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A Metal‐Chelating Piezoelectric Sensor Chip for Direct Detection and Oriented Immobilization of PolyHis‐Tagged Proteins
Author(s) -
Chen HsiuMei,
Wang WeiCheng,
Chen ShengHorng
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049968f
Subject(s) - nitrilotriacetic acid , chemistry , chelation , biosensor , metal ions in aqueous solution , substrate (aquarium) , metal , lysis , selectivity , x ray photoelectron spectroscopy , combinatorial chemistry , chromatography , inorganic chemistry , chemical engineering , organic chemistry , biochemistry , oceanography , engineering , geology , catalysis
A metal‐chelating piezoelectric (PZ) chip for direct detection and controlled immobilization of polyHis‐tagged proteins has been demonstrated. The chip was prepared by covalently binding a hydrogel matrix complex of oxidized dextran and nitrilotriacetic acid (NTA) ligand onto an activated alkanethiol‐modified PZ crystal. The resulting chip effectively captured Ni 2+ ions onto its NTA surface, as disclosed by the resonant frequency shift of the crystal and an X‐ray photoelectron spectroscopy analysis. The real‐time frequency analysis revealed that the bare NTA chip was nonfouling, regenerable, and highly reusable during continuous repetitive injections of ion solutions and binding proteins. In addition, the chip displayed good long‐term reusability and storage stability. The individual binding studies of a polyHis‐tagged glutathione‐ S ‐transferase and its native untagged form on various metal‐charged chips revealed that Co 2+ , Cu 2+ , and Ni 2+ ions each had different immobilization ability on the NTA surface, as well as their binding ability and selectivity with the tagged protein. As a result, the tagged protein immobilized on the Ni 2+ ‐charged chip can actively be bound with its antibody and substrate. Further, the quantitative analyses of the tagged protein in crude cell lysate with a single Ni 2+ ‐charged chip and of its substrate with a protein‐coated chip were also successfully demonstrated. Therefore, this study initiates the possibilities of oriented, reversible, and universal immobilization of any polyHis‐tagged protein and its functional study using a real‐time PZ biosensor.