Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces.
We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps.
Matveeva
Evgenia
E
Center for Fluorescence Spectroscopy, Department of Biochemistry Molecular Biology, University of Maryland at Baltimore Medical School, 725 West Lombard Street, Baltimore, MD 21201, USA.
Gryczynski
Zygmunt
Z
Malicka
Joanna
J
Gryczynski
Ignacy
I
Lakowicz
Joseph R
JR
D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D005453:Fluorescence; D005898:Glass; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D012680:Sensitivity and Specificity; D012834:Silver / Q000737:chemistry*; D013057:Spectrum Analysis
D000906:Antibodies; D012834:Silver