- Dental plaque is one of the well-characterized biofilms in the human body. Oral bacterial species play vital roles in maintaining healthy bacterial homeostasis as well as causing oral infections. Many of the oral diseases are caused by opportunistic pathogens, and therefore, the bacterial metabolic activities become important in dictating their survival and pathogenesis. Chemical changes related to the bacterial metabolic activity are limited to their local environment, which extends only up to a few hundred micrometers away from their biofilm surface. To study those chemical changes, a highly precise analytical technique which can reach to the bacterial local environment is required. Scanning electrochemical microscopy (SECM) is an ideal tool to study bacterial local chemical changes because of its noninvasive nature and its ability to reach into the bacterial local environment without disturbing the bacterial metabolism or their local environment. To use SECM for bacterial metabolic studies, SECM probes with ultra-micro electrodes (UME) which can quantify metabolites or metabolic byproducts with high selectivity and sensitivity are required.
Streptococcus mutans (S. mutans) is the main contributing species for dental caries formation, one of the most prevalent chronic diseases among human across the globe. They catabolize sugars (sucrose, glucose) producing lactic acid, which decreases the pH of their local environment. For probing the metabolic behavior of this bacteria, highly selective, ultra-micro enzymatic glucose and lactate sensors have been developed. Glucose oxidase and lactate oxidase enzymes were covalently immobilized on to 25 µm Platinum (Pt) UMEs modified with a new enzyme immobilization matrix. New immobilizing matrix was made up of functionalized carbon nanotubes and ionic liquid composite. At 37 oC, in artificial saliva solutions, the developed glucose sensor shows a sensitivity of 94.44 (±18.55) µA.mM-1.cm-2 (0.1mM -1.0 mM) with a linear range, up to 4.0 mM and the lactate sensor shows a sensitivity of 139.8 (±10.43) µA.mM-1.cm-2 (10 µM to 0.25 mM) with a linear range up to 1.0 mM. These new sensors have fast response times (~2.0 S) which make them ideal to use in SECM probes for real-time metabolite quantification.
S. mutans’s glucose uptake behavior provides information on their metabolic rate and the effect of their metabolism on other oral bacterial species. To quantify glucose consumption of S. mutans bacteria, a new SECM probe with a glucose microsensor was fabricated by selectively modifying one of the 25 µm Pt UME of a dual-tip SECM probe into the glucose sensor. It was observed that S. mutans glucose consumption decreased the local glucose concentration by 81 %. In a mixed diet condition, where both glucose and sucrose were available, bacterial glucose consumption decreased by 73 %, showing that, the bacteria preferentially uptake sucrose over glucose. The glucose concentration profile extended up to about 600 µm - 800 µm from the biofilm surface, creating a nutrient competitive environment for other species.
Oral bacterial metabolic activity results in multiple chemical changes in their local environment. To study the bacterial metabolic activity related local chemical changes in bacterial environment, multifunctional SECM probes were designed. S. mutans consume carbohydrates producing lactic acid, increasing their local lactate concentration and decreasing the local pH. To quantify their glucose consumption and corresponding local pH change, a new Pt-glucose-pH SECM probe was designed. For this, a SECM probe consists of three 25 µm Pt UMEs were fabricated. One UME was modified into a glucose sensor, and the second UME was modified into a pH sensor by depositing iridium oxide (IrOx). Third Pt UME was used to obtain the probe approach curve. Similarly, for simultaneous quantification of local lactate concentration and corresponding pH, new Pt-lactate-pH probe was fabricated by combing the lactate microsensor, and IrOx deposited pH sensor. S. gordonii bacteria increase local peroxide concentration upon glucose catabolism. To quantify their glucose consumption and corresponding peroxide production, glucose-peroxide-Pt SECM probe was fabricated by combining the glucose sensor with a platinized-Pt peroxide sensor. These newly designed multifunctional SECM probes were successfully used for simultaneous quantification of multiple analytes in the bacterial local environment.