Tris-nitrilotriacetic Acid Sensor Chips For Bioanalyses By Surface Plasmon Resonance

Surface plasmon resonance(SPR)has been widely used as a label-free,real-time and sensitive technique for studying biomolecular interactions.Owing to the relatively high cost of the SPR chips and the uncontrollable orientation of biomolecules immobilized through covalent linking to the carboxymethylated(CM)-dextran chips,it is of significance to develop regenerable SPR sensor chips that yield a uniform molecular orientation.This thesis describes the development of a new tris-nitrilotriacetic acid(Tris NTA)sensor chip that is regenerable and onto which protein molecules can be immobilized stably and uniformly.The specific research findings are summarized as follows:1.We studied the performance of the Tris-NTA sensor chips,which includes p H,density,stability,regeneration times,and protein immobilization conditions.The optimal p H of TrisNTA was 9.3,and the final exact concentration was 1 m M(kinetic analisis)and 5 m M(concentration determination).We demonstrated that the Tris-NTA chip not only retains Histagged proteins more strongly than its mono-NTA counterpart,but also orients them more uniformly than protein molecules coupled to carboxymethylated dextran films.The reproducibility of the sensor chips remain high even after 92 cycles of repeated His-tagged protein G binding and EDTA regenerations.We found that,at neutral p H,the surface of the Tris-NTA contains many negative charges.Consequently,the amount of His-protein immobilized can be adjusted based on the protein isoelectric point(p I)and by varying the p H value of the carrier solution.The development of this chip provides a new sensor and platform for biologists and chemists.2.By using the binding between protein G and immunoglobulin(IgG)molecules as the system,we accurately and rapidly quantified the total amount of IgGs in sera by using the initial association phases of the sensorgrams collected from His-tagged protein G immobilized onto the tris-NTA chip to construct the calibration curve.We also established the criteria for selecting the optimal time for constructing the calibration curve.Our method is highly reproducible(with relative standard deviations all less than 2%)and three orders of magnitude more sensitive than immunoturbidimetry.Reliable kinetic data in a five-channel SPR instrument can also be rapidly obtained by using a low density of His-tagged protein.The experimental parameters and procedures optimized in this study should help expand the range of SPR applications involving His-tagged proteins.