Laminar Flow Microarray Patterning by Perpendicular Electrokinetic Focusing

This paper describes a method to pattern microarrays in a closed microfluidic device. Two perpendicular laminar flow streams can operate in terms to sequentially coat the surface of a flow-chamber with parallel lanes in two directions. Two perpendicular sample streams can be controlled in position and width by applying electrokinetic focusing, for which each of the two streams is sandwiched between two parallel laminar flow streams containing just a buffer solution. Electroosmotic flow allows a simple chip design without any moving parts being involved. With this device configuration it is possible to define an array of up to 169 spots on a surface area of 1 mm2

Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry

In this paper, we are evaluating the strategy of sorting peptides/proteins based on the charge to mass without resorting to ampholytes and/or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS.Korea Institute of Science and Technology. Intelligent Microsystems CenterMassachusetts Institute of Technology. Center for Environmental Health SciencesNational Institute of Environmental Health Sciences (Grant No. P30-ES002109)United States. National Institutes of Health (grant R21 EB008177

Synchronized, continuous-flow zone electrophoresis

A new method for performing continuous electrophoretic separation of complex mixtures in microscale devices is proposed. Unlike in free-flow electrophoresis devices, no mechanical pumping is requiredboth fluid transport and separation are driven electrokinetically. This gives the method great potential for on-a-chip integration in multistep analytical systems. The method enables us to collect fractionated sample and tensfold purification is possible. The model of the operation is presented and a detailed description of the optimal conditions for performing purification is given. The chip devices with 10-μm-deep separation chamber of 1.5 mm × 4 mm in size were fabricated in glass. A standard microchip electrophoresis setup was used. Continuous separation of rhodamine B, rhodamine 6G, and fluorescein was accomplished. Purification was demonstrated on a mixture containing rhodamine B and fluorescein, and the recovery of both fractions was achieved. The results show the feasibility of the method

Proteomics of industrial fungi: trends and insights for biotechnology

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis

Sweeping flow electrophoresis (SFE): A new continuous separation technique

In this paper, a new principle for continuous electrophoretic separation is introduced. An electrokinetically pinched sample flow is swept through a microfluidic chamber. The wavelengths of the resulting sine-waves are in direct relation with the electrophoretic mobility of the compound. The principle is applied to separate Rhodamine B and Fluorescein