Dynamic single-molecule sensing by actively tuning binding kinetics for ultrasensitive biomarker detection

The ability to measure many single molecules simultaneously in large and complex samples is critical to the translation of single-molecule sensors for practical applications in biomarker detection. The challenges lie in the limits imposed by mass transportation and thermodynamics, resulting in long assay time and/or insufficient sensitivity. Here, we report an approach called Sensing Single Molecule under MicroManipulation (SSM(3)) to circumvent the above limits. In SSM(3), single-molecule binding processes were dynamically recorded by surface plasmon resonance microscopy in a nanoparticle-mediated sandwich scheme. The binding kinetics between analyte and probes are fine-tuned by nanoparticle micromanipulations to promote the repetitive binding and dissociation. Quantifying the heterogeneous lifetime of each molecular complex allows the discrimination of specific binding from nonspecific background noise. By digitally counting the number of repetitive specific binding events, we demonstrate the direct detection of microRNAs and amyloid-β proteins with the limit of detection at the subfemtomolar level in buffer and spiked human serum. Together with the nanoparticle micromanipulation to promote the transportation rate of analyte molecules, the assay could be performed within as short as 15 min without the need for preincubation. The advantages over other single-molecule sensors include short assay time, compatible with common probes and ultrasensitive detection. With further improvement on the throughput and automation, we anticipate the proposed approach could find wide applications in fundamental biological research and clinical testing of disease-related biomarkers.