Limit of detection of Raman spectroscopy using polystyrene particles from 25 to 1000 nm in aqueous suspensions

Raman spectroscopy is an analytical method capable of detecting various microorganisms and small particles. Here, we used 25-1000 nm polystyrene particles in aqueous suspensions, which are comparable in size to viral particles and viral aggregates, to determine the limit of detection of a confocal Raman microscope. We collected Raman spectra using a 785 nm wavelength laser with a power of 300 mW and a 10 s exposure time, with a 5X objective lens. We detected the most prominent peak of the polystyrene particles at 1001 cm^-1, corresponding to the ring breathing mode. We established the minimum and maximum limit of detection (LOD_minand LOD_max) using a partial least squares (PLS) model. The LOD of the smallest size of 50 nm was identified as 7.47 × 10¹²–7.64 × 10¹²particle/mL, and for the largest size of 1000 nm, 6.09 × 10⁸–6.24 × 10⁸particle/mL. We demonstrated that Raman spectroscopy was non-destructive under these conditions by comparing the particle size before and after collecting Raman spectra using dynamic light scattering. Due to their size similarity to viral particles and viral aggregates, this systematic characterization of polystyrene particles provides detailed information on their Raman spectral signatures in aqueous suspensions. These findings establish a foundation for using Raman spectroscopy for the detection of small particles in aqueous suspensions and highlight its potential as a tool for real-time monitoring in vaccine manufacturing.