Sensitive bioimaging in microfluidic channels on the plasmonic substrate: Application of an enhanced fluorescence based on the reverse coupling mode

Grating-coupled surface plasmon-field enhanced fluorescence (GC-SPF) was applied to biosensing. Although the greatest enhancement of GC-SPF on our plasmonic chips compared with that on a glass slide was found to be 40 times, this was due to the enhanced excitation field. Therefore, grating-coupled fluorescence using a reverse coupling mode is here explored to achieve further 4–5 times enhancement. As a result of using both the excitation field enhanced by grating-coupled surface plasmon resonance and the directional emission enhanced by reverse coupling mode, an increase in fluorescence of more than 110 times compared with that on the glass slide was recorded. The reverse coupling mode was also applied to the sensitive fluorescence microscopic imaging of Cy5-streptavidin (Cy5-SA) in microfluidic channels on a two dimensional nanohole array substrate. We performed a Cy5-SA concentration series analysis in which the plasmonic substrate demonstrated 26.3× enhancement of sensitivity and a limit of detection (LOD) of ca. 100 pM, which is at least one order of magnitude lower than in glass slides with identical surface chemistry. This plasmonic nanostructure will be invaluable for colorimetric detection in applications such as microfluidic enzyme-linked immune-sorbent assay (ELISA) device, and portable microarray biosensing, because the optical setup can be simplified.

► Grating-coupled surface plasmon-field enhanced fluorescence was applied to biosensing.
► The reverse coupling mode contributed to the sensitive fluorescence imaging.
► Plasmonic chip and microfluidic channels achieved a sensitive imaging under microscope.