Simultaneous Chemical Mapping of Live Biofilm Microenvironmental pH and Hydrogen Peroxide in Real Time with a Triple Scanning Electrochemical Microscopy Tip

Dental plaque biofilm is a complex ecosystem. The distribution of microbial species in the biofilm is heavily influenced by local chemical interactions that result from diverse metabolic activities and the nature of the released molecules. As a relevant example, H₂O₂-producing bacteria can antagonize disease-associated bacteria, leading to the maintenance of a healthy oral microbiome. Herein, we report the development of a triple-sensor (redox, pH, and H₂O₂) scanning electrochemical microscopy (SECM) tip capable of simultaneously mapping the pH and H₂O₂ concentration produced by a dental plaque-derived multispecies biofilm grown on hydroxyapatite. The pH sensor of the triple SECM tip showed a near Nernstian slope of −71.1 ± 2 mV/pH (N = 3), whereas the H₂O₂ sensor showed a slope of −0.052 ± 0.002 nA/μM H₂O₂ at pH 7.2 and a detection limit of 1.0 ± 0.2 μM (N = 7). There is no significant difference in the sensitivities of H₂O₂ sensors at pH 6.2, 7.2, and 8.2 at 95% CI (N = 7). The pH and H₂O₂ sensors demonstrated excellent reversibility with response times of 3 and 5 s, respectively, along with reliable stability over 4 h at 37 °C. The sensors did not show any cross talk between pH and H₂O₂ concentration ([H₂O₂]) measurements, highlighting the accuracy and versatility of the SECM tip. Simultaneous chemical imaging of pH and [H₂O₂] across the biofilm revealed a clustered distribution of local H₂O₂ concentrations, ranging from 0 to 17 μM. Conversely, the local pH remained constant at 7.2. The relation of local chemical profiles and the distribution of bacterial species within the oral microbiome was experimentally investigated in the context of bacterial H₂O₂ antagonism. The benefit of clustered H₂O₂ production was that the total area of H₂O₂ produced by smaller clusters was 67% more than that of a single cluster with the same starting number of bacteria. Thus, this triple SECM tip can potentially be used to study local molecular mechanisms that result in dysbiosis of the oral microbiome.