Ultrasensitive Detection of α-Fetoprotein by Total Internal Reflection Scattering-Based Super-Resolution Microscopy for Superlocalization of Nano-Immunoplasmonics.

Superlocalization of immunoplasmonic nanotags on antibody-bound gold-nanoislands (GNIs) along the x and y coordinates was determined using total internal reflection scattering-based super-resolution microscopy (TIRS-SRM) at subdiffraction limit resolution. Individual immunoplasmonic nanotags (20 nm silver nanoparticles) and 100 nm GNIs were selectively acquired in the evanescent field layer by wavelength-dependent plasmonic scattering using two illumination lasers (405 and 635 nm, respectively). α-Fetoprotein (AFP), a liver cancer-related model protein, was immobilized as a target molecule on the GNI arrays. The centroid position of a localized immunoplasmonic nanotag on the GNI was resolved at less than 10 nm of spatial resolution by applying 2D Gaussian fitting to its point spread function. This method showed enhanced sensitive quantification with a limit of detection (LOD) of 7.04 zM (1-2 molecules of AFP/GNI), which was 100-5000000000 times lower than detection limits obtained with previous AFP detection methods. Furthermore, the method was also successfully applied to quantify AFP molecules at the single-molecule level in human serum samples. The wavelength-dependent TIRS-SRM method was demonstrated to be an effective tool for superlocalizing individual protein molecules and interactions in nanoscale regions and was a reliable method for the ultrasensitive quantitative detection of disease-related protein molecules as a nanosensor and for diagnosis at the single-molecule level.