Fig. 1 Nanowire-induced electrostatic collection of urine EVs followed by in situ extraction of EV-encapsulated miRNAs. (A) Schematic illustrations for urine EV collection 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 μm. (C) A schematic illustration and an inset illustration on the lower left showing a cross-sectional image for growing nanowires from the buried nanowires (nanowire-embedded PDMS), and a vertical cross-sectional image of the nanowire-embedded PDMS. Scale bar, 1 μm. (D) A schematic illustration and an inset illustration on the lower left showing a cross-sectional image for bonding the nanowire-embedded 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 μm) 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 to lysis buffer. Scale bar, 1 μm.
Fig. 2 In situ extraction of miRNAs using the nanowire-anchored microfluidic device. (A) Scatterplot of normalized intensities of miRNAs extracted from the collected 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 of miRNA 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.
Fig. 3 EV collection onto the nanowires. (A) A schematic illustration for the experimental process and calculation of collection efficiency. (B) Size distribution of the urinary 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 μm. (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 96-well 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 fluorescence intensity 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 of nanowires and a 96-well plate (n = 24 and 3, respectively).
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 easy comparison 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 of each miRNA corresponding to the color gradation.
Fig. 5 Down-regulated and overexpressed miRNAs extracted from Fig. 4 between noncancer donors and each cancer donor. Extracted miRNAs were the second 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.
- Table 1 Comparison among three methodologies.
Fabricated device ExoQuick Ultracentrifugation Collected objects Exosomes
Microvesicles
EV-free miRNAsExosomes
MicrovesiclesExosomes Collection mechanism Electrostatic interaction
between nanowire surface
charge and collected objectsPolymer-based capture of objects ranging
from 60 to 180 nm in diameter, according to
the kit manufacturer’s instruction manualBalance between applied
forces and density of the
collected objectsSample volume 1 ml 1 ml 1 ml for small RNA quantification
20 ml for urinary miRNA profilingProcessing time 40 min 870 min 300 min Collection efficiency (small RNA yield) 0.194 ± 0.028 ng/μl 0.120 ± 0.015 ng/μl 0.159 ± 0.077 ng/μl Extraction species of urinary
miRNAs being identified749, 822, 1111 (n = 3) 337, 355, 491 (n = 3) 171, 261, 352 (n = 3)
200 to 300**From Cheng et al. (31).
- Table 2 Potential cancer-related urinary miRNAs as indicated in Fig. 5.
Blank spaces in the “Biological functions” column indicate that biological functions of the miRNAs have not been reported.
Cancer types (down-regulation/overexpression) miRNA names Biological functions Lung (down-regulation) miR-3127-3p* miR-3130-5p* miR-3131* miR-3141* miR-3150b-5p* miR-3151-3p* miR-3151-5p* miR-3154* 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 and
metastasis (60)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 cell proliferation (63) miR-3129-5p 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 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 miR-337-3p 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 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 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 miR-6875-5p miR-6878-5p miR-7112-3p miR-7703 miR-7848-3p miR-7856-5p Pancreatic (overexpression) let-7i-3p miR-183-5p Promoting cancer proliferation, invasion, and
metastasis (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 Tumor suppressor (53–57) Liver (overexpression) 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 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-4703-3p 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) 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 miR-7849-3p 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.
Supplementary Materials
Supplementary 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 μm.
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-anchored microfluidic device.
data S1. Logarithmic signal intensities in noncancer miRNAs with those in cancer miRNAs.
Additional Files
Supplementary Materials
This PDF file includes:
- 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 μm.
- 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.
- Legend for movie S1
- Legend for data S1
Other Supplementary Material for this manuscript includes the following:
Files in this Data Supplement:
- fig. S1. Schematic of the fabrication procedure for nanowires anchored into PDMS.
