Primer exchange reaction-based dual-signal amplified nano-flow cytometry and fluorescence analysis of MUC1- positive...

Nano-flow cytometry (nFCM) is a single-particle analytical technique for the detection, sizing, and phenotyping of extracellular vesicles (EVs). However, nFCM analysis of small-sized EVs (<100 nm) or those with low-abundance antigen markers for immunostaining remains a major challenge. In this work, we developed a primer exchange reaction-based dual-signal amplified nano-flow cytometry (PER-DSA nFCM) method for the specific detection of MUC1-positive EVs and the phenotyping of exosomes derived from different pancreatic cancer cell lines. The mechanism relies on the specific binding of a DNA hairpin to the MUC1 protein on the EVs surface, followed by the two-dimensional expansion of the opened hairpin via primer exchange reaction to enlarge individual EVs. The enlarged EVs with extended DNA polymers can hybridize with multiple molecular beacons, emitting strong fluorescence, which can be detected by nFCM through the amplified laser side-scattering signal and fluorescence signal. Conventional fluorescence measurement can be also employed for bulk concentration quantification to the expanded EVs. Using PER-DSA nFCM, we successfully distinguished exosomes secreted from pancreatic cancer cell lines HPAC and BxPC-3, which exhibit different surface MUC1 expression levels. Bulk analysis of HPAC exosomes via post-amplification solution fluorescence measurement yielded a good linear quantitative response to concentration of EVs in the range of 5 × 10⁵-7.5 × 10⁶ particles μL^-1, with a detection limit of 1.25 × 10³ particles μL^-1. Applying this assay in plasma biopsy for diagnosing pancreatic cancer, the receiver operating characteristic curve presented a sensitivity of 77.8% and a specificity of 93.3% at the best cutoff, with an area under the curve of 0.844. The proposed method is simple, rapid, and sensitive for the analysis of MUC1-positive EVs, and can be easily modified to detect EVs expressing other surface protein markers, holding potential for clinical applications.