單一DNA分子拘限在奈米狹縫的靜力學與動力學研究

Abstract

本論文聚焦在探索被奈米流道拘限的單一生物分子物理，目標在於發展新穎奈米尺度系統的生物檢測分析技術。標準的微製成技術可以製造出數十奈米級的狹縫型奈米流道，這個尺度已小於雙股螺旋DNA的本徵長度（~ 50奈米）。奈米流體元件具有在奈米尺度下精準操控生物分子的動態構型與傳輸。本研究結果顯示構型因空間限制的熵變化與DNA和奈米流道邊界的交互作用主導著受拘限雙股螺旋DNA分子的靜力與動力學特性。本研究使用全視野螢光顯微鏡觀測DNA分子的構型、形狀、鍊擴散能力與鍊長鬆弛行為，結果並與尺度理論預測作比較。 我們系統性的研究DNA分子拘限在尺度趨近Kuhn長度(~ 100奈米)的狹縫型流道的鍊長相依特性。在本實驗中使用長度界於4微米至75微米的螢光標定單一DNA分子。從測量二維的鍊迴旋半徑分布發現DNA分子展現高度的非等向性的形狀(甚至是環形的DNA也具備此特性)且此特性與鍊長無關。在我們的量測中顯示DNA 的形狀特性藉於二維與三維系統之間。鍊伸長量與迴旋半徑的靜力學尺度律為 <r

,Rg> ~ L^ν，我們觀察到 νr

= 0.65 ± 0.02 與 νRg = 0.68 ± 0.05。這些值靠近三維與二維理論預測的平均值。鍊伸長量與轉動量的鬆弛時間尺度分析結果界於Rouse模型 (良溶劑，奈米狹縫) 與Zimm 模型 (良溶劑，本體溶液) 之間。我們證明被拘束在高度靠近Kuhn長度的奈米狹縫下的DNA分子構型與鍊鬆弛展現著準二維的行為。 本論文系統性的研究 λ-DNA 拘限在奈米狹縫下，範圍從中拘束(h = 780奈米 ~ λ-DNA 在本體溶液中的迴旋半徑) 至強拘束範圍 (h = 20奈米 << 雙股螺旋DNA的本徵長度) 的拓墣相依性。線型與鬆弛-環形DNA的鍊長量測在狹縫高度為140奈米 (~lk) 時產生一個戲劇性的變化，DNA 的構型變化從”顆粒鍊” (de Gennes : Kuhn長度 < h < DNA 在本體溶液中的迴旋半徑) 轉變成”反射型鍊” (Odijk :h < Kuhn長度)。當降低奈米狹縫的高度可以觀察到線型與鬆弛環形 λ-DNA 的形狀特性從三維行為改變至二維行為。而環形DNA的擴散能力大於線型DNA的擴散能力，這意味著環形DNA的流體動力半徑小於線型DNA。伸長量變化的鬆弛時間的尺度分析與流道深度的相依性 (τ

~ h^-0.44) 並不符合de Gennes (τ

~ h^-1.17) 的顆粒理論。 本研究建立一種新穎的方法透過被製造於奈米狹縫中的奈米高度-障礙物可以捕獲和拉伸單一雙股螺旋DNA。DNA分子通常物理性的吸附並延展圍繞在障礙物上，這些圓柱型障礙物或是奈米狹縫的邊牆舉有與DNA相同電性的電荷。這個象相可以與預期的靜電力與空乏作用所造成的現象做比較。DNA 的捕獲現象僅發生在奈米狹縫高度小於雙股DNA的Kuhn長度。被吸附的DNA鍊伸長靜尺度律與鍊長的相依性為 <rnear> ~ N^0.81，這個結果靠近一維方形通道拘束的鍊長相依性 理論值。在準二維奈米狹縫中的牆-捕獲DNA展現著準一維的構型與動力學特性。被奈米狹縫中的柱狀體捕獲的DNA可以透過直流電場壓縮或解壓縮DNA的構型，DNA在圓柱陣列間的傳輸特性展現著捕獲-逃脫的機制。

This thesis focuses on the exploration of individual confined polymer physics that arise in nanofluidic channel with the goal to developing novel diagnostic bioanalysis technology in such nanoscale systems. Standard microfabrication technologies can fabricate slit-like nanofluidic channels with dimension down to few tens nanometers. This is below the persistence length of double-strand DNA (ds-DNA) p~50 nm. The technique provides to precisely manipulate the dynamics, conformation and transport of biomolecules in nanoconfinement. As a result of restricted conformation entropy and DNA-wall interaction that can govern the static and dynamics properties of confined single ds-DNA. The conformation, shape, chain diffusivity and chain relaxation are characterized using fluorescence wide-field microscopy and compared with scaling theoretical predictions. The chain length dependence of DNA properties confined in nanoslit with dimension close to the Kuhn length lk (~ 100 nm) is systematically investigated. Fluorescently labeled single DNA molecules with contour lengths L ranges from 4 to 75 microns are used in the experiments. The distributions of the chain radius of gyration and the two-dimensional asphericity are measured. It is found that the DNA molecules exhibit highly anisotropic shape even for circular-form and the mean asphericity is chain length independence. The shape anisotropy of DNA in our measurements is between three dimensional (3d) and two dimensional (2d). The static scaling law of the chain extension and the radius of gyration <r

,Rg> ~ L^ν are observed with νr

= 0.65 ± 0.02 and νRg = 0.68 ± 0.05. These results are close to the average value between two (νr, Rg = 0.75) and three (νr, Rg = 0.6) dimensional self avoiding walk theoretical value. The scaling of the extensional and rotational relaxation time are between Rouse, good solvent and Zimm, good solvent dynamics in nanoslits and the bulk solution, respectively. We show that the conformation and chain relaxation of DNA confined in a slit close to the Kuhn length lk exhibit the quasi-two dimensional behaviors. The topological dependence ofλ-DNA (48.5kbp) confined in nanoslit ranging from moderate confinement (channel height h = 780 nm ~ radius of gyration of λ-DNA in bulk solution) to strong confinement (h = 20 nm << p) are systematically investigated. There is a drastic change of measured extension of linear rl and relaxed-circular rc of λ-DNA at h = 140 nm ~ lk. The conformation change from“blob-chain” (de Gennes regime : lk < h < Rg,bulk) to “reflecting chain” (Odijk regime : h < lk). The shape properties of both linear and relaxed circular λ-DNA, which the behaviors change from 3d to 2d with decreasing channel height, are observed. The diffusivity of relaxed circular DNA is larger than linear DNA in nanoslit, which implies the hydrodynamic radius of circular DNA is smaller than linear DNA. Scaling of extensional relaxation time with channel height (τ

~ h^-0.44) does not agree with de Gennes blob theory (τ

~ h^-1.17), which is strictly applicable when slit height is larger than the Kuhn length. A novel method to trap and stretch individual ds-DNA molecules using nano-height obstacles in the nanoslit channel is demonstrated. The DNA molecules are unusually physically adsorbed and extend around obstacles such as cylindrical posts or sidewalls of the same charge, in contrast to the expected behavior based on electrostatic and depletion interactions. This trapping occurs only when h is less than lk of ds-DNA. The static scaling law of the chain extension for adsorbed DNA of length N follows a power law <rnear> ~ N^0.81, close to the N dependence in one dimensional square channel confinement. The wall-trapped DNA appears to have one dimensional conformation and dynamics in the nanoslit that is quasi-two dimensional. The conformation of DNA trapped around the posts can be compressed and decompressed using DC electric field and the transport of DNA between post arrays exhibits the trapping-escaping behavior.