Particle flow assays for fluorescent protein microarray applications.
Microarray technology has brought a paradigmatic change in bioanalytics. However, highly sensitive and accurate assays are still needed for a real breakthrough. We present a simple and generic approach for fluorescent signal amplification with fluorescent microparticle labels. The assay principle was demonstrated using a reverse array model consisting of spots of bovine serum albumin with a small fraction of the proteins biotinylated. Specific binding of streptavidin coated fluorescent microparticles to the spots was promoted by applying a controlled continuous microparticle flow. The surface bound beads were visualized and quantified with confocal microscopy images. Comparison with standard fluorescent and flow discrimination assays has revealed several advantages of our approach. First, non-specific particle binding could be reduced to less than 1 particle/spot making therefore the visualization of single biomolecular bonds possible. Second, the amplification scheme presented here is generic and can be applied to any fluorescent microarray. Furthermore, this assay makes use of a biotin-streptavidin linkage and can therefore be applied to all kind of assays. Finally, single fluorescent microbeads can be easily visualized with standard optical equipments, so that no high performance equipment is required.
Bally
Marta
M
Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH and University of Zurich, Gloriastrasse 35, Zurich, Switzerland.
Dhumpa
Raghuram
R
Vörös
Janos
J