Biosensors Based On Odorant-binding Proteins(OBPs)

As superior chemical sensing and detection systems,the remarkable olfactory systems of different organisms could sensitively and selectively detect trace amount of odorant molecules in a complex environment.Inspired by the olfaction,researchers devoted to developing olfactory biosensors in vitro that imitate the organisms' sensing ability for the applications and developments in the fields of environmental monitoring,food detection and disease diagnosis.However,the widely-used biomaterials,such as the olfactory tissues,cells and receptors,were either difficult to obtain or difficult to maintain their activities for a long time,which hindered the development of the olfactory biosensors.As one kind of extracelluar olfaction proteins,odorant-binding proteins(OBPs)could be expressed and purified in large quantities.Meanwhile,OBPs showed high affinities and excellent stabilities when interacting with odorant molecules.Therefore,developing OBP-based biosensors has important practical significance and application prospect to the olfactory sensing researches.In this study,biosensors based on OBPs from Apis cerana cerana(ASP2),Bactrocera dorsalis(BdorOBP2)and human(OBP2a and OBP2b)were developed.Through immobilizing the OBPs on the electrodes,different semiochemicals could be sensed through electrochemical techniques.With the help of impedance models and molecular docking technology,interactions between OBPs and odorant molecules could be analyzed.Meanwhile,the developments and applications of OBP-based biosensors were discussed.The main contents and contributions of this thesis are given as following:1.Olfactory biosensors based on insect odorant-binding proteins were designed and developed for semiochemicals detection.Insects have remarkable olfactory systems,which are closely related to the abilities of the odorant-binding proteins binding to odor molecules.In this thesis,after being successfully expressed and purified,OBPs of Apis cerana cerana(ASP2)and Bactrocera dorsalis(BdorOBP2)were immoblized on the interdigitated electrodes through either physical adsorption or self-assembled monlayer to develop the olfactory biosensors.Different semiochemicals were detected by the biosensors through electrochemical impedance.Compared with other kinds of OBPs-based biosensors,the biosensor in the study showed a relatively wider detection ranges with a good sensitivity and a low detection limit.2.Interactions between the odorant-binding proteins and semiochemicals were analyzed through molecular docking and an impedance model that was developed with the proteins' three-dimensional structures.The central hydrophobic pocket in the three-dimensional structure of OBPs was the main place for interacting with hydrophobic molecules.Thus,conformation changes of OBPs play important roles in binding or releasing the molecules.In this thesis,electrochemical impedance model of protein skeleton and the hydrophobic cavity were developed to analyze the correlations between the sensor impedance changes and the protein conformation changes.Combined with the results of molecular docking,the key amino acid residues in the hydrophobic cavities that played important roles in binding semiochemicals,were distinguished,which laid a foundation to investigate the specific binding functions of OBPs.3.Biosensors based on human odorant-binding proteins were developed for detecting fatty acids with different lengths of carbon chains.As one of the most important components and energy storage materials,fatty acids are also regarded as a kind of important lipids.In this thesis,utilizing the binding properties of human OBP2a to fatty acids,we designed an OBP-based biosensor for fatty acids detection.Through electrochemical reduction,graphene oxide was modified on the carbon working electrode to form a sheet that comprised graphene domains with residual oxygen-containing functional groups,which could increase the electrode conductivity and be used for protein immobilization.For fatty acid sensing,docosahexaenoic acid,linoleic acid,and lauric acid,were sensitively detected through cyclic voltammetry.Results suggested that human OBP2a showed higher binding affinities to the medium-and long-chain fatty acids,which could be used for analyzing fat acids with different lengths of carbon chains.4.Utilizing the anodic aluminum oxide nanopore arrays,biosensors based on odorant-binding proteins were developed for detecting benzaldehyde and fatty acids.As a filtering membrane,the nanopore membrane employed nanoscale channels that highly sensitive due to the large surface-to-volume ratio,to sense target molecules in solution.In this thesis,utilizing the binding properties between human OBP2b and odorant molecules,such as benzaldehyde,a nanopore based biosensor was developed with the three electrode system.After immobilizing OBPs on nanopores as the sensing membrane,the nanopores were placed between the working electrode and the reference/counter electrodes,which lead the nanopores to be the only connection for ions and fluid transferring between the two parts of electrochemical devices.Stimulated by benzaldehydes,lauric acid,and docosahexaenoic acid,protein conformation changes could induce the current changes that flowed through the nanopore arrays.Through electrochemical impedance testing,the biosensor could sensitively detect benzaldehyde and fatty acids at different concentrations.