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Proceedings Paper

Ultrasensitive detection in optically dense physiological media: applications to fast reliable biological assays
Author(s): Evgenia G. Matveeva; Ignacy Gryczynski; Klaus W. Berndt; Joseph R. Lakowicz; Ewa Goldys; Zygmunt Gryczynski
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Paper Abstract

We present a novel approach for performing fluorescence immunoassay in serum and whole blood using fluorescently labeled anti-rabbit IgG. This approach, which is based on Surface Plasmon-Coupled Emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bio-affinity surface. Effective coupling range for SPCE is only couple of hundred nanometers from the metallic surface. Excited fluorophores outside the coupling layer do not contribute to SPCE, and their free-space emission is not transmitted through the opaque metallic film into the glass substrate. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal is discussed. The kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately 2- and 3-fold, respectively (compared to buffer), resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Both glass and plastic slides can be used for SPCE-based assays. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood, without any need for washing steps.

Paper Details

Date Published: 27 February 2006
PDF: 9 pages
Proc. SPIE 6092, Ultrasensitive and Single-Molecule Detection Technologies, 60920K (27 February 2006); doi: 10.1117/12.648112
Show Author Affiliations
Evgenia G. Matveeva, Univ. of North Texas, Health Science Ctr. (United States)
Univ. of Maryland, Baltimore (United States)
Ignacy Gryczynski, Univ. of North Texas, Health Science Ctr. (United States)
Klaus W. Berndt, Becton Dickinson Diagnostic Systems (United States)
Joseph R. Lakowicz, Univ. of Maryland, Baltimore (United States)
Ewa Goldys, Macquarie Univ. (Australia)
Zygmunt Gryczynski, Univ. of North Texas, Health Science Ctr. (United States)
Univ. of Maryland/Baltimore (United States)

Published in SPIE Proceedings Vol. 6092:
Ultrasensitive and Single-Molecule Detection Technologies
Jörg Enderlein; Zygmunt K. Gryczynski, Editor(s)

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