Supersensitive Detection of the Norovirus Immunoplasmon by 3D Total Internal Reflection Scattering Defocus Microscopy with Wavelength-Dependent Transmission Grating.

Norovirus (NoV) is a major foodborne pathogen, and even low levels of virus can cause infection and gastroenteritis. We developed a supersensitive NoV sensor that detects NoV group-I capsid protein (NoVP) via three-dimensional (3D) total internal reflection scattering defocus microscopy (TIRSDM) with wavelength-dependent transmission grating (TG). The combination of evanescent wave scattering and TG significantly enhanced the detection sensitivity and selectivity of NoVP in first-order spectral images (n = +1) by minimizing spectroscopic interference and background noise. In particular, wavelength-dependent 3D defocused TG imaging (3D TG-TIRSDM) separated silver nanotag and gold nanoplate signals on a NoVP immunoplasmon chip along the x, y, and z coordinates simultaneously. Additionally, the use of wavelength-dependent TG increased the spectral resolution by 5-fold along the xy-axis and 1.4-fold along the z-axis compared to conventional 3D TIRSDM at the subdiffraction limit. The NoVP sensor exhibited a lower limit of detection of 820 yM, which is 29 000 times better than the previous potentiometer method, and a wide dynamic detection range of 820 yM to 92.45 pM (R = 0.9801). This new method could be applied to detect various pathogenic viruses during the initial stage of infection.