On-chip Micro- and Nanofluidic Electrokinetic Injection and Separation for PEGylation Analysis

We present an experimental study of micro- and nanofluidic electrokinetic injection and separation in borosilcate channels as a method for characterizing size and zeta potential of biomolecules--specifically polyethlylene glycol (PEG), keyhole limpet hemocyanine (KLH), and pegylated KLH. While pegylation (the conjugation of proteins with PEG) is an established technique for enhancing a protein's therapeutic properties, reliable characterization of these conjugations by traditional analysis techniques (i.e. gel-electrophoresis, zetasizer) remains a challenge. Using a three-step electrokinetic sequence (load, gate, and inject), FITC labeled species and a fluorescein tracer dye are injected into a channel where they separate according to differences in electrophoretic mobility. We find the average absolute mobility of pegylated subunit KLH in 1 micron channels to be 56\% that of unpegylated subunit KLH. In a 250 nm channel, we measure a 33\% shift in the average absolute mobility of PEG dendrimers as compared to measurements in a 1 micron channel. These results begin to demonstrate how a micro- and nanofluidic-based approach might address the demand for effective and accessible nanoparticle characterization platforms.