Concentration-enhanced bead based CA 19-9 ELISA. (a) Step 1: Functionalized beads were drawn into the inlet microchannels by applying a negative pressure at the outlet reservoir. The 6-8 μm beads were trapped in front of the 5 μm microfabricated constrictions. Step 2: Substrate molecules were injected into the inlet microchannels, and were enzymatically converted into charged fluorescent product molecules in the bead pack. When a voltage was applied between the inlet microchannels and the side channels, the product molecules were electrokinetically accumulated and attained a high local concentration. The sensitivity of fluorescence detection was significantly enhanced in this region. (b) Without electrokinetic accumulation, only the top channel which contained beads with high surface concentration of antigen displayed visible increase in product fluorescence. In the accumulation region, it was easy to differentiate the difference in product fluorescence between each channel. (c) Dose response curve of concentration enhanced bead based CA 19-9 ELISA. Square markers represent intensities with product accumulation, while cross markers represent intensities without product accumulation. Sensitivity enhancement due to product accumulation was ~ 65 fold.