A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays

This work describes a concept for extending the dynamic range of quantification in an affinity biosensor assay by using a set of ligands with different affinities toward a common analyte. Three synthetic, biotinylated polypeptides capable of binding a model protein analyte with different affinities (10−9 M ≤ Kd ≤ 10−7 M) were immobilized in a microarray format on a gold slide covered with an oligo(ethylene glycol)-containing alkane thiolate self-assembled monolayer. A five-element affinity array, comprising single-peptide spots as well as spots where peptides were immobilized in mixtures, was realized by means of piezodispensation. Imaging surface plasmon resonance was used to study binding of the analyte to the different spots. The lower limit of analyte quantification was ∼3 nM and the corresponding upper limit was increased by more than an order of magnitude compared to if only the highest affinity ligand would have been used. Affinity array sensors with multiple ligands for each analyte are particularly interesting for omitting dilution steps and providing highly accurate data in assays where several analytes such as disease biomarkers with extremely variable concentrations are quantified in parallel.