| The biosensor based on Quartz Crystal Microbalance(QCM)has proven to be powerful and efficient tools for real-time and label-free detection of biomolecular interactions,permitting the determination of the kinetics and affinity parameters of the interaction,including the association rate constant,dissociation rate constant,and affinity constant.It has been widely used in a variety of areas,such as scientific research,drug development and disease diagnosis.However,it is increasingly acknowledged that a more complete understanding of the interaction of biomolecules requires not only the kinetic and affinity information but also the thermodynamic properties,which play a very important role in the study of the mechanism of biomolecule recognition.Thus far,thermodynamic studies of the biomolecule interaction mainly focus on purified biomolecules and there are no reports about the thermodynamic analysis of the interactions on intact cell surfaces.In addition,QCM cell biosensor is normally fabricated by growing adherent cells on the sensor surface.Compared with the adherent cells,the suspension cells are naturally live in suspension,which cannot be directly attached or grown onto a surface.In view of these problems,this study consists of the following two parts:1.A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor was developed,where the binding events take place at the cell surface,more closely mimicking a biologically relevant environment.In this study,human colon adenocarcinoma cells(KM-12)and human ovary adenocarcinoma cells(SKOV-3)grew on the sensor chip surface without fixation.The cell chip is of excellent stability,reproducibility,and specificity.The association and dissociation between the cell surface carbohydrates and a range of lectins were monitored in real time for evaluation of cell surface glycosylation.Furthermore,the thermodynamic parameters of the interaction between lectins and cell surface glycan,such as the changes of entropy,enthalpy and Gibbs free energy,as well as the transition state information,were obtained by determination of the temperature dependence of the affinity constant,association rate constant and dissociation rate constant,combined with van't Hoff plot analysis and Eyring plot analysis.This method features the advantages of the relatively low consumption of samples,high detection sensitivity and simultaneous collection of kinetic and thermodynamic data.This methodology give interesting complementary information to isothermal titration calorimetry(ITC),since only the physical association and dissociation of the two molecular species are monitored,whereas calorimetric assays the total heat effects during the interaction,including heat of dilution or mixing.This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface,which may have impacts on disease diagnosis and drug discovery.2.A novel polydopamine(PDA)suspension cell chip based on QCM biosensor was developed.Only a simple immersion of the QCM gold chip in an aqueous dopamine solution could lead to spontaneous deposition of a PDA film onto the chip surface.The suspension cells was directly immobilized onto PDA surface for the first time,giving the PDA suspension cell chip.The cell surface glycosylation of human acute monocytic leukemia cells(THP-1),human acute T cell leukemia cells(Jurkat)and human chronic myelogenous leukemia cells(K562),as well as the kinetic parameters of the interaction between Con A and THP-1 cells were evaluated.This study provides a new method for the immobilization of suspension cells,giving insight into the nature of the surface glycosylation of suspension tumor cells and their complex molecular recognition.In addition,the PDA suspension cell chip,fabricated via very simple procedures,can be easily reused,which will make the QCM biosensor analysis more efficient and cost effective. |
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