Development of electrochemical biosensors for human infectious diseases diagnosis

Human infectious diseases affect a large portion of population worldwide and significantly impact the health system and the human quality of life. For all infectious diseases the rapidity of diagnosis and the choice of the correct treatment is a key element for their management. In particular, it is crucial in healthcare associated infections (HAIs), especially when dealing with immunocompromised patients, because these types of infections can lead to a sepsis state. The early identification of the etiological elements plays a crucial role in the infection management, but also it is important to consider the products of the biochemical pathways triggered by the innate and adaptive system which can be useful biomarkers to obtain valuable information regarding the ongoing of the infection or the treatment efficiency. Unfortunately, the conventional techniques are not able to promptly detect these targets, so there is an urgent need to encourage companies and the scientific community to conceive new detection strategies to be implemented in portable devices allowing a rapid, specific and selective identification in the clinics. Electrochemical sensors are promising devices able to respond point of need applications due to their short response time, low-cost, simplicity in fabrication and high potential sensitivity. In this sense, the present work describes the development of two electrochemical biosensors, for bacteria and antibodies detection. First, the development of a CRISPR/cas12a based biosensor for DNA bacterial detection using EIS as transduction technique is described, this system exploited the programmability and selectivity of Cas12a/gRNA endonuclease system. A gram negative (Escherichia coli) and a gram-positive (Staphylococcus aureus) bacterial species known to be associated to BSIs were detected by comparing the charge transfer resistance (Rct) signal change of before and after the enzymatic activity in a label free ssDNA modified gold electrode surface. The second part of the project was aimed to develop a voltametric sensor for the single-step detection of antibodies. This biosensor used a collisional detection mechanism provided by a rationally designed Y-shaped DNA nanostructure scaffold. The sensor was tested using three different model systems (to detect anti-digoxigenin antibody, anti-dinitrophenol antibody and streptavidin), showing the ability to detect the targets at low nanomolar concentrations with a low response time (less then 15 min) in undiluted biological matrices (artificial saliva and human serum). This system may become a promising tool for the rapid quantification of antibodies and small molecules in complex samples, as it does not require any sample pretreatment. In this study two biosensors offering information about different aspects of human infectious disease management were developed. Their use as point of care devices may reduce the time between the diagnosis and treatment. As a whole, these systems may represent useful tools helping the healthcare professional’s decision and improving the quality of the global health surveillance and the clinical practice