CN112844325B - Specific DNA hydrogel material for extracting uranium from seawater and preparation method thereof - Google Patents
Specific DNA hydrogel material for extracting uranium from seawater and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a specific DNA hydrogel material for extracting uranium from seawater, which comprises the following steps: preparation of circular DNA: uniformly mixing a phosphorylated single-chain DNA template and a primer 1, then annealing, and adding ligase and a ligase buffer solution to synthesize circular DNA; preparation of cyclic prepolymer: taking circular DNA, polymerase buffer solution, bovine serum albumin, sodium chloride solution and deoxynucleotide triphosphate according to a certain proportion, mixing, and performing rolling circle replication to obtain a circular prepolymer; preparing DNA hydrogel: adding a primer 2 and a primer 3, continuing to perform rolling circle replication, and then performing enzyme inactivation operation to obtain the DNA hydrogel material. The DNA hydrogel material prepared by the method has good selectivity on uranium extraction, can resist coexisting interference ions, particularly has selectivity on uranyl ions which is 17.95 times of that of vanadium ions, has good tensile property and can be recycled.
Description
Technical Field
The invention relates to the field of new material preparation, in particular to a specific DNA hydrogel material for extracting uranium from seawater and a preparation method thereof.
Background
Nuclear energy has long been of great interest as one of the most mature environmentally friendly energy sources, and uranium resources in terrestrial uranium ores are estimated to be used only for nuclear power plants for less than a century as fuels for the nuclear power industry, without considering the increasing energy consumption. The uranium reserve in seawater is about 45 hundred million tons, and the uranium reserve is estimated to be 1000 times of that of terrestrial uranium ores, so that thousands of years of nuclear energy can be ensured for sustainable power generation. Therefore, extraction of uranium resources from seawater is of great importance to sustainable development of nuclear energy. However, extraction of uranium resources from actual seawater is a challenging task due to the complex marine environment, low uranium concentration (3.3ppb), complex interfering ions, and severe marine biofouling.
The first partial scholars have been devoted to research on excellent uranyl-binding functional groups such as amidoxime groups, which can efficiently extract uranium from seawater, and although the application of amidoxime-based uranium extraction materials greatly improves the uranium extraction efficiency, they are considered to be the most promising candidates for extracting uranium from seawater. However, in practical uranium extraction, the application of uranium is limited by serious interference of other metal ions in seawater, for example, the binding competitiveness of vanadium ions and amidoxime groups is even higher than that of uranyl. Therefore, the development of a novel uranium extracting material having a function of specifically recognizing uranyl ions is imminent.
The DNA hydrogel material is a novel biological macromolecular functional material, and has been widely applied to the fields of chemical detection, biological analysis and biomedicine due to the stable structure, flexible design, easy synthesis and modification, and excellent codeability and stimulus responsiveness. However, no research report about a DNA hydrogel material for specifically extracting uranium is seen at present.
Disclosure of Invention
In view of the above, the invention provides a preparation method of a specific DNA hydrogel material for extracting uranium from seawater, which solves the problem that the existing ammoxim-based material is interfered by other metal ions, especially vanadium ions, in the uranium extraction process, so that the selectivity is low.
The invention discloses a preparation method of a specific DNA hydrogel material for extracting uranium from seawater, which comprises the following steps:
s101 circular DNA preparation: uniformly mixing a phosphorylated single-stranded DNA template and a primer 1 according to a molar ratio of 1:1, then annealing, adding a ligase and a ligase buffer solution, and reacting for 2 hours at 20 ℃ to synthesize circular DNA;
s102 preparation of cyclic prepolymer: mixing circular DNA, polymerase buffer solution, bovine serum albumin, sodium chloride solution and deoxynucleotide triphosphate according to a certain proportion, adding water for dilution, reacting for 4 hours at the temperature of 30 ℃, and performing first round of rolling circle replication to obtain a circular prepolymer;
s103 DNA hydrogel preparation: adding a primer 2 and a primer 3 into the cyclic prepolymer system in the step S102, continuing to react at 30 ℃ for 24h to perform a second round of rolling circle replication, and then performing enzyme inactivation to obtain the DNA hydrogel material.
Preferably, in step S101, the sequence table of the DNA template is 5 '-phospate-TCGTTTGATGTTCCTAACGTACCACACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGTCATAGAGGCATTGGCTG-3';
the sequence listing of primer 1 is 5'-TAGGAACATCAAACGACAGCCA-3'.
Preferably, in step S101, the annealing is specifically to heat at 95 ℃ for 2min, then cool to 65 ℃ and heat for 2min, and cool to 60 ℃ at a cooling rate of 1 ℃/1min, and then cool to 20 ℃.
Preferably, in step S101, the adding amount of the ligase is 1. mu.L, and the adding amount of the ligase buffer is 2. mu.L.
Preferably, in step S102, the amount of circular DNA is 50nM, the amount of polymerase is 10U, the amount of polymerase buffer is 10. mu.L, the amount of bovine serum albumin is 2. mu.L, the concentration of sodium chloride solution is 800mM, and the amount of addition is 2. mu.L; the deoxynucleotide triphosphate concentration was 25mM and the amount added was 4. mu.L.
Preferably, in step S103,
the sequence table of the primer 2 is 5'-ACGTACCACACGTCCATCTCT-3';
the sequence table of primer 3 is 5'-ATAGTGAGTCATAGAGGCAT-3'.
Preferably, in step S103, the amount of primer 2 is 1. mu.L, and the amount of primer 3 is 1. mu.L.
Preferably, in step S103, the enzyme inactivation is specifically carried out at 65 ℃ for 10 min.
The preparation method of the specific DNA hydrogel material for extracting uranium from seawater provided by the invention is characterized in that DNA is used as a raw material, and a rolling ring replication polymerization reaction is used under the action of polymerase to obtain the DNA hydrogel, so that a novel adsorbent is provided for extracting uranium from seawater, the operation method is simple and easy to implement, the equipment is less in use, the adsorption time is short, and the practical application potential is large. The specific DNA hydrogel material obtained by the invention has good specificity and selectivity on uranium, can resist coexisting interfering ions, particularly has the selectivity on uranium which is 17.95 times that on vanadium, has good tensile property and can be recycled.
Drawings
FIG. 1 is a schematic diagram of a specific DNA hydrogel material for extracting uranium from seawater according to the present invention;
FIG. 2 is an electron microscope scanning image of a specific DNA hydrogel material for extracting uranium from seawater according to the present invention;
FIG. 3 is a schematic diagram of the stretching effect of a specific DNA hydrogel material for extracting uranium from seawater according to the present invention;
FIG. 4 is a diagram of selective uranium extraction of a specific DNA hydrogel material for extracting uranium from seawater according to the invention.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The first embodiment is as follows: a preparation method of a specific DNA hydrogel material for extracting uranium from seawater comprises the following steps:
s101 circular DNA preparation: uniformly mixing a phosphorylated DNA template and a primer 1 in a molar ratio of 1:1, then placing the mixture in a PCR amplification instrument, heating for 2min at 95 ℃, then cooling to 65 ℃, heating for 2min, cooling to 60 ℃ at a cooling rate of 1 ℃/1min, then gradually cooling to 20 ℃, annealing, then adding 1 mu L of ligase (T4-DNA) and 2 mu L of ligase (10 XT 4-DNA) buffer solution, and incubating for 2h at 20 ℃ to synthesize circular DNA; wherein the sequence table of the DNA template used in the step S101 is 5 '-phospate-TCGTTTGATGTTCCTAACGTACCACACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGTCATAGAGGCATTGGCTG-3';
the sequence table of the primer 1 is 5'-TAGGAACATCAAACGACAGCCA-3';
s102 preparation of cyclic prepolymer: 50nM circular DNA, 10U polymerase (1)
DNA), 10. mu.L of polymerase buffer solution (
DNA), 2. mu.L of bovine serum albumin (10 XBSA), 2. mu.L of a 800mM sodium chloride solution and 4. mu.L of 25mM deoxynucleotide triphosphate (DNA: (A) ((B))dNTPs), adding water to make the volume of the whole system be 100 mu L, reacting for 4h at 30 ℃ for first round rolling ring replication to obtain a cyclic prepolymer;
s103 DNA hydrogel preparation: subsequently, 1. mu.L of primer 2 and 1. mu.L of primer 3 were added to the S102 system, and the reaction was continued at 30 ℃ for 24 hours to perform a second round of rolling circle replication. The solution obtained above was reacted at 65 ℃ for 10min to inactivate the enzyme to obtain a DNA hydrogel material, which was dried and used, as shown in FIG. 1, and the DNA hydrogel of this example was a white filament in water. Wherein the primer 2 used in this step: 5'-ACGTACCACACGTCCATCTCT-3', respectively;
primer 3: 5'-ATAGTGAGTCATAGAGGCAT-3' are provided.
1. The specific DNA hydrogel material prepared in the first example was subjected to characterization test, as shown in the scanning electron microscope image of fig. 2, the microscopic morphology of the DNA hydrogel was composed of nano-scale petals composed of interacting flower-like structures, as shown in the tensile property test of fig. 3, the DNA hydrogel showed high tensile properties, and after being remolded into DNA hydrogel fibers at 90 ℃, the length thereof could be stretched by more than five times.
2. The specific DNA hydrogel material prepared in the first example is subjected to simulated seawater uranium extraction test:
weighing 5mg of the DNA hydrogel material prepared in the first embodiment, soaking the DNA hydrogel material in 20mL of simulated seawater with uranium concentration of 8ppm, with adsorption time of 30min, measuring the uranium concentrations before and after adsorption by using an ICP-OES inductively coupled plasma optical emission spectrometer, and calculating the adsorption capacity according to the following formula:
where qt represents the amount of uranium extracted during time t, and C0 represents the initial uranium concentration; ct represents the uranium concentration at time t; v represents the volume of test seawater; m represents the mass of the adsorbent. The measured extraction rate of the DNA hydrogel material to uranium in laboratory simulated seawater is 13.96mg/g, and good use repeatability can be shown through cyclic adsorption-desorption uranium extraction (the eluent is 0.1M EDTA).
3. To the embodimentsA test of extracting uranium from natural seawater is carried out on the prepared specific DNA hydrogel material, which comprises the following steps: 10mg of the dried DNA hydrogel was added to 100L of filtered seawater, and the test was repeated six times at 25 ℃. The natural seawater is added for 1L min-1Was flowed through the DNA hydrogel and the uranium concentration in the seawater was analyzed every 12h using inductively coupled plasma mass spectrometry. Experiments show that the DNA hydrogel also shows high selectivity to uranyl in natural seawater. The adsorption capacity of DNA hydrogel to uranium is 18.95 times that of vanadium ion (as shown in fig. 4), while existing amidoxime-based adsorbents only show 0.25 to 2.5 times selectivity to vanadium.
Comparative examples one to three: the comparative examples I to III are different from the example I in that polymerization temperatures used for the preparation of DNA hydrogel in the step S103 are different, and polymerization temperatures of 26 deg.C, 28 deg.C and 34 deg.C are used for the comparative examples I to III, respectively.
TABLE 1 Effect of different polymerization temperatures on the uranium extraction Performance of DNA hydrogels
| Item | Comparative example 1 | Comparative example No. two | Example one | Comparative example No. three |
| Reaction temperature (. degree.C.) | 26 | 28 | 30 | 34 |
| Uranium extraction amount (mg/g) | 8.93 | 10.42 | 13.96 | 10.85 |
Comparative examples four to seven: comparative examples four to seven are different from example one in that polymerization time for preparation of DNA hydrogel in step S103 was different, and polymerization temperatures of comparative examples four to seven were 12h, 16h, 20h and 30h, respectively.
TABLE 2 Effect of different polymerization times on the uranium extraction Performance of DNA hydrogels
Comparative examples eight to eleven: comparative examples eight to eleven differ from example one in that the effects of different amounts of raw materials (BSA, NaCl, dNTPs) on the uranium extraction performance of the DNA hydrogel in the preparation of the cyclic prepolymer in the step S102 are as shown in table 3, and other conditions are the same as in example one.
TABLE 3 influence of different raw material dosages on the uranium extraction performance of DNA hydrogel
| Item | Comparative example eight | Comparative example No. nine | Comparative example ten | Example one | Comparative example eleven |
| BSA | 1μL | 1μL | 2μL | 2μL | 3μL |
| NaCl(800mM) | 1μL | 1μL | 2μL | 2μL | 3μL |
| dNTPs(25mM) | 1μL | 2μL | 2μL | 4μL | 2μL |
| Uranium extraction amount (mg/g) | 6.32 | 7.86 | 8.76 | 13.96 | 10.26 |
As shown by experimental results, the DNA hydrogel shows high-specificity selective adsorption on uranyl ions. It is possible that the ligands of the phosphate may now form six coordination bonds between the oxygen atom in the phosphate group and the uranium atom in the uranyl group, possibly due to their specific helical space structure and the coordination synergy of the phosphate.
In conclusion, by adopting the preparation method of the specific DNA hydrogel material for extracting uranium from seawater provided by the invention, the DNA hydrogel is obtained by ring-forming prepolymerization and then rolling ring replication polymerization reaction, so that a novel adsorbent is provided for extracting uranium from seawater, the operation method is simple and easy to implement, the equipment is less in use, the adsorption time is short, and the practical application potential is large. The specific DNA hydrogel material obtained by the invention has good specificity and selectivity on uranium, can resist coexisting interfering ions, particularly has the selectivity on uranium which is 17.95 times that on vanadium, has good tensile property and can be recycled.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A preparation method of a specific DNA hydrogel material for extracting uranium from seawater is characterized by comprising the following steps:
s101 circular DNA preparation: uniformly mixing a phosphorylated single-stranded DNA template and a primer 1 according to a molar ratio of 1:1, then annealing, adding a ligase and a ligase buffer solution, and reacting for 2 hours at 20 ℃ to synthesize circular DNA;
s102 preparation of cyclic prepolymer: mixing circular DNA, polymerase buffer solution, bovine serum albumin, sodium chloride solution and deoxynucleotide triphosphate according to a certain proportion, adding water for dilution, reacting for 4 hours at the temperature of 30 ℃, and performing first round of rolling circle replication to obtain a circular prepolymer;
s103 DNA hydrogel preparation: adding a primer 2 and a primer 3 into the annular prepolymer system in the step S102, continuing to react at 30 ℃ for 24 hours to perform a second round of rolling ring replication, and then performing enzyme inactivation operation to obtain a DNA hydrogel material;
in the step S101, the sequence table of the DNA template is 5 '-phospate-TCGTTTGATGTTCCTAACGTACCACACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACATAGTGAGTCATAGAGGCATTGGCTG-3'; the sequence table of the primer 1 is 5'-TAGGAACATCAAACGACAGCCA-3';
in the step S102, the adding amount of the circular DNA is 50nM, the adding amount of the polymerase is 10U, the adding amount of the polymerase buffer solution is 10 muL, the adding amount of the bovine serum albumin is 2 muL, the concentration of the sodium chloride solution is 800mM, and the adding amount is 2 muL; the concentration of the deoxynucleotide triphosphate is 25mM, and the addition amount is 4 mu L;
in the step S103, the sequence table of the primer 2 is 5'-ACGTACCACACGTCCATCTCT-3'; the sequence table of primer 3 is 5'-ATAGTGAGTCATAGAGGCAT-3'.
2. The method for preparing a specific DNA hydrogel material for uranium extraction from seawater according to claim 1, wherein in the step S101, annealing is specifically to heat at 95 ℃ for 2min, cool to 65 ℃ and heat for 2min, cool to 60 ℃ at a cooling rate of 1 ℃/1min, and then cool to 20 ℃.
3. The method for preparing a specific DNA hydrogel material for uranium extraction from seawater according to claim 1, wherein in step S101, the adding amount of the ligase is 1 μ L, and the adding amount of the ligase buffer is 2 μ L.
4. The method for preparing a specific DNA hydrogel material for uranium extraction from seawater according to claim 1, wherein in step S103, the addition amount of primer 2 is 1 μ L, and the addition amount of primer 3 is 1 μ L.
5. The method for preparing a specific DNA hydrogel material for uranium extraction from seawater according to claim 1, wherein in step S103, the enzyme inactivation is specifically performed for 10min at 65 ℃.
6. A specific DNA hydrogel material for extracting uranium from seawater, which is prepared according to the method of any one of claims 1 to 5.
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