Chem-mdbpdevelopment of chemistry-based multiplexing diagnostic beadplatforms

Resumo

Biotechnology and nanotechnology are two fields in their splendour moments with excellent scientific publications, large and unprecedented investments, and technological developments that are constantly revolutionising science and impacting positively to society. The development and utilisation of smart materials and nanomaterials have meant a before and after in different scientific fields. Novel technologies have emerged as a consequence, generating an invaluable impact both in economic terms and knowledge. In this context, this doctoral thesis aims to take advantage of smart materials and novel technologies to create bead-based platforms, focusing on the diagnosis field, by enabling both cellular and molecular assays, using chemical-based technologies. These bead-based platforms which allow both type of assays, comprise polystyrene particles, novel nucleic acid and metal chemistries and different analytical platforms. Regarding the molecular assays, the bead-based platform comprises commercially available polystyrene-based magnetic microparticles and Chem-NAT technology, which is a chemical-based PCR-free technology for nucleic acid detection with single base resolution. The Chem-NAT technology employs peptide nucleic acids probes with an abasic position (DGL probes) whose sequences are fully complementary to the target nucleic acids. This is due to the fact that target nucleic acids act as template of a thermodynamically controlled and specific dynamic incorporation of the reactive aldehyde-modified nucleobases (Smart-NB) into the abasic position, through the formation of a reversible covalent bond, an iminium specie, that is thereupon reduced to an stable tertiary amine. Due to the thermodynamic control given by the target nucleic acid this technology is highly selective and specific, avoiding the presence of false positive results. This Chem-NAT technology was used for both quantitative and qualitative applications: (i) Quantitative application: The synthesis and optimisation of DGL probes for the direct detection and quantification of miR-21 using fluorescence-based readout platforms is presented through dynamic chemistry labelling (DCL). The DGL probes optimisation steps were performed on flow cytometry, whilst the direct detection and quantification was performed on a fluorescence-based microplate reader. MiR-21 was successfully profiled and quantified from tumour cells and from plasma from patients with non-small cell lung cancer (NSCLC) in advances stages. Moreover, it was assessed the multiplexing capability of the platform by distinguishing miR-21 and miR-122 within the same sample. (ii) Qualitative approach: The capability of the bead-based platform for testing single nucleotides polymorphisms (SNP) of KRAS is presented. The KRAS WT sequence and the mutated G13C KRAS sequence were successfully identified, within the same sample, by flow cytometry. On the other hand, the bead-based platforms designed for cellular based assays are presented. Monodispersed cross-linked amino-functionalised polystyrene nanoparticles were functionalised with a fluorophore and a metal, giving rise to metallofluorescent nanoparticles, to achieve multi-modal applications for diagnosis. This dual combination allowed the nanoparticles to be employed for imaging techniques, chemical reactions, and mass-based technologies. They were used as imaging probes for confocal microscopy and/or for flow cytometry. Furthermore, the properties of the fluorophore, like the fluorescence lifetime, allowed their employment as probes in fluorescence life-time imaging. Moreover, the presence of metals, especially palladium, confers additional features without quenching the fluorophore. For instance, palladium nanoparticles can act as catalysts since they are capable of catalysing chemical reactions. Finally, the nanoparticles can be used as mass-tag reagents for mass cytometry along with flow cytometry, what converts them in dual probes. Through the combination of pure palladium isotopes, the metallofluorescent nanoparticles were employed as mass-tag reagents for mass cytometry barcoding. In the doctoral thesis the first proof of concept (PoC) of the mass-based barcoding with two different metallofluorescent nanoparticles, and the first assays for live cell barcoding using three different metallofluorescent nanoparticles were developed. These two assays demonstrate the high capability, the non-toxicity, the specificity, and resistance of the metallofluorescent nanoparticles to be used as live cell barcodes in mass cytometry and flow cytometry.