Fluorescence sensitivity and linearity.

<p>(A) Different concentrations of calcein were introduced into a microfluidic cartridge and the corresponding change in photoamplifier voltage was recorded. The circles indicate the mean signal amplitude across all reaction wells for three different chips (red, green, and blue). As expected, the relationship between calcein concentration and photoamplifier output is linear (R² = 0.983). Limit of detection can be evaluated by determining when this trend line crosses the noise floor of the instrument at a given bandwidth. At a time resolution (BW⁻¹) of 2 min, the µBAR can distinguish changes in calcein concentration of 30 pM, which is orders of magnitude below the fluorophore concentrations typically used in nucleic acid amplification reactions. (B) 50 µM steps of calcein are clearly visible in the photoamplifier output and this output remains stable for a constant concentration. (C) Due to the nature of the illumination scheme and the lack of optics, there is some nonuniformity of sensitivity across the 6×10 reaction chamber array. However, we take this into account and normalize each well by its mean sensitivity, shown here. In this graphic, the illumination LEDs and sample inlets are located above the topmost row.