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Engineering Nanowire-Mediated Cell Lysis for
Microbial Cell Identiﬁcation
Takao Yasui,*,†,‡,§ Takeshi Yanagida,*,⊥,∥ Taisuke Shimada,† Kohei Otsuka,† Masaki Takeuchi,†Kazuki Nagashima,⊥ Sakon Rahong,‡,# Toyohiro Naito,¶ Daiki Takeshita,† Akihiro Yonese,†
Ryo Magofuku,† Zetao Zhu,⊥ Noritada Kaji,‡,§,∇ Masaki Kanai,⊥ Tomoji Kawai,*,∥and Yoshinobu Baba*,†,‡,○,◆
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†

Department of Biomolecular Engineering, Graduate School of Engineering and ‡ImPACT Research Center for Advanced
Nanobiodevices, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan§
Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 4-1-8
Honcho, Kawaguchi, Saitama 332-0012, Japan⊥
Institute of Materials Chemistry and Engineering, Kyushu University, 6-1 Kasuga-Koen, Kasuga, Fukuoka 816-8580, Japan∥
Institute of Scientiﬁc and Industrial Research, Osaka University, 8-1 Mihogaoka-cho, Ibaraki, Osaka 567-0047, Japan#
College of Nanotechnology, King Mongkut’s Institute of Technology Ladkrabang, Chalongkrung Rd., Ladkrabang, Bangkok 10520,
Thailand¶
Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510,
Japan∇
Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, Moto-oka 744, Nishi-ku,
Fukuoka 819-0395, Japan○
Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu 761-0395, Japan◆
College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, 80708 Kaohsiung City, Taiwan, R.O.C.
S Supporting Information*

ABSTRACT: Researchers have demonstrated great promise forinorganic nanowire use in analyzing cells or intracellular components.
Although a stealth eﬀect of nanowires toward cell surfaces allows
preservation of the living intact cells when analyzing cells, as acompletely opposite approach, the applicability to analyze intracellularcomponents through disrupting cells is also central to understanding
cellular information. However, the reported lysis strategy is
insuﬃcient for microbial cell lysis due to the cell robustness andwrong approach taken so far (i.e., nanowire penetration into a cell
membrane). Here we propose a nanowire-mediated lysis method for
microbial cells by introducing the rupture approach initiated by cell
membrane stretching; in other words, the nanowires do not penetrate
the membrane, but rather they break the membrane between the nanowires. Entangling cells with the bacteria-compatible and ﬂexible
nanowires and membrane stretching of the entangled cells, induced by the shear force, play important roles for the nanowire-mediatedlysis to Gram-positive and Gram-negative bacteria and yeast cells. Additionally, the nanowire-mediated lysis is readily compatible withthe loop-mediated isothermal ampliﬁcation (LAMP) method because the lysis is triggered by simply introducing the microbial cells. We
show that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identiﬁcation assay(just introducing a premixed solution into a device), resulting in visual chromatic identiﬁcation of microbial cells. This approach allows
researchers to develop a microﬂuidic analytical platform not only for microbial cell identiﬁcation including drug- and heat-resistancecells but also for on-site detection without any contamination.

KEYWORDS: nanowires, microﬂuidics, nanowire-mediated lysis, microbial cell, intracellular analysis,
loop-mediated isothermal ampliﬁcationInorganic nanowires have been found to hold great promise
for analyzing cells or intracellular components,1 by suchtechniques such as three-dimensional intracellular record© 2019 American Chemical Society

Received: November 25, 2018
Accepted: January 25, 2019
Published: February 13, 2019
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Figure 1. Schematic illustrations for nanowire-mediated lysis of microbial cells. There is a nanowire-mediated membrane rupture of microbial
cells, followed by DNA extraction from the microbial cells.

ing,2 syringe-injectable electronics,3 intracellular component
isolation4 and separation,5 and intracellular reagent delivery.6
These studies have utilized features of the nanowires, including
their nanometer-scale diameter size, rigidity, high aspect ratio,
and capability to function as electrophysiological probes.2−10
These studies were aimed at preserving the intact living cells andintracellular components via a stealth eﬀect of nanowires toward
cell surfaces and components, for example, nanowire penetration into cells without compromising cell viability. As acompletely opposite approach, an analysis of intracellularcomponents through lysing cells is an important way to obtaincellular information in molecular biology, especially for
microbial cells. However, the applicability of penetration of
nanowire tips into microbial cells has been thought to be
doubtful due to their toughness to external forces (Young’s
modulus, ∼100 MPa),11,12 and therefore, this limitation has
hindered the versatility of the inorganic nanowires for analyzing
cells or intracellular components.
Microbial cell lysis is an essential analytical methodology for
microbial genome sequencing from single cells,13,14 since
genomic DNA analysis from single microbial cells is expected
to uncover and broaden uncharted branches of the tree of life,that is, microbial dark matter. Among the existing lysis methods
for microbial cells,15,16 thermal, chemical, optical, electrical, andmechanical lysis in a microﬂuidic platform are candidates for thelysis applicability to all types of microbial cells. However, theselysis methods have suﬀered from various issues, including:insuﬃcient thermal diﬀusion;17 diﬃcult regent delivery anddiﬃcult reagent separation;18,19 expensive and complex
setups;20,21 bubble formation;22,23 and complex nanofabrication
processes.24−27 A concept for nanowire-mediated microbial cell
lysis is expected to provide a good way to address the inherentdiﬃculties in these lysis methods. Use of nanowires adds anadditional feature to conventional cell lysis, leading tosigniﬁcantly enhanced lysis performance and overcoming theabove issues.
A nanowire-assisted cancer cell lysis by utilizing ZnO
nanowires (ca. 100 nm in diameter) has been reported by Kim
et al.28 and So et al.,29 in which they used nanowires to anchor
cells and ruptured the cells anchored in the nanowires by theshear force. Their studies and that of Ning et al.30 were intended
to rupture the cells via a cytotoxic eﬀect of nanowires toward thecell surfaces, that is, by nanowire penetration into cells withtearing of the cell membrane28,29 or inducing apoptosis,30 orboth of them. Since cancer cells are soft (Young’s modulus, ∼1kPa),31 it is considered that the cell rupture has been initiated bypenetration of nanowire tips into the cellular membrane. The
reported lysis strategy has had the intention of fabricating morerigid nanowires for nanowire penetration into a cell membrane.
However, this strategy is insuﬃcient for microbial cell lysis dueto the cell robustness. The rigidity of microbial cells does notallow nanowires to penetrate into the cell membrane, andtherefore, from the perspective of the lysis applicability to alltypes of microbial cells, our strategy to accomplish nanowiremediated cell lysis should be based on a diﬀerent rupture
mechanism rather than the conventional mechanism (apenetration of nanowire tips into cells).
Here we propose a nanowire-mediated lysis method for
microbial cells by introducing the rupture approach initiated by
the cell membrane stretching; in other words, the nanowires do
not penetrate the membrane, but rather they break themembrane between the nanowires. Figure 1 shows a schematicof our proposed concept to rupture microbial cells by stretching
the cell membrane via the thin and bacteria-compatible
nanowires, that is, nanowire-mediated mechanical lysis. Thisrupture mechanism is initiated by cell adsorption onto thenanowires, it proceeds by cell entanglement with the nanowires,and it is completed by non-uniform stretching of the cell
membrane in the spaces between the nanowires while applying
external driving forces (an electric ﬁeld and ﬂuid ﬂow), leading
to irreversible membrane rupture. The role of the external
driving forces is to provide an external force to press the cells
into the spacing between nanowires, thus enhancing the shear
force applied to the cell membrane. A key factor for membranerupture is the degree of stretching that is determined by a
combination of cell rigidity,32−34 material compatibility withbacteria for their adsorption, nanowire ﬂexibility for bacteria
entanglement, and the shear force for membrane stretching; themechanism is inherently diﬀerent from the nanowire tip
penetration mechanism. The rupture mechanism evokes animage that the more bacteria-compatible materials and ﬂexible
nanowires would have a stronger ability to rupture microbial
cells. Since it is well-known that SiO2 has more biocompatibility35 and thinner nanowires have higher ﬂexibility,36,37 weanticipated that the thinner nanowires with an SiO2 surface
coating would signiﬁcantly enhance bacteria adsorption andentanglement. In this paper, ﬁrst, we fabricated 30 nm diameterand 2 μm length nanowires (∼60 wires/μm2) with the SiO2
surface coating (10 nm diameter SnO2 nanowires with a 10 nm2263

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Figure 2. DNA extraction of microbial cells using nanowires in a microchannel. (a) A schematic illustration showing nanowire-mediated lysis.
(b) A ﬂuorescence image showing DNA extraction from a single cell of B. subtilis; scale bar, 10 μm. (c) A ﬂuorescence image showing DNA
extraction from a single cell of E. coli; scale bar, 10 μm.

Figure 3. Antibacterial test for SiO2 and SnO2 substrates. (a) Bacteria recovery rate for SiO2 and SnO2 substrates. Error bars show the standard
deviation for a series of measurements (N = 3). (b, c) Confocal microscope images that show E. coli on SiO2 substrates, respectively, without andwith 10 mL PBS washing process; scale bars, 30 μm. Some bacteria were observed on SiO2 substrates even though they underwent the washingprocesses. The washing processes could not remove all bacteria from the SiO2 substrates.

Nanowire-Mediated Lysis Mechanism (Bacteria-Compatibility, Flexibility, and External Driving Forces). Basedon our proposed concept (Figure 1), the present nanowiremediated lysis has three steps: adsorbing cells onto thenanowires, entangling cells with the nanowires, and stretching
the cell membrane in the spaces between the nanowires while
applying external driving forces. We carried out a JapaneseIndustrial Standards (JIS)-based antibacterial test (JIS
Z2801:2010, which is based on ISO 22196, Supporting
Information) for bacteria compatibility of nanowire materials.
Then, we examined the eﬀect of nanowire ﬂexibility (speciﬁcally,
the wire diameter) on bacteria entanglement by SEM
observations of microbial cells on the nanowires with diﬀerent
diameters, and we investigated the eﬀect of the external driving
forces (applied electric ﬁelds) on membrane rupture by
ﬂuorescently observing the rupture event. Considering thefeatures of the present nanowires and the eﬀect of the external
driving forces, we concluded that the present nanowiremediated lysis could extract microbial DNA molecules via thethree steps.
The ﬁrst step for adsorbing cells onto the nanowires wasevaluated by the antibacterial test showing that SiO2 and SnO2
materials (the nanowire shell and core materials) had goodbacteria compatibility, and SiO2 enhanced bacteria adsorption(Figure 3). For fabrication of SnO2 substrates, a 100 nm-thick
SnO2 layer was deposited on a SiO2 substrate (50 × 50 mm2) byatomic layer deposition (Figure S5). The antibacterial test

thick SiO2 shell) (Figure S1) to facilitate membrane stretching,
and we followed that stretching by rupture of microbial cells.

RESULTS AND DISCUSSIONDNA Extraction via Nanowire-Mediated Lysis. To
demonstrate that the DNA extraction via nanowire-mediatedrupture is applicable to microbial cells (Figure 2a), we used
Bacillus subtilis and Escherichia coli as a typical example of Grampositive and Gram-negative bacteria, respectively (Supporting
Information). The Gram-positive bacteria have strong resistivity
to physical damage due to their thick (20−80 nm)
peptidoglycan layer,38 and the Gram-negative bacteria havestrong resistivity to chemical damage due to their outermembrane.39 The cells were introduced by capillary force
onto the nanowires in a microchannel, and the cells contactedwith the oxide nanowire surface (Figure S2). Various imaging
results showed that the cells were not ruptured by the capillary
force (Figure S3). After applying an electric ﬁeld of 500 V/cm,
the nanowires exhibited DNA extraction from B. subtilis and E.
coli (Figure 2b,c). We determined the molecules extracted by theobserved cell lysis were DNA molecules via monitoring the timeseries data of ﬂuorescence intensity of the extracted moleculeswith ﬂuorescence labeling (Figure S4 and Supplementary Movie
1). These results highlight our demonstration of a methodology
for nanowire-mediated lysis to extract microbial DNA moleculesby 30 nm diameter nanowires while applying an electric ﬁeld.
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Figure 4. FESEM images of B. subtilis deposited onto nanowires (left, 30 nm diameter; right, 110 nm diameter). (a, b) FESEM images showing
nanowires of each diameter and 2−3 μm length; scale bars, 1 μm. The inset FESEM images are an enlarged image of each diameter nanowire;
scale bars, 100 nm. (c, d) FESEM images that show embedding of B. subtilis onto each diameter nanowire; scale bars, 1 μm. (e, f) FESEM imagesof each diameter nanowire after B. subtilis deposition; scale bars, 1 μm.

SiO2 was comparable to or less than that for SnO2, but thesurface coating of SiO2 could signiﬁcantly enhance bacteria
adsorption. It is noteworthy that the present nanowire materials(core/shell: SnO2/SiO2) have bacteria compatibility andenhance microbial cell adsorption onto the nanowires, leading
to the eﬃcient initiation of the nanowire-mediated lysis.
The second step for entangling cells with the nanowires wasevaluated by the SEM observations showing that the nanowire
ﬂexibility (diameter) had an eﬀect on bacteria entanglement(Figure 4, Figures S6−S10). We fabricated SnO2 nanowires (10nm in diameter) with a 10 nm or a 50 nm-thick SiO2 shell layer
for ﬂexible or rigid nanowires (Figure 4a,b, Figures S6−S8).
These SEM images also showed that the nanowires could havebacteria compatibility. The 30 nm diameter nanowires (core/shell: 10 nm SnO2/10 nm SiO2) easily deformed their structure
by sticking to the bacteria through their mechanical ﬂexibility

evaluated whether the substrate had a bactericidal or a bacteriacompatible activity based on colony formation. After exposing
each substrate type, we determined the bacteria formed around100 colonies on SiO2 substrates and over 250 colonies on SnO2
substrates, and we calculated their respective bacteria recovery
rate as around 30% and 100% (Figure 3a); SnO2 substrates
seemed to have better bacteria-compatible activity than SiO2
substrates. However, since the antibacterial test has underlying
assumptions that give little thought to bacteria adsorption, thesubstrates with a bacteria adsorption property seem to havefalse-positive bactericidal activity (a low bacteria recovery rate).
In the antibacterial test, some bacteria were not fully removed
but rather were attached onto the SiO2 substrates after washingprocesses (Figure 3b,c). Considering the well-known biocompatibility of SiO235 and bacteria adsorption onto SiO2
substrates, we could conclude that bacteria compatibility for
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Figure 5. Nanowire-induced microbial cell membrane rupture. (a) A schematic illustration that shows embedding of B. subtilis onto thenanowires in a microchannel. (b) A schematic illustration that shows emitted ﬂuorescence intensity of ﬂuorescence molecules by combining
them with DNA molecules inside the bacteria after the B. subtilis membrane ruptured under application of an electric ﬁeld. (c) Time-coursemonitoring of ﬂuorescence intensity inside a single B. subtilis using nanowires with a 30 nm diameter. The time 0 s was an interaction point
between the cells and nanowires. The microbial cells in the microchannel without nanowires were moved electrophoretically to the anode.
Error bars show the standard deviation for a series of measurements (N = 3). (d) Time-course monitoring of ﬂuorescence intensity inside asingle B. subtilis using nanowires with a 110 nm diameter. Error bars show the standard deviation for a series of measurements (N = 3).

could not rupture it (Figure 5d). The strength of the electricﬁeld can control the strength of the pressing force, and then theelectric ﬁeld can control the degree of membrane stretching; the375 V/cm ﬁeld is strong enough to make the membrane rupture
irreversible, but a ﬁeld strength <250 V/cm is not. Since themicrobial cell membrane in the microchannel with 110 nm
diameter nanowires and without them under the applied electricﬁeld was not ruptured (it was just moved electrophoretically tothe anode), the combination of entangling bacteria with the
ﬂexible nanowires and making the shear force in the spacing
between the nanowires via applying the external driving forces
was essential to the completion of the nanowire-mediated lysisof microbial cells.
Nanowire-Mediated Lysis Using Fluid Flow. Since cell
adsorption onto the nanowires, cell entanglement with the
nanowires, and then cell membrane stretching by the shear force
in the spacing between the nanowires are important factors fornanowire-mediated mechanical lysis, we conceived the idea ofusing ﬂuid ﬂow instead of the applied electric ﬁeld for extracting
and collecting DNA molecules for downstream assays. The
nanowire device for ﬂuid ﬂow consisted of nanowires on fusedsilica substrates and a polydimethylsiloxane (PDMS) microchannel (height, 10 μm) with herringbone structures (height, 3μm) (Figure 6a,b). The microchannel with herringbone
structures can induce chaotic mixing ﬂow,40 ensuring good
convection of solutions. We anticipated that the convection ﬂow
could force the microbial cells to contact and entangle with the
nanowires and make the shear force in the spacing between(Figure 4c,e, Figure S9); on the other hand, the 110 nm
diameter nanowires (core/shell: 10 nm SnO2/50 nm SiO2) did
not structurally deform by sticking to the bacteria because of
their mechanical stability (Figure 4d,f, Figure S10). Althoughthese SEM images were taken after drying out the samples, it islikely that the mechanical ﬂexibility of the bacteria-compatible
nanowires when present in a liquid environment should behave
similarly with respect to bacteria entanglement. These results
highlight the behavior that the microbial cells introduced bycapillary force onto the 30 nm diameter nanowires are absorbed
onto the nanowires and then are entangled with the nanowires,leading to progression of the nanowire-mediated lysis.
The third step for stretching the cell membrane was evaluatedby monitoring the time series data of ﬂuorescence intensity oflabeled DNA molecules showing that the external driving forces(applied electric ﬁelds) could rupture the membrane of theentangled bacteria with the 30 nm diameter nanowires or the
100 nm diameter nanowires (Figure 5). It should be noted that
we used DNA staining ﬂuorescence molecules, which could not
go inside the intact cell membrane (Figure 5a). On the other
hand, if the microbial cell can be ruptured solely by the shear
force occurring in the spacing between the nanowires under theapplied electric ﬁeld, the ﬂuorescence molecules should go
inside the cells from the ruptured membrane and dye nucleic
acids inside the cells (Figure 5b). For the applied electric ﬁeld of
375 V/cm, the 30 nm diameter nanowires could rupture themicrobial cell membrane (Figure 5c); on the other hand, under
the same applied electric ﬁeld, the 110 nm diameter nanowires
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Figure 6. Nanowire-mediated lysis using ﬂuid ﬂow for extracting and collecting DNA molecules. (a) A schematic illustration showing the
nanowire device including nanowires and the chaotic mixer. (b) An FESEM image showing the herringbone structures; scale bar, 100 μm. (c)FESEM images showing the nanowire structures (length: 2, 4, and 7 μm); scale bars, 10 μm. (d) Extracted DNA concentration from S. cerevisiae
cells using the nanowire devices and a microﬂuidic device with the chaotic mixer (without nanowires). The control had no S. cerevisiae cells.
Error bars show the standard deviation for a series of measurements (N = 3). (e) Extracted DNA concentration from S. cerevisiae cells using the
microﬂuidic device with the chaotic mixer (without nanowires) on the Peltier heater at 0, 25, 37, 65, and 94 °C. The control had no S. cerevisiae
cells. Error bars show the standard deviation for a series of measurements (N = 3). (f) Extracted DNA concentration from S. cerevisiae cells using
the nanowire devices and the microﬂuidic device with the chaotic mixer (without nanowires) on the Peltier heater at 94 °C. The control had noS. cerevisiae cells. Error bars show the standard deviation for a series of measurements (N = 3). (g) An FESEM image showing a lysed S. cerevisiae
cell after nanowire and thermal lysis; scale bar, 1 μm. The lysed cell was highlighted as dark pink, and the boundary between the nanowire andno-nanowire regions was highlighted as the yellow dotted line.

Saccharomyces cerevisiae as a typical example of yeast cells(Supporting Information) which have thick cell walls (200−300nm)41,42 against any environmental invasions; the cell walls
impose labor-intensive and time-consuming diﬃculties whentreated by yeast cell lysis. Nanowire-mediated lysis using ﬂuid
ﬂow allowed us to extract and collect DNA molecules solely byintroducing yeast cells, and the extracted amount of DNA
molecules had a relationship to nanowire length (Figure 6d).
When we used the more densely distributed nanowires (∼90−
110 wires/μm2),9 these were the branched SnO2 nanowires

them, leading to membrane stretching and cell rupture (FigureS11). In further anticipation that the nanowire-mediated lysis
performance is closely related to the nanowire ﬂexibility, weused the thinner and longer (and therefore, more ﬂexible)
nanowires; these were the SnO2 nanowires without the SiO2
shell (diameter and length, 10 nm and 2−7 μm, respectively,
Figure 6c), since thinner nanowires have higher ﬂexibility,36,37
and SnO2 had bacteria compatibility (Figure 3). To demonstratethat the DNA extraction via ﬂow-based nanowire-mediated lysis
is applicable to much harder microbial cells, we used
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Figure 7. Microbial cell identiﬁcation using the identiﬁcation system. (a) A photo of sample tubes after DNA ampliﬁcation using LAMP in them.
While 200 E. coli cells exhibited the green ﬂuorescence color when using the nanowire device at 94 °C or a commercially available kit (heated at
94 °C for 20 min after adding lysis solution), 200 E. coli cells showed no ﬂuorescence color when using a device without nanowires at 94 °C (onlythe chaotic mixer) and when heating at 94 °C for 20 min in the sample tubes (without the nanowire device). The control had no E. coli cells. (b)A schematic illustration showing the bacteria identiﬁcation system including the nanowire device and DNA ampliﬁcation device. Bacteria
detection can be realized by introducing a bacteria suspension into the system. (c) A photo of the DNA ampliﬁcation devices after 60 min. The
control had no E. coli cells. In the other device, 200 E. coli cells exhibited the green ﬂuorescence color.

without the SiO2 shell (length, 7 and 2 μm for ﬁrst and second
growth, respectively, Figure S12), the extracted amount of DNA
molecules was similar to the amount without nanowires. Since
the entanglement with the nanowires is a key factor for cell lysis,
which is realized by the deformation of the nanowires, it isreasonable that the densely distributed nanowires cannot
deform in the conﬁned space and lead to the degradation ofcell lysis performance; and therefore, controlling the density
distribution of the nanowires is important to achieve a highperformance on nanowire-mediated lysis. These results revealed
that the nanowire ﬂexibility was closely related to the nanowiremediated lysis performance. The applicability of ﬂow-based
nanowire-mediated lysis to yeast cells could expand diversity of
our methodology on the issues of the lysis applicability to alltypes of microbial cells and the downstream assay applicability
to quantitative DNA ampliﬁcation.
Furthermore, our concept for cell lysis based on oxide
nanowire features and ﬂuid ﬂow led us to the idea to incorporate
thermal lysis into nanowire-mediated lysis based on the highthermal conductivity of oxide nanowires. As previously
reported,17 thermal lysis under continuous ﬂow conditions has
been hindered by the ineﬃciency of thermal energy transferfrom substrate to buﬀer solution due to low thermal
conductivity and the ease of thermal diﬀusion from the buﬀer
solution to the PDMS microchannel. Since the thermal
conductivity of SnO2 is much higher than that of buﬀer solution,
the nanowires could directly provide thermal energy to yeast
cells during the cell adsorption, cell entanglement, or both ofthem; this concept is completely diﬀerent from conventional
thermal lysis and is another approach that can be facilitated bynanowire-mediated cell lysis. Thermal energy supplied by the
nanowires could denature the cell walls and assist the shear force
induced by ﬂuid ﬂow, leading to eﬃcient cell ruptures.
Compared to only thermal lysis (Figure 6e), the nanowiremediated thermal and mechanical lysis signiﬁcantly improved

the extraction eﬃciency of genomic DNA from yeast cells(Figure 6f). After quantifying extracted DNA concentrations(Supporting Information), we obtained their FESEM images bypeeling the nanowire device oﬀ the PDMS microchannel; theseobserved results allowed us to conﬁrm the yeast cells were
ruptured on the nanowires (Figure 6g). These results have led us
to believe that nanowire-mediated lysis will be a powerful toolthat oﬀers a strategy to add an additional feature to conventional
cell lysis, leading to signiﬁcantly enhanced lysis performance andovercoming the inherent issues.
Simple, Rapid, and One-Step Identiﬁcation Assay
System. Next, we showed good compatibility of the present
nanowire-mediated lysis method with DNA ampliﬁcation anddetection for microbial cell identiﬁcation. For the identiﬁcation
assay, we used a DNA ampliﬁcation method based on the loopmediated isothermal ampliﬁcation (LAMP),43 which could
amplify our target genes isothermally (Supporting Information).
Extracted DNA samples were taken at the nanowire device
outlet after introduction of E. coli-containing solution into thedevice, and they showed a chromatic change in the sample tube
(Figure 7a). Two hundred E. coli cells showed green
ﬂuorescence color when using the nanowire device at 94 °C,
and this was similar to the result seen in the nanowire-mediatedlysis for yeast cells (Figure 6f). On the other hand, 200 E. coli
cells showed no ﬂuorescence color when using only thermal
lysis, such as a chaotic mixer device at 94 °C (no nanowires) andwhen heating at 94 °C for 20 min in the sample tubes (without
the nanowire device); these observations also were similar to
those of thermal lysis for yeast cells (Figure 6e). Since 200 E. coli
cells showed the green ﬂuorescence color when using acommercially available lysis solution at 94 °C for 20 min,nanowire-medicated cell lysis played a similar role to chemicallysis with the lysis objective of 200 E. coli cells. These results
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ACS Nanosolid (VLS) growth technique (Figure S1). First, a 250 nm-thickchromium layer was deposited on fused silica substrates using an RF
sputtering apparatus (Sanyu Electron Co.) (Figure S1a,b). Positive
photoresist TSMR V50 (Tokyo Ohka Kogyo Co.) was spin-coated on
the substrates (Figure S1c). Then the microchannel pattern, with alength and width of 8000 and 100 μm, respectively, was formed byphotolithography. After development of the resist (Figure S1d), thepatterned area was etched by immersing in chromium etchant solution(H2O:Ce(NH4)2(NO3)6:HClO4, 85:10:5 by weight percent) for 5 min(Figure S1e). The microchannel was etched using a reactive ion etchingsystem (Samco Co.) under CF4 gas ambient (Figure S1f). The
microchannel depth was controlled to 2 μm. After cleaning to remove
resist and metal residues (Figure S1g), the inlet and outlet via holes (2mm in diameter) for the microﬂuidic system were drilled with anultrasonic driller (Shinoda Co.). We also patterned metal catalysts todeﬁne the spatial position of the nanowires within the microchannel. A
10 nm-thick chromium layer was deposited on the substrates (FigureS1h). Electron-beam positive resist ZEP520 A7 (Zeon Corp.) was
coated on the microchannel by spin-coating (Figure S1i), and then thepattern was drawn by an electron beam lithography apparatus (Sanyu
Electron Co.). After developing the resist (Figure S1j), the chromium
layer was patterned by using the chromium etchant solution (FigureS1k). A 3 nm-thick gold layer for nanowire growth was deposited on thesubstrates using a DC sputtering system (Sanyu Electron Co.) (FigureS1l). Then the chromium layer was removed using chromium etchantsolution (Figure S1m). The SnO2 nanowires were grown from gold
catalyst particles that were deposited in a layer formed using a pulse
laser deposition (PLD) system (Figure S1n). Details of the nanowire
fabrication conditions can be found elsewhere.48 A 10 nm or a 50 nmthick SiO2 shell was deposited onto the SnO2 nanowires using RF
sputtering (Figure S1o). Finally, the microchannel was sealed using a
fused silica cover plate (130 μm thick) by the literature method (FigureS1p).8,10,49
Bacillus subtilis Preparation. We cultured B. subtilis spore(ATCC6633) in 2 mL of 2.5% LB medium (Sigma-Aldrich, Co.)overnight at 37 °C using a multi shaker (Tokyo Rikakikai Co., Ltd.) at ashaking speed of 125 rpm. Then, the B. subtilis was applied to LB agar
medium (2% agar (Wako Pure Chemical Industries, Ltd.) in 2.5% LB
medium), and colonies were allowed to form. After forming thecolonies, they were put into 2 mL of fresh 2.5% LB medium andcultured overnight at 37 °C using the multi shaker (shaking speed, 125rpm). Next, we added 10 μL of the cultured B. subtilis to 2 mL of fresh
2.5% LB medium, and we continued the culturing for 5 h at the same
temperature and shaking speed. Then, we removed 1 mL of the B.
subtilis solution and centrifuged it for 10 min at 25 °C and relative
centrifugal force (rcf) of 5000g. We discarded 1 mL of the supernatant,then added 1 mL of 10 mM phosphate buﬀer solution, and centrifuged
this for 10 min at the same temperature and rcf. After that, we discarded
1 mL of the supernatant again, then added 1 mL of 10 mM phosphatebuﬀer solution, and centrifuged for 10 min for a third time at the same
temperature and rcf. Finally, we discarded 1 mL of supernatant and thensuspended the B. subtilis in 500 μL of 10 mM phosphate buﬀer solution.
We measured optical density (OD) and concentrations of the B. subtilis
solution by using a POLARstar OPTIMA plate reader (BGM Labtech.
Ltd.) at 584 nm and a qNano particle analyzer (Meiwafosis, Co., Ltd.).
The B. subtilis solution concentrations were diluted as desired insubsequent experiments.
Escherichia coli Preparation. E. coli DH5α competent cells(Nippon Gene Co., Ltd.) were applied to LB agar medium (1.5% agar
in 2.5% LB medium), and colonies were formed. The formed colonies
were put in 2 mL of 2.5% LB medium and cultured overnight at 37 °C
with the multi shaker (shaking speed, 125 rpm). Then, we measured the
OD of the E. coli solution using the plate reader and diluted the E. coli
using 10 mM phosphate buﬀer to 0.4 of the measured OD. We
centrifuged the diluted E. coli solution (10 min; 25 °C; 5000g),
discarded the supernatant, and suspended the cells in 10 mM phosphatebuﬀer. The concentration (3.9 × 108 E. coli cells/mL) in the suspended
solution was measured using the qNano particle analyzer.
Observation of B. subtilis under a Microscope. We stained thenucleic acid of the B. subtilis using 1 μM of SYTO-9 (Life Technologies,

bial cell lysis and the nanowire device had good compatibility
with the LAMP method.
Finally, for a simple, rapid and one-step identiﬁcation assay
system, we combined the nanowire device with a DNA
ampliﬁcation device based on the LAMP method (Figure 7b,
Supplementary Movie 2). A silicon tube was connected between
the outlet of the nanowire device and the inlet of the DNA
ampliﬁcation device. According to the kit manufacturer’s
instruction manual, DNA polymerase is highly activated at 65°C when heated for 15−60 min and inactivated at 80 °C whenheated for 5 min; therefore, we decided to use a Peltier heater
operating at 65 °C. Since the condition of being at 94 °C for 20
min required by the commercially available lysis solution
interferes with the DNA polymerase activity, only the nanowiremediated lysis allows us to premix the LAMP solution with E.
coli-containing solution before introducing it into the identiﬁcation system. Thermal lysis at 65 °C showed no signiﬁcantlysis performance for yeast cells (Figure 6e); therefore, achromatic change of the LAMP solution should occur when the
nanowire-mediated lysis is well-performed on E. coli cells.
Conversely, a chromatic change of the LAMP solution should
not occur when E. coli cells are not in solution or the nanowiremediated lysis does not work well. The identiﬁcation systemshowed the green ﬂuorescence color within 60 min (Figure 7c)after introduction of 4 E. coli cells/μL in a 50 μL sample volume,in which the LAMP solution was premixed with E. coli before
introducing it into the identiﬁcation system. A scenario based on
nanowire-mediated mechanical lysis, nanowire-mediated thermal lysis, or both of them can explain the present results. The
present concept based on nanowire-mediated lysis has a great
potential to achieve simple, rapid, and one-step assay for
identiﬁcation of microbial cells.

CONCLUSION
In summary, we demonstrated a nanowire-mediated lysis
method which ruptures cells for Gram-positive and -negative
bacteria and yeast cells. Membrane stretching of the microbial
cells caused by bacteria-compatible and ﬂexible nanowires plays
an important role in attaining nanowire-mediated lysis. In thispaper, we demonstrated only nanowire-mediated mechanicaland thermal lysis; however, researchers can provide nanowiremediated chemical, optical, and electrical lysis via use of surfacemodiﬁed nanowires,44 a nanowire laser45 or a waveguide,46 andnanowire electrodes,47 respectively. These nanowire-mediatedlysis methods have a potential to overcome the inherent issues
related to chemical, optical, and electrical lysis. Additionally, thenanowire-mediated lysis is readily compatible with various
analysis tools because the lysis is triggered by simply introducing
the microbial cells. We demonstrated identiﬁcation of microbial
cells based on a chromatic change just by introducing premixed
solution into the identiﬁcation assay system, which could
perform DNA extraction, ampliﬁcation and detection, sequentially. This level of versatility cannot be attained by othermethodologies. Since the present method allows users to
perform lysis of microbial cells, such nanowire-mediated lysis
will oﬀer a strategy to develop analytical systems not only for cell
identiﬁcation but also for on-site detection in a closed system
without any contamination.
MATERIALS AND METHODS
Fabrication of Nanowires in a Microchannel. For fabrication of
nanowires in a microchannel, we utilized a conventional photolithography process, electron beam lithography, and a vapor−liquid−
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ACS NanoLtd.) for 30 min. Then, we centrifuged the B. subtilis solution (10 min;
25 °C; 5000g). After discarding the supernatant, we added 100 μL of
phosphate buﬀer. Finally, we stained the cell membrane using 5 μg/mLof FM1-43FX (Life Technologies, Ltd.). We observed the B. subtilis
with a confocal microscope DMI6000B (Leica, Ltd.) (Figure S2a−c).
The excitation wavelengths of the microscope laser were 488 and 514
nm for SYTO-9 and FM1−43FX, respectively, and their respective
observed emission wavelengths were 495−520 nm and 600−650 nm.
We also used the JSM-7610F (Jeol) to capture the ﬁeld emission
scanning electron microscopy (FESEM) images, one example of whichis shown in Figure S3d.
DNA Extraction from B. subtilis on Nanowires in a Microchannel. We added 500 nM YOYO-1 (Life Technologies, Ltd.) to 3.0
× 108 B. subtilis cells/mL in phosphate buﬀer. Then, we supplied 1 μL of
3.0 × 106 B. subtilis cells/mL to the sample reservoir on the nanowire
device (Figure S2). After ﬁlling the channel with the 3.0 × 106 B. subtilis
solution, we removed 0.8 μL of the solution and then supplied 2 μL of
phosphate buﬀer containing 500 nM YOYO-1. The high voltage forelectrophoresis was controlled from an HVS448 high voltage sequencer(LabSmith) through a platinum electrode which was precisely
positioned on the stage of an Eclipse TE-300 inverted microscope(Nikon). A high-pressure mercury lamp was used as an optical source toilluminate DNA molecules at the detection point, and the emitted
ﬂuorescence was collected with a C7190−43 EB-CCD camera(Hamamatsu Photonics K.K.) through a 100×/1.40NA objective
lens. The excitation wavelength was 450−490 nm, and the emission
wavelength was 520 nm. All images were recorded with a DSR-11
digital videocassette recorder (Sony), and then the ﬂuorescenceintensity was analyzed by Cosmos32 image-processing software(Library Co., Ltd.).
DNA Extraction from E. coli on Nanowires in a Microchannel.
We added 500 nM YOYO-1 to 3.0 × 108 E. coli cells/mL. Then, we
supplied 1 μL of 3.0 × 106 E. coli cells/mL to the sample reservoir on the
nanowire device. After ﬁlling the channel with the 3.0 × 106 E. coli
solution, we removed 0.8 μL of the solution and then supplied 2 μL of
phosphate buﬀer containing 500 nM YOYO-1. The high voltage forelectrophoresis was controlled from the high-voltage sequencer
through the platinum electrode which was precisely positioned on
the stage of the inverted microscope. The high-pressure mercury lamp
was used as the optical source to illuminate DNA molecules at thedetection point, and emitted ﬂuorescence was collected with the EBCCD camera through the 100×/1.40NA objective lens. The excitation
laser wavelength range was 450−490 nm, and the observed emission
wavelength was 520 nm. All images were recorded with the digital
videocassette recorder, and then the ﬂuorescence intensity was
analyzed by the Cosmos32 image-processing software.
Antibacterial Test. We carried out a Japanese Industrial Standards(JIS)-based antibacterial test (JIS Z2801, ISO 22916) for SiO2 andSnO2 substrates. Each substrate was immersed in ethanol for 10 min
before use. Polyethylene ﬁlm squares (40 × 40 mm2) to facilitatebacteria solution spreading onto the substrates were also cleaned inethanol before their use. A 250 μL aliquot of suspended microbial cells(1.0 × 106 cells/mL) was dropped onto the substrates which had been
put into sterile Petri dishes (AGC Techno Glass Co., Ltd.), and then thepolyethylene ﬁlm squares were placed onto the substrates to spread thesuspended cells over the entire surface. The Petri dishes with thesubstrates were transferred into highly humid containers, into which
200 mL sterile water had been added, and the cells were incubated at 35°C for 24 h. After incubation, the substrates were cleaned using SCDLP
culture medium (Eiken Chemical Co., Ltd.). Next, 10 mL of SCDLP
culture medium was introduced in between the substrates and thepolyethylene ﬁlms, and then the medium was recovered. After that, thesubstrates were rinsed by the recovered medium, and then the medium
was recovered again. This rinsing process was repeated at least ﬁvetimes. Next, the recovered medium was diluted 100-fold, and then 1 mLof the 100-fold diluted recovered medium (totally, 1000-fold dilution)
was mixed with 15 mL of 0.035% Pearlcore nutrient agar, which had
been kept at 48 °C, and this medium mixture was put into sterile Petridishes. After solidifying at room temperature, the dishes were put into
an incubator at 35 °C for 48 h. A series of measurements (N = 3) weredone for SiO2 and SnO2 substrates, and 94/84/30 and >250/>250/>250 colonies were observed for SiO2 and SnO2 substrates,
respectively. The bacteria recovery rate (%) was calculated using therelationship: (observed colonies/250) × 100 (Figure 3a). For confocal
microscope images without PBS washing process (Figure 3b), after
peeling oﬀ the polyethylene ﬁlm, 250 μL of PBS including SYTO9(volume ratio: PBS, 1000 μL; SYTO9, 3 μL) was introduced onto thesubstrates. Finally, another polyethylene ﬁlm was placed covering the
SiO2 substrates before incubating at 35 °C for 5 min, followed by
confocal ﬂuorescence observation. For confocal microscope images
with the PBS washing process (Figure 3c), after peeling oﬀ thepolyethylene ﬁlm, 10 mL of PBS, instead of SCDLP culture medium,was introduced onto the substrates, and then, 250 μL of PBS including
SYTO9 (volume ratio: PBS,1000 μL; SYTO9, 3 μL) was introduced
onto the substrates. Finally, another polyethylene ﬁlm was placed
covering the SiO2 substrates before incubating at 35 °C for 5 min,followed by confocal ﬂuorescence observation.
SEM and STEM Imaging and EDS Mapping. The morphology
and the composition of SnO2/SiO2 core−shell nanowires werecharacterized by FESEM, scanning transmission electron microscopy(STEM), and energy dispersive X-ray spectroscopy (EDS). For the
cross-sectional SEM-EDS analysis, we utilized an accelerating voltage of
10 kV. For the single-nanowire STEM-EDS analysis, we used an
accelerating voltage of 30 kV. The EDS mapping 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 Kα (1.739 keV), Sn Lα(3.443 keV), and O Kα (0.525 keV) were chosen to construct theelemental mapping images. The SEM-EDS analysis of B. subtilis
deposited onto SnO2/SiO2 core−shell nanowires was done at anaccelerating voltage of 5 kV. We put B. subtilis solution onto nanowires
on the fused silica substrates and evaporated the solution, and then we
observed the SEM images of B. subtilis. The SEM images were takenfrom raw samples, while the 10 nm Pt deposited samples weresometimes used to discriminate the surface morphologies. The EDS
mapping 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
Kα (1.739 keV), Sn Lα (3.443 keV), O Kα (0.525 keV), and C Kα
(0.277 keV) were chosen to construct the elemental mapping images.
Fabrication of Nanowire Device for Fluid Flow. For fabricationof the microchannel with herringbone structures, negative photoresistof SU-8 (3005, Microchem Corp.) was coated on a 3-in. Si wafer(Cretec Kogyo) using a spinner. After baking at 95 °C for 3 min, themicrochannel was patterned by photolithography. The microchannel
had a length, width, and height of 4000, 200, and 10 μm, respectively.
Then, the substrate was baked at 65 °C for 1 min and next at 95 °C for 2
min. The baked substrate was set on the spinner and coated again with
3005 SU-8. The coated substrate was heated at 95 °C for 2 min, andherringbone structures were patterned by photolithography. Afterbaking at 65 °C for 1 min and 95 °C for 1 min, the SU-8 pattern wasdeveloped using SU-8 developer (Microchem Corp.) and rinsed with 2propanol. Polydimethylsiloxane (PDMS) was poured into the SU-8
mold. After degassing, the mold was heated at 80 °C for 2 h. Nanowires
were fabricated on fused silica substrates by the VLS method. The
nanowire diameter was about 10 nm. The nanowire length wascontrolled by the VLS growth time. The nanowires on the substrate andthe fabricated PDMS microchannel were plasma-treated and bonded toeach other at 80 °C.
Saccharomyces cerevisiae Preparation. S. cerevisiae (S288C,
Open Biosystems) was applied to YPD (yeast extract-peptone-dextrose,
MP Biomedicals, Llc.) agar medium (2% agar in 5% YPD medium), andcolonies were allowed to form. After forming the colonies, a single
colony was put into 5 mL of fresh 5% YPD medium and cultured for 12
h at 30 °C using the multi shaker (shaking speed, 240 rpm). Next, weadded 1 mL of the cultured S. cerevisiae to 3 mL of fresh 5% YPD
medium, and we continued the culturing for 4 h at the same
temperature and shaking speed. Then, we measured OD of S. cerevisiae
solution using the plate reader at 600 nm and diluted the S. cerevisiae
solution using 5% YPD medium to 0.6 of the measured OD. We
centrifuged the diluted S. cerevisiae solution (3 min; 25 °C; 15000g),
discarded the supernatant and suspended the cells in 10 mM phosphate
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ACS Nanosystem. Then, 50 μL of the 60 μL mixed solution was introduced into
the bacteria identiﬁcation system at a ﬂow rate of 5 μL/min. The system
was put on the Peltier heater and heated at 65 °C (Figure 7b). After 60
min, 1 μL of SYBR Green I was introduced into the DNA ampliﬁcation
device.
The Bacteria Identiﬁcation Assay System. The mold for the
DNA ampliﬁcation device was fabricated by an RVS-R1 3D printer(Real Vision Systems Inc.). The mold was 3 mm in diameter and 2 mm
in height. PDMS was poured into the silanized mold and treated as
mentioned in the Fabrication of Nanowire Device for Fluid Flow
section. The bacteria identiﬁcation assay system was fabricated byconnecting the nanowire device and the DNA ampliﬁcation device
using a silicon tube (i.d. 0.64 mm, o.d. 1.19 mm, Helix Medical Llc.).
Peltier Heater. The heater for the microﬂuidic device wascomposed of a TEC-12708 Peltier element, an HLE power supply,
an SP5R7−576 controller, and an SPT-67 thermocouple sensor (allmanufactured by Nihontecmo Co. Ltd.). The thermocouple sensor was
ﬁxed onto the Peltier element using thermal grease. The controller
maintained the temperature at 65 or 94 °C with the accuracy of ±0.3 °C
by proportional-integral-derivative control.

buﬀer. The concentration (1.8 × 107 S. cerevisiae cells/mL) in thesuspended solution was measured using the cell counter.
Quantifying Extracted DNA Concentrations from S. cerevisiae Cells. Extracted DNA concentrations from S. cerevisiae cells were
calculated from measurements by a spectrophotometer (NanoDrop,
Thermo Fisher Scientiﬁc Inc.). We centrifuged 200 μL of the S.
cerevisiae (1.8 × 107 cells/mL) solution (3 min; 25 °C; 15000g),
discarded the supernatant, and suspended the cells in 200 μL of 2.5 mg/
mL Zymolyase (Nacalai Tesque Inc.). Then, we incubated thesuspended solution for 10 min at 37 °C, 10 min at 94 °C, 5 min at−80 °C, and 15 min at 94 °C. We centrifuged the solution (3 min; 25°C; 15000g) and recovered the supernatant as extracted DNA solution.
We applied quantitative PCR (qPCR) to the recovered solution anddepicted a calibration curve for cycle number vs extracted DNA
concentrations. We quantiﬁed extracted DNA concentrations fromyeast cells via the calibration curve.
Loop-Mediated Isothermal Ampliﬁcation (LAMP) Method.
The LAMP method is an isothermal DNA ampliﬁcation method, and it
uses four kinds of primers and six targeted genes. Mixing samples,
primers, DNA polymerase, and substrates were at a constanttemperature (around 65 °C) when the LAMP reaction was initiated.
Ampliﬁcation eﬃciency was high enough to produce 109−1010 copies
for 15−60 min. Speciﬁcity was also high enough to distinguish whether
the targeted genes were in samples or not. The LAMP method was doneusing an E. coli detection kit (Eiken Chemical Co., Ltd.). According to
the kit manufacturer’s instruction manual, each E. coli detection was
performed as follows.
Nanowires at 94 °C (Figure 7a): 50 μL of E. coli-containing solution
was introduced into the nanowire device at a ﬂow rate of 5 μL/min. The
device was put on the Peltier heater and heated at 94 °C. Fifteen μL of
the 50 μL E. coli-containing solution was collected from the outlet of thedevice and mixed with 15 μL of LAMP solution in a sample tube. The
mixed solution was heated at 65 °C. After 60 min, 1 μL of SYBR Green I
was introduced into the sample tube.
Commercially available kit (Figure 7a): 50 μL of E. coli-containing
solution and 200 μL of lysis solution (Eiken Chemical Co., Ltd.) weremixed in a sample tube. The mixed solution was heated at 94 °C for 20
min. Fifteen μL of the heated solution and 15 μL of LAMP solution
were mixed in another sample tube. This second mixed solution washeated at 65 °C. After 60 min, 1 μL of SYBR Green I was introduced
into the sample tube.
Without nanowires at 94 °C (Figure 7a): 50 μL of E. coli-containing
solution was introduced into a device with the chaotic mixer (without
nanowires) at a ﬂow rate of 5 μL/min. The device was put on the Peltier
heater and heated at 94 °C. Fifteen μL of the 50 μL E. coli-containing
solution was collected from the outlet of the device and mixed with 15
μL of LAMP solution in a sample tube. The mixed solution was heatedat 65 °C. After 60 min, 1 μL of SYBR Green I was introduced into thesample tube.
94 °C for 20 min (Figure 7a): 50 μL of E. coli-containing solution washeated at 94 °C for 20 min. Fifteen μL of the heated solution and 15 μL
of LAMP solution were mixed in another sample tube. The mixed
solution was heated at 65 °C. After 60 min, 1 μL of SYBR Green I was
introduced into the sample tube.
Control (no cells) (Figure 7a): 50 μL of solution without E. coli wasintroduced into the nanowire device at a ﬂow rate of 5 μL/min. The
device was put on the Peltier heater and heated at 94 °C. Fifteen μL of
the 50 μL E. coli-containing solution was collected at the outlet of thedevice and mixed with 15 μL of LAMP solution in a sample tube. The
mixed solution was heated at 65 °C. After 60 min, 1 μL of SYBR Green I
was introduced into the sample tube.
200 Escherichia coli cells (Figure 7c): 30 μL of E. coli-containing
solution and 30 μL of LAMP solution were mixed outside the bacteria
identiﬁcation system. Then, 50 μL of the 60 μL mixed solution was
introduced into the bacteria identiﬁcation system at a ﬂow rate of 5 μL/
min. The system was put on the Peltier heater and heated at 65 °C(Figure 7b). After 60 min, 1 μL of SYBR Green I was introduced into
the DNA ampliﬁcation device.
Control (no cells) (Figure 7c): 30 μL of solution without E. coli and30 μL of LAMP solution were mixed outside the bacteria identiﬁcation

ASSOCIATED CONTENT
S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsnano.8b08959.
(1) Schematic of the fabrication procedure for nanowires
in a microchannel; (2) schematic illustrations forexperimental procedure of nanowire-mediated lysis; (3)B. subtilis imaging; (4) the extracted DNA molecules
showing electric ﬁeld response; (5) an FESEM image and
EDS elemental mappings of SnO2 ﬁlm; (6) FESEM
images, EDS elemental mappings, and water contact angleof 30 nm diameter nanowires; (7) FESEM images, EDS
elemental mappings, and water contact angle of 110 nm
diameter nanowires; (8) a TEM image and STEM imagesof nanowires; (9) FESEM images of B. subtilis deposited
onto 30 nm diameter nanowires; (10) FESEM images of
B. subtilis deposited onto 110 nm diameter nanowires;(11) schematic illustrations for DNA extraction using
ﬂuid ﬂow and DNA recovery eﬃciency from the device;(12) nanowire-mediated lysis using the branched nanowires (PDF)
Movie 1: The electrical responsiveness evinced as DNA
elongation and contraction with and without an applied
electric ﬁeld, respectively (MOV)
Movie 2: A simple, rapid and one-step bacteria
identiﬁcation assay system (MOV)AUTHOR INFORMATION
Corresponding Authors*E-mail: yasui@chembio.nagoya-u.ac.jp.
*E-mail: yanagida@cm.kyushu-u.ac.jp.
*E-mail: kawai@sanken.osaka-u.ac.jp.
*E-mail: babaymtt@apchem.nagoya-u.ac.jp.
ORCID

Takao Yasui: 0000-0003-0333-3559
Takeshi Yanagida: 0000-0003-4837-5701
Kazuki Nagashima: 0000-0003-0180-816X
Noritada Kaji: 0000-0002-9828-873X
Author Contributions

T. Yasui, T. Yanagida, T.S., K.N., N.K., T.K., and Y.B. plannedand designed the experiments. T. Yasui, T. Yanagida, K.O., M.T.,
and S.R., performed DNA extraction via nanowire-mediated2271

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lysis and data analyses. T. Yasui, K.N., T.S., A.Y., R.M., and Z.Z.
performed the antibacterial test using fabricated nanowires anddata analyses. T. Yasui, T. Yanagida, M.T., and K.N. performed
FESEM observations. T. Yasui, T. Yanagida, M.T., S.R., andT.N. performed bacteria identiﬁcation assay using the nanowire
device with a DNA ampliﬁcation device based on the LAMP
method. T. Yasui, T. Yanagida, T.S., K.O., M.T., K.N., S.R., T.N.,
D.T., A.Y., R.M., Z.Z., N.K., and M.K. fabricated theexperimental setups. T. Yasui, T. Yanagida, K.N., and N.K.
wrote the paper.
Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS
This research was supported by PREST (JPMJPR151B), JST,the JSPS Grant-in-Aid for Young Scientists (A) 17H04803, the
ImPACT Program of the Council for Science, Technology, andInnovation (Cabinet Oﬃce, Government of Japan), the JSPS
Grant-in-Aid for Scientiﬁc Research (A) 16H02091, and the
Cabinet Oﬃce, Government of Japan and the Japan Society for
the Promotion of Science (JSPS) through the Funding Program
for World-Leading Innovative R&D on Science and Technology(FIRST Program), 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 ResearchCenter for Materials and Devices.”
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