TY - JOUR
T1 - Directional surface plasmon-coupled emission
T2 - Application for an immunoassay in whole blood
AU - Matveeva, Evgenia G.
AU - Gryczynski, Zygmunt
AU - Malicka, Joanna
AU - Lukomska, Joanna
AU - Makowiec, Slawomir
AU - Berndt, Klaus W.
AU - Lakowicz, Joseph R.
AU - Gryczynski, Ignacy
N1 - Funding Information:
This work was supported by the National Center for Research Resources (RR-08119), the National Institute of Biomedical Imaging and Bioengineering (EB-1690), Philip Morris USA and Philip Morris International, and the Biomolecular Interaction Technology Center (University of New Hampshire).
PY - 2005/9/15
Y1 - 2005/9/15
N2 - We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. 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 bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. 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 was studied. It was demonstrated that 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 two- and threefold, respectively, as compared with 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. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. 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 with no need for washing steps.
AB - We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. 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 bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. 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 was studied. It was demonstrated that 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 two- and threefold, respectively, as compared with 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. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. 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 with no need for washing steps.
KW - Background suppression
KW - Fluorescence immunoassay
KW - Silver film
KW - Surface plasmon-coupled emission
KW - Whole blood
UR - http://www.scopus.com/inward/record.url?scp=23944457867&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2005.07.005
DO - 10.1016/j.ab.2005.07.005
M3 - Article
C2 - 16091280
AN - SCOPUS:23944457867
SN - 0003-2697
VL - 344
SP - 161
EP - 167
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -