量測奈米粒子之布朗運動用於檢測齒舌蘭輪斑病毒

Abstract

近年來，為了提升蘭花品質與他國競爭，蘭花病毒的檢測成為我國急需發展的新技術之一。本研究使用一種新的方法來檢測蘭花病毒，以台灣常見的齒舌蘭輪斑病毒作為主要研究對象，首先，將對齒舌蘭輪斑病毒具有專一性的多株抗體分別接在300 nm和500 nm的微粒子上，接著將其分別混入具有不同濃度的鞘蛋白與齒舌蘭輪斑病毒，並使用微粒子追蹤測速儀量測其布朗運動的情況，藉由觀察布朗運動的變化，檢測齒舌蘭輪斑病毒是否存在。為了驗證抗體球製備方法的正確性，使用穿透式電子顯微鏡做輔助判斷，以便檢驗粒徑增加與抗體球捕捉抗原的情況。根據實驗結果得知，製備抗體球的方法正確，且此新方法所能檢測到的最低病毒濃度為0.1 μg/mL，此時的粒子數與齒舌蘭輪斑病毒數比為1：1.5，最高病毒濃度為32 μg/mL，此時的粒子數與齒舌蘭輪斑病毒數比為1：480，反應時間約為8分鐘，若是以組成齒舌蘭輪斑病毒的最小分子：鞘蛋白作為檢驗對象，則反應時間縮短至3分鐘。此種感測方式的優點為使用上簡單，檢驗過程快速，易與微流系統於晶片上進行整合；除此之外，還能夠解決傳統酵素連結免疫吸附法低靈敏度的困擾，並改善反轉錄聚合酶連鎖反應需要專業人員判別的問題。

The thesis reports on an approach for detecting Odontoglossum ringspot virus (ORSV) by using micro-particle-tracking-velocimetry (micro-PTV). The ORSV is one of the major viruses in orchids, for example, Phalaenopsis, which is the most important economic plant and export good in Taiwan. To sensing the ORSV, the anti-ORSV proteins are modified on the 300 nm and 500 nm fluorescent beads and then separately mixed with the coat proteins of the ORSV and ORSV samples. The nanobeads suspended in samples were traced and statistically analyzed their Brownian velocity in real-time by micro-PTV. Through observing the variation of Brownian velocity of the nanobeads, the number of anti-ORSV and ORSV conjugation can be quantitatively estimated. Moreover, the image of the ORSV and coat proteins conjugating with nanobeads were captured through transmission electron microscope (TEM) to demonstrate the approach. In the experiment, the detection limit is exhibited to the ratio nanobeads to coat proteins of 1 to 1.5 in 8 minute. Based on our current Brownian motion measurement technique using micro-PTV, the detection limit of the concentration of 0.1 μg/ml can be found. The novel method provides higher sensitivity than enzyme-linked immunosorbent assay (ELISA) and easier operating than reverse transcription polymerase chain reaction (RT-PCR) technique, which are currently major orchid virus detection methods. Further, because of the potential that less device fabrication request, it shows higher capability on integrating with any microfluidic device for making the portable device with disposable chips.