0_7503 Rapid detection of viruses in biological or environmental samples represents an unmet need for the containment of infectious diseases [1].  The main diagnostic tools exploited in the recent SARS-CoV-2 outbreak were based on the detection of viral genome in centralized laboratories and virus proteins by rapid lateral-flow devices. An effective method to quantify the amount of viral particles exhaled by a person or present in swabs, saliva, or environment is still missing. In this framework, we designed a novel label-free optical biosensor [2] enabling the enumeration of single virion particles binding on antibody or aptamers immobilized on a surface in a micro-array format. The detection of single nano-particles with diameter of 100 nm over a wide field of view is achieved by a fine tuning of the interference between the light scattered by the particle and the double reflection of an anti-reflective coating. In this regime, nanoparticles are detected not through the light they diffuse, but through the light they subtract to the incoming wave because of diffusion. This term originates from the often overlooked radiation reaction effect on an oscillating dipole. A trough-the-objective detection scheme was realized with simple optical components and a narrow spectra LED source was employed to record time-lapse videos [2]. By choosing the correct wavelength and by using custom image-difference analysis algorithm we could quantify, by enumeration, virus-like nanoparticles and delineate statistics of contrast, rates of binding events and binding lifetime for every experiment at different analyte concentrations. After a careful optimization, different variants of SARS-CoV-2 virions interacting with antibodies and aptamers immobilized on different spots were then tested in the biosensor. For all tested variants, the aptamers showed larger affinity but lower specificity relative to the antibodies. The combination on the biosensor surface of different probes and a fine tuning of the analysis technique resulted in a low limit of detection (c.a. fM) as well as a linear 3 order of magnitude dynamical range.[1] Nava, G., Zanchetta, G., Giavazzi, F. & Buscaglia, M. “Label-free optical biosensors in the pandemic era”. Nanophotonics 11, 4159–4181 (2022).[2] Nava, G. et al. “Digital Detection of Single Virus Particles by Multi-Spot, Label-Free Imaging Biosensor on Anti-Reflective Glass”. Small 2300947, (2023).