Artificial mini-enzymes on nanogold surfaces

Nanomaterials (NMs) have drawn great interest over the last decades in a variety of research areas, including biosensing, catalysis and diagnostics. In the field of enzyme immobilization, goldcontaining nanomaterials (AuNMs) play a central role as excellent platforms due to a high surface area-to-volume ratio as well as with a versatile surface chemistry. Especially gold nanorods have been identified as promising candidates for the construction of highly effective biosensors.1 In this context, the conjugation of AuNMs with artificial heme-enzymes, known as Mimochromes (MCs),2 enables the construction of versatile hybrid bio-systems. Indeed, preliminary results on Fe(III)-Mimochromes demonstrated their successful immobilization onto gold electrode surfaces and conjugation to AuNPs while retaining redox and catalytic properties.3,4 Driven by these interesting results, the work here presented focused on the construction of bionanoconjugates, selecting the artificial heme-peroxidase Fe(III)-MimochromeVI*a (FeMC6*a) as the biomolecular building block and gold nanorods (AuNRs) as the target support. The immobilization was achieved by carrying out two different approaches: on one hand, FeMC6*a was derivatized with lipoic acid, in order to be directly grafted on the surface of AuNRs (Figure 1). On the other hand, the SPAAC (strain-promoted azide-alkyne cycloaddition) chemistry guaranteed the fast-covalent immobilization of the mini-enzyme modified with a pegylated spacer to carry an aza-dibenzocyclooctyne (DBCO) moiety. In this case, AuNRs were properly modified to expose azide moieties (Figure 1). The two methodologies proved to be easy and efficient, allowing the attachment of several copies of FeMC6*a to gold nanorods. The catalytic properties of the resulting hybrid bio-nanomaterials were evaluated using model oxidation reactions. Both approaches afforded functional bio-nanoconstructs, which retained peroxidase activity, although decreased catalytic performances were observed, if compared to that of the freely diffusing FeMC6*a. However, the developed methods represent a proof of concept that the artificial metalloprotein FeMC6*a can be firmly anchored on nanomaterials and support the development of stable and functional FeMC6*a-AuNMs conjugates. [1] Y. Lin, M. Zhao, Y. Guo, X. Ma, F. Luo, L. Guo, B. Qiu, G. Chen, Z. Lin, Sci Rep 2016, 6 (1), 37879. [2] L. Leone, M. Chino, F. Nastri, O. Maglio, V. Pavone, A. Lombardi, Biotechnol. Appl. Biochem. 2020, 67 (4), 495. [3] G. Zambrano, E. Ruggiero, A. Malafonte, M. Chino, O. Maglio, V. Pavone, F. Nastri, A. Lombardi, Int. J. Mol. Sci. 2018, 19, 2896. [4] G. Zambrano, M. Chino, E. Renzi, R. Di Girolamo, O. Maglio, V. Pavone, A. Lombardi, F. Nastri, Biotechnol. Appl. Biochem. 2020, 67 (4), 549.