Handcrafted Metal Enzymes as Convenient Reporters in Biosensors

Leveraging their inherent metal cores, metalloproteins assume pivotal roles in orchestrating intricate natural transformations. This fundamental importance extends to invaluable applications in in vitro diagnostics and biosensing, encompassing immunoassays and electrochemical sensors 1. Bolstered by recent strides in computational protein design, our endeavors have yielded bespoke enzymes, marking significant milestones in the field 2. Through diverse strategies, we have crafted compact yet potent models housing multiple metal sites. This exposition shines a spotlight on chosen designs, elucidating their potential within the domain of sensing technology. We first show that by asymmetrization of the heme environment and precise positioning of the residues at the distal site, with Fe-Mimochrome 6*a (FeMC6*a), we reached unprecedented selectivity in hydrogen peroxide activation leading to a peroxide sensor 3, a catalytic nanoconjugate 4, a signal enhancer in lateral-flow immunoassays 5, and efficient dye degradation for bleaching applications 6. Finally, we describe our efforts in bioelectronics by coupling a photosensitizing zinc porphyrin with a 3kDa iron-sulphur designed protein, leading to the first fully artificial light-harvested electron cascade 7. In perspective, our designed metalloproteins will be equipped for the next-generation of biosensors. Financial support by the Campania Region (Programma Operativo FESR Campania 2014 - 2020, Asse 1), and MUR (Next Generation EU – Piano Nazionale di Ripresa e Resilienza, PNRR-Mission 4 Component 2 Investment 1.4 “Agritech National Research Center” – Spoke 2, and PRIN project SEA-WAVE 2020BKK3W9) is gratefully acknowledged. 1. Hussein, H. A.; Hassan, R. Y. A.; Chino, M.; Febbraio, F. Point-of-Care Diagnostics of COVID-19: From Current Work to Future Perspectives. Sensors 2020, 20 (15), 4289. 2. Leone, L.; Chino, M.; Nastri, F.; Maglio, O.; Pavone, V.; Lombardi, A. Mimochrome, a Metalloporphyrin-Based Catalytic Swiss Knife. Biotechnology and Applied Biochemistry 2020, 67 (4), 495–515. 3. Zambrano, G.; Nastri, F.; Pavone, V.; Lombardi, A.; Chino, M. Use of an Artificial Miniaturized Enzyme in Hydrogen Peroxide Detection by Chemiluminescence. Sensors 2020, 20 (13), 3793. 4. Zambrano, G.; Chino, M.; Renzi, E.; Di Girolamo, R.; Maglio, O.; Pavone, V.; Lombardi, A.; Nastri, F. Clickable Artificial Heme-Peroxidases for the Development of Functional Nanomaterials. Biotechnology and Applied Biochemistry 2020, 67 (4), 549–562. 5. Renzi, E.; Piper, A.; Nastri, F.; Merkoçi, A.; Lombardi, A. An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays. Small 2023, 2207949. 10.1002/smll.202207949 6. Chino, M.; La Gatta, S.; Leone, L.; De Fenza, M.; Lombardi, A.; Pavone, V.; Maglio, O. Dye Decolorization by a Miniaturized Peroxidase Fe-MimochromeVI*a. International Journal of Molecular Sciences 2023, 24 (13), 11070. 7. Chino, M.; Di Costanzo, L. F.; Leone, L.; La Gatta, S.; Famulari, A.; Chiesa, M.; Lombardi, A.; Pavone, V. Designed Rubredoxin Miniature in a Fully Artificial Electron Chain Triggered by Visible Light. Nat Commun 2023, 14 (1), 2368.