Quantification of Antibiotic Diffusion in Biofilms Using Gold Nanostar Surface‐enhanced Raman Spectroscopy

Abstract The increased resistance to antibiotics shown by bacteria in biofilms is believed to be partly the result of the limited penetration of antibiotics. However, there are no well‐established techniques which allow quantitative, label‐free monitoring of antibiotic transport in biofilms. Here, it is shown that surface‐enhanced Raman spectroscopy (SERS) with gold nanostars (NS) can be used for the detection of levofloxacin (Levo) in Staphylococcus aureus biofilms at clinically relevant concentrations. Ex situ studies showed that although matrix interference reduced the sensitivity compared to aqueous solutions, quantitative detection remained possible. With intact biofilms, monitoring the SERS signals from layers of NS embedded at specific depths allowed the time‐dependence of the penetration of Levo from the surface to the embedded layer to be measured and the diffusion coefficient of Levo to be calculated. The measured value of D = 2.79 ± 0.79 × 10 ⁻⁹ cm ² s ⁻¹ is over three orders of magnitude lower than in aqueous solutions. This work is the first demonstration that SERS can be a powerful method for investigating antibiotic transport in biofilms, offering new insights into resistance mechanisms and supporting the development of more effective antimicrobial strategies.