Monovalent Targeting Nanotag-Mediated Single-Molecule Counting by SP-ICP-MS.

Accurate single-molecule quantification of biomolecular targets is essential for ultrasensitive bioanalysis and early disease diagnostics. Single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) enables the detection of individual metal nanoparticles with high accuracy, sensitivity, and excellent elemental selectivity, making it an attractive platform for nanoparticle-based bioanalysis. However, its application to single-molecule counting of biomolecules remains limited. Herein, we report a monovalent nanotag-mediated strategy for single-molecule counting of biomolecules by using SP-ICP-MS. We constructed monovalent DNA-functionalized gold nanoparticles (mDNA-AuNPs), each carrying a single DNA strand designed for target-specific DNA hybridization or aptamer-mediated protein recognition, thereby enabling well-defined 1:1 interaction with target molecules and AuNP release. We first validated the feasibility of this strategy using DNA targets via a strand displacement-mediated release mechanism. We subsequently extended the method to thrombin detection through a binding-induced aptamer conformation switching and nanotag release process. This strategy provides a promising platform for single-molecule counting and expands the applicability of SP-ICP-MS for ultrasensitive biomolecular detection.