Development Of His-tag Protein Chips And Suspension-cell Chips Based On QCM Biosensor And Their Applications

Biosensor based upon the Quartz Crystal Microbalance (QCM)technology is a simple,efficient and high resolution mass sensing device for measuring biomolecule interactions inreal time without any labeling toobtain the affinity and kineticdata of theinteractions,whichprovides a powerful means for studying the process and explaining themechanism of biomolecular interactions. It has been widely used in avarietyofareas, such asmolecular biology, drug development, disease diagnosis, environmental monitoring, and foodsafety. In this study, a two-dimensional (2D) and a three-dimensional (3D) His tag proteinchip were fabricated for oriented and reversible immobilization of His tagged proteins onQCM biosensor surface, which can be used for protein-protein interaction studies, and a novelsuspension-cell chip based on QCM biosensorwas developed for label-free evaluationof theinteractions between protein and cell surface carbohydrates,which will facilitatethe QCMbiosensor’s widespread use.1) A2D and a3D His tag protein chip were fabricated for oriented and reversibleimmobilization of His tagged proteins on QCM biosensor surface, which can be used forlabel-free and real-time detection of the interaction of the His tagged protein and itsinteracting protein.The binding activity of immobilizedHis tagged proteinsvia amine couplingand His tag capture, as well as the propertyof2D and3D His tag protein chip, such asimmobilization capacity, binding activity and specificity of theimmobilized protein wascompared, whichprovides important reference for the choice of sensor chips and theoptimization of immobilization method in biomolecular interaction analysis.Regeneration ofthe His tag protein chip can be optionally performed using conditions to remove the Histagged protein as well as the analyte for the next cycle of His tagged protein immobilization,or only remove the analyte, leaving the His tagged protein intact on the surface for the nextcycle of analyte binding, which was more efficient and cost effective.In addition, the kineticparameters of the interaction of His tagged protein with its interacting protein were evaluatedon the His tag protein surface, providing more in-depth information about the interaction,such as the association and dissociation rate constant as well as the affinity constant.2) A novel suspension-cell chip based on QCM technology was developed forlabel-free evaluationof the interactions between protein and cell surface carbohydrates in real time,where thesuspension cancer cells were captured onto a Con A-coated quartzcrystalvia theinteraction of cell surface carbohydrates and Con A. The glycosylation of Jurkat (Humanacute lymphocytic leukemia cell line) and K562(Human acute myelocytic leukemia cell line)cell surface, as well as the binding kinetics ofthe interaction with lectins was evaluated.Thissuspension-cell chip allows us to directly determine thebinding kinetics ofprotein-carbohydrate interactions on cancer cell surfaces in the native environment, providinginsight intothe nature of glycoconjugates exposed on cell surfaces, which mayhave impacts onthe study of the molecular recognition on cellsurfaceandthediscoveryofdrugs,aswellasonthediagnosisofdiseases.