Bioconjugation of enzymes and proteins on multifunctional and nanostructured solid supports for biomolecular interactions monitoring

Biosensors are interesting tools in various biotechnological fields, because of their versatility in fields such as diagnostics, development of biomedical devices and monitoring of pollutants in drinking water. Biosensors are constituted by an optical (or electrical, mechanical, etc.) transducer, which constitutes the sensitive element, and a biomolecular probe, which gives specificity to the device, because it is able to selectively bind target molecules. Both elements are the subject of this thesis, that studies the properties of different materials and characterization techniques for biosensing devices development. Nanostructured materials have physical and chemical properties arising interesting peculiarities (such as photoluminescence, reflectivity, etc.) that make them the fundamental building blocks for the development of the next generation of tools and devices for biotechnological applications. In particular, the following two types of nanostructured material were studied: • porous silicon (PSi); • gold nanoparticles (AuNPs). Further item of the present thesis is the bioconjugation of molecular bioprobes. It is well known that there are several methods of bioconjugation useful in the development of nano-bio-systems for sensor applications. Self-assembling materials, found in nature, are extremely interesting as functional interfaces between the artificial transducers and the living world. These interfaces do not require any chemical procedure, nor special equipment for their deposition. Along with the self-assembling materials, essential is the study of the use of aminosilanes and crosslinker. Moreover, it is well known that an optimization study of bioprobes immobilization is a hot topic in biosensors study. Therefore, objectives of this PhD project are: • Study of spotting conditions of bioconjugation onto nanostructured materials by two strategies: o immobilization of biomolecules by covalent approach (using aminosilane and crosslinker); o immobilization by non-covalent approach due to self-assembling of proteins; • Development of biosensors for biomedical and environmental applications. Molecular interactions monitored by label-free optical techniques performed onto different nanostructured materials found application in both biomedical and environmental monitoring fields. Interesting devices were finally obtained: • glucose interaction monitoring with an affinity constant of about 40mM; • PSA-Anti/PSA interaction monitoring with a sensitivity of 37±2 cm-1/mgL-1; • reversible device for lead ions detection in aqueous environment were developed, with a LOD of 2ppb (5 times lower then World Health Organization legal limit for drinking water); • naked eye assay for arsenic ions speciation (also at 85μM)