Abstract
BACKGROUND: The local pH change mediated by the pathogenic bacterial species Streptococcus mutans plays a significant role in the corrosion of hydroxyapatite (HA) present in the tooth in the dynamic oral cavity. The acid produced by the bacteria decreases the local pH and releases Ca(2+) ions from the HA. We studied the bacteria-mediated demineralization of HA by scanning electrochemical microscopy (SECM) after growing S. mutans biofilm on HA for 7 days. RESULTS: We notably developed a triple-function SECM-compatible tip that could be positioned above the biofilm. It can also measure the pH and [Ca(2+)] change simultaneously above the biofilm-HA substrate. The triple-function SECM tip is a combination of a potentiometric pH sensor deposited with iridium oxide and a dual-function carbon-based Ca(2+) ion-selective membrane electrode with a slope of 67 mV/pH and 34.3 mV/log [Ca(2+)], respectively. The distance-controlled triple-function SECM tip monitored real-time pH and [Ca(2+)] changes 30 μm above the S. mutans biofilm. The high temporal resolution pH data demonstrated that after approximately 20 min of sucrose addition, S. mutans started to produce acid to titrate the solution buffer, causing a pH change from 7.2 to 6.5 for HA and from 7.2 to 5 for the glass substrate. We observed that, after 30 min of acid production, ∼300 μM of Ca(2+) ions were increased at pH 6.5 above the biofilm surface as a result of the pH change in the local microenvironment. After the release of Ca(2+) from HA, the pH environment again shifted toward the neutral side, from 6.5 to 7.2. Therefore, precipitation of Ca(2+) happens at the top of the biofilm, thus corroding the HA from underneath. For a glass substrate, in contrast, no Ca(2+) ions were released, and the pH did not change back to 7.2. We were able to observe the dynamics of the HA demineralization-remineralization process simultaneously with our newly developed triple-function SECM tip or microprobe. SIGNIFICANCE: This technique could notably advance the study of similar complex processes, such as bacteria-mediated corrosion in biomedical and environmental contexts.