Reflected light microspectroscopy for single-nanoparticle biosensing.

Conventional and dark-field microscopy in the transmission mode is extensively used for single plasmonic nanoparticle (NP) imaging and spectral analysis. However, application of the transmission mode for realtime biosensing to single NP poses strict limitations on the size and material properties of the microfluidic system. This article proposes a simple optical technique based on reflected light microscopy to perform microspectroscopy of a single NP placed in a conventional, nontransparent liquid delivery system. The insertion of a variable spot diaphragm in the optical path reduces the interference effect that occurs at the NP-substrate interface and improves the signal-to-noise ratio in NP imaging. Using this method, we demonstrated spatial imaging and spectral analyses of 60-, 80-, and 100-nm single gold NPs. A single-NP sensor based on a 100-nm NP was used for real-time measurement of bulk refractive index changes in the microfluidic channel and for detection of fast dynamic poly(ethylene glycol) attachment to the NP surface. Finally, electrochemical single-particle microspectroscopy was demonstrated by using a methylene blue electroactive redox tag. The proposed optical approach is expected to significantly improve the miniaturization and multiplexing capabilities of high-throughput biosensing based on single NP.