Interfacing nanomaterials and biological systems: development and applications
UNIVERSAL IDENTIFIER: http://hdl.handle.net/11093/3666
UNESCO SUBJECT: 2210 Química Física
DOCUMENT TYPE: doctoralThesis
Nanomaterials (NMs) present uniquely physical, chemical and mechanical properties that made them very interesting for biological applications. Their interface with biological systems results in a synergistic combination that provides new properties and applications that surpass their individual components. This hybrids systems that combine biotic-abiotic entities, take advantage of the physicochemical properties and sensing capabilities of the NMs, and the biological properties of living cells as their programmable gene expression, self-replication and movement. FunNanoBio group (former Colloid Chemistry group) and Synthetic Biology group, where this thesis was carried out, are focused on the synthesis of nanoparticles with well-defined and tunable optical properties, and the engineering of biological systems respectively, aiming the development of novel hybrid systems with improved capabilities. In this thesis, we focus on the applications of noble metal nanoparticles, upconversion nanoparticles (UCNPs) and polymeric nanoparticles and their interface with different biological systems for generating biosensors, optogenetic actuators and bioimaging tools. This thesis is structured into five chapters. Chapter 1 gives a general introduction about the nanomaterials that will be used in the different works presented within the subsequent chapters and their possibilities at the interface with human and bacterial cells. In Chapter 2, we describe the intimin system used for bacterial display of different lectins, such as Galectin-3 (Gal3) and Mannose Binding Lectin (MBL) proteins. We study the functionality of these displayed proteins once expressed on the surface of bacteria through adhesion assays to ligand-coated surfaces and polymeric nanoparticles. Chapter 3 aims to generate hybrid systems based on gold nanoparticles and bacterial cells, by means of protein-protein interactions. Two strategies were investigated: (i) A covalent approach mediated by the SpyCatcher/SpyTag system and (ii) a light-mediated reversible approach employing pMagHigh and nMagHigh optogenetic photoswitches. To this aim, we explored the intimin display system for expressing either SpyTag or nMagHigh on the surface of bacterial cells. The bacteria-NP hybrids would be obtained upon incubation of the cells expressing SpyTag or nMagHigh with gold nanoparticles conjugated with SpyCatcher or pMagHigh partners, respectively. Chapter 4 is dedicated to the other optogenetic tool presented in this manuscript. Here we describe the use of UCNPs as blue light sources to trigger oligomerization of CRY2olig-mCherry expressed in HeLa cells. UCNPs-based substrate was fabricated through layer-by-layer technique and used to grow and transfect HeLa cells with plasmid expressing the CRY2olig-mCherry optogenetic module. The capabilities of the UCNPs-based substrate to trigger oligomerization of CRY2olig-mCherry, once expressed in HeLa cells through the upconversion of near infrared irradiation into blue light, were investigated. Chapter 5 deals with the biofunctionalization of Au nanocapsules encoded with different Raman active molecules with specific antibodies, to obtain highly efficient SERS tags, which enable multiplex detection and imaging of three tumor-associated biomarkers, allowing the discrimination of A431 tumor and 3T3 2.2 non-tumor cells.
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