Research On Biomimetic Olfactory Cell And Receptor-based Biosensors

Biological olfactory systems can recognize and discriminate thousands of distinct odors with very high sensitivity and specificity. With the progress of research on olfactory mechanisms and the advancements of modern biomedical sensing technologies, by imitating the biological mechanisms of olfaction, many types of biomimetic olfactory-based biosensors have been developed by the combination of olfactory functional components with various secondary sensors. Olfactory functional components, such as olfactory sensory neurons (OSNs), natural olfactory receptor proteins (ORPs), ORPs expressed in heterologous cell systems, are used as sensitive elements in biomimetic olfactory-based biosensors. Similar to the olfactory system in vivo, biomimetic olfactory-based biosensors are characterized with high sensitivity, low detection limit and excellent selectivity. They have great potential commercial prospects and promising applications in such fields as biomedicine, environmental monitoring, pharmaceutical screening and the quality control of food and water.At present, with the developments of multi-discipline and technologies in biology, electronics, materials and informatics, great progress has been achieved in the research of biomimetic olfactory-based biosensors. They have shown great developmental potentials. But many problems still exist in the studies of biomimetic olfactory-based biosensors, such as how to gain a great deal of functional olfactory components, how to monitor their responses to the specific stimulations with high-throughput and multivariate analysis, how to improve the sensitivity and specificity of olfactory-based biosensors to broaden their applications. To address these issues, this thesis developed novel biomimetic olfactory-based biosensors at the cell and molecule level, investigated the performances of these olfactory-based biosensors and their applications in biomedicine. Firstly, at the cell level, we developed a single-cell based biosensor for the studies of olfactory transduction mechanisms, in which OSNs were served as sensitive elements and light-addressable potentiometric sensor (LAPS) was used as secondary sensors. Secondly, at the molecule level, an olfactory receptor protein of C. elegances, ODR-10, was expressed on the plasma membrane of a heterologous cell system, human embryonic kidney (HEK)-293 cells. Quartz crystal microbalance (QCM) was used to confirm the odor recognition function of ODR-10. To achieve a cell line that can stably express ODR-10, we primarily studied the expression of ODR-10 in human breast cancer cell line MCF-7 cells. Then the plasma membrane fraction of MCF-7 cells was extracted and served as sensitive elements, which contains the expressed ODR-10. It was used to develop ORPs-based surface acoustic wave (SAW) biosensors, which is a kind of gas sensitive biosensors. Finally, by combining the technologies of modern miniaturization, we explored the possibility of applying micromaching technologies to the development of biomimetic olfactory-based biosensors.The major contents and contributions of this thesis are given in the following aspects:1. On the basis of single-cell-based LAPS biosensor, we applied the olfactory transduction mechanisms to the research of biomimetic olfactory-based biosensors. The inhibitory effect of MDL12330A to the intracellular olfactory signal transduction pathway was applied to confirm that the recorded signals by LAPS are originated from the responses of OSNs to odor stimulations, which were cultured on the surface of LAPS chip. By this way, we demonstrated the signals recorded by LAPS are originated from the olfactory signals of ORNs. It is also indicated this biomimetic olfactory-based biosensor can be used in the research of olfactory transduction mechanisms.2. We proposed a novel method for the sensitivity enhancement of LAPS and single-cell hybrid biosensor by the utilization of enhancer. The enhancive effect of LY294002 to the intracellular olfactory signal transduction pathway was used to enhance the responses of ORNs to odor stimulations, thus the sensitivity of the biosensor was enhanced. The results show that the sensitivity of this biosensor can be enhanced by LY294002 by about 1.5 folds. It is suggested that this method is also applicable to other kinds of OSNs-based biosensors for cellular activity detection, such as microelectrode array (MEA) and field effect transistor (FET).3. By DNA recombinant technologies, an olfactory receptor protein of C. elegants, ODR-10, was functionally expressed on the plasma membrane of a heterologous cell system, HEK-293 cells. Its expression and sub-cellular location were analysed. Its odor recognition function was demonstrated by QCM. The results indicated that ODR-10 expressed in HEK-293 cells still maintain its function of recognizing its natural ligand, diacetyl. It is suitable to be used as sensitive elements in the development of biomimetic olfactory-based biosensor.4. We achieved a MCF-7 cell line, which can stably express ODR-10. Its function of odor recognition was demonstrated by the method of monitoring the concentration of intracellular Ca2+. Then the membrane fraction of MCF-7 cells, which contains the expressed ODR-10, was extracted and used as sensitive elements. By the utilization of SAW sensors as secondary sensors, we developed a SAW-based gas sensitive biosensor. The results show that this ORPs-based SAW biosensor can monitor the interactions between ORPs and odor molecules effectively, consequently realizing the detection of specific odors.