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Application of chemiluminescence and bioluminescence to nanomaterials

Functionalization of NiO Thin Films with His-Tag Proteolytic and Bioluminescent Enzymes for Sensor Applications

Adrianne M. M. Brito1, Gabriel F. Pelentir2, Marcos P. C. Ramos3, Victor Oliveira3, Marcelo Y. Icimoto3, Denise Criado4, Vadim Viviani2, Iseli L. Nantes4

1UNIVERSIDADE FEDERAL DO ABC, Centro de Ciências Naturais e Humanas, Brazil, 2Universidade Federal de São Carlos, Depto de Física, Química e Matemática, Campus Sorocaba, Brazil, 3Universidade Federal de São Paulo UNIFESP, 4UNIVERSIDADE FEDERAL DO ABC

E-mail: ilnantes@gmail.com

Immobilization of enzymes on films, nanoparticles, and liposomes is largely used to produce reusable catalysts and sensors. In the present study, a new system of enzyme immobilization was developed to take advantage of the well-known affinity of histidine to divalent metal ions. Therefore, a thin film of Ni was deposited by the evaporation technique (Prest Vacuo model PV0450) of Ni flake 99.99% on glass substrates. Ni film was oxidized upon entering air in the vacuum chamber immediately after the deposition. The NiO films were calcinated using a tubular furnace (Thermo Scientific, Lindberg Blue M) open to the atmosphere at 400◦C for 3 h. Micro-Raman spectroscopy (Horiba-Jobin-Yvon model T64000 spectrometer) was carried out to identify the vibrational properties of the NiO thin film. The morphology of the NiO films was studied by scanning electron microscopy (SEM) using a JEOL JSM-6010LA microscope. Two potentially applicable sensing and diagnosis enzymes were chosen for immobilization and expressed with a tail of six histidine residues (His-tag). The bioluminescent green-blue emitting Amydetes vivianii firefly luciferase (Amy-WT) linked with the N-terminal ZZ portion of protein A (chimeric ZZ-Amy) and the neuronal enzyme prolyl oligopeptidase (POP) were dispersed on the NiO films and incubated for two hours. The film surfaces were rinsed with water to remove unlinked protein. The binding of the chimeric ZZ-Amy and POP on the NiO surface was characterized by Fourier Transform Infrared (FTIR), which evidenced the enzyme binding in a concentration-dependent manner. The functionalized films exhibited the finger-print protein bands amide I (1600-1800 cm-1), amide II (1470-1570 cm-1), amide III (1250-1350 cm-1), and amide A (3300-3500 cm-1). These results show NiO films as a promising platform for enzyme immobilization and application based on the His-tag affinity for coordinated metal ions.

Keywords: his-tag, NiO, luciferases, prolyl oligopeptidase, sensor

Acknowledgments: FAPESP 2017/02317-2, CNPq 2021/313111-9.

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