SCIENCE ADVANCES | RESEARCH ARTICLEAPPLIED SCIENCES AND ENGINEERING

Unveiling massive numbers of cancer-related
urinary-microRNA candidates via nanowires
Takao Yasui,1,2,3* Takeshi Yanagida,4,5* Satoru Ito,1 Yuki Konakade,1 Daiki Takeshita,1Tsuyoshi Naganawa,1 Kazuki Nagashima,4 Taisuke Shimada,1 Noritada Kaji,1,2,3 Yuta Nakamura,1Ivan Adiyasa Thiodorus,1 Yong He,4 Sakon Rahong,2,6 Masaki Kanai,4 Hiroshi Yukawa,1,2
Takahiro Ochiya,7 Tomoji Kawai,5* Yoshinobu Baba1,2,8,9*

INTRODUCTION

MicroRNAs (miRNAs) inside extracellular vesicles (EVs), including
exosomes, microvesicles, and apoptotic bodies (1–4) [where someauthors have used EVs additionally to encompass all membrane vesicles, including the three types of vesicles with microsomes (4, 5)], withdiameters of 40 to 5000 nm, have been found in various body fluidsof both healthy subjects and patients with malignant diseases (6–20).
The difference in the EV-encapsulated miRNAs between the two groupsof people may represent a warning sign for various disease scenarios(20). Because EV encapsulation of miRNAs has an advantage of being
able to lower the ribonuclease effect on RNA degradation (21), the miRNAs
inside the EVs are much more stable than free-floating RNAs. This promotes a switch from free-floating miRNA analysis to EV-encapsulated
miRNA analysis.
Conventionally, three major methodologies have been used for
EV collection (4): ultracentrifugation or differential centrifugation,
immunoaffinity-based capture, and size exclusion chromatography.
Some emerging methodologies have been reported as promising alternatives, including polymer precipitation (22), microfluidic-based platforms (23–26), and size-based filtration (27). However, none of the1

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. 2ImPACT Research Center
for Advanced Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya
464-8603, Japan. 3Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho,
Kawaguchi, Saitama 332-0012, Japan. 4Institute of Materials Chemistry and Engineering, Kyushu University, 6-1 Kasuga-koen, Kasuga, Fukuoka 816-8580, Japan. 5Instituteof Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka-cho, Ibaraki,
Osaka 567-0047, Japan. 6College of Nanotechnology, King Mongkut’s Institute ofTechnology Ladkrabang, Chalongkrung Road, Ladkrabang, Bangkok 10520, Thailand.
7
Division of Molecular and Cellular Medicine, National Cancer Center Research Institute,5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. 8Health Research Institute, National
Institute of Advanced Industrial Science and Technology, Takamatsu 761-0395, Japan.
9College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan, Republicof China.
*Corresponding author. Email: yasui@chembio.nagoya-u.ac.jp (T. Yasui); yanagida@cm.kyushu-u.ac.jp (T. Yanagida); kawai@sanken.osaka-u.ac.jp (T.K.); babaymtt@apchem.
nagoya-u.ac.jp (Y.B.)
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existing methodologies for collecting EV-encapsulated miRNAs havesatisfied the requirements for simple and noninvasive urine-based early
disease diagnoses and timely medical checkups. This is because theconcentration of EVs in urine is extremely low (<0.01 volume %) (28).
For example, ultracentrifugation as the most commonly used method
has allowed researchers to collect urine EVs, perform the following extraction of EV-encapsulated miRNAs (29, 30), and identify 200 to 300
urinary miRNAs (31). Although more than 2000 human miRNAs havebeen discovered, it is unclear whether the other 90% of the miRNA species do not present in urine because of their tissue-specific functions or
because they are simply undetectable by the ultracentrifugation method
due to their low abundance. The second assumption presents a motivation for researchers to realize urine-based early disease diagnoses andtimely medical checkups using the urinary miRNAs; the small population of identified urinary miRNAs cannot reflect the variety of diseases
possible. An alternative methodology is needed.
Here, we propose a nanowire-based methodology for collecting urine
EV–encapsulated miRNAs that unveils massive numbers of urinary
miRNAs of different sequences. This methodology moves researchers
toward the goal of miRNA-based noninvasive and simple early diseasediagnoses and timely medical checkups from urine. Although nanowires
have shown great potentials for analyzing properties of cells or intracellular components (32–39), none of the previous studies have dealt
with applications to collect EVs. We consider utilization of a surface
charge, a relatively large surface area, and mechanical stability of the
nanowires within a microchannel. This methodology essentially allows
us to perform EV-encapsulated miRNA analysis with a small sample
volume and short treatment time; that is, collecting the EVs requires
only 1 ml of urine and 20 min. The mechanical stability of nanowires
anchored into poly(dimethylsiloxane) (PDMS) during lysis buffer flow
is effective for efficient in situ extraction of the urine EV–encapsulated
miRNAs within 20 min; more species of miRNAs of different sequences(around 1000 types) can be extracted from collected EVs than by conventional methods.
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Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based,
simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the developmentof these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a
microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various
miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being
extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchoredinto substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key
mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify
urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies(bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept
will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical
checkups for cancer.

Copyright © 2017
The Authors, somerights reserved;
exclusive licensee
American Association
for the Advancementof Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
NonCommercial
License 4.0 (CC BY-NC).

SCIENCE ADVANCES | RESEARCH ARTICLERESULTSTo realize our methodology for collecting urine EV–encapsulated
miRNAs, we developed nanowires that were anchored into microfluidic
substrates; these nanowires can play an important role as a solid phasefor electrostatic collection of EVs followed by in situ extraction of EVencapsulated miRNAs (Fig. 1A and movie S1).
Nanowire-embedded PDMS substrate
We fabricated the nanowire-embedded PDMS substrate in four steps(40) (fig. S1): First, we grew nanowires from a thermally oxidized
chromium layer on Si substrates; second, we poured uncured PDMS
onto the grown nanowires; third, we buried nanowires into the PDMS
after curing and peeling off the PDMS with the nanowires (Fig. 1B); andfourth, we grew nanowires from the buried nanowires, which we named
as the nanowire-embedded PDMS substrate (Fig. 1C). Vertical cross-sectional field-emission scanning electron microscopy (FESEM)
images of the buried nanowires demonstrated that nanowires were
uniformly and deeply buried into PDMS with their heads slightly
emerged (Fig. 1B), and the heads provided growth points for the second
nanowire growth (Fig. 1C and fig. S2). Comparing the energy dispersive
x-ray spectroscopy (EDS) elemental mapping of a cross-sectional FESEM
image for nanowire-free PDMS with that for nanowire-buried PDMS
(fig. S3), we confirmed that ZnO nanowires were buried into PDMS.
In addition, a vertical cross-sectional and an overview FESEM image
and EDS elemental mapping of a cross-sectional FESEM image showedthat the second nanowire growth occurred at the buried nanowires;
hence, we had successfully fabricated the nanowire-embedded PDMS
substrate. To enhance contact events between the nanowires and theEVs and to avoid any pressure drop events, we used a microfluidic
herringbone-structured (41) PDMS substrate, which ensures good
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Fig. 1. Nanowire-induced electrostatic collection of urine EVs followed by in situ extraction of EV-encapsulated miRNAs. (A) Schematic illustrations for urine EVcollection and in situ extraction of urine EV–encapsulated miRNAs using a nanowire-anchored microfluidic device. (B) A schematic illustration (gray rods, nanowires;transparent cyan areas, PDMS) and an inset illustration on the lower left showing a cross-sectional image (yellow and blue represent nanowires and PDMS, respectively)
for buried nanowires after poring, curing, and peeling off PDMS, and a vertical cross-sectional FESEM image of buried nanowires; nanowires and PDMS are highlighted
as yellow and blue, respectively, and the white dotted line indicates a PDMS edge. Scale bar, 1 mm. (C) A schematic illustration and an inset illustration on the lower leftshowing a cross-sectional image for growing nanowires from the buried nanowires (nanowire-embedded PDMS), and a vertical cross-sectional image of the nanowireembedded PDMS. Scale bar, 1 mm. (D) A schematic illustration and an inset illustration on the lower left showing a cross-sectional image for bonding the nanowireembedded PDMS substrate to the microfluidic herringbone-structured PDMS substrate, an image of the nanowire-anchored microfluidic device (bonding the nanowire-embedded
PDMS and the microfluidic herringbone-structured PDMS substrates) with PEEK tubes for an inlet and an outlet (scale bar, 1 cm), and a laser micrograph of the microfluidic
herringbone structure on PDMS (scale bar, 1 mm). (E) A schematic illustration of the nanowire-embedded PDMS (gray rods, nanowires; transparent cyan areas, PDMS), and an
overview of FESEM image for the nanowire-embedded PDMS (scale bar, 1 mm) after being exposed to lysis buffer. (F) A schematic illustration of nanowires on the Si substrate(gray rods, nanowires; dark cyan areas, Si substrate; faded cyan areas, Cr layer), and an overview of FESEM image for the nanowires on the Si substrate after being exposed tolysis buffer. Scale bar, 1 mm.
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convection and diffusion of solutions, with a larger channel height(50 mm) than the nanowire length (2 mm) as a cover for the nanowireembedded PDMS substrate (Fig. 1D). A nanowire-anchored microfluidic device for in situ extraction of urine EV–encapsulated miRNAs
was finalized by bonding the nanowire-embedded PDMS substrate to
the microfluidic herringbone-structured PDMS substrate and connecting polyether ether ketone (PEEK) tubes for introducing and collecting
urine samples (Fig. 1D). The nanowires anchored into PDMS showedmechanical stability when exposed to lysis buffer, thus preventing
nanowires peeling off from the substrates as occurred for the nonanchored nanowires (Fig. 1, E and F, and fig. S4).

Fig. 2. In situ extraction of miRNAs using the nanowire-anchored microfluidic device. (A) Scatterplot of normalized intensities of miRNAs extracted from thecollected EVs on nanowires in the device versus the ultracentrifuged EVs. Each point corresponds to a different miRNA type (that is, species). The boundary between
pink and cyan represents the same level of miRNA expression for the two approaches. (B) Histogram of miRNA species for nanowires (pink) and ultracentrifugation
extraction (cyan). Error bars show the SD for a series of measurements (n = 3). (C) Scatterplot of normalized intensities of miRNAs extracted from the collected EVs using
the nanowire-anchored microfluidic device versus miRNA expression extracted from the collected EVs when using a commercially available kit. Each point corresponds
to a different miRNA type (species). The boundary between pink and gray represents the same level of miRNA expression for the two approaches. (D) Histogram ofmiRNA species for nanowires (pink) and the commercially available kit (gray). Error bars show the SD for a series of measurements (n = 3). a.u., arbitrary units.
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Microarray analysis of miRNA expression
Microarray analysis of miRNA expression (2565 types) showed that insitu extraction using the device provided a greater variety of species ofmiRNAs (around 1000 types) compared to the conventional ultracentrifugation method or a commercially available kit (ExoQuick-TC,
polymeric precipitation method) (Fig. 2 and fig. S5A). In situ extraction
within 40 min (collection, 20 min; extraction, 20 min) of urine EV–
encapsulated miRNAs was demonstrated by introducing 1 ml of urine
sample followed by 1 ml of lysis buffer into the device. In comparison,
the miRNA extraction from the ultracentrifuged EVs of 20 ml of urine
was demonstrated by suspending the EVs in 1 ml of lysis buffer, and thisrequired more than 5 hours for collection and extraction. Because it has
been reported that the kit showed superiority to other EV isolation
methods in terms of small RNA yield (fig. S5, B and C) (22), we also
demonstrated the miRNA extraction from the collected EVs of 1-ml
urine using the kit by suspending the EVs in 1 ml of lysis buffer, andthis required more than 14 hours for collection and extraction. The
scatterplot and histogram revealed that the miRNA expression levelfrom the device was much higher than that from ultracentrifugation(Fig. 2, A and B), despite the fact that the consumed volume of urine
for the device was 20 times less. Normally, the miRNA expression levelfrom ultracentrifugation should be higher than that from the device dueto the 20 times larger volume; however, the results were completely
opposite: Compared to ultracentrifugation, the device showed a fivefold higher miRNA expression level (Fig. 2A) and a greater variety of
extracted species of miRNAs (749, 822, and 1111 types versus 171, 261,
and 352 types) (Fig. 2B). Moreover, the scatterplot and histogram
revealed that the miRNA expression level from the device was muchhigher than that from the kit (Fig. 2, C and D), despite the fact that theconsumed volume of urine for the device was the same as that of the kit.
The device showed a fourfold higher miRNA expression level (Fig. 2C)
and a larger variety of extracted species of miRNAs (749, 822, and 1111
types versus 337, 355, and 491 types) (Fig. 2D). We concluded that our
methodology was superior to other methodologies regarding miRNA

SCIENCE ADVANCES | RESEARCH ARTICLE
extraction efficiency (Fig. 2), treatment time (fig. S5, A and B), and small
RNA extraction efficiency (fig. S5C).

Fig. 3. EV collection onto the nanowires. (A) A schematic illustration for the experimental process and calculation of collection efficiency. (B) Size distribution of theurinary free-floating objects in the untreated urine. Error bars show the SD for a series of measurements (n = 3). (C) Size distribution of the urinary free-floating objects
in the flow-through fraction of the urine being processed by the device (pink) and in the ultracentrifuged urine (cyan). Error bars show the SD for a series of measurements (n = 3). (D) Fluorescently (PKH26) labeled EVs collected on nanowires. Red denotes PKH26-labeled EVs on nanowires. Scale bar, 500 mm. (E) An FESEM image of
nanowires after introduction of PKH26-labeled EVs. White arrows indicate collected EVs. Scale bar, 200 nm. (F) Detection of EVs in urine on nanowires (pink) and a 96well plate (cyan) using an antibody of CD63 or CD81. The measured concentration of the urinary free-floating objects was 1.4 × 108 ml−1. N.D. indicates fluorescenceintensity was not detected. The black dotted line shows the signal level at 3 SD above the background. Error bars showing the SD for a series of measurements ofnanowires and a 96-well plate (n = 24 and 3, respectively).
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EV collection capability
To elucidate that in situ extraction using the device could provide amuch larger variety of species of miRNAs, we confirmed the capabilityof the device to collect EVs; the device showed highly effective EV collection (Fig. 3). Considering that the total volumes of urinary freefloating objects in the untreated urine, in the flow-through fraction of
the urine being processed by the device (input volume, 1 ml), and in theultracentrifuged urine (input volume, 20 ml) were 3.4 ml, 8.8 nl, and11.1 nl, respectively, we estimated the collection efficiency of the objects
on the device to be more than 99% and that volume was larger thanthe volume achieved by ultracentrifugation (collected EV volume
was calculated by subtracting object volume in processed urine fromits volume in untreated urine) (Fig. 3, A to C). After observing fluorescently labeled EVs, we obtained their FESEM images by peeling the
nanowire-embedded PDMS off the microfluidic herringbone-structured
PDMS substrate; these observed results allowed us to confirm that thefree-floating objects collected by the nanowires included EVs (Fig. 3, C
and D). To further confirm whether the free-floating objects collectedby the nanowires included EVs, we detected CD63 and CD81 membrane proteins expressed on the EVs; these are well-known membraneproteins expressed on exosomes (2, 42–44). Fluorescence intensityfrom collected urine EVs (concentration, 1.4 × 108 ml−1) onto nanowires or plate wells of a 96-well plate showed that only nanowires
could detect the membrane proteins (Fig. 3E). Significantly, this indicated the efficient collection of EVs onto the nanowires in the device.
We prepared ZnO/Al2O3 core-shell nanowires by covering the ZnO
nanowires with about a 10-nm-thick Al2O3 layer (fig. S6), which resulted in a nearly neutrally charged surface (that is, slightly positive or
slightly negative) at pH 6 to 8 due to the isoelectric point value of
Al2O3 (ca. 7.5) (45, 46). We used these ZnO/Al2O3 core-shell nanowires to roughly estimate that the nanowire surface charge was thedominant effect for EV collection (fig. S7). Because urine EVs havea negatively charged surface at pH 6 to 8 (fig. S8), it makes sense that
the ZnO nanowires with a relatively large surface area and a positively
charged surface at pH 6 to 8 due to the isoelectric point value of ZnO(ca. 9.5) (47, 48) could achieve highly efficient EV collection. These
results highlighted the idea that ZnO nanowires could collect urinary
free-floating objects with diameters up to 200 nm, including EVs, withmore than 99% collection efficiency.

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Benefit of nanowire-based collection
Regarding the reason why in situ extraction using the device wassuperior to ultracentrifugation extraction or kit-based extraction,
we considered two possible scenarios: In the first scenario, the device
could collect exosomes and microvesicles, and in the second, only thedevice could collect EV-free miRNAs. The measured concentrationand volume obtained from adding the objects collected by ultracentrifugation, that is, the exosomes (9.0 nl; fig. S9A), to the uncollected objects (11.1 nl; Fig. 3C) seemed inconsistent with theircalculated concentration and volume (3.4 ml; Fig. 3B). This implied thatultracentrifugation could collect mainly exosomes (4, 5, 29), although
there is still discussion that current purification methods do not allow
full discrimination between exosomes and microvesicles (1). Ultracentrifugation might have a tendency to fuse and rupture most microvesicles by pressing them to the interior walls of the ultracentrifugation
tubes, and this process would force microvesicles to release miRNAs
into the surrounding solution. Because the EV collection mechanism
by ultracentrifugation is based on a balance between applied forcesand density of the collected objects, the experimental conditions forultracentrifugation do not allow for the collection of released miRNAs.

Table 1. Comparison among three methodologies.
ExoQuick

Ultracentrifugation

Exosomes
Microvesicles
EV-free miRNAs

Exosomes
Microvesicles

Exosomes

Electrostatic interaction
between nanowire surface
charge and collected objects

Polymer-based capture of objects ranging
from 60 to 180 nm in diameter, according to
the kit manufacturer’s instruction manual

Balance between applied
forces and density of thecollected objects

Sample volume

1 ml

1 ml

1 ml for small RNA quantification
20 ml for urinary miRNA profiling

Processing time

40 min

870 min

300 minCollection efficiency (small RNA yield)

0.194 ± 0.028 ng/ml

0.120 ± 0.015 ng/ml

0.159 ± 0.077 ng/ml

Extraction species of urinary
miRNAs being identified

749, 822, 1111 (n = 3)

337, 355, 491 (n = 3)

171, 261, 352 (n = 3)200 to 300*

Collected objects

Collection mechanism*From Cheng et al. (31).

Fig. 4. In situ extraction of cancer-related miRNAs using the nanowire-anchored microfluidic device; heat maps of the miRNA expression array for noncancer
lung, pancreatic, liver, bladder, and prostate cancer donor urine samples (n = 3). For intuitive understanding of the expression level of each miRNA and easycomparison between each group, we used color gradations showing signal intensity variation. Black, logarithmic signal intensity of 5; blue, logarithmic signal intensity
less than or equal to 2; and yellow, logarithmic signal intensity greater than or equal to 8. Each column in the heat maps represents the logarithmic signal intensities ofeach miRNA corresponding to the color gradation.
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Fabricated device

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On the other hand, the collection mechanism by nanowires is based onelectrostatic interactions between positively charged nanowires andnegatively charged objects; the nanowires could collect exosomes andmicrovesicles. Furthermore, the capability of nanowires to collectEV-free miRNAs was conceivable because nucleic acids, including
miRNAs, are known to have a negatively charged surface property
at pH 6 to 8; for ultracentrifugation and the kit, this was impossible.
Although the collection efficiency of miRNAs onto nanowires was notsatisfactory, the recovery rate by introducing lysis buffer was almost
100% (fig. S9B). The positively charged surface nanowires would pro-vide a considerable benefit for collecting negatively charged objects inurine, including exosomes, microvesicles, and EV-free miRNAs.
Considering the features of collection efficiency, selectivity of obtained sample, and ability to collect urinary miRNAs (Table 1), we concluded that the device offers high collection efficiency, low sample
selectivity (exosomes, microvesicles, and EV-free miRNAs), and highcollection ability; ultracentrifugation offers low collection efficiency,
high sample selectivity (only exosomes), and low collection ability; andthe kit provides the three features at intermediate levels (probably
exosomes and microvesicles). Compared to the charge-based isolation

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Fig. 5. Down-regulated and overexpressed miRNAs extracted from Fig. 4 between noncancer donors and each cancer donor. Extracted miRNAs were thesecond smallest logarithmic signal intensities in one group larger than the three pulsing second largest logarithmic signal intensities in the other group. The
symbols − and + show noncancer and cancer donors, respectively. Pink lines highlight minimums in one group that were larger than the three pulsing maximums in
the other group, giving highlighted miRNAs an edge over other miRNAs. Green and orange lines highlight cancer-specific down-regulated miRNAs and overexpressed
miRNAs, respectively.
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Table 2. Potential cancer-related urinary miRNAs as indicated in Fig. 5. Blank spaces in the “Biological functions” column indicate that biological functionsof the miRNAs have not been reported.
Cancer types (down-regulation/overexpression)

miRNA names

Lung (down-regulation)

miR-3127-3p*

Biological functions

miR-3130-5p*
miR-3131*
miR-3141*
miR-3150b-5p*
miR-3151-3p*
miR-3151-5p*
miR-3154*

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miR-3160-3p*
miR-3160-5p*
miR-378a-5p

Suppressing cell proliferation and inducing
apoptosis (52)

miR-520c-3p

Tumor suppressor (53–57)

miR-526b-3p

Tumor suppressor (58)miR-3150a-3p
miR-3162-5p
miR-4254
Lung (overexpression)miR-3117-5p*
miR-3118*
miR-3121-3p*
miR-3121-5p*
miR-3126-5p*
miR-3128*
miR-3133*
miR-3134*
miR-3136-3p*
miR-3136-5p*
miR-3139*
miR-3142*
miR-3143*
miR-3145-3p*
miR-3163*

Inhibiting non–small cell lung cancer cell
growth (59)miR-3166*
miR-3167*
miR-16-1-3p

Repressing gastric cancer cell invasion andmetastasis (60)continued on next page

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Cancer types (down-regulation/overexpression)

miRNA names

Biological functions

miR-424-3p

Potential metastasis-related miRNAs (61)miR-519c-5p
miR-525-5p
miR-551b-5p
miR-558

Promoting tumorigenesis (62)miR-921
miR-942-3p
miR-3126-3p
miR-3127-5p

Reducing non–small cell lung cancer cellproliferation (63)miR-3129-5p

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miR-3144-5p
miR-3150a-5p
miR-3152-5p
miR-3155a
miR-3157-3p
miR-3159
miR-3165
miR-3678-3p
miR-4321
miR-4521

Significantly up-regulated miRNA in cancer
stem cells (64)miR-4800-3p
miR-4999-5p
miR-5096
miR-5187-5p
miR-6874-5p
Pancreatic (down-regulation)

miR-372-3p*

Tumor suppressor (65)

miR-378b*
miR-520b*

Inhibiting cellular migration and invasion (66)

miR-1266-3p*
miR-3605-5p*
miR-3612*
miR-4645-3p*
miR-4694-3p*
miR-4752*
miR-6816-3p*
miR-8087*
let-7f-2-3p
continued on next page

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Cancer types (down-regulation/overexpression)

miRNA names

Biological functions

miR-15a-3p

Inducing apoptosis in human cancer cell lines (67 )miR-20a-3p
miR-33b-3p
miR-34c-5p

Tumor suppressor (68)miR-93-5p
miR-130a-5p
miR-135a-5p

Inhibiting tumor metastasis (69)

miR-135b-5p
miR-185-5p

Tumor suppressor (70)

miR-203a-3p
miR-302d-5p
Tumor suppressor (71)

miR-378c
miR-422a

Down-regulated in colon cancer (72)

miR-449c-5p
miR-483-5p
miR-506-3p

Inducing differentiation (73)

miR-511-5p
miR-520c-3p

Tumor suppressor (53–57)

miR-654-3p
miR-668-5p
miR-670-5p
miR-671-3p
miR-744-3p
miR-1178-3p
miR-1254
miR-1284

Down-regulated in lymph node metastatic sites (74)miR-1323

Modulating radioresistance (75)

miR-2116-5p
miR-2355-3p
miR-3132
miR-3138
miR-3164
miR-3186-3p
miR-3189-3p
miR-3198
miR-3200-5p
miR-3657
continued on next page

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miR-337-3p

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Cancer types (down-regulation/overexpression)

miRNA names

Biological functions

miR-3667-5p
miR-3680-5p
miR-3692-5p
miR-3713
miR-3921
miR-3936
miR-4273

Increasing colorectal cancer risk (76)miR-4299
miR-4306
miR-4316
miR-4319

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miR-4421
miR-4429
miR-4435
miR-4441
miR-4473
miR-4506
miR-4633-5p
miR-4658
miR-4733-5p
miR-4733-3p
miR-5004-3p
miR-5194
miR-5197-5p
miR-5571-5p
miR-6083
miR-6717-5p
miR-6720-5p
miR-6767-3p
miR-6781-3p
miR-6811-3p
miR-6821-3p
miR-6828-5p
miR-6832-5p
miR-6837-3p
miR-6841-5p
miR-6853-5p
miR-6871-3p
continued on next page

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Cancer types (down-regulation/overexpression)

miRNA names

Biological functions

miR-6875-5p
miR-6878-5p
miR-7112-3p
miR-7703
miR-7848-3p
miR-7856-5p
Pancreatic (overexpression)

let-7i-3p
Promoting cancer proliferation, invasion, andmetastasis (77)miR-202-5p

Increasing TGFBR1 and TGFBR2 protein expressions (78)miR-409-5p

Promoting tumorigenesis (79)

miR-4661-5p
miR-4800-3p
miR-5587-5p
Liver (down-regulation)

let-7i-2-3p
miR-520c-3p

Liver (overexpression)

Tumor suppressor (53–57)

miR-4521*
let-7c-3p
let-7i-5p
miR-16-1-3p

Repressing gastric cancer cell invasion and metastasis (60)miR-26a-1-3p
miR-28-5p

Suppressing insulin-like growth factor 1 expression (80)miR-105-5p
miR-195-3p
miR-200b-5p
miR-219a-2-3p
miR-297

Promoting cell proliferation and invasion (81)miR-300

Inhibiting pituitary tumor transforming gene
expression (82)miR-330-3p

Promoting invasion and metastasis (83)miR-374b-5p
miR-431-5p
miR-454-5p

Promoting tumorigenesis (84)miR-513c-5p
miR-548ax
miR-593-5p
miR-623
miR-664a-5p
continued on next page

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miR-183-5p

SCIENCE ADVANCES | RESEARCH ARTICLE

Cancer types (down-regulation/overexpression)

miRNA names

Biological functions

miR-942-3p
miR-1205
miR-1276
miR-1288-3p
miR-1297

Promoting cell proliferation (85)miR-3678-3p
miR-4283
miR-4295

Promoting cell proliferation and invasion (86)

miR-4439
miR-4524b-5p

miR-4768-5p
miR-4800-3p
miR-5187-5p
miR-5696
miR-7161-5p
Bladder (down-regulation)

let-7f-2-3p
miR-520c-3p

Tumor suppressor (53–57)

miR-4783-5p
Bladder (overexpression)miR-16-1-3p

Repressing gastric cancer cell invasion and metastasis (60)miR-23b-3p

Regulating chemoresistance of gastric cancer cell (87)

miR-28-5p

Suppressing insulin-like growth factor 1 expression (80)

miR-92a-2-5p
miR-142-3p

Promoting malignant phenotypes (88)miR-195-3p

Promoting tumorigenesis and inhibiting apoptosis (89)miR-196b-5p
miR-299-3p

Reducing Oct4 gene expression (90)miR-492
miR-513b-5p
miR-601
miR-619-5p
miR-1285-3p
miR-3155a
miR-3162-5p
miR-3678-3p
miR-4283
miR-4295

Promoting bladder cancer cell proliferation (91)continued on next page

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miR-4703-3p

SCIENCE ADVANCES | RESEARCH ARTICLE

Cancer types (down-regulation/overexpression)

miRNA names

Biological functions

miR-4311
miR-4531
miR-5096
miR-5187-5p
Prostate (down-regulation)

miR-15a-3p

Inducing apoptosis in human cancer cell lines (67)miR-135b-5p
miR-520c-3p

Tumor suppressor (53–57)

miR-4783-5p

Prostate (overexpression)miR-4531*
miR-28-5p

Suppressing insulin-like growth factor 1 expression (80)miR-103a-2-5p
miR-105-5p

Inhibiting the expression of tumor-suppressive genes (92)

miR-124-3p

Regulating cell proliferation, invasion, and apoptosis (93)miR-151a-5p

Tumor cell migration and invasion (94)miR-151b
miR-200a-5p
miR-300

Promoting cell proliferation and invasion (95)

miR-424-3p
miR-519c-5p
miR-551b-5p
miR-617
miR-873-3p
miR-921
miR-1288-3p
miR-3124-5p
miR-3155a
miR-3917
miR-4283
miR-4727-3p
miR-5096
miR-5187-5p
miR-6074
miR-6874-5p
miR-6892-5p*Marks showing miRNAs highlighted by pink lines in Fig. 5.

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miR-7849-3p

SCIENCE ADVANCES | RESEARCH ARTICLE
approach for EVs (49), by which protamine precipitation of EVs is performed using protamine/polyethylene glycol in an overnight incubation
at 4°C (similar to ExoQuick), we can say that our device has an advantage of providing 40-min in situ extraction of urine EV–encapsulated
miRNAs (collection, 20 min; extraction, 20 min) at room temperature.
Moreover, compared to the charge-based isolation approach for freenucleic acid (50), which uses chitosan polymer with amine groups thatprovide a positively charged surface only below pH 6.3, we can say thatour device has an advantage of an assured positively charged surface at
pH 6 to 8 of urine. Although the exact collection mechanism of EVs bynanowires in the present work must be rather complex, our findings
indicated that the mechanically stable anchoring of nanowires anchoredinto PDMS during lysis buffer flow and the electrostatic collection of EVs(and, moreover, EV-free miRNAs) onto ZnO nanowires were two key
mechanisms, and they will lead to realization of early disease diagnoses
and timely medical checkups based on urine miRNA analysis.

Yasui et al., Sci. Adv. 2017; 3 : e1701133

15 December 2017

DISCUSSION

To summarize, we have demonstrated that our nanowire-anchored microfluidic device, which had bonding of the nanowire-embedded PDMS
and the microfluidic herringbone-structured PDMS substrates, could
achieve higher efficiency for in situ extraction of urine EV–encapsulated
miRNAs compared to the most popular conventional method of ultracentrifugation. Surprisingly, the device could extract a much larger
variety of species of miRNAs than ultracentrifugation despite the factthat the device uses a smaller sample volume and shorter treatment timethan the latter method. The positively charged surface nanowires played
an important role in the highly efficient EV collection, and themechanical stability of nanowires anchored into PDMS during lysis
buffer flow had an important impact on in situ extraction of miRNAs.
We could find cancer-related miRNAs from urine samples of just 1 ml
for not only urologic malignancies but also nonurologic ones. Although
we need to perform further trials for biomarker recognition, thepresent results have led us to believe that our developed approach
will be a powerful tool that offers a new strategy for researchers to
discover cancer-related miRNAs in urine samples for future medical
applications and to perform urinary miRNA-based diagnosis fortimely medical checkups.

MATERIALS AND METHODS

Fabrication procedure for nanowires anchored into PDMS
After cleaning the Si (100) substrates (Advantech Co. Ltd.) (fig. S1A),
positive photoresist (OFPR8600, Tokyo Ohka Kogyo Co. Ltd.) was
coated on the Si substrates, and then the channel pattern was formedby photolithography (fig. S1B). A 140-nm-thick Cr layer was depositedon the substrate by sputtering (fig. S1B). After removal of the photoresist (fig. S1D), the Cr layer was thermally oxidized at 400°C for
2 hours; the thermally oxidized Cr layer was a seed layer for ZnO
nanowire growth. The ZnO nanowires were grown by immersing thesubstrate in a solution mixture of 15 mM hexamethylenetetramine(HMTA; Wako Pure Chemical Industries Ltd.) and 15 mM zinc nitratehexahydrate (Thermo Fisher Scientific Inc.) at 95°C for 3 hours (fig. S1E).
The nanowires grown on the substrate were cleaned using Millipore
water and allowed to air-dry overnight in a vacuum desiccator. Then,PDMS (Silpot 184, Dow Corning Corp.) was poured onto the nanowiregrown substrate, followed by curing (fig. S1F). After peeling off the
PDMS from the substrate, the nanowires were transferred to the PDMS
from the substrate (fig. S1G). The transferred nanowires were uniformly
and deeply buried into PDMS with their heads slightly emerged (fig.
S1H), and the heads provided growth points for the second nanowire
growth. The second nanowire growth was carried out by immersing the
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Finding undiscovered miRNAs from urine samples of various
cancer donors
Finally, we demonstrated the potential of in situ extraction of urine
EV–encapsulated miRNAs using the device to find previously undiscovered miRNAs from urine samples of various cancer donors(Fig. 4). Because the device allowed us to analyze many species of miRNAs
from a 1-ml urine sample, we compared a heat map of miRNA expression for cancer and noncancer donor urine samples. To the best of ourknowledge, we have reported the first case of unveiling massive
numbers of potential cancer-related miRNAs of different sequencesin 1-ml urine samples. miRNAs from urine samples had different expression levels; comparison between cancer and noncancer donor urine
samples showed some down-regulated and overexpressed miRNAs(Fig. 5 and Table 2). The heat maps suggested that a combination ofsome down-regulated and overexpressed miRNAs could be a candidate
for a new cancer indicator, which is contrary to the stereotype thinkingof depending on a single miRNA biomarker.
Although we need to make more trials for clear recognition of statistically significant down-regulated and overexpressed miRNAs (51)based on comparisons with published results (52–95), we can say thatthe device mainly unveils two types of miRNA groups: miRNAs with
unreported biological functions and miRNAs with previously reported
biological functions. In addition, the former can be divided into two
subgroups: potential cancer-related miRNAs and artifacts presumably
derived from EV-free miRNAs. The latter also has two subgroups: miRNAs
with functions that positively correlate with disease outcomes, such as
miR-520c-3p (tumor suppressor, down-regulated miRNAs in urine
from all cancer patients) (53–57), and miRNAs with a counterintuitive
function-disease relationship, such as miR-16-1-3p (repressing gastriccancer cell invasion and metastasis, overexpressed miRNAs in urine
from liver and bladder cancer patients) (60). For the counterintuitivecase, three possibilities were considered: The first possibility was thatpatients had potentially corresponding risks; the second possibility
was that current studies about a correlation between miRNA functions
and disease risks could not cover a relationship between the miRNAs
concerned and cancer types; and the third possibility was that the miRNAs were also referable to artifacts. Complete medical checkups before
urine sampling, further studies about the correlation, and more trials
will offer answers for these possibilities. We also compared the overexpressed miRNAs with previously reported urinary miRNAs (shown ascell-free and exosome-derived top 25 up-regulated miRNAs) (96),which were extracted using a commercially available kit from 4 ml ofurine from bladder cancer patients, and concluded that 20 miRNAs,
such as miR-4454, were found in our results (data S1); however, thesemiRNAs were not identified as candidates for a cancer indicator. This isbecause we could also find the miRNAs in the results for noncancer
subjects, and we could not find any significant difference in logarithmic
fluorescence intensity between samples from noncancer subjects andbladder cancer patients for the miRNAs. More trials would allow us
to decide whether these miRNAs can be identified as candidates andwhether the remaining five miRNAs can be found (to show a statistically significant difference with 95% reliability and 5% sampling error for a population size of more than 1 million, we need to conduct
384 trails).

SCIENCE ADVANCES | RESEARCH ARTICLEPDMS in the mixed solution of 15 mM HMTA and 15 mM zinc nitratehexahydrate at 95°C for 3 hours (fig. S1I). After the nanowire-embedded
PDMS substrate was cleaned using Millipore water and allowed toair-dry overnight in the vacuum desiccator, we measured nanowire
diameters and spacing between nanowires using FESEM (SUPRA
40VP, Carl Zeiss).

EDS elemental mappings of cross-sectional FESEM images
Elemental mappings of PDMS without nanowires, PDMS with buried
nanowires, and PDMS-anchored ZnO nanowires were obtained by
FESEM (JSM-7610F, Jeol) equipped with the EDS function. We used
accelerating voltage conditions of 5 and 30 kV for the top-view images
and the cross-sectional images, respectively. The images were 512 × 384
pixels and the delay time for each pixel was 0.1 ms. The images were
integrated for 100 cycles. The peaks of Si Ka (1.739 keV) and Zn La(1.012 keV) were chosen to construct the elemental mapping images.
Elemental mapping of ZnO/Al2O3 core-shell nanowires was also performed by FESEM equipped with the EDS function at an accelerating
voltage condition of 30 kV. For preparation of a scanning transmission
electron microscope (STEM) specimen, we first cut off the nanowires
from the substrate using an ordinary cutting blade, and then they werecollected and transferred onto the TEM grid (Cu mesh with carbon
microgrid; Jeol) by the contact printing technique. The STEM images
were 512 × 384 pixels and the delay time for each pixel was 0.1 ms. The
images were integrated for 100 cycles. The peaks of Zn Ka (8.630 keV),O Ka (0.525 keV), and Al Ka (1.486 keV) were chosen to construct theelemental mapping images.
In situ extraction of urine EV–encapsulated miRNAs using
the nanowire-anchored microfluidic device
Commercially available urine (single donor human urine; Innovative
Research Inc.) was centrifuged (15 min, 4°C, 3000g) to remove apoptotic
bodies (5) before use. Then, a 1-ml urine sample aliquot was introduced into the nanowire-anchored microfluidic device at a flow rateof 50 ml/min using a syringe pump (KDS-200, KD Scientific Inc.). The
miRNA extraction from the collected EVs on nanowires was performed using Cell Lysis Buffer M [20 mM tris-HCl (pH 7.4), 200 mM
sodium chloride, 2.5 mM magnesium chloride, and 0.05 w/v% NP-40
substitute; Wako Pure Chemical Industries Ltd.] introduced at a flow rateof 50 ml/min by the syringe pump. We used the same lysis buffer at a flowrate of 50 ml/min for the experiment to study peeling-off of the nanowires(Fig. 1, I to K, and fig. S4).
Yasui et al., Sci. Adv. 2017; 3 : e1701133

15 December 2017

EV collection followed by miRNA extraction in urine using acommercially available kit
Commercially available urine (single donor human urine) was centrifuged (15 min, 4°C, 3000g) to remove apoptotic bodies (5) before
use. The EVs were collected from a 1-ml urine sample according to
the kit (ExoQuick-TC, System Biosciences Inc.) manufacturer’s
instruction manual. Finally, we applied the lysis buffer to extract themiRNAs.
Microarray analysis of miRNA expression
We used Toray’s 3D-Gene (Toray Industries) human miRNA chips for
miRNA expression profiling. The miRNA solution extracted by lysis
buffer was purified using the SeraMir Exosome RNA Purification Column Kit (System Biosciences Inc.) according to the kit manufacturer’s
instruction manual. Fifteen microliters of purified miRNA was analyzed
for miRNA profiling using a microarray and the 3D-Gene Human miRNA
Oligo chip ver.21 (Toray Industries). Microarray analysis of miRNA expression containing 2565 human miRNA probes showed that each signal intensity corresponded to one miRNA type. The expression level ofeach miRNA was expressed as the background-subtracted signal intensity of all the miRNAs in each microarray. For a comparison of miRNA
expression level between the same urine miRNA samples extracted bynanowires and ultracentrifugation or a commercially available kit, signal intensity was globally normalized. We made a scatterplot of globallynormalized intensities greater than or equal to 10 (nanowires versus ultracentrifugation or kit). Each point shows normalized intensities whenusing nanowires and ultracentrifugation or kit for the same miRNAtype. For a comparison of miRNA expression between the same urine
miRNA samples extracted by nanowires and ultracentrifugation or kit,
signal intensities were log2-transformed. For a comparison of miRNA
expression between cancer and noncancer donor urine samples, globallynormalized intensities for each sample were log2-transformed. The normalized intensities were colored black (intensity = 5), blue (intensity ≤2), and yellow (intensity ≥ 8) for the heat maps.
EV zeta potential
After the ultracentrifugation process, EV zeta potential was measured
using a dynamic light-scattering apparatus (Zetasizer Nano ZS, Malvern
Instruments Ltd.).
Size distribution and concentrations of the urinary
free-floating objects
Size distribution and concentration of the urinary free-floating objects
were measured using a nanoparticle analyzing system (NanoSight, Malvern
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Nanowire-anchored microfluidic device for in situ extractionof urine EV–encapsulated miRNAs
A nanowire-anchored microfluidic device for in situ extraction of urine
EV–encapsulated miRNAs was fabricated by bonding the nanowireembedded PDMS substrate and a herringbone-structured PDMS substrate. The herringbone-structured PDMS substrate had a microchannel(width, 2 mm; length, 2 cm; and height, 50 mm) with a 12-mm-highherringbone structure. The surfaces of the nanowire-embedded PDMS
and the herringbone-structured PDMS substrates were treated using aplasma etching apparatus (Meiwafosis Co. Ltd.) and then bonded. Thisbonded device was heated at 180°C for 3 min to achieve strong bonding(fig. S1J). Next, the herringbone-structured PDMS was connected to
PEEK tubes [0.5 mm (outside diameter) × 0.26 mm (inside diameter);length, 10 cm; Institute of Microchemical Technology Co. Ltd.] for an
inlet and an outlet. The microfluidic herringbone structure contributed
to the increase of collection efficiency (fig. S10).

EV collection followed by miRNA extraction in urine
using ultracentrifugation
Commercially available urine (single donor human urine) was centrifuged (15 min, 4°C, 3000g) to remove apoptotic bodies (5), and then theurine was centrifuged (15 min, 4°C, 12,000g) to remove cellular debris(29) before use. Next, a 20-ml urine sample was ultracentrifuged (2 hours,
4°C, 110,000g) (29). After discarding the supernatant, we added 20 mlof 0.22-mm filtered phosphate-buffered saline (PBS; Thermo Fisher
Scientific Inc.) to the collected EVs, and this was ultracentrifugedagain (70 min, 4°C, 110,000g) (29). After discarding the supernatant,
we added 20 ml of 0.22-mm filtered PBS to the collected EVs, and thesample was ultracentrifuged for a third time (70 min, 4°C, 110,000g)(29). Finally, we discarded the supernatant and applied the lysis buffer
to extract the miRNAs.

SCIENCE ADVANCES | RESEARCH ARTICLE
Instruments Ltd.); the concentrations of the urinary free-floating objects
with diameters of up to 200 nm in the untreated urine, in the flowthrough fraction of the urine being processed by the device, and in
the ultracentrifuged urine were 2.6 × 1012, 5.8 × 109, and 3.5 × 109 ml−1,
respectively. Size distribution and concentration of the urinary freefloating objects were also measured using a nanoparticle detector (qNano,
Meiwafosis Co. Ltd.) with a 100-nm nanopore membrane (NP100,
Meiwafosis Co. Ltd.); the concentrations of the urinary free-floating
objects in the untreated urine, in the flow-through fraction of the urine
being processed by the nanowire-anchored microfluidic device, and in
the ultracentrifuged urine were 1.4 × 1012, 2.4 × 1010, and 2.5 × 1010 ml−1,
respectively.

Membrane protein detection
After introduction of EVs, PBS was introduced into a device withnanowires to remove uncollected EVs. Then, we introduced 1% bovine serum albumin (BSA) solution (Kirkegaard & Perry Laboratories Inc.) into the device and let it stand for 15 min. Afterwashing out the device using PBS, we introduced a mouse monoclonalanti-human Alexa Fluor 488–labeled CD63 antibody (10 mg/ml; Santa
Cruz Biotechnology Inc.) or a mouse monoclonal anti-human CD81
antibody (10 mg/ml; Abcam PLC) into the device and then let the antibody solution stand for 15 min. In addition, for CD81 detection, we
washed out the device using PBS, introduced a goat polyclonal antimouse Alexa Fluor 488–labeled immunoglobulin G (IgG) secondary
antibody (5 mg/ml; Abcam PLC) into the device, and then let thesecondary antibody solution stand for 15 min. Finally, we washedout the device using PBS and followed that with fluorescence intensity
observation under a fluorescence microscope (Olympus Co. Ltd.). To
obtain the background value, we used PBS instead of the EV sample. Regarding detection using a 96-well plate (Nunc Co. Ltd.), we
injected the EV samples into the plate wells and let them stand for6 hours, and then we discarded the samples. After washing out theplate using PBS, we applied 1% BSA solution to the plate wells, let it
stand for 90 min, and then discarded it. Again, we washed out theplate using PBS, applied the mouse monoclonal anti-human Alexa
Fluor 488–labeled CD63 antibody (10 mg/ml) or the mouse monoclonal anti-human CD81 antibody (10 mg/ml) into the plate wells,
and let them stand for 45 min. In addition, for CD81 detection, we
washed out the device using PBS, applied the goat polyclonal antimouse Alexa Fluor 488–labeled IgG secondary antibody (5 mg/ml)
into the plate, and let it stand for 45 min. Finally, we washed out
the device using PBS followed by fluorescence intensity observation
using a plate reader (POLARstar OPTIMA, BMG Labtech Japan
Ltd.). To obtain the background value, we used PBS instead of
the EV sample.
Yasui et al., Sci. Adv. 2017; 3 : e1701133

15 December 2017

Urine sample information and in situ extraction of urine
EV–encapsulated miRNAs using the nanowire-anchoredmicrofluidic device
The following urine samples (BioreclamationIVT) were used: noncancer urine (ages 53, 60, and 50), lung cancer urine (age 68, stage 2b; age54, stage 3a; and age 50, stage 3b), pancreatic cancer urine (age 56, stage
2a; age 61, stage 2a; and age 74, stage 3), liver cancer urine (age 49, stage
3; age 64, stage 3a; and age 18, stage 3c), bladder cancer urine (age 63,
stage 1; age 65, stage 1; and age 67, stage 0a), and prostate cancer urine(age 58, stage 4; age 57, stage 2a; and 54; stage 2b). These urine samples
were centrifuged (15 min, 4°C, 3000g) to remove apoptotic bodies (5)before use. Then, a 1-ml urine sample aliquot was introduced into thenanowire-anchored microfluidic device at a flow rate of 50 ml/min using
the syringe pump. The miRNA extraction from the collected EVs on the
nanowires was performed with the lysis buffer introduced at a flow rateof 50 ml/min using the syringe pump.
Identifying urinary miRNAs that could potentially serve as
biomarkers for cancer
According to the Z-score of 1.96 (95% reliability and 5% significancelevel) and the relationship of coefficient of variation (CV) (without
concrete numerical values) versus log2(intensity) provided by Toray,
the 95% confidence interval could be calculated using (average) ±1.96 × (average × CV/100). When we used X% for CV in the relationship log2(intensity) = 3, the upper limit of the confidence interval was
8 + 0.16X. The CV value in log2(intensity) = 5 and 6 seemed to be 0.7X
and 0.5X% from the relationship, and the lower limits of the confidence
interval were 32 − 0.44X and 64 − 0.63X, respectively. Considering the
5% significance level, we found that the CVs for each case were less than
40 and 71%. We did not consider the CV value of more than 70% to bereasonable, and we set a difference of three logarithmic intensities forthe statistically significant threshold in Fig. 5.
SUPPLEMENTARY MATERIALSSupplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/3/12/e1701133/DC1
fig. S1. Schematic of the fabrication procedure for nanowires anchored into PDMS.
fig. S2. Nanowires anchored into PDMS.
fig. S3. FESEM images and EDS elemental mappings corresponding to FESEM images for PDMS
without nanowires, PDMS with buried nanowires, and nanowire-embedded PDMS; scale bars,
1 mm.
fig. S4. Mechanical stability of anchored nanowires and nonanchored nanowires.
fig. S5. Extraction process of miRNAs in urine using the nanowire-anchored microfluidic device,ultracentrifugation, and commercially available kits.
fig. S6. FESEM images and EDS elemental mappings of an STEM image of a single
nanowire.
fig. S7. Detection of EVs on ZnO nanowires, ZnO/Al2O3 core-shell nanowires, and no nanowires
using an antibody of CD9.
fig. S8. Zeta potential of EVs in urine.
fig. S9. Size distribution of EVs collected by ultracentrifugation and EV-free miRNAs collected
onto nanowires.
fig. S10. Size distribution of the urinary free-floating objects.
movie S1. EV collection followed by miRNA extraction in urine using the nanowire-anchoredmicrofluidic device.
data S1. Logarithmic signal intensities in noncancer miRNAs with those in cancer
miRNAs.

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Fluorescent molecule labeling of EVs
We labeled EVs using fluorescent molecules of PKH26 (excitation/
emission = 551/567 nm; Sigma-Aldrich Co. LLC), which could penetrate
into the lipid bilayers of the EVs. We added 5 mg of PKH26 for 1.5 ×108 ml−1 of EVs in 240 ml of 0.22-mm filtered Millipore water. The
PKH26-labeled EVs were introduced into a device with nanowires at
a flow rate of 10 ml/min using the syringe pump and then Millipore water was introduced into the device at the same flow rate to remove uncollected EVs. Finally, we observed the fluorescence of EVs using afluorescence microscope (AZ100, Nikon Corp.). After that, we peeled
off the device and observed the EV-collected nanowires using FESEM(SUPRA 40VP, Carl Zeiss).

Fabrication of ZnO/Al2O3 core-shell nanowires
After fabrication of ZnO nanowires, we covered the nanowires using an
atomic layer deposition apparatus (Savannah G2, Ultratech Inc.). For
Al2O3 deposition, we used trimethylaluminum and H2O precursors
at 150°C for 100 cycles.

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Acknowledgments
Funding: This research was supported by PRESTO (JPMJPR151B); the Japan Science andTechnology Agency; the “Development of Diagnostic Technology for Detection of
miRNA in Body Fluids” grant from the Japan Agency for Medical Research and Development
and New Energy and Industrial Technology Development Organization; the ImPACT
Program of the Council for Science, Technology and Innovation (Cabinet Office, Governmentof Japan); the Japan Society for the Promotion of Science Grant-in-Aid for Scientific
Research (A) 16H02091; the Nanotechnology Platform Program (Molecule and Material
Synthesis) of the Ministry of Education, Culture, Sports, Science and Technology(MEXT); and the Cooperative Research Program of “Network Joint Research Center for Materials

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SCIENCE ADVANCES | RESEARCH ARTICLE
and Devices.” Author contributions: T. Yasui, T. Yanagida, N.K., H.Y., T.O., T.K.,
and Y.B. planned and designed the experiments. T. Yasui, T. Yanagida, S.I., Y.K., D.T., T.N.,
K.N., T.S., N.K., Y.H., S.R., and M.K. fabricated the experimental setups. T. Yasui, S.I., Y.K., D.T., T.N.,
Y.N., and I.A.T. performed EV collection using nanowires and data analyses. T. Yasui,
T. Yanagida, K.N., and N.K. wrote the paper. Competing interests: The authors declarethat they have no competing interests. Data and materials availability: All dataneeded to evaluate the conclusions in the paper are present in the paper and/or the
Supplementary Materials. Additional data related to this paper may be requested
from the authors.

Submitted 13 April 2017
Accepted 16 November 2017
Published 15 December 201710.1126/sciadv.1701133
Citation: T. Yasui, T. Yanagida, S. Ito, Y. Konakade, D. Takeshita, T. Naganawa, K. Nagashima,
T. Shimada, N. Kaji, Y. Nakamura, I. A. Thiodorus, Y. He, S. Rahong, M. Kanai, H. Yukawa,T. Ochiya, T. Kawai, Y. Baba, Unveiling massive numbers of cancer-related urinary-microRNA
candidates via nanowires. Sci. Adv. 3, e1701133 (2017).

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Yasui et al., Sci. Adv. 2017; 3 : e1701133

15 December 201719 of 19

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires
Takao Yasui, Takeshi Yanagida, Satoru Ito, Yuki Konakade, Daiki Takeshita, Tsuyoshi Naganawa, Kazuki Nagashima,
Taisuke Shimada, Noritada Kaji, Yuta Nakamura, Ivan Adiyasa Thiodorus, Yong He, Sakon Rahong, Masaki Kanai, Hiroshi
Yukawa, Takahiro Ochiya, Tomoji Kawai and Yoshinobu Baba

Sci Adv 3 (12), e1701133.
DOI: 10.1126/sciadv.1701133

http://advances.sciencemag.org/content/3/12/e1701133

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