Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis

Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis. We successfully fractionate isoforms of two potential glycoprotein cancer markers, fetuin and prostate-specific antigen (PSA), with 10 min run time, and results are compared qualitatively and quantitatively to conventional slab gel IEF. In the second dimension, functionalized monolithic columns are used to capture and detect targeted analytes from each fraction. We demonstrate rapid two-dimensional fractionation, immunocapture, and detection of C-reactive protein (CRP) spiked in human serum. This rapid, flexible, and automated approach is well-suited for glycoprotein biomarker research and verification studies and represents a practical avenue for glycoprotein isoform-based diagnostic testing.