A Metal-Chelating Piezoelectric Sensor Chip for Direct Detection and Oriented Immobilization of PolyHis-Tagged Proteins

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• 作者：Hsiu-Mei Chen ; Wei-Cheng Wang ; and Sheng-Horng Chen
• 刊名：Biotechnology Progress
• 出版年：2004
• 出版时间：August 2004
• 年：2004
• 卷：20
• 期：4
• 页码：1237 - 1244
• 全文大小：121K
• 年卷期：v.20,no.4(August 2004)
• ISSN：1520-6033

文摘

A metal-chelating piezoelectric (PZ) chip for direct detection and controlled immobilization of polyHis-tagged proteins has been demonstrated. The chip was prepared bycovalently binding a hydrogel matrix complex of oxidized dextran and nitrilotriaceticacid (NTA) ligand onto an activated alkanethiol-modified PZ crystal. The resultingchip effectively captured Ni²⁺ ions onto its NTA surface, as disclosed by the resonantfrequency shift of the crystal and an X-ray photoelectron spectroscopy analysis. Thereal-time frequency analysis revealed that the bare NTA chip was nonfouling,regenerable, and highly reusable during continuous repetitive injections of ion solutionsand binding proteins. In addition, the chip displayed good long-term reusability andstorage stability. The individual binding studies of a polyHis-tagged glutathione-S-transferase and its native untagged form on various metal-charged chips revealedthat Co²⁺, Cu²⁺, and Ni²⁺ ions each had different immobilization ability on the NTAsurface, as well as their binding ability and selectivity with the tagged protein. As aresult, the tagged protein immobilized on the Ni²⁺-charged chip can actively be boundwith its antibody and substrate. Further, the quantitative analyses of the taggedprotein in crude cell lysate with a single Ni²⁺-charged chip and of its substrate witha protein-coated chip were also successfully demonstrated. Therefore, this studyinitiates the possibilities of oriented, reversible, and universal immobilization of anypolyHis-tagged protein and its functional study using a real-time PZ biosensor.