title: Peptide aptamers in label-free protein detection: 1. Characterization of the immobilized scaffold.
abstract: 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 KD = 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.
authors.affiliation: Central Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK. jason.davis@ chem.ox.ac.uk
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Davis
authors.forename: Jan
authors.initials: J
authors.lastname: Tkac
authors.forename: Sophie
authors.initials: S
authors.lastname: Laurenson
authors.forename: Paul
authors.initials: P
authors.lastname: Ko Ferrigno
mesh_terms: D052158:Aptamers, Peptide; D055312:Cystatin A; D015891:Cystatins; D003545:Cysteine; D040081:Protein Array Analysis; D011506:Proteins / Q000032:analysis*; D020349:Surface Plasmon Resonance
chemical_list: D052158:Aptamers, Peptide; D055312:Cystatin A; D015891:Cystatins; D011506:Proteins; D003545:Cysteine