Aberrant expression of protein kinase A (PKA) can cause severe human diseases, including Alzheimer’s disease, cancers, and so on. Development of sensitive methods for the accurate detection of PKA activity and screening of the inhibitors are significantly important for disease diagnosis and drug development. Direct recoding the specific and dynamic interactions between single-molecules has been demonstrated to be a robust strategy for biomolecule assay with ultra-high sensitivity. In this work, we design a new approach for digital counting-based protein kinase activity assay by using plasmonic nanoparticle-assisted single-molecule dynamic binding. This method breaks the concentration-dependent limitation in single-molecule detection. Meanwhile, the localized plasmonic resonance effect at the binding sites can greatly enhance the signal-to-noise ratio of the fluorescent signal from single-molecules and also improve the target capturing efficiency. According to the digital counting results, this method displays good selectivity and sensitivity with a detection limit as low as 0.0001 U/mL for PKA assay, which would find broad applications in drug screening and medical investigations.

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