DEVELOPMENT OF CRISPR-CAS IMMOBILIZATION SRATEGIES FOR THE FABRICATION OF A BIOSENSING PLATFORM

The main focus throughout my PhD course has been the development of different strategies for the immobilization of the CRISPR-Cas enzyme, to create an integrated platform for the detection of DNA samples. In recent years, the CRISPR-Cas based diagnostic is emerging as a promising technology for the detection of infectious disease in point of care (POC) environments because of its high specificity and high sensitivity. Moreover, CRISPR-Cas biosensing platforms allow a rapid time of testing and could be designed to be portable and deployable in various settings, making them suitable for decentralized testing and surveillance. Specifically, the CRISP-Cas 12a isoform possesses peculiar features which make it suitable for diagnostic application. In fact, this enzyme works by forming a complex with an RNA sequence that recognizes a complementary sequence of dsDNA and, upon recognition, this isoform acquires the ability to cut also non-specific sequences of ssDNA. This feature has been exploited to generate a fluorescent signal by adding a short sequence of ssDNA bearing both a fluorophore and a quencher at the two ends. Upon DNA recognition and interaction with the Cas, this short sequence is excised: the fluorophore is separated from the quencher and a signal is generated. One of the challenges of employing CRISPR-based tools in POC settings is still represented by the integration of the enzyme together with all the components needed for the analysis, in a format that would also maintain their stability. Moreover, this platform should be easy to manufacture and, possibly, totally automated. To achieve this goal, a flexible strategy to immobilize the enzyme on different scaffolds, while also preserving its activity is required. Therefore, we focused our research on developing a hydrogel capable of encapsulating all the CRISPR-Cas enzyme system. Hydrogels indeed, with their highly hydrated 3D structure, provide an ideal environment for biological interactions, minimizing surface interactions and allowing entrapped proteins to retain their biological activity. Additionally, we designed the hydrogel for multiple printing into separate plugs, creating distinct environments for the analysis of different targets and samples. A suitable polymeric matrix to produce the hydrogel has been properly selected and optimized. Specifically, a class of synthetic copolymers developed in our lab has been employed. These copolymers derive from a parental copolymer composed of three monomers: N, Ndimethylacrylamide (DMA) N-acyloxy succinimide (NAS) and 3(Trimethoxysylil propyl) methacrylates (MAPS). By post-polymerization modification of the succinimide moieties, different groups which enable click-chemistry reactions were inserted into the polymer chains. These click groups allow crosslinking of the polymer chains forming the hydrogel in a fast and reproducible way in mild conditions. Different concentrations of polymers, different molar fractions of functional groups, catalyzed or non-catalyzed reactions were investigated, together with the employment of different types of crosslinkers. We mainly focused on two different click chemistry reactions, the so called Strain-promoted azide-alkyne cycloaddition (SPAAC) between a dibenzocyclooctyne (DBCO) and azide groups, and the reaction between hydrazine and aldehyde. This latter reaction allowed the formation of a hydrogel with minimal interference with the activity of the enzyme. The integration of this system into a portable device demands effective miniaturization while ensuring that high sensitivity and efficiency are maintained. Achieving this balance is crucial for creating a compact, yet reliable tool that can operate with the same level of precision as larger-scale systems, ensuring accurate results in real-world, point-of-care settings. Compared to others, the microarray platform emerges as a superior choice for its several advantages: their miniaturized format significantly reduces the amount of materials and samples required for analysis, while also offering high multiplexing capacity, enabling the simultaneous analysis of multiple targets in a single experiment. To integrate the hydrogel combined with CRISPR-Cas system into a microarray platform, extensive optimization was undertaken to fine-tune the viscosity and spotting protocol. This process included assessing the hydrogel array's stability, permeability to proteins and DNA fragments, and its capacity to retain the activity of biological probes. In parallel, other formats were investigated, specifically microtiter plates and multi-wells slides, which offer the multiplex capability of the microarray together with a larger volμMe to accommodate the enzyme. The Cas enzyme immobilized in hydrogel plugs demonstrated high specificity in distinguishing between two different targets and yielded promising results when applied to real samples, particularly in the detection of bacterial DNA. Alternative strategies for the immobilization of the CRISPR-Cas enzyme were also explored. By exploiting the presence of a poly-Histidine tag on the C-terminal portion of the Cas enzyme, the Nitrilotriacetic acid (NTA) chemistry, combined with bivalent cation activation, was investigated for the immobilization on the surface of tangential flow filtration (TFF) membranes and on magnetic silica beads. Extensive studies were conducted on the method of introducing NTA onto the surface, as well as on the type of cation used for NTA activation. This approach resulted in optimal enzyme immobilization, highlighting the critical role of probe orientation on the surface to ensure proper functionality.