石墨烯及其奈米複合材料的生物醫學應用

Biomedical Applications of Graphene and Their nanocomposites

吳柏勳

二氧化錫@石墨烯 ； 生物醫學應用 ； 石墨烯 ； 生物感測器 ； 基質輔助雷射脫附質譜儀 ； 細菌生物膜 ； 抗菌效果 ； Bacteria ； MALDI-MS ； GN-MALDI-MS ； Graphene ； SnO2@G ； Antibacterial

中山大學化學系研究所學位論文

2014年

碩士

吳慧芬

繁體中文

在奈米科學的快速發展下，開啟了許多跨領域的研究工作，包含在生物、醫學等。當物質大小到達奈米尺寸，相較於巨觀物質，在物性及化性都會有很大 不同。日本學者田中耕一先生，以基質輔助雷射脫附游離質譜(MALDI-TOF-MS) 獲得諾貝爾化學獎，打開了在生物領域的大門，也在生物大分子分析上成為有力的分析工具。許多文獻中，也利用奈米材料結合MALDI-TOF-MS 的方法開發應用在生物醫學及生物感測中，包含細菌、真菌、細胞等。 (一)、石墨烯修飾上二氧化錫 (Graphene@SnO2)的合成和抗菌活性 石墨烯是一種單層的碳原子，這是一緊密包裝成的二維結構。它可以被廣泛應用於生化應用。石墨烯也可以與不同的奈米物質作結合，作為一種新穎的抗菌型奈米物質。 本章報導合成及抗菌活性的水溶性分散的二氧化錫（SnO2) 修飾在石墨烯 (Graphene) 奈米片，稱Graphene@SnO2。Graphene@SnO2先製備出來，然後用穿透式電子顯微鏡（Transmission Electron Microscopy）、紫外線光譜儀（UV）、傅立葉紅外光譜儀（FTIR）、拉曼光譜儀（Raman）和螢光光譜儀（Fluorescence）檢驗其性質。研究抗菌活性性質部分，先使用綠膿桿菌（Pseudomonas aeruginosa）和金黃色葡萄球菌（Staphylococcus aureus）分別作為革蘭式陰性菌和革蘭式陽性菌的對照組。使用光學性質（OD600）和平板計數方法（Plate counting methods）作為抗菌活性的評估和依據。結果顯示SnO2@Graphene比Graphene具有較高的毒性，高出1~3倍。利用Graphene做為複合式奈米材料的基板，發現Graphene可以抑制綠膿桿菌的抗菌效果高於金黃色葡萄球菌。因協同效應（Synergic effect）是兩者奈米物質組成後，有更強烈的抗菌效果，所以二氧化錫（SnO2）增加石墨烯的毒性對革蘭式陰性菌的抗菌效果達到3.6倍。使用TEM、Fluorescence和MALDI-MS來證實製備出的奈米材料和細菌細胞之間的相互作用。數據顯示，疏水性、靜電、凡得瓦力和氫鍵促成SnO2@Graphene奈米片黏附附著於細菌細胞上，阻斷細胞服用養分，進而導致細菌細胞死亡。未來我們期望此新穎的Graphene做為複合式奈米材料的基板能有效地應用於環境和生醫診斷中。 (二)、利用石墨烯奈米片介導MALDI-MS (GN-MALDI-MS)感測於快速、原位和早期細菌生物膜靈敏的檢測 生物膜是微生物積存在生物表面或非生物表面上的膜。簡單來說，是許多微生物細胞黏附在一起所形成的膜。生物膜最早出現於化石記錄中早期（~32.5億年前）和各種不同有機體古生菌與細菌繁衍及傳承的形式。生物膜的形成是古代原核生物能始終存在地球的一個原因，並且也是在不同生存環境生存的關鍵因素。生物膜結構複雜，且是一動態的系統。生物膜形成代表細菌成長的保護模式，使細菌細胞能在惡劣的環境中生存，也可以不斷分裂並拓展其範圍。生物膜的生存和繁殖機制的影響，是研究學者一直在關注的。無論是傳染病和致病菌的檢測，都會在此應用中說明及討論。 生物膜的組成為蛋白質和醣類（醣蛋白）的磷脂雙分子層，其形態上都呈現雙分子層的片層結構，厚度約5~10 nm。其組成成分主要是脂質和蛋白質，另有少量糖類通過共價鍵結合在脂質或蛋白質上。 此篇應用的研究去做整合：(1).鋁金屬和鈦金屬之細菌晶片具有低成本效益和快速熱處理的優點；(2).本研究製作的細菌晶片是多功能，其有附著的氧化物塗層，大部分的酸無法破壞；所以可以重複使用；(3).本細菌晶片可作為一生物感測器，將其浸入到細菌溶液；從溶液中可捕捉到細菌；並研究其短時間和長時間的變化；(4). 細菌晶片製作後，可以直接作為MALDI-MS plate，可快速、直接、靈敏於細菌生物膜的分析；(5).使用Epifluorescence micrographs(螢光顯微鏡), MALDI-MS (基質輔助雷射脫附質譜儀), Environmental scanning electron microscope (環境掃描式電子顯微鏡) 研究生物膜的生長狀況。最後，利用石墨烯(Graphene)增強MALDI-TOF-MS對於致病性細菌和海洋細菌生物膜(biofilm)的診斷；從中可以發現低濃度的石墨烯奈米片可以提早偵測致病菌的訊號。未來，可以利用石墨烯於MALDI-TOF-MS上，達到早期檢測的方法；並廣泛應用於生醫檢測上。

(1) Synthesis and antibacterial activities of graphene decorated with stannous dioxide We report the synthesis and antibacterial activity of water dispersible stannous dioxide (SnO2) modified with graphene (G) nanosheets. Nanomaterials of G and SnO2@G were prepared and then characterized by transmission electron microscopy (TEM), ultraviolet (UV) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, Raman and fluorescence spectroscopy. The antibacterial activities were investigated using Pseudomonas aeruginosa and Staphylococcus aureus as model strains of Gram negative and Gram positive bacteria, respectively. The antibacterial activities were evaluated using optical density (OD600) and plate counting methods. The results indicated that SnO2@G displayed a higher cytotoxicity than G by 1–3 fold. The G-based nanomaterials inhibited the growth of P. aeruginosa more effectively than for S. aureus. SnO2 increased the cytotoxicity of G against Gram negative bacteria by 3.6 times due to the synergic effect. The interactions between the prepared nanomaterials and bacteria cells were evaluated using TEM, fluorescence spectroscopy and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The data revealed that there were many forces facilitating the SnO2@G nanosheets to adhere to bacteria cells, which block the cells from taking nutrients, and result in cell death. We expect that this novel G-based composite can be effectively applied in the future for environmental and clinical applications. (2) Graphene nanosheet mediated MALDI-MS (GN-MALDI-MS) sensors for rapid, in situ and sensitive detection of incipient biofilm Diagnosis is the first step to treatment, early detection of biofilm thus gains paramount importance. In the current study, a systematic study was conducted to trace the biofilm formation by Staphylococcus aureus and Vibrio alginolyticus on Aluminium, Titanium surfaces and surface modified counterparts with oxide films. The biofilm development on these four substrates has been studied from 1h- 2 month periods. Traditional MALDI-MS has been demonstrated as a potent tool for direct in situ detection of biofilms on material surfaces. In the subsequent part of the study the Graphene nanosheet mediated MALDI-MS (GN-MALDI-MS) approach using our inhouse synthesized Graphene nanosheets was combined with the traditional MALDI-MS study, to lower the LOD. Using this approach early detection of the biofilm was demonstrated to be 1h in case of titanium surfaces and 3h in case of Al surfaces. The results and discussion pertaining to these findings are presented in the following paper.

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