Size Exclusion Chromatography of Microliter Volumes for On-Line Use in Low-Pressure Microfluidic Systems

摘要

We have developed a new cartridge format for on-line size exclusion processing in low-pressure, portable microfluidic devices. The described system allows size exclusion chromatography of microliter volumes (termed (mu)L-SEC) to be performed with ready integration in complex protocols for continuous-flow sample processing and analysis. The refillable cartridge format was employed for the preparation of Bacillus subtilus spores lysed in the presence of a strong reducing agent. While the reducing agent is known to interfere with subsequent fluorescent labeling of the solubilized proteins, the described continuous-flow size exclusion processing allowed complete isolation of interferences from a 10-(mu)L sample in 70 s. Following efficient labeling, the protein sample was injected and separated on-chip using gel electrophoresis. To increase the resolution, speed, and sample capacity of buffer exchange under low-pressure operation (40 psi), parameters such as the size exclusion resin, load volumes, flow rates, buffer composition, and cartridge geometry were optimized and are presented here. The (mu)L-SEC analysis is compatible with automated sample preparation for microfluidic systems and has resulted in significantly increased analysis speed and throughput over benchtop methods. The presented technique has the potential to improve capabilities such as buffer exchange, size fractionation, and high-abundance protein removalsteps that are frequently required prior to on-chip, point-of-care, and mass spectrometric analyses.