Application of zirconium-based metal organic framework as alkaline phosphatase mimic enzyme
An organic framework, metal-based technology, applied in organic chemistry, organic compound/hydride/coordination complex catalyst, preparation of sugar derivatives, etc., can solve the problems of poor stability and high cost of biological enzymes, achieve stable properties, reduce Production cost, the effect that is conducive to large-scale promotion and application
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Embodiment 1
[0031] (a) Preparation of UiO-66 nanoparticles: Dissolve 159 mg zirconium tetrachloride and 102 mg terephthalic acid in 20 mL N,N-dimethylformamide, sonicate the mixture for 5 minutes, and transfer Add 0.3 mL of trifluoroacetic acid to a 25 mL polytetrafluoroethylene liner, and stir for 1 minute. Then the liner is put into the autoclave, at 120 o C for 24 hours. After the reaction, wait for the reactor to cool down to room temperature, collect the product by centrifugation, and wash three times with N,N-dimethylformamide and ethanol respectively. The washed product was vacuumed at 80 o C under the conditions of drying to obtain UiO-66 nanoparticles.
[0032] Various characterizations were carried out on the UiO-66 nanoparticles, such as figure 1 Shown are the synthesized UiO-66 nanoparticles and the UiO-66 nanoparticles in a series of different pH (pH = 2, 4, 6 and 8) HEPES buffer (the pH of the HEPES buffer was adjusted by adding NaOH or HCl). Precise control) The transm...
Embodiment 2
[0041] One of the functions of alkaline phosphatase is to convert the 5'-P end of the RNA fragment into a 5'-OH end, thereby preventing RNA self-ligation. Select the UiO-66 activated by the HEPES buffer with pH = 8, and make a solution of 80 µg / mL with the HEPES buffer with pH = 8, 37 o Under C, take 2970 µL of a series of solutions to hydrolyze 30 µL of 0.01-0.1 mM RNA mimics. Similarly, use a UV-Vis spectrophotometer to detect the absorption of the product p-nitrophenol at 405 nm every two minutes. Such as Figure 8 It was shown that the higher the concentration of RNA mimics, the faster the hydrolysis reaction, and when the concentration of PNPP increased to 0.05 mM, the reaction rate was hardly affected by the concentration of PNPP. This indicates that UiO-66 can mimic alkaline phosphatase for hydrolyzing the P-terminus of RNA.
Embodiment 3
[0043] UiO-66 was used to mimic alkaline phosphatase to detect antigen (mIgG). Take 2 mg UiO-66 in 2 mL water, add 800 μL water dissolved with 400 mM EDC and 100 mM NHS, stir at room temperature to activate the carboxyl group for 1 hour, the activated UiO-66 is dissolved in 100 μL CH 2 Cl 2 Wash once and once with 100 µL acetone and once with 100 µL water. Prepare 1 mg / mL UiO-66 dispersion system with pH = 8 HEPES buffer, take 2 mL and add 20 µL 10 mg / mL rabbit anti-mouse antibody (RIgG), 4 o C for 18 hours. The immobilized RIgG@UiO-66 was washed three times with pH = 8 HEPES buffer, and placed in 4 o C stored in the refrigerator.
[0044] Take a 96 microwell plate, add 100 µL of 10 µg / mL RIgG to each well, 4 o C overnight, washed 4 times with 0.01 M HEPES buffer dissolved in 0.05% Tween 20, added 400 µL of 0.01 M HEPES dissolved in 1% PBS, 37 o C for 40 min. Wash with 0.01 M HEPES buffer dissolved in 0.05% Tween 20, add 100 µL of different concentrations of mIgG (100, ...
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