Electrokinetic Gradient-Based Focusing Mechanisms for Rapid, On-Chip Concentration and Separation of Proteins.

Abstract

Biochemical assays have seen a trend toward miniaturization as researchers strive to develop faster, more sensitive, and less expensive assays. Sample enrichment is often required in assays where dilute sample concentrations fail to meet instrumental limits of detection. This work describes three independent microfluidic techniques developed for electrokinetic concentration and separation of protein samples along imposed gradients by exploiting molecular properties unique to individual species. All three methods are embodied in microdevices with simple fabrication requirements, and are operable with solely the application of an external electric field. In temperature gradient focusing (TGF) via Joule heating, focusing is achieved by balancing the bulk fluid flow against the temperature-dependent electrophoretic velocity of an analyte. Here a temperature gradient is induced by exploiting Joule heating along a variable-width microchannel embedded in a simple PDMS-glass device. Focusing is demonstrated with fluorescent dyes, BSA, and insulin, with concentration factors >500 achievable in <10 minutes. A theoretical and numerical analysis captures the transport and heat transfer behavior of this device in a quasi-1D model. Numerical simulations show good agreement with experimental results. Next, a microscale immobilized pH gradient (μIPG) is photopolymerized in a glass microdevice for rapid isoelectric focusing (IEF) of proteins, marking the first on-chip realization of IPG-IEF methodology. Immobilines are linearly distributed via diffusion across the IPG segment prior to polymerization, and a numerical solver predicts the resulting pH profile. Focusing along a pH 3.8 – 7.0 μIPG is demonstrated in < 20 minutes with resolving power of ΔpImin ≈ 0.040. Finally, polyacrylamide porosity gradients are generated by linearly varying the acrylamide monomer and bisacrylamide crosslinker concentrations along a channel prior to polymerization. Microscale pore limit electrophoresis (μPLE) is demonstrated, in which proteins are separated based on their pore limit – the pore size at which their migration is nearly halted due to their molecular size. The “effective” pore limit is shown to be logarithmically dependent on the molecular weight of the protein. The inherent stacking effect of this process leads to improved peak resolution along the gel, as well as a means to preconcentrate dilute samples. Concentration factors >40,000 are demonstrated.