間距可調整之快速且高靈敏度微粒式數位微流體螢光免疫分析晶片

Abstract

相較傳統孔盤式免疫分析法，數位微流體晶片因具有較高的表面積與體積比，應用於微粒式免疫分析可提升靈敏度。本實驗室於先前研究中針對免疫反應後的訊號偵測方式首先提出透過聚集磁珠提升訊號強度的方法，但同時亦發現數位微流體晶片上下兩板間的間距不同，會影響磁珠聚集後的訊號強度及穩定性；另外，根據文獻指出，提高間距可提升液滴分離或合併傳輸前後的效率，但間距對免疫分析的影響，過去並未多加著墨，本研究首次探討間距對免疫反應操控及訊號偵測的影響，並整合提出間距可調整免疫分析晶片。 在介電溼潤式微流體晶片上，本研究針對三種電極面積於不同間距下對液滴傳輸及分割的現象進行探討，選出可提升免疫反應操控效率的間距；亦探討在不同間距聚集磁珠所測得之訊號影響，選出強度強且穩定之間距，並分別使用此兩間距於免疫分析中的免疫反應及螢光偵測以驗證操控時間縮短以及訊號提升的效果。為方便比較本研究的檢測結果與先前研究於未調整間距的檢測結果，沿用人體腫瘤壞死因子受器1 (sTNF-R1)作為免疫分析的偵測目標。 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.