以介電泳技術改良微粒免疫分析晶片

Abstract

本論文的研究重點在於如何結合並應用介電濕潤(EWOD)、介電泳(DEP)及微粒免疫分析法(Bead-based ELISA)於微流體晶片中，並提出三相螺旋電極圖樣的新設計，可將螢光微粒聚集成團以增強螢光訊號，達到降低晶片偵測極限的主要目標。三相螺旋電極圖樣設計為三條並行旋轉向內的方型螺旋，當以由外到內的順序分別在此三條電極上施加交流電145伏特，頻率為100千赫茲的電訊號時，直徑6微米的螢光微粒會受到負介電泳力(nDEP)的影響，趨於弱電場梯度區域；重複此順序數次後，螢光微粒會逐步被移至中央，最後會聚集於三相螺旋電極的中心。螢光微粒聚集完畢後，經由倒立式螢光顯微鏡觀察結果及影像軟體分析螢光訊號後，可從實驗數據中發現，聚集後的螢光訊號比起聚集前，確實有顯著性的提升。 在微粒免疫分析法中，選定IL-18作為樣品，先利用生物素接合IL-18小鼠抗體將其固定於卵白素修飾微粒上，再加入IL-18兔抗體以及山羊抗兔螢光抗體，最後可得到一接有IL-18的螢光微粒，此螢光微粒被使用於以上所提到的微流體晶片中。此外利用介電濕潤原理，僅需要一微液滴即可完成聚集螢光微粒之實驗，一方面可減少樣品消耗量，另一方面可固定每次觀察到的螢光微粒數量。在本實驗中，微液滴大小僅有6.25 奈升，在3分鐘內可將螢光微粒聚集完畢。

This research describes a new method to integrate electrowetting on dielectric, dielectrophoresis and bead-based ELISA into a microfluidic chip with innovative 3-phase spiral electrodes. A single droplet containing randomly dispersed polystyrene microbeads conjugated with fluorescent dye is first moved by electrowetting to 3-phase spiral electrodes. The fluorescent microbeads are then concentrated by DEP force at 145 VRMS over 100 kHz. It is shown that the proposed microfluidic chip can evidently enhance the fluorescent intensity even there are only very few fluorescent microbeads (10 to 100) in a droplet whose volume is only 6.25 nL. The total operation time of the concentrating process is less than 3 min and the results are captured by inverted fluorescence microscope and are analyzed by image analysis software. The fluorescence intensity is enhanced evidently after concentrating fluorescent microbeads from the experiment data. IL-18 is determined to be a sample in bead-based ELISA. The process is described as follows; first of all, IL-18 is immobilezed on streptavidin microbead by using biotin anti IL-18 rat IgG and then, IL-18 rabbit IgG and anti rabbit goat IgG conjugated with PE are added sequentially in order to conjugate fluorescent dye with IL-18. Finally, these fluorescent microbeads are used in above microfluidic chip for concentrating. In addition, the usage of EWOD will decrease the sample consumption and control the numbers of concentrated microbeads. In this research, fluorescent microbeads will be concentrated in a 6.25 nanoliter droplet within 3 minutes.