2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/649252摘要：生物分子交互作用的隨機行為是所有生命訊息的起源。長久以來的研究皆植基於巨觀尺度下測量一群分子的動態反應所得到的平均訊號配合時域統計分析而獲得瞭解，主要的限制是因為微量分子所釋放的訊號未能被傳統的儀器所偵測。近年來隨著越來越多種材料的奈米結構表現出與bulk 材料時嶄新的物理性質，顯微觀測與製程技術朝奈米尺度發展，生物感測器的尺度也隨著奈米化，這使得陣列式單分子偵測(array-based singlemolecule detection)變為可能。在分子檢測中一項十分重要的工作在於如何將巨觀(macro scale)尺度下所量測到的平均分子動態行為參數與微觀(micro scale)下個別的分子動態行為，透過統計分析的隨機亂數模型建立起定性與定量的關係。在我們先前的研究裡，成功地使用沾筆式奈微影系統(Dip-Pen Nanolithograpy, DPN)製作出每個點50 nm 的10x10 奈米陣列, 並且固定strepavidin 於製作的50 奈米點陣列的大小；為能達到統計上有效的誤差值，我們建立了一個二維影像亂數分析模擬程式，模擬結果顯示需建構足夠的隨機分析的陣列為15*15的矩陣以上(<3%CV)。我們所建構的多功能暗場顯微鏡可順利進行奈米尺度結構之量測觀察，利用不同容量的光碟的表面結構，我們可以確認在SNR=3 的條件下解析度為120nm，並成功在鍍有50 奈米金膜的藍光碟片上進行表面電漿子共振頻譜量測可定性觀察到蛋白質分子固定前後的顯著差異。然而，在奈米尺度下，材料表面活性易因缺陷而大幅地提升，而發展生物奈米晶片要經過分子固定化，反應流道的建立等多重步驟，需要進一步發展快速且簡便的製程與提升奈米解析度與適合遠場觀測技術以實現奈米尺度下的單一分子隨機行為的統計模型的目標。基於上述的初步研究成果與經驗，本計畫擬使用plasmonic nanolithography 取代原先DPN 製做生物奈米陣列樣版的方法，使用DPN 於後續的多種生物分子塗佈標的優點，對奈米生物晶片進行加工。生物分子預計採用有接抗原之奈米粒子(直徑>=50 奈米)與ALV 病毒顆粒(約100 奈米)達到在一個直徑50 奈米的陣列點上因為空間限制只允許單一分子交互事件的發生。在觀測的架構部分，除了嘗試以高NA 物鏡在穿透式架構下改善原先暗場顯微鏡的解析度外，達到直接觀測100 奈米下的標的物目標之外，也將跟法國Ecole Normale Supérieure de Cachan, Prof. Dominique Chauvat 與台大李世光教授合作的Radially-polarized SPRM 進行合作量測，以掃描或二維影像方式觀測奈米陣列上生物分子反應的動態折射率變化，達到非標識(non-labeling)的觀測。最後取得的訊號將與傳統使用表面電漿共振測量分子動態反應做比較，以驗證奈米尺度下生物分子的隨機行為模型。此一整合奈米製程與光學檢測技術之Stochastic Array 研究在目前全世界的研究中尚在起步中，本團隊在長期合作努力下有希望獲得實用的突破在高度創新的生物感測器領域獲得領先地位。<br> Abstract: The stochastic behavior of biomolecular interaction is the origin of the entire livingmessages. For a long time, the related research to understand this knowhow is based on theaverage signals of spatial and temporal summations from a group of biomolecularinteractions, because the signals released from those biomolecules with an extreme lowconcentration cannot be detected by conventional devices. Recently, as more and morenanostructures present some novel properties, which are different from the properties of thesame materials in bulk, the techniques for microscopy and fabrication are toward to the scaleof nanometer. The size of biosensor also develops to this level, which makes the array-basedsingle molecule detection possible.In molecular diagnostics, how to connect the average behavior of biomolecular interaction atmacro-view with single molecule recognition at micro-view is a critical issue for moleculardiagnosis by using stochastic model to do the qualitative and quantitative assay. In ourprevious work, we have done a successful work to make a 10 by 10 digital nanoarray with10 nm diameter per dot by dip-pen nanolithography (DPN) and immobilized the straptavidinon the dot site. We have established a simulation model for the events of biomolecularinteraction occurred in a two-dimension matrix, the result shows that the array size should beover 15 by 15 (< 3% CV) to provide sufficient events to analyze. In the multi-function darkfield microscopy we built, the resolution for observing nanostructure can be identified byoptical disks with various capacities, and it is 120 nm at SNR=3 so far. The apparentdifference with and without labeling streptavidin on a blu-ray disc coated with 50 nm goldfilm can be recognized by qualitative analysis of absorption spectra due to surface plasmonresonance. However, the activity of materials under 100 nm is largely increasing due todefect. Developing nano-biochips need multi-steps, such as fabricating nanopatterns,immobilizing biomolecules and making microfludic channel, so a fast and simple fabricationand a nanometer-resolution of far-field microscopy is necessary to realize this goal forstochastic behavior of single molecular interaction under nanomenter scale.Based on the above preliminary results and experiences, this project propose to use surfaceplasmon (SP) nanolithography to replace the original method that is DPN to make thetemplate for the biomolecular nanoarray. Then, this chip will be processed by DPN that hasan advantage of depositing various molecules at one time. The target molecules will be theartificial nanoparticles with a diameter of lager than 50 nm or AIV virus particle (diameter isabout 100nm), which makes one site of dot (50 nm) react only with one particle. That is,there is only one event that will take place on one site due to the limitation of dot size.Regarding the observation, except to use the high NA objective lens to improve theresolution of microscopy for detecting the structure under 100 nm, we will measure theseevents using racially polarized surface plasmon resonance (SPRM), which is the co-workresult by Prof. Zyss (Ecole Normale Supérieure de Cachan, France), Prof. Chauvat (EcoleNormale Supérieure de Cachan, Fance) and Prof. CK Lee (Institute of Applied Mechanics,NTU), to measure the reflective index change caused by biomolecular interaction withscanning or imaging the 2-D image for non-labeling observation. Finally, this experimentaldata will compare to that measured by conventional SPR method to identify the stochasticmodel of biomolecular interaction under nanometer scale. It is hopeful to obtain practicalbreakthrough and to obtain the leading ship in the field of biosensor based on our long-termcooperation and hard work.沾筆式微影術電漿共振奈米結構單分子交互作用蛋白質陣列軸向極化光雙光子顯微鏡dip-pen lithographyplasmon resonancenanostructuresingle molecular interactionprotein arrayradial polarizationtwo photon microscopy