title: Electrochemical detection of the 2-isobutyl-3-methoxypyrazine model odorant based on odorant-binding proteins: the proof of concept.
abstract: We developed an electrochemical assay for the detection of odorant molecules based on a rat odorant-binding protein (rOBP3). We demonstrated that rOBP3 cavity binds 2-methyl-1,4-naphtoquinone (MNQ), an electrochemical probe, as depicted from the decrease of its electrochemical signal, and deduced the dissociation constant, KdMNQ=0.5(±0.2)μM. The amount of MNQ displaced from rOBP3 by 2-isobutyl-3-methoxypyrazine (IBMP), a model odorant molecule, was measured using square-wave voltammetry. The release of MNQ by competition led to an increase of the electrochemical response. In addition, this method allowed determination of the dissociation constant of rOBP3 for IBMP, KdIBMP=0.5(±0.1)μM. A negative control was performed with a non-binding species, caffeic acid (CA). The determined binding affinity values were confirmed using a fluorescent competitive binding assay and isothermal titration microcalorimetry. This electrochemical assay opens the way for designing robust, reliable and inexpensive odorant biosensors.
authors.affiliation: Institut de Chimie Moléculaire de l'Université de Bourgogne, ICMUB, UMR CNRS 6302, 9, Avenue Alain Savary, BP 47870, 21078 Dijon Cedex, France; Centre des Sciences du Goût et de l'Alimentation, UMR INRA 1324, UMR CNRS 6265, Université de Bourgogne, 17 rue Sully, 21065 Dijon, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Barou
authors.affiliation: Centre des Sciences du Goût et de l'Alimentation, UMR INRA 1324, UMR CNRS 6265, Université de Bourgogne, 17 rue Sully, 21065 Dijon, France.
authors.forename: Maud
authors.initials: M
authors.lastname: Sigoillot
authors.affiliation: Institut de Chimie Moléculaire de l'Université de Bourgogne, ICMUB, UMR CNRS 6302, 9, Avenue Alain Savary, BP 47870, 21078 Dijon Cedex, France.
authors.forename: Marcel
authors.initials: M
authors.lastname: Bouvet
authors.affiliation: Centre des Sciences du Goût et de l'Alimentation, UMR INRA 1324, UMR CNRS 6265, Université de Bourgogne, 17 rue Sully, 21065 Dijon, France. Electronic address: loic.briand@dijon.inra.fr.
authors.forename: Loïc
authors.initials: L
authors.lastname: Briand
authors.affiliation: Institut de Chimie Moléculaire de l'Université de Bourgogne, ICMUB, UMR CNRS 6302, 9, Avenue Alain Savary, BP 47870, 21078 Dijon Cedex, France. Electronic address: rita.meunier-prest@u-bourgogne.fr.
authors.forename: Rita
authors.initials: R
authors.lastname: Meunier-Prest
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002109:Caffeic Acids / Q000737:chemistry; D002138:Calibration; D002151:Calorimetry / Q000379:methods; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011719:Pyrazines / Q000032:analysis*; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D024483:Vitamin K 3 / Q000737:chemistry / Q000378:metabolism
chemical_list: D002109:Caffeic Acids; D055767:Immobilized Proteins; D011719:Pyrazines; D018035:Receptors, Odorant; D011994:Recombinant Proteins; C045648:odorant-binding protein; D024483:Vitamin K 3; C034376:2-isobutyl-3-methoxypyrazine; C040048:caffeic acid
keywords: Electroanalysis; Electrochemistry; Fluorescence; Microcalorimetry; Olfaction