Plastic versus glass support for an immunoassay on metal-coated surfaces in optically dense samples utilizing directional surface plasmon-coupled emission.
We compared plastic (polycarbonate) and high-quality glass support materials for gold-coated slides, when performing a model immunoassay against rabbit IgG using fluorescently labeled (AlexaFluor-647) anti-rabbit IgG, and detecting surface plasmon-coupled emission (SPCE) signals. Both, glass and plastic slides were simultaneously coated with a 48-nm layer of gold and protected with a 10-nm layer of silica. The maximum SPCE signal of AlexaFluor-647 was only two- to three-fold smaller on plastic slides than on glass slides. A small difference in the SPCE angles on glass (theta (F) = 55 degrees ) and plastic (theta (F) = 52.5 degrees ) slides was observed and can be explained with a slightly smaller refractive index of the plastic. We have not found any difference in the angle distribution (sharpness of the fluorescence signal at optimal SPCE angle) for the plastic slide compared to the glass slide. The kinetics of binding was monitored on the plastic slide as well as on the glass slide. Optically dense samples, a 4% red blood cell suspension and a 15% hemoglobin solution, are causing a reduction in the immunoassay SPCE signal by approximately 15% and three times, respectively, and the percentage of the reduction is the same for plastic and for glass slides. We believe that plastic substrates can be readily used in any SPCE assay, with only marginally lower total signal compared to high-quality glass slides.
Matveeva
Evgenia G
EG
Center for Fluorescence Spectroscopy, University of Maryland at Baltimore, School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, Maryland 21201, USA. evgenia_matveeva@hotmail.com
Gryczynski
Ignacy
I
Malicka
Joanna
J
Gryczynski
Zygmunt
Z
Goldys
Ewa
E
Howe
Joseph
J
Berndt
Klaus W
KW
Lakowicz
Joseph R
JR
D000818:Animals; D001773:Blood Cells / Q000276:immunology; D005455:Fluorescent Antibody Technique; D015200:Fluoroimmunoassay / Q000379:methods*; D005898:Glass / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006454:Hemoglobins / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D055095:Optics and Photonics; D011076:Polycarboxylate Cement / Q000737:chemistry*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D013499:Surface Properties
D006454:Hemoglobins; D007074:Immunoglobulin G; D011076:Polycarboxylate Cement; C005506:polycarbonate; D006046:Gold