Md. Harvey et al., Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool - Nanomolar human serum albumin quantitation, J CHROMAT B, 754(2), 2001, pp. 345-356
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.