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- Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reductionPublication . Gomes, Filipa O.; Maia, Luísa B.; Cordas, Cristina; Moura, Isabel; Delerue-Matos, Cristina; Moura, José J.G.; Morais, SimoneUnderstanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b3 and non-heme FeB, respectively. The determined sensitivity (-4.00 × 10-8 ± 1.84 × 10-9 A/μM and - 2.71 × 10-8 ± 1.44 × 10-9 A/μM for NO and O2, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O2, respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O2, while the O2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors.
- Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic 3 bilayer nanocompositePublication . Gomes, Filipa O.; Maia, Luísa B.; Loureiro, Joana A.; Pereira, Maria Carmo; Delerue-Matos, Cristina; Moura, Isabel; Moura, José J.G.; Morais, SimoneAn 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.
- Nitric Oxide Detection Using Electrochemical Third-generation Biosensors - Based on Heme Proteins and PorphyrinsPublication . Gomes, Filipa O.; Maia, Luísa B.; Cordas, Cristina; Delerue-Matos, Cristina; Moura, Isabel; Moura, José J. G.; Morais, SimoneNitric oxide radical (NO) is a signalling molecule involved in virtually all forms of life. Its relevance has been leading to the development of different analytical methodologies to assess the temporal and spatial fluxes of NO under the complex biological milieu. Third‐generation electrochemical biosensors are promising tools for in loco and in vivo NO quantification and, over the past years, heme proteins and porphyrins have been used in their design. Since there are some limitations with the biorecognition element directly adsorbed onto the electrode surface, nanomaterials (carbon nanotubes, gold nanoparticles, etc.) and polymers (cellulose, chitosan, nafion®, polyacrylamide, among others) have been explored to achieve high kinetics and better biosensor performance. In this review, a broad overview of the field of electrochemical third‐generation biosensors for NO electroanalysis is presented, discussing their main characteristics and aiming new outlooks and advances in this field.
- Third-generation electrochemical biosensor based on nitric oxide reductase immobilized in a multiwalled carbon nanotubes/1-n-butyl-3-methylimidazolium tetrafluoroborate nanocomposite for nitric oxide detectionPublication . Gomes, Filipa O.; Maia, Luísa B.; Delerue-Matos, Cristina; Moura, Isabel; Moura, José J.G.; Morais, SimoneNitric oxide (NO) has a crucial role in signaling and cellular physiology in humans. Herein, a novel third-generation biosensor based on the Marinobacter hydrocarbonoclasticus metalloenzyme (nitric oxide reductase (NOR)), responsible for the NO reduction in the denitrifying processes, was developed through the direct adsorption of a new nanocomposite (multiwalled carbon nanotubes (MWCNTs)/1-n-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF4)/NOR) onto a pyrolytic graphite electrode (PGE) surface. The NOR direct electron transfer behavior (formal potential of -0.255 ± 0.003 V vs. Ag/AgCl) and electrocatalysis towards NO reduction (−0.68 ± 0.03 V vs. Ag/AgCl) of the PGE/[MWCNTs/BMIMBF4/NOR] biosensor were investigated in phosphate buffer at pH 6.0. Large enzyme loading (2.04 × 10−10 mol/cm2), acceptable electron transfer rate between NOR and the PGE surface (ks = 0.35 s-1), and high affinity for NO (Km = 2.17 μmol L-1) were observed with this biosensor composition. A linear response to NO concentration (0.23–4.76 μmol L-1) was perceived with high sensitivity (0.429 μA/μmolL-1), a detection limit of 0.07 μmol L-1, appropriate repeatability (9.1% relative standard deviations (RSD)), reproducibility (6.0–11% RSD) and 80–102% recoveries. The biosensor was stable during 1 month retaining 79–116% of its initial response. These data confirmed that NOR incorporated in the MWCNTs/BMIMBF4 nanocomposite can efficiently maintain its bioactivity paving a new and effective way for NO biosensing.
- Cork - a natural material for linalool controlled releasePublication . Sousa, Sara; Silva, Mário; Gomes, Filipa O.; Domingues, Valentina; Delerue-Matos, CristinaControlled release of aromatic mixtures to the atmosphere is a requirement for scented systems for indoor applications. The product must smell nice, but also be able to last, slowly releasing the perfume over time. Several adsorption materials have been used, for this purpose. In this study, cork was investigated as a potential perfume adsorbent for application in scented drawer sachets and equivalent products. Cork was selected due to its adsorption properties and because it is a natural, renewable, sustainable material. Granulated cork is a significant by‐product in cork industries and it was chosen for adsorption in this work. Linalool, an enantiomeric monoterpene alcohol and one of the main components of several essential oils, was selected as the model compound for adsorption studies. Activated carbon (AC) was used as the reference material. The sorption of linalool to granulated cork and AC was evaluated by HS‐SPME‐GC‐FID. The linalool isotherm on cork was shown to follow a Brunauer‐Deming‐Deming and Teller, Type IV model. The isotherm data on AC can be adjusted to Langmuir and Freundlich models. A maximum adsorption capacity of 3.9x103μg/g was achieved for AC. Desorption studies were performed. Linalool was still released from granulated cork after three equilibrium stages of desorption, whereas only two desorption values were obtained for AC from the equilibrium with highest linalool concentration. Thus, AC demonstrated good adsorption but not good desorption properties. Sorption and desorption studies of linalool from granulated cork, showed that granulated cork could be an excellent material allowing controlled release of the aroma.