New tools for the study of transport kinetics and micro-localisation of critical ions in biofilms

Aim: To develop new approaches to study micro-localisation and dynamic ion flux in biofilms using fluorescent nanosensors. Introduction: Bacterial biofilms are implicated in the onset of many oral diseases such as caries, gingivitis and halitosis. These biofilms, often referred to as dental plaque, are attached to both oral hard and soft tissues and whilst daily brushing of the teeth removes a significant amount of these biofilms, their rapid re-growth and the presence of undisturbed biofilm located in areas not amenable to brushing means that plaque is ubiquitous. A typical dental plaque consists of a multi-species biofilm containing both aerobic and anaerobic species. Acidophiles such as Streptococcus mutans metabolise fermentable sugars to acids, particularly lactic acid. The pH of these plaques is therefore variable on diet and it has been repeatedly shown that the pH of plaque falls rapidly after consumption of sugar, rising after a period of time as the acid is neutralised by salivary buffers. During this low-pH period the enamel surface of the tooth is vulnerable to dissolution of the calcium-containing mineral hydroxyapatite that makes up 95% of the enamel composition. Repeated pH challenge of the enamel eventually leads to the formation of a carious lesion and ultimately cavitation of the tooth, requiring dentist-intervention (drilling and filling). The process of enamel dissolution though is dependent on many factors other than pH, including the concentration of calcium and fluoride ions within the biofilm. Other ions, such as zinc, which is present in many types of toothpaste, may also have an impact on both plaque ecology and enamel dissolution. Current models of oral plaques are useful tools that have been used to understand the concentrations of the critical ions (Zn2+, H+, F-, and Ca2+) in plaque in total, although a detailed understanding of both the transport kinetics under dynamic conditions and micro-localisation of the ions within the biofilm have yet to be determined. It is generally believed that the plaque is able to act as a reservoir for these ions, although the form and location of the ions within the plaque has yet to be determined. Research Programme: In this work we will incorporate fluorescent chemo-sensing nanoparticles (PEBBLE nanosensors) within model oral biofilms grown on enamel and map, in real-time, concentration profiles of the critical ions. PEBBLEs sensitive to zinc, calcium and pH have already been prepared and a fluoride-sensitive PEBBLE will be developed as part of this work. Ion-concentrations will be mapped both in static biofilms, and in dynamic biofilms, where glucose will be used to generate acid in situ, mimicking a real carious challenge. Further the effects of added fluoride and zinc ions on the biofilm calcium concentration will be examined under a variety of conditions, including investigation of approaches that increase plaque-fluoride uptake. It is believed that this work will contribute greatly towards the understanding of ion-transport through biofilms in general, and in oral biofilms in particular. This work should also enable a more thorough understanding of the caries process and how oral healthcare actives can influence caries development.