H1N1為A型流感病毒的其中一種亞型，過去數十年來已成為一種具危害性的全球傳染性威脅。由於傳播速度極快，因此迫切需要開發精確且快速的檢測方法。本研究利用磁珠作為中繼點進行與單一適體以及兩種抗體進行類酵素結合免疫吸附分析法之檢測，並提出了一新型數位液滴微流體平台來對H1N1病毒進行檢測。在此平台上含有磁珠的液滴藉由電磁力驅動於無結構式的超疏水表面上。藉由不同層級的疏水性改質，液滴可以輕易地在此無阻力表面上進行控制以及定位而無須任何的物理性結構限制。作為驅動的電磁力可以針對三種液滴及磁珠的操作模式進行調整，包括移動含有磁珠的液滴、進行兩個液滴的混合以及從液滴中進行磁珠的萃取。與先前的數位微流體相比，此平台的磁通量可調性使得系統在液滴與磁性粒子的操控上擁有更高的靈活性。除此之外，磁珠也作為酵素結合免疫吸附分析法的媒介，透過與適體及抗體的連結以特異性來檢測H1N1病毒。而帶有HRP的二級抗體能夠活化酪胺分子使其不穩定，並且與周圍的蛋白質快速共價鍵結進行酪胺訊號放大技術。藉由此技術能夠將帶有螢光的酪胺分子濃縮聚集在目標分子附近提高檢測訊號且避免增加背景值。根據實驗，本研究之檢測濃度極限大約為0.032血球凝集力單位/反應，對於臨床診斷來說是足夠靈敏的，且整體檢測時間小於40分鐘。此應用類酵素結合免疫吸附分析法的數位液滴微流體平台以每顆液滴20 μL的有效反應體積有效降低H1N1病毒樣品和試劑(抗體、酪胺分子和洗滌緩衝液(去離子蒸餾水、磷酸鹽緩衝鹽水)的消耗。這是首次建立一個針對A型流感H1N1病毒檢測的電磁力驅動數位液滴微流體平台。

H1N1, a subtype of influenza A virus, has emerged as a global threat in the past decade. Due to its highly infectious nature, an accurate and rapid detection assay is urgently required. Therefore, this study presents a new type of digital microfluidic platform for H1N1 virus detection by utilizing a one-aptamer/two-antibodies assay and magnetic beads as the substrate. The droplets containing magnetic beads were driven by electromagnetic forces on a structure-free, super-hydrophobic surface to automate the entire assay. With different levels of hydrophobic modification, the droplets could be easily controlled and positioned without any assisted structure. The tunable electromagnetic forces could be adjusted for three kinds of operating modes for the manipulations of beads and droplets in a simple and automatic process. The manipulations include movement of droplets containing magnetic beads, mixing of two droplets and beads extraction out of droplets. When compared with previous studies, the manipulations of droplets and magnetic particles in this study are more flexible as they can be easily adjusted by tuning the magnetic flux density. Furthermore, the magnetic beads also served as a three-dimensional substrate for the new enzyme-linked immunosorbent assays (ELISA)-like assay. The magnetic beads were conjugated with aptamers, which have high specificity towards H1N1 virus such that they could be specifically isolated and detected. The horseradish peroxidase (HRP)-conjugated secondary antibody activates the tyramide-tetramethylrhod amine, a core type of the tyramide signal amplification (TSA) technology, and the activated tyramide becomes extremely unstable and rapidly covalently bonds with target proteins such that tyramide can be concentrated in the vicinity of the target molecule without any increase in background value. Therefore, a large amount of fluorescence signal will be displayed on the target molecule. The limit of detection (LOD) was experimentally found to be 0.032 hemagglutination units (HAU)/reaction within 40 min, which is sensitive enough for clinical diagnostics. This kind of digital microfluidic platform with ELISA-like assay could effectively decrease the consumption of samples and reagents such that the volume of all the droplets including H1N1 sample, antibodies, tyramide-tetramethylrhod amine and wash buffers (i.e. deionized distilled water and phosphate buffered saline) was only 20 μL. This is the first time that a digital microfluidic platform was demonstrated such that the entire diagnostic process for influenza H1N1 virus could be performed by using electromagnetic forces.

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