We present an integrated microfluidic system consisting of a label-free biosensor of a guided-mode resonance filter (GMRF) and a microfluidic channel with a micropost filter. The GMRF was fabricated through replica molding using an ultraviolet-curable polymer and a plastic substrate. An array of microposts (a diameter and height of 26.5 and 56 μm, respectively, and a spacing between 7.5 and 9.5 μm), fabricated on a silicon substrate through photolithography, was used as the filter. A double-sided tape was used to laminate the GMRF and a microfluidic chip such that the integrated device provides two functions: filtration of the cell debris and quantification of the in-cell protein concentration. By measuring the changes in the resonant wavelength from the GMRF, the detection of β-actin in an unprocessed lysed cell sample was demonstrated; the cell debris was separated using the micropost filter to prevent false measurement.

This paper describes the effect of bead aggregation in an immunoassay system and the aggregation-magnified post-assay fluorescence for ultrasensitive immunodetection on digital microfluidics.ABSTRACT | Several strategies are currently employed to enhance the detection limit of bead-based assays, but all these approaches improve the sensitivity by varying the assay procedures chemically or biologically. In previous digital microfluidic setups for bead-based immunoassay, the magnetic beads were suspended for detection. We investigated the effect of bead aggregation in such an immunoassay system and propose the aggregation magnified post-assay fluorescence for ultrasensitive immunodetection on digital microfluidics (AMPFLUID). The detection signal and sensitivity are further enhanced even at the post-assay stage without altering the original assay protocol on employing magnetically triggered post-assay aggregation of beads in a digital microfluidic setup followed by processing of the fluorescent signal. This method is shown to enhance the fluorescent signal with increased consistency and sensitivity after appropriate chargecoupled device (CCD) calibration. This method of on-chip detection allows the fulfilment of consumption of a volume at the nanoliter level and a limit of detection in the range picogram/mL. In our sTNF-RI model immunoassay, only 2.5 nL of sample is required; a detection limit 15 pg/mL is achieved.The decreased uncertainty of the measure is indicated by the error bars and coefficient of variation.

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Here, we describe a technique to manipulate a low number of beads to achieve high washing efficiency with zero bead loss in the washing process of a digital microfluidic (DMF) immunoassay. Previously, two magnetic bead extraction methods were reported in the DMF platform: (1) single-side electrowetting method and (2) double-side electrowetting method. The first approach could provide high washing efficiency, but it required a large number of beads. The second approach could reduce the required number of beads, but it was inefficient where multiple washes were required. More importantly, bead loss during the washing process was unavoidable in both methods. Here, an improved double-side electrowetting method is proposed for bead extraction by utilizing a series of unequal electrodes. It is shown that, with proper electrode size ratio, only one wash step is required to achieve 98% washing rate without any bead loss at bead number less than 100 in a droplet. It allows using only about 25 magnetic beads in DMF immunoassay to increase the number of captured analytes on each bead effectively. In our human soluble tumor necrosis factor receptor I (sTNF-RI) model immunoassay, the experimental results show that, comparing to our previous results without using the proposed bead extraction technique, the immunoassay with low bead number significantly enhances the fluorescence signal to provide a better limit of detection (3.14 pg/ml) with smaller reagent volumes (200 nl) and shorter analysis time (<1 h). This improved bead extraction technique not only can be used in the DMF immunoassay but also has great potential to be used in any other bead-based DMF systems for different applications.