De Masi, Alessandra (2020) Microfluidic Synthesis and Engineering of Hydrogel-based Biosensor for In-gel Immunoassays. [Tesi di dottorato]

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Recently hydrogel microparticles have been widely used as a versatile tool to perform beads-based assays due to their hydrophilic, biocompatible and highly flexible chemical and physical properties. Since the chemical properties of the polymeric network can be tuned, the gel can be easily functionalized with any type of biomolecule. Late studies show an ever-increasing interest in performing microparticles-based sandwich immunoassays, using primary antibodies to capture a specific biomarker and a fluorescent secondary antibody to show the binding event. In order to adapt this scheme to hydrogels, where diffusion represents the limiting factor, it is necessary to increase the porosity of the network and avoid non-specific interactions between gel and biomolecules. The following PhD project aims to design, synthesize and characterize PEG-based hydrogels microparticles able to detect specific biomarkers with high sensitivity in complex fluids such as serum, blood and urine. Hydrogel microparticles thus realized show a high sensitivity due to their capability to concentrate the target in a small volume, amplifying the signal. This PhD project can be divided into several steps: • Design and microfluidic synthesis of the hydrogels microparticles • Characterization of the hydrogel microparticles • Assay optimization and specificity tests • Realization of a microfluidic device to perform assays on chip Microfluidics offers a high-throughput platform for synthesizing uniform and monodisperse polymeric microspheres in one step. In particular, a T-junction glass chip was used to produce 70-75 µm diameter microparticles, polymerized on flow by a UV lamp. PEG microparticles were obtained using light mineral oil mixed with nonionic surfactant Span 80 as continuous phase and PEGDA, Darocur 1173 and N,N′(1,2Dihydroxyethylene)bisacrylamide (DHEBA) as disperse phase. DHEBA is a cleavable crosslinker which can be cut via oxidation, increasing the pore size in order to allow the passage of antibodies and, simultaneously, producing aldehydes that can be used for further conjugation. In order to characterize the hydrogel microparticles, cleavage kinetics and conditions, porosity, aldehydes titration, diffusion and equilibrium partitioning of fluorescent probes (antibodies and 50 nm nanoparticles) have been analyzed. Optimized conjugated hydrogel microparticles were then used to perform a sandwich immunoassay on hIgG. Experimental data show a limit of detection in the picomolar range, a high specificity and selectivity of the assay (in presence of unrelated proteins) and good results even in complex fluids such as serum and urine. To detect low-molecular-weight biomolecules, which are not suitable for a sandwich system, a bead-based competitive assay has been developed and optimized. The experimental data confirm a limit of detection in the picomolar range. In order to speed up all the incubation and washing steps, the assay has been implemented inside a microfluidic chip, which can host up to five particles in parallel. Preliminary studies on the capture of fluorescent probes and washing volumes and time have been performed.

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