以布朗動態法研究DNA受正向力影響產生電泳速差之分離策略

Abstract

在生物醫學與分子分析領域中，依據DNA片段長度進行高效率分離是基因定序、疾病檢測與微生物分型等技術的基礎。傳統的脈衝場凝膠電泳（PFGE）雖可處理大型DNA片段，但操作耗時，且裝置系統不易擴展。相較之下，微流道DNA分離技術因具快速、便利與低成本等優勢，近年來被視為具高度潛力的替代方案。目前主流微流道設計多以障礙物（如圓柱結構）誘發DNA碰撞來實現尺寸篩選，但在提升電場強度以加快分離速度時，DNA往往無足夠時間恢復至鬆弛態，導致碰撞效果降低並產生穿隧效應。為克服此限制，本研究提出以DNA流變性質為基礎的正向應力(normal stress)分離策略。 當 DNA 處於拉伸且彎曲構型時，內部產生的彈性回復力即為正向應力，會使分子朝向曲率中心位移。由於長鏈 DNA 受正向應力影響更為顯著，偏移量亦較短鏈更大，達成長短DNA分離。然而，僅以偏移量作為分離依據，效果易受擴散作用削弱。因此，本研究轉而以正向應力影響 DNA 電泳速度作為分離策略，也就是利用DNA 受正向力作用後，偏移至電場強度不同之區域，進而產生電泳速度差異，最終實現分離效果。根據此策略，本研究設計具 Y 型結構之微流道，驅動正向應力使DNA產生偏移，並透過流道結構影響電場，放大電場強度差異，進而增強 DNA 電泳速差。本研究之微流道長度為1mm，內含20排Y 型結構，DNA每次通過Y 型結構所造成的速度變化將累積於時間維度，最終形成顯著通過時間差異。考量 Y 型結構排列對電場分佈之影響，亦比較交錯型與平行型兩種排列對分離行為之影響。 本研究以布朗動態法（Brownian Dynamics）模擬 DNA 於微流道中運動，驗證此分離策略之可行性。模擬結果顯示，透過黛博拉數（Deborah number, De）評估 DNA 在不同操作條件下所受正向應力之相對強度，能有效預測分離效果隨電場變化之趨勢。此外，影響 DNA 電泳速度的主要因素包括：(1)電場強度分佈、(2)電力線走向，以及 (3) DNA 在 y 方向的初始位置。其中，電場強度分佈決定DNA偏移量與速度之關係，並證實速度差異主要來自正向應力所致。電力線走向則影響 DNA 受正向力作用之規律性，交錯型裝置中，電力線走向可能導引 DNA快速穿越 Y 型結構，減少受正向力影響的機會。而平行型裝置雖無電力線走向問題，但 DNA 於 y 方向初始分佈差異，仍可能引發穿隧效應，削弱分離效果。 為解決穿隧效應問題，本研究在原有平行型裝置之入口處引入檔板結構，有效阻斷穿隧路徑，並顯著提升分離表現。優化後結果指出，僅長度為1 mm的檔板型裝置，即可達到與15 mm圓柱障礙陣列相近之分離效能。未來若能整合DNA於y方向聚焦的技術，將可進一步發揮本設計之潛力。本研究針對正向應力分離機制提出具體之微流道結構與電場設計方向，為實驗裝置開發提供理論依據與設計構想。

In the fields of biomedicine and molecular analysis, efficient separation of DNA fragments based on their length forms the foundation for technologies such as gene sequencing, disease diagnosis, and microbial typing. Although conventional pulsed-field gel electrophoresis (PFGE) is capable of handling large DNA fragments, it is time-consuming and difficult to scale. In contrast, microfluidic DNA separation technologies have emerged as promising alternatives in recent years, offering advantages such as speed, convenience, and low cost. Most existing microchannel designs achieve size-based separation by inducing DNA collisions through obstacles (e.g., cylindrical posts). However, under high electric field strength aimed at accelerating separation, DNA often lacks sufficient time to relax to its equilibrium state, resulting in reduced collision efficiency and tunneling effects. To overcome this limitation, this study proposes a separation strategy based on the rheological properties of DNA, specifically utilizing normal stress. When DNA is in a stretched and curved conformation, the internal elastic restoring force generates normal stress, driving the molecule toward the center of curvature. Longer DNA chains experience more significant normal stress effects, resulting in greater lateral displacement compared to shorter chains, thus enabling size-based separation. However, relying solely on displacement for separation is susceptible to diffusion effects. Therefore, this study adopts a strategy based on differences in electrophoretic velocity induced by normal stress. Under the influence of normal stress, DNA is displaced into regions of varying electric field strength, leading to differences in electrophoretic velocity and ultimately achieving separation. Based on this principle, a microchannel with Y-shaped structures is designed to induce lateral migration through normal stress and to amplify electric field strength differences through channel geometry, thereby enhancing velocity differences between DNA molecules. The microchannel used in this study is 1 mm in length and contains 20 rows of Y-shaped structures. The velocity changes induced each time DNA passes through a Y-shaped structure accumulate over time, ultimately leading to significant differences in passage time. Considering the influence of Y-structure arrangement on electric field distribution, this study further compares the separation behavior between staggered and parallel configurations. Brownian Dynamics simulations are employed to verify the feasibility of this separation strategy by analyzing DNA migration within the microchannel. The simulation results demonstrate that evaluating the relative magnitude of normal stress acting on DNA under different operating conditions through the Deborah number (De) effectively predicts the trend of separation performance with varying electric fields. Moreover, the primary factors influencing DNA electrophoretic velocity include: (1) electric field strength distribution, (2) direction of electric field lines, and (3) the initial y-direction position of DNA. Among these, the electric field strength distribution determines the relationship between lateral displacement and velocity and confirms that velocity differences primarily arise from normal stress. The direction of electric field lines affects the regularity of DNA experiencing normal stress; in staggered configurations, the electric field lines may guide DNA to pass rapidly through Y-shaped structures, reducing opportunities for normal stress effects. In contrast, parallel configurations do not exhibit issues related to field line direction; however, initial distribution differences in the y-direction may still induce tunneling effects, weakening separation performance. To address the tunneling issue, this study introduces a baffle structure at the entrance of the parallel configuration to effectively block tunneling pathways, thereby significantly enhancing separation performance. The optimized results indicate that a baffle structure with a length of only 1 mm can achieve separation efficiency comparable to that of a 15 mm cylindrical post array. Further integration with y-direction focusing techniques could potentially maximize the performance of this design. This study provides concrete microchannel structures and electric field design strategies based on the normal stress separation mechanism, offering theoretical guidance and design concepts for future experimental device development.