鑑於奈米量子點(Quantum Dot, QD)的獨特光學性質被視為良好的供體，於螢光能量共振轉移(Fluorescence Resonance Energy Transfer, FRET)的應用具有許多優勢，然而在低濃度時會出現FRET假訊號。本文探討如何結合交流電荷作用以主動的方式調控QD的發光行為，期望不僅能消除假訊號，並且開發出可信度更高的FRET分子檢測新手段。首先，我利用交流電場在城垛型的氧化銦錫(Indium Tin Oxide, ITO)微米電極陣列上匯集QDs，接著再以同樣方式將接有螢光受體的單股DNA(single stranded DNA, ssDNA)集濃與事先匯集好的QDs在電場作用下進行FRET檢測，同時觀察並比較QDs在鍵結ssDNA前後的發光響應。
本文第三章，透過交流電滲流(AC Electro-Osmosis, ACEO)以及介電泳(Dielectrophoresis, DEP)的手段，捕捉QDs將之固定於電極表面上，發現QD沿著電極邊緣和電極中央的聚集最為明顯，並改變電場的頻率歸納出QDs的最佳發光型態，以此發光型態接續進行FRET檢測，在施加電場後1秒顯示出明顯的FRET訊號。
第四章透過開關電場的手段觀察QD及FRET的發光響應，發現在電極邊緣處，已鍵結的QD開電場時訊號降低，關電場時增加，然而FRET訊號呈現相反的響應，原因是開電場時供體QD將自身能量轉移給受體放出FRET訊號，使得QD的發光強度降低而FRET增加。另外發現未鍵結QD的響應在電極邊緣/中央的響應與外加電場反相/同相。在電極邊緣的QD受到局部電場的作用聚集，沿著電場方向排列呈現向序性聚集，產生反相響應，也就是在電場開啟/關閉時，QD的螢光強度衰退/增加；同相的響應發生在QD聚集較無向序性的電極中央處，在電場開啟/關閉時，螢光強度增加/衰退，而對應於後者的FRET響應，也觀察到電極中央區的FRER訊號在開關電場時隨著時間呈現持續成長和衰退，這表明QD和FRET的發光是通過QD的組裝和電場交互作用的結果，而電極邊緣上的QD比電極中央的QD更有序，這也表明QD和FRET發射響應都可以通過適當的QD組裝且透過交流場來調控其響應的表現。
第五章，我們認為QD的訊號表現以及FRET效率皆取決於電極位置，以及開和關電場的作用。為了瞭解交流電荷對是否會影響FRET效率，我們採用光激發螢光光譜儀(Photoluminescence, PL)和時間解析光激發螢光光譜儀(Time-Resolved Photoluminescence, TRPL)針對QD的放射光譜和螢光生命週期進行量測，於此章節介紹PL和TRPL儀器架設和操作方法，並透過實驗校正、規劃，準確測量未鍵結/已鍵結QD的光學性質。
第六章呈現開關電場下PL以及TRPL的測量結果，第一部分介紹未鍵結QD的螢光生命週期隨電場變化的響應：純QD受電場作用後螢光生命週期變短，而在連續開/關電場下未鍵結QD螢光生命週期縮短/增長。第二部分說明QD的放射光譜於鍵結受體ssDNA後藍移5nm，在電場的作用下，發現已鍵結QD的螢光生命週期比未鍵結的短，然而在關閉電場後卻發現其螢光生命週期居然比未鍵結的QD還長，這暗示了在交流電場的作用下存在了反向FRET的機制，將受體Cy5獲得的能量反饋給供體QD。
第七章根據第四章以及第六章的實驗結果，分成三個部分進行探討。第一部分研究未鍵結QD在受電場作用後，QD的載子於能帶間的穿隧效應，造成螢光生命週期變短。後續觀察到QD與ssDNA鍵結後光譜藍移很可能是因為QD表面電荷受鍵結改變影響能階變化，產生了藍移的結果。第二部分探討為何已鍵結QD在受電場作用時發光強度降低，螢光生命週期反而增加，我基於前者的穩態螢光以及後者的脈衝螢光進行討論。雖然QD受電場作用後詳細的放光機制，以及反向FRET發生的原因都尚不清楚，我仍於第三部分提出動力學模型來建立發光表現如何相互依賴供體QD和受體，該模型表明，即使在沒有電場時， 由於QD與受體鍵結後其放射光譜會藍移，傳統利用QD 鍵結前後的螢光生命週期來度量FRET效率可能會導致錯誤結果。除此之外，當於電場下存在反向FRET時，供體 QD與受體 Cy5的發光將相互影響，供受體個別的發光強度以及生命週期都包含了對方的貢獻，無法如傳統的方式做簡單分割。以上這些發現也暗示了以往的計算的FRET公式都需要做修正，儘管如此，由於供受體的發光響應彼此耦合，且這又取決於QD的組裝排列以及供受體的濃度以及施加電場的條件，這些響應不僅可作為FRET指紋，也為將來設計兼備高敏度及主動調控功能的FRET分子感測器帶來新的可能。

SUMMARY

Quantum-Dot-based Fluorescence Resonance Energy Transfer (QD-FRET) is a robust technique for probing target molecules. However, it often suffers from weak emissions and false signals. Here we utilize AC electrokinetic effects to overcome these shortcomings. Not only can FRET signals be amplified very rapidly by trapping QDs and target ssDNAs, but also we observe unusual quenching/re-bursting of both QD and FRET signals in response to alternate switching on/off of the applied electric field. The latter is further accompanied by a “blue shift” of the emission spectrum of the QDs. As these peculiar photoluminescence responses are tunable and sensitive to samples, they can serve as new dynamic FRET fingerprints for more accurate FRET sensing at the chip scales.

Keywords: Quantum Dot, Fluorescence Resonance Energy Transfer, AC Electro-kinetics, Fluorescence lifetime, DNA probing

INTRODUCTION

Semiconductor quantum dots (QDs) have been emerged as excellent donors for Fluorescence Resonance Energy Transfer (FRET) applications because of their unique photonic and optical properties. However, false FRET signals can still occur at low sample concentrations. In this thesis, I explore the use of AC electric fields in regulating the photoluminescent behavior of QDs, hoping that not only can false FRET signals be eliminated but also a more reliable FRET probing strategy can be developed to detect target molecules. In the experiment, I first use an AC electric field to trap QDs on castellated indium tin oxide (ITO) microelectrode arrays. I then concentrate FRET-acceptor-tagged single-stranded DNAs (ssDNAs) onto the as-trapped QDs and perform the corresponding FRET sensing also under an AC field. Both the photoluminescent responses of bare QDs and bound QDs are inspected and compared.

MATERIALS AND METHODS

Making use of the joint effects of AC electro-osmosis (ACEO) and dielectrophoresis (DEP), a successful trapping and immobilization of QDs can be realized. The trapping is found most pronounced along the edges and in the centers of the electrodes. The corresponding FRET probing is also carried out, showing an apparent FRET buildup within 1 second upon the application of an electric field.
To better understand how the emission of trapped QDs changes with an AC field and the corresponding FRET process, I employ both Photoluminescence (PL) spectroscopy and Time-Resolved Photoluminescence (TRPL) spectroscopy to measure the emission spectrum and lifetime for QDs. I provides details for how I set up the instruments, carry out the measurements, and conduct a series of testings for correctly quantifying the photoluminescent characteristics of bare/bound QDs with/without fields in the context.

RESULTS AND DISCUSSION

The emissions of QD and FRET are observed under alternate switches of an AC field. I show the result in Figure 1, it is found that the emission of the bound QDs on the electrode edges becomes decreased/increased when the field is on/off, whereas the corresponding FRET emission is completely the opposite. The emission of the bare QDs on the electrode edges also shows the same response, whereas that in the electrode centers shows an increase/decrease when the field is on/off. In the latter case, I find that the observed FRET response shows either grow or decay with time during alternate switches of the applied field. These distinct responses of QD and FRET may be attributed to the fact that QD trapped on the electrode edges appear more ordered than those in the electrode centers. This suggests that both QD and FRET emissions can be mediated by AC fields with an appropriate assembly of QDs.
I use PL and TRPL to measure bare QDs and bound QDs. In Table 1, I find that the lifetime of the bare QDs trapped on the electrode edges becomes decreased/increased when an AC field is on/off. But after trapping ssDNA onto the QDs, I observe a 5nm blue shift for the QD emission spectrum in Figure 2. In Table 2, the lifetime of the bound QDs in the presence of an AC field is also found shorter than that of the bare QDs. But when turning off the field, I find that, much to my surprise, the lifetime of the bound QDs does not become longer. Rather, it turns to be shorter, contrary to the response of the bare QDs. The longer lifetime for the bound QDs in the presence of an AC field suggests that there is a backward FRET from the acceptors on the ssDNAs to the donor QDs, then backward FRET kinetic model shows in Figure 3.
I provide plausible explanations for the experimental findings. These explanations consist of three parts. In the first part, I explain why the lifetime of the bare QDs becomes shorter in the presence of an AC field in terms of interband carrier tunneling and transport. The observed blue shift of the QD’s PL after binding is likely attributed to surface-charge-mediated quantum confinement effects. In the second part, I provide an explanation for why the bound QDs can undergo quenching but display a longer lifetime from different points of views based on steady-state fluorescence in the former and pulsed fluorescence in the latter. Although the detailed mechanisms of the AC-mediated QD emission and the origin of the backward FRET are still not clear, in the third part I postulate a kinetic model that allows me to establish a relationship between the QD and the acceptor emission dynamics. The model suggests that because of the blue shift of the QD emission after binding, even in the absence of electric fields, the common use of QD’s quenching lifetimes before and after binding for quantifying the FRET efficiency will lead to an erroneous result. In addition, when a backward FRET exists in the presence of an electric field, both the lifetimes of QD and acceptor will no longer be independent quantities but be coupled to each other. These suggest that all the commonly used FRET formulas have to be revised.

CONCLUSION

Since the photoluminescent responses due to such coupling will strongly depend on the concentrations and assemblies of QD and acceptor as well as on the applied field condition, they may not only provide unique fingerprints but also spur more sensitive and tunable means for target molecule detections using FRET.

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