Figure 4. Detection of 5hmC in ssDNA using SWCNT nanopore.

(a) 5hmC modification scheme: oxidation28 of 5hmC to 5fC followed by oxidative condensation with 1,2-phenylenediamine to afford 5C–Bzim. The R group is DNA. (b) A typical multilevel current signature and diagram showing molecular mechanism of DNA2–Bzim translocation through SWCNT nanopore. Level 1: DNA2–Bzim entering the nanopore before Bzim-modified part reaches the pore; level 2: the Bzim part entering and sliding through the nanopore; level 3: translocation of the rest of DNA2–Bzim. The mean duration of 118 single-spike events is 406±111 μs. (c) Histograms of the conductance differences between level 1–3 and the open-pore level G₀ in b (118 events in total). (d) New type of current signature caused by the translocation of DNA5–Bzim, which contains double 5hmC sites. The mean duration of 110 double-spike events is 536±161 μs. All the data were acquired in the buffer of 1 M KCl and 10 mM Tris, pH 8.0, with the transmembrane potential held at +40 mV.