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.
Background: In facial reanimation via microneurovascular muscle transfer, dual-nerve reinnervation of the muscle capitalizes on the synergistic effects of spontaneous motion from cross facial nerve grafting (CFNG) and increased excursion from masseteric nerve transfer. Two-stage approaches that delay masseteric nerve transfer until the time of the muscle flap increase spontaneity by maximizing muscle reinnervation from the CFNG. While this 2-stage, dual-nerve approach has been described in adults, we present a series of pediatric patients who underwent this reconstructive technique. Methods: We retrospectively reviewed all pediatric patients who underwent 2-stage, dual-nerve reconstruction with CFNG and ipsilateral masseteric nerve transfers. Procedures were performed between 2004 and 2016 by 2 surgeons at a single centre. Degree of facial paralysis before and after surgical intervention was measured using House-Brackmann scores. Results: Nine patients with a mean age of 8.6 (range: 5-15 years) years at time of surgery underwent 2-stage, dual-nerve reconstruction. Average time between CFNG and transfer of the free gracilis with masseteric nerve transfer was 13.3 (SD 2.4) months. Mean follow-up was 27.3 months (SD 25.7). Patients demonstrated initiation of voluntary movement on paralyzed side by 3.6 months (SD 0.6) on average, with 3 patients demonstrating spontaneous movement at 3 months. Conclusion: The 2-stage, dual innervation technique using CFNG and delayed ipsilateral masseteric nerve transfer with a free gracilis is a promising reconstructive option to maximize spontaneous expression in pediatric patients. Validated, objective scoring systems for excursion are needed before meaningful comparisons can be made to other reconstructive strategies.
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