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Abstract While nanoscale quantum emitters are effective tags for measuring biomolecular interactions, their utilities for applications that demand single-unit observations are limited by the requirements for large numerical aperture (NA) objectives, fluorescence intermittency, and poor photon collection efficiency resulted from omnidirectional emission. Here, we report a nearly 3000-fold signal enhancement achieved through multiplicative effects of enhanced excitation, highly directional extraction, quantum efficiency improvement, and blinking suppression through a photonic crystal (PC) surface. The approach achieves single quantum dot (QD) sensitivity with high signal-to-noise ratio, even when using a low-NA lens and an inexpensive optical setup. The blinking suppression capability of the PC improves the QDs on-time from 15% to 85% ameliorating signal intermittency. We developed an assay for cancer-associated miRNA biomarkers with single-molecule resolution, single-base mutation selectivity, and 10-attomolar detection limit. Additionally, we observed differential surface motion trajectories of QDs when their surface attachment stringency is altered by changing a single base in a cancer-specific miRNA sequence. 

Assays utilizing fluorophores are common throughout life science research and diagnostics, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve higher signal‐to‐noise. We describe how the synergistic coupling of plasmonic and photonic modes can significantly boost the emission from fluorophores. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) with the absorption and emission spectrum of the fluorescent dye, a 52‐fold improvement in signal intensity is observed, enabling individual PFs to be observed and digitally counted, where one PF tag represents one detected target molecule. The amplification can be attributed to the strong near‐field enhancement due to the cavity‐induced activation of the PF, PC band structure‐mediated improvement in collection efficiency, and increased rate of spontaneous emission. The applicability of the method by dose‐response characterization of a sandwich immunoassay for human interleukin‐6, a biomarker used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease is demonstrated. A limit of detection of 10 fg mL⁻¹and 100 fg mL⁻¹in buffer and human plasma respectively, is achieved, representing a capability nearly three orders of magnitude lower than standard immunoassays.