奈米金粒增強侷限型表面電漿共振感測器結合微流道晶片用於病原體核酸檢測

Abstract

侷限型表面電漿共振感測技術因其無需標記、即時性和增強光學信號等特性，在生物感測領域引起了廣泛關注。然而，局限型表面電漿共振感測器的靈敏度不夠高，無法量測到低濃度的生物分子。在本研究中，我們提出了加入奈米金粒 （AuNPs）作為信號增強源來提高表面電漿共振感測器靈敏度的方法。為了實現這一目標，我們利用週期為125 nm、直徑為80 nm的陽極氧化鋁（AAO）模板製備奈米金圓盤陣列於玻璃基板上。在沉積金層之前，我們在玻璃基板上修飾上了一層硫醇基丙基三甲氧基矽烷（MPTMS）薄膜，以提高金與玻璃的黏附性，接著沉積20 nm厚的金，最後剝離陽極氧化鋁，我們便會得到一片具有奈米金盤陣列的陽極氧化鋁感測器。將微流道黏附在感測器上，並連接到LabSmith微流道控制系統，我們便能實現自動化的檢測。在本研究中，我們將陽極氧化鋁感測器應用於感測炭疽真菌（Colletotrichum gloeosporioides，C. gloeosporioides）的基因序列，在沒有奈米金粒增強訊號的情況下，檢測極限為2.5 nM。為了進一步提高檢測極限，我們加入了經ssDNA修飾的奈米金粒（直徑為100 nm），並採用了與目標DNA競爭奈米金盤陣列上探針的檢測方法。值得注意的是，若目標DNA濃度落在0.025 nM至2.5 nM範圍內，目標DNA造成的紅移量在沒有奈米金粒增強的情況下無法區分出差異，在加入了奈米金粒之後，紅移量的差異可被提高並呈現和濃度具有相依關係，使得檢測極限可以到達0.025 nM。本研究提出此競爭型檢測方法結合了微流道系統，實現了可重複且準確的目標檢測。這對於生物分析技術在診斷和生物醫學研究等領域的發展具有重要意義。

The localized surface plasmon resonance (LSPR) sensing technique has gained attention in biosensing due to its label-free, real-time, and optical characteristics. However, LSPR suffers from limited sensitivity. In this thesis, we propose enhancing the sensitivity of the LSPR sensor by incorporating gold nanoparticles (AuNPs) to leverage the plasmonic coupling effect. We used anodic aluminum oxide (AAO) with 125 nm periodicity and 80 nm diameter as the template. After coating the glass substrate with (3-Mercaptopropyl)trimethoxysilane (MPTMS), we deposited a 20 nm gold layer and performed AAO lift-off, resulting in an AAO-LSPR sensor. Furthermore, we integrated the sensor with a microfluidic channel and a LabSmith microfluidics controller to enable automated target detection. In this research, we functionalized the nucleic acid sensor with single-stranded DNA (ssDNA) probes specific to Colletotrichum gloeosporioides (C. gloeosporioides) genetic DNA detection. To improve the limit of detection, we introduced 100 nm diameter ssDNA-modified AuNPs and employed a competitive assay approach with the target DNA. The results showed a clear linear relationship between target concentrations ranging from 0.025 nM to 2.5 nM. Notably, differentiating concentrations within this range without the AuNP enhancement proved exceedingly challenging. The proposed competitive detection method, combining microfluidic systems, enables repeatable and accurate target detection, which holds significant importance for the development of bioanalytical techniques in diagnostics and biomedical research.