Capacitively coupled contactless conductivity detection for microfluidic capillary isoelectric focusing

We report capacitively coupled contactless conductivity detection (C4D) of proteins separated by microfluidic capillary isoelectric focusing (μCIEF). To elucidate the evolution of negative conductivity peaks during focusing and seek IEF conditions for sensitive conductivity detection, numerical simulation was performed using a model protein GFP (green fluorescence protein) and hypothetical carrier ampholytes (CAs). C4D was successfully applied to the μCIEF by optimizing assay conditions using a simple and effective pressure-mobilization approach. The conductivity and fluorescence signals of a focused GFP band were co-detected, confirming that the obtained negative C4D peak could be attributed to the actual protein, not the non-uniform background conductivity profile of the focused CAs. GFP concentrations of 10 nM-30 μM was quantified with a detection limit of 10 nM. Finally, the resolving power was analyzed by separating a mixture of R-phycoerythrin (pI 5.01), GFP-F64L (pI 5.48), and RK-GFP (pI 6.02). The conductivities of the three separated fluorescence proteins were measured with average separation resolution of 2.06. We expect the newly developed label-free μCIEF-C4D technique to be widely adopted as a portable, electronics-only protein-analysis tool.