Abstract: Conventional methods mainly rely on ensemble-averaged bulk characterization, which remains challenging for identifying low-abundance endogenous impurities generated during synthesis. Here, we developed a biological nanopore-based single-molecule strategy for the direct detection of truncated-sequence impurities in customized oligonucleotide samples. Using an aerolysin nanopore to analyze a customized poly(dA)9 sample, we observed three clearly distinguishable characteristic peaks in the nanopore current fingerprint spectrum. Among them, the peaks with smaller current blockages matched those of poly(dA)4 and poly(dA)3, indicating the presence of additional oligonucleotide species besides the target product. In addition, the relative peak area ratios remained nearly constant over the data acquisition period from 0.5 to 10 min, demonstrating that this method can provide stable impurity signals within 1 min and enable rapid analysis of sample compositional heterogeneity. This study expands the application of nanopore technology in the quality evaluation of real biomolecular products and offers a single-molecule analytical strategy for impurity detection in oligonucleotides and other biomolecules.

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