The Fundamental Researches On Fast Detection Technology Based On Molecular Recognition

Sweet sensation is the result of a cascade of complex, physiological, chemical and biological events, starting as the sweet molecules reach (enter into) the bud taste cells. Sweet is one of the favorite basic tastes for human. Although there have many contributions from calcium imaging, electrophysiology and NMR, the tangible mechanism of interaction between sweeteners and taste receptor have not been entirely elucidated at the cell level. Recently, the high sensitivity isothermal titration calorimetery (ITC) has been used to explore the elaborate sweet sensation machanism. The new method gives us a novel idea from the thermodynamics view to insight into this puzzle. Initially, in this sweet sensation thermodynamics research, it is necessary for us to assure that cell resources have stable sweet receptors. And therefor, a reliable method to obtain taste cells from mice has been established by our laboratory, lamentedly, it hasn't satisfy the needing of thermodynamics research in aspect of quantity. Thus, it is critical ly important to find a model cell, which can replace the primary taste cell and has the function of sweet sensation.On the basis of previous studies and our efforts, the NCI-H716 cell could sense taste due to that this cell has sweet receptor, signaling elements and function. After the identification of the model cell, the experimental parameters and optimal data analysis method which were related to the interaction study have been explored. As a consequence, the interaction mechanism between the cells and sweeteners has been analyzed.The results are shown as follows:(1) Immunohistochemistry, immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR) were used to determine the NCI-H716 cell that can substitute the taste cell because this cell has sweet receptor, signaling element and function. The results from immuno and RT-PCR showed that taste downstream signaling factors (a-gustducin and PLCβ2) and sweet receptor proteins were expressed by NCI-H716 cell. Furthermore, calcium imaging can be performed to demonstrate that NCI-H716 cell can response to sweeteners.(2) NCI-H716 cell was considered as the experimental object, the optimal titration parameters, including buffer, temperature and titration models, were determined for sweet sensation thermodynamic experiments: buffer,10 mmol/L HEPES; temperature, and 310K; titration modes, positive. Moreove, the data from the titration cannot be analyzed by MicroCal ORIGIN Software, thus the suitable date analysis method was determined:deduct sweetener dilution heat and comparative interaction total quantity of heat method.(3) Preliminary study on sweet sensation thermodynamic using NCI-H716 cell were carried out by the experiments between model cells and different concentration sugar solution obtained from nine types sweeteners. In contrast to taste cells, NCI-H716 cell significantly distinguish different concentration sugar solution, and the thermaldynamic curves of NCI-H716 were more stable. Also, nine types of sweeteners can be divided easily. Consequently, relevant thermodynamic parameters were obtained by fitting methods. Present studies show that NCI-H716 cell can be used to explore sweet sensation mechanism.