Integrated Preconcentration SDS−PAGE of Proteins in Microchips Using Photopatterned Cross-Linked Polyacrylamide Gels

Abstract

The potential of integration of functions in microfluidic chips is demonstrated by implementing on-chip preconcentration of proteins prior to on-chip protein sizing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two polymeric elements-a thin (approximately 50 microm) size exclusion membrane for preconcentration and a longer (approximately cm) porous monolith for protein sizing-were fabricated in situ using photopolymerization. Contiguous placement of the two polymeric elements in the channels of a microchip enabled simple and zero dead volume integration of the preconcentration with SDS-PAGE. The size exclusion membrane was polymerized in the injection channel using a shaped laser beam, and the sizing monolith was cast by photolithography using a mask and UV lamp. Proteins injected electrophoretically were trapped on the upstream side of the size exclusion membrane (MW cutoff approximately 10 kDa) and eluted off the membrane by reversing the electric field. Subsequently, the concentrated proteins were separated in a cross-linked polyacrylamide monolith that was patterned contiguous to the size exclusion membrane. The extent of protein preconcentration is easily tuned by varying the voltage during injection or by controlling the sample volume loaded. Electric fields applied across the nanoporous membrane resulted in a concentration polarization effect evidenced by decreasing current over time and irreproducible migration of proteins during sizing. To minimize the concentration polarization effect, sieving gels were polymerized only on the separation side of the membrane, and an alternate electrical current path was employed, bypassing the membrane, for most of the elution and separation steps. Electrophoretically sweeping a fixed sample volume against the membrane yields preconcentration factors that are independent of protein mobility. The volume sweeping method also avoids biased protein loading from concentration polarization and sample matrix variations. Mobilities of the concentrated proteins were log-linear with respect to molecular weight, demonstrating the suitability of this approach for protein sizing. Proteins were concentrated rapidly (<5 min) over 1000-fold followed by high-resolution separation in the sieving monolith. Proteins with concentrations as low as 50 fM were detectable with 30 min of preconcentration time. The integrated preconcentration-sizing approach facilitates analysis of low-abundant proteins that cannot be otherwise detected. Moreover, the integrated preconcentration-analysis approach employing in situ formation of photopatterned polymeric elements provides a generic, inexpensive, and versatile method to integrate functions at chip level and can be extended to lowering of detection limits for other applications such as DNA analysis and clinical diagnostics.

Transport phenomena in nanofluidics

“…We emphasize that this is fundamentally different from the preconcentration mechanism using an electric field through nanochannels, resulting in the enrichment of both co-ions and counterions at the cathodic side of the nanometer-sized aperture. In addition to these charge-based preconcentration schemes, the enrichment of molecules at the entrance of apertures due to size exclusion has been demonstrated ͑Khandurina et al., 1999;Song et al, 2004;Foote et al, 2005;Hatch et al, 2006; Here we focus on charge-based preconcentration using an electric field through a nanochannel, which is connected by two microchannels ͓see Fig. 21͑a͔͒.…”

Towards autonomous lab-on-a-chip devices for cell phone biosensing

“…The in situ patterning of polyacrylamide gels has been reported for electrophoretic transport systems (Hatch et al, 2006;Herr et al, 2007;Kondapalli et al, 2011), which as well as magnetic beads used as the solid phase to capture antibodies and as carriers of target antigens are inspiring configurations (Inglis et al, 2004;Lien et al, 2008), if could be adapted to non-electrically interfaced transport.…”

Integrated microfluidic preconcentrator and immunobiosensor

“…We use an in situ photopolymerized nanoporous membrane (Song et al 2004a, b;Hatch et al 2006) in our integrated glass microfluidic device for the preconcentration step. Song et al (2004a) have shown high concentration factors (four orders of magnitude local concentration) using these nanoporous membranes.…”

“…The in situ fabrication technique allows for easy integration with total analysis systems. Our membranes are fabricated in polyacrylamide as it is hydrophilic, biocompatible, and shows minimal non-specific adhesion (Hatch et al 2006). The pore size of acrylamide gels can be easily adjusted by changing the percentage of monomer components (Holmes and Stellwagen 1991a, b).…”

“…Figure 1 shows the integrated microfluidic biosensor with the inset showing the concentration membrane. The membrane is nanoporous and is made using polyacrylamide at the intersection of the glass channels by an in situ photopolymerization technique (Hatch et al 2006;Song et al 2004a, b). We use liposomes, which can encapsulate a very large number of fluorescent dye molecules in their core for signal amplification in the biosensor.…”