(240b) Microscale Isoelectric Fractionation Membranes for Rapid Sample Purification and Enrichment

Here we present microscale isoelectric fractionation and enrichment (μIFE) ? a novel method for simultaneously separating and concentrating proteins from complex sample matrices. In μIFE, species are fractionated into zones based on their isoelectric point (pI ? the pH at which a protein has no net charge) by electrophoretically driving them through microchannels supporting a serial array of thin (~50μm-wide) pH-specific polyacrylamide membranes. Membranes are fabricated via photopolymerization of aqueous monomer solutions titrated to precise pH levels with acrylamido buffering compounds. Multiple membranes can be fabricated simultaneously by adopting our previously-developed technique for establishing and photopolymerizing immobilized pH gradients (IPGs) [1]. Rather than polymerizing a continuous IPG, membrane regions are exposed to polymerizing UV light through a photomask. Up to 20 membranes at unique pH values can be implemented in the current device design.

Stacking within each pI zone results in several orders-of-magnitude enrichment of fractionated proteins within minutes. Individual fractions can then be accessed and manipulated for further on-chip processing and analysis. We demonstrate integration of μIFE with our previously-developed electrophoretic immunoassay platform. Such integration enables isolation of trace analytes and minor isoforms from high-abundance proteins in biological matrices which complicate immuno-analysis and curb limits of detection.

This work provides a novel, ampholyte-free, microchip-based isoelectric focusing mechanism that enables improved sample isolation and accessibility over other on-chip approaches [2]. Integrating μIFE with sensitive and specific analysis platforms on-chip promises clear quantitative pictures of low abundance proteins and their subtle modifications from miniscule volumes of complex biological samples, and will enhance the limits of detection of next-generation biological diagnostic devices.

[1] G.J. Sommer, A.K. Singh, A.V. Hatch. ?On-Chip Isoelectric Focusing Using Photopolymerized Immobilized pH Gradients? Anal. Chem., 2008, 80, 3327-3333.

[2] G.J. Sommer and A.V. Hatch. ?Review: Isoelectric Focusing in Microfluidic Devices? Electrophoresis, 2009, 30, 742-757.