Programming nanopore ion flow for encoded multiplex microRNA detection

Abstract

Many efforts are being made in translating the nanopore into an ultrasensitive single-molecule platform for various genetic and epigenetic detections. However, compared with current approaches including PCR, the low throughput limits the nanopore applications in biological research and clinical settings, which usually requires simultaneous detection of multiple biomarkers for accurate disease diagnostics. Herein we report a barcode probe approach for multiple nucleic acid detection in one nanopore. Instead of directly identifying different targets in a nanopore, we designed a series of barcode probes to encode different targets. When the probe is bound with the target, the barcode group polyethylene glycol attached on the probe through click chemistry can specifically modulate nanopore ion flow. The resulting signature serves as a marker for the encoded target. Therefore counting different signatures in a current recording allows simultaneous analysis of multiple targets in one nanopore. The principle of this approach was verified by using a panel of cancer-derived microRNAs as the target, a type of biomarker for cancer detection.

Figure 1

(a) Barcode modulation of nanopore ionic current and multiplex detection of nucleic acid. Each target miRNA is hybridized with a PEG-labeled DNA probe. Upon being trapped in the nanopore, the PEG on the hybrid specifically regulates the nanopore current, thereby generating a signature for target identification. (b) Conjugation of a DNA probe with a PEG barcode through one-step copper(I)-catalyzed click chemistry.

Figure 2

(a, b) Signatures for miR-155 (miRNA) hybridized with an unlabeled probe (P0) (a) and a PEG24-labeled probe (P24) (b). The PEG label allows generating a distinct current profile compared with the block using unlabeled probe. (c) Histogram showing the current amplitude of each stage in the signature in panel b. (d) Molecular configurations for sequential stages in b. When a miR-155·P24 hybrid is trapped in the pore, the single-stranded lead first enters the β-barrel (Ia). As the lead threads in the pore, the PEG label moves into the β-barrel to further reduce the blocking level (Ib). The lead with the PEG label is pulled by the electric field to induce the unzipping of the miR-155·P24 hybrid and continuously slides in the β-barrel while unzipping occurs. The PEG label finally slides out of the pore, resulting in higher pore conductance (Ic). After unzipping and probe translocation, the dissociated miR-155 temporarily resides in the nanocavity (II) and finally translocates through the β-barrel (III) to terminate the block.

Figure 3

Modulation of miRNA·probe blocking level by PEGs of different lengths. (a) PEG length-dependent blocking level (I/I₀) for stage Ib of the signatures (as illustrated in Figure 2b) for four miRNAs: miR-155 (●), miR-182-5p (■), miR-210 (▲), miR-21 (▼). The four colored arrows mark an optimized miRNA/probe combination: miR-155·P0 (red), miR-182-5p·P3 (green), miR-210·P8 (blue), and miR-21·P24 (purple). The four miRNA·probe hybrids demonstrate maximally separated blocking levels, enabling accurate detection of multiplex miRNA species. (b) Distinct signatures for the four optimized miRNA·probe hybrids (miR-155·P0, miR-182-5p·P3, miR-210·P8, and miR-21·P24) marked in panel a. (c) Duration of four miRNA·probe signatures with untagged (black bar) and barcoded (gray bar) probes. Melting temperatures of the four miRNAs are also shown (red circles). The block duration is positively correlated to the miRNA·probe melting temperature. Method for obtaining block duration is described in S1 in the SI.

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.