DSpace collection: 博碩士論文

Synthesis of Biotinylated Silatrane via Huisgen Cycloaddition reaction for Bioconjugation Properties in Biosensors

title: Synthesis of Biotinylated Silatrane via Huisgen Cycloaddition reaction for Bioconjugation Properties in Biosensors abstract: 生物素是一種重要的生物分子，在細胞內多種關鍵代謝反應中扮演重要角色。特別是，生物素與親和素的結合是蛋白質與配體之間最強的非共價相互作用，展現出在生物感測器應用方面的潛力。然而，生物素在基底表面的非特異性結合會增加，導致生物感測器應用數據偏差。矽烷類化合物因其在水相中優異的化學穩定性以及透過跨環N→Si配位鍵調控的水解特性，成為一種有效的表面修飾化合物。這種結合可以控制水解並在表面形成自組裝單分子層。本研究首次報導了一種新型材料－生物素-矽烷（Bi-SiT），以矽烷和生物素為基礎的優異性能，利用Huisgen環加成反應合成。疊氮化物與炔烴的反應已成為合成具有高抗菌活性的1,2,3-三唑類化合物最有效的方法之一。此外，本研究將Bi-SiT與磺基甜菜鹼矽烷（SBSiT）混合，以提供一種可用於修飾的防污功能化表面。本研究透過疊氮矽烷（N=N=N）與生物素-乙炔（C≡C）的反應合成了生物素-矽烷。利用核磁共振氫譜（1H NMR）和傅立葉變換紅外光譜（FT-IR）證實了生物素-矽烷的結構。 Bi-SiT和SBSiT混合塗層的結果表明，塗覆在矽片上的塗層具有高親水性和均勻性，這突顯了其優異的防污和功能化性能。這些性能透過測量水接觸角、塗層厚度、X射線光電子能譜（XPS）和粗糙度得到驗證。此外，利用石英晶體微天平耗散監測（QCM-d）、橢圓偏振光譜和原子力顯微鏡（AFM）證實了表面上存在生物素蛋白。將矽烷環與生物素生物分子結合的生物素-矽烷將成為生物偶聯和生物感測器領域很有前景的材料.;Biotin is known as a biomolecule that plays important roles in a variety of critical metabolic reactions in cells. Especially, avidin-biotin binding is the strongest non-covalent interaction between a protein and ligand that demonstrates the potential for biosensor applications. However, biotin can be increased the non-specific binding on the surface of substrate that led to the mistaken data for biosensor applications. Silatranes are effective compounds for surface modification because of their excellent chemical stability in the presence of water and modulated hydrolysis characteristic through a transannular N→Si dative bond. The binding can controll the hydrolysis and form self-assemble monolayers film on the surface. This study reports for the first time that a new material biotin-silatrane (Bi-SiT) is synthesized based on the combination of the outstanding properties of silatrane and biotin using Huisgen cycloaddition chemistry. The reaction of azides with alkynes has become one of the most efficient methods to synthesize 1,2,3-triazoles that display a high antimicrobial activity. Additionally, Bi-SiT has been mixed with sulfobetaine silatrane (SBSiT) to provide an antifouling and functionalised surface for modification. In this study, the synthesis of biotin-silatrane was carried out by reacting azide-silatrane (N=N=N) and biotin-acetylene (C≡C). The structures of biotin-silatrane is confirmed by 1H nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectroscopies. The results for mixed coating between BiSiT and SBSiT showed the high hydrophilicity and uniform layers that emphasize the antifouling and funtionalised properties of the surface after coating on silicon wafer, determined by measuring water contact angle, thickness, X-ray Photoelectron Spectroscopy (XPS) and roughness. In addition, the detection of avidin on the surface was confirmed by Quartz crystal microbalance with dissipation monitoring (QCM-d), ellipsometry and Atomic Force Microscopy (AFM). Biotin-silatrane with the integration of silatrane ring and biotin biomolecule will be a promising material for bioconjugation, and biosensors.

Novel Trimethylamine N-oxide-Based Zwitterionic Hydrogels for Biomedical Application

title: Novel Trimethylamine N-oxide-Based Zwitterionic Hydrogels for Biomedical Application abstract: 由於具有高水合性、電荷中性以及優異的抗污行為，兩性離子水凝膠近年來在生醫材料、抗污塗層與生物感測器等領域受到廣泛關注。兩性離子材料能與水分子形成強烈作用並有效結合水分子，使其即使在複雜環境條件下，仍能展現優異的含水能力與抗污特性。在眾多兩性離子材料中，三甲胺N-氧化物（Trimethylamine N-oxide, TMAO）是一種天然存在於極端環境海洋生物中的滲透調節物質，因其獨特的水合行為與抗污特性，近年來引起研究者的高度關注。本論文提出一種完全以 TMAO 為基礎的兩性離子水凝膠，其由海鮮來源之 TMAO 單體與一種新穎且特別設計的兩性離子交聯劑——氧化甲基雙（ 2- 丙烯酰氧基乙基）胺（ Methyl-bis(2-acryloyloxyethyl)amine oxide, NOBA）所合成。該水凝膠展現良好的機械穩定性、優異的耐離子強度能力、卓越的抗污性能以及良好的抗凍特性，這些特性可歸因於強烈的水–聚合物交互作用以及水凝膠網絡中水分狀態的有效調控。此外，初步研究結果顯示，TMAO 水凝膠在魚類腐敗偵測方面具有潛在的生物標記應用價值，其特徵表現為水凝膠隨腐敗過程產生明顯膨潤並伴隨顏色變化，顯示其在食品安全生物感測領域具有良好的應用前景。;With the characteristics of increased hydration, charge neutrality, and strong antifouling behavior, zwitterionic hydrogels have attracted growing interest for use in biomedical materials, antifouling coatings, and biosensors. The unique ability of zwitterionic materials to interact with and bind water molecules leads to enhanced water retention and antifouling properties, even under complex environmental conditions. Among zwitterionic materials, trimethylamine N-oxide (TMAO), a naturally derived osmolyte found in marine organisms living in extreme conditions, has attracted interest in recent years due to its unique hydration and antifouling properties. This thesis presents the preparation of a fully TMAO-based zwitterionic hydrogel synthesized from a seafood-derived TMAO monomer and a novel, specifically designed zwitterionic cross-linker, Methyl-bis(2-acryloyloxyethyl)amine oxide (NOBA). This hydrogel exhibits favorable mechanical stability, strong resistance to ionic strength, excellent antifouling attributes, and good anti-freezing properties, which can be ascribed to the strong water-polymer interactions as well as the water state control in the hydrogel network. In addition, the initial results indicate that the TMAO hydrogel exhibits promising bio-tagging properties for detecting fish spoilage, as evidenced by visible expansion and color changes, suggesting strong potential for bio-sensing in food safety.

製備載藥型纖維蛋白凝膠以應用於肌肉組織工程;Preparation of Drug-Loaded Fibrin Gel for Muscle Tissue Engineering Application

title: 製備載藥型纖維蛋白凝膠以應用於肌肉組織工程;Preparation of Drug-Loaded Fibrin Gel for Muscle Tissue Engineering Application abstract: 體積性肌肉缺損（Volumetric Muscle Loss, VML）常因大範圍組織破壞與神經再支配不足，導致骨骼肌結構與功能無法有效恢復。雖然近年組織工程技術可藉由細胞片或生物反應器促進肌管排列與分化，但移植後因缺乏及時且穩定的神經支配，仍容易發生肌纖維萎縮與功能退化。因此，如何在維持肌肉結構完整性的同時，促進神經分化與神經肌肉接點（neuromuscular junction, NMJ）的形成，仍為肌肉組織工程中亟待解決的關鍵問題。本研究提出一種結合「肌肉細胞片轉移」與「神經生長因子（nerve growth factor, NGF）持續釋放」之策略，利用纖維蛋白凝膠（fibrin gel, FG）作為可降解且具生物相容性的藥物載體，以建立具神經誘導潛力的神經肌肉共培養系統。首先，透過調控纖維蛋白原、凝血酶及離子條件，製備具有適當力學性質之 FG，並評估其成膠行為與壓縮模數。隨後，將 NGF 包埋於 FG 中進行釋放行為分析，並觀察其對 PC12 細胞神經分化的影響。在生物反應器培養條件下，將排列良好之 C2C12 肌肉細胞片，成功利用載藥型 FG 進行細胞片轉移。最後以神經細胞種植於載有肌管細胞片的水膠進行共培養，來評估NMJ形成的效果，結果顯示，NMJ 後突觸相關基因（如 CHRNE、RAPSN 及 CHRNA1）表現顯著上調，而神經結構相關基因亦呈現分化趨勢。免疫螢光染色進一步顯示，神經軸突與乙醯膽鹼受體簇集於肌管表面呈現空間共位，顯示本系統已啟動具 NMJ 特徵之神經肌肉整合過程。綜合以上結果，本研究證實載藥型纖維蛋白凝膠結合肌肉細胞片轉移，可在提供神經誘導微環境，以促進神經分化並啟動 NMJ 相關分子與結構特徵。此研究成果可作為未來應用於體積性肌肉缺損修復與神經肌肉組織工程之重要基礎。;Volumetric muscle loss (VML) often results in impaired muscle regeneration due to extensive tissue damage and inadequate reinnervationof the affected muscles. Although tissue-engineered muscle constructs can be generated using cell sheet and bioreactor-based approaches, the lack of effective neural integration remains a major limitation. In this study, we developed a nerve growth factor (NGF) loaded fibrin gel (FG) which can be used not only to transfer myotube sheets from the bioreactor but also to sustain the delivery of NGF, promoting neurogenesis. To prepare FG with the appropriate mechanical properties, the concentrations of fibrinogen, thrombin, and NaCl were tuned, and the gelation behavior and compressive modulus were evaluated. Then, NGF was loaded into FG, and its release profile was monitored, and its effects on PC12 neurite outgrowth/neuronal differentiation were evaluated. When FG was used to harvest C2C12 myotubes from the bioreactor, the transferred myotubes on FG maintained good morphology and high viability. Finally, PC12 cells were co-cultured with the myotube sheets on the FG to evaluate the efficiency of NMJ formation. Gene expression analysis revealed upregulation of postsynaptic NMJ-related markers, including CHRNE, RAPSN, and CHRNA1, under coculture conditions. Immunofluorescence staining of βIII-tubulin and α-bungarotoxin further demonstrated spatial colocalization of neuronal projections and acetylcholine receptor clusters on myotubes. These results indicate that NGF-loaded FG combined with muscle cell sheet transfer provides a neural inductive microenvironment that promotes neural differentiation and initiates NMJ-associated molecular and structural features. This platform may serve as a foundation for future neuromuscular tissue engineering applications.

探討拉伸與電刺激對肌管分泌肌肉激素之影響;The effects of mechanical stretch and electrical stimulation on the secretion of myokines from myotubes

title: 探討拉伸與電刺激對肌管分泌肌肉激素之影響;The effects of mechanical stretch and electrical stimulation on the secretion of myokines from myotubes abstract: 本研究旨在建立一套可同時施加電刺激與機械拉伸刺激之體外生物反應器系統，以探討不同刺激模式對肌管分泌肌肉激素(myokine)與基因調控之影響。為達成可拉伸且具導電性之培養基材，本研究以導電高分子聚吡咯(PPy)塗佈於聚二甲基矽氧烷(PDMS)表面而得到高拉伸性導電薄膜，並用於構建生物反應器。將C2C12成肌細胞培養於薄膜表面並分化為肌管後，首先建立電刺激條件，分別評估定電壓定電場刺激(DC)與電脈衝刺激(EPS)對肌肉激素分泌之影響，於確認DC刺激可有效誘導反應後，再進一步探討其與循環拉伸刺激(1 Hz 、10 %拉伸量)結合之複合刺激效果。利用酵素免疫分析法(ELISA)量測肌肉激素蛋白質分泌量，並以即時定量聚合酶連鎖反應(qPCR)分析肌肉激素相關基因表現。結果顯示，電刺激與機械拉伸刺激皆能顯著誘導C2C12肌管分泌肌肉激素，其中以IL-6之反應最為明顯。在電刺激模式比較中，DC在誘導IL-6分泌之效果優於EPS，顯示即便非典型的生理放電型態，定電場仍能有效促進肌肉激素分泌，突顯電性環境在肌肉激素調控中的重要性。進一步比較複合刺激與單一刺激後發現，複合刺激雖然可促進肌管之肌肉激素基因轉錄及分泌，但相較於對應之單一刺激，整體反應未呈現加成效應，顯示電刺激與機械刺激之調控可能受訊號整合機制限制。綜合而言，本研究成功建立一套可同時施加電刺激與機械拉伸刺激之體外肌肉模型，並證實不同刺激模式可差異性調控肌肉激素之分泌與基因轉錄，其中發現定電壓定電場刺激亦能促進肌肉激素分泌為本研究的重要貢獻之一。本研究所建立之平台可作為未來探討運動模擬、肌肉代謝及肌肉相關疾病研究之實驗工具與基礎。;This study aimed to establish an in vitro bioreactor system capable of simultaneously applying electrical stimulation and mechanical cyclic stretch to investigate how different stimulation modes regulate myokine secretion and gene expression in myotubes. To achieve a stretchable and conductive culture substrate, a highly extensible conductive film was fabricated by depositing the conductive polymer polypyrrole (PPy) onto the surface of polydimethylsiloxane (PDMS) and integrated into the bioreactor platform. C2C12 myoblasts were cultured on the conductive film and differentiated into myotubes. We first established the electrical stimulation conditions by comparing constant-voltage direct-current electric field stimulation (DC) with electrical pulse stimulation (EPS) in terms of their effects on myokine secretion. After confirming that DC stimulation elicited a robust response, we further evaluated the effects of DC combined with cyclic stretch (1 Hz, 10% strain). Myokine protein secretion was quantified using enzyme-linked immunosorbent assay (ELISA), while myokine-related gene expression was analyzed by quantitative real-time polymerase chain reaction (qPCR). The results demonstrated that both electrical stimulation and mechanical stretch significantly promoted IL-6 myokine expression in C2C12 myotubes. Among the electrical stimulation modalities, DC stimulation induced a greater increase in IL-6 secretion than EPS, indicating that even non-physiological stimulation patterns, such as constant electric fields, can effectively enhance myokine secretion and highlight the critical role of the electrical microenvironment in myokine regulation. Further comparison between combined and single stimulation conditions revealed that although cyclic stretch promoted myokine gene transcription and secretion, the combined stimulation did not produce a synergistic effect, suggesting that the regulatory effects of electrical and mechanical stimulation may be constrained by intracellular signal integration mechanisms. In summary, this study successfully established an in vitro platform that allows simultaneous application of electrical and mechanical stimulation to myotubes and demonstrated that different stimulation modalities differentially regulate myokine secretion and gene transcription. Notably, the finding that DC stimulation can promote myokine secretion represents a key contribution of this work. The developed platform provides a valuable experimental tool for future studies on exercise simulation, muscle metabolism, and skeletal muscle-related diseases.