Polymer Microchips Integrating Solid-Phase Extraction and High-Performance Liquid Chromatography Using Reversed-Phase Polymethacrylate Monoliths

Abstract

Polymer microfluidic chips employing in situ photopolymerized polymethacrylate monoliths for high performance liquid chromatography separations of peptides is described. The integrated chip design employs a 15 cm long separation column containing a reversed-phase polymethacrylate monolith as a stationary phase, with its front end seamlessly coupled to a 5 mm long methacrylate monolith which functions as a solid phase extraction (SPE) element for sample cleanup and enrichment, serving to increase both detection sensitivity and separation performance. In addition to sample concentration and separation, solvent splitting is also performed on-chip, allowing the use of a conventional LC pump for the generation of on-chip nano-flow solvent gradients. The integrated platform takes advantage of solvent bonding and a novel high-pressure needle interface which together enable the polymer chips to withstand internal pressures above 20 MPa (~2,900 psi) for efficient pressure-driven HPLC separations. Gradient reversed-phase separation of fluoresceinlabeled model peptides and BSA tryptic digest are demonstrated using the microchip HPLC system. On-line removal of free fluorescein and enrichment of labeled proteins are simultaneously achieved using the on-chip SPE column, resulting in a 150-fold improvement in sensitivity and a 10-fold reduction in peak width in the following microchip gradient LC separation.

Polymeric-Based In Vitro Diagnostic Devices

“…It is only attacked by nonpolar organic solvents, such as hexane and toluene [29]. This chemical resistance is critical for chip-based sample extraction and separation systems that require multiple washing, loading, elution, and recondition processes [30,31]. It is also crucial for various bioprocessor, biosensing, monitoring, and screening applications that require multiple chemical reaction processes or a harsh operating environment [32].…”

“…The integrative character of LOC systems allows for the performance of on-chip sample preparation and serial separation for IVD applications. For example, polymer microfluidic chips employing in situ photopolymerized polymethacrylate monoliths for high-performance liquid chromatography separations of peptides have been described [30]. In this procedure, the integrated chip design employs a 15-cm-long separation column containing a reversed-phase polymethacrylate monolith as a stationary phase, with its front end seamlessly coupled to a 5-mmlong methacrylate monolith that functions as a solid-phase extraction (SPE) element for sample cleanup and enrichment, serving to increase both detection sensitivity and separation performance (Fig.…”

Recent advances in micro-scale and nano-scale high-performance liquid-phase chromatography for proteome research

“…As shown in Fig. 3, Liu et al [49] prepared polymethacrylate monoliths in microfluidic chips, in the format of 5-mm-long solid-phase extraction (SPE) coupled with 15 cm-long reversed phase nano-RPLC. By use of this equipment online removal of free fluorescein, and enrichment and separation of labeled proteins were achieved simultaneously, resulting in 150-fold improvement on sensitivity by SPE, and tenfold reduction in peak width by microchip gradient LC separation.…”

“…In this technique, ion exchange, for example SCX, WAX, or WCX-WAX mixture, and RPLC particles, are packed in tandem in a single capillary and enable the first and second dimensions of the separation, respectively. With this system, approximately [49] 420 μg of the soluble fraction, 440 μg of the lightly washed insoluble fraction, and 490 μg of the heavily washed insoluble fraction from the Saccharomyces cerevisiae strain BJ5460 were analyzed by fully automated 15-step chromatography and a total of 1,484 proteins were identified [65]. High detection sensitivity for low-abundance proteins, for example transcription factors and protein kinases, was also demonstrated.…”

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.…”