Attomolar Sensitivity in Single Biomarker Counting upon Aqueous Two-Phase Surface Enrichment

Abstract

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 Forster 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.

Digital and Absolute Assays for Low Abundance Molecular Biomarkers

“…(c) Enhancement of mass transport of trace targets to surfaces using an aqueous two-phase system of saline (salt phase) and poly(ethylene glycol) (PEG phase) that condenses the targets to the surface. 52 concentration. Analytical challenges are greater for proteins than for nucleic acids in part due to the sheer diversity of the proteome compared with the genome, with protein isoforms, posttranslational modifications, dynamically changing conformations, and natively interacting molecules like heterophilic antibodies all potentially impacting capture efficiency by bioaffinity tags.…”

“…Li et al showed that an immiscible fluid can be used to concentrate attomolar nucleic acid solutions above a capture surface for digital counting, an approach that may be more amenable to simple fluidic systems (Figure 7c). 52 Gao and co-workers also observed that probe capture could be enhanced by cycling solutions above a capture surface with a gas phase through draining, which replenishes the "depletion zone" of targets in the solution proximal to the capture surface. 53 Xiong et al showed that microscopic stir bars that enhance convective transport can substantially increase capture efficiency in surface capture assays.…”

“…(f) Photonic crystal capture surface propagates evanescent waves from line-scanned excitation beams to enhance the emission of proximal QDs for digital counting of surface-captured targets in an excess of unbound QD probes in solution . Adapted with permission from refs , , and . Published 2019 and 2022 by Springer Nature under a Creative Commons Attribution 4.0 International License and Copyright 2021 American Chemical Society.…”

“…A well-developed approach is to use dispersible microbeads to capture targets in solution for concentration into smaller volumes by sedimentation, , although this can add considerable complexity to processing workflows. Li et al showed that an immiscible fluid can be used to concentrate attomolar nucleic acid solutions above a capture surface for digital counting, an approach that may be more amenable to simple fluidic systems (Figure c) . Gao and co-workers also observed that probe capture could be enhanced by cycling solutions above a capture surface with a gas phase through draining, which replenishes the “depletion zone” of targets in the solution proximal to the capture surface .…”

“…Counts correspond in time for target collisions with the surface, assuming instantaneous, irreversible binding. (c) Enhancement of mass transport of trace targets to surfaces using an aqueous two-phase system of saline (salt phase) and poly(ethylene glycol) (PEG phase) that condenses the targets to the surface 52. (d) Nanoporous substrates eliminate nonslip layers on surfaces for more efficient surface-capture.…”