Direct Single-Molecule Detection of mRNA–LNP Drugs in Blood

Messenger RNA (mRNA) therapeutics, particularly those encapsulated in lipid nanoparticles (LNPs), hold significant promise for applications such as vaccines and gene therapies. However, concerns regarding their potential misuse in contexts such as sports doping underscore the need for robust detection platforms. Here, we present direct-SATORI, a fully automated, amplification-free platform capable of detecting mRNA-LNPs directly from blood at the single-molecule level. By integrating CRISPR-Cas13a, magnetic bead-based RNA enrichment, and microchamber technology, the platform achieves high sensitivity, with limits of detection of 0.4 fM for unmodified and 3.5 fM for N1-methylpseudouridine (m¹Ψ)-modified mRNA encoding erythropoietin, a common target of conventional gene doping strategies aimed at enhancing oxygen transport. The platform completes detection within approximately 10 min and demonstrates cross-species compatibility, including equine and human plasma. In vivo validation in horses showed peak detection at 1 h postadministration, with signal clearance by 48 h. These findings highlight the potential of direct-SATORI as a rapid, sensitive, and practical tool for monitoring mRNA-based doping agents, offering a promising solution for future antidoping surveillance and regulatory enforcement.