Enhanced detection sensitivity of carcinoembryonic antigen on a plasmonic nanoimmunosensor by transmission grating-based total internal reflection scattering microscopy.

Carcinoembryonic antigen (CEA) is a glycoprotein associated with colorectal carcinomas and is commonly used as a clinical tumor marker. Enhanced detection sensitivity for the assay of CEA molecules was achieved on a plasmonic nanoimmunosensor by wavelength-dependent transmission grating (TG)-based total internal reflection scattering microscopy (TIRSM). The plasmonic nanoparticles were placed in an evanescent field layer on a glass nanoimmunosensor that produced evanescent wave scattering by the total internal reflection of light from two lasers. The light scattered by target protein (CEA)-bound 20-nm silver nanoparticles (plasmonic nanoprobes) was collected and spectrally isolated in first-order spectral images (n=+1) by a TG (70 grooves/mm). The combination of evanescent wave scattering and TG ​significantly enhanced the detection sensitivity and selectivity due to the minimized spectroscopic interference and background noise. The TG-TIRSM method detected the CEA molecules at concentrations down to 19.75zM with a wide linear dynamic range of 19.75zM-39.50nM (correlation coefficient, R=0.9903), which was 45 to 1.25×10 times lower than the detection limits and 2×10 to 2×10 times wider than the dynamic ranges of previous assay methods. In particular, by simply changing the antibody of the target molecule, this technique can be used to detect various disease-related protein biomarkers directly in human biological samples at the single-molecule level.