4781

Sensors and Materials, Vol. 35, No. 10 (2023) 4781–4788
MYU Tokyo
S & M 3430

Effect of Valinomycin on the Respiration Activityof Zebrafish Embryos Using a Large-scale-integration-based
Multiple Amperometric Biosensor
Kazuki Terao,1 Masato Suzuki,1,2* Ryota Kunikata,3 Atsushi Suda,3 Kumi Y. Inoue,4
Kosuke Ino,5 Tomokazu Matsue,6 and Tomoyuki Yasukawa1,2**
1Graduate School of Science, University of Hyogo, 3-2-1 Koto, Kamigori, Ako, Hyogo 678-1297, Japan
2Advanced Medical Engineering Research Institute, University of Hyogo,

3-264, Kamiya-cho, Himeji, Hyogo 670-0836, Japan

3Japan Aviation Electronics Industry, Ltd., 3-1-1 Musashino, Akishima, Tokyo 196-8555, Japan

4Faculty of Engineering, Graduate Faculty of Interdisciplinary Research, University of Yamanashi,

4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan

5Graduate School of Engineering, Tohoku University,

6-6-11 Aoba, Aramaki, Aoba, Sendai, Miyagi 980-8579, Japan
6Head Office of Enterprise Partnerships, Tohoku University,
468-1 Aoba, Aramaki, Aoba, Sendai, Miyagi 980-8545, Japan(Received May 1, 2023; accepted September 13, 2023)

Keywords: electrode array, zebrafish embryo, respiration activity, electrochemical imaging, toxicity test

A large-scale-integration-based multiple amperometric biosensor (Bio-LSI) was used toobtain images of oxygen concentration around zebrafish embryos and monitor their respiration
activity after valinomycin at different concentrations was injected. The regions with a low
reduction current of oxygen coincided with the embryo positions. The reduction current
decreased immediately after adding valinomycin owing to the increase in the respiration activityof the embryos. The decrement of the reduction current increased with valinomycin
concentration. The estimation system is very simple because no alignment is required between
the embryos and the electrodes arrayed at the bottom of the chamber. In general, the use ofzebrafish embryos for toxicity tests has been expected as an alternative for animal tests.
However, a relatively long time is required to estimate the mortality and morphological changesof the embryos caused by chemical substances by a standardized method. The use of the present
system is expected to allow the rapid and simple sensing of the effects of chemical substances on
the respiration activity of zebrafish embryos.

1.

Introduction

New chemical substances such as medical supplies, agricultural chemicals, and cosmetics
have been continuously developed to improve our quality of life. The effects of these substanceson human health, ecological systems, and the environment should be estimated as a common
challenge for the whole world. The Organisation for Economic Co-operation and Development*Corresponding author: e-mail: suzuki@sci.u-hyogo.ac.jp**Corresponding author: e-mail: yasu@sci.u-hyogo.ac.jp

https://doi.org/10.18494/SAM4493

ISSN 0914-4935 © MYU K.K.
https://myukk.org/

4782

Sensors and Materials, Vol. 35, No. 10 (2023)(OECD) has standardized estimation methods for these chemicals and products. There is the fishembryo toxicity OECD test guideline 236 (TG236) for estimating the effects of chemical
substances by using zebrafish (Danio rerio) embryos.(1) The toxicity of chemical substances is
estimated on the basis of the mortality and morphological changes of the embryos, when theembryos are cultured in sea water containing the chemical substances for 96 h. TG236 wasdeveloped as an alternative for the acute fish toxicity test (e.g., OECD TG203) to overcome
ethical issues from the perspective of animal protection. In Europe, fish embryos at non-feeding
developmental stages are categorized as non-animal by the European Union, Directive 2010/63/
EU.(2)The use of zebrafish embryos for toxicity tests has been expected as an alternative for animal
tests(3–5) because the human genes related to diseases and drug metabolisms have 84% homology
with zebrafish genes.(6,7) The toxicity tests of 31 chemical substances using zebrafish embryos
showed that their developmental toxicities were in agreement with animal tests with accuracies
higher than 80%.(8) Thus, the development of rapid and simple methods for estimating the effects
of chemical substances will lead to an alternative for ecological and animal tests.
Techniques for the simultaneous estimation of different concentrations of chemical
substances applied to zebrafish embryos using microfluidic devices have been reported.(9–12)
However, the morphological changes observed by optical microscopy were used to estimate theeffects of chemical substances on embryos. The quantitation of metabolic activity can be usefulfor the accurate estimation of the physiological effects of chemical substances on embryos. The
oxygen consumption of embryos by respiration is one of the candidates for the estimation ofphysiological effects.(13) The oxygen consumption that depends on mitochondrial activity is a
powerful indicator to discover cellular information such as metabolism and apoptosis. For
example, the oxygen consumption of embryos was measured using a fluorescence molecule withoxygen sensitivity and immobilized on microwells with single embryos.(14) Furthermore, acommercially available extracellular flux analyzer was used to estimate the toxicity ofchemicals to embryos at different developmental stages by indicating the oxygen consumption(mitochondrial activity).(15) The oxygen consumption of zebrafish embryos, as well asmorphological changes, has been recognized as an important factor for estimating the effects ofchemicals on embryos.
In this study, the oxygen consumption of zebrafish embryos in the presence of valinomycin atdifferent concentrations was monitored using the reduction currents of oxygen obtained by achip of large-scale-integration-based multiple amperometric biosensors (Bio-LSI).(16) The use of
the chip with an array of 400 microelectrodes (20 × 20) in a 5 × 5 mm2 region allows individual
current responses to be obtained at each electrode, and thus the time courses of current images
that reflect the distribution of the concentration of electrochemical species immediately above
the electrode array can be obtained. Until today, Bio-LSI devices have been applied to themonitoring of the enzyme activities of embryoid bodies by amperometry(17) and potentiometry,(18)the imaging of dopamine release from the spheroids of PC12 cells,(19) multiple immunosensing,(20)the motion tracking of microorganisms,(21) the detection of multiple cell functions,(22) the
fabrication of hydrogels at a localized area,(23) the detection of oxygen generated from spinachby photosynthesis,(24) and the estimation of endothelial permeability.(25) Our group reported theoxygen consumption of a single zebrafish enclosed in a small container by determining theSensors and Materials, Vol. 35, No. 10 (2023)

4783

electrochemical reduction of oxygen.(26) In this work, the decrease in oxygen concentration
caused by the respiration of embryos arranged on the electrode array was monitored with a BioLSI chip by adding valinomycin at different concentrations to determine its effect on the
respiration activity of embryos. Valinomycin acts as an uncoupler to inhibit the synthesis ofadenosine triphosphate (ATP) by oxidative phosphorylation. The continuous monitoring ofoxygen concentration around embryos as images of reduction currents of oxygen allows for theestimation of respiration activity. In this system, the effect of valinomycin can be rapidly andsimply estimated compared with the OECD tests because the respiration activity changes
immediately after valinomycin is injected, and no difficult decision concerning the mortality
and morphological changes of each embryo by the naked eyes is required. In addition, no precise
control of the positions of the embryos on the electrode array is required, because themicroelectrodes arranged under the embryos are used to obtain the reduction current of oxygen.

2.

Materials and Methods

The Bio-LSI chip comprises 400 (20 × 20) platinum electrodes. The fabrication of the chip
was detailed in a previous report.(16) The rows in the electrode array were named lines 1–20 fromabove, and the columns were named columns a–t from the left. Electrodes were covered by an
insulating layer (5 mm thick) of negative photoresist (SU-8) to define the regions exposed tosolutions (50 mm diameter). The distance between the centers of neighboring electrodes was 250
mm. A layer with an acrylic chamber (5 × 5 mm2, 5 mm thick) and a layer with a type “H”
fluidic channel were arranged on the electrode array [Fig. 1(A)]. The width of the type “H”
channel and the lengths of the vertical and horizontal lines of the type “H” channel were set at 6,30, and 10 mm, respectively. The chamber and channel were filled with artificial sea water (0.6mL, SEALIFE, Nihonkaisui Co., Ltd., Tokyo). A platinum coil inserted at the bottom left of thetype “H” channel and a Ag/AgCl electrode inserted at the top right were used as the counter andreference electrodes, respectively. Furthermore, two tubes were inserted at the top left andbottom right to inject and remove the artificial sea water containing valinomycin at different
concentrations, respectively. The flow rate of the artificial sea water was 0.4 mL/min. Thus, thesolution in the chamber and fluidic channel can be replaced with the solution containing
valinomycin in 1.5 min.

Fig. 1. (Color online) (A) Image of Bio-LSI chip with square chamber and type “H” fluidic channel. (B) Crosssectional illustration of oxygen consumption of zebrafish embryos arranged on Bio-LSI chip.

4784

Sensors and Materials, Vol. 35, No. 10 (2023)

The breeding facility of the zebrafish was illuminated for 14 h and exposed to darkness for 10
h in a day. The temperature of the artificial sea water for the zebrafish was maintained at 26 ℃.
Fertilized embryos were cultured in the artificial sea water at 28.5 ℃ for 48 h. The embryoswith a diameter of approximately 1 mm were put on the electrode arrays of chips.
A CMOS camera (VEX120, WRAYMER) was placed above the electrode array in thechamber to confirm the positions of the embryos. The potential of −0.5 V vs Ag/AgCl wasapplied to 400 electrodes at the same time for the same duration (36 min) to obtain the reduction
currents of oxygen [Fig. 1(B)]. The reduction currents of oxygen obtained by the electrodes near
the embryos decrease with the decrease in oxygen concentration owing to the consumption ofoxygen by the embryos. The reduction currents of oxygen were used to investigate the effect ofvalinomycin on the respiration activity of the embryos.

3.

Results and Discussion

3.1

Imaging of oxygen concentration around embryos

Three zebrafish embryos were arranged on the electrode array in the chamber filled withartificial sea water. The potential of −0.5 V was applied to 400 electrodes to obtain amperometricresponses for reducing the oxygen dissolved in the artificial sea water. The artificial sea watercontaining 5.0 mmol/L valinomycin was then injected from the tube inserted at the top left of thetype “H” channel, and at the same time, the artificial sea water in the chamber and type “H”
channel was removed by the tube inserted at the bottom right of the type “H” channel at thesame rate. Figures 2(A) and 2(D) show the image of three embryos on the electrode array and theimage of reduction currents of oxygen, respectively. The centers of embryos a, b, and c were at
the electrodes 3c, 3g, and 16l, respectively [Fig. 2(A)]. The reduction currents of oxygen obtainedby the electrodes in the vicinity of the centers of the embryos were lower than those obtained by
the other electrodes without an embryo [Fig. 2(D)]. The whitish regions in the current image,Fig. 2. (Color online) (A)–(C) Images of three embryos on electrode array and (D)–(F) images of reduction
currents of oxygen (A, D) before the solution containing 5.0 μmol/L valinomycin was injected, and (B, E) 1.5 min
and (C, F) 6.5 min after the injection of the solution containing 5.0 μmol/L valinomycin was stopped.

Sensors and Materials, Vol. 35, No. 10 (2023)

4785

which indicate low currents, coincided with the positions of embryos a and b. The result suggeststhat the concentration of oxygen near the embryos decreased with increasing consumption ofoxygen by the embryos. In addition, the decrease in reduction current may also be attributable tothe inhibition of the oxygen diffusion by the embryos.
We injected the artificial sea water containing 5.0 mmol/L valinomycin within 1.5 min.
Figures 2(B) and 2(C) show the optical images of the embryos and Figs. 2(E) and 2(F) show
current images 1.5 and 6.5 min after the injection was stopped, respectively. In the current
images, the whitish regions gradually spread in all directions and the whitish intensity wasemphasized. The results indicate that the oxygen concentration around the embryos decreased
with the addition of valinomycin owing to the increase in the respiration activity of the embryos.
In the presence of valinomycin that cancels the membrane potential as an uncoupler to theoxidative phosphorylation, the embryos should consume more oxygen than in the absence ofvalinomycin to maintain ATP production. Therefore, the effect of valinomycin on the embryos
can be qualitatively estimated from the images of the reduction currents of oxygen. Furthermore,no precise control of the positions of the embryos is required to estimate the respiration activityof the embryos. When the microelectrode was applied to the electrochemical estimation, thepositional relationship between the electrode and the embryo should be regulated to obtain theelectrochemical responses. In the present system, the introduction of the embryos to the chamber
above the electrode array allows the arrangement of the embryos immediately above some of the
400 electrodes.
No current response was observed for embryo c in the current image [Fig. 2(D)], although
embryo c was on electrode 16l. This is due to the transfer of embryo c on the electrode array.
Embryos grow to be young fish 48 h after fertilization. The young fish in embryo c actively
repeated the rotational motion and the swing of the tail, resulting in the random repeat of the
movement and stillness on the electrode array. Embryo c moved to the right side of the image
after passing electrode 16l and reached electrode 18q [Fig. 2(B)]. Then, embryo c again started tomove toward the top of the image and reached electrode 2s [Fig. 2(C)]. When embryo c was onelectrodes 18q and 2s without the spatial movement, decreases in the reduction currents ofoxygen were observed [Figs. 2(E) and 2(F)]. However, the embryos that moved on the electrodearray cannot be applied to this work because of the disturbance of the diffusion layer of oxygen
formed on the electrodes by the movement of the embryos and the insufficient formation of the
diffusion layer of oxygen around the embryos.
3.2

Investigation of the respiration activity of embryos in the presence of valinomycin

We investigated the amperometric responses of oxygen after injecting valinomycin atdifferent concentrations as shown in Fig. 3(A). The potential of −0.5 V was applied to 400
electrodes at zero time, as shown in Fig. 3(A). The reduction currents reached almost the steadystate 30 s after the potential was applied. The artificial sea water containing valinomycin atdifferent concentrations was then injected 1.5 min after the potential was applied. The currents
were plotted as averages of reduction currents obtained by four electrodes immediately below
the arranged embryos and normalized with the average responses obtained immediately after the4786

Sensors and Materials, Vol. 35, No. 10 (2023)

Fig. 3. (A) Normalized reduction currents of oxygen obtained by the electrodes immediately under the embryos
upon injection of valinomycin at different concentrations. Concentrations of valinomycin: (a) 0, (b) 0.05, (c) 0.5, and(d) 5.0 μmol/L. (B) Normalized currents of oxygen as a function of the concentration of injected valinomycin. The
number of trials for 0 and 5.0 μmol/L was six, and that for the other concentrations was three.

injection was stopped at 3 min. The embryos arranged around the center of the electrode array
were used to remove the inhibition of the formation of the diffusion layer of oxygen around thearranged embryos by the walls of the chamber. The normalized currents obtained for the first 3
min were unstable owing to the disturbance of the diffusion layer generated by the reduction ofoxygen at the 400 electrodes caused by the injection of the solution. When the artificial sea water
without valinomycin was injected, the reduction currents showed a gradual decrease to reach analmost steady state in 10 min after stopping the injection. The decrease was attributed to theformation of the diffusion layer over the electrodes. When the solutions containing valinomycin
were injected, the reduction currents also decreased and were lower than that obtained byinjecting the solution without valinomycin. This is because of the decrease in the concentrationof oxygen caused by the increase in the respiration activity of the embryos. The average currents
between 30 and 35 min were used as responses for the calibration curve. Figure 3(B) shows thesecurrent responses as a function of the concentration of valinomycin. The results indicate that the
responses decrease with increasing valinomycin concentration owing to the decrease in oxygen
concentration caused by the increase in the respiration activity of the embryos. A goodcorrelation between the valinomycin concentration and the current response was obtained in
nano- and micromolar orders of valinomycin.
3.3

Investigation of the frequency of tail swing in the presence of valinomycin

We also investigated the frequency of the tail swing of young fish embryos after the injectionof valinomycin. The frequency was calculated at minute intervals and normalized with thenumber at the first minute during the measurements of the reduction current of oxygen. The
number at the first minute after the potential of −0.5 V was applied was found to be 10 times on
average. The standard deviation for three embryos was 2.8 times. When the solution in thechamber was replaced with the solution containing 5.0 mmol/L valinomycin from 1.5 to 3.0 min,the frequency of the tail swing increased and became four times that observed before

Sensors and Materials, Vol. 35, No. 10 (2023)

4787

Fig. 4. Normalized frequency of the movement (tail swing) of young zebrafish embryos after the injection ofvalinomycin. Concentration of valinomycin: (white circle) 0 and (black circle) 5.0 μmol/L.

valinomycin was added (black circle in Fig. 4). The increment obtained by adding valinomycin
was larger than that obtained by replacing the solution without valinomycin (white circle in Fig.
4). Therefore, this increase could be due to the stress applied to the embryos with the decrease inoxygen concentration caused by the enhancement of the respiration activity. However, a slightincrease in swing frequency was observed by replacing the solution without valinomycin. Thismay be due to the decrease in oxygen concentration around the embryos caused by the
consumption of oxygen by the electrochemical reaction with the 400 electrodes. The resultshows the possibility of estimating the effects of chemical substances on the respiration activityof the embryos. In addition, there was no significant difference in the frequency of tail swing
with and without a low valinomycin concentration.

4.

Conclusions

The distribution of the oxygen concentration around the embryos was indicated as images of
the reduction currents of oxygen based on the electrode array of Bio-LSI. The reduction currentsof oxygen decreased immediately after adding valinomycin that acts as an uncoupler andreached a steady state within 10 min. The currents decreased with increasing concentration ofvalinomycin. The estimation system is very simple because no alignment is required between
the embryos and the electrodes arrayed at the bottom of the chamber. The monitoring of the
reduction currents allows for the rapid and simple estimation of the effect of valinomycin on the
respiration activity of the embryos. Moreover, the frequency of the tail swing of young fishembryos also increased after adding valinomycin. Therefore, monitoring the reduction currentsof oxygen and the frequencies of tail swing can be useful for estimating the effects of chemical
substances on human health, ecological systems, and the environment. Oxygen was consumed
not only by the embryos, but also by the 400 electrodes. In this system, the decrease in the
reduction current of oxygen consumed by the embryos must be measured under low
concentration conditions due to oxygen consumption at the 400 electrodes. Thus, the systemshould be improved to decrease the consumption of oxygen by electrodes.

4788

Sensors and Materials, Vol. 35, No. 10 (2023)

Acknowledgments
This work was partly supported by JSPS KAKENHI, grant number 20H02771.

References
1 T. Braunbeck, B. Kais, E. Lammer, J. Otte, K. Schneider, D. Stengel, and R. Strecker: Environ. Sci. Pollut. Res.
22 (2015) 16247. https://doi.org/10.1007/s11356-014-3814-7
2 Official Journal of the European Union, DIRECTIVE 2010/63/EU OF THE EUROPEAN PARLIAMENTAND OF THE COUNCIL of 22 September 2010 on the protection of animals used for scientific purposes (Textw i t h E E A r e l e v a n c e) : h t t p s : //e u r- l e x . e u r o p a . e u / L e xU r i S e r v/ L e xU r i S e r v.
do?uri=OJ:L:2010:276:0033:0079:en:PDF (accessed September 2023).
3 Y. Nishimura, S. Murakami, Y. Ashikawa, S. Sasagawa, N. Umemoto, Y. Shimada, and T. Tanaka: Congenit.
Anom. 55 (2015) 1. https://doi.org/10.1111/cga.12079
4 Y. Fernandes, D. M. Buckley, and J. K. Eberhart: Biochem. Cell Biol. 96 (2018) 88. https://doi.org/10.1139/bcb2017-0122
5 K. Brotzmann, A. Wolterbeek, D. Kroese, and T. Braunbeck: Arch. Toxicol. 95 (2021) 641. https://doi.
org/10.1007/s00204-020-02928-7
6 K. Howe, M. Clark, C. Torroja, et al.: Nature 496 (2013) 498. https://doi.org/10.1038/nature12111
7 T. Uechi and N. Kenmochi: Pharmaceuticals 12 (2019) 151. https://doi.org/10.3390/ph12040151
8 K. C. Brannen, J. M. Panzica-Kelly, T. L. Danberry, and K. A. Augustine-Rauch: Birth Defects Res. Part B Dev. Reprod. Toxicol. 89 (2010) 66. https://doi.org/10.1002/bdrb.20223
9 F. Yang, C. Gao, P. Wang, G.-J. Zhang, and Z. Chen: Lab Chip 16 (2016) 1106. https://doi.org/10.1039/
c6lc00044d
10 A. Khalili and P. Rezai: Brief. Funct. Genomics 18 (2019) 419. https://doi.org/10.1093/bfgp/elz006
11 P. Bansal, A. Abraham, J. Garg, and E. E. Jung: BioChip J. 15 (2021) 42. https://doi.org/10.1007/s13206-02100012-5
12 D. Wlodkowic and O. Campana: Environ. Sci. Technol. 55 (2021) 3505. https://doi.org/10.1021/acs.est.0c07688
13 C. L. Souders, X. Liang, X. Wang, M. Ector, Y. H. Zhao, and C. J. Martyniuk: Aquat. Toxicol. 199 (2018) 162.
https://doi.org/10.1016/j.aquatox.2018.03.031
14 S.-H. Huang, K.-S. Huang, C.-H. Yu, and H.-Y. Gong: Biomicrofluidics 7 (2013) 064107. https://doi.
org/10.1063/1.4833256
15 K. D. Stackley, C. C. Beeson, J. J. Rahn, and S. S. L. Chan: PLoS One 6 (2011) 0025652. https://doi.org/10.1371/
journal.pone.0025652
16 K. Y. Inoue, M. Matsudaira, N. Nakano, K. Ino, C. Sakamoto, Y. Kanno, R. Kubo, R. Kunikata, A. Kira, A.
Suda, R. Tsurumi, T. Shioya, S. Yoshida, M. Muroyama, T. Ishikawa, H. Shiku, S. Satoh, M. Esashi, and T.
Matsue: Lab Chip 15 (2015) 848. https://doi.org/10.1039/c4lc01099j
17 K. Ino, T. Onodera, M. T. Fukuda, Y. Nashimoto, and H. Shiku: ACS Sensors 4 (2019) 1619. https://doi.
org/10.1021/acssensors.9b00344
18 Y. Kanno, K. Ino, C. Sakamoto, K. Y. Inoue, M. Matsudaira, A. Suda, R. Kunikata, T. Ishikawa H. Abe, H.
Shiku, and T. Matsue: Biosens. Bioelectron. 77 (2016) 709. https://doi.org/10.1016/j.bios.2015.10.021
19 H. Abe, K. Ino, C. Z. Li, Y. Kanno, K. Y. Inoue, A. Suda, R. Kunikata, M. Matsudaira, Y. Takahashi, H. Shiku,
and T. Matsue: Anal. Chem. 87 (2015) 6364. https://doi.org/10.1021/acs.analchem.5b01307
20 T. Hokuto, T. Yasukawa, R. Kunikata, A. Suda, K. Y. Inoue, K. Ino, T. Matsue, and F. Mizutani: Biotechnol. J.
11 (2016) 838. https://doi.org/10.1002/biot.201500559
21 K. Ino, K. Kanno, K. Y. Inoue, A. Suda, R. Kunikata, M. Matsudaira, H. Shiku, and T. Matsue: Angew.
Chemie - Int. Ed. 56 (2017) 6818. https://doi.org/10.1002/anie.201701541
22 Y. Kanno, K. Ino, H. Abe, C. Sakamoto, T. Onodera, K. Y. Inoue, A. Suda, R. Kunikata, M. Matsudaira, H.
Shiku, and T. Matsue: Anal. Chem. 89 (2017) 12778. https://doi.org/10.1021/acs.analchem.7b03042
23 K. Ino, M. Terauchi, M. Gakumasawa, N.Taira, A. Suda, R. Kunikata, T. Matsue, and H. Shiku: Sens. Actuator.
B 277 (2018) 95. https://doi.org/10.1016/j.snb.2018.08.135
24 S. Kasai, Y. Sugiura, A. Prasad, K. Y. Inoue, T. Sato, T. Honmo, A. Kumar, P. Pospíšil, K. Ino, Y. Hashi, Y.
Furubayashi, M. Matsudaira, A.,Suda, R. Kunikata, and T. Matsue: Sci. Rep. 9 (2019) 12234. https://doi.
org/10.1038/s41598-019-48561-y
25 K. Ino, H.-J. Pai, K. Hiramoto, U. Utagawa, Y. Nashimoto, and H. Shiku: ACS Omega 6 (2021) 35476. https://doi.org/10.1021/acsomega.1c04931
26 T. Yasukawa, M. Koide, N. Tatarazako, R. Abe, H. Shiku, F. Mizutani, and T. Matsue: Anal. Chem. 86 (2014)
304. https://doi.org/10.1021/ac402962f