Analytical
Methods
View Article Online

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

PAPER

View Journal | View Issue

Cite this: Anal. Methods, 2021, 13, 337

ZnO/SiO2 core/shell nanowires for capturing CpG
rich single-stranded DNAs†
Marina Musa, a Takao Yasui, *abc Kazuki Nagashima, bd Masafumi Horiuchi,aZetao Zhu,a Quanli Liu,a Taisuke Shimada, a Akihide Arima, aTakeshi Yanagida de and Yoshinobu Baba*acf
Atomic layer deposition (ALD) is capable of providing an ultrathin layer on high-aspect ratio structures withgood conformality and tunable ﬁlm properties. In this research, we modiﬁed the surface of ZnO nanowires
through ALD for the fabrication of a ZnO/SiO2 (core/shell) nanowire microﬂuidic device which we utilized
for the capture of CpG-rich single-stranded DNAs (ssDNA). Structural changes of the nanowires whilevarying the number of ALD cycles were evaluated by statistical analysis and their relationship with thecapture eﬃciency was investigated. We hypothesized that ﬁnding the optimum number of ALD cycles
would be crucial to ensure adequate coating for successful tuning to the desired surface properties,
besides promoting a suﬃcient trapping region with optimal spacing size for capturing the ssDNAs as theReceived 19th November 2020
Accepted 18th December 2020

biomolecules traverse through the dispersed nanowires. Using the optimal condition, we achieved highcapture eﬃciency of ssDNAs (86.7%) which showed good potential to be further extended for the

DOI: 10.1039/d0ay02138e

analysis of CpG sites in cancer-related genes. This ﬁnding is beneﬁcial to the future design of core/shell

rsc.li/methods

nanowires for capturing ssDNAs in biomedical applications.

Introduction
Extraordinary properties of nanostructured materials have led
to their evolution as zero-dimensional nanoparticles,1,2 onedimensional nanotubes and nanowires,3,4 two-dimensional
nanosheets5 and nanoplates,6 and three-dimensional nanoballs7 and nanoowers.8 Among these nanomaterials, nanowires have shown good potential in various applications due totheir exceptionally high surface-to-volume ratio and strong
structure.9,10 Owing to these advantages, their applications havebeen broadened to include the recognition of biomolecules11–15
and aptamer-incorporated nanowires based on single-strandedaDepartment of Biomolecular Engineering, Graduate School of Engineering, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. E-mail: yasui@chembio.
nagoya-u.ac.jp;
babaymtt@chembio.nagoya-u.ac.jp;
Tel:+81-52-789-4611;+81-52-789-4664bJapan Science and Technology Agency (JST), Precursory Research for Embryonic
Science and Technology (PRESTO), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012,
JapancInstitute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japand

Department of Applied Chemistry, Graduate School of Engineering, The University ofTokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japane

Institute for Materials Chemistry and Engineering, Kyushu University, 6-1 KasugaKoen, Kasuga, Fukuoka 816-8580, Japanf

Institute of Quantum Life Science, National Institutes for Quantum and Radiological
Science and Technology, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan† Electronic supplementary
10.1039/d0ay02138e

information(ESI)

available.

This journal is © The Royal Society of Chemistry 2021

See

DOI:

DNAs (ssDNAs) or RNA oligonucleotides have received considerable interest in biosensing,16–18 disease diagnoses19,20 anddrug deliveries.21–23 Among the materials studied, ZnO nanowires grown by a hydrothermal method have been widely used
due to their several advantages which include low fabrication
cost, low temperature-driven growth, high fabrication yield24
and feasibility for tuning structural properties of the nanowires;
hence ZnO nanowires have the promise of applications indiverse elds.25–27 However, some researchers reported a toxicity
risk when using ZnO based materials;28–31 and hence, surface
coating on ZnO nanowires, especially by the widely used silicon
dioxide (SiO2),32,33 has become a countermeasure to prevent
dissolution of Zn2+. SiO2 based materials have been widely usedin solid-phase extraction of nucleic acids,34,35 a common
method for the adsorption, purication and isolation of nucleic
acids which relies on a reversible interaction between the
nucleic acids and the solid support. Hence we chose to fabricate
ZnO/SiO2 (core/shell) nanowires for capturing ssDNAs.
For fabrication of the ZnO/SiO2 (core/shell) nanowires, ZnO
nanowires were grown through a hydrothermal method followed by surface modication through atomic layer deposition(ALD). ALD is a robust vapor phase method based on sequential,
self-limiting reactions enabling the formation of ultrathin layerlms with precise thickness and good uniformity.36 Although
ALD is a powerful technique for achieving coating thicknesses
at the nanometer level, it is crucial to ensure conformal coatingof the whole nanowire framework as the conformal coating
determines the success of tuning to the desired surface

Anal. Methods, 2021, 13, 337–344 | 337

View Article Online

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

Analytical Methods

Paper

properties.37,38 We furthered our investigation by varying thenumber of ALD cycles to 10, 25, 55, 100 and 200 cycles which is
expected to cause remarkable changes to the structure of the
nanowire framework due to the increase in coating thickness,and nally, to inuence the capture eﬃciency of ssDNAs.
The ZnO/SiO2 nanowires were embedded in a microchip
device that was integrated with a syringe pump system andapplied for capturing CpG rich ssDNAs. The uncaptured ssDNAs
were collected in a centrifugation tube and then quantiedusing qPCR. ssDNAs with the CpG rich sequence have becomea binding material of interest especially for aptamers which canbe further extended as capture probes for the recognition of
DNA methylation on CpG sites, a widely known biomarker incancer-related genes.39,40 As capturing occurs while the ssDNAs
traverse through the nanowire arrays under an applied owrate, there should be a suitable nanowire spacing in the trapping region to capture them. Although the dispersedly distributed nanowires are foreseen as a good platform for the trapping
region, we supposed that for capturing small biomolecules suchas ssDNAs, there should be an optimum spacing between the
nanowires to achieve maximum capture eﬃciency.

wafer (Advantech Co., Ltd., Japan). The silicon wafer was spincoated with 3 mL SU-8 3005 (Nippon Kayaku., Ltd., Japan),baked at 95  C for 3 min and then subjected to photolithography with UV energy of 200 mJ cm2 for two microchannel
patterns formation on one silicon wafer. This was followed bypost-baking at 65  C and 95  C for 1 min and 2 min, respectively.
Next, the patterns were developed using SU-8 developer (Nippon
Kayaku Co., Inc.), rinsed with isopropyl alcohol (IPA) (Kanto
Chemical Co., Inc., Japan) and by silanization treatment withtrichloro(1H,1H,2H,2H-peruorooctyl)silane (Merck KGaA,
Germany) for 2 h. The resulting silicon wafer which had two
photolithographed-SU-8 microchannel patterns with dimensions of 10 mm length, 5 mm width and 10 mm height was later
used as a mold for the PDMS microchannel. A mixture of Silpot184 (Dow Corning Toray Co., Ltd.) and curing agent Catalyst
Silpot184 (Dow Corning Toray Co., Ltd.) at a ratio of 10 : 1 waspoured into the mold, a vacuum pressure was applied to remove
air bubbles and the mold was heated at 80  C for 2 h. Aerpeeling oﬀ the PDMS microchannel, the inlet and outlet holes
were then made using a 0.5 mm hole puncher (Harris Uni-Core,
USA).

Methodology

Zinc oxide nanowire microuidic device fabrication

PDMS microchannel mold fabrication
The mold for fabricating a polydimethylsiloxane (PDMS)
microchannel was formed on a 3-inch N-type <100> siliconFig. 1a illustrates the steps (i) to (x) for the nanowire microuidic device fabrication process. A pre-cleaned fused silica
substrate with 20 mm  20 mm  0.5 mm dimensions (Crystal
Base Co., Ltd., Japan) (step i of Fig. 1a) was spin-coated with

Fig. 1 Schematic illustrations of the ZnO/SiO2 nanowire microﬂuidic device fabrication process. (a) Steps (i) to (x) in the microﬂuidic device
fabrication. (b) Details of ALD (step viii) using tris(dimethylamino)silane (TDMAS) and ozone (O3). (c) Surface modiﬁcation done to obtain diﬀerent
layer thicknesses by varying the number of ALD cycles as 10, 25, 55, 100 and 200 cycles.

338 | Anal. Methods, 2021, 13, 337–344

This journal is © The Royal Society of Chemistry 2021

View Article Online

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

Paper
1,1,1,3,3,3-hexamethyldisilazane (OAP, Tokyo Ohka Kogyo Co.,
Ltd., Japan) and then with OFPR8600 (Tokyo Ohka Kogyo Co.,
Ltd.) (steps ii and iii, respectively). Next (step iv), photolithography was performed to make two areas for the nanowire growthwhich each had a length of 10 mm and width of 5 mm. The
pattern areas were developed using NMD-3 solution (Tokyo
Ohka Kogyo Co., Ltd.). An RF-sputtering machine (SVC-700RF I,
Sanyu Electron Co., Ltd., Japan) was utilized to sputter a seedlayer of ZnO (thickness, 130 nm) on the substrate for 10 min(step v). Hydrothermal growth of ZnO nanowires was performedby immersing the substrate into a mixture of 40 mM hexamethylenetetramine (HMTA, Wako Pure Chemical Industries,
Ltd., Japan) and 40 mM zinc nitrate hexahydrate (Thermo
Fisher Scientic Inc.) followed by heating at 95  C for 3 h (stepvi). The OFPR8600 was then removed using acetone (Kanto
Chemical Co., Inc.) leaving the nanowires on the desired areas(step vii).
Aer fabrication of ZnO nanowires, an ALD system(Savannah G2, Ultratech Inc., USA) was used to deposit a thinlayer of SiO2 to fabricate core/shell nanowires (Fig. 1a, step viii).
The conditions used were: precursors, tris(dimethylamino)silane (TDMAS) and ozone; temperature, 150  C; number of ALD
cycles, 10, 25, 55, 100 and 200. The resulting fused silica
substrate with nanowires was then attached with the PDMS
microchannel (step ix). The surfaces of the PDMS and thenanowire-grown substrate were rst treated using a plasma
etching apparatus (Meiwafosis Co., Ltd., Japan) and then heatedat 180  C for 2 min to assist the bonding process. A 0.5 mm
diameter polyetheretherketone (PEEK) tube (Institute ofMicrochemical Technology Co., Ltd., Japan) was then inserted
into the inlet hole and into the outlet hole (Fig. 1a, step x).
FESEM and STEM-EDS characterization of nanowires
A eld emission scanning electron microscope (FESEM) (SUPRA
40VP Carl Zeiss, Germany) was utilized to characterize thesurface morphology of the nanowires. Elemental mappings of
the ZnO/SiO2 core/shell nanowires were obtained using a scanning transmission electron microscope (JSM-7610F, JEOL,Japan) equipped with an energy dispersive X-ray spectrometer(STEM-EDS) operated at an acceleration voltage of 30 kV. The
images were obtained with 512  384 pixels at a scan rate of 0.1
ms and they were integrated for 100 cycles. The peaks of Zn Ka(8.630 keV), O Ka (0.525 keV), and Si Ka (1.739 keV) were used toconstruct the images (ESI Fig. 1†). The statistical analysis of thecoating thickness, diameter, length, aspect ratio and spacing
between nanowires was performed with an image processing
program, Image J using the images from FESEM and STEMEDS. Because of the dispersed distribution of nanowires,
spacings between them were measured at a low height (evaluation at 700 nm from the nanowire bottom) and at an upper
region (evaluation at 700 nm from the nanowire top).
Zeta potential measurement
The zeta potential of 50 ng mL1 DNA in Millipore water solution
was measured at 25  C using a dynamic-light scattering spectrophotometer (ZETASIZER Nano-ZS Malvern Panalytical, UK).

This journal is © The Royal Society of Chemistry 2021

Analytical Methods
ZnO/SiO2 nanowires were fabricated on a glass substrate(26 mm wide  37 mm long  1.0 mm thickness) followed byzeta potential measurement (ELSZ-2000, Otsuka Electronics,
Japan) in Millipore water at 25  C.
Capture experiment of ssDNA using the nanowire microuidic
deviceThe ssDNAs, primers and probes used in this experiment wereobtained from Invitrogen, Themo Fisher Scientic, Inc.; theirsequences were: (i) ssDNA, 50 -ATACGCGTACTGCGGTCGCGATCGCGCTCTCGCGCTGACGGTGCGTCGCGCGTACGCGATT30 ; (ii) forward primer, 50 -ACGCGTACTGCGGTCG-30 ; (iii) reverse
primer, 50 -GCGTACGCGCGACG-30 ; and (iv) probe, 50 -FAMATCGCGCTCTCGCGCTGACGG-30 . 50 ng mL1 of DNA wasprepared by dissolving the stock DNA in Millipore water. The
capture experiment was performed using a syringe pumpsystem (KDS-200, KD Scientic Inc., USA) at a ow rate of 5
mL min1. 50 mL of Millipore water was introduced prior to
sample introduction to remove any possible contaminates.
Then, 50 mL of 50 ng mL1 DNA was introduced into the inlet ofthe microuidic device and the recovered amount was collected
in a 1 mL centrifuge tube.
Quantications of DNA
The recovered ssDNAs were analyzed using a PIKOREAL 96 realtime polymerase chain reaction (RT-PCR) system (Thermo
Fisher Scientic Inc.). A mixture containing 1 mL DNA solution,
3.5 mL Millipore water, 5 mL TaqMan® Gene Expression MasterMix (Applied Biosystems, Thermo Fisher Scientic Inc., USA)and 0.5 mL of the Custom TaqMan® Gene Expression Assays(Applied Biosystems, Thermo Fisher Scientic Inc.) was pipetted into a 96-well reaction plate, sealed with an optical seal
(Applied Biosystems, Thermo Fisher Scientic) and RT-PCR wasrun. The protocol for RT-PCR was carried out using the cycling
conditions 2 min at 50  C, 10 min at 95  C, 50 cycles of 15 s at
95  C and 1 min at 60  C. The amount of captured DNA wascalculated by deducting the amount of recovery from the input
DNA.

Results and discussion
Surface modication using ALD
Surface modication of the nanowire through ALD for the
fabrication of core/shell nanowires has been proven able toimprove surface adsorption of target molecules.41–43 In thepresent research, we grew ZnO nanowires by a hydrothermal
method followed by surface modication using ALD to fabricate
the ZnO/SiO2 (core/shell) nanowires. Fig. 1b schematicallyshowed details for fabrication of the core/shell nanowires using
ALD. The reactant and co-reactant used were tris(dimethylamino)silane (TDMAS) and ozone (O3), respectively. The process
was initiated with the exposure of the rst reactant to hydroxylated ZnO nanowires followed by nitrogen (N2) purging to
remove the unreacted chemical species. The next reaction
involved the introduction of co-reactant as the oxygen source
followed by N2 purging which resulted in monolayer formation.

Anal. Methods, 2021, 13, 337–344 | 339

View Article Online

Analytical Methods

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

The sequence was repeated for the following numbers of ALD
cycles: 10, 25, 55, 100 and 200 to yield nanowires with diﬀerent
coating thicknesses (Fig. 1c). The sequential deposition of thereactant and co-reactant led to the ultrathin lm with good
conformality.
Characterizations and statistical analysis of ZnO/SiO2 whilevarying number of ALD cycles
STEM-EDS was utilized to obtain a more detailed analysis forthe composition of ZnO/SiO2 nanowires. Successful formationof a very thin SiO2 lm can be observed starting from 10 cycles
followed by increment in lm thickness as the number of ALD
cycles further increased until 200 cycles were reached (Fig. 2aand ESI gure†). The resulting SiO2 thickness had a linear
relationship with the number of ALD cycles with an approximate growth rate of 0.13 nm per cycle (Fig. 2b). Surface
morphology of the nanowires was characterized using FESEM
which indicated an increase in the size of nanowires withincreasing number of ALD cycles (Fig. 2c).
The dependence of nanowire structural properties on
number of ALD cycles was investigated by statistical analysis ofimportant parameters which include the diameter, length,
aspect ratio and spacing between nanowires. Fig. 3aPapersummarizes the size distribution of bare ZnO nanowires andthe maximum peak was for the diameter range of 60–90 nm andfor the length range of 1.6–1.8 mm. A consistent increase in the
diameter and length of the nanowires could be observed withincreasing number of ALD cycles suggesting uniform deposition of SiO2 lm was provided by ALD (Fig. 3b). Aspect ratios, inthe range of 15 to 19, were decreasing as the number of ALD
cycles increased in Fig. 3c. Due to the dispersed distribution of
the nanowires (Fig. 2c), statistical analysis of available spacing
between nanowires was done randomly at lower and upper
regions. A decreasing trend in spacing between nanowires wasobserved as ALD cycle numbers increased for spacing in therange of 15.9 nm to 34.9 nm and 43.5 nm to 60.9 nm, at thelower and upper regions, respectively (Fig. 3d). Increasing thenumber of ALD cycles resulted in the increase in nanowire
diameter with a drop in aspect ratio, and eventually reduced thespacing volume between the nanowires.
Inuence of SiO2 coating thickness on the capture eﬃciency
of ssDNAs
Since nding the optimum number of ALD cycles is crucial toensure conformal coating of lm layer on the whole nanowire
framework, we further investigated the impact of the number ofFig. 2 Characterizations of metal oxide nanowires. (a) STEM micrographs of ZnO/SiO2 nanowires fabricated with varying number of ALD cycles:
10, 25, 55, 100 and 200 cycles. (b) Plot of the SiO2 thickness as a function number of ALD cycles. (c) FESEM images of ZnO/SiO2 nanowires
fabricated with 10 cycles, 25 cycles and 55 ALD cycles: (upper row) top and (lower) cross-sectional views.

340 | Anal. Methods, 2021, 13, 337–344

This journal is © The Royal Society of Chemistry 2021

View Article Online

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

Paper

Analytical Methods

Statistical analysis of fabricated nanowires. (a) Size distribution of bare ZnO nanowires. (b) Diameter and length. (c) Aspect ratio. (d)Spacing between nanowires.

Fig. 3

ALD cycles on the capture eﬃciency of ssDNAs. We observedthat ZnO/SiO2 (core/shell) nanowires fabricated by 10 ALD
cycles showed slight improvement of the capture eﬃciency(Fig. 4a and ESI Fig. 2†). The increasing trend of the capture
eﬃciency could be observed from 0 to 55 cycles followed bya declining trend until 200 cycles. The zeta potential of ZnO/
SiO2 nanowires while changing the number of ALD cycles wasmeasured in neutral pH to investigate the net surface charges
which might be correlated to the changes in capture eﬃciency(Fig. 4b). Negative zeta potential values indicate the presence of
SiO2 layers which are negatively charged in neutral pH. The highzeta potential value exhibited at 10 cycles (16.19 mV) was
possibly due to a thin SiO2 layer coating that led to interferenceof charges from the core ZnO nanowires. A consistent trend inzeta potential values, from 24.7 mV to 26.0 mV was observed
for nanowires fabricated by 25 ALD cycles to 200 cycles (Fig. 3b)
leading to remarkable changes in the capture eﬃciency(Fig. 3a). Therefore, we concluded the suﬃcient coating is
crucial to ensure the tuning of surface properties to the desired
materials.
Further increases in the ALD cycle numbers will cause
structural changes to the nanowires and eventually reduce thespacing between the nanowires. Although a higher number ofThis journal is © The Royal Society of Chemistry 2021

ALD cycles resulted in a bigger nanowire size (Fig. 3b), however,
we observed a decline in the capture eﬃciency from 55 cycles(Fig. 4a) onwards possibly due to a more signicant decrease inaspect ratio at 100 and 200 cycles (Fig. 3c), hence the interaction
areas, which are the surface of nanowires available for capturing
the ssDNAs, were limited. Besides, the low aspect ratio iscorrelated with the increase in the diameter of nanowires
resulting in smaller spacing between nanowires. Consideringthat the gyration radius of the ssDNA is Rg < 10 nm, there should
be suitable spacing between the nanowires as interactions are
more likely to occur among the nanowires on the periphery of
the array as the ssDNAs traverse through the framework. We
supposed that the capture eﬃciency of the ssDNAs was governed by the spacing between nanowires (Fig. 3d) which
decreased with increasing ALD cycle numbers.
Here, we proposed a model for capturing ssDNAs based on
the structural eﬀect while varying ALD cycle numbers (Fig. 4c).
Apart from the interactions between SiO2 and the nucleobase
side,44 interactions are possible with the phosphate side basedon the theory of hard and so acids and bases although thesurface of ssDNA and SiO2 are negatively charged. This is corresponding with previous method, which reported on the DNA
adsorption on several metal oxide nanoparticles with negative

Anal. Methods, 2021, 13, 337–344 | 341

View Article Online

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

Analytical Methods

Paper

Fig. 4 Inﬂuence of numbers of ALD cycles on DNA capture eﬃciency. (a) Capture eﬃciency of ssDNAs on ZnO/SiO2 nanowires fabricated for
diﬀerent numbers of ALD cycles. Experimental conditions: 50 mL of 50 ng mL1 DNA solution; ﬂow rate 5 mL min1. (b) Zeta potential of ZnO/SiO2
nanowires fabricated for diﬀerent numbers of ALD cycles at pH ¼ 7. Error bars show the standard error for a series of measurements (N ¼ 3). (c)Proposed model explaining the eﬀect of structural changes on DNA capture.

charged surface.45,46 Low capture eﬃciency at 10 cycles (64.3%)
and 25 cycles (69.0%) was possibly due to an unsatisfactory
trapping region allowing some of the ssDNAs to traverse
through the nanowires without being captured. The highest
capture eﬃciency was achieved at 55 cycles (86.7%) due to the
suﬃcient optimal spacing as a sieving region. Further increasein ALD cycle number from 100 cycles (78.3%) to 200 cycles(73.2%) resulted in a decline in the capture eﬃciency due tosmall spacing for capturing ssDNAs, hence the capture might beinhibited and occurring only at the top of the nanowires. We
hypothesized that the design of the nanowire framework with
an optimum trapping region will promote capturing as the
ssDNAs traverse through the nanowire arrays, hence ensuringhigh capture eﬃciency.

enhance the capture eﬃciency of ssDNAs. In order to obtain the
maximum capture eﬃciency, nding the optimum number of
ALD cycles is crucial to ensure adequate coating of the wholenanowire framework with the desired materials and to provide
a suitable trapping region to capture the ssDNAs. We proposedthat in the design of the framework, an optimum spacing
between nanowires is crucial to ensure a suitable trapping
region as the ssDNAs traverse through the nanowires. Besides,
by using the optimum condition, higher capture eﬃciency(86.7%) was achieved compared to our previously reported
study (60%).47 This research provides fundamental ndings
based on the design of the core/shell nanowire framework in
microuidic devices which will be useful in future developmentof applications for high capture eﬃciency of ssDNAs.

Conclusion

Author contributions

In this research, we proved surface modication by coatinga thin SiO2 layer on ZnO nanowires using ALD was able to

M. M., T. Yasui, N. K., T. Yanagida and Y. B. planned anddesigned the experiment. M. M. and N. K. performed the342 | Anal. Methods, 2021, 13, 337–344

This journal is © The Royal Society of Chemistry 2021

View Article Online

Paper
experiments. M. M., T. Yasui, N. K., M. H., Z. Z., Q. L., T. S., A. A.
analyzed data. M. M. and T. Yasui wrote the paper.

Conﬂicts of interest

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

There are no conicts to declare.

Acknowledgements
This research was supported by PRESTO (JPMJPR19H9), Japan
Science and Technology Agency (JST), the JSPS Grant-in-Aid for
Scientic Research (S) 18H05243, the JSPS Grant-in-Aid for
Scientic Research (B) 18H02009, the JSPS Grant-in-Aid for
Exploratory Research 20K21124, the JSPS Grant-in-Aid for
Scientic Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems”, and research grants fromeach of the following: the Murata Science Foundation,
Advanced Technology Institute Research Grants 2019, Foundation of Public Interest of Tatematsu, the Nitto Foundation, theG-7 Scholarship Foundation, 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 the “Network Joint
Research Center for Materials and Devices”.

References
1 J. N. Tiwari, R. N. Tiwari and K. S. Kim, Prog. Mater. Sci.,
2012, 57, 724–803.
2 P. K. Mishra, H. Mishra, A. Ekielski, S. Talegaonkar and
B. Vaidya, Drug Discovery Today, 2017, 22, 1825–1834.
3 C. Zhang, Y. Yan, Y. S. Zhao and J. Yao, Annu. Rep. Prog.
Chem., Sect. C: Phys. Chem., 2013, 109, 211–239.
4 R. C. Arbulu, Y. Jiang, E. J. Peterson and Y. Qin, Angew.
Chem., Int. Ed., 2018, 57, 5813–5817.
5 M. Wang, X. Liu, P. Song, X. Wang, F. Xu and X. Zhang, Int. J.
Biol. Macromol., 2019, 128, 621–628.
6 S. Shi, X. Chen, J. Wei, Y. Huang, J. Weng and N. Zheng,
Nanoscale, 2016, 8, 5706–5713.
7 M. Guo, J. Balamurugan, X. Li, N. H. Kim and J. H. Lee, Small,2017, 13, 1701275.
8 W. Lei, D. Liu, J. Zhang, P. Zhu, Q. Cui and G. Zou, Cryst.
Growth Des., 2009, 9, 1489–1493.
9 S. Rahong, T. Yasui, T. Yanagida, K. Nagashima, M. Kanai,
A. Klamchuen, G. Meng, Y. He, F. Zhuge, N. Kaji, T. Kawaiand Y. Baba, Sci. Rep., 2014, 4, 5252.
10 T. Yasui, S. Rahong, K. Motoyama, T. Yanagida, Q. Wu,
N. Kaji, M. Kanai, K. Doi, K. Nagashima, M. Tokeshi,
M. Taniguchi, S. Kawano, T. Kawai and Y. Baba, ACS Nano,
2013, 7, 3029–3035.
11 T. Shimada, T. Yasui, A. Yokoyama, T. Goda, M. Hara,
T. Yanagida, N. Kaji, M. Kanai, K. Nagashima, Y. Miyahara,
T. Kawai and Y. Baba, Lab Chip, 2018, 18, 3225–3229.
12 Z. Lin, Y. Li, J. Gu, H. Wang, Z. Zhu, X. Hong, Z. Zhang, Q. Lu,
J. Qiu, X. Wang, J. Bao and T. Wu, Adv. Funct. Mater., 2018,28, 1802482.

This journal is © The Royal Society of Chemistry 2021

Analytical Methods
13 M. Nuzaihan, U. Hashim, M. K. M. Arshad, S. R. Kasjoo,
S. F. A. Rahman, A. R. Ruslinda, M. F. M. Fathil, R. Adzhri
and M. M. Shahimin, Biosens. Bioelectron., 2016, 83, 106–114.
14 T. Adam and U. Hashim, Biosens. Bioelectron., 2015, 67, 656–
661.
15 J. Wang and N. Hui, Sens. Actuators, B, 2019, 281, 478–485.
16 M. Liu, Q. Yin, Y. Chang, Q. Zhang, J. D. Brennan and Y. Li,
Angew. Chem., Int. Ed., 2019, 58, 8013–8017.
17 H. M. Meng, H. Liu, H. Kuai, R. Peng, L. Mo and X. B. Zhang,
Chem. Soc. Rev., 2016, 45, 2583–2602.
18 J. Kong, J. Zhu, K. Chen and U. F. Keyser, Adv. Funct. Mater.,
2019, 29, 1807555.
19 M. Liu, X. Yu, Z. Chen, T. Yang, D. Yang, Q. Liu, K. Du, B. Li,
Z. Wang, S. Li, Y. Deng and N. He, J. Nanobiotechnol., 2017,15, 1–16.
20 X. Meng, Z. Liu, Y. Cao, W. Dai, K. Zhang, H. Dong, X. Fengand X. Zhang, Adv. Funct. Mater., 2017, 27, 1605592.
21 O. Boyacioglu, C. H. Stuart, G. Kulik and W. H. Gmeiner,
Mol. Ther.–Nucleic Acids, 2013, 2, e107.
22 Y. Wan, L. Wang, C. Zhu, Q. Zheng, G. Wang, J. Tong,
Y. Fang, Y. Xia, G. Cheng, X. He and S. Y. Zheng, Cancer
Res., 2018, 78, 798–808.
23 M. Mie, R. Matsumoto, Y. Mashimo, A. E. G. Cass andE. Kobatake, Mol. Biol. Rep., 2019, 46, 261–269.
24 N. A. Alshehri, A. R. Lewis, C. Pleydell-Pearce andT. G. G. Maﬀeis, J. Saudi Chem. Soc., 2018, 22, 538–545.
25 Q. Liu, T. Yasui, K. Nagashima, T. Yanagida, M. Hara,
M. Horiuchi, Z. Zhu, H. Takahashi, T. Shimada, A. Arimaand Y. Baba, J. Phys. Chem. C, 2020, 124, 20563–20568.
26 X. Zhao, K. Nagashima, G. Zhang, T. Hosomi, H. Yoshida,
Y. Akihiro, M. Kanai, W. Mizukami, Z. Zhu, T. Takahashi,
M. Suzuki, B. Samransuksamer, G. Meng, T. Yasui, Y. Aoki,
Y. Baba and T. Yanagida, Nano Lett., 2019, 20, 599–605.
27 Q. Liu, T. Yasui, K. Nagashima, T. Yanagida, M. Horiuchi,
Z. Zhu, H. Takahashi, T. Shimada, A. Arima and Y. Baba,
Anal. Sci., 2020, 36, 1125–1129.
28 R. S. Alghsham, S. R. Satpathy, S. R. Bodduluri, B. Hegde,
V. R. Jala, W. Twal, J. A. Burlison, M. Sunkara and
B. Haribabu, Front. Immunol., 2019, 10, 2604.
29 K. H. Müller, J. Kulkarni, M. Motskin, A. Goode, P. Winship,
J. N. Skepper, M. P. Ryan and A. E. Porter, ACS Nano, 2010, 4,
6767–6779.
30 S. L. Chia and D. T. Leong, Heliyon, 2016, 2, e00177.
31 M. Ramasamy, M. Das, S. S. A. An and D. K. Yi, Int. J.
Nanomed., 2014, 9, 3707.
32 M. Afsal, C. Y. Wang, L. W. Chu, H. Ouyang and L. J. Chen, J.
Mater. Chem., 2012, 22, 8420–8425.
33 H. W. Kim, H. S. Kim, H. G. Na and J. C. Yang, Vacuum, 2012,
86, 789–793.
34 G. R. M. Duarte, C. W. Price, J. L. Littlewood,
D. M. Haverstick, J. P. Ferrance, E. Carrilho and
J. P. Landers, Analyst, 2010, 135, 531–537.
35 P. E. Vandeventer, J. S. Lin, T. J. Zwang, A. Nadim, M. S. Johaland A. Niemz, J. Phys. Chem. B, 2012, 116, 5661–5670.
36 R. W. Johnson, A. Hultqvist and S. F. Bent, Mater. Today,
2014, 17, 236–246.

Anal. Methods, 2021, 13, 337–344 | 343

View Article Online

Published on 21 December 2020. Downloaded by The University of Tokyo on 1/30/2021 7:35:30 AM.

Analytical Methods
37 J.-H. Kim, A. Katoch and S. S. Kim, Sens. Actuators, B, 2016,
222, 249–256.
38 T. G. Ulusoy, A. Ghobadi and A. K. Okyay, J. Mater. Chem. A,
2014, 2, 16867–16876.
39 D. R. Masser, D. R. Stanford, N. Hadad, C. B. Giles,
J. D. Wren, W. E. Sonntag, A. Richardson and
W. M. Freeman, Age, 2016, 38, 1–14.
40 S. Catuogno, C. L. Esposito and V. De Franciscis,
Pharmaceuticals, 2016, 9, 69.
41 T. Shimada, T. Yasui, A. Yonese, T. Yanagida, N. Kaji,
M. Kanai, K. Nagashima, T. Kawai and Y. Baba,
Micromachines, 2020, 11, 610.
42 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,

344 | Anal. Methods, 2021, 13, 337–344

Paper
H. Yukawa, T. Ochiya, T. Kawai and Y. Baba, Sci. Adv.,
2017, 3, e1701133.
43 C. Guan, X. Wang, Q. Zhang, Z. Fan, H. Zhang and H. J. Fan,
Nano Lett., 2014, 14, 4852–4858.
44 B. Shi, Y. K. Shin, A. A. Hassanali and S. J. Singer, J. Phys.
Chem. B, 2015, 119, 11030–11040.
45 B. Liu, L. Ma, Z. Huang, H. Hu, P. Wu and J. Liu, Mater.
Horiz., 2018, 5, 65–69.
46 R. Pautler, E. Y. Kelly, P. J. J. Huang, J. Cao, B. Liu and J. Liu,ACS Appl. Mater. Interfaces, 2013, 5, 6820–6825.
47 H. Takahashi, T. Yasui, K. Shinjo, N. Kaji, A. Okamoto and
Y. Baba, 22nd International Conference on Miniaturized
Systems for Chemistry and Life Sciences, MicroTAS 2018,
Chemical and Biological Microsystems Society, 2018, pp.
1886–1888.

This journal is © The Royal Society of Chemistry 2021