Single-bead-based consecutive biochemical assays using a dielectrophoretic microfluidic platform

In this paper, we describe a method for performing single-bead-based, consecutive biochemical assays on a dielectrophoretic microfluidic chip using the interaction of FITC-labeled streptavidin with biotin-coated 2-μm polystyrene (PS) beads as a model system. A microfluidic device with four sets of electrodes on a glass slide was used to trap a single bead at the center of each set of electrodes through negative dielectrophoresis. Each set of electrodes was composed of four electrodes arranged in a cross-like manner. The electrodes were prepared using a simple process consisting of UV lithography and aluminum etching. A laser-induced fluorescence system detected the fluorescence emission from the FITC-labeled streptavidin conjugated with the biotin-coated PS beads. We investigated the effects of (i) the magnification/numerical aperture of the objective used for collecting fluorescence signals and (ii) the cut-off frequency of the low-pass digital filter, which filtered out high-frequency noise, on the signal-to-noise ratio. The average measured fluorescence maximum, determined from 10 consecutive fluorescence signals, increased with increasing concentrations of streptavidin (up to 0.3 μg/mL); it leveled off when the concentration of streptavidin was greater than 0.3 μg/mL. The limit of detection (signal-to-noise ratio = 3.0) was 8.3 ng/mL.