Renzi, Emilia (2023) Artificial metalloenzymes for the construction of functional nanostructures. [Tesi di dottorato]

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Abstract

In the last decades, diverse and powerful strategies have been developed for the design of artificial metalloenzymes (ArMs), as customized catalysts able to perform natural and unnatural reactions, often overcoming their natural counterpart. In this field, Mimochromes (MCs), consisting of a metal-containing porphyrin core embedded within two synthetic peptides, set the gold standard. Apart from their outstanding catalytic result in solution, MCs can also be successfully anchored on gold nanosurfaces, while retaining structural properties and catalytic potential. The latest scaffold of the MCs’ series, MC6*a, is extremely versatile, accessing customized catalytic activities upon insertion of a different metal ion. Into the porphyrin core. Insertion of an iron ion affords Fe(III)-MimochromeVI*a (FeMC6*a), provided with high catalytic versatility and enhanced performances in a simple miniaturized scaffold (Mw 3.5 kDa, radius of gyration ≈ 1 nm). Experimental data has proven the ability of FeMC6*a to outperform natural and artificial biocatalysts in catalyzing several oxidation reactions. The repertoire of application of FeMC6*a can be further widened through its interaction with nanomaterials, with the aim of preparing functional nanoconjugates. Nanomaterials are now in trend for the immobilization of natural enzymes by virtue of enticing physico-chemical properties. Gold nanomaterials (AuNMs) provide a high enzyme loading, due to the large surface-area-to-volume ratio, possess a versatile surface chemistry as well as tunable sizes and shapes. Apart from isotropic AuNMs, throughout the years, interest has moved toward the use of anisotropic AuNMs (including nanostars, nanorods, and triangular nanoprisms), intriguing for building optical devices and biosensors, by virtue of plasmon-related optical response dependent upon their size and shape. In this scenario, the substitution of natural biocatalysts with artificial metalloenzymes, tailored ad-hoc by design and endowed with a reduced size, may be envisaged as a significant step forward in broadening the practical use of immobilized enzymes. Driven by this fascinating background, the present Ph.D. thesis has been aimed at the development of functional nanoconjugates using FeMC6*a as a biomolecular component. This goal was pursued by addressing two main aspects: investigation of FeMC6*a behaviors in catalysis when immobilized on differently shaped gold nanomaterials (described in Part A) and the application of immobilized FeMC6*a in biosensor technology (described in Part B). Part A The conjugation of FeMC6*a with both isotropic (gold nanoparticles) and anisotropic (gold nanorods and triangular nanoprisms) nanomaterials was investigated, studying the optimal conditions for interfacing this catalytically active metalloprotein on the target nanosupports. First, using AuNPs, the conjugation was achieved by means of two different approaches: i) FeMC6*a was derivatized with lipoic acid, in order to be directly grafted on the surface of AuNPs, affording FeMC6*a-LA@AuNPs; ii) click chemistry (SPAAC) guaranteed the fast-covalent immobilization of the mini-enzyme modified with a pegylated spacer to carry an aza-dibenzocyclooctyne (DBCO) moiety. In this case, AuNPs were properly modified to expose azide moieties. The two methodologies proved to be efficient for the attachment of several copies of FeMC6*a to AuNPs, affording stable nanoconjugates endowed with peroxidase activity, which catalytic behavior was assayed toward the oxidation of model substrates in the presence of H2O2. All the results showed that the support shape has a significant effect on the catalytic behavior of the immobilized FeMC6*a, resulting in a 5-fold and 3-fold enhancement of the turnover frequency when using AuNRs and AuNTs, respectively, instead of AuNPs. Overall, even if the resulting activity of the nanoconjugates is lower with respect to the free FeMC6*a, the prepared nanoconjugates retained the intrinsic peroxidase activity of the mini-enzyme and displayed good turnover frequencies and catalytic efficiencies. Part B The results obtained in Part A prompted the exploitation of FeMC6*a-based gold nanoconjugates for practical applications. Fascinated by the worldwide spread of AuNP-based lateral flow immunoassays, during the period abroad spent at the Catalan Institute of Nanoscience and Nanotechnology, in the research group of Prof Arben Merkoçi, the peroxidase activity of AuNP-FeMC6*a was exploited as a strategy to obtain catalytic signal amplification in sandwich immunoassays on lateral flow strips, for the detection of Human-IgG. The recognition of the analyte by the capture and detection antibodies was first evidenced by the appearance of a red color in the test line, due to the accumulation of AuNPs. Subsequent FeMC6*a-assisted oxidation of a chromogenic substrate on the line increased the test line color, improving the sensitivity of FeMC6*a-based LFiA with respect to a conventional assay and in control experiments, where it was replaced by HRP, the natural counterpart. In conclusion, the research activities carried out demonstrate that simple, properly designed scaffolds can mimic and, in some exciting cases, overcome the kinetic performance of the natural counterparts, providing nanoconjugates catalytically active on different nanosupports and in diverse environments. Therefore, the use of FeMC6*a is a cutting-edge strategy for the development of advanced functional tools with applications in biosensing and biocatalysis.

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