Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Single-Protein Molecular Interactions on Polymer-Modified Glass Substrates for Nanoarray Chip Application Using Dual-Color TIRFM

  • Kim, Dae-Kwang (Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University) ;
  • Lee, Hee-Gu (Cellomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Jung, Hyung-Il (Department of Biotechnology, Yonsei University) ;
  • Kang, Seong-Ho (Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University)
  • Published : 2007.05.20
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Abstract

The immobilization of proteins and their molecular interactions on various polymer-modified glass substrates [i.e. 3-aminopropyltriethoxysilane (APTS), 3-glycidoxypropyltrimethoxysilane (GPTS), poly (ethylene glycol) diacrylate (PEG-DA), chitosan (CHI), glutaraldehyde (GA), 3-(trichlorosilyl)propyl methacrylate (TPM), 3'-mercaptopropyltrimethoxysilane (MPTMS), glycidyl methacrylate (GMA) and poly-l-lysine (PL).] for potential applications in a nanoarray protein chip at the single-molecule level was evaluated using prismtype dual-color total internal reflection fluorescence microscopy (dual-color TIRFM). A dual-color TIRF microscope, which contained two individual laser beams and a single high-sensitivity camera, was used for the rapid and simultaneous dual-color detection of the interactions and colocalization of different proteins labeled with different fluorescent dyes such as Alexa Fluor® 488, Qdot® 525 and Alexa Fluor® 633. Most of the polymer-modified glass substrates showed good stability and a relative high signal-to-noise (S/N) ratio over a 40-day period after making the substrates. The GPTS/CHI/GA-modified glass substrate showed a 13.5-56.3% higher relative S/N ratio than the other substrates. 1% Top-Block in 10 mM phosphate buffered saline (pH 7.4) showed a 99.2% increase in the blocking effect of non-specific adsorption. These results show that dual-color TIRFM is a powerful methodology for detecting proteins at the single-molecule level with potential applications in nanoarray chips or nano-biosensors.

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References

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