Detection of Bacterial Activity via Reduction of Prussian Blue Thin Films as Probed by Raman Spectroscopy

Biomarker-free and in situ detection of microbes and microbial biofilms is an important challenge. Prussian Blue (PB) has emerged as a potential material for a variety of biological applications. It is safe, sustainable, and also versatile, being employed as nanocarriers for drug delivery, antidotes for Cs and Tl poisoning, in photothermal therapy, and for the detection of live-dead bacteria. In this work, the ability of electrodeposited Prussian Blue (PB) thin films to detect bacterial activity was investigated using <i>Staphylococcus aureus</i> biofilm growth on the PB surface. The conversion of PB to Prussian White (PW), the reduced form of PB, due to the metabolic activity of <i>S. aureus</i>, was tracked using Raman microspectroscopy, enabling both time- and spatially resolved effects to be observed during biofilm growth. These results reveal that Raman spectroscopy can detect the onset of the reduction of the PB films after about 2.5 h of incubation time with <i>S. aureus</i> at 10⁷ CFU mL^-1. In addition, localized Raman mapping of the PB surface reveals that the level of PB reduction varies across the surface in response to the local bacterial biofilm presence and activity. This work shows that Raman spectroscopy detection of the reduction of PB films by bacterial activity is a sensitive, direct, and nondestructive way of sensing the presence of live and metabolically active microbes in a localized way.