Research On Olfactory Receptor-based Biomimetic Molecular And Cell-based Biosensors

The well evolved biological olfactory system has excellent capacity of detecting and discriminating thousands of odorants with diverse chemical structures and properties from the complex environment, which plays an important role in the life of most creatures. Inspired by the biological olfactory system, biomimetic olfactory sensors have become a hot research focus in the field of chemical sensors, which utilize various biological components as the sensitive elements, such as olfactory receptors, olfactory cells, and olfactory tissues. Biomimetic olfactory sensors have great application prospects in many fields for the detection of specific odorants with high sensitivity. Besides, they also provide a novel research platform for the basic research of olfactory mechanisms. In this thesis, recent advances in biomimetic olfactory sensors were well reviewed, and the biological basis of olfactory system was also introduced briefly. Based on olfactory transdution mechanisms, biomimetic olfactory sensors were investigated from the molecular aspect to the cellular network level, including an olfactory receptor-based biomimetic molecular sensor and an olfactory receptor cell-based biomimetic sensor. The cellular network has also been developed on the surface of microelectrode array (MEA) chip.The major contributions of this thesis include the following four parts:1. Preparation of functional olfactory receptor proteins based on molecular cloning technologyThe expression plasmid pcDNA3.1(+)/his6-tag/rho-tag/odr-10was constructed using standard molecular cloning techniques, and was used to transfect the HEK-293cells. The olfactory receptor protein, ODR-10, was expressed and located onto the plasma membrane of HEK-293. RT-PCR, Western blot, and cell immunofluorescence were conducted to verify the expression and subcellular distribution of ODR-10in HEK-293cells. Intracellular Ca2+imaging was employed to test the function of odorant detection. All the results demonstrated that functional olfactory receptor protein was successfully expressed on the plasma membrane of HEK-293cells, which is suitable to be used as sensitive elements of biomimetic olfactory biosensors.2. Development of olfactory receptor-based biomimetic molecular sensors To improve the immobilization efficiency of olfactory receptor ODR-10on the sensitive surface of quartz crystal microbalance, an aptamer-assisted method was proposed to capture His6-tagged ODR-10specifically, which realized the simultaneous immobilization and purification processes in one step. The processes of ODR-10immobilization were also well-characterized using electrochemical techniques and atomic force microscope. The results demonstrated that this aptamer-assisted protein immobilization method improved the performances of olfactory receptor-based biomimetic sensors, and the detection limit was as low as1.5ppm.3. Development of bioengineered olfactory receptor cell-based biomimetic sensorsBy expressing olfactory receptor ODR-10on the cellular membrane of primary olfactory sensory neurons, excitable bioengineered olfactory receptor cells are suitable to detect odorants specifically. Light addressable potentiometric sensor (LAPS) was used as the secondary transducer to detect odorant-induced extracellular firing signals. The results demonstrated that bioengineered olfactory cells could specifically respond to diacetyl, the natural ligand of ODR-10. Furthermore, different concentrations of diacetyl can also be discriminated by this biosensor.4. Methods for the construction of controlable cellular networks were proposed.The olfactory cellular network has excellent innate capacity of signal processing and information encoding, which has great potential to improve the performance of biomimetic olfactory sensors. Currently, the major obstacle in this field is the specific growth of cellular networks on the sensor chip. In this thesis, DNA molecules and1,6-hexanedithiol were employed to capture the cells onto the specfic electrode sites of microelectrode array (MLA) chip, which was the first step to culture cellular networks on the sensor chip. Besides, different cellular network layouts could also be realized by changing the microelectrodes arrangement of MFA chip.