本論文主要利用超快雷射加工系統之非線性多光子激發來進行三維(three-dimensional，3D)光微影製程(photolithography)，經由調控飛秒雷射加工功率和掃描速率，製作具氧化石墨烯(graphene oxide quantum dot，GOQD)的蛋白質3D微結構。主要機制為利用光活化劑(photoactivator)對雙光子吸收(two-photon absorption，TPA)，進而誘發蛋白質間雙光子交聯(two-photon crosslinking，TPC)反應，其中孟加拉玫瑰素(rose Bengal，RB)作為光活化劑。首先我們使用明膠為基材，其可為細胞外基質(extracellular matrix，ECM)材料。TPA後可產生高活性的單態氧(singlet oxygen)，其單態氧會活化蛋白質上的官能基，蛋白質間形成共價鍵鍵結。進一步為了防止在製作3D微結構過程中加工溶液中GOQDs因RB造成聚集，我們添加牛血清蛋白(bovine serum albumin，BSA)作為分散劑以防止聚集結晶產生，使其溶液中的GOQDs得以長時間穩定地均勻分散於水溶液中。另外，為了避免在TPC過程中誘發嚴重的熱損傷及產生大量的氣泡，而造成所製作出的微結構遭到破壞，因此選擇RB最大TPA之激發波長以降低雷射強度，並以較快的掃描速率進行重複的書寫，避免大量的熱累積於結構中。最後，成功地製作出上述蛋白質與GOQD之複合微結構，並透過雙光子激發螢光(two-photon excited fluorescence，TPEF)顯微鏡與掃描式電子顯微鏡(scanning electron microscope，SEM)來檢視3D結構的表面型貌。

In this study, three dimensional (3D) graphene oxide quantum dot (GOQD)-doped protein microstructures were successful fabrication via 3D photolithography that adopted nonlinear multiphoton excitation of an ultrafast laser machining system by adjusting the power and scan rate of the femtosecond laser. The main mechanism of the multiphoton-induced fabrication is use rose Bengal (RB) as the photoactivator that was activation by two-photon absorption (TPA) of laser energy to start up the two-photon crosslinking (TPC) of protein. First, gelatin as an extracellular matrix (ECM) material was adopted as a base material in TPC. After TPA of RB, an activated RB was generated singlet oxygen to activate proteins that transformed into free radicals and crosslinked with proteins by the covalent bonding. In addition, in order to prevent the aggregation of GOQDs that was resulted from canceling the electric charge by RB during the fabricating 3D TPC microstructure, bovine serum albumin (BSA) as a dispersant was added into the fabrication solution to provide a long term dispersive stability of GOQDs in fabrication solution. Furthermore, excitation wavelength of the TPC fabrication was chosen for achieving superior TPA of RB to reduce the laser power, and was adopted the fabrication method of higher scan rate with repeated writing to prevent the photothermal damage during the TPC processing. Finally, we had successfully fabricated 3D GOQD-doped protein microstructures and also examined surface profiles of 3D GOQD-doped protein microstructures from the two-photon excited fluorescence imaging and scanning electron microscopy.

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