Fine discrimination of volatile compounds by graphene-immobilized odorant-binding proteins

Abstract We describe the fabrication and performance of a biosensor for odorants, using wildtype and engineered mutants of the Italian honeybee (Apis mellifera ligustica) odorant binding protein 14 (OBP14), immobilized onto a reduced graphene oxide field-effect transistor (rGO-FET). The binding properties of the protein when immobilized on the biosensor are similar to those measured in solution, thus providing a method for measuring affinities to small molecules as an alternative to the current fluorescence assay. Out of the 14 chemicals tested, the best ligands for wildtype OBP14 were eugenol, homovanillic acid and related compounds sharing a phenol-methoxy backbone. Other chemicals, including methyl eugenol, showed affinities to OBP14 100–1000 times lower. We have also tested two mutants of OBP14. The first, bearing a HisTag at its N-terminus for better orientation on the sensor surface, showed only minor differences in its binding properties for chemicals when compared to the wildtype. The second contained an additional disulfide bond between helices α3 and α6, thus reducing the dynamics of OBP14 and leading to a higher affinity for eugenol. These data also demonstrate that it is feasible to produce biosensors with desired ligand specificities by introducing selected mutations into the structure of OBPs or other active proteins.

[1]  Andrey Rzhetsky,et al.  A Spatial Map of Olfactory Receptor Expression in the Drosophila Antenna , 1999, Cell.

[2]  W. Knoll,et al.  Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements , 2013, European Biophysics Journal.

[3]  Shun Mao,et al.  Graphene-based electronic biosensors , 2017 .

[4]  Jing-Jiang Zhou,et al.  Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth, Plutella xyllostella , 2013, PloS one.

[5]  C. Cambillau,et al.  Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules. , 2012, Insect biochemistry and molecular biology.

[6]  Hyun Seok Song,et al.  Single‐Carbon‐Atomic‐Resolution Detection of Odorant Molecules using a Human Olfactory Receptor‐based Bioelectronic Nose , 2009 .

[7]  Tai Hyun Park,et al.  Bioelectronic nose with high sensitivity and selectivity using chemically functionalized carbon nanotube combined with human olfactory receptor. , 2012, Journal of biotechnology.

[8]  Mohammad Yusuf Mulla,et al.  Capacitance-modulated transistor detects odorant binding protein chiral interactions , 2015, Nature Communications.

[9]  J. Clardy,et al.  Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein-bombykol complex. , 2000, Chemistry & biology.

[10]  Christoph Nowak,et al.  The extended growth of graphene oxide flakes using ethanol CVD. , 2013, Nanoscale.

[11]  E. Guittet,et al.  Structural basis of the broad specificity of a general odorant-binding protein from honeybee. , 2009, Biochemistry.

[12]  Robert L. Metcalf,et al.  Chemical ecology of Dacinae fruit flies (Diptera: Tephritidae). , 1990 .

[13]  C. Cambillau,et al.  The crystal structure of odorant binding protein 7 from Anopheles gambiae exhibits an outstanding adaptability of its binding site. , 2011, Journal of molecular biology.

[14]  Angelo Gazzano,et al.  Differential expression of odorant-binding proteins in the mandibular glands of the honey bee according to caste and age. , 2011, Journal of proteome research.

[15]  P. Pelosi,et al.  Two Odorant-Binding Proteins Mediate the Behavioural Response of Aphids to the Alarm Pheromone (E)-ß-farnesene and Structural Analogues , 2012, PloS one.

[16]  M. Wogulis,et al.  The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism. , 2006, Biochemical and biophysical research communications.

[17]  W. Knoll,et al.  Insights into structural features determining odorant affinities to honey bee odorant binding protein 14. , 2014, Biochemical and biophysical research communications.

[18]  P. Pelosi,et al.  Discrimination of alarm pheromone (E)-beta-farnesene by aphid odorant-binding proteins. , 2009, Insect biochemistry and molecular biology.

[19]  Wolfgang Knoll,et al.  Graphene-based liquid-gated field effect transistor for biosensing: Theory and experiments. , 2015, Biosensors & bioelectronics.

[20]  W. Leal,et al.  Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori , 1999, FEBS letters.

[21]  Oh Seok Kwon,et al.  Polypyrrole nanotubes conjugated with human olfactory receptors: high-performance transducers for FET-type bioelectronic noses. , 2009, Angewandte Chemie.

[22]  Jing Zhang,et al.  Impedance sensing and molecular modeling of an olfactory biosensor based on chemosensory proteins of honeybee. , 2013, Biosensors & bioelectronics.

[23]  C. Cambillau,et al.  Revisiting the Specificity of Mamestra brassicaeand Antheraea polyphemus Pheromone-binding Proteins with a Fluorescence Binding Assay* , 2001, The Journal of Biological Chemistry.

[24]  Tai Hyun Park,et al.  Cell-based olfactory biosensor using microfabricated planar electrode. , 2009, Biosensors & bioelectronics.

[25]  J. Moore,et al.  Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions. , 2000, Methods.

[26]  R. Karlsson,et al.  Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. , 1997, Journal of immunological methods.

[27]  P. Pelosi,et al.  Soluble proteins in insect chemical communication , 2006, Cellular and Molecular Life Sciences CMLS.

[28]  A. Scaloni,et al.  Chemosensory proteins of Locusta migratoria , 2003, Insect molecular biology.

[29]  Stefan Schütz,et al.  Electronic Olfactory Sensor Based on A. mellifera Odorant‐Binding Protein 14 on a Reduced Graphene Oxide Field‐Effect Transistor , 2015, Angewandte Chemie.

[30]  A. Cornel,et al.  Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants , 2010, Journal of Chemical Ecology.

[31]  P. Xu,et al.  Drosophila OBP LUSH Is Required for Activity of Pheromone-Sensitive Neurons , 2005, Neuron.

[32]  Biomedical Vibrational Spectroscopy and Biohazard Detection Technologies , 2004 .

[33]  P. Pelosi,et al.  Binding properties of a locust's chemosensory protein. , 2002, Biochemical and biophysical research communications.

[34]  H. Breer,et al.  A receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus , 2009, International journal of biological sciences.

[35]  L. Vosshall,et al.  Molecular architecture of smell and taste in Drosophila. , 2007, Annual review of neuroscience.

[36]  Wolfgang Knoll,et al.  In situ antibody detection and charge discrimination using aqueous stable pentacene transistor biosensors. , 2011, Journal of the American Chemical Society.

[37]  Tai Hyun Park,et al.  A bioelectronic sensor based on canine olfactory nanovesicle-carbon nanotube hybrid structures for the fast assessment of food quality. , 2012, The Analyst.

[38]  D. Cheng,et al.  An overview of odorant-binding protein functions in insect peripheral olfactory reception. , 2011, Genetics and molecular research : GMR.

[39]  H. Biessmann,et al.  The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes , 2010, PloS one.

[40]  W. Leal,et al.  Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. , 2013, Annual review of entomology.

[41]  P Luginbühl,et al.  NMR structure reveals intramolecular regulation mechanism for pheromone binding and release , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[42]  E. Grosse-Wilde,et al.  A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro. , 2006, Chemical senses.

[43]  L. Riddiford,et al.  Pheromone binding and inactivation by moth antennae , 1981, Nature.

[44]  M. Benetti,et al.  Detection of odorant molecules via surface acoustic wave biosensor array based on odorant-binding proteins. , 2013, Biosensors & bioelectronics.

[45]  Tai Hyun Park,et al.  Nanovesicle-based bioelectronic nose platform mimicking human olfactory signal transduction. , 2012, Biosensors & bioelectronics.

[46]  W. A. Johnson,et al.  The Odor Specificities of a Subset of Olfactory Receptor Neurons Are Governed by Acj6, a POU-Domain Transcription Factor , 1999, Neuron.

[47]  A. Scaloni,et al.  Structural analysis and disulfide-bridge pairing of two odorant-binding proteins from Bombyx mori. , 1999, Biochemical and biophysical research communications.

[48]  P. Pelosi,et al.  Pheromone binding proteins enhance the sensitivity of olfactory receptors to sex pheromones in Chilo suppressalis , 2015, Scientific Reports.

[49]  R. Zhao,et al.  Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster , 2003, Nature Structural Biology.

[50]  Tai Hyun Park,et al.  Ultrasensitive flexible graphene based field-effect transistor (FET)-type bioelectronic nose. , 2012, Nano letters.

[51]  Josep Samitier,et al.  A novel detection strategy for odorant molecules based on controlled bioengineering of rat olfactory receptor I7. , 2007, Biosensors & bioelectronics.

[52]  R. Anholt,et al.  Functional dissection of Odorant binding protein genes in Drosophila melanogaster , 2011, Genes, brain, and behavior.

[53]  Wolfgang Schuhmann,et al.  Graphene-based field effect transistors as biosensors , 2017 .

[54]  R. Vogt,et al.  3.15 – Molecular Basis of Pheromone Detection in Insects , 2005 .

[55]  Sindhuja Sankaran,et al.  Odorant binding protein based biomimetic sensors for detection of alcohols associated with Salmonella contamination in packaged beef. , 2011, Biosensors & bioelectronics.

[56]  I. Iovinella,et al.  New fluorescent probes for ligand-binding assays of odorant-binding proteins. , 2014, Biochemical and biophysical research communications.

[57]  P. Pelosi,et al.  Soluble proteins of chemical communication: an overview across arthropods , 2014, Front. Physiol..

[58]  A. Scaloni,et al.  Soluble proteins of chemical communication in the social wasp Polistes dominulus , 2003, Cellular and Molecular Life Sciences CMLS.