Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping

Gene doping is an increasing challenge in sports, demanding highly sensitive and specific detection tools beyond the limitations of the current amplification-dependent methods. Here, an innovative amplification-free clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) 12a assay integrated with osmotically tunable double emulsion (DE) droplets is reported for rapid and ultrasensitive gene doping detection. Target DNA and CRISPR/Cas12a complexes are encapsulated within DE droplets, where osmotic shrinkage rapidly concentrates the reaction components, thereby enhancing the fluorescent signal intensity without nucleic acid amplification. This platform enables the detection of the human erythropoietin (hEPO) gene at unprecedented attomolar levels within 30 min, achieving a 25-fold improvement in sensitivity compared with that of nonshrinkable formats. Notably, the assay demonstrated a robust and specific performance in complex serum samples with minimal matrix interference. This novel approach offers a rapid, reliable, and inherently contamination-free solution for gene doping surveillance with broad potential for versatile amplification-free nucleic acid diagnostics.