Development of an innovative user-friendly colorimetric biosensor based on aptamerfunctionalized nanomaterials for the detection of staphylococcus aureus from different biological sources

Staphylococcus aureus (S. aureus) is a common Gram-positive pathogen causing a wide spectrum of diseases ranging from minor wound to life-threatening infections such as endocarditis, septicemia, pneumonia and toxic shock syndrome. Furthermore this pathogen is present in inadequately treated food and, consequently, widely involved in many cases of enterotoxin-related food poisoning worldwide. Nowadays, antibiotic-resistant strains of S. aureus are seriously challenging global monitoring platforms not only in clinical settings such as in hospitals, but also in whole communities. In some regions the local healthcare organizations failed to control the above infections inducing fatal consequences. For these reasons, rapid methods to identify S. aureus in biological samples are of paramount importance, allowing for a prompt antimicrobial therapy. Currently, the most important detection methods are classical cultures and biochemical tests. However they are time-consuming and not highly sensitive. A novel and highly performing approach for diagnostic applications is offered by aptamers, tailored DNA or RNA segments acting as artificial recognition elements that are able to recognize conserved epitopes on the surface of a bacterium. Moreover, the recent progress in the aptamer selection procedure broadens the library of applicable aptamers ensuring their application for high-throughput screening of a large variety of bacteria. Aptamers interacting with S. aureus are already known, thus providing a solid base for developing promising solutions for this important issue. Taking advantage of this knowledge, in this project we intend to tackle two principal drawbacks of the aptamer-based biosensor available to date: the lack of materials for high density and functional surface immobilization of aptamers and the restriction in the user-friendly read-out of the detected molecules. In order to promote the surface linking, we will adopt as supports
nanostructured titanium and zirconium dioxide
synthesized in our laboratory and we will prepare a detection system that is expected to work
without the use of any expensive detection equipment
. In our previous work we have demonstrated that the increase of nanoscale roughness significantly promotes the absorption of proteins and, in very preliminary tests, we have verified that also aptamers well adhere on nanostructured titanium dioxide supports. We have also verified that they can be deposited by exploiting the microarray technique allowing a rapid and cost effective screening of a large library of various aptamers at different concentrations and/or substrates having spatially confined morphological and chemical properties. However, for a full exploitation of this high-throughput approach and a simple recognition of the presence and the amount of Staphylococcus species, we will use a colorimetric switch which is activated when the aptamer links to the Staphylococcus target. Our strategy will consist in: i) the use of labeled peptide/s with a solvatochromic dye absorbing in the visible region; ii) the hybridization of labeled-peptides with aptamers, that can be then immobilized on nanostructured substrates; iii) the displacement of the labeled-peptides due to the higher affinity of the aptamer for S. aureus and the consequent colorimetric switch.