Improving sensitivity in micro-free flow electrophoresis using signal averaging

Microfluidic free-flow electrophoresis (microFFE) is a separation technique that separates continuous streams of analytes as they travel through an electric field in a planar flow channel. The continuous nature of the microFFE separation suggests that approaches more commonly applied in spectroscopy and imaging may be effective in improving sensitivity. The current paper describes the S/N improvements that can be achieved by simply averaging multiple images of a microFFE separation; 20-24-fold improvements in S/N were observed by averaging the signal from 500 images recorded for over 2 min. Up to an 80-fold improvement in S/N was observed by averaging 6500 images. Detection limits as low as 14 pM were achieved for fluorescein, which is impressive considering the non-ideal optical set-up used in these experiments. The limitation to this signal averaging approach was the stability of the microFFE separation. At separation times longer than 20 min bubbles began to form at the electrodes, which disrupted the flow profile through the device, giving rise to erratic peak positions.