Lim, Chang Hwan and Cho, Hyung Min and Choo, Jaebum and Neff, Silke and Jungbauer, Alois and Kumada, Yoichi and Katoh, Shigeo and Lee, Eun Kyu (2009) Fluorescence-based peptide screening using ligand peptides directly conjugated to a thiolated glass surface. Biomedical Microdevices, 11 (3). pp. 663-669.
Functional peptides from peptide libraries are frequently screened using an array format. We report here results of a feasibility study of fluorescence-based peptide screening using an array format on surface-modified glass. The surface of an amine-coated glass slide was modified to contain thiol groups by iminothiolane treatment. The Îµ-amine of the C-terminal lysine from a ligand peptide was iodinated and then spotted onto the thiolated glass surface to covalently conjugate the ligand peptide to the surface via a thioether bond. This covalent immobilization allowed the ligand peptides to withstand washing steps by tightly adhering to the glass surface and confining their subsequent binding reactions within a spotted area. Two representative peptides were used as the ligand peptides; a âtargetâ (positive) heptapeptide that could specifically bind to trypsin, and a âcontrolâ (negative) hexapeptide that had no binding affinity with trypsin. When fluorescein isothiocyanate-labeled trypsin was reacted with the ligand peptides, the target peptide demonstrated distinctively higher (ca. 8.7-fold) fluorescence intensity that was easily differentiated from the control peptide by a fluorescence scanner. A separate experiment using a quartz crystal microbalance confirmed that the difference in binding mass (ca. 9.1-fold) was very close to that seen in fluorescence intensity. These results suggested a quantitative, 1:1 correlation between mass and fluorescence signals. Furthermore, a smaller spot volume and a higher ligand peptide concentration resulted in higher fluorescence signal intensity. This study provides information on the potential for using fluorescence-based screening of functional peptides on a glass array format.
|Subjects:||Physical Science > Nano objects|
Material Science > Nanostructured materials
Material Science > Nanochemistry
|Deposited On:||25 Aug 2010 11:28|
|Last Modified:||25 Aug 2010 11:28|
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