Research On Taste Cell Model And Biomimetic Taste Cell Network-based Biosensors

Taste receptor cells are the basic elements for taste sensations to realize taste information processing, such as the chemical-electrical signal transformation. Studies indicated that taste receptor cells would produce action potentials elicited by sour, sweet, bitter, salty and umami tastants. The firing patterns encode taste features. There are some similarities between the responses from taste receptor cells and taste nerve fibers. Thus, the action potentials produced by taste receptor cells may be the key components of taste information transferred to the taste fibers. The electrical activities from taste receptor cells play dominate and important role in taste information processing at periphery.Ion channel study can help reveal the function and mechanism of the excitability of taste receptor cells at the molecular level. There are several types of voltage-gated ion channels on the membrane of taste receptor cells. Such evidence further shows that voltage-gated ion channels are crucial in taste information encoding and processing as the molecular basis.The responses elicited by tastants from taste receptor cells can be recorded with specific electrophysiological techniques, such as whole-cell patch clamp, cell-attached patch clamp and extracellular recording technology based on patch clamp. However, so far we have little knowledge about taste information conveyed by firing patterns of action potentials and the corresponding ionic mechanism, partially due to the limitations of experiment tools. These evidences indicate that it is necessary to utilize multiple approaches, including the computational model and simulation, and biomimetic cell-based sensors to study the taste function and mechanisms at cellular and sub-cellular levels.Based on the whole-cell and single-channels recordings, we describe and analyze the dynamic properties and mechanisms of ligand-gated and voltage-gated channels. Finally, a computational electrophysiological model of taste cells is constructed. We simulate the temporal firing patterns upon sour stimulus, and analyze the sour sensation mechanism and firing characteristics. With the development of MEMS technology and the advantage of optoelectronic technology, we utilize LAPS (Light Addressable Potentiometric Sensor) as the platform for investigation of taste sensation and firing response. Surface potential at any spot on LAPS could be recorded with laser scanning, which improves the flexibility and feasibility of cell-based biosensor. LAPS chip also conquers the disadvantage of low density of cultured taste cells. It can be used for electrical and chemical signal detection from taste cells upon tastants.The major contents and contributions of this thesis are as follows:1. We built a dynamic model of aicd-sensing PKD1L3/2L1 channels and analyzed the mechanism of OFF response with offset of sour stimulus. Base-activating mechanism dedicates to the OFF response of PKD1L3/2L1 channels. It plays inhibitory roles with pH and calcium concentration dependency. In addition, we established that the channel activities are inhibited by protons.2. A PKD2 single-channel model was constructed. The simulation results indicated that an OFF response could be induced with sour stimulus washout, which was proved by the whole-cell recordings of PKD2L1. From the model and experiments, we confirm that OFF response can be induced with single transfection of PKD2/2L1. Moreover, it further supports the base-activating mechanism based on the single-channel model and patch clamp experiments.3. A whole-cell computational model of acid-sensing taste cell was constructed for the first time. Simulation results demonstrate that there are ON and OFF firings upon sour tastants. There should be three possibilities, which could be proved by the experiments with MEA chip. Besides, in simulation we found that calcium dependent inhibiton of PKDL channels plays a negative feedback role in sour sensation.4. Based on single-cell hybrid LAPS, we developed a taste single-cell based biomimetic sensor. There should be both functionally typeⅡandⅢtaste cells cultured on chip. Combined with taste cell model and the couple model of cell and chip, we simulated the extracellular potentials upon sour stimulus. Experimental results indicated that taste information could be encoded in the periphery. ATP has both enhanced and inhibitory effects on type III taste cells. We also labeled the taste cells cultured on LAPS specifically with immunoflurecence technique. In conclusion, taste-cell based LAPS could detect the ion and molecule changes in the environment.5. With the addressability and flexibility of LAPS chip, we developed a taste cell network-based biomimetic sensor for detection of the neurotransmitter 5-HT and investigation of taste cell-to-cell communication. The detection limit of this sensor was 3.3×10-13 M and the sensitivity was 19.1 mV per concentration decade (19.1 mVp5-HT). Upon the stimuli of sour and mix tastants, the local concentration of 5-HT released from taste cell and taste epithelium is about 10-9 M. Meanwhile, we utilize immunofluorescent imaging technique to confirm that there are both functional typeⅡand typeⅢcells in isolated cluster of taste cells.