aet00 | ACSJCA | JCA11.1.4300/W Library-x64 | research.3f (R4.1.i3 HF01:4938 | 2.1) 2018/08/24 11:08:00 | PROD-WS-116 | rq_4055654 |

2/06/2019 14:54:04 | 9 | JCA-DEFAULT

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Spatially Resolved Chemical Detection with aNanoneedle-Probe-Supported Biological
Nanopore

4

Kan Shoji,†,‡ Ryuji Kawano,† and Ryan J. White*,‡,§

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Articlewww.acsnano.org†

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi,
Tokyo 184-8588, Japan‡§
7 Department of Chemistry and Department of Electrical Engineering and Computer Science, University of Cincinnati, Cincinnati,
8 Ohio 45221-0172, United States
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S Supporting Information*

ABSTRACT: In this article, we describe the quantitative characterization of a gold nanoneedle ion channel probe anddemonstrate the utility of this probe for spatially resolved detection of a small molecule using ion channel activity. Our
report builds on recent reports of Ide and co-workers, who reported the use of an etched gold wire modiﬁed with apoly(ethylene) glycol monolayer as a support for a lipid bilayer and subsequent single ion channel recordings. Although
this nanoneedle electrode approach was reported previously, in our report, we investigate the eﬀects of several operationalparameters on the performance of the ion channel measurement and electrochemical phenomenon that occur in the
nanoconﬁned space between the supported bilayer and the gold electrode. More speciﬁcally, we address the eﬀects oflength of the supporting monolayer and the composition of the electrolyte baths on channel current measurements andprovide a quantitative description of what carries current at the working electrode (double-layer charging). In addition,
we demonstrate the ability to control the direction of protein insertion (tip side vs bath side) with freely diﬀusing protein,which has not been previously reported, with the former method (tip side) enabling single-molecule detection of βcyclodextrin (βCD) using a reconstituted α-hemolysin channel. Finally, anticipating future use of a nanoneedle-based
biological nanopore probe in a scanned-probe microscopy, we demonstrate the ability to quantify and spatially resolve theconcentration of βCD molecules in a microﬂuidic channel. We believe, in the long term, the described nanoneedle-based
biological nanopore probe can be employed in, for example, scanning ion conductance microscopy using ion channels.
KEYWORDS: nanopore, resistive pulse, α-hemolysin, imaging, nanoneedle, lipid bilayer membraneR

provide reproducible pore sizes dictated by the protein 40
structure and have thus found widespread use as resistive- 41
pulse detectors.
42
To employ biological nanopores for sensing purposes, a 43
stable lipid bilayer membrane is required. Whereas the painting 44
method21 and the Montal−Mueller method22 are generally 45
used, resulting in the formation of a membrane spanning across 46
a pore in a hydrophobic substrate, these methods are time- 47
consuming and the resulting membrane is unstable to 48

esistive-pulse nanopore sensors that measure blockage
currents caused by single molecules binding to, ortranslocating through, a nanopore provide single28
29 molecule sensing with high sensitivity, temporal resolution,
1−6
These
30 signal-to-noise ratio, and molecular selectivity.
7−9
and sensors
31 sensors have been applied as DNA sequencers
5,10−13and proteins.14−17
32 for biomolecules including miRNAs
18,19
typically fabricated by exposing thin
33 Solid-state nanopores
34 membranes such as silicon nitride and carbon to a high-energy
20
35 electron beam or biological nanopores, which comprise pore36 forming proteins reconstituted into a lipid bilayer membrane,
37 are key examples of the types of nanopores utilized in this38 sensing strategy. Although both show promise for sensing
39 applications, biological nanopores (i.e., protein channels)
26
27© XXXX American Chemical Society

Received: December 21, 2018
Accepted: February 6, 2019
Published: February 6, 2019
A

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Figure 1. Schematic illustrations of the nanoneedle-based nanopore probe. Probes are composed of an electrochemically etched gold
microwire that is modiﬁed with a thiolated PEG layer. The probe is immersed across an oil/lipid aqueous interface to form a bilayer at theelectrode surface. (Left) Bath-side protein insertion is achieved by incorporating protein in the aqueous bath layer. In this conﬁguration,
βCD in the bath solution do not interact with αHL. (Right) Conversely, with tip-side insertion, αHL inserts from the aqueous solution
reservoir between the bilayer and gold surface. In this conﬁguration, bath-side βCD molecules enter the trans side of the αHL pores,
resulting in single-molecule binding events in the current−time trace.

Figure 2. Measured conductance through the αHL pore is aﬀected by (top and bottom) the length of the supporting PEG monolayer and(left and right) insertion side. For example, median conductance values when using a short PEG (282.35 g/mol) at an applied potential of
100 mV were 0.86 and 0.38 nS using bath-side and tip-side insertion methods, respectively. In contrast, the values were 1.00 and 1.15 nS(bath side and tip side) when a longer PEG layer (3000 g/mol) was employed. The observation of multiple step increases in channel current
is consistent with additional protein insertions (acting as resistors in parallel).

measurements. For example, a droplet contact method23−25 52
and droplet interface bilayers26,27 that form bilayer membranes 53
by contacting droplets that are surrounded by a lipid 54

49 vibrationaland high voltage (≳250 mV) perturbations. As
50 such, numerous developments of methods for the formation of
51 lipid bilayer membranes have been reported for channel
B

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55 monolayer have been reported as a simple and easy method tobath solution on the opposite side of the membrane from the 117
nanoneedle. In addition to protein insertion orientation, here 118
we demonstrate how the length of the PEG monolayer aﬀects 119channel conductance and how asymmetric salt concentrations 120
can overcome current drift presumably caused by the 121
capacitive charging of the double layer at the surface of gold 122
nanoneedle electrodes.
123
Eﬀects of PEG Monolayer Length and Protein 124
Insertion Direction on Channel Measurements. The 125
length of the PEG monolayer used to modify the surface of the 126
gold nanoprobe has noticeable eﬀects on the stability, 127
conductance, and overall performance of the protein channel 128
measurement. Speciﬁcally, we investigated the eﬀects of two 129
diﬀerent thiolated PEGsa short (MW = 282.35; length = 130∼1.8 nm) and long (MW = 3000; length = ∼24 nm) PEG 131
monolayer by evaluating the pore conductance. The direction 132
of αHL pores should also be considered in order to apply the 133
nanopore-based lipid bilayer platform to chemical detection 134
because the binding and/or translocation of molecules can 135
depend on the direction of the pore. For example, single- 136
stranded DNAs and RNAs more easily translocate from the cis 137
side of αHL pores, whereas βCDs do not bind the αHL pores 138
when entering from the cis side.37 Figure 2 shows typical 139
channel currents of αHL pores and histograms of the initial 140
pore conductance with diﬀerent directions and PEG lengths. 141
The observed channel currents of αHL pores were successfully 142
obtained with insertion of αHL from both directions (tip or 143
bath side) and both PEG lengths tested.
144
When the αHL nanopores were reconstituted into the lipid 145
bilayer from the bath side, a similar pore conductance was 146
obtained regardless of the PEG length. The median initial pore 147
conductances of αHL with the short and the long PEG- 148
modiﬁed nanoneedles are 0.86 ± 0.21 and 1.00 ± 0.17 nS, 149
respectively, in 1 M KNO3 symmetric salt conditions (Figure 1502). Although the initial pore conductance with the short PEG 151
is not much diﬀerent than that of the long PEG, we observe a 152
decay of the channel current several seconds after pore 153
reconstitution when using the shorter PEG (Figure 2 and 154
Figure S1). We hypothesize that the current decay is caused by 155
the much smaller volume of the tip-side PEG layer when 156
employing the short PEG. The ion concentration in the PEG 157
layer after the channel current measurements can be estimated 158
by the volume of the PEG layer and the charge ﬂux. The 159
volume of the PEG layer can be calculated from the geometry 160of the nanoneedle and the PEG length. The shape of the 161
nanoneedle is the combination of a cone and a cylinder with a 162
cone angle of 20° and the cylinder diameter of 250 μm (Figure 163
S2). The total height of the nanoneedle is deﬁned as the height 164of the lipid/oil mixture in the bath chamber and is ∼2 mm. As 165
a result, the surface area of the nanoneedle can be calculated as 166
1.30 mm2, and the volumes of the PEG layer with the short 167(1.8 nm) and the long (24 nm) PEG are 2.4 and 31.2 pL, 168
respectively. The charge ﬂux during the ﬁrst 30 s and the initial 169
number of ions in the PEG layer with the long PEG-modiﬁed 170
nanoneedles are 66.9 pC/s and 1.88 × 1013, respectively. As a 171
result, the concentration change in ion composition in the 172
PEG layer during the ﬁrst 30 s is ∼2.1%. Because the volume 173
with the short PEG is about 13 times smaller (2.4 pL), the 174
concentration change of ions in the tip-side volume of the 175
PEG-modiﬁed nanoneedle would be more than 10 times larger 176
than that of the long PEG-modiﬁed nanoneedle, leading to a 177
more rapid current decay.
178

56 prepare

stable lipid bilayer membranes. Glass micro- and28−30
57 nanopore membranes
also provide a stable platform to
58 support lipid bilayers across small (<20 μm) oriﬁces for
59 protein channel measurements. In recent years, glass pipet
31−35
60 supported lipid bilayers serve as ion channel probesfor
61 the development of imaging probes employed in scanning ion
62 conductance microscopy (SICM). For example, we recently
63 reported the development of single-barrel imaging probes that64 support a lipid bilayer membrane and reconstituted α65 hemolysin (αHL) channels as a means to obtain both chemical
32
66 and ion concentration proﬁle information using a SICM.
67 Baker and colleagues reported similar strategies employing
33,34
68 both single- and double-barrel probes.
69
In an another approach, Ide and co-workers introduced the
70 use of a polyethylene glycol (PEG)-modiﬁed, etched gold
71 electrode to support lipid bilayers and thus protein channel
36,37
72 measurements.
In their measurements, ion channel
73 proteins were immobilized on the tip of the gold nanoneedles,
74 and the channel activities of a single ion channel were
75 measured. The advantage of this approach is that the bilayer is
76 formed by pushing two lipid monolayer leaﬂets together, one
77 oriented tail-out on the surface of the PEG-modiﬁed electrode78 and the other at an oil/lipid aqueous interface. As such, the79 lipid bilayer is not suspended across a pore oriﬁce, rather it
80 separates a small volume aqueous reservoir between the lipid
81 bilayer and gold electrode (tip side) and a bath reservoir on
82 the opposite side of the membrane. The bilayer formation is
83 relatively facile compared to the methods described above and84 requires only that the gold electrode be immersed across this85 oil/lipid aqueous interface.
86
Although Ide and co-workers demonstrated the utility of the
87 PEG-modiﬁed gold nanoneedles for lipid bilayer membrane
88 formation and channel measurements, in this article, we
89 quantitatively characterize the performance of the probe, study
90 the eﬀects of probe surface chemistries, and demonstrate
91 control over the direction of freely diﬀusing protein insertion.
92 We ﬁnd that the current through the protein channel and at
93 the gold electrode is supported by double-layer charging at the
94 electrode surface. Furthermore, we optimize the PEG length
95 and the salt concentration of electrolyte baths for stable
96 current recordings and apply the probe for spatially resolved
97 chemical detection. The nanoneedle-probe-supported bio98 logical nanopore promises to improve the spatial resolution
99 of, for example, bio-SICM probes because of the nanometer
100 scale tip size.
101 RESULTS AND DISCUSSION
102 Theetched and modiﬁed gold nanowire, when immersed

103 across a lipid/oil aqueous interface, supports a lipid bilayer
104 membrane that is able to allow single ion channel measuref1f2

105 ments (Figures 1 and 2). Similar to previous reports by Ide and35,36
106 co-workers,
the PEG-layer-modiﬁed electrode is able to107 support a lipid bilayer as veriﬁed by the insertion of αHL

108 channels into the lipid bilayer. In this study, we demonstrate
109 that the protein channel can be incorporated from either the
110 tip side (tip-side insertion) or from the bath side (bath-side
111 insertion) as the previous study only demonstrated bath-side
35,36
112 reconstitution.
Tip-side insertion involves incorporating
113 thechannel from the aqueous solution within the PEG

114 monolayer between the bottom leaﬂet of the bilayer membrane
115 andelectrode surface (Figure 1). Conversely, bath-side
occurs when αHL monomers are present in the

116 insertionC

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Figure 3. Tip-side insertion method allows the detection of bath-side βCD. (Top) Single-βCD events are indicated by resistive pulses at∼85% of the open channel current. (Bottom left) βCD binding events block, on average 82% of the open channel current, with a mean dwell
time of 7.8 ms. However, over the course of the experiment, (bottom right) the blocking current and number of events decreased. In
addition, we observed a reproducible decay in the open channel current discussed below.
179

Protein conductance using tip-side insertion exhibited atypical current−time trace indicating single blockade events as 214
S7βCD enters an αHL pore. The scatter plots of blocking 215
current magnitude and duration caused by S7βCD binding 216
events are also shown in Figure 3. S7βCD binding events were 217
clearly observed using the long PEG-modiﬁed nanoneedle- 218
based biological nanopore probe with the tip-side insertion of 219
αHL nanopores (the correct orientation for S7βCD binding 220
events).
221
Eﬀects of Electrode Double-Layer Charging on 222
Observed Current−Time Traces. The open channel 223
current, in symmetrical 1.0 M KNO3 solutions, is not stable 224
over several seconds. More speciﬁcally, we observe a 225
reproducible drift in the open channel current over the time 226
scale of seconds to minutes (Figure 3). In addition, the 227
frequency of S7βCD binding events dramatically decreased 228
after several seconds of recording (Figure 3). We hypothesize 229
that the decay of the open channel current is caused by 230
charging up the electric double layer of the surface of the gold 231
nanoneedle (which likely carries the current at the gold 232
electrode). The decrease of S7βCD binding events also relates 233
to the double-layer charging. As the potential drop decreases 234
across the nanopore by charging up the electric double layer, 235
the electrophoretic force acting on the S7βCD molecules drops 236
and the frequency of S7βCD binding events also decays.41 To 237
better understand the observed current decay, the gold 238
nanoneedle-based biological nanopore probe can be described 239
as an equivalent circuit in which the capacitance of the electric 240
double layer and the resistance of the αHL nanopore are 241
connected in series (Figure S4). The time-dependent current 242
i(t) response should follow eq 1.
243

180 much stronger dependence on PEG length. With the tip-side
181 insertion of αHL, the median initial pore conductance of αHL
182 is 0.38 ± 0.10 and 1.15 ± 0.19 nS for short and long PEG,

183 respectively (Figure 2). The pore conductance with the short
184 PEG is much lower than what is observed with the longer
185 PEG.
186

We hypothesize that the pore conductance relates to the

187 extent of physical blocking of the protein pore aperture, which
188 changes with the direction of insertion and the length of PEG.
189 This is because the distance between the pore opening and the
190 surface of the nanoneedle changes with PEG length and the
191 direction of the nanopore. The long PEG layer, ∼24 nm in
192 length, is much longer than the cis portion of the protein, thus
193 there is space for ions to move freely to and from the aperture
194 of hemolysin. On the other hand, the short PEG length is only

195 ∼1.8 nm which, regardless of protein orientation, creates an
196 impediment to ion ﬂow through the protein channel, leading

197 to a lower overall observed conductance value. This eﬀect is

198 exacerbated when using tip-side insertion of αHL nanopores,
199 which led to conductance values that were much lower than

200 those of the bath-side-inserted αHL because the length of the
201 short

PEG (∼1.8 nm) is smaller than the extramembrane38
We also measured the channel
203 current of SLO nanopores with the long PEG-modiﬁed
204 nanoneedle (Figure S3). Although these channels are much
205 larger (>10 nm) in comparison to αHL nanopores, we
206 successfully measured channel currents. From this result, the207 long PEG-modiﬁed nanoneedle allows us to utilize other kinds
208 of biological nanopores that have larger pore diameter.
S7βCD Detection Using the Nanoneedle-Based Nano209
210 pore Probe. To demonstrate the resistive-pulse chemical
211 detection abilities of the nanoneedle-based probe, similar to a
37,39,40
we detected binding events
212 traditional bilayer cup setup,
213 of S7βCD molecules with αHL pores. Figure 3 shows the
202 portion of αHL (∼6 nm).

f3

i(t ) =

V −t / R pCdeR(1) 244

In eq 1, V is the applied potential between the gold 245
nanoneedle and the reference electrode, and Rp and Cd indicate 246
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Figure 4. Introduction of asymmetric salt concentrations (tip side/bath side) circumvents the decay in open channel current. (Top) Typicalcurrent−time traces of αHL nanopores with βCD in the asymmetric salt condition (tip side/bath side = 1 M/0.2 M) exhibits a much morestable open channel current while still allowing for single-molecule measurements. (Bottom left) Rate of open channel current decay (I/I0)
is a function of the asymmetric salt conditions. In addition (bottom right), the binding event frequency is aﬀected by the current decay andsalt conditions. At a tip side/bath side ratio of 1 M/0.2 M, the βCD binding event frequency remains constant during the time frame of anexperiment where, at ratios of 1:1 M and 3:1 M, event frequency decreases throughout the experimental time scale.
247 theresistance of the nanopore and the capacitance of theshown in Figure 4. With the salt condition of 3 M/1 M and 1 280
M/1 M KNO3, the channel currents decreased more than 10% 281
over 5 s. Each then decayed to ∼70% of the initial value over 282∼30 s. The event frequencies of S7βCD binding also dropped 283
to half the initial frequencies (i.e., in ﬁrst 5 s), gradually 284
decreasing afterward. With the salt condition of 1 M/0.2 M 285
KNO3, on the other hand, the channel current was almost 286
maintained at the initial value, and the decay within the ﬁrst 30 287
s was less than 5%. In addition, S7βCD binding event 288
frequency was maintained at around the initial event frequency 289(2.20 ± 0.27/s). As the decay of the channel current and the 290
event frequency are hypothesized to be caused by double-layer 291
charging at the electrode interface, a higher pore resistance can 292
decrease the RC time constant. The pore resistances of the 293
αHL nanopore with asymmetric salt conditions of 3 M/1 M 294
and 1 M/0.2 M KNO3 that are calculated from the initial open- 295
level channel current was 0.8 and 4.0 GΩ, respectively. The 296
decay rate of the calculated open channel current with 4.0 GΩ 297
pore resistance is very close to one of the measured currents 298
with the asymmetric salt condition of 1 M/0.2 M KNO3 299(Figure S4). As a result, the decays of the channel current and 300
event frequency were extended with the salt condition of 1 M/ 301
0.2 M KNO3, due to the higher pore resistance. As a future 302
plan, the use of the equilibrium electrodes such as the Ag/AgCl 303
electrode as the nanoneedles would improve the current decay 304
caused by double-layer charging without the need to use 305
asymmetric salt conditions.
306
The frequency of S7βCD binding events is a function of the 307
concentration of S7βCD (Figure 5). We recorded current− 308 f5
time traces in bath solutions of S7βCD at concentrations of 10, 309
50, 100, and 200 μM using the long PEG-modiﬁed nanoneedle 310
probe and asymmetric salt condition of 1 M/0.2 M KNO3 311(tip/bath solution). The event frequencies with the S7βCD 312

248 double layer, respectively. The applied potential is +100 mV,

249 and the pore resistance when the αHL is reconstituted from
250 the tip side and the lipid bilayer was formed by the long PEG251 modiﬁed gold nanoneedle is 0.87 GΩ. The capacitance of the
252 gold nanoneedle is calculated by the capacitance per unit area
253 of the double layer of the gold surface and the surface area of
254 the gold nanoneedle immersed in the lipid/oil mixture. The
255 capacitance perunit area of the double layer of the PEG-

2 42
256 modiﬁed gold surface is expected to be ∼5 μF/cm . Because

257 the shape of the nanoneedle tip is a combination of a circular

258 cone and cylinder, the surface area is calculated as πrnm +259 2rwπh, where rn is the radius of the nanoneedle, m is the length
260 of the conical generatrix, rw is the radius of the gold wire, and h
261 is the length of the cylinder. The estimated surface area is 1.3
2
262 mm . The calculated time-dependent circuit current should
263 decay to ∼60% of the initial value in 30 s. This time response is

264 similar to the recorded current decay experimentally (Figure
265 S4).

Furthermore, the potential across the nanopore is
as i(t) × Rp, and the potential also decays to267 ∼60% of the initial value in 30 s. As a result, the268 electrophoretic force acting on the S7βCD molecules that is
269 dependent on the electric ﬁeld would be reduced and the event
270 frequency also decayed.
Improvement of Decays of Channel Current and
271
272 Event Frequency. To improve the observed decay in channel
273 current and event frequency, we applied asymmetric salt
274 conditions to the tip and bath solutions. The typical recorded
275 channel currents with asymmetric salt conditions of 3 M/1 M
276 and 1 M/0.2 M KNO3 (tip/bath solution) are shown in Figure277 S5 and Figure 4. Normalized channel currents and numbers of
278 S7βCD binding events every 5 s with salt conditions of 1 M/1
279 M, 3 M/1 M, and 1 M/0.2 M KNO3 (tip/bath solution) are
266 calculated

f4

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relatively high concentration, the calibration curve did not pass 333
through the origin.
334
Determination of S7βCD Concentration in the Micro- 335
channel with a Nanoneedle-Based Nanopore Probe. 336
The nanoneedle probe enables spatially resolved concentration 337
proﬁle measurements, demonstrated using a microﬂuidic 338
channel device to generate a linear concentration gradient 339
proﬁle. The microchannel comprised two main parallel 340
channels and a narrow perpendicular channel connecting the 341
two main channels. The concentration gradient of S7βCD is 342
generated in the narrow channel by ﬂowing the solutions with 343
higher and lower S7βCD from each inlet of the main channels. 344
The simulated concentration mapping in the microchannel and 345
graph of S7βCD concentration in the narrow channel are 346
shown in Figure 6. The S7βCD concentration in the narrow 347 f6channel changes linearly between the two main channels, and 348
the S7βCD concentrations at open spaces (x = 5.75, 11.75, and 349
17.75 mm) are 150, 100, and 50 μM, respectively. The 350
frequencies of the S7βCD binding events also estimated at 351
2.48, 1.95, and 1.42/s using the calibration curve.
352
The typical recorded channel currents and the plots of the 353
event frequency of S7βCD binding events at each open space 354(x = 5.75, 11.75, and 17.75 mm) are shown in Figure 6. S7βCD 355
binding events were obtained at each open space, and the 356
event frequency was clearly diﬀerent at each open space. The 357
event frequencies at each open space (x = 5.75, 11.75, and 358
17.75 mm) were 2.94 ± 0.33, 1.96 ± 0.09, and 1.18 ± 0.48/s 359(n > 3), respectively. From these results, the concentrations at 360
each open space were calculated as 192, 101, and 27 μM. 361
Although the gradient of the ﬁt curve was larger than the 362
simulated value, the measured S7βCD concentrations followed 363
a linear trend (Figure 6). The larger gradient indicates that the 364
concentration distribution in the narrow channel did not reach 365
static state (Figure S6).
366

CONCLUSION
367
In conclusion, we improved the operational parameters of the 368
nanoneedle-based ion channel recording method that Ide and 369
co-workers previously reported and applied it as the local 370
nanopore sensing method by combining with microﬂuidic 371
technologies. First, we optimized the length of the modiﬁed 372
PEG to obtain a suitable conductance of αHL nanopores and 373
controlled the direction of αHL insertion to allow detection of 374
target chemicals. As a result, the gold nanoneedle modiﬁed 375
with the long PEG provided the expected pore conductance of 376
αHL for both directions of the αHL pore. We also 377
demonstrated detection of binding events of S7βCD molecules 378
in αHL pores with the biological nanopore probe when αHL 379
proteins were inserted from the tip side. Although the binding 380
events were detected, the channel current of the open level and 381
the event frequency of S7βCD binding decayed in a few 382
seconds. To prolong these decays, we applied the asymmetric 383
salt condition of 1 M/0.2 M KNO3 to the solutions of the tip 384
and the bath sides. As a result, the event frequency and the 385
channel current were maintained at initial values more than 30 386
s, and the event frequency was 2.20 ± 0.27/s with the S7βCD 387
concentration of 100 μM and the applied potential of 100 mV. 388
The event frequency was linearly changed between the 389
concentration range of 10 to 200 μM. Finally, we 390
demonstrated the ability to achieve spatially resolved measure- 391
ments by measuring the S 7 βCD concentration in a 392
concentration gradient, generated in the microchannel, with 393
the nanopore probe. We believe that our nanoneedle-based 394

Figure 5. Frequency of βCD binding events is a function of theconcentration of βCD in the bath-side solution. The graph shows
the calibration curve of the βCD concentration and the event
frequency. More speciﬁcally, the event frequency is linearly related
to the concentration of βCD (n > 3).
313 concentration of 10, 50, 100, and 200 μM in the initial 30 s

314 after the single pore reconstitution are 0.94 ± 0.39 (n = 4),
315 1.31 ± 0.05 (n = 3), 2.20 ± 0.27 (n = 5), and 2.91 ± 0.22/s (n316 = 4), respectively (Figure 5 and Table S1). The number of

317 blocking signals caused by the S7βCD binding clearly increased

increasing S7βCD concentration. The event frequency
319 versus the S7βCD concentration relationship was linearly ﬁtted
2
2
320 with a high correlation factor (R ) (mean R value of 0.96, n =318 with

321 3) in the range from 10 to 200 μM S7βCD concentration

322 (Figure 5), and the intercept of the calibration curve does not
323 pass through the origin. Previously, we reported the detection

324 of βCD binding events with an αHL nanopore. The nature of
325 the calibration curve is a function of the concentration window
326 employed.

For example, when using a low concentration

327 window (10−50 μM), the calibration curve passes through the
328 origin. At high concentration regions (0.5−5 mM), the binding
329 event frequency is underestimated on account of not being
330 ableto distinguish closely spaced events, and as such, the40
331 calibration curve does not pass through the origin.
In the332 present

study, as we measured βCD binding events at a

F

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Figure 6. Nanoneedle-based nanopore probe enables spatially resolved measurements. The concentration gradient of βCD was generated byﬂowing the solution with and without βCD to the H-shaped microchannel, and the βCD concentration was measured with the nanoneedlebased nanopore probe. (Top) βCD concentration was simulated by the hydrodynamic simulation. The concentration gradient of βCD was
linearly generated in the narrow channel, and the frequency of the βCD binding events was calculated from the calibration curve. (Bottom
left) Typical current−time traces of αHL nanopores at each point. (Bottom right) Event frequency decreased with increasing the x distance,
and the measured βCD concentration shows the linear gradient.
the chemical for nanopore sensing. DL-Dithiothreitol (DTT; Sigma- 421
Aldrich) was used as the reducing agent for SLO nanopores.
422
Preparation of a PEG-Modiﬁed Gold Nanoneedle. A gold 423
nanoneedle (Figure S2) was fabricated by electrochemical etching of 424
the gold wire in a 2.0 M CaCl2 solution (distilled water). A carbon 425
rod with 3 mm diameter was used as a counter electrode, and a 426
function generator (GFG-8219A; GW Insteck) was used to control 427
the input voltage magnitude and frequency. The 1 mm tip of the gold 428
wire was vertically immersed in the CaCl2 solution. Then, a sine wave 429
AC voltage was applied between the gold wire and the counter 430
electrode until the current reached zero, typically in 60 min. The 431
typical conditions of the applied voltage were AC voltage of 12 432
Vpeak to peak (p‑p) at 1 kHz with a DC oﬀset of 9.5 V.
433
The nanoneedle was modiﬁed with thiol-PEG to prepare a 434
hydrophilic surface suﬃcient to support a lipid bilayer membrane. 435
First, the gold nanoneedle was electrochemically cleaned to prepare a 436
ﬁne gold surface. See Supporting Information for details of the 437
cleaning method of the gold nanoneedle. Then, the tip of the gold 438
nanoneedle was immersed in 60 mg/mL thiol-PEG solution (in 439
ethanol) for 2 h at room temperature. Finally, the nanoneedle was 440
rinsed with ethanol and deionized water.
441
Formation of a Lipid Bilayer Membrane. A bath solution of 442
two immiscible layers that are aqueous electrolyte solutions (1 M 443
KNO3, 20 mM sodium phosphate buﬀer, pH 7.4) and the oil/lipid 444
mixture (20 mg/mL DPhPC for αHL nanopores or DPhPC/ 445
cholesterol = 7:3 (mol/mol) for SLO nanopores in n-decane) was 446
prepared. The layered solution consisting of the oil/lipid mixture in 447
the top layer and the aqueous electrolyte solution in the bottom layer 448

395 nanopore sensor platform has a potential to allow much higher
396 spatial resolution as a scanning probe for use in bio-SICM.
397 EXPERIMENTAL METHODS

Reagents and Chemicals. Calcium chloride (CaCl2; Sigma398
399 Aldrich) was used for electrochemical etching to fabricate a gold
400 nanoneedle. A 250 μm diameter gold wire of 99.95% purity (Alfa
401 Aesar) was used as a material of the gold nanoneedle. Thiol-dPEG4402 acid (MW 282.35; Quanta BioDesign) and O-(3-carboxypropyl)-O′403 [2-(3-mercaptopropionylamino)ethyl]propylethylene glycol (MW404 3000; Sigma-Aldrich) were used as short and long thiolated
405 polyethylene glycol (thiol-PEG), respectively. 1,2-Diphytanoyl-sn406 glycero-3-phosphocholine (DPhPC; Avanti Polar Lipid), cholesterol
407 from ovine wool (Chol; Avanti Polar Lipid), and n-decane (Merck
408 Millipore Corp.) were used as the lipid/oil solution. Potassium nitrate
409 (KNO3; Sigma-Aldrich) and sodium phosphate buﬀer (pH 7.4) made410 with sodium dihydrogen phosphate (NaH2PO4·2H2O; Sigma411 Aldrich) and sodium hydrogen phosphate (Na2HPO4·2H2O; Sigma412 Aldrich) were used as the aqueous electrolyte solution. The buﬀer
413 solution was prepared using ultrapure water from a Milli-Q (Merck
414 Millipore Corp.) resist of 18.2 MΩ at 25 °C. α-Hemolysin (αHL;
415 Sigma-Aldrich) isolated from Staphylococcus aureus and Streptolysin O
416 (SLO; Sigma-Aldrich) isolated from Streptococcus pyogenes were
417 obtained as a monomer protein in the form of a powder. For use,
418 samples were diluted to the designated concentration using a buﬀered
419 electrolyte solution and stored at 4 °C. Heptakis(6-O-sulfo)-β420 cyclodextrin heptasodium salt (S7βCD; Sigma-Aldrich) was used as
G

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ACS Nano
449 is formed, and a lipid monolayer is formed at the interface between
450 these two layers. The PEG-modiﬁed gold nanoneedle was dipped in
451 the electrolyte solution for 10 min and then immersed in the bath452 solution. The tip-side aqueous solution is drawn from the solution
453 into which the tip is dipped prior to being inserted into the bath. A
454 lipid bilayer membrane is formed on the tip when the tip is pushed
455 through the interface of these two layers. The movement distance was
456 manipulated with a three-axis manual micromanipulator (Newport
457 Corp.).
Channel Current Measurements of Biological Pores. We
458
459 applied two lengths of PEG and two insertion directions of αHL pores
460 to the nanoneedle-based lipid bilayer platform and measured channel
461 currents of each pattern. Thiol-PEGs with molecular weight of 282.35
462 and 3000 g/mol were used as a “short” and “long” length PEG,
463 respectively. The direction of αHL pores was controlled by placing
464 αHL in either aqueous electrolyte solution of bath or tip side. To
465 reconstitute from the tip side, the nanoneedle was dipped in the
466 electrolyte solution (1 M KNO3, 20 mM sodium phosphate buﬀer,
467 pH 7.4) containing 30 mM αHL for 10 min before immersing the468 nanoneedle into the bath solution. From bath side, the aqueous
469 electrolyte solution with 15 mM αHL was used as the bath solution.
470 For the reconstitution of SLO nanopores, SLO was dissolved in the
471 electrolyte solution (0.2 M KNO3, 10 mM DTT, 20 mM sodium
472 phosphate buﬀer, pH 7.4) at 32 nM, and it is used as the bath473 solution. The quasi-reference-counter Ag/AgCl electrode was also
474 immersed in the aqueous electrolyte solution in the bath chamber,
475 and a potential of +100 mV was applied to the tip side. Channel
476 currents were monitored using a patch clamp ampliﬁer (PICO 2,
477 Tecella) with a 7.9 kHz low-pass ﬁlter at a sampling frequency of 40
478 kHz. Analyses of channel signals were performed using pCLAMP
479 version 10.7 (Molecular Devices).
S7βCD Detection and Analysis. To demonstrate the chemical
480
481 detection using the nanoneedle-based nanopore probe, we added
482 S7βCD at a concentration of 100 μM to the bath aqueous solution
483 (trans side). We used the long PEG-modiﬁed nanoneedle-based
484 nanopore probe, and αHL was reconstituted from the tip side. A
485 potential of +100 mV was applied to the tip side (cis side), and486 channel currents were monitored with the patch clamp ampliﬁer. For
487 the analysis of the S7βCD binding events, the channel current data
488 with the single αHL nanopore of the initial 30 s after the pore489 reconstitution were utilized.
S7βCD Detection with Asymmetric Salt Conditions. To
490
491 extend the decays of the channel current of αHL pores and increase
43
492 the frequency of S7βCD binding events, we applied asymmetric salt
493 conditions in aqueous electrolyte solutions of the tip and bath sides.
494 Salt conditions of 3 M/1 M KNO3 and 1 M/0.2 M KNO3 (tip/bath)495 were applied, and the channel currents at +100 mV were measured.
496 The long PEG-modiﬁed nanoneedle-based nanopore probe was used,
497 and αHL was reconstituted from the tip side. The concentration of
498 S7βCD in the bath aqueous solution (trans side) was 100 μM.
Detection of S7βCD Concentration Inside a Concentration
499
500 Gradient Microchannel. To demonstrate our nanoneedle-based
501 nanopore probe could achieve spatially resolved chemical sensing, we
502 measured S7βCD concentration in a concentration gradient micro503 channel. As the concentration microchannel, an H shape micro504 channel was prepared (Figure S7). The microchannel is composed of
505 two main channels, and a narrow channel connects two main
506 channels. The widths of the main and the narrow channels are 1.0 and
507 0.25 mm, respectively, and the depth is 0.8 mm. The concentration
508 gradient is generated in the narrow channel by ﬂowing the solutions
509 with diﬀerent chemical concentration to each main channel.
First, the concentration distribution of S7βCD in the microchannel
510
511 by ﬂowing solutions with 0 and 200 μM S7βCD to the inlets 1 and 2
512 was simulated with COMSOL Multiphysics 5.2a (COMSOL Inc.)
513 using the microﬂuidic module. The density and viscosity of the
514 solution and the diﬀusion coeﬃcient were set to 1.0 g/mL, 1.0 mPa·s,−92515 and 1.0 × 10 m /s, respectively. The ﬂow rate was set to 100 μL/h
516 for each inlet. The microchannel was fabricated by cutting acrylic
517 sheets with a laser cutter (VLS3.50, Universal Laser Systems, Inc.)
518 and bonding with an acrylic cement (Weld-on). In order to insert thenanoneedle probe into the narrow channel by the micromanipulator, 519
open spaces (1 mm square) on the narrow channel were constructed. 520
Each solution (0.2 M KNO3, 20 mM sodium phosphate buﬀer, pH 521
7.4) with and without S7βCD was ﬂowed to the channel, which is 522
ﬁlled by 100 μM S7βCD solution (0.2 M KNO3, 20 mM sodium 523
phosphate buﬀer, pH 7.4) at 100 μL/h by injecting and sucking with a 524
syringe pump (YSP-202, YSP Co., Ltd.). After 15 min from ﬂowing 525
the solutions, the oil/lipid mixture (20 mg/mL DPhPC in n-decane) 526
was added to the open spaces, and the nanoneedle-based nanopore 527
probe in which the long PEG was modiﬁed and αHL was 528
reconstituted from the tip side was inserted to the narrow channel 529
passing through the oil/lipid solution. A potential of +100 mV was 530
applied to the tip side, and channel currents were monitored with the 531
patch clamp ampliﬁer.
532

ASSOCIATED CONTENT

533

S Supporting Information*

534

The Supporting Information is available free of charge on the 535
ACS Publications website at DOI: 10.1021/acsnano.8b09667. 536
Cleaning procedures of the gold nanoneedle, SEM 537
images of gold nanoneedles, design and photograph of 538
the microchannel, comparison of current decays 539
between the long and short PEG, equivalent circuit of 540
the nanoneedle-based nanopore probe and the calcu- 541
lated decay rates, and typical current−time traces of 542
αHL nanopore with the asymmetric salt condition of 3 543
M/1 M (PDF)
544

AUTHOR INFORMATION

545

Corresponding Author546*E-mail ryan.white@uc.edu.

547

ORCID

548

Ryuji Kawano: 0000-0001-6523-0649
Ryan J. White: 0000-0003-0849-0457

549

Notes

551

The authors declare no competing ﬁnancial interest.

552

550

ACKNOWLEDGMENTS
553
This material is based upon work supported by the National 554
Science Foundation under CHE 1608674 and Japan Society 555
for the Promotion of Science (JSPS) research Fellow, and 556
KAKENHI (Grant No. 17K19138) from the MEXT Japan. We 557
thank Dr. Henry S. White and Dr. Martin Edwards for helpful 558
discussions on the current decay.
559
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ACS Nano XXXX, XXX, XXX−XXX