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The biological function of proteins is dictated by the formation of supra-molecular complexes that act as the basic machinery of the cell. As such, measuring the properties of protein species in heterogeneous mixtures is of key importance for understanding the molecular basis of biological function. Here, we describe the combination of analytical microfluidic tools with liquid chromatography for multidimensional characterisation of biomolecules in complex mixtures in the solution phase. Following chromatographic separation, a small fraction of the flow-through is distributed to multiple microfluidic devices for analysis. The microfluidic device developed here allows the simultaneous determination of the hydrodynamic radius, electrophoretic mobility, effective molecular charge and isoelectric point of isolated protein species. We demonstrate the operation principle of this approach with a mixture of three unlabelled model proteins varying in size and charge. We further extend the analytical potential of the presented approach by analysing a mixture of interacting streptavidin with biotinylated BSA and fluorophores, which form a mixture of stable complexes with diverse biophysical properties and stoichiometries. The presented microfluidic device positioned in-line with liquid chromatography presents an advanced tool for characterising multidimensional physical properties of proteins in biological samples to further understand the assembly/disassembly mechanism of proteins and the nature of complex mixtures.

Microelectromechanical systems (MEMS) have enabled the development of a new generation of sensor platforms. Acoustic sensor operation in liquid, the native environment of biomolecules, causes, however, significant degradation of sensing performance due to viscous drag and relies on the availability of capture molecules to bind analytes of interest to the sensor surface. Here, we describe a strategy to interface MEMS sensors with microfluidic platforms through an aerosol spray. Our sensing platform comprises a microfluidic spray nozzle and a microcantilever array operated in dynamic mode within a closed loop oscillator. A solution containing the analyte is sprayed uniformly through picoliter droplets onto the microcantilever surface; the micrometer-scale drops evaporate rapidly and leave the solutes behind, adding to the mass of the cantilever. This sensing scheme results in a 50-fold increase in the quality factor compared to operation in liquid, yet allows the analytes to be introduced into the sensing system from a solution phase. It achieves a 370 femtogram limit of detection, and we demonstrate quantitative label-free analysis of inorganic salts and model proteins. These results demonstrate that the standard resolution limits of cantilever sensing in dynamic mode can be overcome with the integration of spray microfluidics with MEMS.