Fabrication And Application Of Biochips Based On QCM Biosensor

As a real-time and label-free detection technique,Quartz Crystal Microbalance(QCM)biosensor has been widely used in many fields,including biotechnology,environmental monitoring,drug discovery,disease diagnosis and food safety.Surface functionalization of sensor chips is the core of QCM technology,which provides the tailored functional surfaces for the targeting molecules and facilitates a fast and efficient analysis on the interaction of proteins,carbohydrates,lectins,and other biological components in life science processes.In this thesis,three-dimensional carboxymethyl dextran sensor chip,three-dimensional branched-chain carbohydrate chip,polydopamine carbohydrate chip,and cell chip based on QCM biosensor were prepared,respectively.1)In this research,three-dimensional(3D)carboxymethyl dextran sensor chips were successfully fabricated via surface modification using dextrans with different molecular weight(100kDa,500kDa and 2000kDa,respectively).The immobilization capacity of proteins on the three 3D chip surfaces was evaluated using Anti-h myoglobin 7005 as a model protein,and compared with that of a 2D carboxyl surface using a QCM biosensor.The results showed that the protein immobilization capacity of the 3D carboxymethyl dextran(2000kDa)surface exceeded 4 times the capacity of the 2D surface,and 2 times the capacity of the 3D carboxymethyl dextran(500kDa)surface.The 3D carboxymethyl dextran chip surface still had a high biological activity even after 10 cycles of regeneration.Moreover,the 2D and 3D carboxymethyl dextran(100kDa)surface were not capable of detecting binding events for small molecule oligo(0.67kDa).In contrast,the binding of biotinylated oligo(0.67kDa)reached 2.5Hz on the 3D carboxymethyl dextran(500kDa)chip surface,and 8.1 Hz on the3 D carboxymethyl dextran(2000kDa)chip surface.The results confirmed that the 3D carboxymethyl dextran(2000kDa)surface has great potential applications for characterizing the interactions between small molecule drugs and proteins.2)In this study,a versatile and efficent three-dimensional(3D)branched-chain carbohydrate chips were successfully fabricated,in which an alkynyl-branch molecule modified QCM chip surface was firstly obtained by 4-azido-2,3,5,6-tetrafluorobenzoic acid(PFPA)based photo-insertion chemistry and amide conjugation strategy,and subsequently 3D dendritic mannose and galactose modified chips were prepared via “click chemistry” on the surface of the chips.The 2D straight-chain carbohydrate chip,as a control,was prepared using the same chemical approaches.The obtained chips were used to study carbohydrate-protein interactions using QCM biosensor.The results showed that the 3D branched-chain surface not only supplied more specific binding site but also revealed significant cluster effect,which significantly improved the detection sensitivity compared with the 2D straight-chain chip.Moreover,after several surface regeneration,the 3D branched-chain carbohydrate chip surface still retained high activity.3)A novel QCM carbohydrate chip integrated with a polydopamine(PDA)layer was developed,which was further utilized in the investigation of the carbohydrate-lectin interactions.Polydopamine coating on the surface of QCM gold chip was achieved by immersing the QCM chip with an aqueous dopamine solution at p H 8.5,followed by Michael addition reaction or Schiff reaction with amino modified carbohydrate molecules,resulting in a PDA coated carbohydrate chip.Using this chip,the interactions between carbohydrates including mannose and n-acetyl glucosamine with five lectins were studied,in which kinetic of interaction between mannose and Con A was further investigated.PDA coated carbohydrate chip could maintain high activity even after several regeneration cycles.Moreover,the PDA layer on the gold chip could be easily removed,which allows the repeat use of the gold chip,and thus provides an economic method of gold chip modification.The PDA coating strategy in this research provides a simple and versatile method towards carbohydrate chip functionalization,which is of great importance in the development of QCM biosensor and investigations of biomolecules interactions.4)In this study,a versatile cell chip based on QCM biosensor was developed.A compound containing short-chain glycol and biotin was synthesized and incubated with cells to achieve cell surface biotinylation.Thereafter,streptavidin was immobolized on the QCM carboxyl surface,and the cell chip was fabricated via biotin-streptavidin specifical interactions.This strategy could be used for surface modification of both the glass surface and the sensor chip surface.Moreover,an adherent cell chip as well as a suspension cell chip could be prepared by the approach.Using this cell chip,the interactions between a series of lectins and cell surface glycosylation of human acute lymphoblastic leukemia cells(Jurkat)were detected in real time based on the QCM biosensor.This method could therefore provide a convenient and efficient method for the study of cell surface glycosylation.