Detection method of intracellular glucose concentration in living cells based on enzyme-amorphous metal-organic framework complex
A metal-organic framework, glucose concentration technology, applied in measurement devices, material excitation analysis, fluorescence/phosphorescence, etc., can solve problems such as increased complexity, cell puncture, complex operations, etc. Detect simple effects
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Embodiment 1
[0057] Embodiment 1, the preparation of enzyme-amorphous metal-organic framework complex
[0058] Add 1mL of zinc acetate solution with a concentration of 20mM to 1mL of 2-methyl-1H imidazole solution with a concentration of 80mM (solvent is water), and keep stirring to form a precipitate for about 30s. Add 80μL of 0.5mg / mL GOx (Glucose oxidase) aqueous solution, continue to stir for 30 minutes, centrifuge, wash 3 times with deionized water, freeze-dry to obtain glucose oxidase-amorphous metal-organic framework compound dry powder, the obtained quality is about 1.5 ~ 2mg.
[0059] The scanning electron microscope SEM picture of the glucose oxidase-amorphous metal-organic framework complex prepared in this example is as follows figure 1 shown by figure 1 It can be seen that the main body shape of the obtained composite is spherical, and the particle size is between 100-200 nm.
[0060] The X-ray diffraction pattern of the glucose oxidase-amorphous metal-organic framework comp...
Embodiment 2
[0064] Example 2. Using the prepared enzyme-amorphous metal-organic framework complex to measure the intracellular activity of human breast cancer cell MCF-7 cell population, human gastric cancer cell population MGC803, mouse breast cancer cell population 4T1, and mesenchymal stem cell population MSC glucose concentration
[0065] To the prepared glucose oxidase-amorphous metal-organic framework complex, pH 7.4, 10 mM phosphate buffer solution was added and resuspended to a turbid solution with a concentration of 1.5-2 mg / mL. In order to fully disperse the glucose oxidase-amorphous metal-organic framework complex, use a water bath to sonicate for about 10s. Add 1 μL, 10 mM 2',7'-dichlorodihydrofluorescein-acetoacetate-DMSO solution to 1 mL, pH 7.4, 10 mM phosphate buffer.
[0066] Take 5 μL of the resuspended glucose oxidase-amorphous metal-organic framework complex solution, add 45 μL of pH 7.4, concentration of 10 mM phosphate buffer, and then add 50 μL of 2',7'-dichlorodih...
Embodiment 3
[0071] Embodiment 3, drawing fluorescence intensity-glucose concentration standard curve
[0072] Human breast cancer cell line MCF-7, mouse breast cancer cell line 4T1, gastric cancer cell line MGC803 and human normal liver cell line L-02 were collected respectively.
[0073] Taking human breast cancer cell MCF-7 as an example, trypsin was added to a culture dish in which human breast cancer cell groups were cultured for 3 minutes, and then culture medium was added to neutralize the reaction. The cell suspension was centrifuged (1000rpm), washed with phosphate buffered saline, and repeated 3 times. Subsequently, 500 μL of phosphate buffer was added to resuspend the cells and counted using a Millipore Sceptor hand-held cell counter. After the counting was completed, the cells were ultrasonically disrupted for 5 minutes in an ice-water bath using a hand-held ultrasonic cell disruptor. After the cell lysate was centrifuged, the supernatant was collected. Add 20, 40, 60, 80, a...
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