Molecularly imprinted polymer microsphere for detecting dengue fever NS1 protein, and application of molecularly imprinted polymer microsphere
A molecular imprinting and polymer technology, applied in the chemical field, can solve the problems of low concentration, complex structure, and difficulty in imprinting polymers, and achieve the effect of strong selectivity and high sensitivity
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Embodiment 1
[0046] Example 1: Selection of dengue fever NS1 proteolysis fragments and establishment of UPLC-MS detection method
[0047] According to the dengue NS1 protein sequence combined with trypsin, the polypeptide fragment (RTTTASGKLITE (SEQ ID NO.1)) was screened out as a potential template. Use methanol to prepare a mother solution of polypeptide fragments with a concentration of 10mg / 10mL. The mother liquor was stored in a refrigerator at 4°C. Dilute the mother solution of the above peptide fragments 1000 times with 0.1% formic acid aqueous solution to prepare a 1mg / L, 250mL standard solution, store it in a refrigerator at 4°C, and then dilute it into a series of solutions for UPLC-MS detection.
[0048] Take a small tube of dengue fever NS1 protein reagent containing 4 μg / 10 μL, add 1 mL of methanol to completely dissolve the protein in it and reconstitute the volume, label it as dengue fever NS1 protein stock solution, and store it in a -8°C refrigerator. Every time you use ...
Embodiment 2
[0054] Example 2: Synthesis of Molecularly Imprinted Surface Polymer Material of Dengue Fever NS1 Proteolysis Fragment
[0055] 1. Synthesis of three functional siloxane monomers
[0056] Add 1mmol of methacrylic acid, 1mmol of N-tert-butylacrylamide and 1mmol of 4-vinylimidazole to three 25mL round-bottomed flasks, and add 1mmol of 3-mercaptopropyl to the three round-bottomed flasks. Triethoxysilane and 5mL of methanol solvent were added with triethylamine to adjust the pH to 8.0, and reacted in a water bath at 60°C for 6h. The three reaction products are sequentially labeled S-M, S-T and S-V. For the subsequent characterization of functional monomers, use a nitrogen blower to set the temperature at 50°C to sweep the product for half an hour, distill off the methanol solvent and triethylamine, and leave the oily siloxane functional monomer.
[0057] 2. Imprinting polymerization on the surface of silica spheres
[0058] Using the RTTTASGKLITE (SEQ ID NO.1) polypeptide seque...
Embodiment 3
[0061] Embodiment 3: solid phase extraction experiment
[0062] Take 10 μL of dengue fever-positive serum samples, add 1.0 mL of acetonitrile to precipitate the protein, centrifuge at 3000 r / min, redissolve the precipitated protein with 1.0 mL of methanol, and store it in a -8 °C refrigerator. The enzymatic hydrolysis method of serum precipitated protein is consistent with the proteolytic method in Example 1, take 100 μ L dengue fever positive patient serum precipitated protein solution, add 1 mL 50 mmol / L ammonium bicarbonate aqueous solution to it as solvent and buffering agent, add 100 μ L 0.3mol / L L dithiothreitol solution, shake evenly and incubate in a constant temperature incubator at 37°C for 1 hour. After the reaction stopped, add 100 μL 0.3mol / L iodoacetic acid solution, incubate in a dark room at 37°C for 1 hour, take it out, add 200 μL 0.3mol / L dithiothreitol solution and 200 μL 100ng / L bovine trypsin, place in Incubate in a constant temperature incubator at 37°C ...
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