Ultrasensitive detection in optically dense physiological media: Applications to fast reliable biological assays

Evgenia G. Matveeva, Ignacy Gryczynski, Klaus W. Berndt, Joseph R. Lakowicz, Ewa Goldys, Zygmunt Gryczynski

Research output: Contribution to journalConference article

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.

Original languageEnglish
Article number60920K
JournalProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume6092
DOIs
StatePublished - 29 Jun 2006
EventUltrasensitive and Single-Molecule Detection Technologies - San Jose, CA, United States
Duration: 21 Jan 200624 Jan 2006

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Biological Assay
Assays
Immunoassay
Fluorophores
blood
Rabbits
Serum
Blood
serums
immunoassay
rabbits
Buffers
Antigens
antigens
Silver
Glass
Anti-Idiotypic Antibodies
Hemoglobins
buffers
Immunoglobulin G

Cite this

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title = "Ultrasensitive detection in optically dense physiological media: Applications to fast reliable biological assays",
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.",
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Ultrasensitive detection in optically dense physiological media : Applications to fast reliable biological assays. / Matveeva, Evgenia G.; Gryczynski, Ignacy; Berndt, Klaus W.; Lakowicz, Joseph R.; Goldys, Ewa; Gryczynski, Zygmunt.

In: Progress in Biomedical Optics and Imaging - Proceedings of SPIE, Vol. 6092, 60920K, 29.06.2006.

Research output: Contribution to journalConference article

TY - JOUR

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T2 - Applications to fast reliable biological assays

AU - Matveeva, Evgenia G.

AU - Gryczynski, Ignacy

AU - Berndt, Klaus W.

AU - Lakowicz, Joseph R.

AU - Goldys, Ewa

AU - Gryczynski, Zygmunt

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