以磁珠聚集法提升數位微流體式免疫晶片之螢光偵測能力

Abstract

先前多數文獻提及免疫檢測晶片設計微粒操控部分，僅進行前處理的混合、清洗等步驟，並無後端以聚集操控微粒方法提升訊號靈敏度相關論述而本研究開發之數位微流體蛋白質免疫檢測晶片，主要以介電濕潤原理操控微液珠並以磁力聚集液珠內抗原磁珠載體，以磁力聚集方法降低偵測極限並提高靈敏度為本研究最大特點。 在此待測物為螢光標記人類腫瘤壞死因子受器1 (TNFR1)，為降低背景雜訊，在倒立螢光顯微鏡平台進行CCD黑平衡調校，以確保背景訊號為0，並找到最適合的螢光曝光時間範圍是60~80 ms，實驗結果顯示，最適曝光時間內較高螢光曝光時間，靈敏度變高而偵測極限降低。將抗原磁珠以磁力聚集後比起聚集前有更好的偵測靈敏度，為進一步驗證在聚集偵測抗原磁珠在提升偵測靈敏度及降低偵測極限上的效能，同時以流式細胞儀進行，以TNFR1為抗原的螢光磁珠免疫檢測對照組，實驗結果流式細胞儀偵測極限為146 pg/ml，消耗偵測體積為50 μl；微流體晶片偵測極限為31.47 pg/ml， 偵測體積為2.5 nl。結果顯示以磁珠聚集法，在改善螢光偵測效能，有提升靈敏度，降低偵測極限及樣品消耗量小的三大優點。

Most of the references proposed that manipulating micro-beads to get involved in mixing and washing of immunoassay pre-process, but few about detecting sensitivity enhancement. In the research, a novel digital micro-fluidic chip (DMF chip) for immunoassay had been developed, which can create sample droplet in nano-scale by electro-wetting on dielectric (EWOD) force and aggregating bio-conjugated beads by magnetic force. The features of DMF chip are high sensitivity and low limit of detection (LOD). Take fluorescent labeled tumor-necrosis factor receptor-1 (TNFR1) as a detecting target antigen. Firstly, calibrate CCD black balance to the proper range of fluorescent exposure time from 60 to 80 ms to reduce the interference of unwanted background noise. Then the result indicated that the longer fluorescent exposure time in the proper range the more fluorescent sensitivity and the lower LOD. In addition, the fluorescent detecting sensitivity can also be enhanced by aggregating bio-conjugated magnetic beads. Finally, take flow cytometry as a TNFR1 detecting control group to prove the functions of high fluorescent sensitivity and low LOD on DMF chip. The results of flow cytometry shows that LOD is 146 pg/ml and detecting sample consumption is 50 μl. In contrast with the flow cytometry, LOD and detecting sample consumption of DMF chip platform are 31.47 pg/ml and 2.5 nl. In summary, the advantages of DMF chip in the research are high fluorescent detecting sensitivity, low limit of detection and sample consumption.