19

生体物質を探索するための多機能サブマイクロピペットの開発
高見知秀，西山北斗，渡辺悠，大見春奈，

太田望月,小野茉奈美，渡部みなみ，宮下一帆，
三井大虎，米田里緒,澁谷

興，阿久津祐介，

金子直暉，大友千恵，井上瑞紀,小澤眞美子，
真柄英之，小川修一，虻川匡司
Development of Multi-Functional Submicropipettes
for Exploring Biomaterials
Takami Tomohide, Nishiyama Hokuto, Watanabe Yu,
Omi Haruna, Ohta Mizuki, Ono Manami, Watabe Minami,
Miyashita Kazuho, Mitsui Taiga, Yoneda Rio,
Shibuya Kyo, Akutsu Yusuke, Kaneko Naoki, Ohtomo Chie,
Inoue Mizuki, Ozawa Mamiko, Magara Hideyuki,
Ogawa Shuichi, and Abukawa Tadashi
抄録和訳
本総説は、工学院大学ナノ化学研究室で平成 28 年度から令和 5 年度までに行ってきた、直径 1 マイクロメートル以下のガラスピペットであるサブマイクロピペット
に関する卒業論文と修士論文をまとめたものである。ガラス製サブマイクロピペッ
トは細胞手術やナノ加工など様々な用途に使用されている。ここでは、その内径の空間分解能を利用した、イオン選択的検出、抽出、吸引などの多機能サブマイクロ
ピペットについて解説する。さらに、ピペットの内部が複数に分かれていることで、抽出、吸引、測定などが同時に行うことが可能となるマルチバレルサブマイクロピ
ペットを用いた研究についても解説する。
Abstract
This review paper summarizes the bachelor theses and the master thesis
works between 2016 and 2023 fiscal years in Nanotechnology Chemistry工学院大学研究論叢

20

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Laboratory, Kogakuin University, described on glass submicropipettes, thepipettes with their diameters less than one micrometer. Glass submicropipettes
have been used for cell surgery, nanofabrication, and various applications. Here
we show the capabilities of the multi-functional submicropipettes, such as ionselective detection, extraction, aspiration, with the spatial resolution of their innerdiameters. We also show the work on multi-barrel submicropipettes that can beused for simultaneous operation such as extraction, aspiration and measurements.

1. Preface
Glass submicropipettes have been used for various applications such as cell surgery,
nanofabrication, and microscopy1. We
have been exploring the capabilities ofglass submicropipettes. In this review
paper, our attempts to extend thefunction of glass submicropipettes bydeveloping the component methods inorder to realize beetle robot “nanomosquito”2, with showing the concept
flag in Fig. 1. The design of the nanomosquito is inspired from the beetle
STM developed by Besocke. The quote“Learn from nature and create what is
not in nature” is by Dr. Heinrich Rohrer
who invented STM.

Fig. 1

 chematic concept of a “nano-mosquito”;
S
three-legged beetle-type robot with asubkicropipette. See Ref. 2 for moredetails on the nano-mosquito. The
illustrations were partly prepared by
Miyuki Miyata.

2. Preparation of micro- and nano-pipettes
The glass pipettes were prepared with filament-based submicropipette puller（P-97/
IVF, Sutter Instrument）. A glass capillary loaded into the puller bar is softened byheating with a filament to a certain degree before the hard pull, and the capillary is thenseparated into two glass submicropipettes. This machine has four parameters to control
the heat, pull, and time to adjust the shape of the as-prepared submicropipettes3.
（1）HEAT: the output power of the filament heat, and consequently the amount of energy
supplied to the glass capillary. It is noted that the HEAT value corresponds
approximately to the puller filament temperature in ℃.

生体物質を探索するための多機能サブマイクロピペットの開発

21（2）VELOCITY: the velocity at which the puller bar must be moving before the hard pullis executed.
（3）DELAY: the timing of the start of the hard pull relative to the deactivation of the laser.
（4）PULL: the force of the hard pull.
Though there are slight differences between instruments, submicropipettes withdiameters of 0.3-1 µm can be successfully prepared by synergistically adjusting these fourparameters. Borosilicate glass is usually used to prepare these submicropipettes because
borosilicate glass is easy to control the shape of the pipettes and cheap. As the heating
temperature （HEAT value） was increased, the taper length of the resulting
submicropipette tended to increase. Settings with a HEAT value exceeding 785 were not
performed because of the risk of damage to the heating filament of the puller device. Also,
when the HEAT value was lower than 740, the glass capillary was not heated sufficiently
to soften the glass capillary, and thus could not be fabricated4,5.
Sutter’s manual3 mentions that the tip inner diameter becomes smaller as the HEAT
value is increased. The desired result is obtained when the temperature is increased too
much because the tip inner diameter exceeds the softening point of borosilicate glass bymuch more than 730℃. The stability of the tip inner diameter obtained depends on theheating temperature. However, too high a temperature will not give the desired results
because it is well above the softening point of borosilicate glass, 730℃. On the other hand,
too low a temperature below the softening point will result in a very unstable tip. On theother hand, setting too low a temperature below the softening point results in unstableconditions for making the tip bore, resulting in failure to burn out the tip. Therefore, theoptimum heating temperature for 3-step pulling is considered to be 742, which is close to
the softening point as the HEAT value4,5.
Next, we will discuss the 4-step pulling. In making submicropipettes with pullers, fourstep pulls are not mentioned in the Sutter manual3, because in general, more than fourstep pulls are difficult and unstable results have been obtained. In this study, we also triedvarious 4-step pulling conditions, but the tubing burned out before the 4th step, making it
impossible to fabricate a stable pipette or even a pipette. In this situation, we succeeded inpulling the pipette in four steps and obtained a stable parameter of 845 ± 5 nm. The
reason why this 4-step parameter was obtained is thought to be that the problem of theinability to burn out the tubules at low HEAT was solved by lowering the VELOCITY
and DELAY values, and the tubules were burnt out firmly even at low temperatures4,5.
On the other hand, it is possible that not only heating temperature but also dust, oil, andhumidity adhering to the fine tube may affect the variation of the tip inner diameter.

工学院大学研究論叢

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However, since a large amount of experimental data would be required to demonstrate theeffects of humidity and other factors, there is room for further investigation of these factors4,5.
Although the taper length tends to increase as the HEAT value is increased, thedifference is not as great as expected and does not change at temperatures above 770℃.
Therefore, it is considered difficult to further increase the taper length while maintaining
the tip inner diameter in the 3-step pulling process. Therefore, it is expected that the
4-step pulling method, which requires a greater number of pulls, will result in a longer
taper length than the 3-step pulling method, though there is room for further investigationof this issue. In anyway, we found stable parameters to produce submicropipettes asshown in Tables 1 and 24,5.
4, 5

Table 1 S
 table parameter to achieve505 ± 20 nm pipettes using 3-stage process .
HEAT

PULL

VELOCITY

DELAY

1st

742

0

30

250

2nd

732

0

40

250

3rd

722

30

55

250

Table 24, 5

Stable parameter to achieve845 ± 5 nm pipettes using 4-stage process .
HEAT

PULL

VELOCITY

DELAY

1st

727

0

10

230

2nd

717

0

20

230

3rd

717

0

30

230

4th

707

30

40

230

We considered that the application of this 4-step pull parameter provides the key tosearching for parameters to stably fabricate “multi-channel pipettes,” such as the two-part“theta pipette”, which is difficult to fabricate in the past and is not shown in the Sutter
manual3.
The suitable parameters to fabricate dual-barrel submicropipettes were not clear andno reports on these parameters to fabricate double-barrel pipettes with high accuracy andhomogeneity. Kaneko found that these parameters determine the stability and accuracy ofthe pipette fabrication; unsuitable parameters sometimes resulted in the half-open of the
double-barrel or the standard deviation of the inner diameter is more than 10%6. We tried
32 kinds of the combination of the parameters, and more than three times fabrications
were conducted for each parameter. In this way, we have obtained a parameter tofabricate double-barrel borosilicate submicropipette at 1 µm, as shown in Table 36. The
average total inner diameter of the prepared micropipettes was 513 ± 25 nm. With our生体物質を探索するための多機能サブマイクロピペットの開発

23

efforts, the limit of the small diameters and the short taper length of the double-barrel
glass pipettes were 0.5 µm and 5.2 mm, respectively, using our filament-type puller. Laserpuller could fabricate small-diameters and shorter-taper double-barrel submicropipettes6.
Table 3

6

The best parameters for the fabrication of 1 µm double-barrel pipette .
HEAT

PULL

VELOCITY

DELAY

1st

761

0

30

200

2nd

751

0

40

200

3rd

751

50

50

200

We also investigated the influence of the direction of the glass tube set in the puller.
We tried（1）perpendicular,（2）horizontal, and（3）45°tube setting by using theparameter shown in Table 1, and the standard deviations were（1）5.07%,（2）7.09%, and（3）18.10%, respectively. Therefore, we concluded that perpendicular setting of the glass
tube is suitable for the stable fabrication of the double-barrel submicropipettes6.

3. Observation of the tip top of submicropipettes by scanning electron
microscopy
The observation of the edge of insulators such as glass usually suffered from charge up
during the observation with electron beam by scanning electron microscopy（SEM）. Here
we demonstrate the method to prevent tip top glass pipettes from charge up.

Fig. 2

T op view （left） and side view（right）of the copper sample stage
for the observation of the tip topof glass pipettes; the schematic
drawings（upper）and the photos
7（lower）.

Fig. 3

 canning electron microscope image ofS
the tip top of the glass submicropipette,
7observed at the tilted angle of 15°.

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7
Ono used conventional antistatic spray（Elegard® manufactured by Lion Inc.）.

Among the components of Elegard®, those involved in antistatic properties arepolyethylene glycol and alkyl trimethyl ammonium salt, and they pretend like ionic liquids.
T h u s, s t r o n g C o u l o m b f o r c e s a c t b e t w e e n t h e n i t r o g e n i o n s o f t h e a l k y ltrimethylammonium salt and the oxygen ions of the polyethylene glycol, and they exhibit
the same properties as an ionic liquid.
Moreover, Ono prepared a copper sample stage to observe the tip top of glass
7
submicropipettes at the suitable angle 15°. Figure 2 shows the photo of the preparedsample stage. Figure 3 shows a typical SEM image of the glass submicropipettes. The
sample stage allows the glass submicropipettes to be tilted 15°from the vertical whenviewed from the side, as shown in Fig. 3, making it easier to observe the tip with SEM7.

4. Evaluation of inside of submicropipettes with gas flow method, using
Gomperz function for simulating the shape of submicropipettes
The outer shape of the fabricated submicropipettes can be examined with SEM
observation. However, the inner shape and cleanliness cannot be observed because
electron is not transparent to glass. Therefore, we have developed the gas flow method toinvestigate the inner shape and cleanliness of sub-micropipettes8 12.
-

11

Fig. 4 How to fit the shape of submicropipette with Gompertz curve .

For the simulation of the gas flow through glass submicropipettes in order toinvestigate the inner shape and cleanliness, we need a function to fit the shape of生体物質を探索するための多機能サブマイクロピペットの開発

25

submicropipettes. We found a suitable function of Gompertz function,
G（x）= c exp［−exp（a−bx）］,
where a, b, and c are the constant parameters. The Gompertz function is commonly used
for the growth curve with its applications such as economical management and population
growth model11,12. Figure 4 shows how we fit the Gompertz function to the shape ofsubmicropipette11. In this way, we have established a method to evaluate the fabricatedsubmicropipettes with non-distractive measurement combined with the shape
simulation11,12.

5. Various ion-selective electrodes prepared in glass pipettes
Son reported separate detection of sodium and potassium ions by preparing ionselective membrane（ISM）in submicropipettes13, and Deng reported calcium-ion ISM inglass submicropipettes14. We have ever prepared cesium15,16 and chlorine17 ISMs. We have
explored the signal detection system using rock-in18 or common patch cramp amplifier19,20.
Nishiyama developed a method to prevent ISM from mixing with water during the
preparation process15,16. Figure 4 shows the photos showing the process to fabricate the
ISM in submicropipette. It is noted that the photos show upside down. First, pure water
was filled in the submicropipette to the shank end using a micro-syringe（A-1）. Then, wepoured ca. 0.01 μL cyclohexane（Sigma-Aldrich, purity >99.0%, CAS 110-82-7）on thewater（A-2）.After making the cyclohexane solvent layer on the water, 10 μL of（i）32 mg
of poly（vinyl chloride）（PVC）（Sigma-Aldrich, high molecular weight, CAS 9002-86-2）forthe main resource of the membrane,（ii）62 mg of 2-Nitrophenyloctylether（NPOE,
Dojindo, purity >99.0%, CAS 37682-29-4）for the plasticizer,（iii）5 mg of 1,3alternate
thiacalix［4］biscrown-6,6 for the cesium ion-selective ionophore, and（iv）1 mg of tetrakis［3,5-bis（trifluoromethyl） phenyl］borate, sodium salt（TFPB, Dojindo, purity >99.0%,
CAS 79060-88-1）for the anion repeller, which were dissolved in 1 mL tetrahydrofuran（THF, Sigma-Aldrich, purity >99.9%, with 250ppm dibutyl-hydroxytoluene（BHT）for thestabilizer, CAS 109-99-9）, which we call the mix solution “PVC solution” in the caption ofFig. 5 （B-1）, and were poured in the submicropipette.

The solution put insubmicropipette was dried to cure the PVC membrane（B-2）. It is noted that thecyclohexane solvent was finally transpired with THF on the drying process. In this way,using cyclohexane before pouring the solution is the improved point in this study15,16.

26

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Fig. 5 P hotos of submicropiette tube during the curing process of PVC
membrane inside the submicropipette.（A-1）: After pouring the PVC
solution on the distilled water covered with cyclohexane.（A-2）: Five
minutes later after taking the photo（A-1）.（B-1）: After pouring the PVC
solution on the distilled water, without using cyclohexane.（B-2）: Five15, 16minutes later after taking the photo（B-1） .

6. Double-barrel ion-selective electrodes
Kaneko found the best parameter to fabricate double-barrel submicropipettes6. Akutsu
succeeded in preparing different ISEs in each barrel of double-barrel submicropipettes21,
which we reported the results in eJSSNT22.
Akutsu discovered a way to prepare double-barrel submicropipettes to avoid theformation of bubbles in the ISM caused the disturbance of the ion-selective signal 21. Even
after confirming the absence of bubbles with an optical microscope, bubbles sometimes
appear when the dried ISM is checked again with an optical microscope, which resulted inno signal detected as well. For solving the bubble formation problem, the tip of a doublebarrel pipette was immersed in pure hot water（90 ℃）during the dry process to form
ISM in the double-barrel pipette. Even if there are no bubbles after drying, the ISM might
peel off from the inner glass wall when the electrolyte was filled in the pipette, which used

生体物質を探索するための多機能サブマイクロピペットの開発

27

to occur in the case that the adhesive area between the ISM and the inner wall of theglass pipette was small21. This problem could be solved by fabricating the ISM closer tothe tip, as reported previously23, and by reducing the filling volume of the ISM solution to1 μL23.

7. Double-barrel micropipette used for tuning folk non-contact microscope
Yoneda developed frequency-modulation atomic force microscope（FM-AFM）with adouble-barrel submicropipette, by modifying an existing SPM apparatus（JEOL JSPM5200）24. A theta-tube submicropipette was glued to a quartz tuning folk（QTF）oscillator,
and the Q value was estimated. The operation of the external device was confirmed bydetermining the resonance frequency of the QTF with pipette. An image based on
frequency modulation was obtained as FM-AFM using a double-barrel submicropipette.
Figure 6 shows the block diagram of the FM-AFM with a double-barrel submicropipette.
The developed apparatus would be a future study of simultaneous action of measurement
and fabrication.

Fig. 6 chematics of FM-AFM with double-barrel submicropipette developed in ourS
24laboratory .

工学院大学研究論叢

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8. Automatic injection to living cells
Yokogawa Electric Corporation developed
Single Cellome™ Unit, SU10, which would be a
promising tool to explore molecular dynamics
in living cells25. We rent the apparatus from
Yokogawa, and tested the injection offluorescent molecule into a HeLa cell, one of
the famous cancer cell.

Figure 7 showst y p i c a l r e s u l t s o f a u t o m a t i c i n j e c t i o n,
conducted by a master course student, after
learned how to use from Yokogawa Electric
Corporation for several hours24.

9. Remarks
We have developed component methods
to realize automatic injection robot, “nano2

mosquito” , by integrating the developed
methods of pipette fabrication4 6, evaluations-

Fig. 725

 emonstration of SU-10 ; injection
Dof FITC, fluorescent molecule, to24
HeLa cell .

by SEM 7 and gas flow method8 12, ISEs for-various ions15 20, double-barrel ISE micropipettes21,22, combination of FM-AFM with QTF24,-

and automatic injection system developed by Yokogawa Electric Corporation24,25. We
promise that the developing tool will contribute to the development of various fields suchas cell engineering and nano-fabrication.
Acknowledgments
We would like to thank Prof. Atsushi Sekiguchi of Kogakuin University for the
discussion of vacuum conductance. We are also grateful to Dr. Fumitaka Kamachi of
Yokogawa Electric Corporation for providing us with the nanopipettes. This work was
performed under the Cooperative Research Program of the Network Joint ResearchCenter for Materials and Devices（No. 20211087）, and JSPS KAKENHI Grant Number
19K05275.

生体物質を探索するための多機能サブマイクロピペットの開発

29

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2

T. Takami, X. L. Deng, J. W. Son, B. H. Park, and T. Kawai, Jpn J. Appl. Phys., 51, 08KB12（2012）.

3

A. Oesterle, “P-1000 & P-97 Pipette Cookbook”, Rev.E（Sutter Instruments, 2009, Novato CA）.

4

三井大虎,令和二年度工学院大学先進工学部生命化学科卒業論文（2021）.

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三井大虎,高見知秀,工学院大学研究報告（in Japanese）,130, 1-11（2022）.

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金子直暉,令和三年度工学院大学先進工学部生命化学科卒業論文（2022）.

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小野茉奈美,令和元年度工学院大学先進工学部生命化学科卒業論文（2020）.

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太田望月,平成三十年度工学院大学先進工学部応用化学科卒業論文（2019）.

9

宮下一帆,平成三十一年度（令和元年度）工学院大学先進工学部応用化学科卒業論文（2020）.

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澁谷

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大友千恵,令和四年度工学院大学先進工学部生命化学科卒業論文（2023）.

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T. Takami, C. Ohtomo, N. Kaneko, K. Shibuya, K. Miyashita, M. Ohta, R. Yoneda, M. Ozawa,

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J. W. Son, T. Takami, J.-K. Lee, B. H. Park, and T. Kawai, Appl. Phys. Lett., 99, 033701（2011）.

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H. Magara, S. Ogawa, and T. Abukawa, e-J. Surf. Sci. Nanotech., 21, 257-261（2023）.

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渡部みなみ,平成三十一年度（令和元年度）工学院大学先進工学部生命化学科卒業論文（2020）.

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渡辺

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井上瑞紀,令和五年度工学院大学先進工学部応用化学科卒業論文（2024）.

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阿久津祐介,令和三年度工学院大学先進工学部生命化学科卒業論文（2022）.

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T. Takami, Y. Akutsu, N. Kaneko, R. Yoneda, H. Magara, S. Ogawa, and T. Abukawa, e-J. Surf. Sci.

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E. J. Kang, T. Takami, X. L. Deng, J. W. Son, T. Kawai, and B. H. Park, J. Phys. Chem. B, 118, 5130

悠,平成二十九年度工学院大学工学部応用化学科卒業論文（2018）.

Nanotech., 21, 17-23（2023）.
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25

SU-10 Automatic injection system, Yokogawa Electric Corporation（https://www.selectscience.net/
products/single-cellome-unit-su10/?prodID=226106）
.

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30

工学院大学研究論叢

第 61 − 1 号

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東北大多元物質科学研究所 技術職員）
日本大学生産工学部 准教授）
東北大多元物質科学研究所 教授）