Droplet Digital Enzymatic Recycling for Single‐Molecule Homogeneous Immunoassay

The merge of droplet microfluidics with single-molecule homogeneous immunoassay (SiMHoI) provides a transformative tool for biomedical diagnosis with high sensitivity, simplified workflow and reduced reagent consumption. Existing droplet-based SiMHoIs use polymerase chain reaction for single-molecular protein signal amplification; and thus, suffer from the intrinsic deficiencies of intensive instrument dependence, high background interference, and redundant reaction time. To tackle these limitations, an enzymatic recycling (ER) reaction is introduced into the microdroplets as an isothermal amplification mechanism. This approach utilizes antibody- or aptamer-labeled nucleic acid probes to capture trace amounts of targets confined in the microdroplets at a single- or sub-single-molecule level. Single-target fluorescence signal is then amplified via an in-droplet ER reaction with molecular beacons. This strategy eliminates the reliance on thermal cycling, and achieves selective droplet illumination within 20 min. The self-noise suppression mechanism of the in-droplet ER reaction enhances the positive-to-negative ratio nearly 20-fold compared to the bulk-phase reaction and reduce the detection limit to 10⁻¹⁸ m. Clinical validation with human serum specimens shows excellent consistency with flow cytometry (P > 0.05). The isothermal signal amplification approach proposed here upgrades current droplet-based molecular diagnostic platforms, and enables an engineering solution for detection of ultra-low-abundance biomarkers at preclinical stage.