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Issue 10, 2012
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Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay

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Abstract

The proper orientation and lateral spacing of antibody molecules are a crucial element for an on-chip immunoassay in which the antibody or its antigen-binding fragments are immobilized on a solid surface. We covalently immobilized a modified protein G (Cys-protein G: protein G with only an N-terminal cysteine) on a dendron-coated surface to control its orientation and lateral spacing simultaneously. The cysteine-specific immobilization of Cys-protein G through the N-terminal cysteine resulted in 2.2-fold higher binding efficiency of Cys-protein G to IgG2a capture antibody than its random immobilization via lysine residues. The lateral spacing of 3.2 nm due to the surface modification with the 9-acid dendron molecule contributed to a 1.5-fold increase in the antibody-binding ability of Cys-protein G. Topographic images of atomic force microscopy exhibited a uniform coverage of Cys-protein G molecules immobilized on the thiol-reactive 9-acid dendron surface and homogeneous distribution of antibody bound to Cys-protein G. In the sandwich immunoassay, the control of the orientation of Cys-protein G led to 10-fold higher detection capability for rIL-2 compared with the randomly oriented protein G. The synergistic advantage of the unidirectional orientation and homogeneous lateral spacing of Cys-protein Gs on the dendron-coated surface can be applied to the development of more sensitive and reproducible antibody microarrays.

Graphical abstract: Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay

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Publication details

The article was received on 21 nóv. 2011, accepted on 01 mar. 2012 and first published on 05 apr. 2012


Article type: Paper
DOI: 10.1039/C2AN16137K
Analyst, 2012,137, 2421-2430

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    Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay

    E. Kim, C. Shim, J. W. Lee, J. W. Park and K. Y. Choi, Analyst, 2012, 137, 2421
    DOI: 10.1039/C2AN16137K

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