The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays. In particular, the devices and methods of the invention are useful in screening large numbers of different compounds for their effects on a variety of chemical, and preferably, biochemical systems.

High throughput screening assay systems in microscale fluidic devices

Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. Various genes, up-regulated in atherosclerosis but also by psychosocial stress, are controlled by the transcription factor nuclear factor κB (NF-κB). Therefore, NF-κB is a good candidate to convert psychosocial stress into cellular activation. Volunteers were subjected to a brief laboratory stress test and NF-κB activity was determined in peripheral blood mononuclear cells (PBMC), as a window into the body and because PBMC play a role in diseases such as atherosclerosis. In 17 of 19 volunteers, NF-κB was rapidly induced during stress exposure, in parallel with elevated levels of catecholamines and cortisol, and returned to basal levels within 60 min. To model this response, mice transgenic for a strictly NF-κB-controlled β-globin transgene were stressed by immobilization. Immobilization resulted in increased β-globin expression, which could be reduced in the presence of the α1-adrenergic inhibitor prazosin. To define the role of adrenergic stimulation in the up-regulation of NF-κB, THP-1 cells were induced with physiological amounts of catecholamines for 10 min. Only noradrenaline resulted in a dose- and time-dependent induction of NF-κB and NF-κB-dependent gene expression, which depended on pertussis-toxin-sensitive G protein-mediated phosphophatidylinositol 3-kinase, Ras/Raf, and mitogen-activated protein kinase activation. Induction was reduced by α1- and β-adrenergic inhibitors. Thus, noradrenaline-dependent adrenergic stimulation results in activation of NF-κB in vitro and in vivo. Activation of NF-κB represents a downstream effector for the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response.

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A mechanism converting psychosocial stress into mononuclear cell activation.

Monolithic columns in high-performance liquid chromatography.

An organic microdrop (∼1.3 μL) is suspended inside a flowing aqueous drop from which the analyte is extracted. The drop-in-drop system is achieved by a multitube assembly. The aqueous phase is continuously delivered to the outer drop and is aspirated away from the bottom meniscus of the drop. After the sampling/extraction period, a wash solution replaces the sample/reagent in the aqueous layer, resulting in a clear outer aqueous drop housing a colored organic drop containing the extracted material. This also results in an automatic backwash. The color intensity of the organic drop, related to the analyte concentration, is monitored by a light-emitting diode based absorbance detector. After the analytical cycle, the organic drop is removed and replaced by a new one. The performance of the system is illustrated with the determination of sodium dodecyl sulfate (a methylene blue active substance) extracted as an ion pair into chloroform. This unique microextraction system is simple and flexible, permits autom...

Analytical chemistry in a drop. Solvent extraction in a microdrop.

Micrometre-scale analytical devices are more attractive than their macroscale counterparts for various reasons. For example, they use smaller volumes of reagents and are therefore cheaper, quicker and less hazardous to use, and more environmentally appealing. Scaling laws compare the relative performance of a system as the dimensions of the system change, and can predict the operational success of miniaturized chemical separation, reaction and detection devices before they are fabricated. Some devices designed using basic principles of scaling are now commercially available, and opportunities for miniaturizing new and challenging analytical systems continue to arise.

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Johan C. Kraak

Papers

Simple and fast solvent extraction system for selective and quantitative isolation of adrenaline, noradrenaline and dopamine from plasma and urine.

Capillary zone electrophoretic separations of proteins in polyethylene glycol-modified capillaries

Comparison of five methods for the study of drug-protein binding in affinity capillary electrophoresis.

Study of protein-drug binding using capillary zone electrophoresis.

Solvent-generated ion-exchange systems with anionic surfactants for rapid separations of amino acids.