Martone, Nicoletta (2022) Platform for multifunctional particles in biosensing applications. [Tesi di dottorato]

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Abstract

Microgels are versatile materials due to their hydrophilic, bio-friendly, and highly tunable nature, making them applicable in various biomolecule detection. The microgels-based platform is composed of multifunctional particles which possess multiple components and properties tunable to fulfil the requirements of the applications. The chemical flexibility of hydrogel microparticles and microgels allows the embedding of several building blocks into their structure during the synthesis. Anchoring groups on particles are used to immobilize probes of diverse natures such as antibodies, enzymes, or oligonucleotide strands. Particles active for the detection of a target or more than one biomarker can be obtained. The high biocompatibility and antifouling properties of hydrogel make possible the employing of the microgels-based platform in biosensing and inspired the development of several sophisticated applications, such as biosensors. My PhD project aims to widen the applications of the microgels-based platform towards new goals in the biosensing field to have innovative bioassays. Microgels can be produced with multiple functions thanks to their chemical flexibility. During their synthesis, different building blocks can be introduced into the reactor allowing their co-polymerization with the cross-linking agent. Core microgels with different physical and chemical properties are produced according to the desired properties to confer. The microgels are used to develop an ultrasensitive and innovative immunoassay for the detection of new biomarkers such as proteins and antibodies. Nowadays, immunoassays are the most spread analytical procedures used to measure proteins in biological samples. Analytes and disease biomarkers are usually present in fluid and serum at subfemtomolar concentrations. For this reason, methods and devices featured by both sensitive target detection and broad analytical ranges are required. As the first application in the immunoassay field, core microgels with Rhodamine dye as an optical barcode are adopted as the carrier of both anti-Fab antibodies and the HRP enzyme. Magnetic particles functionalized with anti-Fc antibodies are involved as capture particles. When the target antigen IgG is present, the two antibodies used can form a sandwich structure capturing the target. Microgels bound to beads can be separated magnetically from the sample. Particle clusters are formed and visible by a confocal laser scanning microscope (CLSM). The cluster area increases as the target concentration increase too. After the interaction, unbound microgels are separated from the solution and used to produce a colourimetric signal for target detection. Increasing the target concentration in samples, the number of free microgels in the solution decreases. The colourimetric signal produced by HRP-microgels free in solution decreases too. Single-entity detection approaches and miniaturized structures are increasingly spreading to overcome the limits linked to sensitivity. The flexibility of the particle based platform allows for decreasing the number of particles involved in the assay. The application of the single-entity method and the use of microfluidic devices can lead to the development of bioassays characterized by ultrahigh sensitivity.

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