Electrophoretic Concentration of Proteins at Laser-Patterned Nanoporous Membranes in Microchips

Abstract

Laser-patterning of nanoporous membranes at the junction of a cross channel in a microchip is used to integrate protein concentration with an electrokinetic injection scheme. Upon application of voltage, linear electrophoretic concentration of charged proteins is achieved at the membrane surface because buffer ions can easily pass through the membrane while proteins larger than the molecular weight cutoff of the membrane (>5700) are retained. Simple buffer systems can be used, and the concentration results constitute outward evidence that the uniformity of buffer ion concentration is maintained throughout the process. Local and spatially averaged concentration are increased by 4 and 2 orders of magnitude, respectively, upon injection with moderate voltages (70-150 V) and concentration times (100 s). The degree of concentration is limited only by the solubility limit of the proteins. The porous polymer membrane can be used repeatedly as long as care is taken to avoid protein precipitation.

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.…”

Collection, focusing, and metering of DNA in microchannels using addressable electrode arrays for portable low-power bioanalysis

“…In addition to geometry, the electric field in the vicinity of the channel intersection can also be manipulated (pinched injection) to further define the injected zone size and shape (6). Other variations on the basic T-injector configuration involve the incorporation of microporous (7) and nanoporous (8) membranes, thin film electrodes (9), optical gating (10,11), and locally narrow channel geometries (12). Stacking at the interface between the sample and a background buffer has also been performed in T-injector geometries (13)(14)(15)(16)(17).…”

Capillary electrophoresis of proteins 2003–2005

“…Fluorescently labeled ovalbumin could have been detected at initial concentrations of 100 fmol by combining field-amplified injection and the membrane preconcentration [22]. A laser-patterned nanoporous membrane was prepared on a microchip at a junction of a cross channel to integrate electrokinetic injection and concentration of protein analytes [23]. While smaller ions can pass through the membrane, proteins of molecular mass above the membrane cutoff (5700) are retained.…”

“…A number of CE steps and components that are used in fused-silica capillaries have been transferred and successfully tested on a microchip including preconcentration on porous silica membrane [22,23], automated derivatization [29], microreactor with immobilized proteases and other proteins [181,182], UV detector [183,184], chemiluminescence detector [34,185], multiwavelength LIF detector [186], UV LIF detector [98], wall coatings [82,85], affinity CE [129], CIEF [80], and sheathless electrospray for MS [113,114].…”