Functional magnetic bead, preparation method and method for specifically capturing circulating tumor cells of colorectal cancer
A colorectal cancer and tumor cell technology, applied in the field of chemical biology, can solve the problems of not being able to mass-produce, low antibody expression, and high price, and achieve the effect of short time, good specificity, and simple preparation method
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[0037] The present invention also provides a preparation method of functionalized magnetic beads, comprising:
[0038] 1) Wash the MBs, then add EDC and NHS for activation to obtain activated MBs;
[0039] 2), the activated MBs were magnetically sorted to remove the supernatant, washed with HEPES buffer, then added with HEPES buffer containing UEA-I, and reacted at room temperature;
[0040] 3) After the reaction, remove the supernatant by magnetic separation, wash with HEPES buffer to remove unreacted UEA-I, suspend the modified MB-UEA-I in HEPES buffer, and save for future use.
[0041] According to the present invention, first add MBs to the centrifuge tube, wash with MES buffer solution for 3 to 5 times, then add EDC and NHS for activation; the activation temperature is preferably room temperature, and the activation time is preferably 30 to 120 min. The concentration of the MES buffer solution is preferably 100 mmol / L, the pH value is 5.5-6.7, and the mass mg of the MBs:...
Embodiment 1
[0058] Embodiment 1 Preparation of functionalized magnetic beads MB-UEA-I
[0059] Take 100μL of MBs (5mg / mL) into a 2mL centrifuge tube, add 1mL of MES buffer to wash three times, remove the supernatant by magnetic separation each time, then add 100mmol / L EDC and 100mmol / L NHS 500μL each, and place at room temperature Vibrate on a shaker to activate for 1h (160rpm).
[0060] After the activation, wash with HEPES buffer three times, then add 1 mL of HEPES (containing 0.025 mg of UEA-I), and place on a shaker at room temperature for 3 h (160 rpm).
[0061] After the coupling reaction, wash with HEPES buffer three times to remove unreacted lectin, and finally add 1 mL of HEPES buffer to suspend MB-UEA-I, and store at 4°C for future use.
[0062] figure 1 It is the SEM characterization diagram before and after MBs modification of UEA-I in Example 1 of the present invention. (a) is the SEM characterization image of MBs, and (b) is the SEM characterization image of MB-UEA-I. fi...
Embodiment 2
[0063] Example 2 MB-UEA-I efficiently and specifically captures tumor cells
[0064] Experimental group: about 100 SW480 cells were added to 1 mL of complete medium, and then 0.05 mg of the functionalized magnetic beads MB-UEA-I prepared in Example 1 were added.
[0065] Control group: Add about 100 HCT116 cells and NCM460 cells to 1 mL of complete medium, then add 0.05 mg of the functionalized magnetic beads MB-UEA-I prepared in Example 1; add about 100 SW480 cells to 1 mL To the complete medium, add 0.05 mg of unmodified MBs.
[0066] Set up the above four groups of experiments, each with three parallel experiments. After the cells were mixed with MB-UEA-I or MBs, they were incubated on a shaker at 37°C for 45min (150rpm). After the reaction, the captured cells were obtained by magnetic separation, and the supernatant was added to a 48-well plate, and after standing for 20 min, the uncaptured cells in the supernatant were counted using an inverted microscope. In addition,...
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