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ArticleBiological Nanopore Probe: Probing of Viscous Solutions in a
Conﬁned Nanospace
Masaki Matsushita, Kan Shoji, Natsumi Takai, and Ryuji Kawano*
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ABSTRACT: This paper describes a nanospace probing systemconstructed with a pore-forming toxin and a hairpin DNA(hpDNA) molecule. The single hpDNA molecule can be inserted
and can move in the conﬁned nanospace of the alpha-hemolysin(αHL) pore. The molecular motion of the hpDNA can be
determined based on the ﬂuctuation of the blocking current via
channel current measurements. Using this system, we investigatedthe eﬀect of viscosity of the aqueous solution in the macrospace(bulk) and in the conﬁned nanospace with a small molecule(glycerol) and a polymer (PEG600). The molecular motion of the
hpDNA in the nanospace diﬀered in glycerol and PEG600
solutions, while the viscosity remained the same in the bulk
solution. The fundamental factors for the viscosity in glycerol and PEG600 solutions are hydrogen bonding and the entanglement ofpolymer chains, respectively. This diﬀerence in factors becomes signiﬁcant in conﬁned nanospaces, and our system allows us toobserve its eﬀect. Additionally, we constructed a spatially resolved nanopore probe integrated into a gold nanoneedle. The αHL−
hpDNA nanoprobe system was constructed with the nanoneedle and can be used to monitor the nanospace with nanometer spatial
resolution.

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INTRODUCTION
The physicochemical properties of aqueous solutions arestrongly inﬂuenced by temperature and pressure. Speciﬁcally,
these solutions have freezing and boiling points that depend on
the ambient temperature and pressure. It is also known that thesize of the space they occupy signiﬁcantly aﬀects their
physicochemical properties, referred to as conﬁnementeﬀects.1−7 For instance, the phase transition of aqueous
solutions in nanospace exhibits unusual behavior owing to
conﬁnement eﬀects; water freezes and generates cubic ice at −13°C in 9 nm diameter silicate pores.4 The mechanism for thisbehavior is explained by the theory on depression of the freezing
point of water below a critical conﬁnement length scale (ca. 2.5nm), described by continuum thermodynamics and the Gibbs−
Thomson eﬀect.2,8,9 In contrast, the freezing temperature ofwater inside an isolated carbon nanotube with a diameter ofapproximately 1 nm is much higher than the theoreticalprediction.8
The conﬁnement eﬀect can be investigated experimentallyfrom either of two viewpoints: one focuses on the kinds ofmaterials that can be used to construct the conﬁned nanospace,
and the other focuses on estimation of the physicochemical
properties of solutions in the conﬁned space. With respect to thetype of materials, porous silica samples (Spherisorb or Gasil)
have been used as nanospace conﬁning materials in earlystudies.1−4 Subsequently, various materials, such as sol−gel
glasses, silica gels, zeolites, polymers, carbon, metal−organic© 2020 American Chemical Society

framework, and reversed micelles, have been used to make theconﬁned nanospace.1 In addition, nanoﬂuidic technology has
been used for extended nanospaces.10,11 The size of the conﬁned
space of porous materials ranges from 1 nm to several microns.
Using these materials, physicochemical properties such as
supercooling or nucleation behavior have been estimated andthe anomalous properties due to conﬁnement studied using
methods such as NMR,10,12 neutron scattering,1,3,5,7 thermal
calorimetry,13,14 and molecular dynamic simulations.15,16 Spectroscopy can reveal the behavior of the molecule inside theconﬁned space but requires highly transparent materials. In
addition, the low sensitivity of spectrometry to water molecules
is an issue. Further, the data obtained from conventional porous
materials are the ensemble average and have a wide distribution
owing to the size distribution of the nanospace. Recently,
isolated carbon nanotubes have been used as a conﬁning
material with known size.6,8,17 The phase transition of water innanotubes observed by Raman spectroscopy was found to besensitive to the carbon nanotube diameter, and the results
revealed large temperature variations of freezing transition (byas much as 100 °C) in this system.8 Although isolated and single
Received: November 27, 2019
Revised: February 5, 2020
Published: February 7, 2020

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ArticleFigure 1. Illustration of the nanopore probe. (a) A typical current through the nanopore probe and schematic illustration of the αHL nanopore and
hpDNA states. (b) Lipid bilayers are prepared by the droplet contact method. αHL is reconstituted in the lipid bilayer, and hpDNA is inserted into thepore. (c) Photograph of the device for measuring channel currents.

■

METHODS
Reagents and Chemicals. All aqueous solutions wereprepared with ultrapure water from a Milli-Q system (Millipore,
Billerica, MA, USA). The reagents were as follows: 1,2diphytanoyl-sn-glycero-3-phosphocholine (DPhPC; Avanti
Polar Lipids, Alabaster, AL, USA), n-decane (Wako Pure
Chemical Industries, Ltd., Osaka, Japan), glycerol (Wako Pure
Chemical Industries, Ltd., Osaka, Japan), polyethylene glycol
600 (PEG600; Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan), potassium chloride (KCl; Nacalai Tesque), potassium
nitrate (KNO3; Sigma-Aldrich), calcium chlorite (CaCl2;
Sigma-Aldrich, St. Louis, MO, USA), and O-(3-carboxypropyl)-O′-[2-(3-mercaptopropionylamino) ethyl]propylethylene
glycol with molecular weight 3000 (thiol-PEG; Sigma-Aldrich,
St. Louis, MO, USA).
Buﬀered electrolyte solutions were prepared from ultrapure
water, which was obtained from a Milli-Q system (Millipore,
Billerica, MA, USA). αHL (Sigma-Aldrich, St. Louis, MO,
USA), obtained as a monomer protein isolated from Staphylococcus aureus in the form of a powder, was rehydrated at aconcentration of 1 mg/mL in ultrapure water and stored at −80°C. For use, samples were diluted to 0.5 μM using a buﬀered
electrolyte solution and stored at 4 °C. Hairpin DNA(GTTCGAACGTTTTCGTTCGAAC) synthesized by FASMAC Co., Ltd., Kanagawa, Japan, in the form of a powder, wasdissolved at a concentration of 100 μM in ultrapure water andstored at −20 °C as the stock solution.
Lipid Bilayer Preparation and Reconstitution Hemolysin. Bilayer lipid membranes (BLMs) were prepared using a
microfabricated device (Figure 1c), as previously reported.37,38
BLMs can be simultaneously formed in this device by the droplet
contact method. First, the DPhPC (lipids/n-decane, 10 mg/mL) solution (2.3 μL) was poured into each chamber. Next, thebuﬀer solution (4.7 μL) without αHL and hpDNA was poured

nanospaces have an essential role in studying conﬁnementeﬀects, the development of conﬁning materials and probing
methods is still challenging.
Here, we report on a single conﬁned space using a biological
nanopore, alpha-hemolysin (αHL) (Figure 1). αHL is a poreforming toxin produced by Staphylococcus aureus and forms amushroom-like nanopore structure in the lipid bilayer with acylindrical vestibule of 5 nm height and 2.6 nm diameter(volume is ca. 27 nm3 = 27 yoctoL) inside the nanopore (Figure1b).18,19 This nanopore protein has been used for DNA
sequencing20−23 and single molecule detection19,24−33 bymeasuring the ionic current through the nanopore. A singlestranded DNA (ssDNA) can pass through this nanopore,
whereas double-stranded DNA (dsDNA) cannot because thediameter of the restricted region of the nanopore is 1.4 nm.19
Using this unique geometrical feature, Vercoutere et al. studied
the orientation of hairpin DNA (hpDNA) with nine base pairs in
the nanopore at the single molecule level.34−36 They reported
that hpDNA is trapped in the vestibule and mainly shows three
distinctive current signals, upper level (UL), intermediate level(IL), and lower level (LL), as shown in Figure 1a. These threecurrent levels reﬂect the three diﬀerent orientations of hpDNA
in the vestibule.36 Based on this information, we propose that the
movement of hpDNA in the nanopore could also signal thesolution properties in the conﬁned space; for instance, thesolution viscosity in the conﬁned space can be investigated byanalyzing the blocking current signals. In this study, we used two
diﬀerent viscous solutions with a similar viscosity range(speciﬁcally, glycerol and PEG600) and analyzed the movementof hpDNA in order to understand the eﬀect of viscosity in the
conﬁned nanospace. Based on our results, nanopore probes
comprised of nanopores with hpDNA will be a useful tool forobserving the physical chemistry of conﬁned nanospaces.
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ArticleFigure 2. Result of nanopore probe measurement of pore conductance. (a, b) Typical current and time traces in the presence of (a) glycerol and (b)PEG600 (Vapp = +120 mV). (c) The conductance in each viscosity of glycerol (blue line) and PEG600 (red line). The black-dashed line is prediction
from bulk viscosity. (d) Dwell time of each solution. The dwell time is from pore opening to hpDNA insertion.

into each recording chamber. The buﬀer solution (4.7 μL) with
αHL (ﬁnal concentration: 0.01 μM) and hpDNA (ﬁnalconcentration: 5 μM) was poured into each ground chamber.
In this study, the buﬀer solution (1 M KCl, 10 mM MOPS, pH
7.0) was used for all droplets. A few minutes after adding the
buﬀer solution, the two lipid monolayers connected and formed
BLMs, and αHL formed nanopores by reconstitution in the
BLMs. When the BLMs ruptured, they were reformed by tracingwith a hydrophobic stick between two droplets.
Channel Current Measurements and Data Analysis.
Channel current was monitored using a Pico patch-clamp
ampliﬁer (Tecella, Foothill Rantch, CA) connected to eachchamber. Ag/AgCl electrodes were already present in each
droplet when the solution was added to the chambers. A
constant voltage of +120 mV for most of the experiments or+100 mV for the nanoneedle experiments was applied to therecording chamber, and the other chamber was connected to
ground. The solution conductivity and viscosity were used froman electrochemical handbook and the previous reports,39,40
respectively. Reconstituted αHL in BLMs allowed ions to passthrough a nanopore under the voltage gradient, and channel
current signals were obtained. When hpDNA was present in the
ground chamber, the αHL nanopore was blocked by thecaptured hpDNA, and the channel current decreased toapproximately half that of the open state and exhibited three
individual current states. The UL, IL, and LL states were
determined by selecting the peak top using Origin software.
When IL and LL states were unseparated in the blocking current

analysis, we determined each state using the duration time
(Figure S2). We subsequently analyzed the blocking amplitude
and the duration time of the three states. The signals weredetected using an 8 kHz low-pass ﬁlter at a sampling frequencyof 40 kHz. Analysis of channel current signals and duration time
was performed using pCLAMP ver. 10.5 (Molecular Devices,
CA, USA) and Excel (Microsoft, Washington, USA) software.
All data were presented as mean values ± SE, where each
duration time was taken using the natural logarithm. Channel
current measurements were conducted at room temperature (22± 2 °C). All operations were conducted from three to ﬁve times(3 < N < 5) with diﬀerent nanopores, and all DNA transitions
were observed from 400 to 1700 times (400 < n < 1700) in eachnanopore. The deﬁnitions for “dwell time of open level” and“event frequency”, appearing in Figure 3, and “dwell time” and“ratio of dwell time”, appearing in Figure 4, are as follows:
Dwell time of open level: time from the open pore ofαHL to the blocking by an hpDNA
Event frequency: number of transition events per second
Dwell time: mean dwell time of each state
Ratio of dwell time: total dwell time of each state/time ofall states■

RESULTS AND DISCUSSION
Nanopore Conductance in Glycerol and PEG Solution.
Prior to performing the hpDNA experiment, the conductance ofαHL nanopores in glycerol and PEG600 solutions was initially2412

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ArticleFigure 3. (a, b) Typical transition signal of (a) glycerol and (b) PEG600 in each solution (Vapp = +120 mV). (c) Typical histogram of blocking currents
at each transition level in glycerol solution (2.0 mPa s). (d) Event frequency of hpDNA transition in each solution.

dependence of the conductance with glycerol and PEG600 andthe predicted value from the bulk solution. The predicted value
provides the bulk conductance of both the glycerol and PEG600
solutions theoretically. The conductance in both solutions
shows an inverse proportion with the viscosity as well as thepredicted value. Interestingly, although the pore conductance inthe glycerol solution was lower than that of the predicted value,
the conductance of the PEG600 solution was higher than thepredicted value even at the same viscosity. The mobility of ionsor small molecules should decrease in the conﬁned nanospacebecause the surface area of the wall in the nanospace is large, andthe experimental results support the low mobility of watermolecules in the nanospace of the glycerol solution.42 In
contrast to the PEG600 system, there are three possible reasonsfor the higher conductance: (1) the concentration (c) of ions in
the nanospace is diﬀerent in bulk solution, (2) the mobility ofions (μ) in the nanospace is diﬀerent in bulk solution, or (3)both 1 and 2. Next, we attempt to reveal the mechanism for theabove-mentioned phenomenon using hpDNA in terms of thesethree hypotheses.
Entry Kinetics of hpDNA into the αHL Pore. Figure 2d
presents the time from “αHL pore opening” to “hpDNA
entrance into the pore vestibule”, called the “dwell time of theopen level”, of each solution. In the case of the glycerol solution,
the dwell time was virtually constant in this viscosity range. In
contrast to the PEG600 solution, the entrance time shortenedwith increasing viscosity. The hpDNA rapidly entered thenanopores even in the higher viscosity solution. This

examined in order to compare their behavior in bulk solution.
Parts a and b of Figure 2 show the open channel amplitudes ofthe αHL pore in glycerol and PEG600 solutions. The open
currents decrease with increasing viscosity in both solutions.
The current and voltage curves in each solution are also shownin Figure S1. The conductivity in the bulk and in the nanopore
can be theoretically deﬁned as follows41
Bulk:
σbulk = ∑ (niziμi )iNanopore:.

ij Vpore yz
zz
Gpore = F jjjjz ∑ (ziciμi )j d pore 2 zz
k
{ i
μ=

zie
6πrη

where σbulk is the conductivity (S cm−1) in bulk solution. Gpore isthe conductance (S) of the nanopore. n, z, c, μ, r, and η are thecarrier density, the charge of the carrier, the concentration, themobility, the Stokes radius, and the viscosity, respectively, of theith mobile species. F is Faraday’s constant, and Vpore and dpore are
the volume and length of the nanopore. According to theseequations, the conductance has an inverse proportional
relationship with the viscosity. Figure 2c depicts the viscosity2413

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ArticleFigure 4. Dwell time of each state (UL, IL, and LL, Vapp = +120 mV). (a) Average time of all events in each state. Dwell time = mean dwell time of eachstate. (b) Ratio of each dwell time in total blockings. Sum of the entire time of each state. For example, ratio of LL state = (time of total LL state)/(timeof all states). (c) Schematic illustration of the glycerol and PEG600 systems. The L value indicates the approximate size of each molecule.

phenomenon would be explained by a depletion force. Possibly
due to the low concentration of PEG600 in the inner space of thepore, the hpDNA existing near the pore entrance should bepressed by the circumambient PEG molecules into thenanopore, resulting in its rapid entry.
A low PEG concentration state in the pore can well explain thehigher conductance of the PEG system because the ions (K+ andCl−) in the pore have high mobilities and/or high concentrations in the pore. From another viewpoint, the diﬀerence ofmolecular phase between the inside and outside of the pore canbe considered as the separation mechanism in the PEG600
system. Phase separation in conﬁned spaces, especially in living
cells, has been reported in cell biology.43 The conﬁned
nanospace can induce phase separation. The measurement
results for the PEG600 solution indicate that there may be
diﬀerent phases present: a high polymer condensed phase in theoutside bulk and a low polymer condensed phase in the inside
pore. Considering these results, we concluded that our
hypothesis of “(3) both 1 and 2” provides a reasonable
explanation for the unusual conductance phenomenon of thenanospace in the PEG600 aqueous environment.
Modulation of hpDNA in the Nanopore Reﬂects theDiﬀerent Nanoenvironments between the Glycerol and
PEG600 Solutions. It has been previously reported that an
hpDNA with nine base pairs having a GC terminus has threestable current states: upper (UL), intermediate (IL), and lower
levels (LL).35 These states reﬂect three diﬀerent interactions
between the terminus bases and the pore wall in the αHLvestibule (see Figure 1a). In the case of UL, the terminus of
hpDNA, GC in this case, does not interact with the inside wall of
the pore, orienting along with the central pore region, resulting
in a large current ﬂowing. IL is observed when one of the ends of
hpDNA interacts with the restriction region of αHL and shows
medium current blocking. LL, the deepest current state, occurs
when both termini interact with the pore vestibule and shows
the highest current blocking state.36

The hpDNA with GC terminus also showed clear transition
signals in both glycerol and PEG600 solutions after injection
into the nanopore; the typical current and time traces arepresented in Figure 3a and b. The transition amplitudes became
smaller with increasing viscosity. The histogram of the blocking
current also indicates the three states, UL, IL, and LL, even in
the viscous solutions of glycerol and PEG600; the typical resultis shown in Figure 3c, and all histograms are shown in Figure S2.
Next, we analyze the viscosity dependence of the transitionevent frequency of [IL] ⇆ [UL] ⇆ [LL] (Figure 3d). Thistransition reﬂects the molecular motility in the conﬁned
nanospace, as also reported previously.44 In the case of theglycerol solution, the event frequency became slow, suggestingthat each state became stable with increasing viscosity(increasing the concentration of glycerol). The energy barrierof the transition reaction rose in the more viscous solution. On
the other hand, in the PEG600 solution, the frequency decreased
from the 1 to 1.5 mPa s solution. However, the frequency was
subsequently virtually constant or slightly increased. This result
indicated that hpDNA moved independently of the viscosity
changing with the PEG600 solution in the nanospace.
We next focus on the transition events of [IL] ⇆ [UL] ⇆[LL] and estimate the inner environment of the nanopore withtwo diﬀerent viscous solutions. It has been proposed that thesethree states correspond to the diﬀerence in the number of thehydrogen bonds between the terminus hpDNA and therestricted region of the αHL pore: UL, IL, and LL have zero,one, and two hydrogen bonds, respectively.35 Figure 4a shows
the dwell time of each state in both the glycerol and PEG600
solutions. In the glycerol solution, the dwell time of all threestates became longer with increasing viscosity. Probably due tothe eﬀect of the increasing viscosity, each state should become
stable; in other words, the energy barrier of the transition may
become high, inhibiting the state transition reaction. These
results correspond to the results from the transition analysis, asmentioned above. In contrast to the glycerol system, the dwell2414

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time of the LL and UL states becomes slightly shorter withincreasing PEG600 concentration. This result implies theoccurrence of destabilization of each state by the PEG600
addition. However, the change is too small to make a clear
description. To analyze the details on the dwell time, the ratio of
each dwell time in the total signal appearance was analyzed, as
shown in Figure 4b. The reduction of LL in the glycerol system islarger than that in the PEG600 system, and the UL slightly
increases in glycerol compared with the PEG600 system. Thistendency may indicate that a glycerol molecule inhibits theinteraction between hpDNA and the αHL wall, probably
because of its hydrogen bonding ability. In contrast, the PEG has
weak hydrogen bonding ability and it can make each hpDNA
state stable.
The two viscous systems in the conﬁned nanospace are
diﬀerentiated schematically in Figure 4c. Glycerol (as a smallsized molecule) exists in the nanospace as in the bulk solution,and each molecule interacts via hydrogen bonding in the narrow
area, inhibiting the mobility of ions and hpDNA. On the other
hand, in the PEG600 system (as a polymer), the number ofmolecules in the nanospace is less than that in the bulk solution,and each polymer interacts with an entanglement of the polymer
chains, relatively not inhibiting the mobility of ions and hpDNA.

Tokyo 184-8588, Japan; orcid.org/0000-0001-6523-0649;
Email: rjkawano@cc.tuat.ac.jp
Authors

Masaki Matsushita − Department of Biotechnology and Life
Science, Tokyo University of Agriculture and Technology,
Koganei-shi, Tokyo 184-8588, Japan
Kan Shoji − Department of Biotechnology and Life Science, Tokyo
University of Agriculture and Technology, Koganei-shi, Tokyo
184-8588, Japan; Department of Chemistry, University ofCincinnati, Cincinnati, Ohio 45221, United States; orcid.org/
0000-0002-7198-9683
Natsumi Takai − Department of Biotechnology and Life Science,
Tokyo University of Agriculture and Technology, Koganei-shi,
Tokyo 184-8588, Japan

Complete contact information is available at:https://pubs.acs.org/10.1021/acs.jpcb.9b11096
Author Contributions

M.M. and R.K. conceived the original idea. M.M., K.S., and N.T.
conducted the experiments and analyzed the data. R.K. and
M.M. wrote the paper. All authors have given approval to theﬁnal version of the manuscript.

■

Notes

CONCLUSIONSIn summary, we observed and characterized the single conﬁned
nanospace of an αHL nanopore with hpDNA. Using this system,
we measured the channel conductance and molecular motion of
hpDNA in the αHL nanopore and compared the macro- andnanospace using two diﬀerent viscous molecules: glycerol and
PEG600. Two important ﬁndings were made: (1) The channel
conductance in the PEG system was higher than that in theglycerol system, suggesting that the concentration of PEG in thenanospace is lower than that in the macrospace. This result
implies high ion mobility or concentration in the nanospace. (2)In the nanospace, glycerol and PEG600 have diﬀerent eﬀects on
the molecular motion of hpDNA even when their viscosities are
the same in the bulk solution. These diﬀerences are due todiﬀerences in the mechanism of viscosity between glycerol and
PEG600: the viscosity induced by glycerol is due to hydrogenbonding-based interactions, while the viscosity induced by
PEG600 is due to entanglement of the polymer chains. These
diﬀerences can be observed clearly by using the biological
nanopore and hpDNA.
As the next step, we have integrated this system with a gold
nanoneedle to construct a spatially resolved nanopore probe, as
we recently proposed.45 The hpDNA was able to be inserted
into the αHL pore, as indicated by the blocking current (FigureS3). This system can be further improved for applications suchas living cell imaging.

■

The authors declare no competing ﬁnancial interest.

■■

ACKNOWLEDGMENTS
This work was partly supported by KAKENHI (Grant Nos.
17K19138, 19H00901) from MEXT, Japan.
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ASSOCIATED CONTENT* Supporting Information
sı

The Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acs.jpcb.9b11096.
The device fabrications and detailed experimental data are
available (PDF)■

ArticleAUTHOR INFORMATION

Corresponding Author

Ryuji Kawano − Department of Biotechnology and Life Science,
Tokyo University of Agriculture and Technology, Koganei-shi,2415

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