advances.sciencemag.org/cgi/content/full/3/12/e1701133/DC1

Supplementary Materials for
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, Yoshinobu Baba
Published 15 December 2017, Sci. Adv. 3, e1701133 (2017)
DOI: 10.1126/sciadv.1701133

The PDF file includes:
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fig. S1. Schematic of the fabrication procedure for nanowires anchored intoPDMS.
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, andnanowire-embedded PDMS; scale bars, 1 μm.
fig. S4. Mechanical stability of anchored nanowires and nonanchored nanowires.
fig. S5. Extraction process of miRNAs in urine using the nanowire-anchoredmicrofluidic device, ultracentrifugation, and commercially available kits.
fig. S6. FESEM images and EDS elemental mappings of an STEM image of asingle 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-freemiRNAs collected onto nanowires.
fig. S10. Size distribution of the urinary free-floating objects.
Legend for movie S1
Legend for data S1Other Supplementary Material for this manuscript includes the following:(available at advances.sciencemag.org/cgi/content/full/3/12/e1701133/DC1)

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movie S1 (.mov format). EV collection followed by miRNA extraction in urine
using the nanowire-anchored microfluidic device.
data S1 (Microsoft Excel format). Logarithmic signal intensities in noncancer
miRNAs with those in cancer miRNAs.

fig. S1. Schematic of the fabrication procedure for nanowires anchored into PDMS. (a-g) The
nanowires are anchored into PDMS by utilizing lithographic techniques and a PDMS curing process.
Components include Si substrate (dark cyan), OFPR8600 photoresist (pink), Cr layer (faded cyan),
nanowires (gray) and PDMS (transparent cyan). (h) A schematic illustration (gray rods, nanowires;transparent cyan areas, PDMS) for buried nanowires after poring, curing and peeling off PDMS. (i)A schematic illustration for growing nanowires from the buried nanowires (nanowire-embeddedPDMS). (j) A schematic illustration for bonding the nanowire-embedded PDMS substrate to themicrofluidic herringbone-structured PDMS substrate.

fig. S2. Nanowires anchored into PDMS. (a) An overview FESEM image of the nanowires; scalebar, 1 µm. (b) Diameter distribution of the nanowires. Mean diameter of the nanowires was 150 nm.
(c) Spacing distribution between the nanowires. A spacing was defined as the closest spacing
between two nanowires, which was measured from cross-sectional SEM images. Mean spacing
between the nanowires was 200 nm.

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 μm. (a-c) Vertical cross-sectional FESEM images of PDMS without nanowires, PDMS
with buried nanowires, and nanowire-embedded PDMS, respectively. (d-f) EDS elemental mappings
corresponding to the cross-sectional FESEM images in figs. S2a-c; Si and Zn are highlighted as blueand green. (g) An overview FESEM image of PDMS with buried nanowires. (h) An EDS elemental
mapping corresponding to the overview FESEM image in fig. S2g; Si and Zn are highlighted as blueand green.

fig. S4. Mechanical stability of anchored nanowires and nonanchored nanowires. (a, b)
Schematic illustrations of the nanowires anchored into PDMS and nanowires on the Si substrate
when exposed to lysis buffer. Components include nanowires (gray), PDMS (transparent cyan), Si
substrate (dark cyan), and Cr layer (faded cyan). The nonanchored nanowires were fabricated on thethermally oxidized chromium layer on the Si substrate. (c, d) An overview of FESEM images for thenanowires anchored into PDMS and the nanowires on the Si substrate; scale bars, 1 µm. (e, f) Anoverview of FESEM images for the nanowires anchored into PDMS and the nanowires on the Si
substrate when exposed to lysis buffer; scale bars, 1 µm. After exposure to lysis buffer, thenanowires anchored into PDMS did not peel off, while the nonanchored nanowires did.

fig. S5. Extraction process of miRNAs in urine using the nanowire-anchored microfluidic
device, ultracentrifugation, and commercially available kits. (a) Experimental procedures for
microarray analysis of miRNA expression. (b) Experimental procedures for small RNA
quantification using the Qubit® microRNA assay kit (Thermo Fisher Scientific). After miRNA
extraction using each methodology, we performed RNA purification and small RNA quantification.
(c) Small RNA quantification using each methodology. Since the Qubit® microRNA assay kit could
quantify small RNAs (~20 nucleotides or base pairs), not only miRNAs, we used the small RNA
concentration as the y-axis. These data implied that the washing step did not affect RNA collection
efficiency. Error bars show the standard deviation for a series of measurements (N ≥ 3).

fig. S6. FESEM images and EDS elemental mappings of an STEM image of a single nanowire.
Zn, O, and Al are highlighted as green, red, and orange for single elemental mappings. Zn and Al arehighlighted as green and red for dual elemental mappings. (a) A ZnO single nanowire; scale bars,
500 nm. (b) A ZnO/Al2O3 core-shell single nanowire; scale bars, 500 nm. (c) A ZnO/Al2O3
core-shell single nanowire; scale bars, 100 nm.

fig. S7. Detection of EVs on ZnO nanowires, ZnO/Al2O3 core-shell nanowires, and nonanowires using an antibody of CD9. The black dotted line shows the signal level at 3 standard
deviations above the background. Error bars showing the standard deviation for a series ofmeasurements of ZnO nanowires, ZnO/Al2O3 core-shell nanowires, and no nanowires (N=40, 24,and 24, respectively). Pink and cyan circles show EV concentrations of 109 and 1010 particles/mL,respectively. After introduction of EVs, PBS was introduced into three different devices to remove
uncollected EVs. The first device had ZnO nanowires, the second had ZnO/Al2O3 core-shellnanowires, and the third had no nanowires. Then, we introduced 1% BSA solution into each device
and let this stand for 15 min. After washing out each device using PBS, we introduced a mousemonoclonal anti-human CD9 (10 µg/mL, Abcam, Plc.), which is also a well-known membraneprotein expressed on exosomes, into each device, and then let this stand for 15 min. Additionally, wewashed out each device using PBS, and introduced a goat polyclonal anti-mouse
AlexaFluor488-labeled IgG secondary antibody (5 µg/mL, Abcam, Plc.) into each device, and thenlet this stand for 15 min. Finally, we washed out each device using PBS and followed that byfluorescence intensity observation under a fluorescence microscope (Olympus, Co., Ltd.). We foundthat ZnO nanowires could collect EVs efficiently while ZnO/Al2O3 core-shell nanowires could
collect EVs only slightly; these findings led us to conclude that the nanowire surface charge was thedominant factor.

fig. S8. Zeta potential of EVs in urine. EV zeta potential was measured using a dynamic lightscattering apparatus (Zetasizer Nano-ZS, Malvern Instruments, Ltd.). Mean zeta potential of EVs
was -28.0 mV.

fig. S9. Size distribution of EVs collected by ultracentrifugation and EV-free miRNAs collected
onto nanowires. (a) The concentration of collected EVs was 1.6×109 mL-1. Error bars show thestandard deviation for a series of measurements (N=3). (b) Collected and released miRNAs
quantities evaluated by the quantitative reverse transcription polymerase chain reaction (qRT-PCR).
As EV-free miRNA, we used a 100-nM miRNA (sequence, uugcauagucacaaaagugauc). The
designation “no treatment” represents the 100-nM miRNA quantity. Recovery rates of
microchannels with and without nanowires were 100 and 23 %, respectively.

fig. S10. Size distribution of the urinary free-floating objects. The urinary free-floating object
size distribution and concentration were measured using a nanoparticle detector (qNano, Meiwafosis
Co., Ltd.) with a 100 nm nanopore membrane (NP100, Meiwafosis Co., Ltd.). (a) The urinary
free-floating objects in the untreated urine; concentration was 1.4×1012 mL-1. (b) The urinary
free-floating objects in the flow-through fraction of the urine being processed by thenanowire-anchored microfluidic device; concentration was 2.4×1010 mL-1 (capturing efficiency,99%). (c) The urinary free-floating objects in the flow-through fraction of the urine being processedby a nanowire-anchored microfluidic device without the herringbone structure (only amicrochannel); concentration was 4.3×1011 mL-1 (capturing efficiency, 61%).

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.