Peptide Aptamers in Label-Free Protein Detection: 1. Characterization of the Immobilized Scaffold

Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer...

Full description

Saved in:
Bibliographic Details
Published in:Analytical chemistry (Washington) 2007-02, Vol.79 (3), p.1089-1096
Main Authors: Davis, Jason J, Tkac, Jan, Laurenson, Sophie, Ferrigno, Paul Ko
Format: Article
Language:English
Subjects:
Analytical chemistry
Aptamers, Peptide
Chemistry
Cystatin A
Cystatins
Cysteine
Exact sciences and technology
General, instrumentation
Peptides
Protein Array Analysis
Proteins
Proteins - analysis
Surface Plasmon Resonance
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be K D = 1.47 ± 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac061863z