Insect odorant receptors (ORs) are believed to be a complex of an odorant binding subunit, OrX, and an ion channel forming subunit, Orco. In our previous study, we showed that the OrX subunit on its ...own in liposomes could detect volatile organic compounds (VOCs) ultrasensitively using Electrochemical Impedance Spectroscopy (EIS). In this study, we investigated the effect of the presence of Orco on the response of the OrX subunit to detect the VOCs. The OrXs - Or10a, Or22a, Or35a and Or71a, together with Orco, were recombinantly expressed, purified and integrated into liposomes. These OrX/Orco liposomes were covalently attached to a gold surface modified with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (NHS/EDC)-activated self-assembled monolayers (SAMs) of 6-mercaptohexanoic acid (MHA). It was demonstrated that the OrX/Orco liposomes could sensitively and selectively detect their ligands by monitoring a change in frequency and impedance signal upon binding with both Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and EIS. Using EIS, three OrXs (Or10a, Or22a and Or35a) showed a shift in their dose-response curves when Orco was co-integrated, reflecting an increase in ligand sensitivity and a decrease in limit of detection (LOD). Or71a in the presence of Orco did not show any improvement in ligand sensitivity as this is a highly tuned receptor which may be already at the sensitivity limit for EIS. The observed enhancement in sensor performance is believed to be an effect of Orco which is stabilizing the OrX in a more active conformation and amplifying charge transfer to result in a greater reduction in impedance.
•The effect of the presence of Orco on the response of the olfactory receptor subunit (OrX) to detect the ligands was investigated.•QCM-D demonstrated that OrX/Orco liposomes were intact on gold surface and could sensitively and selectively detect their ligands but the presence of Orco had no obvious effect on the sensitivity.•EIS demonstrated synergistic enhancement in the performance of the OrX based sensor when Orco was co-integrated, showing an increase in ligand sensitivity (10–1000 fold) and a decrease in LOD.
In this study, in situ vapor phase polymerization (VPP) of (1) 3,4-ethylenedioxythiophene (EDOT) and (2) pyrrole (Py) on an oxidant-impregnated thermoplastic polyurethane (TPU) substrate was ...conducted to produce a (1) PEDOT-TPU hybrid film and (2) PPy-TPU hybrid film. Scanning electron microscopy, atomic force microscopy, and energy dispersive x-ray spectroscopy confirmed the formation of uniform, rough surfaces of PPy and PEDOT on the TPU matrix. Both PPy-TPU and PEDOT-TPU films showed excellent thermal stability across the temperature range of 300 °C–400 °C and enhanced mechanical properties, depicted by very high stress and strain values. The performance of these hybrid films to detect ammonia (NH3) gas was investigated and then optimized under the effects of varying temperature, ammonia concentration, and monomer type used. The PEDOT-TPU and PPy-TPU showed remarkable sensitivity to NH3 gas, with coefficients of variation of 0.9858 and 0.9653, respectively. Particularly, the PEDOT-TPU sensor showed higher responsiveness to NH3 at higher operating temperatures owing to its higher surface roughness and thermal stability. These flexible, stretchable, smart conducting polymer composite materials fabricated using VPP are therefore potential candidates for monitoring toxic and non-toxic gases.
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•PEDOT-TPU and PPy-TPU based ammonia sensors were fabricated by in situ vapor phase polymerization.•The sensors showed high sensitivity, electrical stability, good selectivity, and repeatable response towards NH3.•These smart conducting polymer composite materials are potential candidates for monitoring toxic and non-toxic gases.
Copper membrane monooxygenases (CuMMOs) oxidize ammonia, methane and some short-chain alkanes and alkenes. They are encoded by three genes, usually in an operon of
. We aligned
operons from 66 ...microbial genomes, including members of the
-,
-, and
and the candidate phylum NC10. Phylogenetic and compositional analyses were used to reconstruct the evolutionary history of the enzyme and detect potential lateral gene transfer (LGT) events. The phylogenetic analyses showed at least 10 clusters corresponding to a combination of substrate specificity and bacterial taxonomy, but with no overriding structure based on either function or taxonomy alone. Adaptation of the enzyme to preferentially oxidize either ammonia or methane has occurred more than once. Individual phylogenies of all three genes,
and
, closely matched, indicating that this operon evolved or was consistently transferred as a unit, with the possible exception of the methane monooxygenase operons in
, where the
gene has a distinct phylogeny from
and
. Compositional analyses indicated that some clusters of
operons (for example, the
in gammaproteobacterial methanotrophs and the
in betaproteobacterial nitrifiers) were compositionally very different from their genomes, possibly indicating recent lateral transfer of these operons. The combined phylogenetic and compositional analyses support the hypothesis that an ancestor of the nitrifying bacterium
was the donor of methane monooxygenase (pMMO) to both the alphaproteobacterial and gammaproteobacterial methanotrophs, but that before this event the gammaproteobacterial methanotrophs originally possessed another CuMMO (Pxm), which has since been lost in many species.
Summary
Recently, methanotrophic members of the phylum Verrucomicrobia have been described, but little is known about their distribution in nature. We surveyed methanotrophic bacteria in geothermal ...springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in samples covering a broad temperature range (22.5–81.6°C), but only in acidic conditions (pH 1.8–5.0) and only in geothermal environments, not in acidic bogs or fens. Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic Verrucomicrobia were observed. Those detected in high‐temperature geothermal samples (44.1–81.6°C) grouped with known thermoacidiphilic ‘Methylacidiphilum’ isolates. A second group dominated in moderate‐temperature geothermal samples (22.5–40.1°C) and a representative mesophilic methanotroph from this group was isolated (strain LP2A). Genome sequencing verified that strain LP2A possessed particulate methane monooxygenase, but its 16S rRNA gene sequence identity to ‘Methylacidiphilum infernorum’ strain V4 was only 90.6%. A third group clustered distantly with known methanotrophic Verrucomicrobia. Using pmoA‐gene targeted quantitative polymerase chain reaction, two geothermal soil profiles showed a dominance of LP2A‐like pmoA sequences in the cooler surface layers and ‘Methylacidiphilum'‐like pmoA sequences in deeper, hotter layers. Based on these results, there appears to be a thermophilic group and a mesophilic group of methanotrophic Verrucomicrobia. However, both were detected only in acidic geothermal environments.
Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short-chain alkanes and alkenes. In a metagenome ...constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived protein sequence identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell amplified genomes (SAGs) from propane-enriched samples were screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium encoded enzymes with the potential to support catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies these enzymes in non-nitrifying Betaproteobacteria.
Insect Odorant receptors (OrXs) can be used as the recognition element in a biosensor as they demonstrate high levels of sensitivity and selectivity towards volatile organic compounds. Herein, we ...describe a method to express and purify insect odorant receptors and reconstitute them into artificial lipid bilayers (liposomes). These OrX/liposomes were covalently attached to a gold surface and characterized using quartz crystal microbalance with dissipation monitoring (QCM-D). The interaction of OrX/liposomes immobilized on a gold surface to positive and negative odorants were studied by means of electrochemical impedance spectroscopy (EIS) and QCM-D. The data presented in this article are related to the research article titled “An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants” 1.
Herein, we present that insect odorant receptors reconstituted into the lipid bilayers of liposomes can be successfully immobilized onto a gold surface and selectively and sensitively detect odorant ...molecules. The odorant receptors (OrXs) Or10a, Or22a, and Or71a from the common fruit fly, Drosophila melanogaster, were recombinantly expressed, purified and integrated into nano-liposomes (100–200 nm). These liposomes were covalently attached to the self-assembled monolayers (SAMs) of a 6-mercaptohexanoic acid (MHA)-modified gold surface. X-ray Photo Electron Spectroscopy (XPS) and Quartz Crystal Microbalance with Dissipation (QCM-D) measurements confirmed the successful modification of the gold surface and immobilization of liposomes. Atomic Force Microscopy (AFM) revealed that the liposomes were covalently attached to the surface without any disruption of vesicles. The liposomes tethered to the gold sensor surface were then treated with a range of known ligands of various concentrations. We demonstrated by Electrochemical Impedance Spectroscopy (EIS) that an OrX/liposome EIS sensor can sensitively and selectively detect its known ligand to femtomolar concentrations by detecting a change in electrical signal upon binding. Our study is the first step towards using purified insect odorant receptors alone in biosensors to enable the development of novel ultrasensitive volatile sensors for medical diagnostic, air quality, food safety and border security applications.
•A novel biosensor was fabricated by immobilizing purified insect odorant receptors reconstituted into artificial liposomes on to a gold surface.•XPS and QCM-D confirmed the successful modification of the gold surface and immobilization of the odorant receptor/liposomes.•TEM and AFM revealed the covalent attachment of the odorant receptor/liposomes (100–200 nm) onto the gold surface without any disruption of vesicles.•EIS measurements showed that these insect odorant receptor based biosensors could detect their respective odorants down to femtomolar level.
Herein,
in situ
vapor-phase polymerization (VPP) of pyrrole on an oxidant-impregnated styrene-ethylene-butylene-styrene (SEBS) matrix comprising a three-dimensional sugar particle assembly was used ...to produce a soft and porous polypyrrole (PPy)-SEBS hybrid scaffold. Characterization of the PPy-SEBS hybrid scaffold using field-effect scanning electron microscopy, Fourier-transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, and micro-computerized tomography confirmed the successful uniform and homogenous polymerization of PPy onto the SEBS matrix with a porous morphology. The performance of the hybrid scaffold of different pore sizes as an ammonia sensor under different temperature conditions was evaluated in terms of resistance change. The results showed that the PPy-SEBS scaffolds of larger pore size had higher resistance changes under lower temperature conditions when ammonia (NH
3
) gas was introduced compared to those observed for smaller pore sizes under higher temperature conditions. These scaffolds showed excellent repeatability and reversibility in detecting NH
3
gas with fast response and recovery times of 30 s and 10-15 min, respectively. Moreover, the larger pore size scaffolds polymerized for a longer time possessed a remarkable ability to be applied as strain sensors. These kinds of novel, soft, and porous conductive polymer composite materials produced by VPP will have huge practical applications in monitoring other toxic and non-toxic gases.
Fabrication of a hybrid scaffold from an oxidant-impregnated styrene-ethylene-butylene-styrene (SEBS) matrix comprising a three-dimensional sugar particle assembly by vapor phase polymerization (VPP).
•Polyethylene oxides in electropolymerization forming more compact PPy films.•Linear actuators of PPy-PEO/DBS showing significant increase in strain.•The ion diffusion coefficient and strain rate is ...improved in PPy-PEO/DBS.
In the present paper, we report the electro-chemo-mechanical deformation (ECMD) measurements of pristine polypyrrole (PPy) and polypyrrole/poly(ethylene oxide) composite films doped with dodecylbenzenesulfonate (DBS) under isotonic (constant force) conditions. The PPy/DBS and PPy-PEO/DBS films, the latter made from a polymerization solution containing 5wt% PEO, were actuated potentiodynamically in an aqueous solution containing lithium bis-trifluoromethane-sulfonimide (LiTFSI). The obtained composite films were characterized with scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy and Fourier transform infrared spectroscopy (FTIR). The results confirmed the successful incorporation of PEO within the PPy film. The composite films showed a remarkable improvement in maximum strain achieved (of 7.7%), nearly double that of the pristine PPy/DBS film, had a higher conductivity, higher ion diffusion coefficient and faster strain rate. The ECMD measurements for both the pristine as well as the PEO-composite films showed cation driven actuation involving the migration of Li+ cations in the aqueous electrolyte.
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