Microchip isoelectric focusing with monolithic immobilized pH gradient materials for proteins separation

Abstract

Monolithic immobilized pH gradient (M-IPG) materials were prepared in microchannles by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by the attachment of focused Ampholine onto the surface of porous monoliths via epoxide groups. With M-IPG materials as matrix, FITC-labeled ribonuclease B, myoglobin and alpha-casein were well separated by microchip isoelectric focusing (muCIEF) without carrier amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were applied to drive focused zones for LIF detection. Our experimental results showed that pressure mobilization was preferable with neglectable band broadening, and good peak shape and high detection sensitivity were obtained. All these results demonstrate that muCIEF with M-IPG materials is not only an efficient mode for protein enrichment and separation but also attractive to couple with other CE modes to achieve multi-dimensional separation or MS for further identification, without the interference of mobile CAs.

Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

“…Microchips were fabricated with Type SG2506 glass plates (Shaoguang Microelectronics Corp, Changsha, China), by photolithography, wet chemical etching and room-temperature bonding, as described by our previous method [26]. UV-transparent capillaries (100 m i.d.…”

“…The internal wall surface of UV-transparent fused-silica capillaries and the channel of microchips were first vinylized to enable the covalent attachment of the monolith to the wall, as described in our previous research [26]. For photopolymerization in capillaries, the polymerization mixture containing 15 wt% NAS, 10 wt% PEGDA, 75 wt% CH 3 OH and 1 wt% DMPA with respect to monomers, was purged with N 2 for 30 s to remove dissolved O 2 , and then filled into the capillaries.…”

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient, immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

“…Another promising approach is to immobilize ampholytes onto supporting materials (i.e. monolithic matrix) to form a stable pH gradient [10,11], avoiding the additional steps for ampholyte removal and possibly expanded diffusion of focused zones. …”

Review on recent and advanced applications of monoliths and related porous polymer gels in micro-fluidic devices

“…oligoamino and oligocarboxylic acids, pH 3.5-10.0) on the surface of a porous monolith via epoxide groups to establish a stable pH gradient for on-chip IEF [73]. The monolith was fabricated in situ in a pretreated glass micro-channel (∼100 m × 30 m) by photo-initiated polymerisation from a monomer solution containing glycidyl methacrylate (GMA), AAm, MBAA and AIBN.…”