Difunctional laser crackable probe and preparation method and mass spectrometry application thereof
A dual-function, laser technology, which is applied in the synthesis of laser cleavable probes, mass spectrometry detection and imaging, can solve the problems of single function, lack of universality, cumbersome synthesis methods of cleavable probes, etc., and achieve simple synthesis methods, The effect of universal applicability and simple preparation method
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Embodiment 1
[0056] The synthesis of the dual-functional laser cleavable probe adopts the "one-pot reaction method". The specific preparation method is as follows:
[0057] Add 5mL of 10mM 4-hydroxyethylpiperazineethanesulfonic acid buffer (pH=7.5) into a 50mL round bottom flask, add 5mg concanavalin lectin (or 6.86mg elderberry lectin) and 55μL concentration of 0.45mM 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-hexadecaneoxa-28,29-dithiapentadecanedi Acid di-N-succinimide ester (formula II, when n=7), the reaction mixture was stirred at room temperature for 12 hours, and 33 mL of 15 nM gold nanoparticle solution and 11 mg of potassium carbonate (final concentration of 1.8 mM) were added to the solution. ), continue to stir and react for 12 hours. Subsequently, 2.5 mL of 10 mM mass spectrometry reporter molecule (11-mercaptoundecyl)hexa(ethylene glycol) (or (11-mercaptoundecyl)tetrakis(ethylene glycol)) was added and stirring was continued at room temperature for 24 hours . The mixed ...
Embodiment 2
[0062] The dual-functional laser-cleavable probe prepared in Example 1 was used for in-situ analysis of cell surface glycosides, and the specific steps were as follows:
[0063] Set the concentration to 10 5 / mL of cell suspension was dropped on the surface of ITO conductive glass, and the glass was placed in a cell culture incubator at 37°C, 5% CO 2 Incubate for 24 hours under an atmosphere to allow the cells to grow naturally on the wall. Then take out the glass slide carefully, suck off the culture medium on the glass surface, wash the cells once with PBS phosphate buffer solution, add 1mL paraformaldehyde solution to fix the cells for 10min, after fixing, suck off the paraformaldehyde solution and wash the cells with PBS phosphate buffer solution for three times all over. Then add redispersed in PBS solution (containing 0.1mM Ca 2+ and Mn 2+ ), two laser-cleavable probes (modified with concanavalin and (11-mercaptoundecyl)hexa(ethylene glycol) or elderberry lectin and ...
Embodiment 3
[0066] The dual-functional laser cleavable probe prepared in Example 1 is used for in situ imaging analysis of tissue surface glycosides, and the specific steps are as follows:
[0067]Using the frozen section method, using 15% gelatin aqueous solution as the embedding agent, slice the fresh hepatocellular carcinoma and paracancerous tissues at -20 ° C, the thickness of the slices is 8 μm, and paste them on the surface of ITO conductive glass, cancer tissues and paracancerous tissues Tissues were placed on the same piece of ITO glass. On the tissue section, dropwise redisperse in PBS solution (containing 0.1mM Ca 2+ and Mn 2+ ), a laser cleavable probe at a concentration of 10 nM, and incubated at 37° C. for 1 hour. After incubation, the tissue was washed three times with PBS phosphate buffer to remove unbound probes, and finally washed once with deionized water. Tissues were naturally dried at room temperature and analyzed by laser desorption ionization imaging mass spectr...
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