本論文我提出結合量子點(Quantum Dot, QD)修飾λDNA及交流電荷動力學製備快速且靈敏1-D FRET(螢光共振能量轉移，Fluorescence Resonance Energy Transfer, FRET) 分子感測器，利用表面有修飾stretapvidin的QD作為FRET供體以及螢光分子Alexa 647作為FRET受體。首先我將biotin沿著λDNA骨干連接到λDNA上。QDs透過stretapvidin與biotin鍵結使QD以每間隔200個鹼基對鍵結在λDNA上，再利用介電泳將其補足及拉伸，其中QD不僅作為FRET供體來實現目標ssDNA分子的檢測，還可以作用為目標ssDNA(single strand DNA, ssDNA)分子富集器。此外，我還進一步研究使用此感測器所產生的FRET訊號及FRET效率於交流電場下的響應。主要結果在第4-6章中給出，如下所述。
本論文第四章中，我介紹了我的四極電極設計，並描述如何產生一個高電場來預集濃純QD。這是QD-FRET感測器的可行性的初步測試。本章包含三部分。在第一部分中，我使用微米尺寸的latex顆粒來了解電場的行為和ACEO的流動型態。在第二部分，我研究了QDs和ssDNA如何在不同的交流頻率下被捕獲和聚集。根據第一部分和第二部分的結果，我選擇適當的交流頻率進行FRET檢測。我發現QD的分散頻率很差，形成了大顆的QD聚集，這可能導致量子點的螢光強度被過度增亮。通過電場捕獲QD使得這個問題更糟。雖然FRET信號仍然可以觀察到，但是所觀察到的FRET訊號中會夾雜被過度增亮的QD訊號。這不僅影響FRET強度測量，而且還使我無法確定FRET的效率。
本論文第五章，為了克服QD過曝的問題，我詳細介紹了如何使用QD 修飾之λDNA作為1-D FRET 分子感測器。本章分為三部分，第一部分為在不同的頻率下捕捉λDNA（以 yoyo-1標記）來了解λDNA的介電性質、第二部分為研究如何使有QD 修飾的λDNA在電場的作用下被捕捉及拉伸、第三部分則是第二部分完成拉伸的QD 修飾之λDNA進行FRET感測實驗。我們發現FRET信號不僅可以大大放大，而且可以將FRET效率推高到比正常值10.6％高出90％。我們猜測這種FRET增強歸因於沿著1D幾何形狀的FRET對的激子偶極子的對準，使得被激發的電子更傾向於發生FRET。
本論文第六章， 我們的目的是研究五章中FRET效率異常高的原因，以及探討電場在這一過程中所扮演的角色。本章包含兩個部分對FRET訊號電壓地倚賴性測試及FRET訊號對電場開關的即時響應。我們先演示了FRET訊號在有目標ssDNA分子補充的情況下，調降電壓的瞬間FRET訊號也如階梯式的瞬間減弱，再演示沒有目標ssDNA分子補充的情況下，FRET訊號隨開關電場的瞬間顯示出急劇的上升和下降。顯示了我們整合拉伸有QD 修飾之λDNA及交流電荷動力學製備快速且靈敏1-D FRET 分子感測器是可以利用電場調控的，使其可以作為加速生物分子檢測和定量的更靈敏和準確的工具。

英文延伸摘要
Integration of DNA Stretching and AC Electrokinetics for Preparing a Robust Dynamic FRET Sensing Microdevice
Tzu-Han Liang
Hsien-Hung Wei
Department of Chemical Engineering, National Cheng Kung University

INTRODUCTION
In this thesis, I develop a rapid and sensitive 1-D FRET（Fluorescence Resonance Energy Transfer, FRET）molecular sensor using quantum dot（QD）conjugated λDNA and AC electro-kinetics. Here QD is streptavidin-coated CdSe-ZnS core shell nanocrstal and serves as the FRET donor. I first attach biotin to λDNA biotin along its backbone. QDs are then attached to λDNA through streptavidin-biotin binding every 200 base pairs. This QD- λDNA is stretched and immobilized under AC electric fields, working as a molecular concentrator that can facilitate target ssDNA molecule detection. In addition, I also study how FRET responds to changes of AC electric fields. Main results are given in Chapters 4-6, as summarized below.

MATERIALS AND METHODS
In Chapter 4, I present my quadrupole electrode design and describe how to generate a high electric field for pre-concentrating pure QDs. This is the prelimnary test for the feasibility of QD-FRET sensing. This chapter contain three parts. In the first part, I use micron-sized latex particles to see how the electric field behaves and to envision the AC electrokinetic streaming. In the second part, I study how QDs and ssDNA are trapped and aggregated at different AC frequencies. Based on the results of the first and second parts, I select an appropriate AC frequency to conduct FRET detection.
In Chapter 5, to overcome the above overbrightenig issue, I detail how to prepare QD-conjugated λDNA as a 1-D FRET molecular sensor. This chapter is divided into three parts. The first part is to undertand the AC trapping behavior of λDNA（labeled by yoyo-1）at different frequencies. In the second part, I then study how to capture and stretch QD- λDNA under the actions of an electric field. The third part is the use of stretched QD- λDNA found in the second part to perform FRET sensing.
In Chapter 6, I want to understand why the measured FRET efficiency is unusually high and to explore how electric fields play roles.

RESULT AND DISCUSSION
In Chapter 4, I find that quite frequently QDs are poorly dispersed and form large aggregates, which can result in overbrightening of QDs. Trapping QD by electric fields makes this problem even worse. While FRET signals can still be observed, they sometims are overshadowed by overbrightening QDs. This not only affects FRET intensity measurent, but also prevents me from determining the FRET efficiency.
In Chapter 5, I find that FRET signals can not only be greatly amplified, but also can push the FRET efficiency higher than the normal value of 10.6% to 90%. We speculate that this FRET enhancement is attributed to the alignment of the exciton dipole of the FRET pair along the 1-D geometry, making the excited electrons of the donor QD more inclined to FRET to the acceptor.
In Chapter 6, this chapter report two phenomena:（i）FRET response due to sudden voltage decrease, and（ii）instantaneous FRET switch by turning on/off an electric field. In（i）, I find that even in the presence of a continuous supply of target ssDNA, a FRET signal can undergo an abrupt fall upon a sudden decrease of voltage. In（ii）, there is no continuous supply of target ssDNA, a FRET signal can display a sharp rise and fall when turning the field on and off. These findings suggest that an electric field might participat in the modulation of the FRET process.

CONCLUSION
In overall, this thesis demonstrates that integration of QD conjugated λDNA and AC electrokinetics can be used to prepare a roburst and sensitive 1-D FRET molecular sensors. As this sensor can also be modulated by additional electric fields, it can be used as a more sensitive and accurate tool for expediting biomolecule detection and quantification.

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