micromachines
ArticleMicrofluidic Formation of Double-Stacked Planar
Bilayer Lipid Membranes by Controlling theWater-Oil Interface
Kan Shoji * ID and Ryuji Kawano *Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology,
2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan* Correspondence: kan-shoji@m2.tuat.ac.jp (K.S.); rjkawano@cc.tuat.ac.jp (R.K.);
Tel.: +81-42-388-7187 (K.S. & R.K.)
Received: 19 April 2018; Accepted: 18 May 2018; Published: 22 May 2018


Abstract: This study reports double-stacked planar bilayer lipid membranes (pBLMs) formed usinga droplet contact method (DCM) for microfluidic formation with five-layered microchannels thathave four micro guide pillars. pBLMs are valuable for analyzing membrane proteins and modelingcell membranes. Furthermore, multiple-pBLM systems have broadened the field of applicationsuch as electronic components, light-sensors, and batteries because of electrical characteristics ofpBLMs and membrane proteins. Although multiple-stacked pBLMs have potential, the formationof multiple-pBLMs on a micrometer scale still faces challenges. In this study, we applied a DCM
strategy to pBLM formation using microfluidic techniques and attempted to form double-stacked
pBLMs in micro-meter scale. First, microchannels with micro pillars were designed via hydrodynamic
simulations to form a five-layered flow with aqueous and lipid/oil solutions. Then, pBLMs weresuccessfully formed by controlling the pumping pressure of the solutions and allowing contact
between the two lipid monolayers. Finally, pore-forming proteins were reconstituted in the pBLMs,
and ion current signals of nanopores were obtained as confirmed by electrical measurements,
indicating that double-stacked pBLMs were successfully formed. The strategy for the double-stacked
pBLM formation can be applied to highly integrated nanopore-based systems.
Keywords: microfluidic lipid bilayer formation; multiple lipid bilayers; water-oil phase flow

1. Introduction
Planar bilayer lipid membranes (pBLMs) are powerful tools for measuring ion channels [1,2],
pore-forming proteins [3,4], peptides [5–7], and synthetic channels [8,9], owing to their transport
activities that can be evaluated via electrical measurements. Furthermore, nanopore sensing withbiological nanopores reconstituted into the pBLM offers an opportunity for extremely sensitive andselective biosensors by measuring the change of ion current caused by the reversible binding ortranslocation of a single molecule in a single nanopore. These sensors are attracting attention as a DNA
sequencer [10,11] and a detection method of biomolecules [12,13] because of their high sensitivity
and molecular selectively. Recently, a combination of nanopore sensing and DNA computing [14,15]
and nanopore systems with multiple-stacked pBLMs [16–19] have been reported, and they led tomulti-functional sensors and cascade type nanopore-based calculators. Especially, multiple-pBLM
systems will broaden the field of application such as electronic components, light-sensors, and batteries
because of electrical characteristics of pBLMs and membrane proteins such as high capacitance andresistance of pBLMs and permselectivity of membrane proteins [16–18]. We also reported a stacked
biofuel cell system in which each cell is isolated by pBLMs to apply as the battery of bio-robots [17].

Micromachines 2018, 9, 253; doi:10.3390/mi9050253

www.mdpi.com/journal/micromachines

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Although the multiple-stacked pBLMs have potential, few methods to form multiple-stacked pBLMs
in micro-meter space have been considered. pBLMs are generally prepared using the Montal–Mueller
method, wherein two lipid monolayers formed at the air-water interface are attached in the hole ofa Teflon wall (Figure S1a). This method is very time-consuming, and the membrane is not stable.
Therefore, in the last decade, numerous studies have attempted to use microfluidic technologies toimprove the throughput of the pBLM formation [20–26]. Arrayed pBLMs are automatically formedby sequentially flowing aqueous and lipid/oil solutions through microchannels comprising a main
flow channel and closed chambers (Figure S1b). Furthermore, microfluidic technologies have been
applied not only to provide high-throughput platforms but also to increase the stability of the pBLMs
and to reduce the noise level for electrophysiological measurements, and to be able to flush/refresh
solutions [27–31]. However, these microfluidic methods have not been applied to the formationof multiple-stacked pBLMs. On the other hand, the droplet contact method (DCM) [22,32–36] hasbeen reported to prepare stable pBLMs. Using this method, pBLMs are formed by contacting two
droplets that are surrounded by a lipid monolayer (Figure S1d). The durability of the membranes
formed using DCM is improved; pBLMs can be maintained for a long time (2 weeks) and are stableagainst mechanical vibration [36], because much of the lipid/oil solution is located around the areaof lipid bilayer, and lipid molecules can be supplied and supported to the membrane region (FigureS1d). Furthermore, multiple-stacked pBLMs formed by DCMs have been reported [32,33,37,38].
Although stable pBLMs can be prepared by the DCM-based formation, DCM-based methods havesome disadvantages for stacking pBLMs. First, the distance of each pBLM becomes more than hundredsof micrometers. Because the running time of nanopore-based cascade systems depends on the distance,
we have to miniaturize the nanopore device to improve the running time. However, the electrodes
insertion for measurements of ion currents must be required, and the size of droplets should be bigger
than the one of electrodes. In fact, the size of droplets was about 500 µm in diameter, and the gap ofpBLMs was also 500 µm in the DCM-based microfluidic formation of stacked pBLMs [38]. Second, theinner solution in micrometers scale droplets cannot be exchanged. Although some solution exchanges
and, additionally, chemical injections would be required to develop nanopore-based systems withmultiple chemical reactions and sensing, exchanging the inner solution is very difficult, because
droplets are isolated from other aqueous solutions by being surrounded with an oil solution.
Therefore, we propose an approach for pBLM formation based on the strategy of lipid monolayer
contacting in a microfluidics system (Figure 1a) and present a method for the double-stacked pBLM
formation in a microfluidic device by optimizing the geometry of the guide pillars (Figure 1b). Thismethod promises to prepare narrower gapped pBLM stacks than conventional droplet-based methods
and pBLMs with open flow channels on both sides. To construct double-stacked pBLMs, first, we
proposed to build guide pillars for the formation of a five-layered flow comprising aqueous andlipid/oil solutions. Next, we designed the microchannel with guide pillars using a hydrodynamic
simulation. Then, we formed double-stacked pBLMs in a five-layered microchannel by decreasing theflow pressure after the five-layered flow was established. Formation of stable pBLMs in microchannels
allowed us to form double-stacked pBLMs. Finally, we reconstituted pore-forming proteins in thepBLMs and measured the channel current through the two membranes.

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Figure 1. Concept of our formation method of pBLMs. (a) By applying the strategy of the DCM to
Figure 1. Concept of our formation method of pBLMs. (a) By applying the strategy of the DCM topBLM formation using microfluidic techniques, a pBLM with open flow channels on both sides canpBLM formation using microfluidic techniques, a pBLM with open flow channels on both sides can be
be prepared. (b) Double-stacked pBLMs can be formed using our method.
prepared. (b) Double-stacked pBLMs can be formed using our method.

2. Materials and Methods
2. Materials and Methods
2.1. Regents
Regents andand Chemicals
Chemicals
2.1.
1,2-Diphytanoyl-sn-glycero-3-phosphocholine(DPhPC;(DPhPC;
Avanti
Polar
Lipids,Alabaster,AL,
1,2-Diphytanoyl-sn-glycero-3-phosphocholine
Avanti
Polar
Lipids,Alabaster,
AL, USA)USA)
and hexadecane
ChemicalIndustries,
Osaka,
Japan)
wereusedthelipid/oil
lipid/oilandhexadecane(Wako(Wako
Pure Pure
ChemicalIndustries,
Ltd.,Ltd.,
Osaka,
Japan)
wereusedasasthesolution.
Potassiumchloride(KCl;
NacalaiTesque,Kyoto,
Japan)
and3-morpholinopropane-1solution. Potassium chloride (KCl; Nacalai Tesque, Kyoto, Japan) and 3-morpholinopropane-1-sulfonicsulfonic
acid Nacalai(MOPS; Tesque,Nacalai Kyoto,
Tesque,Japan)
Kyoto,were
Japan)
wereused
as the aqueous(1.0 10
M mM
KCl,
acid
(MOPS;
usedas theaqueoussolution solution(1.0 M KCl,
10
mM
MOPS,
pH
7.0).
The
MOPS
solutionwas
preparedusing
ultrapure
water
from
a
Milli-Q
MOPS, pH 7.0). The MOPS solution was prepared using ultrapure water from a Milli-Q (Merck(Merck Millipore
Corp., Billerica,
MA,USA). Alpha-hemolysin(αHL; Sigma-Aldrich,
St. Louis,
MilliporeCorp., Billerica,
MA, USA).
Alpha-hemolysin(αHL; Sigma-Aldrich,
St. Louis, MO,
USA) MO,
was
USA)
was
obtainedasamonomerproteinisolatedfrom
Staphylococcus
aureus
in
the
form
of
a
powder,obtained as a monomer protein isolated from Staphylococcus aureus in the form of a powder, dissolved
dissolved
at a concentrationof 1inmg/mL
in ultrapure
stored−80use,°C. Foruse, samples
wereat
a concentrationof 1 mg/mL
ultrapurewater, andwater,
storedandat −80 ◦ C.atFor
samples
were diluted
dilutedinthedesignatedconcentrationusingabufferedelectrolyte
solutionandstored
at
4
°C.
SU-8
in the designated concentration using a buffered electrolyte solution and stored at 4 ◦ C. SU-8 3025
3025 (MicroChem
Corp.,
Newton,USA)useda photoresist.Polydimethylsiloxane
Polydimethylsiloxane (PDMS;(PDMS;(MicroChem
Corp.,
Newton,MA,MA,
USA)
waswasused
as asa photoresist.
Sylgard184,
Dow
Corning
Toray
Co.,
Ltd.,
Tokyo,
Japan)
was
used
for
the
materialofmicrochannels.
Sylgard 184, Dow Corning Toray Co., Ltd., Tokyo, Japan) was used for the material of microchannels.
2.2. Concept
Concept ofof Double-Stacked
Double-Stacked pBLM
pBLM Formation
Formation Using
Using Five-Layered
Five-Layered Microchannels
Microchannels
2.2.
The five-layered
five-layered microchannels
confluentarea
joiningallThe
microchannels comprised
comprisedfive
fiveinlets,
inlets,five
fiveoutlets,
outlets,area
area
confluentarea
joiningchannels,andandguidepillars
in theconfluentarea.
In themicrochannels,
double-stacked
pBLMs
are
all
channels,guidepillars
in theconfluentarea.
In themicrochannels,
double-stacked
pBLMs
formedasfollows.
First,
theaqueousandlipid/oilsolutions
flow
to
each
channeltoformafiveare formed as follows. First, the aqueous and lipid/oil solutions flow to each channel to form alayered flowflow
withwith
threethree
aqueoussolutionlayersandandtwotwolipid/oilsolutionlayersin thearea
five-layered
aqueoussolutionlayerslipid/oilsolutionlayersinconfluent
the confluent(Figure2a). Atthat
spontaneously
form atformthe interfaces
betweenbetween
the aqueousarea(Figure2a).
Attime,that lipid
time,monolayers
lipid monolayers
spontaneously
at the interfaces
theandlipid/oilsolutions.
Then,
the
flow
pressures
decreasetostabilizetheinterfaces
betweentheaqueous and lipid/oil solutions. Then, the flow pressures decrease to stabilize the interfaces between
aqueousandandlipid/oilsolutions(Figure2b).2b).
Finally,thethelipidmonolayers
arearecontacted
in in
thethegaps
oftheaqueouslipid/oilsolutions(FigureFinally,lipidmonolayers
contacted
gaps
pillarsbyreducing
theflowpressureofthelipid/oilsolutions(Figure2c).
Asaresult,pBLMs
areof pillars by reducing the flow pressure of the lipid/oil solutions (Figure 2c). As a result, pBLMs areformed,and
andmembrane
membraneproteins
be reconstituted
topBLMs
the pBLMs(FigureTodouble-stacked
form doubleformed,
proteins
cancanbe reconstituted
to the(Figure2d). To2d).
form
stackedusing
pBLMs
abovewemethod,weguideset micro
pillars with hydrophobicity
at thepBLMs
theusing
abovethemethod,set micro
pillarsguidewith hydrophobicity
at the midpointsofmidpointsofthechannelforincreasing
the
area
of
the
hydrophobic
surface.
The
guidepillarshave
the channel for increasing the area of the hydrophobic surface. The guide pillars have a dual functiona
dual
functionis to guidethe lipid/oil
solution
along
their surfaceof theirasfollows.
Oneasisfollows.
to guideOne
the lipid/oil
solutionalong theirsurfacebecauseof their becausehydrophobicity.
hydrophobicity.
Secondthecontact
aperture
forofthearea of two Restricting
lipid monolayers.
Restricting
theSecondis the aperture
foristhearea
twocontact
lipid monolayers.
the contact
area of thecontact
area
of
the
two
lipid
monolayers
mayincreasethestability
of
the
BLMs.
two lipid monolayers may increase the stability of the BLMs.

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(a)

(b)

(c)

(d)

Figure 2. Schemes of double-stacked pBLM formation processes using the five-layered microchannel
Figure 2. Schemes of double-stacked pBLM formation processes using the five-layered microchannel
with guide pillars. (a) Aqueous solutions and lipid/oil solutions flow alternately in each channel, andwith guide pillars. (a) Aqueous solutions and lipid/oil solutions flow alternately in each channel,
the five-layered flow is formed. (b) The flow pressure of all the solutions is decreased. (c) Lipidand the five-layered flow is formed. (b) The flow pressure of all the solutions is decreased. (c) Lipidmonolayers are contacted by decreasing the flow pressure of lipid/oil solutions, and pBLMs aremonolayers are contacted by decreasing the flow pressure of lipid/oil solutions, and pBLMs areformed. (d) Pore-forming proteins are reconstituted to the pBLMs, and the channel current throughformed. (d) Pore-forming proteins are reconstituted to the pBLMs, and the channel current through thethe paralleled pBLMs can be measured.
paralleled pBLMs can be measured.

2.3. Hydrodynamic Simulation of Five-Layered Microchannels
2.3. Hydrodynamic Simulation of Five-Layered Microchannels
The behaviors of aqueous and lipid/oil solutions in the confluent area with and without the
Thepillars
behaviors
aqueous withand lipid/oilin theconfluentareaInc.,withand without
guide
guide
wereofsimulated
COMSOLsolutions
Multiphysics
4.2a(COMSOL
Burlington,
MA,theUSA)pillars
with
COMSOL
Multiphysics
4.2a (COMSOLInc.,
Burlington,
MA, USA)flow
using
usingwere
the simulated
microfluidics
moduleto ascertain
the effectsof the guidepillars
for multi-layered
theformation.
microfluidics
moduleandto ascertain
effectsof theguidepillars
for multi-layered
formation.
The density
viscosity theof theaqueousandlipid/oilsolutionswere set to flow
1.0 g/mLand1.0density
mPa·s andg/mLofandmPa·s,and
respectively.
The boundary
betweenThe
and 0.77
viscositythe3.34
aqueouslipid/oil solutions
weretension
set to 1.0
g/mL theandaqueous
1.0 mPa·s
andlipid/oilwas ·15mN/m [39]. The
angle
betweenthe channel
and theaqueous
and0.77
g/mL solutions
and 3.34 mPas, respectively.
The contact
boundary
tensionbetweenthe aqueous
andlipid/oilsolutionwas
was15
3/4mN/m
π. The[39].
contactwasangle
determinedbythe
observinginterface
betweenaqueoussolutions
Theangle
contact
betweenchanneltheandthe aqueous
solutionwasandlipid/oilsolutionsinaPDMS
microchannel.
The
flow
rate
was
estimatedwiththeconditionof
3/4 π. The contact angle was determined by observing the interface between aqueous and lipid/oil
20 mbarinflow
pressure
and set to 10
μL/hforrateall channels.
The flowof theofaqueousandsolutionsa PDMS
microchannel.
Theflow
was estimatedwithbehaviors
the condition
20 mbarflow
lipid/oilsolutions
weresimulatedwithandwithout
guidepillars,andtheresults
werecompared.
pressure and set to 10 µL/h for all channels. The flow behaviors of the aqueous and lipid/oil solutions
Furthermore,
dependencyof flow
behaviorsthetheguidepillarwere
size wassimulatedfor designingthe
were
simulatedthewithand without
guidepillars,onandresults
compared.
Furthermore,
the guide pillars.
dependency of flow behaviors on the guide pillar size was simulated for designing the guide pillars.
Fabrication
Five-LayeredMicrochannels
Microchannels
2.4.2.4.
Fabrication
ofofFive-Layered
The microchannels were fabricated by standard photolithography techniques. SU-8 3025 was
The microchannels were fabricated by standard photolithography techniques. SU-8 3025 was used
used as the master photoresist. Film masks were fabricated with a spot diameter and drawing pitch
as the master photoresist. Film masks were fabricated with a spot diameter and drawing pitch for a
for a laser of 4 and 1 μm, respectively, for the photomasks. An exposure masking system (UV-KUB
laser of 4 and 1 µm, respectively, for the photomasks. An exposure masking system (UV-KUB 2, KLOE,
2, KLOE, Montpellier, France) was used for UV exposing. The channels were fabricated by PDMS
Montpellier, France) was used for UV exposing. The channels were fabricated by PDMS molding.
molding. PDMS was cured for 2 h at 95 °C. The PDMS channels were bonded to a cover glass (NEOPDMS was cured for 2 h at 95 ◦ C. The PDMS channels were bonded to a cover glass (NEO cover glass,cover glass, Matsunami Glass Ind., Ltd., Osaka, Japan) using plasma etching equipment (FA-1, Samco
Matsunami
Glass
Ind.,
Ltd.,
Osaka,values
Japan)areusing
plasmaetchingequipment
SamcoInc.,flow
Kyoto,
Inc., Kyoto,
Japan).
The
designedshown
in Figure
3 andFigure S3. (FA-1,
The widthof eachJapan).
The
designedvaluesareshownin
Figure3
andFigure
S3.
The
width
of
each
flow
channelchannel was 40 μm. The width and length of the confluent area were 360 and 100 μm, respectively.
was
40tipsµm.ofThewidth
andwerelengthof the confluent
area were
andof100
respectively.
The tips of
The
guide
pillars
designed
1 mm flat shape.
The360heightall µm,channels
was 20 μm.
guide pillars were designed 1 mm flat shape. The height of all channels was 20 µm.

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Figure3. (a)
Design ofof thethe five-layered
five-layered microchannel.
microchannel. (b)Experimental setup
setup forfor double-stacked
double-stacked
Figure 3.
(a) Design(b) Experimental
pBLM
formation.
pBLM formation.

2.5. Microfluidic Experiment of Double-Stacked pBLM Formation
2.5. Microfluidic Experiment of Double-Stacked pBLM Formation
The experimental setup for the double-stacked pBLM formation and the photograph and the
The experimental setup for the double-stacked pBLM formation and the photograph andmicroscopic image of the five-layered microchannel are shown in Figures 3b and 4a.
the microscopic image of the five-layered microchannel are shown in Figures 3b and 4a.
DPhPC/hexadecane(1 mM) was prepared as the lipid/oil solution, and 1 M KCl (10 mM MOPS,
DPhPC/hexadecane (1 mM) was prepared as the lipid/oil solution, and 1 M KCl (10 mM MOPS,
pH 7.0) was used as the aqueous solution. Each solution flowed with a pressure pump (MFCS-EZ,
pH 7.0) was used as the aqueous solution. Each solution flowed with a pressure pump (MFCS-EZ,
Fluigent, Paris, France). The PDMS microchannels were coated with a fluorochemical coating
Fluigent, Paris, France). The PDMS microchannels were coated with a fluorochemical coating solution
solution (NovecTM 1720, 3 M, St. Paul, MN, USA) to uniformize the surface properties of the PDMS
(NovecTM 1720, 3 M, St. Paul, MN, USA) to uniformize the surface properties of the PDMS andand glass surfaces in the microchannel and prevent the adherence of lipid molecules to the surface ofglass surfaces in the microchannel and prevent the adherence of lipid molecules to the surface of thethe microchannel. The microchannels were filled with the solution by insertion with a micropipettemicrochannel. The microchannels were filled with the solution by insertion with a micropipette andand dried at◦100 °C for 20 min. The contact angles of the aqueous solution and the PDMS surface
dried at 100 C for 20 min. The contact angles of the aqueous solution and the PDMS surface before
before and after coating were107° and 105°, respectively. The one of the DPhPC/hexadecane and theand after coating were 107◦ and 105◦ , respectively. The one of the DPhPC/hexadecane and the PDMSPDMS surface before and after coating were
26° and 27°, respectively.
surface before and after coating were 26◦ and 27◦ , respectively.
First, each solution was flowed to form a five-layered flow at 250 mbar of pumping pressures.
First, each solution was flowed to form a five-layered flow at 250 mbar of pumping pressures.
After confirming the formation of five-layered flow, all pumping pressures were gradually reduced
After confirming the formation of five-layered flow, all pumping pressures were gradually reduced
to 20 mbar while maintaining the five-layered flow. Finally, the pumping pressure of the aqueous
to 20 mbar while maintaining the five-layered flow. Finally, the pumping pressure of the aqueous
solutions was fixed at 20 mbar, and the pumping pressure of the lipid/oil solutions was graduallysolutions was fixed at 20 mbar, and the pumping pressure of the lipid/oil solutions was gradually
reduced until interfaces were contacted on the gap of pillars. All pressure control was carried out
reduced until interfaces were contacted on the gap of pillars. All pressure control was carried out
manually. Microchannel observation was performed using an inverted microscope (AXJ-5300TPHFL,
manually. Microchannel observation was performed using an inverted microscope (AXJ-5300TPHFL,
WRAYMER Inc., Osaka, Japan) mounted with a microscopic digital camera (FLOYD, WRAYMERWRAYMER Inc., Osaka, Japan) mounted with a microscopic digital camera (FLOYD, WRAYMER Inc.,
Inc., Osaka, Japan).
Osaka, Japan).
2.6.
the Double-Stacked
Double-Stacked pBLMs
pBLMs
2.6. Reconstitution
Reconstitution ofof Membrane
Membrane Protein
Protein toto theTo
confirm thethe formation
formation ofof double-stacked
double-stacked pBLMs,
pBLMs, pore-forming
pore-forming membrane
To confirm
membrane proteinsproteins were
werereconstituted
reconstituted inin thethe double-stacked
double-stacked pBLMs.
pBLMs. αHL
αHL waswas used
used asas thethe pore-forming
pore-forming membrane
membrane protein.
protein.
Moreover,
M KCl
KCl (10(10 mMmM MOPS,
MOPS, pHpH 7.0)7.0) containing
containing 10
10 nM
nM αHLαHL andand 11 mg/mL
mg/mL bovine
bovine serum
serum
Moreover, 11 M
albumin(BSA;
Sigma-Aldrich,
St.
Louis,
MO,
USA)
was
used
for
all
aqueoussolutions.
BSA
wasalbumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) was used for all aqueous solutions. BSA was
utilized
for preventing
the adhesion
adhesion ofof proteinsproteins inin thethe microchannel.
microchannel. DPhPC/hexadecane
DPhPC/hexadecane (1utilized forpreventing the(1 mM)mM) was
was
used
as
the
lipid/oilsolution.
Ag/AgCl
electrodes
were
used
for
channelcurrent
recording,used as the lipid/oil solution. Ag/AgCl electrodes were used for channel current recording, andand thetheelectrodes
inserted into
into thethe reservoir
reservoir ofof thethe pressurepressure pump.
pump. Channel
Channel currents
currents at
at +120+120 mVelectrodes were
were inserted
mV wereweremonitored
(Pico
2, 2,
Tecella,Foothill
Ranch,
CA,CA,
USA)
withwith
a 7.9akHz
monitored using
using aa patch-clamp
patch-clampamplifier
amplifier(Pico
Tecella,Foothill
Ranch,
USA)
7.9 lowkHz
passfilteratasampling
frequency
of
40
kHz.
low-pass filter at a sampling frequency of 40 kHz.
3. Results and Discussion
3.1. Hydrodynamic Simulation for Designing the Double-Stacked BLMs
To confirm the effect of guide
guide pillars for formation of the five-layered flow and design thegeometries of the microchannels, a hydrodynamichydrodynamic simulation
simulation for the microfluidic behaviors of thegeometries
aqueous and lipid/oil solutions was performed, because the stable five-layered flow of the aqueousand lipid/oil solutions is very important as the pre-state for the stacked pBLM formation in ourMicromachines 2018, 9, 253

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6 of 12aqueous and lipid/oil solutions was performed, because the stable five-layered flow of the aqueous
andlipid/oilis very
as behaviors
the pre-state
forandthewithout
stackedguide
pBLM
formationin ourmethod.
First, solutions
we confirmed
theimportant
microfluidic
withpillars,
as showninmethod.
we confirmed
the microfluidic
withdidandnotwithout
guide pillars,
as shown
inFigure 4 First,
and Video
S1. Without
guide pillars,behaviors
all solutions
flow straightand flowed
to theFigure4 and Video
S1. Without
guidepillars,
all solutions
did not
flowandstraight
and(red)flowedto theother channels,
becausethe surface
tensionbetweenthe aqueous(blue)
lipid/oilsolutionsotherchannels,
becausethesurfacetensionbetweentheaqueous(blue)
andlipid/oil(red)solutionsisis different (Figure 4a). Under microfluidic experiments, the surface tensions strongly influence thedifferent(Figure 4a).
Under microfluidic
experiments,
the surface
tensions strongly
influence thelipid/oil
fluidic
fluidic behaviors
compared
to macro-scaleconditions.
To configurea straight-flowing
behaviors
comparedtomacro-scaleconditions.
To
configureastraight-flowing
lipid/oilsolution,solution, we set the guide pillars at the midpoints of the channels for increasing the areaweofset
the guide pillars
at (Figure
the midpointsof thechannels
the area offlowedhydrophobic
surfaceshydrophobic
surfaces4b). With
guidepillars,fortheincreasing
lipid/oil solutions
along theguide
(Figureguide pillars,
lipid/oilalong ofthetheguidepillars, and a of
five-layered
pillars, 4b).
and Witha five-layered
flowthewasformedsolutions(Figure flowed4b). Becausehydrophobicity
the guideflow
wasformed(Figure4b).
Becauseofthehydrophobicity
of
the
guide
pillars,
thesurfaceforce
pillars, the surface force between the guide pillars and the lipid/oil solutions becomes strong,
betweenpillars and the lipid/oil
solutionsbecomesstrong,resultingin thethestraight-flowing
resultingthe
in guide
the straight-flowing
path to be
maintained.
Somegroupsreported
two or threepathtobemaintained.
Somegroupsreportedthetwoor
three-layered
flowingwithaqueousand oillayered flowing with aqueous and oil solutions by modifying the surface [40] or by fabricating
solutionsbymodifyingthesurface[40]
orbyfabricating
groovesinthechannels[41].
By
comparinggrooves in the channels [41]. By comparing with these reports, we can fabricate our devices muchwithreports, we can fabricate our devices much more simply.
morethese
simply.

(a)

(b)

(c)

(d)

(e)

Figure 4. Simulation results of microfluidic behaviors (a) without guide pillars and with guide pillars
Figure 4. Simulation results of microfluidic behaviors (a) without guide pillars and with guide pillars
with aa width
width ofof (b)(b) 1010 µm,
μm, (c)(c) 2020 µm,
μm, (d)(d) 1010 µm
μm andand aa gap
gap ofof 30
30 µm,
μm, andand (e)(e) 1010 µm
μm andand withwith aa gap
gap of
of
with
10
μm).
In
the
conditionsof(a,c,d),allsolutions
were
jumbled.
Intheconditionsof(b,e),
allsolutions
10 µm). In the conditions of (a,c,d), all solutions were jumbled. In the conditions of (b,e), all solutions
flowed straight,
straight, andand aa five-layered
five-layered flow
flow was
was formed.
formed. Bluecolors show
show thethe aqueousaqueous andandflowedBlue andand redred colors
lipid/oil solutions.
lipid/oilsolutions.

Next, we simulated the geometry of the guide pillars, including the width and gap distance. The
Next, we simulated the geometry of the guide pillars, including the width and gap distance.
pillar width was changed from 10 to 15 μm and from 15 to 20 μm (Figure 4c). In the case of 20 μm
The pillar width was changed from 10 to 15 µm and from 15 to 20 µm (Figure 4c). In the case of 20 µm
width, the straight-flowing lipid/oil solution was disturbed (0.4 to 0.5 s in Figure 4c), because thewidth, the straight-flowing lipid/oil solution was disturbed (0.4 to 0.5 s in Figure 4c), because thewidth was very large. In a similar fashion, straight flow was not observed in the case of a 15 μm width
width was very large. In a similar fashion, straight flow was not observed in the case of a 15 µm width
pillar. From these results, the pillar width should be designed to 10 μm. The gap distance is also
pillar. From these results, the pillar width should be designed to 10 µm. The gap distance is also
important, because this is where the pBLM is formed; we changed the gap distance from 5 toimportant, because this is where the pBLM is formed; we changed the gap distance from 5 to 10 µm
10 μm and from 10 to 30 μm. Figure 4d,e shows the results of the 30 and 10 μm gap system. With aand from 10 to 30 µm. Figure 4d,e shows the results of the 30 and 10 µm gap system. With a 30 µm
30 μm pillar gap, aqueous solution layers break through the lipid/oil layer at the gap; therefore, fivepillar gap, aqueous solution layers break through the lipid/oil layer at the gap; therefore, five-layered
layered flows were not maintained (Figure 4d). With the 10 μm pillar gap, although the aqueous
flows were not maintained (Figure 4d). With the 10 µm pillar gap, although the aqueous layers brokelayers broke the lipid/oil layer at the gap in the initial stage, the five-layered flow formed and was
the lipid/oil layer at the gap in the initial stage, the five-layered flow formed and was maintained
maintained with continuing flow (Figure 4e). As expected, with a 5 μm pillar gap, a five-layered flow
also formed. From these results, we decided on the use of pillar width and gap distances of 10 μm.

Micromachines 2018, 9, 253

7 of 12with continuing flow (Figure 4e). As expected, with a 5 µm pillar gap, a five-layered flow also formed.
From these results, we decided on the use of pillar width and gap distances of 10 µm.
3.2. Formation of Double-Stacked pBLMs Using Five-Layered Microchannels
Based on the optimal conditions determined from the hydrodynamic simulation, we attempted
to form double-stacked pBLMs using five-layered microchannels (Figure 5 and Video S2). First,
a five-layered flow comprising three aqueous and two lipid/oil solution layers was formed using the
microfluidic device (Figure 5a,b). After the five-layered flow formed, the flow speed of all channels
was reduced by decreasing the pumping pressure from 200 to 20 mbar for stabilizing the interfaces(Figure 5c). At that time, the width of the lipid/oil solution decreased in the gap area, and the interfaces
seemed to be stable. Then, the two aqueous–oil interfaces in the gap were contacted and formed a lipid
bilayer (as confirmed in the next section) by decreasing the pumping pressure of lipid/oil solutions(Figure 5d). Furthermore, when we changed the number of pillars from 2 to 4, five-layered flow anddouble-stacked pBLMs were formed (Figure S4, Video S3). While it is very difficult to observe thesurface of pBLMs in our system, because the pBLMs were vertically placed, we expected that theshape of pBLMs is almost square and the pBLMs are surrounded by annulus by reference to a previous
work to form the free-standing pBLM by using a microchannel [31]. By contrast, without guide pillars,
five-layered flow was not formed as expected from the simulation. Previously, some double-stacked
pBLMs have been reported using the droplet-based method in microchannels [32,33,37,38]; our method
has significant advantages (the shorter gap distance (150 µm) of each pBLM is obtained, and 8open
Micromachines 2018, 9, xof 14
flow channels are constructed on both sides of pBLMs). First, we discussed the success rate of thestackedpBLM
formation.
The success
rate
tothe
form
double-stacked
wasnotaround50%. Although
both
sides
of pBLMs
continuously
flow
and
shape
of the pillar pBLMs
tips could
be controlled
due tothe low
five-layered
flow can bewith 100% successrate, almost
all failures
werewasraisedwhen
thespatial resolutionof formed
the photolithography
instruments,
the shorter
lifetime
obtained.
interfaces
were
contacted.
In
this
study,
we
could
notdiscuss
the
shape
of
the
pillaredge
becauseof
However, the stacked pBLMs that are placed in a micro space and have flow channels on their boththe lowerof theTomanufacturingsystems.
improvethe successrate, theof
shape
of
sides
havespatial
never resolution
been reported.
improve the lifetimeofTostackedpBLMs,
improvement
fluidic
pillar should be
However,wehighercan easily
andrapidly reattempt
to form double-stacked
controllability
at optimized.
low flow speed
area andspatialresolution
to get the designed
shape of pillars
pBLMs
by flashingall solutions at equipment.
high pumping pressure flowing.
arerequiredusing state-of-the-art

(b)

(a)

(c)

(d)

Figure 5. Photograph and microscopic images of the microchannel. (a) The photograph of the
five-layered microchannel. The insert shows the microscopic image of the confluent area of the channel.
5555555
The five-layered flow of aqueous and lipid/oil solutions at (b) high pumping pressure (>200 mbar)and (c) low
pumpingpressure (<20mbar). (d) Reconstituted
The parallel arrayof planarlipid bilayers
formed in the
3.3. Channel
Current
Measurementof Nanoporesin theDouble-Stacked
BLMs
micro space. The inset shows the enlarged photograph of the lipid bilayer.

We demonstrated channel current measurements of αHL through double-stacked pBLMs(Figures 6 and S6) to confirm the formation of pBLMs. Although the formation of pBLMs is usuallyconfirmed by measuring membrane capacitances of pBLMs, we evaluated the formation of pBLMs
by the reconstitution of αHL pores, because it is difficult to show the doubled-stacked structure fromthe membrane capacitance. Initially, the current was 0 pA when both or one of the pBLMs were notformed. Then, when both pBLMs formed and αHLs were reconstituted in the double-stacked pBLMs,

Micromachines 2018, 9, 253

8 of 12

Next, the lifetime of the pBLM was examined. The pBLM remained at the interspace between
guide pillars for ca. 2 h (Figure S5). Compared with other methods for the pBLM formation such as
the millimeter scale DCM (2 weeks) [36], the droplet-based method in microfluidic channels (12 h) [38]and freestanding pBLMs (36 h) [30], the lifetime of the pBLMs was shorter. Because the solutions at
both sides of pBLMs continuously flow and the shape of the pillar tips could not be controlled dueto the low spatial resolution of the photolithography instruments, the shorter lifetime was obtained.
However, the stacked pBLMs that are placed in a micro space and have flow channels on their bothsides have never been reported. To improve the lifetime of stacked pBLMs, improvement of fluidic
controllability at low flow speed area and higher spatial resolution to get the designed shape of pillars
are required using state-of-the-art equipment.
3.3. Channel Current Measurement of Nanopores Reconstituted in the Double-Stacked BLMs
We demonstrated channel current measurements of αHL through double-stacked pBLMs (Figure 6
and Figure S6) to confirm the formation of pBLMs. Although the formation of pBLMs is usuallyconfirmed by measuring membrane capacitances of pBLMs, we evaluated the formation of pBLMs
by the reconstitution of αHL pores, because it is difficult to show the doubled-stacked structure fromthe membrane capacitance. Initially, the current was 0 pA when both or one of the pBLMs were notformed. Then, when both pBLMs formed and αHLs were reconstituted in the double-stacked pBLMs,
step-like currents were clearly observed, as shown in Figure 6a. One step indicates a single αHL
reconstitution. In our systems, current can be observed when αHLs form pores in both pBLMs [38].
Next, we estimated the number of pores in each pBLM using the model of equivalent circuit (Figure 7).
In the case of a single pBLM with αHL pores, the electrical circuit of the nanopore system is shownin Figure 7a, and the total resistance (RS ) of the single pBLM with nanopores is calculated using
Equation (1).
RS = RE + RP /n(1)
In Equation (1), RE and RP indicate the resistance of the electrolysis solution and a single nanopore,
respectively, and n is the number of pores. Because the resistance of electrolysis solution is sufficiently
smaller than that of one nanopore, R_E can be ignored. Commonly, the αHL pore has a constantconductance of 1 nS in 1 M KCl, resulting in each step of the ion currents being almost constant at
120 pA under application of 120 mV. As a result of checking the conditions in this study, the ion
current steps were also shown to be almost 120 pA from a single pBLM formed by a three-layered
microchannel (Figure 7b and Figure S7). On the other hand, in the case of double-stacked pBLMs with
αHL pores, the step values were not constant. An equivalent circuit can predict this phenomenon,
described as follows (Figure 7c). The total resistance in the double-stacked pBLMs with nanopores isshown in Equation (2).


RP
RP
+
(2)
R = RE +nAnB
In Equation (2), nA and nB are the number of pores in pBLMa and pBLMb, respectively; RE canbe ignored as in Equation (1), and αHL shows 1 nS conductance (Figure 7d). Using Equation (2), theion current that depends on the number of pores can be calculated, and the inconsistent step current
can be explained by how many αHL reconstituted in two different pBLMs (see detailed description
in Table S1). From these calculation results, we estimated the pore number in each pBLM as shownin Figure 6b,c. In the estimation of pore formation, single pores were initially reconstituted in bothpBLMs, and ~60 pA current was obtained. Then, pores were randomly reconstituted one-by-one ineach of the pBLMs, as shown in Figure 6c. The estimated currents from the model circuit could be
predicted well using the experimental results.

Micromachines 2018, 9, xMicromachines 2018, 9, 253

9 of 129 of 12(a)

Micromachines 2018, 9, x

(b)

(b)

10 of 14

(c)

(a)

(c)

Figure 6. (a) Ion current signals of αHLs reconstituted in the double-stacked pBLMs. The channel

Figure6. 6.(a)(a)Ion
signalsof αHLs
reconstituted
inin
thethedouble-stacked
pBLMs.
The
FigureIoncurrent
current
signals
αHLs
reconstituted
double-stacked
pBLMs.
Thechannel
channelcurrent
signalwas
0 pA before
theofdouble-stacked
pBLM wasformed.
Then, the ion current
increasedcurrent
signalwas
0
pA
beforethedouble-stackedpBLM
wasformed.
Then,
the
ion
current
increasedbycurrent
signal was 0 αHLs
pA before
double-stackedpBLM
wasformed.
Then, theionobtained.
current increased
by reconstituting
in thethedouble-stacked
pBLMs,
andsevenstep signals
were(b)reconstituting
αHLs
in
the
double-stacked
pBLMs,
and
seven
step
signals
were
obtained.
(b)
Histogramby reconstituting
in the(c)double-stacked
stepinsignals
were obtained. (b)Histogram of theαHLs
ion current.
Schematic of the pBLMs,
estimatedand
poresevenconditions
each step.
ofHistogram
the ion current.
of Schematicthe estimatedporeconditions
in conditions
each step. in each step.
of the(c)ionSchematiccurrent. (c)of theestimated
pore

(a)

(b)

(c)

(d)

Figure7.7.(a)(a)Schematic
Schematicand
andelectricalelectricalcircuits
circuitsof
ofthe
the single
single nanopore
nanopore system.
system. (b)Figure(b) IonIoncurrent
currentsignals
signalsof
The current
current steps
steps were
werealmost
almost120
120pA.
pA.(c)(c)Schematics
Schematicsand
andofαHLsαHLsreconstituted
reconstitutedto
to thethe single
single pBLM.
pBLM. The
electricalcircuitsofthedouble-stacked
pBLMs
with
nanopores.
(d)A
schematic
andelectricalcircuitelectrical circuits of the double-stacked pBLMs with nanopores. (d) A schematic and electrical circuit ofofdouble-stacked
the double-stacked
withunder
αHLstheunder
the condition
1 M KCland 120 mV
thepBLMspBLMs
with αHLs
conditionof 1 M KClofsolution
andsolution
120 mV application.
application.

Micromachines 2018, 9, 253

10 of 12

4. Conclusions
In conclusion, we proposed a new approach for a pBLM formation in which the DCM strategy
is applied to the microfluidic formation of pBLMs. We achieved double-stacked pBLMs with open
flow channels on both sides by using a five-layered microchannel comprising aqueous and lipid/oil
solutions. First, we designed five-layered microchannels with hydrophobic guide pillars in the middle
area for formation of five-layered flow using hydrodynamic simulations. The formation of five-layered
flow was confirmed by microfluidic experiments. Then, double-stacked pBLMs were successfully
formed by contacting the lipid monolayers in the interfaces between the aqueous and lipid/oil layers.
We also successfully reconstituted membrane proteins in the pBLMs and measured the ion currents
through the two membranes with nanopores. The ion currents can be estimated from the electricalcircuit of the nanopore system, and the measured ion currents result in the same values as the calculated
currents. We think that our double-stacked pBLM system has the potential to develop high speed andmultiple functional nanopore calculators with multiple pBLMs.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-666X/9/5/253/s1,
Figure S1: Methods for the formation of pBLMs. (a) The Montal–Mueller method, wherein pBLMs are formedby attaching the lipid monolayers in the hole of a Teflon wall. (b) Conventional pBLM formation method using
microfluidic techniques, wherein aqueous and lipid/oil solutions flow sequentially through the microchannel
with small chambers. (c) The pBLMs cannot be paralleled because of the pressure difference between eachflow channel. (d) DCM, in which the pBLM is formed by contacting two droplets surrounded with a lipid
monolayer. The pBLM is supported by the lipid/oil solution, and lipid molecules are supplied to the pBLM;
Figure S2: microscopic images of the microfluidic experiment for the formation of double-stacked pBLMs using
the conventional microfluidic method. These solutions did not flow straight because of the channel resistanceand boundary tension between aqueous and lipid/oil solutions. Scale bars are 50 µm; Figure S3: designs of
five-layered microchannels with eight guide pillars; Figure S4: microscopic images of (a) the microchannel witheight guide pillars and (b) the vertical paralleled pBLMs formed by the channel; Figure S5: microscopic images of
a pBLM (a) before and (b) after 2 h from the pBLM formation; Figure S6: ion current signals of αHL reconstituted
in the vertical paralleled pBLMs. The ion current was changed depending on the pore number in each pBLM;
Figure S7: microscopic images of (a) the three-layered microchannel and (b) pBLMs formed by the channel; Table
S1: Estimated ion current of the nanopores in a double-stacked pBLM. The conductance of nanopores is 1 nS andthe applied voltage is 120 mV (Figure 7d); Video S1: hydrodynamic simulations of microfluidic behaviors withand without guide pillars; Video S2: formation of the double-stacked pBLMs using the five-layered microchannel
with four guide pillars; Video S3: formation of the double-stacked pBLMs using the five-layered microchannel
with eight guide pillars.
Author Contributions: K.S. and R.K. conceived and designed the experiments; K.S. performed the experiments;
all authors approved the final version of the manuscript.
Acknowledgments: This work was partly supported by Japan Society for the Promotion of Science (JSPS) Research
Fellow, Grant-in-Aid for Scientific Research (KAKENHI) (Grant Nos. 15H00803, and 16H06043) from the Ministryof Education, Culture, Sport, Science and Technology (MEXT), Japan and the Okawa Foundation for Information
and Telecommunications.
Conflicts of Interest: The authors declare no conflict of interest.

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