Figure 4.

(a) Simultaneous observation of multiple miRNA·probe blocking levels in a current trace. Each level is for a specific miRNA species. From top to bottom, the four conductance levels are for miR-155·P0, miR-182-5p·P3, miR-210·P8, and miR-21·P24. All miRNAs were at 50 nM. (b) Event amplitude histogram based on over 2000 blocks obtained from the trace in panel a, showing the distinct blocking levels for the four signatures. An extended trace showing more signatures is illustrated in Figure S8a, and the duration–current scattering plot showing the separation of the four signatures is given in Figure S8b. (c, d) Signature frequencies at various miRNA concentrations in multiplex detection. In c, the miR-155 concentration varies while other miRNA concentrations are fixed to 75 nM. Confidential intervals are marked. The frequency of miR-155 signatures increases as its concentration increases, whereas the frequencies of the other three miRNAs remain unchanged and are not influenced by the increasing miR-155 concentration. In d, each miRNA was measured in the presence of other miRNAs at the same concentration. The signature frequencies for all four miRNAs are increased in proportion to their concentrations and do not interfere with each other. Each concentration has been measured based on at least three independent nanopores.