▎ 摘　　要

NOVELTY - Method for detecting aquatic product allergens by using a microfluidic chip involves making a first carrier of a bionic super-wetting interface material, making the second carrier of polydimethylsiloxane (PDMS) material, adding mixed droplet of a certain concentration of crustacean tropomyosin and a certain concentration of fish parvalbumin incubated with colored fluorescent microspheres to the inlet of the first microchannel and irradiating the near-infrared light upwards from the lower surface of the first carrier, where the near-infrared light moves along the direction of the first microchannel to drive the mixed droplets to enter the thin film of the microchamber along the first microchannel. USE - The method is useful for detecting aquatic product allergens. ADVANTAGE - The target protein in the sample to be tested is specifically combined with the protein nucleic acid aptamer with fluorescent mark, which effectively detects whether the food contains water product sensitization source. DETAILED DESCRIPTION - Method for detecting aquatic product allergens by using a microfluidic chip involves (a) making the first carrier of the bionic super-wetting interface material, pasting several mutually spaced microchambers on the lower surface of the first carrier, where each microchamber is a multilayer film superimposed up and down, the film has pore size of 3-5 mum and the film material is polycarbonate, pasting a mask with patterns of the first micro-channel, the second micro-channel and several third micro-channel patterns on the lower surface of the first carrier, where the first microfluidic channel runs through each microchamber and is connected with the second microfluidic channel and the first micro-channel and the second micro-channel are confluent and connected to the inlet of each third micro-channel, preparing second carrier using polydimethylsiloxane (PDMS) material, arranging through holes at the bottom of the second carrier and placing the second carrier on the upper surface of the first carrier with the through hole facing the inlet of the second microfluidic channel, (b) incubating mixed solution formed using crustacean tropomyosin solution and fish parvalbumin solution with green light-emitting colored microspheres, so that the tropomyosin solution and the parvalbumin solution are adsorbed on the microspheres, adding the incubated mixed droplets at the inlet of the first microchannel and irradiating near-infrared light upward from the lower surface of the first carrier, where the near-infrared light moves along the direction of the first microchannel, and drives the mixed droplets to enter the thin film of the microchamber along the first microchannel, (c) adding the tropomyosin nucleic acid aptamer labeled with FAM (RTM: 5-Carboxyfluorescein)fluorescence at the 5' end and the parvalbumin nucleic acid aptamer labeled with Cy3 (RTM: Cyanine dye) fluorescence at the 5' end at the inlet of the first microfluidic channel and using near-infrared light to drive the crustacean tropomyosin nucleic acid aptamer and the fish parvalbumin aptamer into the film of the microchamber along the first microfluidic channel and bind crustacean tropomyosin nucleic acid aptamer to crustacean tropomyosin for forming colored fluorescent microspheres-tropomyosin-aptamer complex and parvalbumin aptamer combined with fish parvalbumin to form a complex of colored fluorescent microspheres-parvalbumin-aptamer, (d) manipulating the release of colored fluorescent microspheres-tropomyosin-aptamer complexes and colored fluorescent microspheres-parvalbumin-aptamer complexes from the microchamber membrane, using near-infrared light to drive the colored fluorescent microsphere-tropomyosin-aptamer complex and the colored fluorescent microsphere-parvalbumin-aptamer complex to move along the first microchannel, feeding oil droplets into the second microchannel through the through holes of the second carrier, using the near-infrared light to drive the oil droplets to move along the second microchannel, merging the colored fluorescent microspheres-tropomyosin-aptamer complex and the colored fluorescent microsphere-parvalbumin-aptamer complex in the first microfluidic channel with the oil droplets in the second microfluidic channel to form water-in-oil droplets and using near-infrared light to drive water-in-oil droplets into one of the third microchannels, (e) adjusting the concentration value of the crustacean tropomyosin solution and the concentration value of the parvalbumin solution, repeating steps (b)-(d) to make several third microchannels correspond one-to-one with water-in-oil droplets of different concentrations, (f) using a fluorescence microscope to take images of water-in-oil droplets in each third microchannel, selecting droplets with green-red or green-blue double fluorescence, analyzing the fluorescence intensity of blue and red in the image, taking the concentration as the abscissa and the fluorescence intensity as the ordinate and drawing the standard curve of crustacean tropomyosin and parvalbumin and (g) detecting whether the sample to be tested contains an aquatic product allergen.