OPENSUBJECT AREAS:
NANOSTRUCTURES
BIOANALYTICAL CHEMISTRY

Received
28 January 2014
Accepted
20 May 2014
Published
11 June 2014

Ultrafast and Wide Range Analysis of
DNA Molecules Using Rigid Network
Structure of Solid Nanowires
Sakon Rahong1*, Takao Yasui2,3,4, Takeshi Yanagida1, Kazuki Nagashima1, Masaki Kanai1,
Annop Klamchuen1, Gang Meng1, Yong He1, Fuwei Zhuge1, Noritada Kaji2,3,4, Tomoji Kawai1& Yoshinobu Baba2,3,4,5
1

Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka-cho, Ibaraki, Osaka 567-0047, Japan,
Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, 3FIRST Research Center for Innovative
Nanobiodevices, Nagoya University, 4Institute of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya
464-8603, Japan, 5Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu
761-0395, Japan.

2

Correspondence andrequests for materials
should be addressed toT.Y. (yasui@apchem.
nagoya-u.ac.jp); T.Y.
(yanagi32@sanken.
osaka-u.ac.jp); T.K.
(kawai@sanken.
osaka-u.ac.jp) or Y.B.
(babaymtt@apchem.
nagoya-u.ac.jp)* Current address:
Institute of Innovation
for Future Society,
Nagoya University.

Analyzing sizes of DNA via electrophoresis using a gel has played an important role in the recent, rapid
progress of biology and biotechnology. Although analyzing DNA over a wide range of sizes in a short time isdesired, no existing electrophoresis methods have been able to fully satisfy these two requirements. Here we
propose a novel method using a rigid 3D network structure composed of solid nanowires within amicrochannel. This rigid network structure enables analysis of DNA under applied DC electric fields for alarge DNA size range (100 bp–166 kbp) within 13 s, which are much wider and faster conditions than thoseof any existing methods. The network density is readily varied for the targeted DNA size range by tailoring
the number of cycles of the nanowire growth only at the desired spatial position within the microchannel.
The rigid dense 3D network structure with spatial density control plays an important role in determining thecapability for analyzing DNA. Since the present method allows the spatial location and density of thenanostructure within the microchannels to be defined, this unique controllability offers a new strategy todevelop an analytical method not only for DNA but also for other biological molecules.

S

ince researchers have revealed the critical role played by information stored within DNA molecules in thecomplex biological activities occurring in living systems1–3, various methodologies have been developed toanalyze DNA molecules4,5. Among them, gel electrophoresis is the most prominent and commerciallyavailable standard method to analyze the size of DNA molecules due to the great controllability of pore sizes
and also the inexpensiveness of electrophoresis devices6. On the other hand, the limitations of gel electrophoresis
have also been pointed out, and they are the difficulty in analyzing relatively long DNA molecules in a short time,
the high skill required for appropriately preparing the gel, and the requirement of gel concentration control foreach targeted DNA size7–9. Recently, data for a wide range of DNA size have been obtained using the hydrodynamic chromatography method10. Although the analysis time using this method is faster than that using gelelectrophoresis, it still takes several tens of minutes for the fastest analysis. To realize an analysis on the timescale of seconds, nanostructure electrophoresis has attracted much attention as a novel method to overcome theabove issues with gel electrophoresis11.
Nanostructure electrophoresis under DC and AC electric fields has been used to analyze DNA molecules by
separating differently sized DNA molecules in a short time, which is impossible in gel electrophoresis12. Since
Volkmuth and Austin13 first proposed to utilize highly-ordered sieving nanostructures as an alternative forconventional randomly-ordered gel or polymer sieving matrices, many lithography-based nanostructures, suchas nanopillar arrays13–16, nanowall arrays17, nanofilter arrays18–21, and nanochannels22–24, have been applied foranalysis of DNA molecules. For example, Kaji et al.15 demonstrated that a nanopillar structure could separate 5components from a mixture of l-DNA (48.5 kbp) digested by HindIII under a DC electric field, however it took
more than 600 s and the DNA size range had to be over 2 kbp. Huang et al.14 also obtained good separation oflarge DNA molecules (61–209 kb) in 15 s, however, the nanopillar arrays required AC electric fields and DNA
size range was limited to over 60 kb. In a third example from a study by Fu et al.20, continuous separation of DNA

SCIENTIFIC REPORTS | 4 : 5252 | DOI: 10.1038/srep05252

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molecules was realized in a wide range (0.1–20 kbp), however it wasessentially impossible to separate 0.1 and 20 kbp at the same time
because of different required separation conditions, which Fu et al.
attributed to different separation regimes for 0.1 and 20 kbp. As
discussed above, intensive efforts have been devoted to developing
new types of nanostructure electrophoresis11,12. Although these artificial nanostructures realize analysis of DNA molecules with fewer
time- and labor-intensive operations25, the use of lithography techniques intrinsically limits the available size range (usually over
1 kbp) and also the formation of three-dimensionally (3D) designed
spatial nanostructures. Nanostructure electrophoresis needs a relatively long time to analyze short DNA molecules due to the sparseness of geometrical nanostructures compared with 3D gel network
structures18,26,27.
A self-assembled nanoparticle array within a microchannel isanother candidate methodology, which offers a 3D-ordered nanoporous sieving matrix28–32. Interesting features of nanoparticle arrays
have been demonstrated by separating DNA molecules under AC
electric fields31. Zeng and Harrison30 reported DNA separations ranging from 100 bp to 2 kbp using the nanoparticle array, and theseparation time was 10 min, and Tabuchi et al.29 showed DNA separations up to 15 kbp within 100 s using nanospheres. Comparedwith lithography-defined nanostructure electrophoresis, the DNA
size range is much wider due to 3D-ordered sieving matrices, however, the spatial controllability of such nanoparticle arrays within
microchannels is rather poor due to the intrinsic difficulties in controlling the spatial positions and forming the sparse structures due tothe naturally occurring aggregation events of nanoparticles. Additionally, these techniques require special instruments such as AC
electric fields generators or a combination of electric fields and pressure application. These limitations, especially as to the spatial controllability, are detrimental to analyze and separate relatively long
DNA molecules (over 20 kbp) in a short time; we have seen a generaldifference between gel electrophoresis and nanostructure electrophoresis. Thus, no existing methods are able to analyze fully a wide
range of DNA molecules in a short time.
To overcome these long-standing issues in DNA analysis, here we
propose a novel electrophoresis method, which utilizes a rigid 3D
network structure composed of solid nanowires only at the desired
position within a microchannel. This method allows analysis of DNA
molecules under DC electric fields in a much wider range and in a
faster time than any other existing methods. Our approach to realize
the rigid 3D network structure at the desired spatial position within
the microchannel is to use cycling nanowire growth by applying avapor-liquid-solid (VLS) method with a metal catalyst. Two noteworthy features of this method are: (1) the spatial selectivity of VLS
nanowire growth and (2) the density control of the network structures by cycling the supply of metal catalyst.

Results and Discussion
The scheme in Figure 1 summarizes the fabrication procedures; also
included are a photo of a fabricated device and SEM and TEM imagesof the nanowire network structure within the microchannel whenvarying the number of growth cycles. We fabricated the microchannel on a fused silica substrate by conventional photolithography andutilized Au catalyst for VLS growth of SnO2 nanowires (typical diameter of 10–20 nm)33–35. It should be noted that we intentionally did
not use electron beam lithography as we wanted to target futurecommercial use. Other details of the fabrication procedures can beseen in Methods and Supplementary Fig. S1. The nanowire surface
can be modified by forming a shell layer on the nanowire sidewall as acore-shell nanowire structure36; and here we utilized the SiO2 shelllayer37, which generates a negatively charged surface on the nanowires to avoid the undesired adhesion of negatively charged DNA.
The network density is readily controllable by varying the number ofgrowth cycles of the nanowires as shown in Figure 1b, due to the
SCIENTIFIC REPORTS | 4 : 5252 | DOI: 10.1038/srep05252

increased number of branched nanowires. Figure 1f shows the poresize distribution data measured from the cross-sectional SEM
images. The average pore size decreases with increasing network
density via the cycling nanowire growth. It should be noted thatthe present method allows users to define the spatial location, surface
properties, density and pore size of the rigid network nanostructure
within the microchannel, which are not attainable by other methods.
Figure 2 and Supplementary Fig. S2 show the electropherogram
data for various DNA mixtures, ranging from 100 bp–166 kbp,
obtained using the fabricated rigid 3D network structure when varying the number of nanowire growth cycles. The clear dependence ofthe electropherogram data on the number of cycles can be seen. In
the microchannel with 1-cycle growth nanowires (Type I), the DNA
mixtures ranging from 10 kbp to 166 kbp were separable; in particular, the DNA mixture of T4-DNA and l-DNA was separable withinjust 3 s with an applied voltage field of 500 V/cm (see Supplementary
Fig. S3), which is the fastest time reported to date. The DNA mixtureswith DNA sizes smaller than 10 kbp could not be separated within
the present experimental range, possibly due to the sparse nanowire
density. The network structure with the 3-cycle growth nanowires(Type II) could well separate DNA mixtures down to 5 kbp. In the
microchannel with the 5-cycle growth nanowires (Type III), theDNA mixtures down to 100 bp were separable within 20 s. Thus,
the nanowire spatial density of the present network structure critically determined the electrophoretic behaviors of different sized
DNA molecules. Denser network structures could separate smaller
DNA molecules, i.e. there was a correlation between the network
density and the separable DNA size range. Since the electrophoretic
mobility determined the capability of DNA separation and analysis,
we measured the mobility of DNA molecules that ranged from100 bp to 166 kbp with an applied DC electric field of 500 V/cm.
Figure 2c shows the mobility data measured for the network structure with 1-, 3- or 5-cycle growth nanowires (see Supporting
Information Movies-1, 2, and 3). These data clearly demonstrate
the significant DNA size dependence on the electrophoretic mobility,
and they also indicate the network density critically affects the size
dependence. The shape of the mobility curves for 1- and 3-cycle
growth nanowires was identical but shifted in absolute value ofmobility (y axis), while the shape of the mobility curve for the 5-cycle
growth nanowires was qualitatively different from other two curves
and it shifted in absolute value of DNA size (x axis). This seems toindicate that there are two different separation mechanisms as to thechange of the pore size in the present network structure. Thus, theseexperimental data highlight that the present network structure composed of solid nanowires is capable of analyzing a wide range of DNA
molecules within a short time.
This DNA size dependence on the electrophoretic mobility is ineffect similar to the trend for gel electrophoresis when varying the gelconcentration6–9. It is well known that a denser gel concentration canseparate shorter DNA base pairs due to the decreased average poresizes. In this sense, the spatial density dependence of the present rigid
3D network structure in the DNA separation is quite similar to thatof gel electrophoresis. However, there is a significant difference
between the present rigid network structure and the soft gel, whichis the capability of the former to separate and analyze relatively long
DNA molecules within a short time. This is similar to the capabilityof nanostructure electrophoresis11–12. Thus, the present rigid network
structures possess the advantages of both the soft gel and the rigid
lithography-defined nanostructures in DNA electrophoresis due totheir tailored network structure density, namely the present network
structure behaves as a hard gel.
When compared with nanostructure electrophoresis, the present
rigid 3D network structure can form 3D confined small pores. The
present rigid 3D network structure can enhance the probabilityof collisions between small DNA molecules and a rigid object
more than conventional lithographically defined nanostructures2

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Figure 1 | 3D nanowire network structure. (a) Schematic illustrations showing nanowire structure after: (I) 1-cycle growth; (II) 3-cycle growth, (III) 5cycle growth; and (lower row) the overall fabrication procedure. The microchannel and nanowire network structure are formed on fused silica substrate.
(b) SEM images (scale bar, 1 mm) of the nanowire network structure showing: (I) 1-cycle growth; (II) 3-cycle growth; and (III) 5-cycle growth. The insets
show vertical cross-sectional SEM images; scale bar, 1 mm. (c) Photograph of a fabricated device; scale bar, 1 cm. (d) SEM image of the nanowire network
structure embedded in the microchannel; scale bar, 10 mm. (e) TEM images of the nanowire network structure; scale bars, 100 nm (left) and 50 nm(right). Black arrows highlight branched positions. (f) Pore size distribution of the nanowire network structure after (I) 1-cycle growth, (II) 3-cycle
growth, and (III) 5-cycle growth.

including nanopillars and nanowalls can15,17,38; these collisions might
possibly enhance Ogston sieving effects (DNA size , the pore size;
mu , exp(-N), where mu is the mobility and N is the DNA length, andmu , mu0exp(-KC), where mu0 is the free solution mobility, K is
the constant of proportionality, and C is the concentration of
the network-forming matrix) for shorter DNA molecules (seeSupplementary Figs. S4a and S4b)39. On the other hand, there would
be an argument against Ogston sieving, which stems from the resultthat the time for transport through the pores (Ttransportation 5 d/(muE), where d is the pore size and E is the applied DC electric field)in the microchannel with the 5-cycle growth nanowires was shorter
than the time for diffusion (Tdiffusion 5 d2/D, where D is the diffusion
SCIENTIFIC REPORTS | 4 : 5252 | DOI: 10.1038/srep05252

coefficient of the DNA molecules) (see Supplementary Fig. S4c)40.
In addition, within the dense network structure, the deformed
DNA molecules might collide with another rigid post before they
contract, and this should affect the electrophoretic mobility andalso the DNA size dependence on the mobility. Since these results
are inconclusive about whether there is Ogston sieving in thematrix, with some evidences in favor and other evidences against
the theoretical description, we can say that the presence of 3D confined small rigid pores in the present network structure seems to be
responsible for the ability to separate DNA mixtures of wider size
ranges than conventional lithographically defined nanostructure
electrophoresis.
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Figure 2 | Separation and mobility data in the network structure. (a) Separation of 166 kbp DNA (20 ng/mL) and 48.5 kbp DNA (50 ng/mL) in
the nanowire network structure with the (I) 1-cycle growth nanowires, (II) 3-cycle growth nanowires, and (III) 5-cycle growth nanowires. The
electropherogram was obtained at 3 mm from the entrance of the microchannel with the nanowire network structure. The applied DC electric field in theseparation channel was 500 V/cm. (b) Separation of (I) 48.5 kbp DNA (20 ng/mL) and 10 kbp DNA (40 ng/mL) in the nanowire network structure withthe 1-cycle growth nanowires at the applied DC electric field of 500 V/cm, (II) 10 kbp DNA (30 ng/mL) and 5 kbp DNA (30 ng/mL) in the nanowire
network structure with the 3-cycle growth nanowires at the applied DC electric field of 500 V/cm, and (III) 1 kbp DNA (30 ng/mL) and 100 bp DNA(30 ng/mL) in the nanowire network structure with the 5-cycle growth nanowires at the applied DC electric field of 500 V/cm. The electropherogram was
obtained at 3 mm from the entrance of the microchannel with the nanowire network structure. (c) Dependence of electrophoretic mobility on DNA sizein the nanowire network structure with 1-cycle growth nanowires (red circles), 3-cycle growth nanowires (blue circles), and 5-cycle growth nanowires(green circles). Error bars show the standard deviation for a series of measurements (N 5 3).

Based on only the pore sizes and the spatial network structure, thepresent network structure is quite similar to that of conventional gel
structures37,41,42. However, our single molecule imaging experiments
revealed a difference between the present network structure andagarose gel regarding the electrophoretic behaviors of 38-kbp
DNA, as shown in the video data of the Supporting Information
Movie 4. Although their pore sizes are controlled to be similar forboth structures, the DNA molecules in the nanowire network structure showed more frequent elongation and contraction by colliding
with the rigid nanowire framework and faster migration than thosein agarose gel. Thus, a scenario based on the pore size alone cannot
explain the present results. One major difference between the present
rigid network structure and the conventional gels is the stiffness of
SCIENTIFIC REPORTS | 4 : 5252 | DOI: 10.1038/srep05252

the network structures. The present network structure is much morerigid than the conventional gel network structures43; for example,
typically the Young’s modulus of agarose gel is 0.1–1 MPa, whereasthat of the solid nanowires is 100 6 20 GPa44,45. Since the deformation of DNA molecules must be larger when softer DNA molecules
collide with a stiffer object, collision with the rigid nanowire network
structure must amplify the DNA deformation more than for the softgel, considering the stiffness of the DNA molecules. Ou et al.46 haveproposed that such mechanical collisions with rigid posts are asimportant as the large sized DNA separation mechanism. Thus,
the different collision behaviors of DNA molecules might cause thedifferent electrophoretic results between the present network structure and the gels, especially for separating mixtures of relatively long4

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DNAs within a short time. The spatial controllability of the present
network structure might also be another factor responsible for the
faster analytical time due to the spatially concentrated DC electricfield. Since the exact separation mechanism of wide-range DNA
mixtures in the present network structure must be rather complex,
such novel DNA electrophoretic behaviors are an interesting subject
for theoreticians and experimentalists alike.

Figure 3 | Separation of multicomponent DNA mixtures in the network
structure. (a) Separation of 166 kbp DNA (20 ng/mL), 48.5 kbp DNA(20 ng/mL), 10 kbp DNA (20 ng/mL), and 5 kbp DNA (20 ng/mL) in the
nanowire network structure with 3-cycle growth nanowires. The
electropherogram was obtained at 1 mm from the entrance of the
microchannel with the nanowire network structure. The applied DC
electric field in the separation channel was 500 V/cm. (b) Separation of
48.5 kbp DNA (20 ng/mL), 20 kbp DNA (20 ng/mL), 5 kbp DNA(20 ng/mL), 1 kbp DNA (20 ng/mL), and 100 bp DNA (40 ng/mL) in the
nanowire network structure with 5-cycle growth nanowires. The
electropherogram was obtained at 1 mm from the entrance of the
microchannel with the nanowire network structure. The applied DC electricfield in the separation channel was 500 V/cm. (c) Separation of lambda
DNA digested by HindIII in the nanowire network structure with 5-cycle
growth nanowires. The electropherogram was obtained at 3 mm from theentrance of the microchannel with the nanowire network structure. The
applied DC electric field in the separation channel was 500 V/cm.
SCIENTIFIC REPORTS | 4 : 5252 | DOI: 10.1038/srep05252

Next we explored the feasibility of using the present network
structure to analyze and separate multicomponent DNA mixtures.
Figure 3a shows the electropherogram data of a mixture composed offour components including T4-DNA (166 kbp), l-DNA (48.5 kbp),
10 kbp-DNA and 5 kbp-DNA separated in the microchannel having
a network structure formed with 3-cycle growth nanowires. The DC
electric field of 500 V/cm was utilized in the electrophoresis. The
multicomponent DNA mixtures are clearly separable using the present network structure device and surprisingly within just 4 s, whichis the fastest time reported so far15. By using a denser network structure formed with 5-cycle growth nanowires, we can analyze andseparate the mixtures composed of even smaller sized DNA molecules 100 bp-DNA, 1 kbp-DNA, 5 kbp-DNA, 20 kbp-DNA, and lDNA (48.5 kbp), as shown in Figure 3b. Under the DC electric fieldof 500 V/cm, the five differently sized DNA molecules are readily
separated within only 7 s, which is also the fastest time reported so
far.
To further demonstrate the capability of the present network
structures for analyzing and separating DNA molecules, we separated l-DNA digested by HindIII, which consists of eight different
DNA size fragments, using the microchannel having the network
structure formed with 5-cycle growth nanowires (Figure 3c).
Measurements were made under an applied DC electric field of
500 V/cm. The DNA fragments were separated as six distinct peaks.
Each peak was assigned to a DNA band as indicated in Figure 3cbased on peak height and comparison with previous data15.
Comparison with the previous top data showed the nanowire network structure had much faster separation time, comparable theoretical plate number and theoretical plate number per column length,
and lower resolution (Table 1). It should be emphasized that thepresent DNA separation is superior to that for the previous data interms of the separation time. The present separation time is 13 s,
which is much faster than the previous top time of 680 s obtainedusing a nanopillar structure15. Thus our experimental data prove thatthe present rigid 3D network structure can well analyze a wide range(100 bp–166 kbp) of multicomponent DNA mixtures within a shorttime, and the results are superior to those of any existing methods5,6,11,12. Since the present concept based on the rigid 3D network
structure in a microchannel has the potential to overcome the longstanding weaknesses in the existing DNA electrophoresis methods,
we expect it will open up new research avenues for not only analyzing
DNA molecules but also for developing analytical methods for various biological molecules, which are not attainable by existing
methods.
In summary, we have demonstrated a novel method based on therigid 3D network structure comprised of solid branched nanowires
within a microchannel, which enables analysis of DNA molecules fora much wider of DNA sizes (100 bp–166 kbp) than any previous
methods within a much shorter time (13 s). The network density isreadily tunable for the targeted DNA size range by controlling thenumber of cycles for nanowire growth only at the desired spatial
position within a microchannel; this level of flexibility cannot be
attained by other methodologies. The rigid 3D network structurewith the spatial density control plays an important role in attaining
better analysis of DNA molecules. Since the present method allows
users to define the spatial location and density of the network nanostructure within the microchannel, such spatial controllability willoffer a new strategy to develop analytical methods not only for
DNA molecules but also for other biological molecules.

Methods
Fabrication of cycling nanowire growth devices. To fabricate cycling nanowire
growth devices, we have utilized a conventional photolithography process, electron
beam lithography, and vapor-liquid-solid (VLS) growth technique for the nanowires(Supplementary Fig. S1). First, a Cr layer (250 nm thick) was deposited on a substrateby RF sputtering (SVC-700LRF, Sanyu Denshi); this layer was a mask for the dry
etching process to fabricate a microchannel. Positive photoresist (TSMR V50, Tokyo

5

SCIENTIFIC REPORTS | 4 : 5252 | DOI: 10.1038/srep05252
Rs5k–10k0.66 6 0.03

Rs1k–5k1.01 6 0.07

5k1500 6 130
6.5 6 0.5
1.5 6 0.1

Rs10k–48.5k
0.35 6 0.01

48.5 k4500 6 400
2.2 6 0.2
4.5 6 0.4

166 k2300 6 700
4.6 6 1.4
2.3 6 0.7

Rs20k–48.5k
0.69 6 0.04

Rs48.5k–166k
0.91 6 0.12
20 k3700 6 660
2.8 6 0.5
3.7 6 0.7
Rs5k–20k
0.67 6 0.02

48.5 k730 6 80
14 6 1.6
0.7 6 0.08

*From Ref. 15.

Not available

125 or 564

Rs2.0&2.3k–4.4k
0.93

2.0&2.3 k

4.4 k

Rs4.4k–6.6k
1.08

6.6 k
600–17000
0.07–2.1
Rs6.6k–9.4k
1.04

9.4 k

Rs9.4k–23k
2.39

23 k

DNA size/bp
Theoretical plate number
Theoretical plate number per column length(3106)/plates m21
Resolution

1k
990 6 80
10 6 0.8
1.0 6 0.08

10 k
1400 6 110
7.1 6 0.6
1.4 6 0.1

Nano pillar*

Rs100–1k0.81 6 0.18

100710 6 40
14 6 0.8
0.7 6 0.04

5k5300 6 820
1.9 6 0.3
5.3 6 0.8

DNA size/bp
125 or 564
2.0&2.3 k
4.4 k
6.6 k
9.4 k
23 k
Theoretical plate number
24000 6 1500
12000 6 1200
2300 6 40
6800 6 900
4100 6 280
2800 6 160
1.2 6 0.1
2.6 6 0.3
13 6 0.2
4.5 6 0.6
7.4 6 0.5
11 6 0.6
HETP (31024)/mm
8.1 6 0.5
3.9 6 0.4
0.8 6 0.01
2.3 6 0.3
1.4 6 0.1
0.9 6 0.05
Theoretical plate number per column length(3106)/plates m21
Rs2.0&2.3k–4.4k
Rs4.4k–6.6k
Rs6.6k–9.4k
Rs9.4k–23kResolution
Rs125 or 564–2.0&2.3k
0.98 6 0.06
0.26 6 0.39
0.52 6 0.03
0.29 6 0.01
0.32 6 0.01

DNA size/bp
Theoretical plate number
HETP (31024)/mm
Theoretical plate number per column length(3106)/plates m21
Resolution

DNA size/bp
Theoretical plate number
HETP (31024)/mm
Theoretical plate number per column length(3106)/plates m21
Resolution

Figure 3c

Figure 3b

Figure 3aTable 1 | Quantification of separation performance using theoretical plate number, height equivalent to a theoretical plate (HETP), theoretical plate number per column length, and resolution in
Figure 3. Each standard deviation was obtained by 3 consecutive runs

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Ohka Kogyo Co.) was spin-coated on the Cr layer. Then the microchannel patternwith a width of 25 mm was formed by photolithography. After development of theresist, the patterned Cr area was etched by immersing in Cr etchant(H2O:Ce(NH4)2(NO3)6:HClO4, 8551055 by weight percent) for 5 min. The
microchannel was formed by etching using reactive ion etching (RIE-10NR, SamcoCo.) under CF4 gas ambient. The microchannel depth was controlled to be 2 mm,
which is larger than the radius of gyration of T4 DNA (970 nm). The inlet and outlet
via holes (1.5 mm diameter) for the microfluidic system were drilled with anultrasonic driller (SOM-121, Shinoda Co.). We also patterned the metal catalysts todefine the spatial position of the nanowires within the microchannel. A Cr layer(10 nm thick) was deposited within the microchannel. Positive resist (ZEP520 A7,
Zeon Corp.) was coated on the microchannel by spin-coating, and then the pattern
was drawn by electron beam lithography (SPG-724, Sanyu Electron Co.). Afterdeveloping the resist, the Cr layer pattern was removed with Cr etchant. An Au metal
catalyst layer (3 nm thick) for nanowire growth was deposited within the
microchannel by DC sputtering. And then the resist was lifted off using
dimethylformamide, acetone and isopropanol, sequentially. The SnO2 nanowires
were fabricated in the microchannel by a pulse laser deposition (PLD) system. Detailsof the nanowire fabrication conditions can be found elsewhere47. To prepare
nanowire branches, the Au catalyst particles were deposited along the nanowire by
DC sputtering and the SnO2 nanowires were grown cyclically using the PLD system toincrease the density of nanowires in the microchannel. The Cr layer was lifted off with
Cr etchant. It should be noted that a 40 nm layer of SiO2 was deposited onto the SnO2
nanowires to avoid charge attraction of the negatively charged DNA molecules.
Finally, the microchannel was sealed using a fused silica cover plate (130 mm thick) by
the literature method17,38.
DNA observations using the cycling nanowire growth devices. The electrophoretic
DNA mobility measurements in the nanowire array structure were done using T4,
l, 20 kbp, 10 kbp, 5 kbp, 2 kbp, 1 kbp, 500 bp, and 100 bp DNA molecules(Nippon Gene Co., Ltd.) stained with the dye, YOYO-1 (Invitrogen), at a dye-tobase pair ratio of 1515 contained in concentrated buffer solution (5 3 TBE;
445 mM Tris-Borate and 10 mM EDTA, pH 8.2, Sigma-Aldrich, Inc.). An
inverted fluorescent microscope (Eclipse TE-300, Nikon) equipped with a highvoltage sequencer (HVS448-1500, Lab Smith) was used to apply the DC electricfields and a Hg lamp was used to observe fluorescently stained DNA molecules.
Fluorescence images were captured with an EB-CCD camera (C7190-43,
Hamamatsu Photonics K.K.) through a 10 3/1.40 NA objective lens (Nikon). The
images were recorded on a DV tape (DSR-11, Sony) and then analyzed by imageprocessing software (Cosmos 32, Library, Tokyo, Japan).
DNA observations using the agarose gel. l-DNA digested Apa I, stainedwith YOYO-1 at a dye-to-base pair ratio of 1515, was observed in apolydimethylsiloxane (PDMS) microchannel (650 mm width; 50 mm height),
which was filled with 5% agarose gel (Pulsed Field Certified Agarose, Bio-Rad
Laboratories, Inc.) in 53 TBE. Under the DC electric field of 300 V/cm, l-DNA
molecules were observed through a 1003/1.40 NA objective lens on an inverted
fluorescent microscope (Eclipse TE-300) equipped with a high-voltage sequencer(HVS448-1500), 488 nm wavelength laser (FLS-488-20), and EB-CCD camera(C7190-43). The images were recorded on a DV tape (DSR-11) and then analyzed
by image-processing software (Cosmos 32).
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Acknowledgments
This research is supported by the Cabinet Office, Government of Japan and the Japan
Society for the Promotion of Science (JSPS) through the Funding Program forWorld-Leading Innovative R&D on Science and Technology (FIRST Program), JSPS
Grant-in-Aid for Scientific Research (A) 24241050, JSPS Grant-in-Aid for Young Scientists

7www.nature.com/scientificreports(B) 25790028 and Nanotechnology Platform Program (Molecule and Material Synthesis) of
the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Author contributions
S.R., T. Yasui, T. Yanagida, T.K. and Y.B. designed the experiments. S.R., T. Yasui,
T. Yanagida, K.N., M.K., A.K., M.G., H.Y., F.Z. and N.K. performed experiments. S.R.,
T. Yasui and T. Yanagida analyzed the data. T. Yasui and T. Yanagida wrote the paper.
All authors discussed the results and commented on the manuscript.

Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports

SCIENTIFIC REPORTS | 4 : 5252 | DOI: 10.1038/srep05252

Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Rahong, S. et al. Ultrafast and Wide Range Analysis of DNA
Molecules Using Rigid Network Structure of Solid Nanowires. Sci. Rep. 4, 5252;
DOI:10.1038/srep05252 (2014).
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