Highly sensitive protein detection by aptamer-based single-molecule kinetic fingerprinting

Sensitive assays of protein biomarkers play critical roles in clinical diagnostics and biomedical research. Such assays typically employ immunoreagents such as monoclonal antibodies that suffer from several drawbacks, including relatively tedious production, significant batch-to-batch variability, and challenges in site-specific, stoichiometric modification with fluorophores or other labels. One proposed alternative to such immunoreagents, nucleic acid aptamers generated by systematic evolution of ligand by exponential enrichment (SELEX), can be chemically synthesized with much greater ease, precision, and reproducibility than antibodies. However, most aptamers exhibit relatively poor affinity, yielding low sensitivity in the assays employing them. Recently, single molecule recognition through equilibrium Poisson sampling (SiMREPS) has emerged as a platform for detecting proteins and other biomarkers with high sensitivity without requiring high-affinity detection probes. In this manuscript, we demonstrate the applicability and advantages of aptamers as detection probes in SiMREPS as applied to two clinically relevant biomarkers, VEGF₁₆₅ and IL-8, using a wash-free protocol with limits of detection in the low femtomolar range (3-9 fM). We show that the kinetics of existing RNA aptamers can be rationally optimized for use as SiMREPS detection probes by mutating a single nucleotide in the conserved binding region or by shortening the aptamer sequence. Finally, we demonstrate the detection of endogenous IL-8 from human serum at a concentration below the detection limit of commercial ELISAs.