低磁珠顆數之數位微流體免疫檢測晶片的研發

Abstract

相較於傳統96孔盤免疫檢測法，因具備較高的表面積/體積比，以數位微流體晶片進行微粒式免疫檢測可提升靈敏度。多數探討微粒式數位微流體免疫晶片之文獻僅針對免疫反應之前處理部分進行探討與改良，偵測方式並未多加著墨；另外，參與免疫檢測之磁珠顆數多為十萬顆以上。本研究探討以數位微流體晶片進行低磁珠顆數之免疫檢測之晶片設計與流程改良。根據文獻指出，愈低磁珠顆數參與免疫反應每顆微粒之訊號強度愈強，可進一步降低偵測極限，但最低僅測試至250顆微粒之反應條件，本研究首次探討低於100顆磁珠之現象。 在介電濕潤式微流體晶片上，本研究針對低磁珠顆數條件下液珠之操控手法進行設計，提出雙向介電濕潤式磁珠清洗流程，並根據其磁珠流失與清洗效率之表現進行改良。本實驗室於先前研究中首先提出以聚集磁珠之方式提升訊號強度之方法，將整合於本研究之反應流程中。為方便比較本研究之檢測結果與先前研究於晶片外檢測之結果，沿用人類腫瘤壞死因子受器1 (sTNF-R1)作為免疫檢測之偵測目標。 文獻中使用之單向介電濕潤式磁珠清洗流程僅能處理高顆數之磁珠，經測試發現，以本研究使用6微米直徑之磁珠而言，至少需要2,550顆才能進行單向介電濕潤式磁珠清洗，少顆數磁珠之條件需以雙向介電濕潤式清洗方法完成。改良後的雙向介電濕潤式磁珠清洗流程應用於少磁珠顆數之反應條件，可僅以1次清洗達到98%的清洗效率，且無磁珠流失。本研究將免疫反應全程在數位微流體晶片上完成，並證明少磁珠訊號增強之效應在磁珠少於100顆時仍存在。相較於本實驗室前期之螢光聚集檢測法，縮短反應時間至小於1小時，待測物試劑消耗降低至200nL以及偵測極限由原先15 pg/mL降低至3.14 pg/mL。

Bead-based immunoassay on digital micro-fluidic (DMF) device leads to higher sensitivity than conventional immunoassay in 96 well plate due to higher ratio of surface/volume. Most literatures of bead-based DMF immunoassay chip were focused on the pre-process of immunoassay, improvement of post-assay detection was rarely investigated. In addition, bead number applied in each immunoassay were mostly higher than 106 beads. Applying low bead number in DMF immunoassay chip is investigated in this research. Lower bead number leads to higher signal intensity on each bead, which lowers detection limit of immunoassay according to literature. But the least bead number reported was 250, the tendency is first evaluated in conditions of bead number under 100 in this research. Dealing with bead washing on-chip with low bead number, techniques of droplet operation with electro-wetting on dielectric (EWOD) are investigated. “Double-sided EWOD bead washing” is developed in this research for low bead number cases by reducing bead loss and improving washing efficiency. Signal enhancement by bead aggregating, which was first proposed by our group, is integrated in the process. In order to compare on-chip results to off-chip ones in prior research, same detection target should be applied. Therefore human soluble tumor necrosis factor receptor-1 (sTNF-R1) is adopted as detection target. Prior researches applied single-sided EWOD bead washing, which could be achieved only with high bead number of magnetic beads. Being tested in this research, if apply 6 μm diameter beads, 2,550 is the least number to achieve single-sided EWOD. Washing with lower bead number could only be achieved by double-sided EWOD. Washing efficiency of the improved double-sided EWOD washing could exceed 98% in only 1 cycle of wash and cause no bead loss. The whole assay process is accomplished on DMF chip, and proved the bead number effect still works as bead number lower than 100. Compared to prior results of our lab, the assay time is reduced into 1 hour, sample consumption is reduced into 200 nL and detection limit is reduced from 15 pg/mL to 3.14 pg/mL.