title: Fluorescent vesicles for signal amplification in reverse phase protein microarray assays.
abstract: Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning.
authors.affiliation: Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH and University of Zurich, 8092 Zurich, Switzerland.
authors.forename: Marta
authors.initials: M
authors.lastname: Bally
authors.forename: Shahida
authors.initials: S
authors.lastname: Syed
authors.forename: Andreas
authors.initials: A
authors.lastname: Binkert
authors.forename: Ekkehard
authors.initials: E
authors.lastname: Kauffmann
authors.forename: Markus
authors.initials: M
authors.lastname: Ehrat
authors.forename: Janos
authors.initials: J
authors.lastname: Vörös
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D005456:Fluorescent Dyes / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology; D008051:Lipid Bilayers / Q000737:chemistry*; D018613:Microscopy, Confocal; D040081:Protein Array Analysis / Q000379:methods*; D011817:Rabbits; D012235:Rhodamines / Q000737:chemistry
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D008051:Lipid Bilayers; D012235:Rhodamines; C022027:lissamine rhodamine B