A microfluidic device for array patterning by perpendicular electrokinetic focusing

This paper describes a microfluidic chip in which two perpendicular laminar-flow streams can be operated to sequentially address the surface of a flow-chamber with semi-parallel sample streams. The sample streams can be controlled in position and width by the method of electrokinetic focusing. For this purpose, each of the two streams is sandwiched by two parallel sheath flow streams containing just a buffer solution. The streams are being electroosmotically pumped, allowing a simple chip design and a setup with no moving parts. Positioning of the streams was adjusted in real-time by controlling the applied voltages according to an analytical model. The perpendicular focusing gives rise to overlapping regions, which, by combinatorial (bio) chemistry, might be used for fabrication of spot arrays of immobilized proteins and other biomolecules. Since the patterning procedure is done in a closed, liquid filled flow-structure, array spots will never be exposed to air and are prevented from drying. With this device configuration, it was possible to visualize an array of 49 spots on a surface area of 1 mm2. This article describes the principle, fabrication, experimental results, analytical modeling and numerical simulations of the microfluidic chip.\ud \ud \ud \u

Proteomics-on-a-chip for Biomarker discovery

In proteomics research still two-dimensional gel electrophoresis (2D-GE) is currently used for biomarker discovery. We applied free flow electrophoresis (FFE) separation technology combined with biomolecular interaction sensing using Surface Plasmon Resonance (SPR) imaging in an integrated proteomics-on-a-chip device as a proof of concept for biomarker discovery

High-throughput surface plasmon resonance imaging-based biomolecular kinetic screening analysis

In this paper we show a high-throughput method to screen the kinetics and affinity constants of many biomolecular interactions simultaneously. During the preparation of the sensor chip, ligands were serially diluted and spotted in a 6 × 4 microarray on the sensor surface. A multi-analyte sample was injected and the real-time surface plasmon resonance (SPR) responses of all the 24 microarray spots were obtained using a commercial SPR imaging instrument. The multi responses of the association and dissociation processes obtained from a single analyte injection are sufficient to calculate the rates and affinity constants of the interactions between three interactant antigen-antibody pairs using a simple monophasic kinetic model. The method drastically reduces the measurement time and cost in the benefit of increased throughput

Free-flow zone electrophoresis and isoelectric focusing using a microfabricated glass device with ion permeable membranes

This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow isoelectric focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow isoelectric focusing could be established. Furthermore, the application of hydrodynamic focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow isoelectric focusing employing a transversal voltage of up to 150 V across the separation chamber.\ud \u

Electrokinetic sorting and collection of fractions for preparative capillary electrophoresis on a chip

A microfabricated device capable of selecting and collecting multiple components from a mixture separated by capillary electrophoresis (CE) is described. This collection is automated and can be easily controlled by a set of rules defined by an operator, enabling fast and consistent operation. The device consists of an electrokinetically steered fluidic network that can be divided into three sections: a CE part, a fractions distribution region and a set of storage channels. Sample fractions leave the CE channel and are detected in the interfacial region by fluorescence intensity measurements. If an upcoming peak is detected, separation is withheld and the potentials are reconfigured to force the fraction into one of the collection channels, where they become available for further processing or analysis. The sequence of separation and collection is repeated until all the bands of interest are captured. A mixture of three fluorescent dyes (Rhodamine 6G, Rhodamine B and Fluorescein) was used to demonstrate the principle. The components were repeatedly separated by means of CE and pooled in their respective storage channels. In comparison to previous developments, the system presented in this paper offers automatic collection of all fractions in a single run. Furthermore, it is possible to run the system in a repetitive mode for accumulative pooling if more fractionated sample is required

Surface plasmon resonance imaging based multiplex biosensor: Integration of biomolecular screening, detection and kinetics estimation

We present a multiplex biosensing method to simultaneously screen targets of interest in a multiple target analyte sample and to extract the binding affinities of all interactant pairs from a single sensor surface using a commercial surface plasmon resonance imaging system. For demonstration, we have prepared our sensor disk with five different ligands varying from low molecular weight antibiotics to high molecular weight human IgG, all immobilized in a microarray format. The multi-target analyte sample was prepared by mixing five antibodies where each one is highly specific for one of the immobilized ligands in a range of concentrations for kinetics estimations. The key advantage of the newly developed approach is that many different types of assays can be performed simultaneously, however, care should be taken to understand the non-specific interactions between different analytes in the sample mixture and unintended ligands, and surface regeneration behavior of different ligand types. Other advantages include reductions in experimental and analysis time, reduced costs, and flexibility since the same microarray can be used for assays with a single target analyte specific for the single ligand. (C) 2010 Elsevier B.V. All rights reserve