藉多光子吸收在三維聚二甲基矽氧烷微流道中固定蛋白質

Abstract

本研究旨在透過多光子吸收(multiphoton absorption, MPA)高解析度的特點，將蛋白質局部固定於三維微流道(microchannel)內。與其他局部固定的技術相比較，本技術可應用於一次成形(one-step)的三維微流道內，因此，除了可將局部固定與三維微流道的特點相結合之外(例如局部固定可在空間範圍內固定多種蛋白質，而三維微流道可提供大量空間做局部固定)，疊層製造(layer-by-layer)的微流道容易產生的問題(例如滲漏)可以被避免。 本研究採用近紅外光的脈衝雷射，以玫瑰紅(rose Bengal, RB)為光活性劑，將牛血清白蛋白(bovine serum albumin, BSA)交聯(crosslinking)於三條重疊的載玻片與PDMS微流道內，並透過帶環氧基的矽烷(epoxysilane)提高BSA交聯結構的固定強度。透過此架設，BSA可被交聯在三條重疊的載玻片微流道的下管壁，以及最上層與中間層的PDMS微流道的下管壁。固定強度測試結果顯示靜置 > 30分鐘後，BSA交聯結構較傾向於固定在有被GPTMS處理的表面上。 單光子吸收(single-photon absorption, SPA)的方法只適合在二維微流道上局部固定蛋白質，MPA的高解析度過去都只用來在二維微流道內製造蛋白質微結構，本研究則首次透過MPA將蛋白質局部固定於不同層的微流道上，這特點可應用於一次成形的三維微流道。除了蛋白質固定以外，未來有機會藉此方法在一次成形的三維微流道內局部地做其他處理。

The present research aims to locally immobilize proteins in 3D microchannels by multiphoton absorption (MPA), which features high spatial resolution. Compared with other local immobilizing techniques, the present technique is applicable to one-step fabricated 3D microchannels. Therefore, besides combining features of both local immobilization and 3D microchannels (e.g. local immobilization allows many protein species to be immobilized in a spatial region, and 3D microchannels provides much space for the local immobilization), potential problems (e.g. leakage) of layer-by-layer fabricated microchannels are avoidable. In the research, bovine serum albumin (BSA) is crosslinked in three overlapping glass or PDMS microchannels by near-infrared pulsed laser with rose Bengal (RB) as the photoactivator. An epoxysilane is used to enhance immobilizing robustness of BSA crosslinked structures. With the setup, BSA can be crosslinked on the bottom walls of all the glass microchannels as well as of the topmost and the middle PDMS microchannels. Tests for immobilizing robustness show that BSA crosslinked structures tend to stick to GPTMS-treated surfaces after incubating for > 30 min. Single-photon absorption (SPA) is appropriate for local immobilization of proteins in only 2D microchannels. High resolution of MPA has been used to only fabricate microstructures of proteins in 2D microchannels. In the present research, MPA is used to immobilize proteins on different layers of microchannels for the first time. The technique can be applied to one-step fabricated 3D microchannels. Besides protein immobilization, localization of other treatments in one-step fabricated 3D microchannels would be feasible with the technique in the future.