Total internal reflection plasmonic scattering-based fluorescence-free nanoimmunosensor probe for ultra-sensitive...

Highly sensitive detection of cancer antigen 125 (CA125) on nanoarray chips was carried out by means of total internal reflection (TIR) microscopy based on fluorescent labeling (i.e., TIR fluorescence microscopy; TIRFM) and fluorescent-free labeling (TIR scattering microscopy; TIRSM). TIR plasmonic scattering of nanoparticles (NPs) as a fluorescence-free immunosensor probe potentially superior to fluorescent probes was applied to quantify CA125 on a nanoarray chip. NP-labeled CA125 (NP-CA125) was immunoreacted on chips, and the TIR scattering illumination of NP-CA125 allowed quantitative TIRSM measurement of wavelength-dependent plasmonic scattering detection of CA125. In addition, Alexafluor 488-labeled CA125 was immunoreacted on the same chips for comparison of detection sensitivity. TIRSM showed less photobleaching and higher photostability and detection sensitivity than TIRFM, as well as a lower limit of detection (LOD), 0.0018U/mL. This LOD was ~144 times lower than that of previously reported detection methods. These results demonstrated that the wavelength-dependent TIR plasmon NPs can be used as an enhanced nanoimmunosensor probe, providing ultra-sensitive fluorescence-free biomolecule detection to enable earliest-stage disease diagnosis.