DNA hydrogel and application thereof in detection of peroxide
A technology of peroxides and hydrogels, which is used in analysis by chemical reaction of materials, material analysis by observing the effect on chemical indicators, determination/inspection of microorganisms, etc.
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Embodiment 1
[0048] The steps for the synthesis of Acrydite-DNA are as follows:
[0049] 1) Synthesis of 6-hydroxyhexylmethacrylamide:
[0050] 6-Amino-1-hexanol (1 g, 8.53 mmol) was added to 20 ml of acetonitrile, and the solution was cooled to 0°C. Triethylamine (2.36mL, 17mmol) was added dropwise to the reaction solution and stirred continuously for 5min. Methacryloyl chloride (2.67g, 2.55mmol 2 ) was slowly added dropwise into the solution, and the mixture was reacted at 0°C for 2h. During the reaction process, the reaction progress was confirmed by spot TLC (ethyl acetate:petroleum ether=5:1). After the reaction was completed, the solvent was removed, and the remaining substance was washed with saturated NaHCO 3 and saturated NaCl to give the product 3 , 80% yield. The extract was dissolved in 10ml ethanol and 15% NaOH (4mL). The reaction solution was stirred for 1 h, and the progress of the reaction was confirmed by point TLC (ethyl acetate: petroleum ether = 5: 1). After the r...
Embodiment 2
[0055] The preparation steps of polyacrylamide hydrogel with DNA as cross-linking agent are as follows:
[0056] 1) Preparation of polyacrylamide chain steps containing DNA sequences
[0057] Add 3 μL of 25% acrylamide and 4.5 μL of 4mM acrydite-DNA to 100mM HEPES buffer, then add 2% ammonium persulfate (APS) and 2% N,N,N',N'-tetramethyl-linked Aniline (TEMED), after mixing, react under vacuum for 15 minutes to obtain polyacrylamide chains containing DNA.
[0058] 2) Preparation steps of DNAzyme:
[0059] Take 1.8μL of 4mM T-DNAzyme-T into a 200μL centrifuge tube, add buffer A (50 mM morpholineethanesulfonic acid (MES) pH 6.5; 100 mM tris-acetate buffer pH 6.5; 40 mM potassium acetate; 1%DMSO; 0.05%Triton X-100). The mixture was kept at 95°C for 5min, and then slowly cooled to room temperature for about 1h to form a DNA quadruplex structure. Add 1.5 μL of 0.6 mM heme and mix it well to form the DNAzyme.
[0060] 3) Preparation of DNA hydrogel:
[0061] Add 4.5 μL of the ...
Embodiment 3
[0064] The steps for detecting peroxides using DNA hydrogels are as follows:
[0065] Determination of hydrogen peroxide:
[0066] Take 40 μL of buffer A in a centrifuge tube, then add about 1 μL of DNA hydrogel and 150 μL of hydrogen peroxide, 3,3',5,5'-tetramethylbenzidine (TMB) Buffer solution B (0.1M citric acid, 0.2M disodium phosphate, 100mM potassium chloride, pH 4.4), mix well and start the reaction. Observe the color change of the hydrogel, and record the results by taking pictures after 50 min. As a control experiment, 40 μL of DNAzyme-heme and heme solution (same composition as buffer A) were prepared in advance, and then 150 μL of hydrogen peroxide (final concentration was 2 mg / L), 3,3',5 , 5'-Tetramethylbenzidine (TMB) buffer solution B, mix well, start the reaction, observe the color change of the solution.
[0067] see test results Figure 4 shown, from Figure 4 It can be seen that the DNA hydrogel catalyzed hydrogen peroxide oxidation of the peroxidase ...
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