Identifying DNA mismatches at single-nucleotide resolution by probing individual surface potentials of DNA-capped nanoparticles

Here, we demonstrate a powerful method to discriminate DNA mismatches at single-nucleotide resolution from 0 to 5 mismatches (χ₀ to χ₅) using Kelvin probe force microscopy (KPFM). Using our previously developed method, we quantified the surface potentials (SPs) of individual DNA-capped nanoparticles (DCNPs, ∼100 nm). On each DCNP, DNA hybridization occurs between ∼2200 immobilized probe DNA (pDNA) and target DNA with mismatches (tDNA, ∼80 nM). Thus, each DCNP used in the bioassay (each pDNA-tDNA interaction) corresponds to a single ensemble in which a large number of pDNA-tDNA interactions take place. Moreover, one KPFM image can scan at least dozens of ensembles, which allows statistical analysis (i.e., an ensemble average) of many bioassay cases (ensembles) under the same conditions. We found that as the χ_n increased from χ₀ to χ₅ in the tDNA, the average SP of dozens of ensembles (DCNPs) was attenuated owing to fewer hybridization events between the pDNA and the tDNA. Remarkably, the SP attenuation vs. the χ_n showed an inverse-linear correlation, albeit the equilibrium constant for DNA hybridization exponentially decreased asymptotically as the χ_n increased. In addition, we observed a cascade reaction at a 100-fold lower concentration of tDNA (∼0.8 nM); the average SP of DCNPs exhibited no significant decrease but rather split into two separate states (no-hybridization vs. full-hybridization). Compared to complementary tDNA (i.e., χ₀), the ratio of no-hybridization/full-hybridization within a given set of DCNPs became ∼1.6 times higher in the presence of tDNA with single mismatches (i.e., χ₁). The results imply that our method opens new avenues not only in the research on the DNA hybridization mechanism in the presence of DNA mismatches but also in the development of a robust technology for DNA mismatch detection.