Abstract
A functional modification of the surface of a 96-well microplate coupled with a thin layer deposition technique is demonstrated for enhanced fluorescence-based sandwich immunoassays. The plasma polymerization technique enabling the deposition of organic thin films was employed for the modification of the well surface of a microplate. A silver layer and a plasma-polymerized film were consecutively deposited on the microplate as a metal mirror and the optical interference layer, respectively. When Cy3-labeled antibody was applied to the wells of the resulting multilayered microplate without any immobilization step, greatly enhanced fluorescence was observed compared with that obtained with the unmodified one. The same effect could be also exhibited for an immunoassay targeting antigen directly adsorbed on the multilayered microplate. Furthermore, a sandwich immunoassay for the detection of interleukin 2 (IL-2) was performed with the multilayered microplates, resulting in specific and 88-fold–enhanced fluorescence detection.
Highlights
The fluorescence-based immunoassay is widely used in biotechnology and clinical testing.Antibodies or antigenic proteins can be labeled with a variety of fluorophores for fluorescence detection
The increased fluorescence intensity can be attributed to an optical phenomenon called metal-enhanced fluorescence (MEF) induced by localized surface plasmon resonance (LSPR) on metal nanoparticles
It was highly possible that the deposition rate for sputtering or plasma polymerization on the well surface of a microplate was different from that of a planar glass slide due to its deep well structure
Summary
The fluorescence-based immunoassay is widely used in biotechnology and clinical testing. A multilayered glass slide modified with a plane metal mirror coated with an optical interference layer is a simple and promising tool for fluorescence enhancement. The multilayered substrate fabricated with approximately 100-nm-thick dielectric LiF as an optical interference layer on the surface of a silver mirror can exhibit up to 400-fold enhancement of fluorescence for Rhodamine B compared with a bare glass slide [9,10]. Al2 O3 [12,13], have been utilized for the optical interference layers Those modified substrates have been applied in many bioassays [14,15,16]. We have recently focused on plasma-polymerized films (PPFs) as excellent alternatives to SiO or Al2O3 for multilayered substrates.
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