Analytical Chemistry, 2004: Electrophoretic Concentration of Proteins at Laser-Patterned Nanoporous Membranes in Microchips
Citation: Song S, Singh AK, Kirby BJ. Electrophoretic Concentration of Proteins at Laser-Patterned Nanoporous Membranes in Microchips, Analytical Chemistry, 76(15): pages 4589-4592. doi pdf
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.
Figures:
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- Figure 1. Cross channel with laser-patterned porous polymer membrane used for the present studies. The channel width and depth are 80 and 20 um. The membrane thickness is roughly 50 um.
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- Figure 2. Membrane schematic and background-corrected images of protein concentration. Microchannel edges have been drawn for clarity: HV, high voltage; PV, pinch voltage (PV ) 0.45 HV); GND, ground. (a) Schematic. S denotes sample, W waste, B buffer, and SW sample waste. (b) Before loading. (c) After loading. (d) During injection.
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- Figure 3. Linear increase in concentration of both BSA and phosphorylase b with the voltage-time product. The increase can be by over 2 orders of magnitude. See Experimental Section for details. “Concentration factor” denotes measured protein concentrations, normalized to the 0.5 uM initial concentration. Measurement uncertainties ((16% for BSA and (21% for phosphorylase b) are dominated by uncertainty in the low signal level of the unconcentrated sample. Concentration measurements were averaged over a measurement window specified in gray in the inset, chosen to be comparable to the channel width and size of injected sample.