不可逆封合微流控芯片中高活性蛋白质阵列的制备

摘要

保持生物分子的高活性是在不可逆封合微流控芯片中构筑微阵列芯片的关键问题.首先,利用MEMS技术和表面修饰方法制作了一种聚二甲基硅氧烷(PDMS)/玻璃芯片.应用光刻技术制作了PDMS盖片上的通道,同时用光刻剥离技术制作了玻璃基片上的金膜图案.进而,使用双官能团修饰剂3-氨丙基三甲氧基硅氧烷(APTMS)在玻璃基体和金膜图案上进行选择性表面修饰以吸附形成蛋白质阵列,并在其上覆盖一层水溶性聚乙烯醇(PVA)来保护蛋白质,既可避免其在加热处理过程中的高温伤害,又能防止在PDMS盖片与玻璃基片进行不可逆封合过程中的氧等离子体轰击造成的活性伤害.然后,通入水溶液冲洗除去PVA膜.使用荧光显微镜和原子力显微镜(AFM)考察蛋白质阵列质量,并结合免疫反应实验和细胞捕获固定实验评估蛋白质阵列的活性.结果表明,使用该方法可在不可逆封合的微流控芯片制作中构筑具有直径为200 μm的高分辨率蛋白质阵列图案,蛋白质保持高的免疫活性,且可用于固定Hela细胞.%The key issue of building mieroarray chips is to maintain high activity of biological molecules during the fabrication process of irreversible bonded microfluidic chips. First, polydimethyl-siloxane(PDMS)/glass chip was prepared utilizing MEMS and surface chemical modification technology. PDMS covered with microchannel was made by using soft lithography, and glass substrate with gold film pattern was obtained by lithography and lift-off technique. Moreover, protein array was immobilized through selective surface modification on the glass substrate and gold film pattern with 3-aminopropy-ltrimethoxysilane (APTMS), one kind of bifunctional linker, and covered by a release-and-protection polyvinyl alcohol (PVA) layer, which can protect activity of the protein array from damage caused by high temperature and oxygen plasma which is used to activate the irreversible bonding of the PDMS cover and glass substrate which can ultimately form a microchip during bonding process. Afterwards, the PVA layer was removed by water. Fluorescence microscope and atomic force microscope (AFM)rnwere used to evaluate the integrity of the protein array, and the activity of the protein array was evaluated by immune reaction and cell capturing. Results show that protein arrays with high-resolution of 200 μm diameter can be obtained by the method presented during the fabrication process of irreversible bonded chip. Meanwhile, protein arrays can keep high immune activity to make Hela cells successfully immobilized on glass substrate.