在脂雙層DNA拉伸平台上架設奈米間隙探測器量測DNA之電訊號

Abstract

現代醫療技術中，如何準確並快速檢測病原體為治療中重要的一環，而現行的技術耗時長導致治療時程拉長。本研究團隊先前已開發出一脂雙層DNA拉伸平台，能有效且快速地辨識λ-DNA之基因圖譜。此基因圖譜平台除了具有低成本、簡易操作、圖譜準確性高、耗時短等優點，也因使用bisPNA標定法而具有較高的序列選擇彈性。 考量到利用觀察顯微鏡下DNA標記點之位置此光學系統其解析度受限於光波長，且受限於在顯微鏡下操作，難以將其微型化。因此，在本研究中我們引入奈米間隙探測器與先前之脂雙層DNA拉伸平台結合，計畫以收集電訊號之方式建立基因圖譜為本研究之目標。 我們首先透過COMSOL Multiphysics軟體模擬，評估利用奈米間隙探測器偵側DNA電訊號之可行性。而後我們先在玻片上以舉離(lift-off)製程鍍出金電極，再利用原子力顯微鏡(AFM)之探針切割出約50 nm寬之奈米間隙，驗證以探針式製作技術製作奈米間隙之可行性。而在裝置製作上，我們發現若是通道高度小於1.5 μm，會導致通道內出現脂球堆積的現象，因此我們透過聚二甲基矽氧烷(PDMS)封頂之設計提高通道高度解決此問題。之後我們在此圖案玻片上舉離出與通道垂直之電極，卻因為曝光時真空度以及光罩圖案之特殊性導致最後電極之寬度增加。此外，我們也觀察到由於乾蝕刻之DNA通道側壁過於垂直，導致舉離之電極有不連續連接的情況，而且AFM探針難以準確切割到側壁轉角處。最後，我們改利用濕蝕刻製作出DNA通道，並在其上舉離出電極後，成功利用AFM探針於側壁轉角處切割出約50 nm的奈米間隙。我們於裝置中架設脂雙層後，以電泳驅動由bisPNA標定之λ-DNA通過奈米間隙，觀察到明顯的脈衝電流訊號。雖然最後無法直接證實為DNA通過時產生之訊號，但此裝置也為本脂雙層拉伸平台轉變為收集電訊號以建立基因圖譜之可能性奠定下基礎。

In modern medical technology, precise and rapid detection of pathogens plays an important role in effective treatment. However, current techniques are often time-consuming, which delay the treatment process. In our previous studies, we have developed a lipid bilayer based DNA extension platform, which capable of efficiently and quickly identifying the genomic profile of DNA. This platform offers advantages including low cost, ease of operation, high mapping accuracy, and short processing time. Moreover, due to the use of the bisPNA labeling method, it provides greater flexibility in sequence selection. Considered that the resolution of using optical system to observe labeled DNA is limited by the wavelength of light and the difficulty of miniaturizing due to the reliance on optical microscopy. Therefore, in this study, we introduce a nanogap detector to our previously established lipid bilayer based DNA extension platform. The goal of this research is to construct genome maps via electrical signal detection. We first used COMSOL Multiphysics software to simulate and confirm the feasibility of detecting DNA electrical signals using a nanogap detector. Subsequently, gold electrodes were patterned on a glass slide using a lift-off process, and 50 nm wide nanogaps were created by cutting with an atomic force microscope (AFM) tip, demonstrating the feasibility of fabricating nanogaps using tip-based nanofabrication. During device fabrication, we found that when the channel height was less than 1.5 μm, vesicles accumulation occurred inside the channel. This issue was resolved by increasing the channel height through a polydimethylsiloxane (PDMS) capping design. We then attempted to fabricate electrodes perpendicular to the DNA channel on the patterned glass using lift-off process. However, due to vacuum conditions during exposure and the specific design of the photomask, the final electrode width increased. Additionally, the vertical sidewall of the dry etched DNA channel led to discontinuities in the lift-off electrode, and the sharp corners made it difficult for AFM tip to precisely cut at the sidewall intersection. As a result, we decided to switch to wet etching to fabricate the DNA channel. After patterning the electrode, we successfully created 50 nm wide nanogap at the corner of the sidewall cutting with AFM tip. After forming lipid bilayers in DNA channel, we used electrophoresis to drive bisPNA-labeled λ-DNA through the nanogap and observed distinct pulsed electrical signals. Although we could not confirm that the signals were generated by the translocation of DNA, this study lays the groundwork for transforming the lipid bilayer DNA extension platform into one capable of generating genome maps via electrical signal detection.