高效能水溶性量子點之製備與其在生物檢測之應用

Abstract

本論文研究係以水溶性量子點硫醇琥珀酸修飾硒化鎘/硫化鋅製備尿素、葡萄糖及三酸甘油脂等生物分子指示劑，更進一步合成了高效能水溶性量子點建立量子點螢光共振能量檢測系統，最終將其應用於生物分子之檢測。 因水溶性量子點螢光強度易受環境中酸鹼值之影響，當量子點暴露於鹼性環境時，其螢光強度隨環境中鹼性強度增加而增強；藉由光譜分析可明顯追蹤其放射光譜產生紅位移之現象，此係因鹼性環境下可減少量子點表面之缺陷與其結構被修飾，造成有效電子-電洞結合率增加所致。利用上述特性，本研究亦探討尿素經尿素酶水解反應後產生鹼性物質，致使螢光產生變化故可藉以分析樣品內尿素之含量。反之，當量子點暴露於酸性環境時，其螢光強度隨酸強度增加而降低，造成其放射光譜產生藍位移之現象，此係酸性環境中量子點表面缺陷增加，而電子-電洞結合率降低所致。另一方面，本研究亦利用葡萄糖氧化酵素催化葡萄糖，因其反應後形成酸性產物，故可藉量子點之螢光變化分析樣品中葡萄糖含量。為增強量子點之螢光強度及脂質被分析物之偵測，本研究藉由光氧化提升量子點螢光效率，並改善其表面疏水性，以提升脂質被分析物之溶解度；本論文亦藉量子點之光活化以結合解脂酵素水解三酸甘油脂，最終利用螢光強度變化以偵測三酸甘油脂。 本研究結合高分子包覆聚二烯丙基二甲基氯化銨與光活化製程可合成量子效率高達48%水溶性且放射波長不同範圍之高效能量子點，此高效能之螢光特性可應用於螢光共振能量轉移系統，建立檢測生物分子之分析模型。本研究利用具高正電荷之聚二烯丙基二甲基氯化銨所修飾量子點與類固醇賀爾蒙酵素以自組裝方式形成複合物，結合已標定有機染料四甲基羅達明的膽酸目標物，探討量子點與有機染料間螢光共振能量轉移現象，並掌握膽酸與類固醇賀爾蒙酵素之鍵結能力，以作為生物分子檢測系統之基礎。

This research focuses on the design and preparation of molecular bioindicators for semi-quantitative determination of urea, glucose, and triglycerides by using water-soluble mercaptosuccinic acid (MSA)-capped CdSe/ZnS quantum dots (QDs) (MSA-QDs). Furthermore, we have also attempted to synthesize high performance water-soluble MSA-QDs and establish a bioassay based on fluorescence resonance energy transfer (FRET) principle that was then utilized in bioassay. The fluorescence intensity of MSA-QDs, highly sensitive to the acidity/basicity of the environment, was discovered to increase with increasing basicity and a red-shifting in emission wavelength has been observed. This observation was attributed to the reduction of surface defects and modified surface structure of MSA-QDs in a basic environment that further increases the recombination probability of holes and electrons. On the contrary, a blue-shifting in emission wavelength was observed when MSA-QDs was exposed to an acidic environment and the fluorescence intensity was found to decrease with increasing acidity, which is attributed to the surface defects formation and lowering probability of electron-hole recombination. Therefore, the assay of urea and glucose has been demonstrated by monitoring the changes of basicity and acidity, respectively, when urea and glucose are hydrolyzed by urease and glucose oxidase in a series of analytes. In order to realize the feasibility of detection for lipid analytes, we have improved the fluorescence intensity and activated the surface hydrophobicity (to enhance the solubility of lipid) of MSA-QDs by carrying out photoactivation under fluorescent lamp irradiation. The assay of triglyceride has been demonstrated by monitoring the change of fluorescence intensity of MSA-QDs when triglyceride is hydrolyzed by lipase in the analytes. We have also demonstrated that a series of highly efficient, wavelength-tunable or multicolored and poly(diallyldimethylammonium chloride) QDs (PDDA-QDs) with quantum efficiency of ~48% can be synthesized through PDDA capping and photoactivation process. The most challenging task of this research is to investigate and utilize the binding affinity between tetramethylrodamine (TMR)-labeled cholic acid (CA) and self-assembled □5-3-ketosteroid isomerase (KSI) by assuming TMR-CA as the energy donor and PDDA-QDs as the acceptor. Based on the principle of fluorescence resonance energy transfer (FRET) from TMR-CA-KSI to PDDA-QDs, our research results exhibit great potential applications in establishing effective assay systems for quantitative determination of a wide variety of biomolecules.