Development of new immunochemical and spectroscopic analytical methods for the characterization and conservation of works of art

Cultural heritage is constituted by complex and heterogenous materials, such as paintings but also ancient remains. However, all ancient materials are exposed to external environment and their interaction produces different changes due to chemical, physical and biological phenomena. The organic fraction, especially the proteinaceous one, has a crucial role in all these materials: in archaeology proteins reveal human habits, in artworks they disclose technics and help for a correct restoration. For these reasons the development of methods that allow the preservation of the sample as much as possible and a deeper knowledge of the deterioration processes is fundamental. The research activities presented in this PhD thesis have been focused on the development of new immunochemical and spectroscopic approaches in order to detect and identify organic substances in artistic and archaeological samples. Organic components could be present in different cultural heritage materials as constituent element (e.g., binders in paintings, collagen in bones) and their knowledge is fundamental for a complete understanding of past life, degradation processes and appropriate restauration approaches. The combination of immunological approach with a chemiluminescence detection and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry allowed a sensitive and selective localization of collagen and elements in ancient bones and teeth. Near-infrared spectrometer and hyper spectral imaging have been applied in combination with chemometric data analysis as non-destructive methods for bones prescreening for the localization of collagen. Moreover, an investigation of amino acids in enamel has been proposed, in order to clarify teeth biomolecules survival overtime through the optimization and application of High-Performance Liquid Chromatography on modern and ancient enamel powder. New portable biosensors were developed for ovalbumin identification in paintings, thanks to the combination between biocompatible Gellan gel and electro-immunochemical sensors, to extract and identify painting binders with the contact only between gel and painting and between gel and electrodes.

Chemiluminescence – based bioanalytical devices for astrobiological, food safety and clinical applications

At the intersection of biology, chemistry, and engineering, biosensors are a multidisciplinary innovation that provide a cost-effective alternative to traditional laboratory techniques. Due to their advantages, biosensors are used in medical diagnostics, environmental monitoring, food safety and many other fields. The first part of the thesis is concerned with learning the state of the art of paper-based immunosensors with bioluminescent (BL) and chemiluminescent (CL) detection. The use of biospecific assays combined with CL detection and paper-based technology offers an optimal approach to creating analytical tools for on-site applications and we have focused on the specific areas that need to be considered more in order to ensure a future practical implementation of these methods in routine analyses. The subsequent part of the thesis addresses the development of an autonomous lab-on-chip platform for performing chemiluminescent-based bioassays in space environment, exploiting a CubeSat platform for astrobiological investigations. An origami-inspired microfluidic paper-based analytical device has been developed with the purpose of assesses its performance in space and to evaluate its functionality and the resilience of the (bio)molecules when exposed to a radiation-rich environment. Subsequently, we designed a paper-based assay to detect traces of ovalbumin in food samples, creating a user-friendly immunosensing platform. To this purpose, we developed an origami device that exploits a competitive immunoassay coupled with chemiluminescence detection and magnetic microbeads used to immobilize ovalbumin on paper. Finally, with the aim of exploring the use of biomimetic materials, an hydrogel-based chemiluminescence biosensor for the detection of H2O2 and glucose was developed. A guanosine hydrogel was prepared and loaded with luminol and hemin, miming a DNAzyme activity. Subsequently, the hydrogel was modified by incorporating glucose oxidase enzyme to enable glucose biosensing. The emitted photons were detected using a portable device equipped with a smartphone's CMOS (complementary metal oxide semiconductor) camera for CL emission detection.