Ultrasensitive norovirus nanoimmunosensor based on concurrent axial super-localization of ellipsoidal point spread function by 3D light sheet microscopy

Abstract

A fluorescence-free ultrasensitive norovirus nanoimmunosensor was investigated based on gradient-fitting super-localization by three-dimensional (3D) dual-view light sheet microscopy (DV-LSM). A highly contagious norovirus was detected by immunoreaction with 100-nm gold nanospots (AuNSs) and 40-nm silver nanoparticles (AgNPs) as imaging probes. Dual-view setup was added to the optical path to simultaneously image AgNP-labelled norovirus and AuNSs in real time. A cylindrical lens and dual-view identified axial distance differences between AuNS and AgNP images as a function of an elliptically distorted point spread function (PSF). Differences between PSFs in each plane at 10 nm slicing intervals were distinguished by gradient-fitting algorithm-based super-localization. This approach showed highly enhanced detection sensitivity and super-localization in the axial direction for norovirus. The axial distance between AuNS and AgNP in norovirus immnosensor was 23 ± 3 nm for 7.8 zM, and the distance increased with increased concentration. The method had an excellent limit of detection (LOD) of 7.8 zM with a wide linear dynamic range of 7.8 zM–240 aM. The LOD was 106 to 2,300,000 times lower than previous methods. Norovirus was detected in lettuce leaf extraction at 14.3 zM with 99.87% recovery. The 3D DV-LSM method with gradient-fitting super-localization was an effective method for ultra-trace norovirus detection at the single-molecule level with highly precise axial resolution.