Grafting odorant binding proteins on diamond bio-MEMS.
Manai R,Scorsone E,Rousseau L,Ghassemi F,Possas Abreu M,Lissorgues G,Tremillon N,Ginisty H,Arnault J-C,Tuccori E,Bernabei M,Cali K,Persaud K C,Bergonzo P
Biosensors & bioelectronics
Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approach based on complexing a histidine-tag located on the protein with nickel allowed control of the proteins' orientation. Evidence confirming protein grafting was obtained using electrochemical impedance spectroscopy, fluorescence imaging and X-ray photoelectron spectroscopy. The chemical sensing performances of these OBP modified transducers were assessed. The second grafting method led to typically 20% more sensitive sensors, as a result of better access of ligands to the proteins active sites and also perhaps a better yield of protein immobilization. This new grafting method appears to be highly promising for further investigation of the ligand binding properties of OBPs in general and for the development of arrays of non-specific biosensors for artificial olfaction applications.
10.1016/j.bios.2014.04.020
Olfactory biosensor for insect semiochemicals analysis by impedance sensing of odorant-binding proteins on interdigitated electrodes.
Lu Yanli,Yao Yao,Zhang Qian,Zhang Diming,Zhuang Shulin,Li Hongliang,Liu Qingjun
Biosensors & bioelectronics
Insects can sensitively and selectively detect thousands of semiochemicals at very low concentrations by their remarkable olfactory systems. As one of the most important olfactory proteins, odorant-binding proteins (OBPs) from insects are the most promising candidates for fabricating biosensors to detect biochemical molecules in the chemical ecology as well as for other biotechnological applications. In this study, we designed an olfactory biosensor by immobilizing OBPs from oriental fruit fly on interdigitated electrodes to detect semiochemicals. After successfully separated and purified, OBPs were immobilized by the special designed polyethylene glycol (PEG), SH-PEG-COOH, to produce a robust sensing membrane. Based on electrochemical sensing, interactions between OBPs and different semiochemicals emitted from host plants of the insect, such as the isoamyl acetate, β-ionone, and benzaldehyde, could be sensitively detected. With related amino acid residues in the hydrophobic cavities distinguished, the interaction forces between semiochemicals and OBPs were analyzed by molecular docking. Integrated biological olfaction proteins of insects, OBPs based biosensors could not only advance the progress in the understanding of chemical communication systems of insects, but also show promising potentials for biosensing applications in many fields.
10.1016/j.bios.2014.09.098
Capacitance-modulated transistor detects odorant binding protein chiral interactions.
Nature communications
Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.
10.1038/ncomms7010
Electronic Olfactory Sensor Based on A. mellifera Odorant-Binding Protein 14 on a Reduced Graphene Oxide Field-Effect Transistor.
Angewandte Chemie (International ed. in English)
An olfactory biosensor based on a reduced graphene oxide (rGO) field-effect transistor (FET), functionalized by the odorant-binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real-time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand-receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K(d)=4 μM to K(d)=3.3 mM. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.
10.1002/anie.201505712
Molecular Basis of Alarm Pheromone Detection in Aphids.
Zhang Ruibin,Wang Bing,Grossi Gerarda,Falabella Patrizia,Liu Yang,Yan Shanchun,Lu Jian,Xi Jinghui,Wang Guirong
Current biology : CB
The sesquiterpene (E)-β-farnesene (EBF) is the alarm pheromone for many species of aphids [1]. When released from aphids attacked by parasitoids or predators, it alerts nearby conspecifics to escape by walking away and dropping off the host plant [2, 3]. The reception of alarm pheromone in aphids is accomplished through a highly sensitive chemosensory system. Although olfaction-related gene families including odorant receptors (ORs) and odorant-binding proteins (OBPs) have recently been identified from aphid genomes [4-6], the cellular and molecular mechanisms of EBF reception are still largely unknown. Here we demonstrate that ApisOR5, a member of the large superfamily of odorant receptors, is expressed in large placoid sensillum neurons on the sixth antennal segment and confers response to EBF when co-expressed with Orco, an obligate odorant receptor co-receptor, in parallel heterologous expression systems. In addition, the repellent behavior of Acyrthosiphon pisum to EBF disappears after knocking down ApisOR5 by RNAi as well as two A. pisum odorant-binding proteins known to bind EBF (ApisOBP3 and ApisOBP7). Furthermore, other odorants that can also activate ApisOR5, such as geranyl acetate, significantly repel A. pisum, as does EBF. Taken together, these data allow us to conclude that ApisOR5 is essential to EBF reception in A. pisum. The characterization of the EBF receptor allows high-throughput screening of aphid repellents, providing the necessary information to develop new strategies for aphid control.
10.1016/j.cub.2016.10.013
Field-Effect Transistor Biosensor Platform Fused with Drosophila Odorant-Binding Proteins for Instant Ethanol Detection.
Lim Cheol-Min,Kwon Jae Young,Cho Won-Ju
ACS applied materials & interfaces
Odorant-binding proteins (OBPs) have attracted considerable attention as sensing substrates for the development of olfactory biosensors. The Drosophila LUSH protein is an OBP and is known to bind to various alcohols. Technology that uses the LUSH protein has great potential to provide crucial information through odorant detection. In this work, the LUSH protein was used as a sensing substrate to detect the ethanol concentration. Furthermore, we fused the LUSH protein with a silicon-on-insulator (SOI)-based ion-sensitive field-effect transistor (ISFET) to measure the electrical signals that arise from molecular interactions between the LUSH and ethanol. A dual-gate sensing system for self-amplification of the signal resulting from the molecular interaction between the LUSH and ethanol was then used to achieve a much higher sensitivity than a conventional ISFET. In the end, we successfully detected ethanol at concentrations ranging between 0.001 and 1% using the LUSH OBP-fused ISFET olfactory sensor. The OBP-fused SOI-based olfactory ISFET sensor can lead to the development of handheld sensors for various purposes such as detecting toxic chemicals, narcotics control, testing for food freshness, and noninvasive diagnoses.
10.1021/acsami.6b15539
Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles.
Zhu Jiao,Arena Simona,Spinelli Silvia,Liu Dingzhen,Zhang Guiquan,Wei Rongping,Cambillau Christian,Scaloni Andrea,Wang Guirong,Pelosi Paolo
Proceedings of the National Academy of Sciences of the United States of America
The giant panda belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda.
10.1073/pnas.1711437114
1-Aminoanthracene Transduction into Liposomes Driven by Odorant-Binding Protein Proximity.
Gonçalves Filipa,Silva Carla,Ribeiro Artur,Cavaco-Paulo Artur
ACS applied materials & interfaces
In this work, the anchorage of pig odorant binding protein (OBP-I) into liposomal membrane was promoted by the fusion of OBP-I with the anchor SP-DS3 peptide and with the (GQ) spacer. The presence of the (GQ) spacer in the construct confers flexibility to the protein and increases the distance between the OBP binding site and the liposomal surface. The engineered proteins, OBP::SP-DS3 and OBP::(GQ)::SP-DS3, were produced in Escherichia coli BL21(DE3) and characterized by circular dichroism spectroscopy and MALDI-TOF. The functionalization of liposomes with the OBP proteins was performed through ethanol injection, and similar liposomal anchorage (∼92-97%) was found for both OBP constructs. The effect of OBPs' proximity to the liposomes membrane on 1-aminoanthracene (1-AMA, model ligand) transduction was evaluated by measuring the amount of 1-AMA transduced into liposomes by fluorescence spectroscopy. While protein flexibility, given by the presence of the (GQ) spacer, seems to influence the binding efficiency, ∼45% for OBP::(GQ)::SP-DS3 and ∼29% for OBP::SP-DS3, the distance between the proteins' binding site and the liposomal membrane determines their ability to transduce the 1-AMA into the liposomes (∼23% for OBP::SP-DS3 and ∼19% for OBP::(GQ)::SP-DS3). The anchorage capacity and proximity effect were confirmed by an experimental control where the wild-type (wt) OBP was added to the liposomes, resulting in low 1-AMA transduction (∼3.5%) and low binding to OBPwt (∼9%). These findings evidence the effect of anchorage, carrier protein's flexibility, and proximity as key features for the entrapment of molecules into the liposomal membrane. The developed OBP-based devices are thus promising anchorage systems for the capture and storage of odors with potential applications in textile and cosmetic industries.
10.1021/acsami.8b10158