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Cite this: DOI: 10.1039/c7nr04215a
Received 13th June 2017,
Accepted 25th September 2017
DOI: 10.1039/c7nr04215a

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MicroRNA detection at femtomolar concentrations
with isothermal ampliﬁcation and a biological
nanopore†
Haolin Zhang,a

Moe Hiratani,b Kentaro Nagaoka

aand Ryuji Kawano*brsc.li/nanoscale

One of the greatest challenges faced by chemists and biologists isthe detection of molecules at extremely low concentrations. This
paper describes a method to detect ultra-low concentrations(1 femtomole) of nucleotides using isothermal ampliﬁcation and abiological nanopore.

Nanopore sensing is a recent and emerging technology for thedetection of single molecules, such as oligonucleotides, thatcan be translocated through a nanochannel of a pore-forming
protein; it has also shown clear blocking currents at the single
molecule level. Since Deamer and co-workers reported theirpioneering work 20 years ago, extensive studies and applications, such as small molecule detection with adapters1 or
aptamers,2 in situ mass-spectroscopy,3 and nanopore sequencing, have been reported.4,5 Furthermore, solid-state nanopores, rather than biological nanopores, have been developedby using an electron beam to form nanopores in silicon
nitride6 or graphene,7 and other materials.8–10 The biological
nanopore alpha-haemolysin (αHL) is a toxin channel secretedby the bacterium Staphylococcus aureus, which has been
mainly used as an oligonucleotide-detecting nanopore because
the size of the pore is comparable to that of single-stranded(ss) DNA or RNA molecules.11–15 Therefore, much eﬀort has
been exerted on using αHL nanopores for ssDNA detection insingle DNA sequencing. In 2015, Oxford Nanopore Co. Ltd
successfully released a portable sequencer using an otherbiological nanopore as a commercialized product; this willlead to the development of extensive practical applications in
nanopore sensing.
One expected nanopore application is the detection of
microRNAs (miRNAs), small non-coding RNAs composed ofaLaboratory of Veterinary Physiology, Cooperative Department of Veterinary
Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology,
Tokyo 183-8509, Japanb
Department of Biotechnology and Life Science, Tokyo University of Agriculture andTechnology (TUAT), 2-24-16 Naka-cho Koganei-shi, Tokyo 184-8588, Japan.
E-mail: rjkawano@cc.tuat.ac.jp† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7nr04215a

This journal is © The Royal Society of Chemistry 201720–30 nucleotides that are produced by two RNase III proteins,
Drosha and Dicer.16,17 Through RNA silencing and post-transcriptional regulation of gene expression, miRNAs are involvedin various cellular processes, including diﬀerentiation, proliferation, migration and invasion. Accumulating evidence
suggests that abnormal miRNA expression in tissues or bloodis related to the development of cancer or other diseases.18 As
a result, the aberrant expression of miRNAs in serum or tissue
has been suggested to have a diagnostic value for several
diseases.19–22 Generally, the methods used for miRNA detection, such as reverse transcription polymerase chain reaction(RT-PCR) and DNA arrays, are quite time-consuming. Several
attempts at miRNA detection using nanopores have been
recently reported,23–26 indicating that the nanopore method is
a promising candidate for simple and rapid miRNA detection.
However, one of the most significant issues in the detection of
miRNAs is their low (femtomolar) concentrations, especiallyduring the early stages of cancer development. Therefore, thedevelopment of ultra-sensitive, simple, and rapid nanopore
sensing methods would be of great significance.
To detect low concentration oligonucleotides using nanopores, two eﬀective techniques have been previously proposed.
The first is a physicochemical strategy based on the asymmetryof electrolytes between the cis and trans sides of the nanopore,enhancing the ionic flow through the nanopore. This flowenhances nucleotide translocation even at low concentrations.
This method, using αHL, facilitated translocation at concentrations as low as 1 pM.25 The second is an electrical strategy
based on dielectrophoretic (DEP) force that is applied at the
opening of a solid (glass) nanopore, which can enhance theoligonucleotide translocation. This method, using a glass
nanopore, facilitated the translocation of dsDNA at concentrations as low as 5 fM.27 In this study, we have attempted toachieve an even lower detection concentration, 1 fM, by combining oligonucleotide amplification and the asymmetricmethod described above. Based on these technologies, we
attempted to measure 1 fM of miR-20a, which is secreted fromlung cancer tissue.28 We constructed this amplification system
based on isothermal amplification29,30 using miR-20a as the

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input and poly-thymines as output molecules. Detailed information on the materials and methods is described in the ESI.†The amplified DNA from 1 fM concentration of miRNA could
be measured by αHL nanopores under asymmetric conditions.
This approach is a promising method for detecting miRNAs at
ultra-low concentrations using nanopores (Fig. 1).
To achieve specific miR-20a amplification, the DNA
template and primer were designed according to the miR-20a
sequence as follows: DNA template, 5′-AAAAAAAAAAAAAAAAAAAAAAAAGATCCTTGTTTTGGAATAAGCACTTTA-3′;
primer,5′-CTACCTGCACTACCAAAA-3′; miR-20a, 5′-UAAAGUGCUUAUAGUGCAGGUAG-3′. The DNA template and primer were partially complementary to miR-20a by 11 and 12 bp, respectively.
As illustrated in Fig. 2, a three-way junction forms between the
DNA template and primer in the presence of miR-20a.
However, stable association is not expected to occur between
the DNA template and primer in the absence of miR-20a, due
to the limited number of complementary nucleotides andresultant low melting temperature. Following a three-way
junction formation, the DNA polymerase Bst binds to the DNA
template and elongates the complementary sequence using
dNTPs as a substrate at 37 °C. A recognition site for thenicking enzyme Nt.AlwI was included in the DNA template,which is highlighted in yellow in Fig. 2B. The elongated complementary sequence is nicked by Nt.AlwI, which then initiates
another cycle of DNA polymerization with the displacement of
the single strand of polyT(20) (Fig. 2B). Therefore, polyT(20) is
generated only in the presence of miR-20a.
To confirm the isothermal amplification reaction, we
performed gel electrophoresis to detect the generation of
polyT(20) with and without miR-20a (Fig. 3A, “+” and “−” lanes).
The polyT(20) band is clearly visible in the presence of miR-20a,but not in its absence. This result suggests miR-20a-specificamplification of polyT(20).
The concentration of polyT(20) generated from isothermal
amplification was measured using an asymmetric nanopore
constructed from αHL for the ultra-sensitive detection of polyT(20), in accordance with a previous report that revealed increasing capture rates when an ionic gradient was establishedacross the pore.31 The 10-fold salt gradient was establishedacross the nanopore by applying a 2 M KCl solution to thetrans side and a 0.2 M KCl solution to the cis side. With theosmotic pressure and a 120 mV voltage between the trans andcis sides, we were able to measure the frequency of polyT(20)translocation from 1 to 1000 pM (Fig. 3B). The current block-Fig. 1 Schematic diagram of miR-20a detection using DNA computing
by isothermal ampliﬁcation and asymmetric nanopore measurement.

NanoscaleNanoscaleFig. 2 (A) Schematic illustration of asymmetric nanopore detection for
the generated polyT(20). (B) Schematic illustration of polyT(20) generation by isothermal enzyme reaction in the presence of miR-20a.

Fig. 3 (A) Gel electrophoresis of polyT(20) generation. M, size marker;+: with miR-20a (100 nM); −: without miR-20a. P: positive control. (B)The calibration curve of polyT(20) detection using the asymmetricnanopore.

ade of polyT(20) was approximately 80% when translocation
occurred, and other short current blockades also occurred due
to collision (approximately 30%) and transient partial entry(approximately 60%).32 A standard calibration curve wascreated based on the event frequency of >80% current blockades, shown in Fig. 3B. According to the capture rate theory ofpolymer translocation, the polyT(20) translated from the nanopore would be separated into three steps with diﬀusion, drift
and translocation.33 The electro-osmotic field would increase

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Nanoscalethe aggregation of polymers near the nanopore, where thecapture rate was linear with the concentration of polymers.33
After conquering the diﬀusion-limited regime, the polymers
pass the energy barrier when successful translocation
occurs.31,33 Even under this asymmetric condition (2 M/0.2 M),
the frequency of signature events was proportional to the DNA
concentration, which was similar to those revealed in previous
reports.2,25
The translocation frequencies of polyT(20) generated by isothermal amplification with or without miR-20a were alsomeasured through the asymmetric nanopore (Fig. 4A). We
measured the frequencies of polyT(20) translocation with 1 to
100 fM miR-20a after 1 hour of isothermal amplification, andthe final concentration of polyT(20) generated from 1 fM
miR-20a was 10.6 pM, which was calculated based on the calibration curve in Fig. 3B. The results suggest that 1 fM target
miRNA can be adequately amplified and transformed into
DNA molecules, which are more stable than RNA molecules.
This high amplification may be partially due to the enzyme
activity of the polymerase/nicking enzymes and the rapid
amplification kinetics of isothermal reactions.34,35 The detection limit could be extended to concentrations lower than
1 fM; however, this would require reaction optimization,
including the types of enzymes used, their concentrations, andthe reaction time. Using the other method of low concentration detection, a nanogap sensor demonstrated the sub-fM
nucleotide detection of long mRNAs electrochemically;36
however, this requires highly sophisticated microfabrications
and may be limited for the detection of miRNAs due to theirCommunication

short length. There are several conventional methods for
miRNA quantification, including real-time polymerase chain
reaction (qRT-PCR), northern blot, and microarray analyses.
Due to several limitations, such as their low sensitivity andspecificity, as well as being time-consuming or expensive, thedevelopment of eﬀective methods for miRNA detection isurgently needed.37,38 Exponential isothermal amplificationand nanotechnology represent a prospective method formiRNA detection. An obvious frequency elevation wasobserved with increased miR-20a concentration (Fig. 4A and
B). The capture rate was 98 238 s−1 nM−1, which was significantly higher than that of a conventional αHL nanopore(0.1 s−1 nM−1)5 or a solid-state nanopore (263 s−1 nM−1).31 The
in vivo miRNA concentration was in the fM range. Therefore,
our method can detect specific miRNAs at fM concentration
without labelling. In the future, we will study the ability of thismethod using a practical in vivo sample.

Conclusions
In summary, we attempted ultra-low concentration miRNAdetection by combining isothermal amplification and nanopore measurement. At miR-20a concentrations of 1 fM to
10 pM, stable DNA polyT(20) was transcribed and amplified.
The output polyT(20) could be measured and quantified bynanopore measurement using the asymmetric solution
method. The detection of 1 fM miRNA has been the lowest
concentration detected by nanopore technology to date. Basedon this methodology, by simply changing the nucleotide
sequences of the DNA template and primer according to thetarget miRNA, diﬀerent miRNAs can be specifically amplified.
For practical application, the present method requires pretreatment, such as miRNA extraction, although the method
does not require the condensation process. Additionally, thesensitivity of the present system may depend on the limitationof isothermal amplification. This system is potentially applicable for the diagnosis of any disease for which a diagnostic
miRNA marker has been established.

Conﬂicts of interest
There are no conflicts of interest to declare.

Acknowledgements
This work was partially supported by KAKENHI (Grant No.
15H00803, 16H04226, and 16H06043) from MEXT, Japan.

Fig. 4 (A) Typical current and time traces (1 min) of generated polyT(20) translocation in the asymmetric nanopore with diﬀerent concentrations of miR-20a. (B) The capture rates of polyT(20) generated from
diﬀerent concentrations of miR-20a. (C) Correlation between miR-20a
and polyT(20) concentrations.

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