Development of electrochemical sensors for microbial biofilm and protease activity detection

Chronic wounds affect a large portion of population worldwide and significantly impact the health system and the patient’s quality of life. Biofilm formation in chronic wounds is associated to the wound healing delay. In addition high levels of protease activity further contribute to the wound chronicization, as proteases degrade the components of the extracellular matrix. Electrochemical sensing systems are promising tools as they combine high sensitivity, fast response-time, low cost, and potential for miniaturization. This study was aimed to the development of two electrochemical sensors for the biofilm monitoring and protease activity assessment, using potentiometry and voltammetry as transduction methods. A potentiometric, low-cost, flexible, graphenic sensor was developed. This proved to be able to monitor in real-time the growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacterial species. The OCP value decreased over time as bacteria accumulated on the sensor surface, displaying a significant negative correlation with respect to the bacterial growth. The development of a voltametric sensor for the detection of protease activity was also been afforded. The protease biosensor used gelatin loaded with a redox mediator as the sensing layer. The proteolytic action and consequent gelatin degradation caused the release of redox mediator and made the electron transfer process more difficult, leading to a decrease of the current peak (ip) value as measured by AC voltammetry. The system response upon gelatin degradation was negatively and significantly correlated to the response measured by the standard azocasein assay.