Application of oxidized graphene modified by polyethylene glycol
A technology of polyethylene glycol and graphene, applied in the direction of biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of failing to meet the requirements of trypsin and trypsin stabilizers, and achieve enhanced activity and stability, Improve activity and synthesize simple effects
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Embodiment 1
[0021] Example 1: Preparation of trypsin specific activator GO-10k- br -PEG-NH 2
[0022] Use 1g of expanded graphite as the initial raw material, add about 30 times the weight of sodium chloride solid and grind until there are no obvious visible particles, add water and suction filter, wash away the sodium chloride, and dry the finely ground graphite for later use. Add about 30mL of concentrated sulfuric acid (98.3%) into the flask, put in dried fine graphite, stir at room temperature for 8 hours, then place the mixture in an ice bath, add about 3g of potassium permanganate, and slowly raise the temperature to 115 ℃, slowly add about 150mL of water, then add about 10mL of 30% hydrogen peroxide and stir, centrifuge to remove the upper aqueous solution, and wash repeatedly until the solution no longer precipitates. Sonicate 5 mL of graphene oxide suspension with a concentration of 3 mg / mL for 30 min to obtain a clear graphene oxide hydrosol, then add NaOH solid at a final con...
Embodiment 2
[0025] Embodiment 2: Study the influence of the polyethylene glycol-modified graphene oxide obtained in Embodiment 1 on trypsin activity.
[0026] Graphene oxide GO-10k- modified with polyethylene glycol obtained in Example 1 br -PEG-NH 2 Interact with trypsin, mix at room temperature for 16min, and then measure the activity of the enzyme, the specific method is: 20μl of 0.1mg / ml trypsin respectively with 20μl of sterile water, 0.15mg / mL, 0.2mg / mL of GO- 10k- br -PEG-NH 2Mix at 25°C for 16 minutes, add 10 μl of 5×PBS to supplement 50 μl, add 50 μl of 5 mg / ml substrate casein for enzymatic hydrolysis reaction at 40°C, 10 minutes, stop the reaction with 100 μl of TCA (0.4M), centrifuge, Take 100 μl supernatant and 100 μl folinol (1:1) to 500 μl Na 2 CO 3 (0.4M) 40°C, 20min color reaction, measure OD 680 .
[0027] The result is as image 3 Shown: The enhancement of trypsin activity was concentration-dependent, when GO-10k- br -PEG-NH 2 When the final concentrations wer...
Embodiment 3
[0031] Example 3: Study the influence of the polyethylene glycol-modified graphene oxide obtained in Example 1 on the thermal stability of trypsin.
[0032] Trypsin and GO-10k- br -PEG-NH 2 The mixed solution was denatured at 70°C and 80°C for 5 minutes respectively, and then the activity of trypsin was measured according to the above enzyme activity assay method, specifically:
[0033] (1) When casein was used as the substrate, trypsin and GO-10k- br -PEG-NH 2 Mix according to the mass volume ratio (mg / mL) of 1:2. In addition, trypsin and sterile water, trypsin and 10k- br -PEG-NH 2 [Mass volume ratio (mg / mL) is 1:10] also mixed with each other, and then the mixture was placed at 70°C and 80°C for 5 minutes, and then the activity of trypsin was measured according to the above method. The result is as Figure 5 Shown: No GO-10k- br -PEG-NH 2 In this case, trypsin is almost denatured and has no activity. However, in GO-10k- br -PEG-NH 2 Under the protective effect o...
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