Development of microarrays for protein and genetic screening with an Electrochemical and Elctrochemiluminescence-based transduction scheme.

Considering the increasing need for sensing elements that is emerging in different fields and applications, particularly in relation to the Internet of Things, electrochemical sensors and biosensors, also for their exceptional attributes, such as being easy-to-operate, economical, sensitive, portable, are candidates to become essential pillars in future scenarios and to play a significant role biomedical and environmental monitoring. Electrochemical and electrochemiluminescent sensors, are devices capable of detecting molecules and biomolecules in solutions and determining the concentration through direct electrical measurements, arising from the change in the redox state of the analyte, and monitored through classical electroanalytical techniques. Remarkably, recent achievements in nanoscience and nanotechnology, have demonstrated the potential for improving greatly both the sensitivity and selectivity of electrochemical sensors and biosensors. In fact, an electrochemical sensor can be miniaturized to a size less than one micrometer or to small-size arrays of nano-electrodes, offering advantages in terms of increased sensitivity and compactness (NEA). The attractiveness of such nanostructured systems resides also in the possibility to immobilized biomolecules on the dielectric surface surrounding each nanoelectrode, instead of on the electrode itself. Therefore, in NEAs, where the electrochemical properties of the electrode and the physical-chemical properties of the dielectric surface may be optimized independently, it may result easier to combine the highly specific molecular recognition mechanisms with high sensitivity (low detection limits). The aim of this work is the development of nanostructured electrochemical and electrochemiluminescence-based sensors for the detection of biomolecules, such as DNA and proteins. The fabrication of arrays of nanoelectrodes was performed by deposition of a thin film of polycarbonate (PC) as insulator on a layer of Boron Doped Diamond (BDD) or Glassy Carbon (GC). NEAs were obtained by creating an array of nanoholes in the polymeric film using two different nanofabrication techniques: electron beam lithography (EBL) and nanoimprint lithography (NIL). These approaches lead to the formation of recessed nanoelectrodes. Particulary, the optimization of the parameters to fabricate NEAs by nanoimprint lithography (NIL) has allowed to reduce time and manufacturing costs. We demonstrate the possibility to efficiently immobilize biomolecules, on the relatively large surface of the PC of our NEAs in order to develop sensitive electrochemical biosensors. Initially, the sensors were tested for the detection of HPV (Human Papilloma Virus) DNA sequences. Later we focused on the optimization of the protocol for the detection of proteins by enzyme immuno-assay with the enzyme Horse Radish Peroxidase (HRP), using Gliadin and Tumor necrosis factor (TNF-α) as target. The process of bio-recognition and detection was carried out by cyclic voltammetry and consists of an immuno-indirect scheme. This method allows to detect a concentration of 0.1 nM for Gliadin fragment and the experiments performed indicate the possibility to further low this limit. The same samples were tested at the Institut des Sciences Molèculaires University of Bordeaux (Group of Nanosystèmes Analitique) using electrochemiluminescence (ECL). In these sensors the transduction scheme involves a secondary antibody labbeled with Biotin, that provides a strong interaction with the complex streptavidin- Ru(bpy)32+. The ECL signal obtained in the presence of the co-reactant tripropylamine (TPrA) was recorded using both a CCD camera and a photomultiplier tube. Furthemore, additional sensors have been prepared using GC as conductive material and PC as insulating layer.