A Universal Photochemical Method To Prepare Carbohydrate Chips Based On Perfluorophenylazide For Study Of Carbohydrate-Lectin Interactions By QCM

The biosensor based on 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.Carbohydrate is one of the most widely distributed organic compounds in nature.Carbohydrate plays an important role in cell communication,apoptosis,immune response and infection by interacting with proteins and other biomolecules.Therefore,it is very important to detect the interaction between carbohydrate and protein.Nowadays,among the various detection methods,the carbohydrate chip technology with the characteristics of high sensitivity,low cost and small sample amount has attract wide attention and become an important means to detect the related biological functions of carbohydrate in life activities.The efficient immobilization of carbohydrates to the chip surface is a primary condition for the preparation of QCM carbohydrate sensor chips.There are usually two strategies to immobilize the carbohydrate on the chip: covalent immobilization and non-covalent immobilization.The application of non-covalent immobilization is limited because of its poor stability.However,the covalent immobilization method connects the carbohydrate to the basal surface by covalent bond,which lead to a higher stability and widely application.In fact,the preparation of carbohydrate chips by covalent immobilization frequently require multi-step synthesis of the carbohydrates,generally not applicable to the direct immobilization of underivatized carbohydrates on chip,where the process for multi-step modification of the carbohydrates is usually time consuming.Therefore,it is thus highly desirable to develop a universal method is applicable to the preparing of carbohydrate chips by directly using the underivatized carbohydrates without any modification.It can be used for carbohydrate-lectin interactions.This paper is based on the above content of the following two aspects of research.1.A universal method to prepare carbohydrate chips.A versatile method to covalent derivatization is based on the specific chemistry of arylazides.Upon thermal activation or light irradiation,the azide functionality in these structures becomes converted to a highly reactive nitrene species that readily inserts into C-H,N-H and C=C bonds.Especially perfluorophenylzaide(PFPA),which produce markedly enhanced insertion yields,have thus been used to thermo-and photochemically introduce functional groups in a range of entities,including proteins,nanostructures,and synthetic polymers.The versatility of the PFPA chemistry makes it an attractive choice for surface modification.Firstly,PFPA derivatives with amino groups were synthesized,and 2 mg/m L dopamine solution was added to the chip for incubation for 1.5 h to prepare PDA chips,The amino-PFPA were immobilized on PDA-coated quartz crystals via Michael addition reactions and Schiff base reactions.Upon thermal activation or light irradiation,The underivatized carbohydrates were inserted into the chip surface,including Mannose,Galactose,Fucose and N-Acetylglucosamine(Glc NAc).Carbohydrate chips were evaluated for the binding to a series of plant lectins,including concanavalin A(Con A),peanut agglutinin(PNA),Ulex europeus agglutinin I(UEA-I),soybean agglutinin(SBA),and wheat germ agglutinin(WGA).In this method,different kinds of carbohydrate chips can be prepared without modification of carbohydrates,and using QCM to detect the interactions between carbohydrate and various kinds of lectins.2.In this study,the kinetics of the prepared carbohydrate chips and their corresponding lectins were studied.In order to understand the interactions between carbohydrate and lectin more comprehensively,we have selected two carbohydrates(Mannose,Fucose)and their corresponding lectins for kinetic studies,and the association rate constant,dissociation rate constant and affinity constant of carbohydrate and lectin can be obtained by kinetic research.We can understand more about the interactions between carbohydrate and lectin.In addition,this method can not only eliminate the tedious modification of carbohydrates,but also reuse the chip after simple cleaning.This method improve the experimental efficiency and reduce the experimental cost,which is of great significance for the development of QCM biosensor and study of biomolecular interactions.