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

Eung Sam Kim, Chang Kyoon Shim, Jae Lee, Joon Won Park, Kwan Yong Choi

Research output: Contribution to journalArticle

14 Citations (Scopus)

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 IgG 2a 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.

Original languageEnglish (US)
Pages (from-to)2421-2430
Number of pages10
JournalAnalyst
Volume137
Issue number10
DOIs
StatePublished - May 21 2012

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immunoassay
Immunoassay
spacing
Proteins
protein
Antibodies
antibody
Cysteine
Immobilization
fold
Molecules
immobilization
effect
Acids
Atomic Force Microscopy
atomic force microscopy
Microarrays
thiol
acid
GTP-Binding Proteins

ASJC Scopus subject areas

  • Analytical Chemistry
  • Spectroscopy
  • Electrochemistry
  • Biochemistry
  • Environmental Chemistry

Cite this

Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay. / Kim, Eung Sam; Shim, Chang Kyoon; Lee, Jae; Park, Joon Won; Choi, Kwan Yong.

In: Analyst, Vol. 137, No. 10, 21.05.2012, p. 2421-2430.

Research output: Contribution to journalArticle

Kim, Eung Sam ; Shim, Chang Kyoon ; Lee, Jae ; Park, Joon Won ; Choi, Kwan Yong. / Synergistic effect of orientation and lateral spacing of protein G on an on-chip immunoassay. In: Analyst. 2012 ; Vol. 137, No. 10. pp. 2421-2430.
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