Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic 3 bilayer nanocomposite

Filipa O Gomes; Luísa B Maia; Joana A Loureiro; Maria Carmo Pereira; Cristina Delerue-Matos; Isabel Moura; José J G Moura; Simone Morais

doi:10.1016/j.bioelechem.2019.01.010

Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic 3 bilayer nanocomposite

Bioelectrochemistry. 2019 Jun:127:76-86. doi: 10.1016/j.bioelechem.2019.01.010. Epub 2019 Feb 5.

Authors

Filipa O Gomes¹, Luísa B Maia², Joana A Loureiro³, Maria Carmo Pereira³, Cristina Delerue-Matos⁴, Isabel Moura², José J G Moura², Simone Morais⁵

Affiliations

¹ REQUIMTE-LAQV, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida, 4249-015 Porto, Portugal; REQUIMTE-LAQV, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal.
² REQUIMTE-LAQV, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal.
³ LEPABE, Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal.
⁴ REQUIMTE-LAQV, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida, 4249-015 Porto, Portugal.
⁵ REQUIMTE-LAQV, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino de Almeida, 4249-015 Porto, Portugal. Electronic address: sbm@isep.ipp.pt.

PMID: 30745281
DOI: 10.1016/j.bioelechem.2019.01.010

Abstract

An enzymatic biosensor based on nitric oxide reductase (NOR; purified from Marinobacter hydrocarbonoclasticus) was developed for nitric oxide (NO) detection. The biosensor was prepared by deposition onto a pyrolytic graphite electrode (PGE) of a nanocomposite constituted by carboxylated single-walled carbon nanotubes (SWCNTs), a lipidic bilayer [1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG)] and NOR. NOR direct electron transfer and NO bioelectrocatalysis were characterized by several electrochemical techniques. The biosensor development was also followed by scanning electron microscopy and Fourier transform infrared spectroscopy. Improved enzyme stability and electron transfer (1.96 × 10^-4 cm.s^-1 apparent rate constant) was obtained with the optimum SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR) ratio of 4/2.5/4 (v/v/v), which biomimicked the NOR environment. The PGE/[SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR] biosensor exhibited a low Michaelis-Menten constant (4.3 μM), wide linear range (0.44-9.09 μM), low detection limit (0.13 μM), high repeatability (4.1% RSD), reproducibility (7.0% RSD), and stability (ca. 5 weeks). Selectivity tests towards L-arginine, ascorbic acid, sodium nitrate, sodium nitrite and glucose showed that these compounds did not significantly interfere in NO biosensing (91.0 ± 9.3%-98.4 ± 5.3% recoveries). The proposed biosensor, by incorporating the benefits of biomimetic features of the phospholipid bilayer with SWCNT's inherent properties and NOR bioelectrocatalytic activity and selectivity, is a promising tool for NO.

Keywords: Direct electron transfer; Enzymatic biosensor; Nitric oxide reductase.

MeSH terms

Biosensing Techniques / methods*
Enzymes, Immobilized / chemistry*
Limit of Detection
Lipid Bilayers / chemistry
Marinobacter / enzymology*
Models, Molecular
Nanotubes, Carbon / chemistry*
Nitric Oxide / analysis*
Oxidoreductases / chemistry*

Substances

Enzymes, Immobilized
Lipid Bilayers
Nanotubes, Carbon
Nitric Oxide
Oxidoreductases
nitric-oxide reductase