毛細管與晶片電泳於DNA之分離及量子點之製備與光電應用

Capillary and microchip electrophoresis for DNA separation as well as synthesis and optoelectronic applications of quantum dots

林泱蔚(Yang-Wei Lin)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.00896

摘要

本論文可區分成兩大主題，第一部分主要是高效率及高再現性之毛細管與晶片電泳分離DNA之技術開發；第二部分著重於奈米粒子量子點之製備及其應用。首先，在毛細管電泳方面，利用線上濃縮技術及增加偵測光徑（氣泡容槽）方式來改善DNA片段靈敏度及分離效率。與傳統毛細管電泳操作模式比較，結果發現對89 bp DNA片段之偵測靈敏度可提升至170倍。此外，於氣泡容槽條件下，亦能提升大片段DNA（> 500 bps）之解析度。為了進一步降低分析時間及改善DNA於管壁吸附問題，於低濃度之篩分聚合物溶液（PEO）中添加少量十六烷基三甲基溴化銨（CTAB）進行DNA之分離。實驗結果發現，DNA吸附情形及與溴化乙錠（EtBr）間作用會隨著CTAB濃度增加而降低。在最佳化條件下，8分鐘內即可完成DNA Marker V及VI（18~2176 bp DNA片段）之分離，且對於18 bp DNA片段之偵測極限可降至2.0 ng/mL。在晶片電泳部分，首先利用聚合物溶液（PVP and PEO）和金奈米粒子（GNPs）來對微流體通道進行動態塗覆。以連續三層塗覆（PVP-PEO- GNPs）之微流體通道，在1.5% PEO(GNPs)聚合物溶液條件下，有效地改善18至2176 bp DNA片段之分離再現性（RSD 2.5%，n = 5）及解析度（R > 1.1）。另外，我們亦利用多層動態塗覆方式（(PEO-PVP)2-PEO(GNPs)）修飾微流體通道。在0.75% PEO(GNPs)溶液條件下，分離DNA Marker V及VI可得到快速（＜3分鐘）、高效率（N > 1 700 000 plates/m）及高再現性（RSD 1.3%，n = 5）之結果。在奈米粒子量子點部分，本論文首先開發出利用雷射輔助-水相合成高量子效率量子點（CdSe）之技術。為了更進一步提升量子效率，除了形成核－殼形式量子點（CdSe@CdS）外，我們亦藉由紫外光照射降低其表面缺陷，成功地將量子點之量子效率提升至80%。此外，利用Stöber process形成核－殼－殼量子點（Silica-QDs-Silica）結構，更能有效地增加其光學、化學穩定性，未來將可應用於生物樣品之檢測及標識。最後，使用layer-by-layer assembly 技術，並且依據量子點間能量轉移和顏色混合之概念，我們可以製作出多色彩量子點薄膜。再結合微影技術，則可將多色彩量子點薄膜之尺度降低至微米範圍（10 um）。而此類薄膜在生物感測器、光子晶體及LED應用方面應極具發展潛力。

關鍵字

DNA分離 ； 毛細管電泳 ； 晶片電泳 ； 量子點 ； 量子點薄膜

並列摘要

This thesis focuses on developing highly efficient and reproducible capillary electrophoresis (CE) and microchip CE (MCE) based techniques for DNA separation and preparing highly fluorescent quantum dots (QDs) for biosensing and optoelectronic devices. First, the sensitivity and resolution of DNA fragments have been optimized in CE, by applying on-line concentration and using a bubble cell (e.g. 300 um in diameter). When compared to that by conventional injection and use of a capillary without a bubble cell, up to 170-fold sensitivity improvements for the DNA fragments have been achieved. The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by CE in conjunction with laser-induced fluorescence (CE-LIF) detection using 0.75% poly(ethylene oxide) (PEO) solution is described. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. Under optimum condition, a mixture of DNA markers V and VI within 8 min at -375 V/cm was separated, with the limit of detection of 2.0 ng/mL based on the peak height for the 18-bp DNA fragment. In microchip electrophoresis for DNA separation section, we have demonstrated a simple method for dynamically coating the wall of the separation channels that were fabricated on poly(methyl methacrylate) (PMMA) plates using poly(vinyl pyrrolidone) (PVP), poly(ethylene oxide) (PEO), and gold nanoparticles (GNPs) in sequence. The three-layer (PVP-PEO-GNPs) coated PMMA chips provide improvements in resolution and reproducibility for DNA separation when using 1.5% PEO(GNPs), allowing the separation of DNA fragments ranging in the size of 18-2176 bp. Besides, multilayer coating of PMMA chips with PEO, PVP, and PEO containing gold nanoparticles [PEO(GNP)] is important for achieve high efficiency. Using a 2-(PEO-PVP)-PEO(GNP) PMMA chip, the separation of DNA markers V and VI by MCE in 0.75% PEO(GNP) was accomplished in 3 min. In QDs section, a simple synthetic route to the preparation of high-quality CdSe QDs in aqueous solution was present. The thermal synthesis, assisted by laser irradiation at 532 nm, allows the preparation of CdSe QDs that possess higher quantum yield. After UV irradiation, the as-prepared core–shell CdSe/CdS QDs become stable and fluoresce strongly in the visible range. For biocomplement, we also prepare highly water-soluble core-shell-shell (CSS) silica–QDs–silica NPs that exhibit greater QYs, photostability, and chemical stability. This feature, together with their narrow emission spectral profile, suggests that the CSS silica–QDs–silica NPs may have a great number of biological applications. QD films are fabricated through layer-by-layer (LBL) assembly using citrate-stabilized CdSe@CdS QDs, 3-mercaptopropionic acid (MPA)-stabilized CdTe QDs, and poly(diallyldimethylammonium chloride) (PDDA). The colors and emission intensities exhibited by the highly fluorescent QD films are readily tunable by controlling the deposition order and the number of bilayers of (PDDA-CdSe@CdS)n and (PDDA-CdTe)n.

並列關鍵字

DNA separation ； capillary electrophoresis ； microchip electrophoresis ； quantum dots ； quantum dots films

參考文獻

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