Affinity Monolith-Integrated Poly(methyl methacrylate) Microchips for On-Line Protein Extraction and Capillary Electrophoresis

Abstract

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

Bioaffinity chromatography on monolithic supports

“…Sun et al reported on an integrated polymethyl methacrylate microchip containing a GMA-EDMA monolith with anti-FITC antibodies for automated pretreatment, extraction and CE separation of proteins [52]. FITC-HSA was used as model analyte to optimize the binding and elution conditions, and stability and performance of the microchip.…”

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

“…Antibody-modified affinity monolithic columns, prepared in micro-fluidic devices by photo-initiated polymerisation, have been recently introduced by workers from the Woolley research group [99][100][101]. The group reported different protocols for the immobilisation of antibodies in GMA-based monolithic columns, embedded in PMMA chips.…”

“…Subsequent treatment of the affinity column with lysozyme in phosphate buffer saline solution (PBS) prevented non-specific adsorption of GFP. Immunoaffinity extraction and separation of FITC-tagged proteins was then carried out within a PMMA chip integrating a 2-mm-long GMA-EGDMA affinity column, placed upstream of a separation channel for on-chip electrophoresis [100]. Immobilisation of the antibody in the affinity column was carried out via direct reaction of the GMA epoxy groups with the amine groups present in the antibody (epoxy method [16]) at 37 • C for 24 h. After anti-FITC immobilisation, the residual epoxy groups were subsequently blocked with Tris buffer (pH 8).…”

Fabrication of a gel particle array in a microfluidic device for bioassays of protein and glucose in human urine samples

“…And the motivation and usability of the system can be described as follows: a device combined with microchips is committed to realizing rapid detection with low consumption, high sensitivity, and accurate definition, while the PEG microcolumns modified to the microfluidic chip and the chip itself spend little and are fabricated easily; thus, it is potential to be applied in analysis of environmental and biological samples. [33][34][35] In our previous work, we developed a PEG hydrogel microcolumn array technique to provide a convenient and suitable platform to perform biochemical assays. 36 PEG is a convenient material for the fabrication of microarrays, because of its non-volatile, recyclable and inert character.…”