在极稀薄磷酸盐缓冲溶液中的溶菌酶－ 蛋白质在奈米矽土及奈米钻石 .doc

在極稀薄磷酸鹽緩衝溶液中的溶菌酶－蛋白質在奈米矽土及奈米鑽石表面上之吸附反應研究 吳為克* 摘　　要 傳統的用紫外線光譜法(UV)檢測蛋白質濃度，濃度至少必須數拾體積摩爾濃度(μM)，譬如: 50μM。為檢測極稀薄蛋白質溶液的濃度，及探討其在奈米矽土(SiO2)及奈米鑽石表面上之吸附反應動力學，在這實驗中是用螢光法，並以直徑約100 nm的奈米矽土及奈米鑽石來當作是吸附表面，選擇 ”溶菌酶”為此蛋白質，靈敏度可以提高至少5000-10000倍。 關鍵字：蛋白質吸附，界面反應動力學，細胞膜反應動力學，螢光光譜，單分子光譜，生物晶片。1. Introduction Biomolecular chain reaction as well as stereospecific oligomerization on a cell membrane has been pionieered by Jovin et al. in 1995 [1]. Biomolecular interface research was initiated and keeps growing as one of the important branches of biomolecular reseaches [2-10]. Lysozyme is one of the proteins, which has been mostly investigated and reported. It contains 6 tryptophanes, which are useful for the spectroscopic detection. UV-absorbance combined with FTIR (Fourier Transformed Infrared Spectroscopy) or/and TIR (Total Internal Reflection) method by use of a prism [11, 12] has been applied usually in the recent 10 years, whatever can resolve the concentration of Lysozyme up to ca. 50-100μM, via Sore band at 409 nm, even the molecular orientation of the adsorbed protein on the surface can be deduced. The absorption capabilities on the surfaces of nanosilica [4, 5, 6], nanodiamond [11, 12, 16] and other materials, as well as the their adsorption mechanisms of proteins, e.g. lysozyme on these surfaces can not be well differentiated by use of these methods. The unimaginable adsorption capabilities of nanosilica and nanodiamond with diameter (?φ) 100 nm - nearly all kinds of proteins in a solution can be completely adsorbed [16], and the detectability of extremely diluted lysozyme solution down to 10 nM are demonstrated by use of the fluorescence method [17-20]. The surface adsorption capabilities as well as adsorption constants of nanosilica and nanodiamond can parallelly be obtained. 2. Experimental procedure Different concentrations of lysozyme (M=14300 g/mole, purity 99.9, Sigma Chemical, USA) of chicken egg white between 10 - 1000 nM as well as 10-5000 nM were prepared in PPBS (Potassiu