BBA - General Subjects 1864 (2020) 129330

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BBA - General Subjects

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Microsystem for the single molecule analysis of membrane transport T

proteins
Rikiya Watanabe
Molecular Physiology Laboratory, RIKEN, Saitama, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: Micro-chamber arrays enable highly sensitive and quantitative bioassays at the single-molecule level.
Biomembrane Accordingly, they are widely used for ultra-sensitive biomedical applications, e.g., digital PCR and digital ELISA.
Membrane protein However, the versatility of micro-chambers is generally limited to reactions in aqueous solutions, although
Single molecule biophysics various functions of membrane proteins are extremely important. To address this issue, microsystems using
bioMEMS
arrayed micro-sized chambers sealed with lipid bilayers, referred to here as a “biomembrane microsystems”,
have been developed by many research groups for the analysis of membrane proteins. In this review, I would like
to introduce recent progress on the single molecule analysis of membrane transport proteins using a biomem-
brane microsystem, and discuss the future prospects for its use in analytical and pharmacological applications.

1. Introduction
The maintenance of an appropriate intracellular environment is a
constant challenge for all living organisms, from prokaryotes to mul-
ticellular eukaryotes. Intracellular homeostasis is in general maintained
by membrane proteins which mediate the translocation of various
compounds across biomembranes [1]. Therefore, the analysis of
transport mechanisms is crucial to understand cell physiology, as well
as the action of drugs [2,3]. The transport of various substrates is
mainly conducted by transport proteins embedded in the cell mem-
brane. For several decades, the functional dynamics of transporters
have been studied using a variety of single-molecule techniques [4,5],
which offer key benefits over macroscopic assay methods, as they un-
lock the ability to quantify transport events [6].
One of the most robust systems used to analyze membrane transport
at the single molecule level is patch clamp recording, which measures
the flux of ions as an electric current under constant electrical voltage
across a membrane. Recent developments have resulted in the auto-
mation of patch clamp recordings, thus enabling massively parallel
analysis of transport activities, e.g. pores and channels [7,8]; however,
transport rates of most transporters (< 102 molecules s−1) are much
smaller than those of ion channels (> 107 molecules s−1), and there-
fore, it is difficult to detect their activities as an electric current.
Moreover, patch clamp recording cannot detect the flux of electrically
neutral substrates, and therefore, the development of a more versatile
system has been long desired.
Recently, microsystems arrayed with micro-sized chambers sealed
with lipid bilayers have been developed by many research groups for
the analysis of membrane transport; in these systems, transport activity
is measured optically based on the accumulation or consumption of a
substrate present in the chambers [9–15]. Microsystems enhance both
sensitivity and throughput, thereby allowing the highly sensitive ana-
lysis of various transporter proteins in a high throughput manner. No-
tably, our “biomembrane microsystem”, which contains > 100,000
arrayed atto- to femto-liter chambers sealed with stable lipid bilayers,
has been used to perform the most sensitive and parallel analysis of
membrane transport, enabling the single molecule analysis of trans-
porters with extremely low transport activities (< 10 molecules s−1)
[5,16]. Moreover, we have successfully demonstrated some physiolo-
gical aspects of the membrane system, such as an asymmetric transbi-
layer phospholipid distribution [16,17], and the modulation of mem-
brane potential across lipid bilayers [18]. Thus, our system paves the
way for understanding the mechanism of membrane transport under
semi-physiologic conditions, as well as allowing for further analytical
and pharmacological applications. In this review, I would like to in-
troduce our current microsystem, and discuss the future prospects for
membrane transport analysis at the single molecule level.
2. Part I. Biomembrane microsystems
Over the past few years, we have developed various “biomembrane
microsystems” in order to carry out the single molecule analysis of
membrane transport proteins under semi-physiological conditions
[5,16–21]. The microsystem consists of a glass substrate containing the

E-mail address: rikiya.watanabe@riken.jp.

https://doi.org/10.1016/j.bbagen.2019.03.016
Received 9 December 2018; Received in revised form 19 March 2019; Accepted 22 March 2019
Available online 26 March 2019
0304-4165/ © 2019 Elsevier B.V. All rights reserved.
R. Watanabe
microarray, a spacer sheet, and a glass block with an access port for
sample injection (Fig. 1a). Microarrays with > 10,000 micro-chambers
(ɸ = 2.4–8.2 μm and h = 0.03–0.5 μm: V = 0.2–71 fL) were fabricated
using soft lithography on a hydrophilic glass substrate coated with
CYTOP (Asahi-glass), a carbon‑fluorine hydrophobic polymer (Fig. 1b).
Notably, in previous studies fluorine polymers have frequently been
used as a support medium for lipid membranes [22]. The fabrication
procedure is comprised of three steps. First, CYTOP is spin-coated onto
a glass substrate of a specified thickness. Photolithography is then used
to pattern mask the structures onto the photo-resist cover on the entire
surface of the CYTOP layer. The resist-patterned substrate is then dry-
etched with O2 plasma using a reactive-ion etching system (RIE) to
expose the glass substrate, resulting in the fabrication of the micro-
chamber. Owing to the treatment with O2 plasma, the bottom and walls
of the micro-chamber become hydrophilic, while the top orifice remains
very hydrophobic. These different surfaces are crucial for forming lipid
bilayers in a high throughput manner (described later).
Lipid membranes are formed on the top orifice of the micro-
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BBA - General Subjects 1864 (2020) 129330
Fig. 1. Biomembrane microsystem.
(a) Components of the system. Top, a glass block
with a sample injection port (l: w: h = 20 mm:
20 mm: 5 mm); middle, a spacer sheet with one side
open (Frame-Seal, BIO-RAD); bottom, a glass slide
with > 10,000 microchambers printed. (b)
Photograph (top) and illustration (bottom) of the
assembled system. The access ports for sample in-
jection are indicated using red circles. (c) Schematic
illustration of the generation of phospholipid mem-
branes. Membrane bilayers were formed over mi-
crochambers via sequential injection of liquids from
the access port. Gray, blue, and yellow are the first
aqueous solution, second aqueous solution, and
chloroform solution containing lipids, respectively.
(d) Fluorescent image of Alexa 647 encapsulated into
microchambers sealed by lipid membranes.
chamber via the sequential injection of liquids from the access port
(Fig. 1c). This technique is similar to the conventional technique known
as the “painting technique,” which forms lipid bilayers through the
deposition of a lipid solution across a small aperture in a hydrophobic
material [12,23]. The experimental procedures used to form the lipid
membrane are comprised of three steps. First, the micro-chambers are
filled with an aqueous solution. A chloroform solution containing
phospholipids, e.g., phosphatidylcholine (PC), phosphatidylethanola-
mine (PE), and phosphatidylserine (PS), is then infused via the access
port. Due to the hydrophobicity of the top orifice, the interface between
water and chloroform is formed over the orifice, where a lipid mono-
layer is then deposited. Finally, a second aqueous solution is infused to
form another lipid monolayer at the interface with the chloroform so-
lution, which is then deposited over the entire surface of the device.
Thus, bilayers are formed over the micro-chambers, whereas mono-
layers are formed elsewhere. It is worth noting that we form lipid bi-
layers using chloroform as a solvent for the lipid, whereas decane or
hexadecane have typically been used in conventional approaches [22].
R. Watanabe BBA - General Subjects 1864 (2020) 129330

A prominent feature of our approach is that the lipid membranes

spontaneously thin down to form bilayers without mechanical pertur-
bation, e.g., hydraulic pressure, which is required for bilayer formation
using decane or hexadecane. This lack of mechanical perturbation is
critically important, because typically more than half of the lipid
membranes are broken by the mechanical perturbation, resulting in an
efficiency of < 30% for bilayer formation using these approaches [22].
Thinning of the lipid membranes is driven by drainage of the organic
solvent over the hydrophobic support. Because chloroform is more
water soluble than decane or hexadecane, and so can be more easily
dissolved in an aqueous solution, it is more easily drained over a hy-
drophobic support, and thus, our approach does not require any me-
chanical perturbation, which contributes to increasing the efficiency of
bilayer formation to over 97% (Fig. 1d).

2.1.1. Membrane potential

Membrane potential, one of the main driving forces for transport by
membrane proteins, is indispensable in cell physiology. For example,
numerous types of ion channels in neurons are controlled by membrane
potential, allowing for signal transmission via intra- or inter-cellular
interfaces. To mimic physiological conditions, a microsystem with a
nano-sized electrode has been developed to modulate membrane po-
tential in a highly quantitative manner [18] (Fig. 2).
The system is fabricated using conventional vacuum metal deposi-
tion and photolithography. First, 500-nm-thick Au layers and 20-nm-
thick chromium adhesion layers are coated onto the glass substrate
using a vacuum evaporator. Following this, CYTOP is spin-coated onto
the Au layer at a thickness of 500 nm. Photolithography is then con-
ducted to pattern-mask structures onto the photo-resist covering the
entire surface of the CYTOP layer. The resist-patterned substrate is then
dry-etched with O2 plasma using a reactive-ion etching system to ex-
pose the Au surface, and regions bare of Au and Cr are then etched
using Au and Cr etchants, respectively. A schematic illustration of the
system is shown in Fig. 2a. The Au and fluororesin layers are used as a
nano-sized electrode to modulate membrane voltage, and to support the
lipid bilayer membrane, respectively. The through-hole structures on
these layers are used as micro-chambers to detect biological reactions
with high sensitivity.
To evaluate membrane voltage, the micro-chambers are filled with a
buffer solution containing DiBAC4 [24], a fluorescent indicator which
increases its fluorescence intensity in proportion to the amplitude of the
membrane voltage, and the lipid bilayers are then formed over the top
orifices as described above (Fig. 2b). As shown in Fig. 2c, the fluores-
cence intensity of DiBAC4 changes following voltage modulation Fig. 2. Membrane protential.
through the nano-sized electrodes. In addition, the change in intensity (a) Photograph (top) and illustration (bottom) of the fabricated microsystem to
of DiBAC4 is highly stable for > 10 min. Thus, a long-term quantitative modulate membrane potential. Microchambers (ϕ = 3 μm) were fabricated on a
modulation of membrane voltage using a nano-electrode has been de- double layer of fluororesin (h = 500 nm) and Au (h = 500 nm). (b) Membrane
monstrated in this microsystem. potential monitoring. A membrane potential indicator, DiBAC4, was en-
capsulated in the chamber. Membrane potential was modulated using elec-
2.1.2. Asymmetric phospholipid distribution trodes on a chamber and a top glass block. (c) Fluorescence intensity of DiBAC4
Most biological membranes possess an asymmetric transbilayer (green) against applied membrane potential using nano-sized electrodes
(black).
phospholipid distribution, the maintenance of which requires en-
dogenous enzymes to expend energy by promoting phospholipid
translocation [25]. In particular, the plasma membrane of most eu- First, an aqueous solution is infused into the flow channel of the system.
karyotes maintains a high degree of asymmetry. For example, PS and PE After this infusion, the micro-chamber is filled with an aqueous solu-
are strictly confined to the inner leaflet, which controls various cellular tion. Second, a lipid solution is then infused to flush away the first
functions, such as signal transduction, membrane fusion, and cell aqueous solution. The chloroform solution used for this process con-
apoptosis [26–28]. Two different methods have been developed to tains a small amount of lipid, and therefore, lipid monolayers, but not
achieve asymmetric phospholipid distributions in our system in order to multilayers, are formed at the top orifice of chambers. Third, a second
reproduce the physiological environment (Fig. 3) [16,17]. chloroform solution containing a different lipid composition, and a
First, a method was developed whereby two lipid monolayers are second aqueous solution are sequentially infused to form a second lipid
independently prepared, and then assembled on the top orifice of the monolayer at their interface, which is then deposited over the entire
micro-chamber to create an asymmetric distribution (top, Fig. 3a) [17]. surface of the system. In this process, the hydrocarbon tails in the
The experimental procedure used for this is comprised of three steps. second lipid layer are zipped together with those of the first lipid layer

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R. Watanabe
to form an asymmetric bilayer over the micro-chamber, whereas
monolayers are formed elsewhere. Notably, this method allows for the
formation of > 100,000 asymmetric lipid bilayer membranes with an
efficiency of over 97% (bottom, Fig. 3a).
A second method for creating an asymmetric distribution of phos-
pholipids involves irradiation with a high-energy laser (Fig. 3b) [16].
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BBA - General Subjects 1864 (2020) 129330
Fig. 3. Assymtery of phospholipid distribution.
(a) Asymmetric lipid bilayer formation using two
compositions of phospholipids. First, an aqueous
solution is injected into the microsystem through an
access port. Second, the first lipid solution con-
taining fluorescent-labeled lipids (fluorescein-DHPE)
is injected. Third, another lipid solution without
fluorescein-DHPE is injected. Finally, a second aqu-
eous solution is injected to flush the second lipid
solution. During this process, asymmetric lipid bi-
layers are formed on the orifices of the micro-
chambers. The bottom panel is the fluorescence
image of fluorescein-DHPE on the asymmetric lipid
bilayers. (b) Asymmetric lipid bilayer formation by
irradiating a high-energy laser. 1) Membrane bi-
layers formed over microchambers were exposed a
laser to 2) photobleach fluorescently labeled phos-
pholipids (red). 3) Fluorescent lipids diffused into
the bilayer from the surrounding area, but were then
restricted to the outer layer. (c) The fluorescence
images of microchambers, TopFluor-TMR-PS (red)
before and after photo-bleaching.
The experimental procedures for this approach are as follows: First,
lipid membranes are formed in the microsystem using a fatty-acid la-
beled fluorescent phospholipid, e.g., 18:1–6:0 TopFluor-TMR-PC, -PS,
-PE or 18:1-6:0 NBD-PS (Avanti, USA). The fluorescence of the bilayer
membrane in the micro-chamber can be clearly seen to be higher than
that of the surrounding monolayers (Fig. 3b, top Fig. 3c). Following
R. Watanabe
this, the fluorescent lipids in the bilayer membranes over the micro-
chambers are photobleached by exposure to a high-energy laser for a
few seconds in order to establish an asymmetric lipid distribution As
shown in the bottom panel of Fig. 3c, the fluorescence in the micro-
chambers gradually recovers over a period of several hundred seconds
to the same level as seen on the surrounding surfaces, confirming that
the membrane over the micro-chambers is contiguous with the mono-
layer in other parts of the system. The recovery of the fluorescence over
the micro-chambers do not exceed the fluorescence of surrounding
surfaces (Fig. 3b), indicating that the vertical translocation of phos-
pholipids between the two layers of the membrane does not occur
during measurement or is very slow, as has been observed in plasma
membranes [29]. In other words, this result shows that photobleaching
generates an asymmetric membrane bilayer in which unbleached
fluorescent-phospholipids are present only in the upper layer. Notably,
the asymmetry is maintained over 2 h after photo-bleaching, and is
moreover reproduced at the same bilayers for many times by laser ir-
radiations. These technical advantages are useful for single molecule
analysis of phospholipid transport proteins, such as flippase, floppase,
and scramblase (see below).
2.1.3. Generation of concentration gradient
Owing to the small volume of the chambers in our system, a minute
quantity of product is detectable, allowing for the direct monitoring of
the individual activities of isolated single membrane proteins in a
highly sensitive and quantitative manner. However, even though the
chambers are highly integrated in the system, it is difficult to conduct
multiple bioassays under different conditions using integrated cham-
bers in parallel, because the composition of the reaction solution en-
capsulated in the chambers is uniform over the entire area of the
system. Recently, this technical issue was addressed by developing a
novel system capable of forming a variety of concentration gradients of
target molecules encapsulated in the chambers (Fig. 4) [21].
In this system, based on the advection diffusion model, a con-
centration gradient of target molecules is formed along the flow
channel via the sequential injection of several liquids from the access
port (Fig. 4a). First, an aqueous solution containing the target mole-
cules is infused into the flow channel to fill the individual chambers, as
described above. Second, a specified amount of a second aqueous so-
lution lacking target molecules (7–12 μL) is infused at a defined flow
rate (0.5–1.5 μL/s) using an electric pipette. In this step, the con-
centration gradients are generated based on the advection-diffusion
process. The aqueous solution first filled in the flow channel is gradu-
ally diluted from the inlet as the second solution for dilution is infused.
Then, the concentration gradients are generated along the flow direc-
tion, i.e., the first solution is not completely diluted because the volume
of second solution is smaller than that of the flow channel. Finally, to
encapsulate the target molecules into the chambers, a chloroform so-
lution containing phospholipids and a third aqueous solution are suc-
cessively infused. After infusion, the lipid-bilayer membrane is formed
on the orifice of the individual chambers, as described above, resulting
in the encapsulation of the target molecules.
To examine the feasibility of this approach, a fluorescent dye (Alexa
488) was used as an indicator of the concentration gradient. The con-
centration gradient was formed using the first or second aqueous so-
lutions, with or without 1 μM Alexa 488, respectively. As expected, the
fluorescence intensity of Alexa 488 encapsulated in the chambers in-
creased along the flow channel (Fig. 4b, c), confirming the formation of
a concentration gradient of target molecules in our system. Further-
more, the concentration gradient of the target molecule, i.e., Alexa 488,
became steeper as the flow rate increased, and/or the volume of the
second aqueous solution decreased (Fig. 4b). Accordingly, the con-
centration gradient in the micro-chamber array can be quantitatively
modulated in proportion to the distance from the access port, L (Fig. 4b,
c). So far, using this system, multiple single-molecule bioassays have
been performed on the system under various conditions.
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BBA - General Subjects 1864 (2020) 129330
3. Part II. Single molecule analysis of membrane transport
proteins
Over the past few years, single molecule analysis has been demon-
strated for a variety of membrane transport proteins, e.g., pores,
channels, ion-pumps, and phospholipid scramblases, using “biomem-
brane microsystems” [5,16–21]. In this section, I would like to in-
troduce the achievements made for the single molecule analysis of
transport proteins.
3.1. Pore: α-hemolysin
A transmembrane pore is a membrane protein that passively
transport substrates along their concentration gradient. The activities of
pores are relatively higher than other transport proteins, e.g., ion-
pumps, because both sides of the permeation pathway are always open,
and as a result, they have been well characterized mechanistically using
patch clamp recordings.
A single molecule transport assay was carried out on our micro-
system using α-hemolysin (Fig. 5) [30], a toxic membrane protein that
binds to lipid bilayer membranes and forms transmembrane nanopores
(ϕ = 1–2 nm) that have been used as various biomedical sensors, e.g.,
in nanopore DNA sequencing [31]. While monomers of α-hemolysin are
soluble in water, once bound to a lipid bilayer, α-hemolysin assembles
into a heptameric ring and forms a nanopore at the center, resulting in
the passive transport of small molecules as small as 1 nm through its
pore.
For the assay, a fluorescent dye, Alexa 488, was encapsulated in the
chambers as the transport substrate, and a low concentration of α-he-
molysin solution (i.e., 1 μg mL−1) was injected into the flow channel
after lipid bilayer formation (Fig. 5a). In this setup, the passive trans-
port activity of α-hemolysin was detectable by analyzing the fluor-
escent intensity of the chamber, because Alexa 488 passively diffuses
out of the chamber through the α-hemolysin pores, resulting in a de-
crease in chamber fluorescent intensity. Notably, a simple physico-
chemical model has been established for the passive transport by α-
hemolysin pores in the microsystem, where diffusion of the dye mole-
cule into the α-hemolysin pore is the kinetic bottleneck, and obeys
Fick's law [5,19]. In this model, the fluorescence intensity, F(t), of Alexa
488 encapsulated in the chamber is expressed by Eq. (1):
N ∙D∙d 2∙π ⎞
F (t ) = Fo ∙exp ⎛−
⎜ ⎟t + F1
⎝ 4∙L∙V ⎠ (1)
where N is the number of α-hemolysin pores reconstituted in the lipid
membrane, D is the diffusion coefficient of Alexa 488, d is the diameter
of the α-haemolysin pore (~1 nm), L is the length of the α-hemolysin
pore (~10 nm), and V is the volume of the chamber. According to Eq.
(1), the fluorescence intensity of the chamber exponentially decreases
due to the passive transport activity of α-hemolysin with a rate constant
proportional to the number of α-hemolysin pores (N).
Fig. 5b, c show typical time courses of the fluorescent signals of
1 μM Alexa 488 encapsulated in 7 fL chambers, where the decay of
fluorescent intensity represents passive transport through the α-hemo-
lysin pore. The response of each individual chamber is not homo-
geneous (Fig. 5b), representing the stochastic formation of α-hemolysin
pores in lipid bilayers. The distribution of the rate constant for fluor-
escent decay, determined by fitting with an exponential function (black
line in Fig. 5c), exhibits four distinctive peaks (Fig. 5d). The intervals
between the peaks are essentially constant, a typical feature of a single-
molecule digital assay [32,33], and each peak corresponds to the ac-
tivity of 0, 1, 2, or 3 α-hemolysin pores.
The rate constant of the passive diffusion through a single α-he-
molysin pore was determined from the peak interval to be
5.5 ± 1.5 × 10−4 s−1. The initial transport flux of a single α-hemo-
lysin pore (v) was thus estimated as v = n · k, where n is the number of
R. Watanabe BBA - General Subjects 1864 (2020) 129330

Fig. 4. Concentration gradient.

(a) Schematic illustration of the concentration gradient of target molecules. The concentration gradients were formed on individual micro-chambers depending on
the distance (L) from the access port for sample injection. The individual micro-chambers were sealed with lipid-bilayer membranes. (b) Concentrations of Alexa 488
in micro-chambers are plotted against the flow rate (top) or volume of the second buffer (bottom) used to generate the concentration gradients. The solid lines
represent linear regressions. (c) Fluorescence image of Alexa 488 (green) encapsulated in micro-chambers (h = 0.5 μm) at each L. The flow rate of liquid was 1.5 μL/s
and the volume of the second buffer solution was 7 μL.

fluorescent dye molecules encapsulated in each chamber, and
n = 7 fL × 1 μM × NA = 4.2 × 103 molecules. The initial transport
flux per α-hemolysin pore was 2.3 molecules s−1, which is much lower
than the detection limit of conventional patch clump recordings (~107
ions s−1). Thus, through the use of the biomembrane microsystem the
highly sensitive detection of passive transport by a transmembrane pore
can be achieved down to the single molecule level.
3.1.1. Ion pump: F-type ATPase
An ion pump is a membrane protein that actively transports ions by
consuming electro-chemical energy, e.g., ATP hydrolysis or ion-motive
force (imf). The activities of ion pumps are relatively low (> 100 mo-
lecules s−1) because one side of the ion permeation pathway is always
closed, i.e., they have to induce conformational changes to open and
close the outward or inward gate on the pathway, and therefore, it is
technically challenging to measure the low activity of ion pumps at the
single molecule level.
A single molecule analysis of an ion pump was carried out on our
microsystem using the F-type ATPase (FoF1) (Fig. 6a), which mediates
proton transport by coupling with ATP hydrolysis [34]. Although
extensive biochemical studies have been conducted for ion-pumps, a
single-molecule analysis has not achieved due to the technical diffi-
culties in detecting slow transport events in highly sensitive manner.
For the assay, RhP-M, a fluorescent pH indicator which increases its
fluorescent intensity under acidic conditions [35], is encapsulated in
the chamber, and a buffer solution containing a low concentration of
FoF1 is injected into the flow channel after lipid bilayer formation.
Following this, ATP is injected to initiate proton pumping. In this setup,
FoF1 molecules with an outward orientation of the catalytic core do-
main can hydrolyze ATP to pump protons into the chamber, decreasing
the pH, and thereby increasing the fluorescent intensity of RhP-M
(Fig. 6a). Fig. 6b, c show typical time courses of fluorescent images
obtained after ATP injection. Notably, some RhP-M molecules bound
nonspecifically to the chamber wall due to its hydrophobicity, ex-
hibiting ring-like shapes in fluorescent images (Fig. 6b). Similarly to the
fluorescence signals obtained from the single-molecule α-hemolysin
assay, the fluorescent signals in this assay show heterogeneity. As ex-
pected, Fig. 6b (left) exhibited discrete signal levels, representing the
stochastic reconstitution of the FoF1 molecules. The active chambers
reached a plateau at approximately 1600, which corresponds to a pH of

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R. Watanabe BBA - General Subjects 1864 (2020) 129330

Fig. 5. Single molecule analysis of pore, α-hemolysin.

(a) Schematic illustration of passive transport of α-hemolysin using the biomembrane microsytem. The fluorescent dye (Alexa 488) diffused out from the chamber via
the pore on the membrane formed by α-hemolysin. (b) Fluorescent images of the passive transport activity in the chambers. The images were recorded just after α-
hemolysin injection (left panel) and 3000 s later (middle panel). The right panel, diff., shows the intensity difference between these two images in the form of a color
gradient. (c) Continuous recording of the passive transport activity of 1 μg mL−1 α-hemolysin. The chambers containing 0 α-hemolysin pores, 1 α-hemolysin pore, 2
α-hemolysin pores, and 3 α-hemolysin pores were plotted as gray, blue, red, and green, respectively. Solid lines represent the fittings with the single exponential
decay: y = C1 · exp.(−k · t) + C2, where k is the rate constant of passive transport. (d) In the histogram, the number of chambers is plotted versus the rate constant of
passive transport, k. The four peaks can be attributed to occupancies of 0, 1, 2, or 3 α-hemolysin pores per chamber. They were fitted to a sum of Gaussians.

approximately 5.4. The transmembrane proton gradient (ΔpH) gener-
ated was approximately 1.7, which is at a level of equilibrium with the
free energy of ATP hydrolysis. At the end of the observation period, a
H+ ionophore, nigericin (arrow in Fig. 6c), was injected into the flow
channel. The active chambers uniformly recovered their fluorescence
toward the original level. This nigericin-induced fluorescence recovery
ensures that the fluorescence increase represents the ATP-driven
proton-pumping activity of FoF1.
To estimate the proton transport rate for FoF1 during the initial
phase, the initial rate is measured from the linear portion of the time-
course (typically from 1500 s to 4000 s) where the fluorescence in-
creases at a constant rate (Fig. 6c). Similarly to the α-hemolysin assay,
the distribution of the proton-pumping rate exhibites three distinct,
regularly spaced peaks, indicating that each peak represented chambers
with 0, 1, or 2 molecules of FoF1 (Fig. 6d). The rate of proton pumping
by FoF1 is estimated from the intervals between the peaks to be
27.5 s−1. Thus, the biomembrane microsystem is the first to achieve the
highly sensitive detection of active transport by an ion pump at the
single molecule level.
3.1.2. Phospholipid scramblase: TMEM16F
Phospholipid scramblase is a membrane protein which translocates
phospholipids between the inner and outer leaflets of cell membranes in
order to disrupt their asymmetric lipid-distribution [36]. The bio-
chemical features of scramblase have not been well characterized be-
cause the purification of intact scramblase is difficult, and moreover,
with a few exceptions, membrane bilayers containing asymmetrically
distributed phospholipids are challenging to construct [17,37,38].
A single molecule analysis of phospholipid scramblase is carried out
using our microsystem [16] and the transmembrane protein 16F
(TMEM16F), a Ca2+-dependent phospholipid scramblase [39] that
mediates PS exposure in activated platelets during blood clotting, and
regulates hydroxyapatite release from osteoblasts during bone miner-
alization (Fig. 7a) [40,41]. Despite its physiological importance, a
single-molecule analysis has not been achieved so far due to the tech-
nical difficulties in preparing asymmetric lipid bilayers and in detecting
lipid translocation in a highly sensitive manner. For the assay, a buffer
solution containing a low concentration of TMEM16F is injected into
the flow channel after lipid bilayer formation. Following this, an
asymmetric distribution of the fluorescent lipid is formed on membrane

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R. Watanabe BBA - General Subjects 1864 (2020) 129330

Fig. 6. Single molecule analysis of ion-pump, F-type ATPase.

(a) Schematic illustration of F-type ATPase (FoF1) active transport. FoF1 pumps the proton from outside to inside the microchambers by hydrolyzing ATP. The
fluorescent pH indicator (RhP-M) is encapsulated in the microchamber for pH monitoring. (b) Fluorescence images of the proton pumping of FoF1 in the chambers.
The images were recorded just after the injection of 200 μM ATP (left panel) and 6000 s later (middle panel). The right panel, diff., shows the intensity difference
between the left and middle panels in the form of a color gradient. (c) Continuous recording of proton pumping. The chambers containing 0 molecules, 1 molecule,
and 2 molecules of active FoF1 were plotted as gray, blue, and red, respectively. (d) In the histogram, the number of chambers is plotted versus the slope of
fluorescence increments from 1500 to 4000 s. The three peaks can be attributed to occupancies of 0, 1, or 2 active FoF1 molecules per chamber. They were fitted with
a sum of Gaussians.

bilayers by irradiation with a high-energy laser, as described above. The
scrambling assay is initiated by injecting 100 μM Ca2+ into the channel
to activate the phospholipid scrambling activity of TMEM16F. Using
this setup, the phospholipid translocation of TMEM16F is detected by
analyzing the fluorescent intensity of the bilayers, because TMEM16F
can transports fluorescent lipids from the outer leaflet to the inner
leaflet of bilayers, resulting in an increase in their fluorescent intensity.
Fig. 7b, c display typical time courses of fluorescent images obtained
after Ca2+ injection, where the fluorescent increase in the bilayers re-
presents the transport of a fluorescent lipid by TMEM16F. The response
of each individual bilayer is not homogeneous (Fig. 7b), showing the
stochastic reconstitution of TMEM16F molecules.
The fluorescent intensity of the bilayers gradually increases to reach
the original intensity level before photobleaching (Fig. 7c), irrespective
of the phospholipid composition of the membranes. Notably, when
active bilayers are laser-photobleached for a second time to re-create an
asymmetric distribution, the bilayers gradually regain their
fluorescence up to their original level in the presence of 100 μM Ca2+,
but not in the presence of 100 μM Ca2+ plus 1.0 μM of the inhibitor
EGCg [42] (Fig. 7c), demonstrating that phospholipid scrambling by
TMEM16F can be triggered more than once in our microsystem.
A kinetic analysis is carried out by conducting the scrambling assay
under various experimental conditions, e.g., different membrane sizes
or at different temperatures. The scrambling catalyzed by TMEM16F
slowes down (Fig. 7d) as the area of the bilayer membrane increased.
Because TMEM16F-mediated scrambling is reversible [39], the reaction
should follow the reaction
k
[Lipidout ] ⇄ [Lipidin]
k (3)
where k is the rate constant, and [Lipidout] and [Lipidin] are the con-
centrations of fluorescent lipids at the outer and inner layers, respec-
tively. Notably, [Lipidout] is considered to be constant because it is
contiguous with a large lipid monolayer in the surrounding areas. Since

8
R. Watanabe BBA - General Subjects 1864 (2020) 129330

(caption on next page)

9
R. Watanabe BBA - General Subjects 1864 (2020) 129330

Fig. 7. Single molecule analysis of phospholipid scramblase, TMEM16F.

(a) Schematic illustration of the phospholipid scrambling by TMEM16F. 1) TMEM16F was incorporated into the asymmetric membrane bilayers with fluorescent
phospholipids (red). 2) Addition of CaCl2 initiated phospholipid scrambling, followed by lateral diffusion of unbleached phospholipids from surrounding areas,
increasing the fluorescence intensity in microchambers. (b) Purified TMEM16F was loaded at 3.4 μg mL−1, and scrambling was initiated with 100 μM CaCl2.
Subsequently, the asymmetric membrane bilayers were again formed by laser irradiation with injection of 1 μM EGCg. Maximum intensity projections of confocal
images of TopFluor-TMR-PS (red) obtained from the same microchambers before (top) and after addition of 100 μM CaCl2 (middle) or 100 μM CaCl2 + 1 μM EGCg
(bottom). Scale bar: 5 μm. (c) Time course of TMEM16F-mediated phospholipid scrambling. Lipid bilayers with (red) or without (gray) TMEM16F were treated with
100 μM CaCl2 (black arrowheads), and fluorescence from TopFluor-TMR-PS was followed over time. Subsequently, the microchambers were re-exposed for a few
seconds to a laser at time points marked in orange, with (lower panel) or without (upper panel) prior injection of 1 μM EGCg (red arrowhead). (d) Time course of
TMEM16F-mediated phospholipid scrambling at different area (S) of the bilayer membrane, 8.6 or 71 μm2. Black curves are fits to the single-exponential function
y = C1 + C2 × (1 – exp.[−k × (t – 1000)]). (e) The rate constant of fluorescence increase (k*) of TopFluor-TMR-PS was plotted against the inverse of membrane area
(1/S). Data in blue, red, and orange were obtained using TopFluor-TMR-PS at 16 °C, 25 °C, and 31 °C, respectively. Black lines are linear regressions. (f) An Arrhenius
plot for TMEM16F-mediated phospholipid scrambling, with k values from Fig. 7e. The solid line represents linear regression, and the inset contains thermodynamic
parameters at 25 °C, as determined from the plot.

the fluorescent intensity over the chamber (FI) is proportional to the
concentration of fluorescent lipids and the membrane area, FI is thus
determined as a function of time t;
FI = FIout + FIin⋅[1 − exp(−k ∗⋅t )]
k 1
k∗ = ⋅ Single-molecule analysis using micro-chamber arrays is an emerging
η S
where FIout and FIin are the fluorescent intensities of the outer and inner
layers, respectively, S is the area of the membrane over the chambers,
and η is the number of lipid molecules (~2.0 × 106/μm2) per unit area
of the monolayer, as determined previously [43]. Fitting the time
course in Fig. 7d to Eq. (4) demonstrates k*, the rate constant of
fluorescent increase at 25 °C. As expected from Eq. (5), k* is inversely
proportional to S (Fig. 7e), demonstrating that k, the rate of TMEM16F-
mediated lipid transport, is 4.5 × 104 lipids/s at 25 °C (Fig. 7f); this
value is ~108-fold faster than the spontaneous vertical lipid flip-flop
across membrane bilayers [29]. Importantly, the k value does not differ
between fluorescence substrates, such as TopFluor-TMR-PS, -PC, and
-PE, indicating that TMEM16F does not distinguish between the head
group moieties of phospholipids, and confirming that TMEM16F non-
specifically scrambles phospholipids.
The TMEM16F-mediated scrambling is temperature dependent
(Fig. 7e), with k values of 1.4 × 104 and 7.1 × 104 lipids/s at 16 °C and
35 °C, respectively. The corresponding Arrhenius plot fitted well to a
linear function (Fig. 7f), indicating that TMEM16F-mediated lipid
transport is governed by a single rate-limiting step, at least from 16 °C
to 31 °C. The thermodynamic parameters of this rate-limiting step are as
follows: ΔG‡ = 47 kJ/mol, ΔH‡ = 82 kJ/mol, and TΔS‡ = 35 kJ/mol at
25 °C (inset, Fig. 7f). The ΔG‡ value for spontaneous vertical lipid flip-
flop is approximately 100 kJ/mol [29], showing that TMEM16F sig-
nificantly reduces the activation free energy. Thus, this biomembrane
microsystem is the first to achieve a highly sensitive detection of
phospholipid transport by phospholipid scramblase at the single mo-
lecule level.
3.1.3. Future prospects
A high-throughput single-molecule analysis of the membrane
transport proteins, α-hemolysin, F-type ATPase, and TMEM16F has
been achieved using the “biomembrane microsystem”. The micro-
system has the following advantages for single molecule analysis: i) a
high sensitivity using ultra-small chambers, ii) high throughput using
chambers in parallel, and iii) a high compatibility for membrane pro-
tein analysis. These features may extend the use of our microsystem for
single molecule analysis toward other membrane transport proteins,
e.g., ion-channels, secondary active transporters, and phospholipid
flippases/floppases. Such studies will reveal the generality and un-
iqueness of the operating principles of membrane transport proteins,
thereby promoting in vitro membrane protein studies.
The currently developed microsystem has several limitations. First,
the concentration of bivalent cations (< 10 mM Ca2+ or Mg2+) and the
size of micro-chambers (ɸ < 100 μm) drastically affect the efficiency of
bilayer formation. In addition, the experimental conditions for recon-
stituting membrane proteins into lipid bilayers are widely varied (not
universal) among protein species and ways of purification. These points
(4)
represent remaining technical challenges for single molecule analysis
using the biomembrane microsystems.
(5)
approach to detect various bio-reactions, e.g., hydrolysis, protein
synthesis, and membrane transport, with a high sensitivity down to the
single-molecule level, enabling biomedical applications, such as digital
PCR and digital ELISA. The biomembrane microsystem described here
has broadened the versatility of micro-chambers to a single molecule
analysis for membrane proteins, which are promising drug targets for
the treatment of various diseases. Thus, this microsystem will enable
further analytical and pharmacological applications, such as use in high
throughput drug screening, and as an early diagnosis tool.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research
(JP17H03660, JP15H05591) from the Japan Society for the Promotion
of Science, a PRESTO Grant (JPMJPR13LC) from the Japan Science and
Technology Agency, and a PRIME Grant (JP17gm0910020) from the
Japan Agency for Medical Research and Development (to R.W.).
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