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

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• 作者：Jason J. Davis ; Jan Tkac ; Sophie Laurenson ; Paul Ko Ferrigno
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：February 1, 2007
• 年：2007
• 卷：79
• 期：3
• 页码：1089 - 1096
• 全文大小：358K
• 年卷期：v.79,no.3(February 1, 2007)
• ISSN：1520-6882

文摘

Protein microarray development is absolutely dependentupon the ability to construct interfaces capable of specific,stable, sensitive, and designable recognition of specificproteins. Peptide aptamers, being peptide recognitionmoieties presented and constrained by a robust scaffoldprotein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplifythe production of microarrays. This paper describes thegeneration and assaying characteristics of a scaffoldprotein adlayer. Orientational control of the scaffoldprotein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with asurface cysteine residue, which leads to the presentationof the scaffold recognition surface to solution. Operationalstability of the system is excellent, with only a minordecrease in detection sensitivity over time (less than 1%h^-1). We use this system to establish a surface plasmonresonance assay offering a limit of detection of 1 nM (150ng mL^-1) and determine the affinity constant of interactionof STM for a cognate antibody to be K_D = 1.47 ± 0.23nM. Thus, we have established a solid foundation for thefuture creation of highly multiplexed peptide aptamermicroarrays that will be compatible with a broad range oflabel-free detection technologies.