Article

Simple Fabrication of Solid-State Nanopores on a Carbon Film
Natsumi Takai 1, Kan Shoji 1,2,*, Tei Maki 1,3 and Ryuji Kawano 1,*
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo,
184-8588, Japan; takai723nt@gmail.com (N.T.); tmaki@jeol.co.jp (T.M.)
2 Department of Mechanical Engineering, Nagaoka University of Technology, Niigata, 940-2188, Japan
3 JEOL Ltd., Tokyo, 196-8558, Japan* Correspondence: kshoji@mech.nagaokaut.ac.jp (K.S.); rjkawano@cc.tuat.ac.jp (R.K.)
1

Abstract: Solid-state nanopores are widely used as a platform for stochastic nanopore sensing because they can provide better robustness, controllable pore size, and higher integrability than biological nanopores. However, the fabrication procedures, including thin film preparation and nanopore formation, require advanced micro-and nano-fabrication techniques. Here, we describe thesimple fabrication of solid-state nanopores in a commercially available material: a flat thin carbon
film-coated micro-grid for a transmission electron microscope (TEM). We attempted two general
methods for nanopore fabrication in the carbon film. The first method was a scanning TEM (STEM)
electron beam method. Nanopores were fabricated by irradiating a focused electron beam on the
carbon membrane on micro-grids, resulting in the production of nanopores with pore diameters
ranging from 2 to 135 nm. The second attempt was a dielectric breakdown method. In this method,
nanopores were fabricated by applying a transmembrane voltage of 10 or 30 V through the carbon
film on micro-grids. As a result, nanopores with pore diameters ranging from 3.7 to 1345 nm wereobtained. Since these nanopores were successfully fabricated in the commercially available carbon
thin film using readily available devices, we believe that these solid-state nanopores offer great utility in the field of nanopore research.
Keywords: nanopore; solid-state nanopore; carbon thin film; dielectric breakdown
Citation: Takai, N.; Shoji, K.;
Maki, T.; Kawano, R. Simple
Fabrication of Solid-State Nanoporeson a Carbon Film. Micromachines
2021, 12, 1135. https://doi.org/
10.3390/mi12091135
Received: 6 August 2021
Accepted: 18 September 2021
Published: 21 September 2021
Publisher’s Note: MDPI stays neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction
Nanopore technology has received extensive attention due to its unique capabilities
on the single-molecule scale, which makes it useful for several applications: single-molecule detection [1–4], nanopore sequencing [5,6], and as nanopore filters [7,8]. With nanopore technology, there are two different types of nanopores, biological and solid-state
nanopores. Biological nanopores are peptide- and protein-based nanopores and are reconstituted into a bilayer lipid membrane. Because biological nanopores can provide amuch smaller pore size and higher spatial resolution compared with solid-state nanopores (ssNPs), they have already been applied as DNA sequencers [1,6,9–15]. On theother hand, glass nanopores [16], track-etched nanopores [17], and ssNPs [18,19] are constructed entirely of artificial materials. Because they have advantages over biological nanopores including controllability of pore size, and mechanical, electrical, thermal, andchemical stability, they are expected to be widely applied as the nanopore sensors withthe highest durability.
The fabrication procedures and characteristics of these nanopores are as follows.
Glass nanopores are formed by pulling glass capillaries with a pipette puller. They arewidely used because they use the cheapest material (under $1 per single glass capillary)
and the simplest fabrication procedure. By contrast, the effective length of these nanopores is longer than ssNPs, and the spatial resolution tends to be lower than other nanopores. Track-etched nanopores are fabricated by initially irradiating an ion beam on
polymer membranes to form damaged tracks and subsequently etching the tracks. In thisMicromachines 2021, 12, 1135. https://doi.org/10.3390/mi12091135

www.mdpi.com/journal/micromachines

Micromachines 2021, 12, 1135

2 of 12method, various geometries of nanopores can be fabricated by changing the tracking process and etching methods. However, because a heavy ion accelerator is required to form
the damaged tracks, the fabrication process is quite costly. A single ssNP can be fabricatedin a free-standing thin membrane by irradiation with ion/electron beam sculpting [20,21],a focused electron beam (EB) with a scanning transmission electron microscope (STEM)[22,23], or using dielectric breakdown (DB) [24–26]. These fabrication methods have already been established for various materials, such as silicon nitride (SiNx) [27,28] and silicon oxide (SiOx) [22,29], alumina materials [30,31], and graphene [32–34] or other 2-D
materials [35,36]. However, before forming a single pore, an ultrathin membrane needs tobe prepared. To fabricate such thin membranes, micro-and nano-fabrication techniques,
including photolithography, chemical vapor deposition, and reactive ion etching are performed, but these fabrication procedures are usually time-consuming and costly. Therefore, a strategy for the simple preparation of the thin membrane at low cost is required forgeneral research use and would facilitate the expansion of nanopore research.
Herein, we propose the use of a commercially available thin carbon film coated on a
micro-grid for TME observation as the thin membrane (Figure 1). The carbon membrane
has a thickness of around 6 or 10 nm and is readily purchasable from various companies.
In addition, carbon membrane with a 2 nm thickness can be purchased from JEOL Ltd.,
Japan, and nanopores with shorter pore lengths than conventional SINx and SiOx nanopores could potentially be fabricated. Besides, they are available at a reasonable pricefrom several companies (around $10 per single micro-grid). Also, because the carbon film
is on the micro-grid for TEM observation, the purchased membrane can be directly applied for nanopore fabrication by the STEM EB method. In addition, the possibility of surface modification and the functionalization of carbon [37] could offer specific sensing[38,39] and signal amplification [40]. We fabricated nanopores in the thin carbon membrane by either the STEM EB method or the DB method (Figure 1). In this paper, we discuss the simplicity and size-controllability of nanopores fabricated by these two methods
by comparing them with conventional methods to fabricate solid-state nanopores.

Figure 1. Schematic illustrations of strategies to fabricate solid-state nanopores in a carbon film ona micro-grid. The nanopores were fabricated by two different methods. One is the “STEM electron
beam method”. In this method, the nanopores were fabricated using a STEM, by irradiating a focused electron beam on the carbon thin film. The other is the “dielectric breakdown method”. In
this method, nanopores were formed by applying a trans-membrane voltage through the carbon
thin film immersed in an electrolyte solution.

Micromachines 2021, 12, 1135

3 of 122. Materials and Methods
2.1. Experimental Materials
Micro-grids consisting of an amorphous carbon thin film coating with a thickness of
6 or 10 nm were purchased from Okenshoji Co., Ltd., Tokyo, Japan, or JEOL Ltd., Tokyo,
Japan. The product No. of each grid is UHR-C10, SHR-C075 (Okenshoji Co., Ltd., Tokyo,
Japan), and 1671 (JEOL Ltd., Tokyo, Japan), and the price was around $10–20 per single
grid. The micro-grids are manufactured by attaching a micro-porous support film to 200
mesh copper grids. The size of the micro-grid hole diameter is between 2.5 and 10 μm.
Since the carbon film is prepared between a water–gas interface, the thickness is extremely
uniform. In addition, because of membrane tension, the carbon film does not distort. 3morpholinopropane-1-sulfonic acid (MOPS, Nacalai Tesque, Kyoto, Japan), potassium
chloride (KCl, Nacalai Tesque, Kyoto, Japan), hydrochloric acid (HCl, FUJIFILM Wako
Pure Chemical Corp., Osaka, Japan), and potassium hydroxide (KOH, FUJIFILM Wako
Pure Chemical Corp, Osaka, Japan) were used as the electrolyte solution. The buffer solution was prepared using ultrapure water from a Milli-Q (Merck Millipore Corp., Burlington, MA, USA) at 18.2 MΩ at 25 °C. For channel current measurements of solid-state nanopores and nanopore fabrications by the DB method, we prepared chambers made frompolydimethylsiloxane (PDMS; Sylgard 184, Dow Corning Toray Co., Ltd., Tokyo, Japan)
in which a Ag/AgCl electrode was incorporated (Figure S1). The micro-grid was sandwiched between 0.2 mm thick polymethyl methacrylate plates (PMMA plates, Mitsubishi
Chemical Corp., Tokyo Japan) with a hole in the center and fixed with glue. Then, thePMMA plate incorporating the micro-grid was sandwiched between PDMS chambers.
2.2. Fabrication of Nanopores by STEM EB Method
For the irradiation with a focused electron beam, Field Emission TEM (FE-TEM)(JEM-2200FS; JEOL Ltd., Tokyo, Japan) with 200 kV acceleration voltage was used. The
spot diameter of the focused electron beam was set to 1.5 nm. We conducted nanopore
fabrication whilst checking the image displayed on the screen of Analysis Station (JEOL
Ltd., Tokyo, Japan), in which an energy dispersive spectroscopy (EDS) analysis system islinked with FE-TEM.
2.3. Fabrication of Nanopores by DB Method
The DB method was performed by applying 10 or 30 V of voltage through the microgrid fixed in the PDMS chamber using an electrochemical analyzer (ALS600DZ, BAS Inc.,Tokyo, Japan) or a DC power supply (DPS-3003, CUSTOM Corp., Tokyo, Japan). We
added 2 M of KCl electrolyte into both chambers and applied the voltage. While applying
the voltage, we monitored the current passing through the carbon membrane using a digital multimeter (CD771, Sanwa electronic instrument). When the monitored current increased rapidly, we turned off the voltage. The pore size was calculated from the current
using Hille’s equation [41]. In the DB method, we investigated the influence of pH, applied voltage, and membrane thickness on the fabrication time and pore size.
2.4. Channel Current Measurements and Data Analysis
For nanopores fabricated by the STEM EB method, an oxygen plasma was exposedto the micro-grids with a plasma etching system (FA-1, Samco Inc., Kyoto, Japan) to prepare a hydrophilic surface before the channel current measurements. Due to the hydrophobicity of carbon films, the plasma treatment must be first applied to increase hydrophilicity and facilitate the entry of water into the nanopores. Next, the micro-grid wasincorporated into the PDMS chambers, and 1 or 0.1 M of KCl solution (10 mM MOPS, pH
7.0) was added to the chambers. Then, 2 V of potential was initially applied through thenanopore to allow passage of the electrolyte solution into the nanopore until the ion current was obtained. In the DB method, the channel currents were measured in the same
solution as the nanopore fabrication (2 M KCl). Channel currents were monitored using a

Micromachines 2021, 12, 1135

4 of 12

patch-clamp amplifier (PICO2, Tecella, Foothill Ranch, CA, USA) with a 4-kHz low-passfilter at a sampling frequency of 20 kHz. Channel current measurements were conducted
at 22 ± 2 °C. Analysis of the channel signals was performed using pCLAMP ver. 10.7 (Molecular Devices, CA, USA).
3. Results
3.1. Nanopores Fabricated by the STEM EB Method
We attempted to fabricate nanopores in the carbon membrane coating on commercially available micro-grids by irradiating it with a STEM electron beam. There have beenprevious reports of nanopore fabrication using STEM EB irradiation of thin films of siliconmaterials or graphene, and precise pore size control has been achieved [19]. In contrast,
there have been few reports of nanopore fabrication in thin carbon membranes as demonstrated in our experiments, and we would like to highlight the novelty of this nanopore
fabrication process.
First, we tried to fabricate various sizes of nanopores (>2 nm in diameter) by increasing the scanning area of the EB using the following procedures. Irradiation with the focused EB on one point of the carbon film for several tens of seconds caused a knock-on
damage to the carbon, resulting in the ejection of carbon. As a result, a small pore wasformed where the EB radiation was applied. Then, the focused EB was scanned in a specified area for tens of seconds up to 90 min, causing the pore to gradually expand (Figure2a,b). This resulted in the fabrication of square-shaped nanopores and successful control
of pore diameter from 2 to 135 nm (Figures 2c and S2). The carbon spectrum was not detected in the nanopore region with the EDS elemental analysis. Thus, we judged that nanopores were completely open in the carbon film. The area was measured from the TEM
images using the “Image J” software, and the pore diameter was calculated from the area
by approximating a circle. Because the scan direction of the EB is fixed in the horizontal
direction, the shape of the EB-scanned area became rectangular. As a result, squareshaped nanopores were constructed. An advantage of this STEM method is that the nanopore fabrication process can be performed while observing the STEM image. Additionally, when nanopores are fabricated with the STEM method in a free-standing SiNx membrane, the free-standing area must be restricted to around 50 μm square to prevent the
breakage of the membrane. On the other hand, in the case of our TEM-grid membrane, ananopore can be fabricated in a larger 3 mm × 3 mm carbon membrane, because there is a
supporting micro-porous layer underneath the carbon thin membrane. In addition, we
tried different EB irradiation patterns to reduce the fabrication time (Figure S3). However,the process detailed above (Figure 2a) was better than these two methods in terms of thepore formation efficiency.
Next, we evaluated the nanopores by obtaining the current and voltage (I-V) nanopore curves (Figures 2d,e and S4). The I-V curve of the 3.4 nm nanopore shows an asymmetric shape, but that of the 10 nm nanopore does not. An asymmetric I-V curve indicatesa conical-shaped nanopore, and such an effect of the pore geometry on the I-V curve is
expected to be observed in small nanopores [42,43]. Conventional ssNPs fabricated byirradiating EB at the same point on SiNx thin membrane [27] provide an hourglass shape.
On the other hand, we can deduce that since the STEM EB was irradiated from one side
and continuously scanned, conical-shaped nanopores were fabricated, resulting in theasymmetric I-V curve for the smaller nanopore. By contrast, a symmetric I-V curve wasobtained from the larger nanopore, although the nanopore owns a conical geometry. In
nanopore sensing, the shape of nanopores significantly influences the sensitivity of molecular analysis because the resistance of nanopores relates to the narrowest zone of thenanopore. Our I-V evaluations demonstrate that the effective length of the nanopores isshorter than the thickness of the carbon thin film, suggesting that they would be useful as
nanopore sensors. However, we found the larger current noise at large absolute values of
the applied potential (Figure S4). Because the ratio of the membrane thickness to area isMicromachines 2021, 12, 1135

5 of 12significantly large (10 nm:7 mm2), mechanical fluctuations in the membrane would
strongly influence the current noise [44]. To improve the current noise, the self-standing
membrane area should be reduced by using a micro-grid with smaller apertures.
From these results, we can see that the STEM EB method allowed us to fabricate nanopores in commercially available carbon thin films. The effective length of the nanoporesshould be shorter than the thickness of the film, whilst the pore size can be precisely controlled over a wide range (2–130 nm in diameter). However, we could not demonstrate
nanopore sensing of DNA molecules with the nanopores. We believe that the adsorptionof DNA molecules on carbon influenced the performance of nanopore sensing. In the future, we need a surface modification on carbon membranes to prevent the adsorption.

Figure 2. Nanopores fabricated by the STEM EB method. (a) The scanning pattern of a focused EB.
The EB was initially irradiated over a small area to fabricate a small pore. Then, the scanning area
was extended to expand the pore. (b) Fabrication processes of a nanopore in a carbon thin film. A
small pore was expanded in size by controlling the scan area of a focused EB. The scale bars indicate
20 nm. (c) Three different sized nanopores: (left to right) 2.0 nm, 8.5 nm, and 45 nm in diameter.
These diameters were calculated by approximating to a circle. I-V curves of nanopores with (d) 3.4
nm (in 1 M KCl solution) and (e) 10 nm (in 100 mM KCl solution) diameter fabricated by the STEM
EB method.

Micromachines 2021, 12, 1135

6 of 123.2. Nanopores Fabricated by the DB Method
Although we demonstrated successful fabrication of nanopores in commerciallyavailable carbon films with high accuracy by the STEM EB method, the STEM EB method
is still labor-intensive since a FE-TEM is expensive, and its operation requires specificskills and training. Therefore, we next applied the DB method for nanopore fabrication ina carbon film to achieve easier and more rapid nanopore fabrication. To the best of ourknowledge, the DB method is the simplest method to fabricate solid-state nanopores inthin films because nanopores can be fabricated by simply applying a voltage through thefilm in an electrolyte solution, as detailed below [19].
First, the application of a transmembrane voltage through the carbon film generates
an electric field inside the carbon film and charges the interfaces with opposite ions. Then,the electric field induces bond breakage, resulting in structural defects in the film. Whenthe structural defects accumulate, a highly localized conductive path forms, and a nanopore is constructed in the film (Figure S5) [25]. In conventional studies of nanopore
fabrication in SiNx thin membranes, nanopores were fabricated when the applied electricfield was larger than the dielectric breakdown strength. On the other hand, because thecarbon membrane with free electrons is a conductive material, the applied transmembrane
voltage cannot be determined with the same strategy used in previous works. Hence, inthe DB experiments for the carbon film, we attempted to apply two different transmembrane voltages of 10 and 30 V. As a result, we obtained current increases after 4.56 ± 1.14
h and 2.60 ± 1.10 h with 10 and 30 V, respectively (Table 1 and Figure 3a,b). In addition,
the current signals could be classified into several main patterns (Figure S6). The first one
was a step-like current increase in which the current rapidly increased and became constant (Figures 3a and S6c). Approximately 60% of the current signals appeared as this signal. In other patterns, for example, gradual increases in the currents (Figure S6a) and current decreases after an initial increase in current (Figure S6b) were obtained. The gradualcurrent increase might indicate that multiple pores were formed [45,46]. In the DB
method, structural defects potentially occur across the whole area where the transmembrane voltage is applied. Since the carbon thin film area is much larger than conventional
nanopore fabrication in SiNx membranes by the DB method, the probability of multiple
pore formation is much higher than when using SiNx. Signals with a decrease in the current would suggest that complete nanopores were not formed. Because of the increase ofleakage currents through the membrane by several discrete structural defects, breakdown
events at a single point did not ultimately occur, and nanopores were not fabricated. On
the other hand, the single step-like current signals indicate the formation of a single nanopore. Therefore, when the single step-like signal was obtained, we halted the voltage afew seconds after increasing the current.

Micromachines 2021, 12, 1135

7 of 12Figure 3. Nanopore fabrication using method 2 (DB + carbon membrane). (a) Current and time traces of the DB experiments with several different nanopore formations estimated from the ion currents. (b) The time of nanopore formation by
applying different voltages (pH 0.5/0.5.) (c) The time of nanopore formation depending on the pH gradations; (cis/trans)means (positive/negative) application of voltages. (d) Schematic diagrams of the proposed mechanism for the break downby pH gradation. (e) I-V curves of the nanopores fabricated by the DB method for different pore sizes.

Although the formation of nanopores in the carbon film was confirmed by measuring
the ion current, the fabrication times of nanopores (4.56 ± 1.14 h @ 10 V and 2.60 ± 1.1 h @30 V) (Table 1 and Figure 3b) were much longer than the conventionally reported nanopore fabrication in SiNx film by the DB method. Therefore, we attempted to improve
the processing time of pore fabrication by changing the pH gradient between the cis andtrans sides of the membrane. The cis/trans refers to positive/negative application of voltage
at the membrane. As shown in a previous report [47], a pH gradient facilitates the accumulation of charged ions on the membrane surface and can shorten the fabrication time
(Figure 3d). As a result of increasing the pH gradient between cis and trans, we were able
to reduce the fabrication time of nanopores to 1.56 ± 1.14 h with the pH gradient (cis/trans)= 0/13 (Figure 3c and Table 1). By contrast, nanopores were not fabricated with a solution
on the cis side above pH 2.2 even when the voltage was applied for 6 h. Although thefabrication time could be reduced to around 2 h, it is still much longer than in SiNx thinmembranes (several minutes). This result is reasonable because carbon has free electrons(conductive material). Therefore, the required magnitude of the electric field that causes
the breakdown event is much higher than for SiNx membranes. One way to reduce thefabrication time for the carbon film would be to increase the applied transmembrane voltage, as in a previous study where the fabrication time decreased exponentially with anincreasing electric field [25]. However, when we applied 30 V to the carbon film with 6
nm thickness, the current increases were too rapid, and the pore size became too large.
Thus, we did not increase the applied voltage by more than 30 V.
Table 1. The relationship between the film thickness, applied voltage, pH, and fabrication time.

Applied Voltage
(V)
10
30
10
10
10

pH (Cis/Trans)
0.5/0.5
0.5/0.5
0/0
0.5/13
0/13

Thickness of the
Membrane (nm)
6
6
10
10
10

Fabrication Time (h)4.56 ± 1.14
2.60 ± 1.10
3.36 ± 1.99
2.42 ± 2.41
1.56 ± 1.14

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Next, we discuss the pore size. Because previous studies have reported that the shapeof nanopores fabricated by the DB method can be assumed to have a cylindrical geometry[25], we calculated the pore diameter using the Hille equation [41], which is a theoreticalmodel that uses the resistance of a cylindrical pore to ion flow, as below.
𝑅𝑅 = �𝑙𝑙 +𝜋𝜋𝜋𝜋 𝜌𝜌
�
2 𝜋𝜋𝑟𝑟 2

where 𝑅𝑅 is the resistivity of the pore, 𝑟𝑟 is the pore radius, 𝑙𝑙 is the length of the pore, and𝜌𝜌 is the resistivity of the electrolyte solution. In the DB method, the length of the pore isdefined as the thickness of the carbon film, and the resistivity of the pore was calculatedby the channel current measurements. We calculated that about 75% of nanopores wereless than 40 nm (3.7 nm–38 nm) in diameter (Figure S7). In others, much larger nanopores
were obtained (maximum: 1345 nm). However, we could not find the fabricated nanopores in the carbon film by TEM observation. Although the area of the thin film is usually restricted to around 50 μm square to obtain stable thin films and easily observe thenanopores, the area of the carbon film was much larger (3 mm in diameter) in this study,
and therefore it was difficult to observe the nanopores by TEM because the nanopores canbe constructed anywhere on the carbon thin film. Thus, we tried to classify single or multiple pore formation from current signals when opening pores. Since multiple nanopores
would not be formed simultaneously, we hypothesized that a single nanopore was constructed when a step-like current increase was obtained. On the other hand, in other cases,
we excluded these nanopores from the current evaluations.
Although we demonstrated successful fabrication of nanopores in the carbon film by
the DB method, the control over pore size still needs to be improved in our system. In theconventional DB method, the pore size can be controlled by setting a threshold current at
which to turn off the applied voltage, to prevent expansion of the existing nanopore withthe applied voltage. In this study, however, the current rapidly increased by around several μA within a second and became constant, and the typical currents had irregular values (Figure 3a). Hence, we could not control the pore size by setting a threshold current
value since we manually controlled the application of the transmembrane voltage. Therefore, in future research, we will build a current feedback program to control the applied
voltage. In addition, even though the application of a pulsed voltage would offer control
over nanopore size, the fabrication time will be much longer, or nanopores will not form
since the bond breaking will not occur by being buffered with the movement of free electrons in the short term.
In addition, the concern that multiple nanopores were constructed on the carbon film
should be dispelled. The use of micro-grids with a smaller aperture or the local DB technique [48] using a fine needle electrode, such as AFM tips, would improve the probabilityof single nanopore formation on the carbon membrane.
We next evaluated the nanopores by measuring the I-V curves. To compare themwith the nanopores fabricated by the STEM EB method, we selected nanopores of 3.7 and10 nm in diameter (calculated using Hille’s equation). Both I-V curves showed a linear
relationship between the applied potential and the channel current, indicating that cylindrical-shaped nanopores were fabricated by the DB method (Figure 3e).
The main advantage of the DB method compared with the STEM EB method is themuch simpler fabrication procedure. Nanopores can be fabricated by simply applying a
DC transmembrane voltage. However, the drawbacks include the reduced control overpore size and the lengthy time required. Although the pore diameters were mostly lessthan 40 nm, much larger nanopores were sometimes obtained and the fabrication time
was more than 1.5 h for any experimental conditions.
Finally, we discuss the comparison between the nanopores fabricated in commercially available carbon thin films and SiNx thin films (Table 2). The advantages of using
carbon films are that the complex fabrication processes for the preparation of thin films
are not required, leading to a simpler nanopore fabrication process. Carbon membranes

Micromachines 2021, 12, 1135

9 of 12can be purchased from companies or can be readily prepared on the micro-grid by transferring a floating carbon thin film. By contrast, although the SiNx thin films can also bepurchased, micro-and nano-fabrication processes are required to prepare the films oneself. In the STEM EB method, there are few differences in the size controllability and thefabrication time of nanopores between the carbon films and the SiNx membranes. In the
DB method, however, the fabrication time is longer and the size controllability of nanopores is lower in the flat carbon films than those of the nanopores in the SiNx thin films.
Overall, the stability of the carbon film is lower than that of SiNx membranes since thearea of the carbon film in a micro-grid is much larger than that of SiNx membranes (3 mmin diameter versus around 50 μm square). The carbon films were occasionally broken
when manipulating the micro-grid. Although the membrane rupture was improved byremoving the static electricity of the device, the larger membrane area influences the mechanical properties. If micro-grids coated by a carbon film with a much smaller surface
area were commercially available, the nanopores fabricated in such a micro-grid would
offer great potential for nanopore sensing.
Table 2. Comparison of fabrication methods of solid-state nanopores.

Methods Materials

TEM EB

Carbonfilm(Microgrid)

DB

Carbonfilm(Microgrid)

TEM EB
DB

Si3N4
SiNx

Pre-Micro Fabrication
Pore Size
Size
Thickness
Membraneand NanoTime(nm)
Stability
Controllability(nm)
Fabrication(min)
2 ~ 135
3.7 ~ 1345
(75% ofnanopores
are less than
40 nm)
2 ~ 201 ~ 25REF.

Excellent−10

Not required

2 ~ 90lowThis work

Poor

−10

Not required

90 ~ 300

lowThis work

Excellent
Good

1010-

Required
Required

0.5 ~
−10

high
high

[23]
[25]

4. Conclusions
In conclusion, we propose the use of commercially available flat thin carbon filmcoated micro-grids as the material for solid-state nanopores. To fabricate nanopores in the
carbon membrane, we applied the STEM EB method and the DB method. We successfully
fabricated solid-state nanopores in the membrane using both methods. In the STEM EB
method, the pore size was precisely controlled, ranging from 2 to 135 nm in diameter, andthe fabrication time was within 90 min for each nanopore. In the DB method, the nanopores can be fabricated with a much simpler experimental setup and procedure compared with the STEM EB method. In contrast, the fabrication time was longer than the
STEM EB method due to the conductivity of the carbon film. In addition, although thepore diameter was less than 40 nm for 75% nanopores, precise control over pore size was
not achieved due to the rapid current increase when opening the pore. We believe that
nanopores fabricated in commercially available carbon thin films could be widely used in
nanopore research due to the lack of requirement of micro-and nano-fabrication for thinfilm preparations.
Supplementary Materials: The following are available online at www.mdpi.com/article/10.3390/mi12091135/s1, Figure S1: A PDMS device for current measurements of a carbon filmcoated TEM grid with a nanopore. (a) A photograph of the top view of the device. (b) A schematic
illustration of the device. A TEM grid and PMMA plates are sandwiched between PDMS chambers
into which Ag/AgCl reference electrodes are incorporated., Figure S2: STEM-HAADF images of theMicromachines 2021, 12, 1135

10 of 12fabricated nanopores at the carbon membrane. Scale bars are 20 nm., Figure S3: Scanning patternsof the focused electron beam and TEM images of carbon membranes. (a) Initially irradiated to forma small pore and further irradiated around the small pore to form the larger nanopore. (b) Irradiatedmany adjacent small pores to ultimately form a large square nanopore. The scale bars indicate 20
nm., Figure S4: Raw current data of TEM nanopores for IV curve measurements., Figure S5: Mechanism of the dielectric breakdown. Ions accumulate at the surface of the membrane when a highvoltage is applied, resulting in defect formation. One of the defects subsequently grows to form athrough-hole of nanometer size., Figure S6: Typical results on the three different conditions of thedielectric breakdown experiment. (a) [KCl] =2 M, Voltage = 10 V, pH (cis/trans) = (0/0), Membranethickness = 10 nm. The typical result shows that the current gradually increases and returns to 0. (b)[KCl] =2 M, Voltage = 10 V, pH (cis/trans) = (0/0), Membrane thickness = 10 nm. The typical resultshows that the current suddenly increases and decreases. (c) [KCl] =2 M, Voltage = 10 V, pH(cis/trans) = (0.5/13), Membrane thickness = 10 nm. The typical result shows that the current suddenly increases and plateaus., Figure S7: A histogram of the pore diameter of nanopores fabricatedby the DB method. The pore diameters were mostly less than 40 nm.
Author Contributions: R.K. conceived the original idea. N.T. conducted all experiments and analyzed the data. K.S. conducted the TEM nanopore experiments and T.M. supported the TEM experiments. R.K., K.S. and N.T. wrote the paper. All authors have read and agreed to the publishedversion of the manuscript.
Funding: This work was partly supported by KAKENHI (Grant No. 17K19138, 19H00901) from
MEXT, Japan.
Conflicts of Interest: The authors declare no conflict of interest.

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