Development of an adjustable-gap mechanism for rapid and ultrasensitive detection on bead-based digital microfluidic fluorescence immunochip
|關鍵字:||微粒式免疫檢測;數位微流體免疫晶片;間距調整;偵測極限;人類腫瘤壞死因子受器1;Bead-based immunoassay;Digital micro-fluidic chip;Gap height adjusting;LOD;sTNF-R1|
經實驗發現磁珠(直徑6 μm)在數位微流體晶片上下電極板間堆疊會降低訊號強度及穩定性，其中，間距10 μm測得強度是25 μm的1.27倍，且其CV值僅3.4%，表示其偵測穩定性高。另外藉實驗選出適合三種電極進行分割及傳輸的間距100 μm，將間距100 μm及10 μm分別應用於免疫反應與螢光偵測兩階段，實驗結果顯示訊號強度提升1.67倍且CV值為4.2%，而免疫反應時間為55分鐘內，相較於先前研究的1小時內，提高間距僅可縮短數分鐘的操控時間，效果並不明顯。為方便比較偵測靈敏度提升的效果，免疫反應階段間距將使用與先前研究相同的25 μm，而螢光偵測一樣使用10 μm。實驗結果顯示透過從降低間距從25μm至10 μm可將各待測抗原濃度訊號提升1.31-1.39倍且更穩定，而偵測極限亦降至0.32 pg/mL。|
Bead-based immunoassay on digital micro-fluidic (DMF) device has higher sensitivity than conventional immunoassay in 96 well plate due to higher ratio of surface area/volume. In previous studies of our group, we first proposed a signal enhancement method by bead aggregation for signal detection of immunoassay, but also found that the intensity and stability of measured signal would be affected in different gap heights,which are the distance between top plate and bottom plate of DMF device. In addition, it is reported that the efficiency of transporting before and after droplet cutting and merging can be improved by increasing the gap height. But, the gap height to the on-chip immunoassay effect was rarely investigated. This research is the first study to explore the gap height effect on immunoreaction and signal detection in immunoassay and propose an adjustable-gap immunochip. For purpose of improving immunoreaction manipulation efficiency and signal intensity and stability in DMF immunoassay, three kinds of electrode area at various gap heights droplet transporting and cutting phenomenon on electro-wetting on dielectric (EWOD), the gap height effect to the signal detection are investigated in this study. Select a gap height for immunoreaction and a gap height for signal detection respectively by the above investigations. Then, apply these two gap heights in immunoassay. In order to compare gap-adjusted results to gap-unadjusted results in previous research, same detection target should be applied. Therefore human soluble tumor necrosis factor receptor-1 (sTNF-R1) is adopted as detection target. The experimental results show that the spatial overlap of beads (diameter of 6 μm) between two plates will decrease signal intensity and stability. The signal intensity of gap height 10 μm is 1.27 times larger than that of gap height 25 μm, and the CV Value of 10 μm is 3.4%, indicating a high detection stability. Gap height 100 μm is much appropriate for proceeding immunoreaction by investigating the droplet transporting and cutting phenomenon in various gap heights. Apply 100 μm and 10 μm gap heights in immunoreaction and fluorescence detection separately, it shows that the signal intensity can be enhanced 1.67 times and the CV Value is 4.2%. The immunoreaction time is within 55 minutes. However, compared to the previous studies of one hour, the effect of decreasing the assay time by increasing the gap height is not quite obvious. Besides, we also investigated the detection limit. For fairly comparing effect of detection sensitivity improvement, immunoreaction gap height changed to 25 μm, same as previous research. And the fluorescence detection gap height is 10 μm. The results show that the signal intensity of each detection target’s concentration can be enhanced 1.31 to 1.39 times by decreasing the gap height from 25 μm to 10 μm, and the Limit of detection value is 0.32pg/mL.