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

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-snglycero-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 Larginine, 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.

Progress in electrochemical detection of neurotransmitters using carbon nanotubes/nanocomposite based materials: A chronological review

“…LOD of 15 nM was reported for the Nafion/Hb/NYPA-MWCNT/GCE electrode in the amperometric determination of varying concentrations of NO. This LOD is lower than the value reported by Wang et al, [239] Santos et al, [61] and Yan et al [60] A biosensor for direct bioelectrocatalysis detection of NO using NO reductase incorporated in carboxylated SWCNT-lipidic bilayer composite was designed by Gomes et al [251] in 2019. The electrode was fabricated by the deposition of a composite made from carboxylated MWCNT, lipidic bilayer (a combination of DOPE, DOTAP, and DSPE-PEG) and NO reductase (NOR) on pyrolytic graphite electrode (PGE) through the solvent casting technique.…”

Electrochemical Biosensors Based on Membrane-Bound Enzymes in Biomimetic Configurations

“…Very recently, Moura and co-workers have reported an interesting third-generation nitric oxide biosensor based on the membrane-bound enzyme nitric oxide reductase [90]. NO is involved in many biological processes, but it is very reactive, thus its quantification requires detection times between 5 and 15 s [91].…”

“…A limit of detection of 0.13 µm was measured, which is adequate for measuring NO evolution in biological processes. The storage stability was good with 83.5% of the initial response was retained after five weeks, which indicated sufficient binding between the three components that were deposited on the electrode that minimized leaching [90]. There are many studies of electrochemical biosensors that are based on transmembrane proteins reconstituted on phospholipid bilayers supported on electrodes, although the vast majority comprises ion-transport proteins or olfactory receptors, not enzymes [57].…”

“…A limit of detection of 0.13 µm was measured, which is adequate for measuring NO evolution in biological processes. The storage stability was good with 83.5% of the initial response was retained after five weeks, which indicated sufficient binding between the three components that were deposited on the electrode that minimized leaching [ 90 ].…”

A Sensitive Sensor Based on Single‐walled Carbon Nanotubes: Its Preparation, Characterization and Application in the Electrochemical Determination of Drug Clorsulon in Milk Samples

“…Due to the differences in their structure, SWCNTs show greater electrical conductivity; the presence of gaps between the carbon layers in the case of MWCNTs structures results in higher resistance of this nanomaterial compared to SWCNTs [23]. In recent years, SWCNTs have found many applications in electrochemistry and electroanalysis of biologically active compounds and other molecules [24][25][26][27][28][29].…”