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 detection

Nitric 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.FG thanks FCT/MCTES for the fellowship grant SFRH/BD/52502/2014, which is financed by national funds and co-financed by FSE. LBM thanks to FCT/MCTES for the CEEC-Individual 2017 Program Contract. This work was supported by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES) and also by the Associate Laboratory Research Unit for Green Chemistry - Technologies and Processes Clean – LAQV, financed by national funds from FCT/MEC (UID/QUI/50006/2019) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265). Funding through REQUIMTE project entitled “NOR-based biosensor for nitric oxide detection in biological and environmental samples” is also acknowledged. The financial support from the European Union (FEDER funds through COMPETE) and National Funds (Fundação para a Ciência e Tecnologia-FCT) through project PTDC/ASP-PES/29547/2017, by FCT/MEC with national funds and co-funded by FEDER, is also acknowledged.info:eu-repo/semantics/publishedVersio

Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reduction

Understanding 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.FG and LBM thank FCT/MCTES for the fellowship grants SFRH/BD/52502/2014 and SFRH/BPD/111404/2015, respectively, which are financed by national funds and co-financed by FSE. CMC acknowledges FCT-MCTES funding through project PTDC/BBB-BQB/0129/2014 (FCT/MCTES). This work was supported by the REQUIMTE, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2013 and UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265 and POCI-01-0145-FEDER-007728), and also by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES). Funding through REQUIMTE project entitled “NOR-based biosensor for nitric oxide detection in biological and environmental samples” is also acknowledged.info:eu-repo/semantics/publishedVersio

Gene Sequence and the 1.8 Å Crystal Structure of the Tungsten-Containing Formate Dehydrogenase from Desulfovibrio gigas

AbstractDesulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H+ by buried waters and protonable amino acids and for CO2 through a hydrophobic channel

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

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.FG thanks to Fundacão para a Ciência e a Tecnologia, MCTES (FCT/MCTES) for the fellowship grant SFRH/BD/52502/2014, which is financed by national funds and co-financed by FSE. LBM thanks to FCT/MCTES for the CEEC-Individual 2017 Program Contract. This work was supported by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES), by FCT/MEC with national funds and co-funded by FEDER, and also by the Associate Laboratory Research Unit for Green Chemistry - Technologies and Processes Clean – LAQV, financed by national funds from FCT/MCTES (UID/QUI/50006/2019). The financial support from the European Union (FEDER funds through COMPETE) and National Funds (Fundação para a Ciência e Tecnologia-FCT) through project PTDC/ASP-PES/29547/2017, by FCT/MEC with national funds and co-funded by FEDER, is also acknowledged. J.A. Loureiro post-doc grant was supported by NORTE-01-0145-FEDER-000005 – LEPABE-2 ECO-INNOVATION, from North Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Construction of effective disposable biosensors for point of care testing of nitrite

© 2015. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/"In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes’ system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40 °C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR’s robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca. -0.4 V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1 – 6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M-1 cm-2 with a linear response range 0.7 – 370 μM.

Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadate

Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68 ± 22 lM and 17 ± 2 lM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2 mM concentration of ‘‘metavanadate’’ solution that contains ortho and metavanadate species, as observed by combining kinetic with 51V NMR spectroscopy studies. Although at 25 C, decameric vanadate (10 lM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 lM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2 mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10 h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18 h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the ‘‘decavanadate’’ interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems

Strategies in the use of light energy by Genipa spruceana Steyerm seedlings subjected to flooding

In an attempt to elucidate strategies in the use of light energy by G. spruceana seedlings subjected to flooding, we investigated the capacity of light capture and use of light energy by G. spruceana in three growing conditions: 1- absence of flooding (SA), 2- partially flooded (PA) and 3- totally flooded (TA). Destructive and non-destructive measurements, such as specific leaf area, chloroplast pigment (chlorophyll and carotenoids) content and fluorescence analyses, were made at regular intervals over a period of 90 days. All parameters decreased in seedlings subjected to flooding. Plants of treatment TA dropped all of their leaves after 30 days of complete submergence. Chloroplast pigment content differed between treatments SA and TA after 30 days from the start of the experiment; whereas SA and PA plants only differed for this variable after 90 days. Plants subjected to flooding (PA and TA) exhibited high dissipation of photochemical de-excitation (DIo/ABS), indicating a limited efficiency of light energy use. This fact was proven by the performance index (PI ABS) only in analyses after 90 days, and no significant difference was verified for PI ABS among treatments up to 30 days. Therefore, considering that G. spruceana seedlings subjected to flooding reduced the chloroplast pigment content more quickly than PI ABS, we suggest that the light energetic flux in G. spruceana seedlings subjected to flooding, in the beginning, is more restricted to a decrease in the structures that captures light (reduction chlorophyll pigment content) than how the photosynthetic apparatus functions (alterations in photochemical efficiency of photosystem II).Na tentativa de elucidar estratégias de utilização da energia luminosa em plantas jovens de Genipa spruceana Steyerm submetidas ao alagamento, nós investigamos a capacidade de captura e uso de energia luminosa em G. spruceana sob três condições de crescimento1- ausência de alagamento (SA), 2- plantas parcialmente alagadas (PA) e 3- plantas totalmente alagadas (TA). Medidas de área foliar específica, teores de pigmentos cloroplastídicos e fluorescência da clorofila a foram feitas em intervalos regulares no período de 90 dias. Todos os parâmetros analisados diminuíram em condições de alagamento (PA e TA). Aos 30 dias, as plantas no tratamento TA sofreram abscisão foliar. Os teores dos pigmentos cloroplastídicos (clorofilas e carotenóides) entre os tratamentos SA e TA diferiram aos 30 dias. Ao passo que, somente foi possível verificar diferenças entre os tratamentos SA e PA aos 90 dias. As plantas submetidas ao alagamento (PA e TA) exibiram alta dissipação de energia de excitação (DIo/ABS) indicando limitada eficiência na utilização da energia luminosa. Este fato foi comprovado pelos resultados do índice de desempenho (PI ABS) somente ao fim do período experimental (90 dias). Mas, não foi verificado diferença para PI ABS entre os tratamentos aos 30 dias. Portanto, considerando que G. spruceana submetidas ao tratamento TA reduziram seus teores de clorofilas mais rapidamente do que decrescem seus PI ABS, sugere-se que o fluxo de energia luminosa em plântulas de G. spruceana sob alagamento total, no início, é mais restringido pelo decréscimo na estrutura de captura de luz (diminuição dos pigmentos cloroplastídicos) do que no funcionamento do aparato fotossintético (alterações na eficiência fotoquímica do fotossistema II)

Dissimilatory nitrate and nitrite ammonification by sulphate-reducing bacteria

Dissimilatory nitrate and nitrite ammonification by sulphate-reducing bacteri