Application of Optical pH Sensors in the Microfluidic Free-flow Isoelectric Focusing of Biomolecules

We present methods for fabrication and application of microfluidic chips for free-flow electrophoresis (FFE) that allow integrated pH sensing. Monitoring of the pH gradient in microfluidic free-flow isoelectric focusing (FFIEF) allows on-line adjustment of the parameters for electrophoresis and evaluation of the isoelectric point (pI) of focused analyte bands almost in real time. The fabrication of these assemblies is demonstrated via multistep photopolymerization or a laser cutting and lamination technique. Integration of pH sensing layers is accomplished via photopolymerization or inkjet printing of hydrogels that contain covalently attached pH probes. These microchips were characterized and employed for the FFIEF of proteins, peptides and plasma. Multispectral microscopic imaging was used to separate the pH sensor and the biomolecule fluorescence. We also show pH sensing in FFE based on time-domain dual lifetime referencing (t-DLR), a microreactor for integrated labelling prior to the FFIEF and the isoelectric focusing of unlabeled compounds.