Nanopore-based detection of circulating
microRNAs in lung cancer patients
Yong Wang1†, Dali Zheng2†, Qiulin Tan1, Michael X. Wang2* and Li-Qun Gu1*
MicroRNAs are short RNA molecules that regulate gene expression, and have been investigated as potential biomarkers
because their expression levels are correlated with various diseases. However, detecting microRNAs in the bloodstream
remains difficult because current methods are not sufficiently selective or sensitive. Here, we show that a nanopore sensor
based on the a-haemolysin protein can selectively detect microRNAs at the single molecular level in plasma samples from
lung cancer patients without the need for labels or amplification of the microRNA. The sensor, which uses a programmable
oligonucleotide probe to generate a target-specific signature signal, can quantify subpicomolar levels of cancer-associated
microRNAs and can distinguish single-nucleotide differences between microRNA family members. This approach is
potentially useful for quantitative microRNA detection, the discovery of disease markers and non-invasive early diagnosis
of cancer.
MicroRNAs are a class of short (18–22 nucleotides)non-coding RNAs that are important in developmentand cell differentiation, the regulation of the cell cycle,
apoptosis and signalling pathways1,2. Since its initial discovery in
Caenorhabditis elegans in 1993 (ref. 3), over 17,000 microRNAs
have been identified across different species, including humans4.
In the cytoplasm, mature microRNAs are associated with an
RNA-induced silencing protein complex to bind with the
3′-untranslated region of target messenger RNAs1,5,6. By either
repressing translation or cleaving the target messenger RNA1,5,6,
these microRNAs regulate around 30% of human gene expression5
at the post-transcriptional level. Aberrant expression of microRNAs
has been found in all types of tumours7,8, and different types
of cancers have distinct microRNA profiles9. Interestingly,
microRNAs can be released from the primary tumour into the
bloodstream in a stable form10. Circulating microRNAs are envel-
oped inside exosomal vesicles and are transferable to and functional
in recipient cells11–13. Therefore, the detection of tumour-specific
circulating microRNAs is useful for the early diagnosis, staging
and monitoring of cancer7,8,10–13.
Quantitative reverse transcription real-time polymerase chain
reaction (qRT-PCR) assays and microarrays have been developed
for the detection of microRNA. However, these methods suffer
from error-prone amplification, cross-hybridization, and a lack of
valid internal controls14,15 because the shortness of the microRNA
sequences makes it difficult to design probes and primers. Other
techniques based on colorimetry, bioluminescence, enzyme turnover
and electrochemistry have been proposed, and nanoparticles,
molecular beacons, deep sequencing16,17 and single-molecule
fluorescence18 have also been applied to microRNA detection (for
reviews, see refs 16, 17). However, many of these methods still
require the labelling and chemical modification of the target or
expensive instruments.
The nanopore is a molecular-scale pore structure that is able to
detect, with high sensitivity, the position and conformation of a
single molecule that is present within the pore lumen19. From the
characteristic change in nanopore conductance, one can electrically
elucidate single-molecule kinetic pathways and quantify the target.
Various nanopore sensors are being developed with broad biotech-
nological applications19–24 (for reviews, see refs 19–23), including
the next generation of DNA sequencing technology25,26. The devel-
opment of nanopore-based microRNA detectors is a novel effort in
this rapidly evolving field, and Wanunu et al. first reported the use
of a 3 nm synthetic pore to quantify the translocation of enriched
microRNAs that were hybridized to a probe27. In this report, we
construct a protein-nanopore-based sensor that enables sensitive,
selective and direct quantification of cancer-associated
microRNAs in the blood and discrimination of single-nucleotide
differences in microRNA family members.
Generation of microRNA signatures in the nanopore
We used the a-haemolysin protein pore, a toxin from Staphylococcus
aureus bacterium28, as the sensor element. The translocation of
single-stranded oligonucleotides through this 2 nm pore has been
studied extensively29–32. However, it is difficult to distinguish the
translocation of different microRNAs because the sequences of all
microRNAs are short and similar in length. One way of overcoming
this challenge is to use a signature that can detect targetmicroRNA in
the mixture. We identified such a microRNA signature signal in the
nanopore using an oligonucleotide probe.
The probe structure is shown in Fig. 1a. The capture domain of the
probe was used to bind the target microRNA by Watson–Crick base
pairing in the solution. Each end (3′ and 5′) of the capture domain
was extended with a poly(dC)30 signal tag. Our first target was
miR-155, a lung-cancer-associated microRNA12,13,33. Figure 1b
illustrates a sequence of nanopore current events in the presence of
miR-155 and its probe, P155, on the cis side of the pore. The boxed
events represent a characteristic type of multi-level block that was
generated by the miR-155.P155 hybrid. This block type was not
observed in the presence of miR-155 or P155 alone in the cis solution.
In a multi-level block (Fig. 1c, left panel), the Level 1 state lasted for
250+58 ms, which is almost equal to the entire event duration,
and significantly reduced the nanopore current, with a relative
residual conductance (g/g0) of 0.15. The Level 1 state was followed
1Department of Biological Engineering and Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, 2Ellis Fischel Cancer Center
and Department of Pathology and Anatomical Sciences, University of Missouri, Columbia, MO 65211, USA; †These authors contributed equally to this work.
*e-mail: gul@missouri.edu; wangmx@health.missouri.edu
ARTICLES
PUBLISHED ONLINE: 4 SEPTEMBER 2011 | DOI: 10.1038/NNANO.2011.147
NATURE NANOTECHNOLOGY | VOL 6 | OCTOBER 2011 | www.nature.com/naturenanotechnology668
© 2011 Macmillan Publishers Limited. All rights reserved.

by a discrete current increase to the Level 2 state, which persisted for
410+20 ms, with a g/g0 of 0.42. Finally, the current dropped to the
Level 3 state and remained there for 270+30 ms before returning
to the full open base level. Similar to Level 1, Level 3 almost fully
reduced the pore current, with a g/g0 of 0.08. The right panel in
Fig. 1c depicts the molecular configurations that correspond to the
multi-level block. The amplitude and duration of Level 1 were
consistent with a configuration in which the miR-155.P155 complex
was trapped in the nanopore at the 2.6 nm cis opening, with either
the 3′ or 5′ signal tag of P155 occupying the 1.6–2.0 nm b-barrel.
Driven by the transmembrane voltage, the signal tag in the
b-barrel induced the dissociation of miR-155.P155. The dissociation
time (the duration of Level 1) was comparable to previously reported
timescales for DNA unzipping in the pore34,35. After unzipping, P155
left the pore through the narrower (trans) opening, and the Level 1
state was terminated. The Level 2 state featured large residue conduc-
tance, which should correspond to a configuration in which mir-155
unzipped from miR-155.P155 and temporarily resided in the nano-
cavity. This result is consistent with a previous finding which
showed that an oligonucleotide trapped in the nanocavity can gener-
ate partial blocks36. The miR-155 in the nanocavity finally passed
through the b-barrel to yield the short-lived Level 3 state. The dur-
ation of Level 3 (270 ms) was consistent with the translocation dur-
ation of miR-155 alone (220 ms) and the timescale for DNA or
RNA translocations in previous studies29–32. The molecular mechan-
ism described above was further proved by the voltage-dependent
durations of Levels 1 and 3. Level 1 was shortened by a factor of 23
to 11 ms and Level 3 by a factor of 2 to 150 ms as the voltage increased
from þ100 to þ180 mV (Fig. 1c, lower panel). This indicated that
the voltage both enhanced the unzipping of miR-155.P155 and
a MicroRNAProbeSignal tag Signal tag
3´ 5´
5´ 3´
b
c
d
e f
150 ms
10
0
pA
Level 1trans
cis
Level 2 Level 3
10 ms
10
0
pA
100 mV
≈
2
1 3
150 mV
180 mV
100

t
r
a
n
s
[m
iR
-1
55
] (
aM
)
by
R
T-
PC
R
1 10
10
cis [miR-155] (nM)
≈
Figure 1 | Capturing single microRNA molecules in the nanopore. a, Molecular diagram of a microRNA (red) bound to a probe (green) bearing signal tags
on each end. b, Sequence of nanopore current blocks in the presence of 100 nM miR-155 and 100 nM P155 in the cis solution. Traces were recorded at
þ100 mV in solutions containing 1 M KCl buffered with 10 mM Tris (pH 8.0). Red boxes represent the multi-level current pattern. c, A typical multi-level
long block (from b) at þ100 mV generated by the miR-155.P155 hybrid. Right panel: diagram showing the molecular mechanism of hybrid dissociation and
translocation. Level 1: trapping of the microRNA.probe hybrid in the pore, unzipping of the microRNA from the probe and translocation of the probe through
the pore. Level 2: unzipped microRNA residing in the pore cavity. Level 3: translocation of the unzipped microRNA through the pore. Lower panel: multi-level
blocks at þ150 and þ180 mV. Increasing the voltage reduced the duration of Levels 1 and 3, which supports the above mechanistic model. d, miR-155 levels
detected by qRT-PCR in trans solutions. Before detection, the pore current was monitored in 0.5 M/3 M (cis/trans) KCl at þ180 mV in the presence of 1mM
P155 and 0.5, 1 or 10 nM of miR-155 in the cis solution. A much higher probe concentration than microRNA was used to enhance their hybridization in the cis
solution (see Supplementary Information S1). e, A single-level block (from b) generated by a trapped miR-155.P155 hybrid that exited the pore from the cis
entrance without unzipping and translocation. f, A spike-like short block generated by the translocation of unhybridized miR-155 or P155 from the cis solution.
NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2011.147 ARTICLES
NATURE NANOTECHNOLOGY | VOL 6 | OCTOBER 2011 | www.nature.com/naturenanotechnology 669
© 2011 Macmillan Publishers Limited. All rights reserved.