High time resolution and high signal-to-noise monitoring of the bacterial growth kinetics in the presence of plasmonic nanoparticles.

Emerging concepts for designing innovative drugs (i.e., novel generations of antimicrobials) frequently include nanostructures, new materials, and nanoparticles (NPs). Along with numerous advantages, NPs bring limitations, partly because they can limit the analytical techniques used for their biological and in vivo validation. From that standpoint, designing innovative drug delivery systems requires advancements in the methods used for their testing and investigations. Considering the well-known ability of resazurin-based methods for rapid detection of bacterial metabolisms with very high sensitivity, in this work we report a novel optimization for tracking bacterial growth kinetics in the presence of NPs with specific characteristics, such as specific optical properties. Arginine-functionalized gold composite (HAp/Au/arginine) NPs, used as the NP model for validation of the method, possess plasmonic properties and are characterized by intensive absorption in the UV/vis region with a surface plasmon resonance maximum at 540 nm. Due to the specific optical properties, the NP absorption intensively interferes with the light absorption measured during the evaluation of bacterial growth (optical density; OD600). The results confirm substantial nonspecific interference by NPs in the signal detected during a regular turbidity study used for tracking bacterial growth. Instead, during application of a resazurin-based method (Presto Blue), when a combination of absorption and fluorescence detection is applied, a substantial increase in the signal-to-noise ratio is obtained that leads to the improvement of the accuracy of the measurements as verified in three bacterial strains tested with different growth rates (E. coli, P. aeruginosa, and S. aureus). Here, we described a novel procedure that enables the kinetics of bacterial growth in the presence of NPs to be followed with high time resolution, high sensitivity, and without sampling during the kinetic study. We showed the applicability of the Presto Blue method for the case of HAp/Au/arginine NPs, which can be extended to various types of metallic NPs with similar characteristics. The method is a very easy, economical, and reliable option for testing NPs designed as novel antimicrobials.