Detection of the Oxygen Consumption Rate of Migrating Zebrafish by

Dec 12, 2013 - equalization system was adapted to detect oxygen in a chamber ... detection of different equalized signals corresponding to respiration...
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Detection of the Oxygen Consumption Rate of Migrating Zebrafish by Electrochemical Equalization Systems Tomoyuki Yasukawa,*,† Masahiro Koide,‡ Norihisa Tatarazako,§ Ryoko Abe,§ Hitoshi Shiku,⊥ Fumio Mizutani,† and Tomokazu Matsue*,⊥,¶ †

Graduate School of Material Science, University of Hyogo, 3-2-1, Kouto, Kamigori, Ako, Hyogo 678-1297, Japan Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1, Ten-nodai, Tsukuba, Ibaraki 305-8577, Japan § Center for Environmental Health Sciences, National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba, Ibaraki 305-8506, Japan ⊥ Graduate School of Environmental Studies, Tohoku University, 6-6-11, Aramaki, Aoba, Sendai, Miyagi 980-8579, Japan ¶ The World Premier International Research Center-Advanced Institute for Materials Research (WPI-AIMR), Tohoku University, 2-1-1, Katahira, Aoba, Sendai, Miyagi 980-8579, Japan ‡

S Supporting Information *

ABSTRACT: A novel measurement system to determine oxygen consumption rates via respiration in migrating Zebrafish (Danio rerio) has been developed. A signal equalization system was adapted to detect oxygen in a chamber with one fish, because typical electrochemical techniques cannot measure respiration activities for migrating organisms. A closed chamber was fabricated using a pipet tip attached to a Pt electrode, and a columnar Vycor glass tip was used as the salt bridge. Pt electrode, which was attached to the chamber with one zebrafish, and Ag electrode were immersed in 10 mM potassium iodide (KI), and both the electrodes were connected externally to form a galvanic cell. Pt and Ag electrodes act as the cathode and anode to reduce oxygen and oxidize silver, respectively, allowing the deposition of insoluble silver iodide (AgI). The AgI acts as the signal source accumulated on the Ag electrode by conversion of oxygen. The amount of AgI deposited on the Ag electrode was determined by cathodic stripping voltammetry. The presence of zebrafish or its embryo led to a decrease in the stripping currents generated by a 10 min conversion of oxygen to AgI. The conversion of oxygen to AgI is disturbed by the migration of the zebrafish and allows the detection of different equalized signals corresponding to respiration activity. The oxygen consumption rates of the zebrafish and its embryo were estimated and determined to be ∼4.1 and 2.4 pmol·s−1, respectively. The deposited AgI almost completely disappeared with a single stripping process. The signal equalization system provides a method to determine the respiration activities for migrating zebrafish and could be used to estimate environmental risk and for effective drug screening.

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measurement for in vivo tissue and oxygen distribution near tissue and living cells.4,5 The measurement of oxygen generation or consumption in plant and animal cells by scanning electrochemical microscopy using a microelectrode as the probe has been previously reported.6−8 Oxygen has also been detected using phosphorescence quenching of organic luminescent complexes and luminescent nanomaterials with molecular oxygen.9,10 However, for these measurement approaches, the interference of ambient air must be eliminated to detect the oxygen consumption of the small cells and organisms.11 Oxygen gradients formed by respiration would disappear and are extremely difficult to detect in the presence of ambient oxygen. Sensitive measurements of oxygen have

uantitative bioanalysis has been the focus for both fundamental and practical importance in understanding cellular processes and single cell functions. Oxygen is a key substrate and energy source for cells and acts as a terminal electron acceptor in the electron transport chain.1 Respiratory oxygen consumption is an indicator of cell viability and the metabolic status in mitochondria and whole organisms. It also helps in the investigation of metabolic pathways caused by various external stimuli. In particular, monitoring of oxygen consumption rates is useful in developing systems to estimate the environmental risk of chemicals and effective drug screening without experiments on animals.2 Therefore, systems that can measure oxygen consumption rates in bacteria, cells, tissues, and organisms could be very useful. Traditionally, Clark-type oxygen sensors have been used to detect oxygen.3 Electrochemical sensors have been miniaturized to improve their spatial resolution and apply them to oxygen © 2013 American Chemical Society

Received: September 16, 2013 Accepted: December 12, 2013 Published: December 12, 2013 304

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been performed in various closed systems using sealable microplates,12 microwell-plates,13 urethane cover-plates,14 and negative photoresist channels.15 Cell based assays are useful to investigate the effects of drugs and toxicities of chemical products,16,17 but the difference in the behaviors of cells and whole organisms to the same stimuli have to be compared for the development of a reliable sensing system. Recently, migrating small organisms such as the zebrafish (Danio rerio)18 and Daphnia magna19 were utilized in environmental risk assessment and drug screening. However, there are no reports of a suitable electrochemical method to determine the respiration levels of migrating organisms, because typical electrochemical techniques are not useful for measuring respiration activities of migrating organisms. Herein, an electrochemical closed chamber was developed to determine the oxygen consumed by a young zebrafish, and this method was used to estimate its respiration activities and compared with the consumption of its embryos. Recently, a dual amplification system based on electrochemical redox cycling and signal accumulation was used to develop a sensitive immunosensing system.20 When a galvanic cell is used, oxidation of redox species generated from labeled enzyme at the anode occurs concurrently with the reduction of Ag ions and deposition of metallic Ag at the cathode. Anodic stripping voltammetry was used to obtain the electrochemical responses, which corresponded to the amount of deposited silver. In this work, this method was oppositely applied for the detection of oxygen in a closed chamber to eliminate the disturbance of the diffusion layer created by migrating fish. Cathodic stripping voltammetry (CSV) of silver iodide (AgI) deposited at the anode was used to obtain the oxygen consumed by the respiration of zebrafish.

Figure 1. (A) Fabricated electrochemical closed chamber. (B) Galvanic cell with a Pt chamber electrode as the cathode and Ag electrode as the anode. Zebrafish were introduced in the cathode. (C) Closed chamber with a single embryo.

Data were evaluated as the average from ten or twenty different experiments. The oxygen consumption rates of the embryos were measured using the same method. The embryo trapped in the closed chamber is shown in Figure 1C. Electrochemical measurements were performed using an HSV-100 electrochemical analyzer (Hokuto Denko, Tokyo, Japan) with a conventional three-electrode system. Ag electrodes with deposited AgI were used as the working electrodes, Ag/AgCl (saturated with KCl) as reference electrodes, and platinum wire electrodes as the auxiliary electrodes, respectively. The Pt and Ag electrodes were polished with an alumina/water slurry and washed with water before AgI was deposited by the galvanic cell reactions. The supporting electrolyte solution was 0.1 M KNO3. The solution was saturated with air and kept at room temperature. The potential sweep rate used for voltammetry was 50 mV·s−1. The potential was stepped from 0.4 to −0.4 V to obtain the reduction currents of oxygen by amperometry. Wild-type Danio rerio were kept at the fish facility of the National Institute for Environmental Studies. Fish were kept at a density of 1 L−1 in recirculating dechlorinated water (conductivity of 32.4 mS·m−1) in a cycle of 14 h of light and 10 h of dark. Water was temperature controlled (26 ± 1 °C). The fish were fed twice daily with Artemia salina and Daphnia magna. Young zebrafishes (body length approximately 2 mm) were used within 12 h of the embryo hatching, and the embryos were used within 12 h of fertilization. Scanning electron microscopy (SEM) was performed using a JSM-6510LA scanning electron microscopy (JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 10 keV to obtain surface images of the electrodes before and after the deposited AgI was removed by CSV.

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EXPERIMENTAL SECTION Figure 1A shows the fabricated electrochemical closed chamber. The top of the 200 μL pipet tip (yellow, WATSON Co., Ltd., Tokyo, Japan) was cut to give an approximately 4 mm inner diameter. The columnar Vycor glass (diameter of 3 mm, length of 4 mm, BAS Inc., Tokyo, Japan) was inserted into the tip and sealed with a heat-shrinkable tube. Approximately 30 μL of dechlorinated water that contained zebrafish was introduced into the pipet. A rod-type Pt electrode (whole of diameter 3 mm, Pt diameter of 1.6 mm, BAS Inc., Tokyo, Japan) was inserted into the pipet to prepare the closed chamber (volume, approximately 30 μL). The fabricated chamber electrode and Ag electrode (diameter of 1.6 mm, BAS Inc., Tokyo, Japan) were immediately immersed into a solution containing 1.0 M potassium nitrate (KNO3) and 10 mM potassium iodide (KI). The two electrodes were connected externally to form a galvanic cell (Figure 1B). The Pt electrode acts as the cathode to reduce oxygen (O2 + 4H+ + 4e− → 2H2O) and the Ag electrode acts as the anode to oxidize silver (Ag + I− → AgI + e−). The galvanic cell was turned on for 10 min to allow deposition of the insoluble AgI, which is the signal source generated by the conversion of oxygen. The initial oxygen concentration of the air saturated with dechlorinated water (2.53 × 10−4 M) was comparable to the external solution. CSV was performed to determine the amount of AgI deposited on the Ag electrode. All electrochemical experiments were performed in the dark Faraday cage to remove the excess light irradiation and electrical noise. The difference in the cathodic charges obtained in the presence and absence of the zebrafish was calculated to estimate the respiration activities. 305

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peaks around −0.3 V to reduce the deposited AgI to metallic Ag. The largest reduction current was observed in the absence of zebrafish (Figure 3Aa), because oxygen molecules in the closed chamber were consumed only by the electrolysis reaction. In contrast, the reduction current obviously decreased in the presence of zebrafish in the chamber (Figure 3Ab). This decrease is because of a decrease in the amount of AgI deposited due to oxygen reduction. The result strongly suggested that the zebrafish consumed oxygen molecules in the closed chamber to reduce the oxygen concentration. Figure 3B shows the charges passed for the potential sweep, and they were easily calculated by time integration of the current response in Figure 3A. The values of the charges and 95% confidential limits were estimated to be 127.0 ± 17.2 and 85.9 ± 10.0 μC in the absence and presence of zebrafish in the chamber, respectively (Figure 3B). Therefore, the results indicate that zebrafish respiration consumes 32.4% of the oxygen in the closed chamber, if it is assumed that there is no oxygen influx into the chamber and no oxygen efflux from the chamber. The average oxygen consumption rate for the zebrafish was estimated using the initial oxygen concentration dissolved in the dechlorinated water (2.53 × 10−4 M). The rate was found to be at least 4.1 pmol·s−1 in this situation. Moreover, after electrochemical conversion with the galvanic cells was performed for 10 min, all the zebrafishes moved in the chamber. Thus, the signal equalization system provides a determination of respiration activities for migrating fish. The oxygen consumption of a zebrafish embryo was also estimated using the same method. By the presence of the embryo in the closed chamber, the cathodic stripping response decreased because of a decrease in the oxygen concentration caused by embryo respiration. Embryo respiration consumed 18.8% of the oxygen in 10 min, which was calculated from the charge passed in Figure 3Ac (103.1 ± 17.7 μC). The difference was recognized as significant by its p value (0.042) in the significance test. The average oxygen consumption rate of the embryo was estimated to be at least 2.4 pmol·s−1. After the electrochemical conversion was performed for 10 min, electrode surfaces with deposited AgI were observed by SEM. Figure 4 shows representative pictures obtained before

RESULTS AND DISCUSSION Linear sweep voltammetry (LSV) was performed to reduce oxygen by using a Pt electrode in the dechlorinated water, which was used as the breeding water. Figure 2a shows the

Figure 2. Linear sweep voltammograms for oxygen in (a) air-saturated dechlorinated water, (b) oxygen removed dechlorinated water, and (c) in 0.1 M air-saturated phosphate buffer.

voltammogram for oxygen in air saturated dechlorinated water. Oxygen reduction starts at 0.2 V (vs Ag/AgCl) and has a reduction peak at −0.1 V. The reduction current significantly decreased in dechlorinated water that was degassed with nitrogen gas for 30 min to remove oxygen (Figure 2b). The current peak obtained by oxygen reduction was negatively shifted by approximately 0.2 V compared to the peak obtained in 0.1 M phosphate buffer with a conductivity of 0.9 S·m−1 (Figure 2c). This may be due to the large solution resistance of dechlorinated water with low conductivity and the increase in the localized pH on the electrode because of proton generation by oxygen reduction. The LSV obtained using the Pt electrode in the closed chamber was nearly identical to the response in Figure 2a. The rest potential of the oxygen/dechlorinated water is approximately 0.31 V, while it is −0.22 V for Ag/AgI. Therefore, oxygen in the closed chamber is spontaneously reduced to form AgI on the Ag electrode in the galvanic cell. Oxygen in the closed chamber was measured by coulometric signal transduction of oxygen to AgI. Figure 3A shows the cathodic stripping voltammograms for detection of oxygen in the closed chamber. In this system, oxygen was reduced at the cathode and silver was oxidized to deposit AgI on the Ag electrode (anode) for 10 min. The reduction currents had

Figure 4. SEM images (A) before and (B) after removal of deposited AgI by CSV.

and after removal of the deposited AgI by CSV. Individual particles were clearly visible on the Ag electrode. Particles with a diameter of 0.1−1 μm were deposited at a density of 5.7 × 107 cm−2 (Figure 4A). The wide distribution of the particles’ sizes indicates the presence of both production and growth processes. Almost all the particles disappeared after CSV was performed (Figure 4B). The results indicate that particles produced by oxygen reduction are almost all converted to the current response of CSV. The influx of oxygen molecules was roughly estimated by amperometry. An aliquot of dechlorinated water (30 μL) that

Figure 3. (A) Cathodic stripping voltammograms for the detection of oxygen in the closed chamber (a) in the absence of a young fish, (b) in the presence of a young fish, and (c) in the presence of an embryo in the chamber. (B) Charges passed for the potential sweeps and 95% confidential intervals in the absence of a young fish (n = 10), in the presence of a young fish (n = 20), and in the presence of an embryo (n = 10). 306

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was degassed with nitrogen gas for 30 min was introduced into the chamber. Oxygen reduction currents were measured with a Pt electrode after the chamber was closed. Each reduction current was obtained 30 s after the potential was stepped and they are plotted in Figure S1 (Supporting Information). The current obtained immediately after the degassed dechlorinated water was introduced was 45% of that obtained from airsaturated dechlorinated water. Atmospheric oxygen may dissolve into the degassed solution during mechanical transfer of the solution. The reduction currents gradually increased and reached 80% after 6 h; subsequently, the reduction currents were almost identical values. These results may indicate that the oxygen influx is low when there is only a slight difference between the oxygen concentrations inside and outside the closed chamber. Moreover, a single amperometric measurement for 30 s consumed oxygen, and less than 1% of the initial amount of oxygen remained in the chamber. The present signal equalization system could be applicable to activity assays of migrating organisms, because the amount of oxygen consumed by respiration is significantly larger than the amount that flowed into the chamber in 10 min. Embryo hatching was also used to investigate the sealing of oxygen influx to the chamber. Zebrafish embryos, six hours postfertilization (hpf), were introduced into the closed chamber with 30 μL of dechlorinated water and incubated for 90 h at 26 ± 1 °C. Images of embryos incubated in the chamber (A) without and (B) with a Pt electrode cover are shown in Figure S2, Supporting Information. We obtained images after the embryos were transferred to the culture dish and then returned to the chamber. Notably, just after the sixth hour of incubation in the chamber without an electrode two-cell embryos developed and hatched larvae after 96 hpf (Figure S2A, Supporting Information). However, when the chamber was covered with the electrode, the embryo growth rate slowed (Figure S2B, Supporting Information). Although the embryos developed, cardiac activity was not observed after 96 hpf. This could be caused by oxygen scarcity because of respiration in the closed chamber. The results also indicate that the present closed chamber intercepts the supply of oxygen.

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CONCLUSION

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ASSOCIATED CONTENT

Letter

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +81-791-58-0171. Fax: +81-791-58-0493. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by Grant-in-Aid for Scientific Research (No. 23106009) on Innovative Areas “Bio Assembler” (Area No. 2305) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and Nakatani Foundation of Electronic Measuring Technology Advancement.

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REFERENCES

(1) Duchen, M. R. Mol. Aspects Med. 2004, 25, 365−451. (2) Papkovsky, D. B. Methods Enzymol. 2004, 381, 715−735. (3) Linek, V.; Vacek, V.; Sinkule, J.; Benes, P. Measurement of Oxygen by Membrane-Covered Probes; Ellis Horwood Ltd.: Chichester, UK, 1988. (4) Buerk, D. G. Methods Enzymol. 2004, 381, 665−690. (5) Bergner, S.; Vatsyayan, P.; Matysik, F.-M. Anal. Chim. Acta 2013, 775, 1−13. (6) Yasukawa, T.; Kondo, Y.; Uchida, I.; Matsue, T. Chem. Lett. 1998, 27, 767−768. (7) Yasukawa, T.; Kaya, T.; Matsue, T. Chem. Lett. 1999, 28, 975− 976. (8) Yasukawa, T.; Kaya, T.; Matsue, T. Anal. Chem. 1999, 71, 4637− 4641. (9) Rumsey, W. L.; Vanderkooi, J. M.; Wilson, D. F. Science 1988, 241, 1649−1651. (10) Papkovsky, D. B.; Ponomarev, G. V.; Trettnak, W.; O’Leary, P. Anal. Chem. 1995, 67, 4112−4117. (11) O’Mahony, F. C.; Donovan, C.; Moore, T.; Davenport, J.; Papkovsky, D. B. Environ. Sci. Technol. 2005, 39, 5010−5014. (12) Alderman, J.; Hynes, J.; Floyd, S.; Kruger, J.; O’Connor, R.; Papkovsky, D. B. Biosens. Bioelectron. 2004, 19 (11), 1529−1535. (13) O’Riordan, T. C.; Buckley, D.; Ogurtsov, V.; O’Connor, R.; Papkovsky, D. B. Anal. Biochem. 2000, 278 (2), 221−227. (14) Koide, M.; Yasukawa, T.; Nagamine, K.; Shiku, H.; Itayama, T.; Matsue, T. Sens. Actuators, B: Chem. 2011, 153 (2), 474−478. (15) Koide, M.; Yasukawa, T.; Horiguchi, Y.; Nagamine, K.; Shiku, H.; Matsue, T.; Itayama, T. Anal. Sci. 2012, 28 (1), 69−72. (16) Pritchard, J. F.; Jurima-Romet, M.; Reimer, M. L. J.; Mortimer, E.; Rolfe, B.; Cayen, M. N. Nat. Rev. Drug Discovery 2003, 2, 542−553. (17) Kenakin, T. Nat. Rev. Drug Discovery 2003, 2, 429−438. (18) Parng, C.; Seng, W. L.; Semino, C.; McGrath, P. Assay Drug Dev. Technol. 2002, 1, 41−48. (19) Sandbacka, M.; Christianson, I.; Isomaa, B. Toxicol. in Vitro 2000, 14 (1), 61−68. (20) Yasukawa, T.; Yoshimoto, Y.; Goto, T.; Mizutani, F. Biosens. Bioelectron. 2012, 37, 19−23.

This is the first report on a method to determine the oxygen consumption rate of migrating organisms. A closed chamber with a Pt electrode was fabricated to eliminate the interference of oxygen diffusion into the chamber. The galvanic cells allow the conversion of oxygen at the Pt electrode (cathode) in the closed chamber to insoluble AgI at the Ag electrode (anode). The amount of AgI that was determined by CSV decreases in the presence of individual zebrafish or its embryo; therefore, the oxygen consumption rate can be estimated on the basis of the decreased stripping response. The rate was determined to be ∼4.1 and 2.4 pmol·s−1 for the zebrafish and embryo, respectively. The technique is applicable for estimating the activity of migrating organisms and is expected to be useful for environmental risk assessments and screening of effective drugs.

* Supporting Information S

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 307

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