Combining affinity selection and specific ion mobility for microchip protein sensing

The sensitive detection of proteins is a key objective in many areas of biomolecular science, ranging from biophysics to diagnostics. However, sensing in complex biological fluids is hindered by non-specific interactions with off-target species. Here, we describe and demonstrate an assay that utilises both the chemical and physical properties of the target species to achieve high selectivity, in a manner not possible by chemical complementarity alone, in complex media. We achieve this objective through a combinatorial strategy, by simultaneously exploiting free-flow electrophoresis for target selection, on the basis of electrophoretic mobility, and conventional affinity-based selection. In addition, we demonstrate amplification of the resultant signal by a catalytic DNA nano-circuit. This approach brings together the inherent solution-phase advantages of microfluidic sample handling with isothermal, enzyme-free signal amplification. With this method, no surface immobilisation or washing steps are required and our assay is well suited to mono-epitopic targets, presenting advantages over conventional ELISA techniques