From longstanding techniques like enzyme-linked immunosorbent assay (ELISA) to modern next-generation sequencing, many of the most sensitive and specific biomarker detection assays require capture of the analyte at a surface. While surface-based assays provide advantages, including the ability to reduce background by washing away excess reagents and/or increase specificity through analyte-specific capture probes, the limited efficiency of capture from dilute solution often restricts assay sensitivity to the femtomolar-to-nanomolar range. Although assays for many nucleic acid analytes can decrease limits of detection (LODs) to the subfemtomolar range using polymerase chain reaction, such amplification may introduce biases, errors, and an increased risk of sample cross-contamination. Furthermore, many analytes cannot be amplified easily, including short nucleic acid fragments, epigenetic modifications, and proteins. To address the challenge of achieving subfemtomolar LODs in surface-based assays without amplification, we exploit an aqueous two-phase system (ATPS) to concentrate target molecules in a smaller-volume phase near the assay surface, thus increasing capture efficiency compared to passive diffusion from the original solution. We demonstrate the utility of ATPS-enhanced capture via single molecule recognition through equilibrium Poisson sampling (SiMREPS), a microscopy technique previously shown to possess >99.9999% detection specificity for DNA mutations but an LOD of only ∼1-5 fM. By combining ATPS-enhanced capture with a Förster resonance energy transfer (FRET)-based probe design for rapid data acquisition over many fields of view, we improve the LOD ∼ 300-fold to <10 aM for an EGFR exon 19 deletion mutation. We further validate this ATPS-assisted FRET-SiMREPS assay by detecting endogenous exon 19 deletion molecules in cancer patient blood plasma.
Keywords: Förster resonance energy transfer; aqueous two-phase system; kinetic fingerprinting; single-molecule fluorescence microscopy; target enrichment; ultrasensitive detection.