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Proceedings Paper

Direct enzymatic hydrolysis and patterning of a chemisorbed peptide thin film
Author(s): David Turner; Mary A. Testoff; Bruce Paul Gaber
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Paper Abstract

We have demonstrated that covalently immobilized enzyme can be used to chemically modify and pattern a chemisorbed peptide film on a solid substrate. The enzyme, alpha- chymotrypsin, was covalently attached to silica and latex spherical beads by glutaraldehyde crosslinking to an amino surface functionality. The fluorescent peptide, suc-ala-ala- phe-AMC (SAAP-AMC) was immobilized to an aminosilane- modified flat silica surface by forming an amide bond to its free carboxylic acid group. SAAP-AMC surfaces were characterized using water wetability, fluorescence spectroscopy and x-ray photoelectron spectroscopy. Upon attack by alpha-chymotrypsin coated beads, the fluorescent AMC group (325 nm ex, 395 nm em) is cleaved from the SAAP- AMC peptide surface and a red-shift occurs in the AMC fluorescence providing a standard marker for enzymatic activity (345 nm ex, 440 nm em). Thus, the signature for alpha-chymotrypsin bead activity against the surface is a reduction in fluorescence intensity from the peptide surface (at 395 nm) and a concomitant increase in fluorescence in the solution above the surface (at 440 nm). Treatment of the SAAP-AMC silica surfaces with alpha-chymotrypsin-beads showed a reduction of the surface fluorescence to background in less than 24 hours, with a corresponding increase of free AMC fluorescence in solution. By restricting the contact region of the beads with the peptide surface we were able to demonstrate chemical patterning of the peptide surfaces.

Paper Details

Date Published: 31 March 1997
PDF: 9 pages
Proc. SPIE 2978, Micro- and Nanofabricated Electro-Optical Mechanical Systems for Biomedical and Environmental Applications, (31 March 1997); doi: 10.1117/12.269971
Show Author Affiliations
David Turner, Naval Research Lab. (United States)
Mary A. Testoff, Naval Research Lab. (United States)
Bruce Paul Gaber, Naval Research Lab. (United States)

Published in SPIE Proceedings Vol. 2978:
Micro- and Nanofabricated Electro-Optical Mechanical Systems for Biomedical and Environmental Applications
Paul Lee Gourley, Editor(s)

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