Preparation method and application of hydrogel/metal organic framework composite carrier
A metal-organic framework, composite carrier technology, applied in biochemical equipment and methods, enzymes, immobilized enzymes, etc., can solve the problems of limited enzyme independence, affecting the spatial conformation of enzymes, etc., to improve catalytic activity and stability, conditions Requires mild, easy-to-achieve results
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Embodiment 1
[0028] The hydrogel / metal organic framework composite (GOx / HRP@pAAm@ZIF-8) with immobilized glucose oxidase and horseradish peroxidase was prepared according to the following steps:
[0029] (1) Weigh 3mg / mL GOx and 5mg / mL HRP, AAm (15%wt), BIS (1%wt), PI (1%wt) to configure 1mL enzyme / pregel mixed solution;
[0030] (2) Using ultraviolet polymerization technology to solidify the enzyme / pregel solution to prepare GOx / HRP@pAAm;
[0031] (3) Soak 200 μL of GOx / HRP@pAAm in 2 mL of Tris-HCl buffer (10 mM, pH=8.5), add 4 mg of dopamine to modify the surface of the hydrogel; react for 1 h under gentle stirring, and collect by centrifugation GOx / HRP@pAAm@PDA, and then washed 3 times with ultrapure water;
[0032] (4) Place the washed GOx / HRP@pAAm@PDA in 2ml 125mM zinc nitrate aqueous solution and incubate at room temperature for 3h; Wash with pure water to get GOx / HRP@pAAm@ZIF-8.
Embodiment 2
[0034] Combined with the oxidation experiment of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), the absorbance corresponding to the maximum absorption peak was recorded by a UV spectrophotometer to detect GOx / HRP@ Catalytic activity of pAAm@ZIF-8. The specific method steps are:
[0035] (1) Phosphate (PBS) with pH=7.4 was used as the buffer solution, the total volume was 300 μL, the reaction temperature was 37° C., and the reaction time was 30 minutes;
[0036] (2) Under the reaction conditions of the above step (1), use four 0.5mL centrifuge tubes, numbered a, b, c, d respectively;
[0037] (3) The preparation method of the solution in centrifuge tube a is: 10 μL ABTS (35 mM), 10 μL glucose (100 mM) and 2 μL GOx / HRP mixture in 278 μL PBS buffer solution with pH=7.4;
[0038] (4) The preparation method of the solution in centrifuge tube b is: take 10 μL ABTS (35 mM), 10 μL glucose (100 mM) and 2 μL GOx / HRP@pAAm in 278 μL PBS buffer solution with pH=7.4;
[00...
Embodiment 3
[0044] The cycle stability of free GOx / HRP, GOx / HRP@pAAm, GOx / HRP@pAAm@PDA, GOx / HRP@pAAm@ZIF-8 system was compared, and then the absorbance corresponding to the maximum absorption peak was recorded by UV spectrophotometer. The specific method is:
[0045] (1) Phosphate (PBS) with pH=7.4 was used as the buffer solution, the total volume was 300 μL, the reaction temperature was 37° C., and the reaction time was 30 minutes;
[0046] (2) Use four 0.5mL centrifuge tubes, add 2 μL GOx / HRP, 2 μL GOx / HRP@pAAm, 2 μL GOx / HRP@pAAm@PDA, 2 μL GOx / HRP@pAAm@ZIF-8 to all four centrifuge tubes;
[0047] (3) After adding the substrate 10 μL ABTS (35 mM) and 10 μL glucose (100 mM) into each test tube for reaction, record the maximum absorption peak at 405 nm with a UV spectrophotometer;
[0048] (4) During the repeated use of the detection of immobilized GOx / HRP, the samples were collected and washed 3 times with PBS (10 mM, pH 7.4) to remove the residual substrate and used for the next reactio...
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