Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool - Nanomolar human serum albumin quantitation

The commercially available dye, NanoOrange, has been investigated asa poten tial tool for clinical diagnostics due to its low cost, ease of use, and ab ility to detect nanomolar concentrations of protein. Virtually non-fluoresc ent in dilute aqueous solutions, NanoOrange fluorescence is enhanced by at least an order of magnitude upon non-covalent interaction with proteins. Th ese features, coupled with the requirement for high throughput assays in th e clinical laboratory has prompted the development of two orthogonal NanoOr ange approaches. Human serum albumin (HSA) was used as a model protein for the development of both 96-well microplate and capillary electrophoresis la ser-induced fluorescence (CE-LIF) assay formats. Dye performance in five co mmonly used buffers of various concentrations and pH indicated considerable flexibility in assay buffer selection, with optimal performance at pH 9.0. A salt concentration study indicated that increasing NaCl concentration ge nerally decreases fluorescence emission and can be minimized by pre-dilutin g biological samples to a final salt concentration of 20-80 mM. Titration o f protein with NanoOrange resulted in optimal HSA-NanoOrange complex format ion utilizing 1x and 2x NanoOrange in the 96-well microplate and CE-LIF app roaches, respectively. A NanoOrange binding model based on rapid signal enh ancement and zero order fluorescence emission kinetics is proposed. The uti lization of NanoOrange in CE-LIF based human serum analysis results in a si gnal-to-background ratio improvement of up to two orders of magnitude. (C) 2001 Elsevier Science B.V. Ail rights reserved.