| Summary: | 碩士 === 國立交通大學 === 機械工程系所 === 103 === 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.
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