整合計時器功能之紙張感測系統

Abstract

本研究開發出整合計時功能之三維紙張檢測平臺(3D-tPADs)，藉由使用便宜的可溶性物質蔗糖(Sucrose)與普魯蘭多糖 (Pullulan) 乾燥在預定設計好的紙張流道上，在接觸反應試劑後改變其於紙張通道流動過程之黏滯性，並加上不同流動距離之因素，使反應試劑於紙張上產生不同流動延遲現象，因此可於不同時間接觸於特定位置預定乾燥置放之染劑，再滲透至上層白色區域而精確地視覺化顯色計時。此外，本研究親水纖維流道設計採三維直流式免疫分析設計，相較於測流式系統，能達到有效降低無效體積能有效的目的，並結合3D列印封裝技術，讓多層試紙能緊密貼合，降低檢測試劑於不同反應纖維素層間之流動所產生之時間以及後續之檢測結果誤差。本平臺之檢測區域並使用羧甲基纖維素與1-(3-二甲氨基丙基)-3-乙基碳二亞胺鹽酸鹽/ N-羥基丁二醯胺進行表面改質，使得高密度捕捉抗體能共價貼附於紙張表面，提升檢測之反應結果穩定性、專一性、以及檢測精度。最後我們將此平臺應用於人類免疫缺乏病毒(Human immunodeficiency virus ; HIV) 衣膜蛋白p24之檢測分析，結果證明可檢測添加p24抗原之血漿偵測極限0.01 ng/mL之結果。吾人預期此具備高檢測靈敏，並結合計時功能之攜帶式試紙分析元件，達到世界衛生組織(World Health Organization ; WHO)所制定的運用於資源匱乏區域之point of care；POC (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users ; ASSURED)檢測標準。

In this study, we have develop a three-dimensional microfluidic paper-based analytical device integrated with timer function (3D-tPADs). When the test reagent contacts with sucrose and pullulan pre-dried onto the designed paper microchannels, the changes of viscosity of reagent flowing in the channels and different flow distances cause fluidic time delay phenomenon occurs in the paper network. In this way, the pre-dried dye at a designated position in the channel will be rehydrated by the passing reagents flow at different times, and thus flow up to the top layer and precisely shows visualized color. Furthermore, we use three-dimensional vertical flow immunoassay design in the hydrophilic cellulose micro-channels, which can greatly reduce dead volume of reagents compared with lateral flow systems. The combination with 3D printed packaging can help multi-layer paper to contact more closely and reduce time differences and detection error caused by the reagent flow among different layers. In addition, the detection cellulose spots are surface modified with sodium carboxymethyl cellulose (CMC) and N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride/ N-Hydroxysuccinimide (EDC/NHS) to generate covalent bonds between high-density capture antibodies and paper surfaces, and therefore improve the stability, specificity, and precision of detection. In order to verify its practice of the paper platform, it is applied to the detection and analysis of Human immunodeficiency virus (HIV) capsid protein p24. The results show that the limit of detection of 0.01 ng/mL of p24-spiked plasma is obtained. We expect this high sensitive, timer-combined, portable paper assay device meets the World Health Organization (WHO) affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users (ASSURED) criteria in resource-limited regions.