螢光共振能量轉移(Florescence Resonance Energy Transfer, FRET)是一項日趨成熟的技術，用來檢測分子之間的鍵結或是探討分子間的作用力，然而因為樣品濃度稀薄的關係，FRET實際上的訊號還很微弱，所以本論文的目標在於去克服這項缺點。本研究的方法是使用奈米量子點(Quantum Dots , QD)做為螢光供體，捕捉受體Alexa647-ssDNA，並嘗試透過主動組裝QD的方式去增益FRET檢測，使用不同的方法去達成目標。
在本論文的第三章中，利用QD修飾於λDNA(QD-λDNAs)上的分子梳去製備FRET感測器。分子梳是利用溶液在微孔洞/微柱陣列與玻璃基板間藉由退水法而完成的技術，發現微柱PDMS印章可以較為成功地利用退水法製備分子梳，而這可以去實現主動組裝QDs於拉伸的λDNA上，有趣的是發現QD-λDNAs分子梳比無修飾螢光的λDNA分子梳較容易拉伸，隨後添加目標分子ssDNA去做FRET實驗，然而經過FRET效率計算後發現線狀的QD-λDNAs分子梳會比懸浮在微柱間的散狀QD來得低。
緊接著第四章，在微柱陣列中設計四角電極針對修飾有yoyo染劑之λDNA以及QD-λDNAs進行電泳操作，發現在微柱陣列中修飾有yoyo染劑的λDNA可以因為勾纏在微柱上而成功被拉伸，但QD-λDNAs並沒有觀察到拉伸現象。
在第五章中則利用微米導電金線和導電玻璃ITO做為電極去捕捉QD，藉由金線旁可以達到放大電場這個優點來進行實驗，發現QD可以因為強大的交流電滲流(ACEO)漩渦被捕捉至金線下緣。
第六章再基於第五章延伸進行FRET實驗，發現FRET強度可以藉由慢慢地將頻率從1kHz往下調整至100Hz而提升，但是相對應的QD強度的下降卻是非單調的響應，在800Hz至500Hz呈現大幅度的下降，但是卻在500Hz至100Hz又呈現上升，前者可能是來自於懸浮在溶液中未鍵結的ssDNA被捕捉至金線上與金線上已存在的QDs鍵結，而後者可能是來自於存在有未鍵結與已鍵結的QD被再次捕捉至金線上。在掃頻的過程當中，原本已經聚集好在金線下緣上的QD會受到電場的影響懸浮至溶液中甚至再受電場被捕捉回來，這種懸浮又再被捕捉回來的過程可以在QD和ssDNA之間提供混合的效果，這不僅能讓更多的ssDNA被QD捕捉，也同時可以增益其鍵結效果。
在第7章中進一步添加微米粒子SiO2透過電場誘導偶極吸引力(FIDA)去輔助捕捉QD和ssDNAs，雖然QD和ssDNAs可以藉由受極化的SiO2夾擊作用而有明顯的聚集，但是因為SiO2的遮蔽效應，FRET訊號相較於第3章中拉伸的QD-λDNAs分子梳低。
本論文呈現出主動組裝QD可能可以促進或是降低FRET訊號，這強烈的取決於如何組裝QD，在這些不同的方法中，利用導電金線捕捉QD的方式具有很大的潛力可以應在未來晶片上的FRET分子檢測。

SUMMARY

This study shows that directional or directed assembly of QDs may promote or discourage FRET performances, strongly depending on how to assemble QDs. There are few methods in this study to make the FRET probing more robust. The first one is talking about preparing of 1-D QD based FRET sensor using DNA molecular combing. The second one is trapping QD using a conducting microwire and its using in facilitating FRET sensing. And the last one is trapping QD using a conducting microwire and microparticles for promoting FRET sensing. Among these different methods, direct trapping of QDs using a conducting microwire seems to be more appealing to expedite FRET molecular sensing at chip scales.
Keyword：FRET、QD、molecular combing、directed assembly、conducting wire

INTRODUCTION

Fluorescence Resonance Energy Transfer (FRET) is a promising tool for probing molecular binding or specific interactions. However, because sample concentrations are typically very low, actual FRET signals are often very weak. This thesis is aimed at overcoming this shortcoming. My approach involves the use of semiconductor Quantum Dot(QD) as the FRET donor in probing target single stranded DNA (ssDNA) tagged with the FRET acceptor Alexa647. To make the FRET probing more robust, I attempt directed assembly of QDs. Several approaches are used to achieve this goal.

METHODS

There are few methods about directional/directed assembly of QDs . The first one is talking about preparing of 1-D QD based FRET sensor using DNA molecular combing. The combing is implemented by having the solution dewetted in between microwell/micropillar arrays and a glass substrate. we find that the combing can be successfully achieved by dewetting through micropillars, allowing us to realize directional assembly of QDs along a stretched λDNA. The second one is trapping QD using a conducting microwire and its using in facilitating FRET sensing. Taking advantage of the fact that the electric field can be greatly amplified by this wire geometry, so that QDs can be trapped beneath the wire due to strong AC electrokinetic swirls therein. And the last one is trapping QD using a conducting microwire and microparticles for promoting FRET sensing. We further add micron-sized SiO2 particles to aid in capturing QDs and ssDNAs through field-induced dipole attraction (FIDA).

RESULTS AND DISCUSSION

In Chapter 3, we prepare FRET nanowires with a molecular comb of QD-conjugated λDNAs (QD-λDNAs), and find that the combing of QD-λDNAs is more attainable than that of bare λDNAs. The subsequent FRET detection is carried out by adding the target ssDNAs. However, in Figure 1, we find that the measured FRET efficiency on the QD-λDNA comb is lower than that of randomly distributed QDs over the substrate. In Chapter 4, using the electrophoretic manipulations for both bare λDNA and QD-λDNAs in micropillar arrays under the influence of a quadrupole electric field. It is found that with the help of the additional hooking effect to the micropillars, λDNAs can be successfully stretched, For QD-λDNAs, however, we do not observe any apparent stretch. we perform a direct trapping of QDs using a conducting microwire in chapter 5. Taking advantage of the fact that the electric field can be greatly amplified by this wire geometry, we find that QDs can be trapped beneath the wire due to strong AC electrokinetic swirls therein. Utilizing the trapped QDs in Chapter 5, in Chapter 6 we perform the subsequent FRET probing. In Table 1, we find that the FRET intensity can be made increased as gradually lowering the frequency from 1kHz to 100Hz. The corresponding QD intensity, however, shows a non-monotonic response. It becomes fading out as lowering the frequency from 800Hz to 500Hz, but shows a significant increase when the frequency is changed from 500 Hz to 100 Hz. The former may come from the replenishment of ssDNAs from the bulk. The latter may be due to the fact that the fresh QDs and as-bound QDs from the bulk can be supplied onto the wire. At all the frequencies, the QDs on the wire may undergo release and re-trapping back and forth around the wire. This release and re-trapping process may in turn render a micro-mixing effect between QDs and ssDNAs, not only allowing more and more ssDNAs to be captured by QDs but also promoting the binding between them. In Chapter 7, we further add micron-sized SiO2 particles to aid in capturing QDs and ssDNAs through field-induced dipole attraction (FIDA) in Figure 2. Utilizing intensified electric fields generated by a conducting microwire, we are able to successfully trap nano-sized particles and molecules with the addition of microparticles. Figure 3 shows Target molecules can be rapidly detected with amplified FRET signals. Although an apparent clustering of QDs and ssDNAs can form along chains of polarized SiO2 particles, the FRET signals appear weaker than those on the stretched QD-λDNAs in Chapter 3 due to the shielding by the SiO2 particles.

CONCLUSION

This thesis shows that directional or directed assembly of QDs may promote or discourage FRET performances, strongly depending on how to assemble QDs. In chapter 3, the FRET efficiency of QD molecular comb is found lower than the theoretical value. Also because it will take a long time to perform FRET sensing, such a comb does not seem advantageous to improve FRET performance. But in terms of making molecular comb, I find combing of QD- λDNA is more attainable than that of bare λDNA. And in chapter 5 and 6, direct trapping of QDs by a microwire is able to produce strong FRET signals despite half success rate. And in chapter 7, the addition of microparticles guarantees the emergence of FRET. However, the FRET signals appear relatively weak due to interferences by the microparticles or possible sample adsorption on the microparticles. Among these different methods, direct trapping of QDs using a conducting microwire seems to be more appealing to expedite FRET molecular sensing at chip scales.

參考文獻
Bensimon, A., Simon, A., Chiffaudel, A., Croquette, V., Heslot, F., & Bensimon, D. 1994 Alignment and sensitive detection of DNA by a moving interface. Science, 265, 2096-2098.
Becker, K., Lupton, J. M., Müller, J., Rogach, A. L., Talapin, D.V., Weller, H., & Feldmann, J. 2006 Electrical control of Förster energy transfer. Nature Materials, 5, 777–781.
Fraden, S., Hurd, A. J. & Meyer, R. B. 1989 Electric-field-induced association of colloidal particles, Physical Review Letters, 63, 2373.
Guan, J., & Lee, L. J. 2005 Generating highly ordered DNA nanostrand arrays. Proceedings of the National Academy of Sciences, 102, 18321-18325.
Guan, J., Yu, B., & Lee, L. J. 2007 Forming highly ordered arrays of functionalized polymer nanowires by dewetting on micropillars. Advanced Materials, 19, 1212-1217.
Hermanson, K. D., Lumsdon, S. O., Williams, J. P., Kaler, E. W., & Velev, O. D. 2001 Dielectrophoretic assembly of electrically functional microwires from nanoparticle suspensions. Science, 294, 1082-1086.
Hilczer, M., Traytak, S., & Tachiya, M. 2001 Electric field effects on fluorescence quenching due to electron transfer. Journal of Chemical Physics, 115, 11249–11253.
Hilczer, M., & Tachiya, M. 2002 Electric field effects on fluorescence quenching due to electron transfer. II. linked donor-acceptor systems. Journal of Chemical Physics, 117, 1759–1767.
Hsieh, S.F., Chang, C.P., Juang, Y.J. & Wei, H.H. 2008 Stretching DNA with electric fields beneath submicron interfacial constriction created by a closely fitting microdroplet in a microchannel, Applied Physics Letters, 93, 084103.
Hu, J., Wang, Z. Y., Li, C. C., & Zhang, C. Y. 2017 Advances in single quantum dot-based nanosensors. Chemical Communications, 53, 13284-13295.
Labit, H., Goldar, A., Guilbaud, G., Douarche, C., Hyrien, O., & Marheineke, K. 2008 A simple and optimized method of producing silanized surfaces for FISH and replication mapping on combed DNA fibers. BioTechniques, 45, 649-658.
Lin, C. H., Guan, J., Chau, S. W., & Lee, L. J. 2008 Experimental and numerical analysis of DNA nanostrand array formation by molecular combing on microwell-patterned surface. Journal of Physics D: Applied Physics, 42, 025303.
Lei, K. F., Wang, Y. H., Chen, H. Y., Sun, J. H., &Cheng, J. Y. 2015. Electrokinetic acceleration of DNA hybridization in microsystems. Talanta, 138, 149–154.
Morgan, H. & Green, N.G. 2003 AC Electrokinetic: Colloids and Nanoparticles. Baldock, UK: Research Studies.
Medintz, I. & Hildebrandt, N. 2014 FRET-Forster Resonance Energy Transfer, Germany: Wiley-VCH, Germany.
Nikiforov T.T. & Beechem J.M. 2006 Development of homogeneous binding assays based on fluorescence resonance enrgy transfer between quantum dots and Alexa Fluor fluorophores. Analytical Biochemistry, 357, 68-76.
Petit, C. A., & Carbeck, J. D. 2003 Combing of molecules in microchannels (COMMIC): a method for micropatterning and orienting stretched molecules of DNA on a surface. Nano Letters, 3, 1141-1146.
Stryer, L. 1978 Fluorescence energy transfer as a spectroscopic ruler. Annual review of biochemistry, 47, 819-846.
Vannoy, C. H., Tavares, A. J., Noor, M. O., Uddayasankar U. & Krull, U. J. 2011 Biosensing with quantum dots: a microfluidic approach, Sensor, 11, 9732-63.
Zhang, C.Y., Yeh, H.C., Kuroki, M.T. & Wang, T.H. 2005 Single-quantum-dot-based DNA nanosensor. Nature Material, 4, 826-831
Zhang, C. Y., & Johnson, L. W. 2007 Microfluidic control of fluorescence resonance energy transfer: breaking the FRET limit, Angewandte Chemie International Edition, 46, 3482-85.
Zhou, R., Chang, H.C., Protasenko, V., Kuno, M., Singh, A.K., Jena, D. & Xing, H. 2007 CdSe nanowires with illumination-enhanced conductivity: Induced dipoles, dielectrophoretic assembly, and field-sensitive emission, Journal of Applied Physics, 101, 073704.
程俊傑，探討以圖案式分子梳技術製備整齊排列之長DNA奈米束，國立成功大學，碩士論文，2009。
林宣甫，於高頻交流電場下膠體粒子與DNA分子之動態組裝，國立成功大學，碩士論文，2009。
王政源，以移動界面法實現分子梳並應用其發展具標的分子診斷功能之一維奈米感測器，國立成功大學，碩士論文，2010。
連政偉，製備含量子點修飾之DNA分子梳並應用其發展具分子探測與檢測功能之一維FRET生物感測器，國立成功大學，碩士論文，2011。
陳信龍，膠體粒子交互作用與單一高分子鏈構形變化之偵測與診斷，國立成功大學，碩士論文，2011。
趙慶安，在交流電場下實現定向組裝奈米粒子和DNA分子來增益FRET感測，國立成功大學，碩士論文，2018。