweezers probe obtained in step (3), an appropriate amount of uranyl ion-specific DNA enzyme chain, and the uranyl ion sample solution to be measured;

(5) detecting the fluorescence signal of the solution obtained in step (4), and using the standard curve to obtain the concentration of uranyl ions in the sample solution.

Wherein, the DNA sequence 4 is specifically: HS-TACCCAAAAAACCT GGCTGCAACTCACTATrAGGAAGAGATGGACGTGACATACGGTACAAAAAACCCTA-FAM.

Wherein, DNA sequence 1-3 is prepared together with DNA sequence 4 in step (3), wherein DNA sequence 1 is: TAGGCTTCGTAAGGTCCACATACATACATACCACCAGCGAGAATGTTCCGT, DNA sequence 2 is: TAGGGTTTTTGTACCGTACCGACGGAACATTCTCGCTGG, and DNA sequence 3 is: TGGACCTTACGAAGCCTAACTAGCCAGGTTTTTTGGGTA.

Preferably, the uranyl ion-specific DNA enzyme chain is: CACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGT.

Preferably, the step (2) specifically includes: mixing the thiolated DNA sequence 4 and the gold nanoparticles at a molar ratio of 1:1 for 12 hours to obtain the gold nanoparticle-modified DNA sequence 4 .

Preferably, step (3) is specifically: by mixing 100 nM DNA sequences 1-4 in 100 mM MES buffer solution (pH 5.5) and 300 mM NaCl, then heating the mixture to 95° C., and cooling slowly to form DNA tweezer probes .

Preferably, step (4) is specifically: mix 30nM uranyl ion-specific DNA enzyme chain and uranyl ion solution to be tested with DNA tweezers in 10mM MES buffer solution (pH 5.5) containing 300mM NaCl, and then mix at 40°C Incubate for 60 minutes.

Preferably, the fluorescence signal in step (5) is the fluorescence signal measured at 500nm to 600nm under excitation at 492nm.

The present invention constructs a DNase-based one-step amplification catalytic DNA tweezers for sensitive fluorescence detection of uranyl ions. DNA tweezers are formed by the hybridization of DNA sequences. Fluorophores and gold nanoparticles (AuNPs) were immobilized at the ends of the two arms of the DNA tweezers, respectively. The two arms of the DNA tweezers are tightly connected by single-stranded DNA, which leads to the quenching of the fluorescent signal. Then, the linker sequence is cleaved by the uranyl ion-specific DNase strand in the presence of uranyl ion-specific DNase strand and uranyl, resulting in restoration of fluorescence intensity. DNase can circularly cleave other DNA tweezers to dramatically increase sensitivity.

The invention creatively combines DNA tweezers and DNA enzymes for metal ion detection, which improves the sensitivity, makes the detection process easy to operate, and reduces the cost.

Description of drawings

Fig. 1 is a schematic diagram of the present invention.

Fig. 2 is a graph of fluorescence signal intensity after changing detection conditions.

Fig. 3 (A) Fluorescence spectra of DNA tweezers for different concentrations of uranyl ions: 0.1nM, 5nM, 10nM, 30nM, 60nM, 100nM, 150nM, 200nM. (B) The relationship between fluorescence intensity and uranyl ion concentration. Inset: calibration plot of fluorescence intensity and uranyl ion between 0.1 nM and 60 nM.

Fig. 4 is a graph of fluorescence signal intensity generated by solutions containing uranyl ions, Ca2+, Mg2+, Pb2+, Sn2+, Hg2+, Zn2+, Cu2+ and Co2+ at the same concentration (60nM).

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments.

As shown in Fig. 1, the principle of the present invention is: the DNA tweezers structure is combined with sequences 1-4. Sequences 2 and 3 are partially complementary to the terminal region of Sequence 1, respectively. They can respectively hybridize with the ends of sequence 1 to form the two arms of DNA tweezers. Then, sequence 4 modified with FAM and AuNPs at both ends could hybridize with single parts of sequences 2 and 3, respectively, to form a complete DNA tweezer structure. The middle part of sequence 4 tightly connects the two arms of the DNA tweezers, resulting in severe fluorescence quenching. The linking region has the same sequence as the substrate strand of the uranyl-specific DNase. It can hybridize with uranyl ion-specific DNase chain to form uranyl ion-specific DNase. The linking region can be cleaved in the presence of uranyl ions, thereby separating FAM and gold nanoparticles. Then, the uranyl ion-specific DNase strands can recombine with other DNA tweezers to form another DNase structure, which then catalyzes the cleavage of the connecting part of the DNA tweezers, and thus, the fluorescent signal is significantly restored. The concentration of uranyl can be quantitatively detected by fluorescence intensity.

The following experiments have verified the feasibility of the detection method of the present invention: the detection results of the optimal detection process are compared with those after changing some conditions of the optimal detection process to prove the feasibility of the method. The optimal detection process is: preparing gold nanoparticles; using the DNA sequence 4 modified by the gold nanoparticles (HS-TACCCAAAAAACCTGGCTGCAACTCACTTrAGGAAGAGATGGACGTGACATACGGTACAAAAAACCCTA-FAM), one end of the DNA sequence 4 is thiolated, and the other end is connected to a fluorescent group; The DNA sequence 4 behind the nanoparticles was used to prepare DNA tweezer probes; the resulting 100 nM DNA tweezer probes, 30 nM uranyl ion-specific DNase chain (CACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGT), and the uranyl ion sample solution to be tested were mixed, and then incubated at 40°C for 60 minutes; the fluorescence signal of the resulting solution was measured (signal of sample 6 in Figure 2). Sample 1 is a blank solution, that is, the solution to be tested does not contain uranyl ions, and the rest of the process is the same as the optimal detection process. In the absence of uranyl ions, the DNA tweezers are still in the "closed" state, so weak fluorescence can be obtained signal (signal of sample 1 in Figure 2). Sample 2 is a sample without uranyl ion-specific DNase chain, and the rest of the process is the same as the best detection process (signal of sample 2 in Figure 2), with a similar fluorescence intensity to sample 1, indicating that there is no uranyl ion-specific DNase chain Strands cannot be formed by uranyl ion-specific DNase, and the linker of sequence 4 is still intact. For sample 3, the uranyl ion-specific DNase chain in the optimal detection process was replaced with a Pb ²⁺ -specific DNase chain: CATTCTCTTCTCCGAGCCGGTCGAAATAGTGAGT, and the rest of the process was the same as the optimal detection process (signal of sample 3 in Figure 2). The reason for the low fluorescence intensity of sample 3 is that the Pb ²⁺ -specific DNase chain cannot form uranyl ion-specific DNase with sequence 4, resulting in that the tweezers cannot be opened when encountering uranyl ions. Sample 4 is the half-reaction time, that is, after mixing with the uranyl ion sample solution to be tested, incubate at 40°C for 30 minutes, and the rest of the process is the same as the best detection process (signal of sample 4 in Figure 2), and the fluorescence intensity is significantly restored . This is because the cleavage reaction has already proceeded to a certain extent within half the reaction time, and part of the tweezers has been opened. In sample 5, the molar ratio of the uranyl ion-specific DNA enzyme strand to the DNA tweezers was changed to 2:10, and the rest of the process was the same as the optimal detection process (signal of sample 5 in Figure 2), because the DNase cleavage reaction was incomplete, and the DNA Lower amounts of enzyme lead to lower fluorescence intensity.

To determine the fluorescence response of uranyl ions, different concentrations of uranyl ions were tested with the formed DNA tweezer probes. As shown in Figure 3A, the fluorescence signal gradually increased with uranyl ions ranging from 0.1 nM to 200 nM. In the range of 0.1 nM to 60 nM between fluorescence intensity and uranyl concentration, a good linear relationship was obtained with a correlation coefficient of 0.993 (Fig. 3B). Based on the 3σ blank standard, the detection limit of this sensitive DNA tweezers was estimated to be 25pM. This detection limit is comparable to other reported DNase-based methods, including fluorometric, colorimetric, and electrochemical methods. The RSD of 0.1 nM uranyl ion for six repeated measurements was 8.8%, indicating that the DNA tweezer probe has a satisfactory reproducibility.

In terms of specificity, change the "sample solution containing uranyl ions" in the above optimal detection process to a solution containing the same concentration (60nM) of Ca2+, Mg2+, Pb2+, Sn2+, Hg2+, Zn2+, Cu2+ and Co2+, and the rest of the detection process With the optimal detection procedure described, the resulting fluorescent signal was negligible (see Figure 4). It can be seen that the fluorescence intensity of other metal ions is much lower than that of uranyl ions. At the same time, experiments have proved that even if the concentration of the above-mentioned interfering ions is 100 times that of uranyl ions, the interference produced by them can be ignored. The good selectivity of this method can be attributed to the strong specificity of the uranyl ion-specific DNase chain.

Detection of uranyl ions in real samples:

The feasibility and applicability of this method for the detection of uranyl ions were evaluated by different water samples (drinking water, tap water and river water). 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. Samples are then tested according to the optimal testing procedure described. The concentration of uranyl in tap water samples determined by this method was 2.9nM, and in river water was 4.7nM. The recoveries determined for the spiked samples ranged from 91.0% to 107.0%. Plus, the RSD ranged from 5.6% to 9.2%. The results show that the DNA tweezers are feasible and can be used for practical water analysis.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, there may be various modifications and changes in the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included within the protection scope of this application.

Claims (5)

1. a detection method of uranyl ion concentration in a solution, comprising the steps of: (1) preparing gold nanoparticles; (2) modifying the DNA sequence 4 with the gold nanoparticles, one end of the DNA sequence 4 is thiolated, and the other end is connected to a fluorescent group, wherein the DNA sequence 4 is specifically: HS-TACCCAAAAAACCTGGCTGCAACTCACTATrAGGAAGAGATGGACGTGACATACGG TACAAAAACCCTA-FAM; (3) prepare DNA tweezers probes with the DNA sequence 4 modified by the gold nanoparticles obtained in step (2); (4) Mix the DNA tweezers probe obtained in step (3), an appropriate amount of uranyl ion-specific DNA enzyme chain, and the uranyl ion-specific sample solution to be tested, wherein the uranyl ion-specific DNA enzyme chain is specifically: CACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGT; (5) detecting the fluorescent signal of the solution obtained in step (4), and utilizing the standard curve to obtain the concentration of uranyl ions in the sample solution, Wherein, DNA sequence 1-3 is prepared together with DNA sequence 4 in step (3), wherein DNA sequence 1 is: TAGGCTTCGTAAGGTCCACATACATACATACCACCAGCGAGAATGTTCCGT, DNA sequence 2 is: TAGGGTTTTTGTACCGTACCGACGGAACATTCTCGCTGG, and DNA sequence 3 is: TGGACCTTACGAAGCCTAACTAGCCAGGTTTTTTGGGTA. 2. The method according to claim 1, characterized in that step (2) is specifically: the thiolated DNA sequence 4 is mixed with gold nanoparticles at a molar ratio of 1:1 for 12 hours to obtain the DNA modified by gold nanoparticles Sequence 4. 3. The method according to claim 1 or 2, characterized in that step (3) is specifically: by mixing the DNA sequence 1-4 of 100nM in 100mMMES buffer solution of pH 5.5 and 300mM NaCl, then the mixture is heated to 95 °C and slowly cooled to form DNA tweezer probes. 4. The method according to claim 3, characterized in that step (4) is specifically: 30nM uranyl ion-specific DNA enzyme chain and uranyl ion solution to be measured and DNA tweezers at pH 5.5 of 10mM containing 300mM NaCl MES buffer solution, and then incubated at 40 °C for 60 min. 5. The method according to claim 3, characterized in that the fluorescence signal in step (5) is the fluorescence signal measured at 500nm to 600nm under 492nm excitation.

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