Development of Oxygen Consumption Analysis with an on-Chip

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Development of oxygen consumption analysis with an on-chip electrochemical device and simulation Kaoru Hiramoto, Masahiro Yasumi, Hiroshi Ushio, Atsushi Shunori, Kosuke Ino, Hitoshi Shiku, and Tomokazu Matsue Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02074 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Analytical Chemistry

Development of oxygen consumption analysis with an on-chip electrochemical device and simulation Kaoru Hiramoto*a, Masahiro Yasumia, Hiroshi Ushioa, Atsushi Shunoria, Kosuke Ino*b, Hitoshi Shikub, Tomokazu Matsuec a

Corporate Engineering Division, Automotive and Industrial Systems Company,

Panasonic Corporation, 1006 Kadoma, Kadomashi, 571-0050, Japan. b

Graduate School of Engineering, Tohoku University, 6-6-11-406 Aramaki-aza Aoba,

Aoba-ku, Sendai 980-8579, Japan. c

WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira,

Aoba, Sendai, 980-8579, Japan. Corresponding authors: Kaoru Hiramoto [email protected] Tel: +81-50-3687-0262 and Kosuke Ino [email protected] Tel: +81-22-795-5872

ACS Paragon Plus Environment


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Abstract The O2 consumption rate of embryos has been attracting much attention as a key indicator of cell metabolisms and development. In this study, we propose an on-chip device that enables the accurate, easy, and non-invasive measurement of O2 consumption rates of single embryos. Pt electrodes and micropits for embryo settlement were fabricated on Si chips via microfabrication techniques. The configuration of the device enables the detection of O2 concentration profiles surrounding the embryos by settling embryos into the pits with a mouth pipette. Moreover, as the detection is based on an electrochemical method, the influence of O2 consumption on the electrodes was also considered. By using a simulator (COMSOL Multiphysics), we estimated the O2 concentration profiles in the device with and without the effects of the electrodes. Based on the simulation results, we developed a normalization process to calculate the precise O2 consumption rate of the sample. Finally, using both the measurement system and the algorithm for the analysis, the respiratory activities of mouse embryos were successfully measured. Keywords: oxygen consumption embryo electrode array on-chip device electrochemical simulation


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Introduction Cell development relies on various metabolic activities. Particularly O2 is one of the most basic energy substrates required to maintain cellular viability. Therefore, cellular O2 consumption has been studied to evaluate cell functions and developmental potentials1-3. Several techniques have been developed to detect cellular O2 consumptions including spectrophotometric4, fluorescence5, and electrochemical techniques such as scanning electrochemical microscopy (SECM)

6-9.

This strategy has been applied to the

evaluation of embryos and was partially successful in increasing the chances of pregnancy.10 During SECM measurements, a probe electrode is scanned to acquire the two- or three-dimensional O2 concentration profile of a sample. Analysis of the O2 flux enables the evaluation of the O2 consumption of single embryos, cell aggregates, and single cells without any damage to the samples6-8. Detection can be performed under conditions that closely resemble those of cell cultures without the use of any markers. Although SECM can evaluate cellular respiratory activity non-invasively, it is difficult to manipulate a fragile probe precisely without training. Moreover, SECM can only determine one sample per measurement and the scanning time is long; thus, its throughput is low. Therefore, a new tool, (e.g. a chip device) that allows fast measurements with easy handling is desirable. We have previously developed a chip device to detect the O2 consumption of an embryo11. Since the working electrodes were arranged on the device, the handling of the device was easy when compared to probe electrodes. By using this device, O2 consumption of two-cell, morula, and blastocyst stages was successfully measured. The previous device detected the O2 concentration near the cells, but the O2 concentration profile was not measured. For the precise evaluation of cellular respiratory activity, an O2 concentration profile should be measured using an electrode array device. Several devices with electrode arrays have been proposed for electrochemical imaging12-17.

Since many electrodes are packed in a small area, a chemical concertation

profile can be monitored. In this study, we utilized an electrochemical chip device specializing in the detection of O2 consumption rate of embryos. By using microfabrication techniques, a micro-pit and Pt microelectrodes were fabricated on a Si substrate. The pit was utilized for trapping the embryo while the electrodes were encircled around the pit to detect O2 concentrations around it. These electrodes were arranged at 120, 170, 220, 300, 400 and 500 µm from the center of the pit so that the O2 concentration profile could be estimated. This architecture only requires the embryo to be placed into a pit and all the measurements proceeds automatically. This device was previously utilized for the evaluation of frozen-thawed human embryos in terms of



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clinical utility for In Vitro Fertilization setting.18 However, electrode array devices affect the O2 concentration profile due to O2 consumption by the electrodes during detection. In this study, the electrode effects were analyzed using simulation analysis to acquire precise O2 consumption rates of the samples. By comparing the simulation and experimental results afforded for mouse embryos, an applicable measurement protocol and a formula for the correction of this measurement were developed. Additionally, the O2 consumption rates of mouse embryos were measured to verify the measurement system of the device. The proof of calculation studied here may support the validity of the previous report18 and shows great potential for application as a new tool for cell analysis.


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Materials and Methods Device fabrication The device design is presented in Fig. 1. The working electrode chip in the device consists of a micro-pit and Pt electrodes. When an embryo was introduced into the pit; an O2 concentration gradient was observed near the embryo as a result of its O2 consumption. The Pt electrodes were used to evaluate the local O2 concentration from the O2 reduction current (Fig. 1A). By using the currents from electrodes placed at different distances from the embryo, the O2 concentration profile could be calculated. First, six concentric Pt rings were fabricated on a SiO2/Si substrate using Ti/Pt sputtering and a dry etching (Fig. 1B). Next, a 500 nm thick SiO2 layer was deposited onto the chip by sputtering. The SiO2 layer was subsequently removed at designated areas by wet etching to expose eight Pt disks (5 µm diameter) at each Pt ring pattern (Fig. 1B). A micro-pit (200 µm diameter and 80 µm depth) was fabricated in the center of the chip via dry etching. Thus the resulting chip comprised a central micropit concentrically surrounded by 48 Pt microelectrodes. The distances between the disk electrodes and the central micropit were 120, 170, 220, 300, 400 and 500 µm (eight electrodes each). The summation of the current from eight disk electrodes was collected at each distance. Pt electrodes, fabricated via Ti/Pt sputtering, were used as reference and counter electrode chips. The top semicircular shape of the Pt electrode served as the reference electrode and the bottom half as the counter electrode. Fig. 1D illustrates the device assembly. One reference-counter chip and five working chips were mounted on a printing board and sandwiched between polystyrene top and bottom plates. The top plate consisted of six open-bottom wells so that the chips on the printing board formed the bottom surface of the well plate. Electrochemical connection was achieved from the back of the printing board through the hollowing of the bottom plate. Experimental setup for the electrochemical measurements The experimental setup is presented in Fig. 2. The device was mounted on a jig plate that connected the device with a multi-channel potentiostat comprising a reference-electrode terminal, a counter-electrode terminal and 30 working terminals. A data acquisition system was mounted on the potentiostat (National Instrument, USB-6216, USA) and controlled by the LabVIEW program. The jig consisted of 32 spring-loaded pin connectors that made contact with the printing board of the device; thus, the device was connected electrically with the potentiostat when mounted and



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locked onto the jig. A transparent warm plate (220×160×7 mm, KITAZATO, Japan) was selected and fixed under the jig. Half the warm plate was employed to maintain the device at 37 °C and the other half of the space was employed to observe embryos in the culture dishes. Therefore, the embryos could be manipulated to the required conditions under a stereomicroscope (Olympus, SXZ12, Japan). Simulation analysis A three-dimensional model comprising a pit and 48 electrodes was prepared using COMSOL Multiphysics (ver. 5.2, COMSOL Inc., USA). A time dependent study was used for time course analysis. The sizes of the pit and the electrodes were identical for all the experiments. Briefly, six ring electrodes with eight disk electrodes (5 µm diameter) each were prepared. The redox current from each ring electrode was then calculated. A sphere (50 µm radius) was set in the center of the pit to represent an embryo. Assuming the sphere as being a mammalian embryo, the O2 consumption rate of the sphere, f (fmol/s), was determined in a range of 0–10 fmol/s so that the input values for the reactions of the sphere were set to 0–0.0191 mol/m3s. The model was filled with 0.209 mM O2. The diffusion coefficient of O2, D (m2/s), was set to 2.1 × 10-9 m2/s. The electrochemical reaction of O2 at the electrodes was assumed to be a four-electron reaction. To estimate the O2 reduction currents, the O2 flux at the electrodes, f Electrode (mol/m2s), was calculated as follows:

f Electrode = k × C Electrode

(1)

where k (m/s) and C Electrode (mol/m3) are the reaction constant and the O2 concentration at the electrodes, respectively. Since the H+ ion concentration is constant in a buffer, the concentration was not set as a variable in the simulation. Constant k was set to X (X = 1.0×10-2, 1.0×10-3, 1.0 × 10-4 or 1.0 × 10-5 m/s) to find a suitable value for k. The current density of O2 reduction, j (A/m2), was calculated as follows:

j = n × F × f Electrode

(2)

where n and F (sA/mol) are the reaction electron number and the Faraday constant, respectively. To determine the current i (A) from the electrode, j was integrated over the electrode using COMSOL.


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The spherical diffusion theory The spherical diffusion theory8 was utilized for the general analyses of the O2 consumption rate of the sample. An O2 concentration profile along the distance from the center of the sample, R (m), is expressed as follows:

C = - (C* - Cs) r/R + C* = -∆C r/R + C*

(3)

where r (m) is the radius of the sample, C (mol/m3) is the O2 concentration at distance R,

C* (mol/m3) is the O2 concentration of the bulk (0.209 mM), Cs (mol/m3) is the O2 concentration at the sample surface, and ∆C is the O2 concentration difference between the surface of the sample and the bulk.

C/C* = i/i*

(4)

where i (A) is the O2 reduction current at distance R, and i* (A) is the O2 reduction current for the bulk O2 concentration. Therefore,

i/i* = - (∆C/C*) r/R + 1

(5)

∆C can be calculated from the slope of a plot of i/i* vs. r/R or C/C* vs. r/R. ∆C is used to determine the O2 consumption rate f (mol/s) of an embryo. By using Fick’s first law, the flux density of O2 at the surface of the sample, fs (mol/sm2), can be determined:

fs = D ∆C/r

(6)

Therefore, the total O2 consumption rate of a spherical sample based on spherical diffusion is expressed as:

fspher = S × fs = 4πr D ∆C

(7)

where S is the surface area of the sphere. When a sample is set in a flat device, as in the present study, semispherical diffusion layer of O2 is formed above the device. Therefore,

f (mol/s) for an embryo that is settled in such a device is expressed as:



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f = S × fs = 2πr D ∆C

(8)

Thus, f can be calculated from ∆C and eq. (8). Here in, we discuss the difference between the value of f calculated from eq. (8) and that of fset used in the simulation. This determines whether the theory can be applied to the proposed device. Embryo culture Vitrified 2-cell mouse embryos (C57BL/6J Jcl) were purchased from Ark Resource Co. (Japan). Freeze-thawing of the embryos was performed according to the manufacturer instructions. After thawing, the embryos were gently introduced into 100 µL KSOM (Ark Resource Co. Japan) droplets pre-incubated in an incubator (CO2 5%, 95% air, 37 °C) and overlaid with 7 mL of mineral oil (The Institute for Assisted Reproductive Medical Technology, Japan). After introduction, the embryos were cultured in the incubator. Measurement of O2 consumption of the embryos First, 700 µL of Gamete buffer (SYDNEY IVF Gamete buffer, G48258, COOK Medical, USA) were introduced into the device with a micropipette to fill the six wells. Next, 2 mL of mineral oil were overlaid onto the whole surface of the wells to avoid evaporation. These solutions were preliminarily heated at 37 °C. For device characterization, P+HEPES (Nakamedical, Japan) and HEPES buffer solutions (Sigma-Aldrich, USA) were also used. A detection flow diagram is displayed in Fig. S1. For initialization of the electrodes, -0.6 and +0.05 V square-wave pulses were applied to each electrode for 14 cycles per two seconds until the curves of the O2 reduction currents became uniformly stabilized. After initialization, -0.6 V pulses were applied for 4 s on each chip to collect the O2 reduction currents as background (reference state measurement). During measurement, the embryos were taken out from the incubator and washed with Gamete buffer. The embryos were then gently transferred to each pit using an aspirator tube assembly (Drummond, USA) under the microscope. During the introduction of the embryos into the device, the potential on the electrodes was held at +0.05 V to avoid electrical noise. After all the embryos were settled, -0.6 V pulses were applied for 4 s to acquire O2 reduction currents (O2 consumption measurement). After detection, the embryos were removed from the pits. In this work, O2 reduction currents at 3 s were used to calculate the O2 concentration profile generated in the device.


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The O2 consumption rate of the mouse embryos at representative growth stages were measured by the device and SECM (CRAS 1.0, Clino Co., Japan). The manufacturer manipulation instructions were followed for SECM measurement. Vitrified two-cell embryos were thawed and cultured in an incubator. At 6 h, 24 h, 48 h, and 72 h after thawing, embryos of typical morphology (two-cell, four-cell, morula, and blastocyst) were selected and the O2 consumption rates were measured.



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Results and Discussion Device characterization using experimental and simulation analyses The proposed device comprised six wells. The well at the far left was modified to hold a reference/counter chip while the other five wells contained working chips to evaluate five embryos simultaneously (Fig. 3A). The working chip consisted of 48 Pt disk electrodes and a micropit for embryo settlement. When an embryo is set in the pit, O2 concentration decrease at the neighborhood of the embryo and distribute to bulk concentration. Therefore, the disks were arranged at six different distances from the pit (120, 170, 220, 300, 400, and 500 µm from the center of the pit) to measure the O2 concentration profile affected by the respiration of the embryo. Moreover, eight disk electrodes were arranged on one ring electrode in rotational symmetry at each distance. As the summation of the current from surrounding eight disk electrodes is collected, the device can afford representative value of each distance even the center of the embryo is not fixed at the central pit. This arrangement can afford precise measurement even the size of the embryo varies or the embryo has cell deviation. By filling the six wells with a measurement solution, the five working chips were connected electrically to the reference/counter chip. The effects of the Pt reference electrode are described in Fig. S2. Cyclic voltammetry was performed with the device in 1 mM ferrocenemethanol solution (Sigma Aldrich, USA.) with a Ag/AgCl reference electrode (BAS Co., Japan), and a Pt reference electrode inserted in the device. When the Pt reference electrode was utilized, the current peak of the oxidation shifted by -0.12 V, compared to that afforded from the Ag/AgCl electrode. The result indicates that the Pt electrode can be applied for use as a quasi-reference electrode with consideration of a slight potential shift. Both the experimental and simulation results were employed to characterize the device. Fig. 4A displays the experimental results of the O2 reduction currents at -0.6 V in Gamete, P+HEPES, and HEPES buffer solutions. Gamete and P+HEPES buffers contain proteins and sugars and are general solutions used for the manipulation of embryos. When using the Gamete and P+HEPES buffers, the O2 reduction currents decreased drastically from -3 nA to -1 nA in 30 s. On the other hand, the current of the HEPES buffer was larger (-12 nA to -7 nA in 30 s). These results indicate that the electrode surface was contaminated with the components, such as proteins, sugars and amino acids in the Gamete and P+HEPES buffers; thus, O2 reduction on the electrodes was disturbed. The simulation results are displayed in Fig. 4B. Four variations in the reaction constant k (1.0×10-2, 1.0×10-3, 1.0 × 10-4 or 1.0 × 10-5 m/s ) were demonstrated.


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In the simulation, the current immediately reached the static state, while it continuously decreased in the experiment. This implies a continuous change in the k value in the experimental buffers due to contamination of the proteins. Although it is ideal to simulate the change in k, the effect of protein contamination is difficult to clarify. Therefore, we compared the current value at 30 s, where the current decrease in the experimental buffers was gradual. Here, we conclude that the Gamete buffer is the best for embryo measurements, as the current reached a near-static state. As a result, the k value was determined for 1×10-4 m/s because the current value under this condition was the closest to that of the Gamete buffer at 30 s. This value was adapted in subsequent simulations. Analysis of the O2 consumption rate of an embryo in the simulation without electrode reactions Fig. 5A exhibits the O2 concentration profile afforded by simulation; 120 s after an embryo with a 50 µm radius was introduced in the micro-pit (fset = 3 fmol/s in this case). An O2 gradient appeared near the embryo and the profile became almost stable at 120 s. Fig. 5B illustrates the relationship between the O2 concentration on the device surface and the distance, R, from the center of the pit. In the analysis, R was defined as illustrated in Fig. 5A. The O2 concentrations near the embryo strictly depended on f. Fig. 5C demonstrates the relationship between C/C* and r/R. According to eq. (5), the C/C* value at x = 0 should be equal to one. However, the x-intercept in the graph differed from the theoretical value, indicating that the chip configuration affected the O2 diffusion layer; thus, the layer disagreed with the theory. Next, the slope of the graph was calculated by the least-square method to determine ∆C. To validate the applicability of the spherical diffusion theory, the value of f, the O2 consumption of the sphere, was calculated from eq. (8). Fig. 5D displays the relationship between the fset and fcalc values, where fcalc is proportional to fset. Even though the embryo was set in the micropit, the fcalc was only 8.6% higher than the actual value, fset. These results indicate that the spherical diffusion theory can be applied to the present chip configuration if the electrodes do not consume O2. In subsequent simulations, f was corrected by multiplying with a geometric factor of 0.914. Analysis of the O2 consumption rate of an embryo in a simulation with electrode reactions We next investigated the influence of O2 consumption of the electrode arrays on the O2 concentration profile of embryo respiration. The O2 consumption rate of the



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embryo was set to fset = 0, 1, 3, 5, 10 fmol/s, and O2 consumption on the electrodes started 120 s after the embryo was set in the pit. Fig. 6A presents the O2 concentration profile at 124 s, where simultaneous O2 consumption by the embryo and O2 consumption on the electrodes occurred. In this case the O2 concentration at the chip surface was generally lower than that afforded in the absence of an electrochemical reaction (Fig. 5A). This indicates that the O2 consumption at the electrode affects the analysis of the respiratory activity. Therefore, we concluded that further normalization should be performed when using the electrode array device. Fig. 6B displays the amperograms of the O2 reduction current in the simulation when the O2 consumption of the embryo was set for fset = 0 and 3 fmol/s. Even though the embryo did not consume O2, an O2 gradient still occurred around the embryo due to the electrode O2 consumption. Since the electrode density near the embryo was high, the O2 concentration near the embryo was lower than that in the areas further away from the sample. Notably, the O2 reduction current at the electrode located at R = 120 µm was higher than that observed at R = 170 µm when fset = 0 fmol/s. This was attributed to the additional O2 flux from the pit. At fset = 3 fmol/s, the effect of the pit was eliminated by the O2 consumption of the embryo; thus, the order of the O2 reduction current was consistent with the location of the electrodes. Fig. 6C illustrates the relationships between i/i* and r/R. The value for i/i* decreased with decreasing R even when fset = 0 fmol/s. This was due to the consumption of O2 by the electrodes located in the vicinity. The effect of the electrodes may lead to overestimation of the actual O2 consumption of an embryo. Thus, we normalized the data by using fset = 0 fmol/s in order to eliminate the influence of the electrode reactions. In brief, as shown in Eq. (4), i/i* is equal to C/C*. Thus C/C* was divided by the corresponding value at 0 fmol/s to acquire the normalized C/C*, so that the effect of O2 consumption due to the electrodes could be eliminated. Fig. 6D displays the plots for normalized C/C* vs. r/R after geometric correction and normalization. These plots were used to determine ∆C from eq. (5). Finally, f was calculated from eq. (8) using ∆C. Fig. 6E presents the relationship between the values of f and fset. The value for f, based on correction and normalization, was very similar to that of fset. On the other hand, the non-normalized f value was 1.4–1.5 fmol/s higher than fset. This indicated that the actual O2 consumption rate of a single embryo can be precisely evaluated by using the above procedure. As the normalization process can eliminate the effect of O2 consumption on the electrodes, it is not necessary for end-users to use the k value to calculate the respiratory activity of embryos.


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Measurement of O2 consumption of embryos O2 consumption measurements were performed with actual mouse embryos according to the flow mentioned above (Fig. S1). To verify the measurement system, embryos at representative growth stages (two-cell, four-cell, morula, and blastocyst) were measured. Gamete buffer was used for the measurement solution. Fig. 7 demonstrates the optical images the embryos and the plots of i/i* vs. r/R and normalized C/C* vs. r/R. The values for i/i* obtained from the O2 reduction current in the reference state measurement are indicated by open circles and those for O2 consumption measurement are indicated by filled circles. The current on each electrode did not always agree with that of the simulation. The O2 reactions on the electrodes were affected not only by the configuration effect demonstrated in the simulation, but also by the initial O2 concentration, temperature, size and quality of the electrodes, insulation layers, and wall of the plastic well. Therefore, we adopted the normalization process demonstrated above in order to remove the initial variations in the O2 reduction current. For normalization, the values in the reference state measurement were assumed as f = 0. Namely, the i/i* values for the O2 consumption measurement were divided by that of the reference state measurement to acquire normalized C/C*. Although the i/i* values differed in each experiment, the normalized C/C* obtained via the above procedure showed reasonable dependence on r/R. The slopes of the normalized C/C* vs. r/R plots indicated that ∆C depends greatly on the developmental stage of the embryo (Fig. 7A-D). The values for ∆C from each plot were as follows; two-cell for -0.011, four-cell for -0.018, morula for -0.024, and blastocyst for -0.032. The developed embryos like morula and blastocyst showed steeper slopes, indicating higher respiratory activities than those of the early stage embryos. By using ∆C, the O2 consumption rate of each embryo was calculated from eq.(8): two-cell (1.5 fmol/s), four-cell (2.5 fmol/s), morula (3.3 fmol/s), and blastocyst (4.5 fmol/s), resulting in reasonable dependence on each developmental stage. In the calculation, the O2 concentration of the bulk, C = 0.209 mM, and the diffusion coefficient of O2, D = 2.1 × 10-9 m2/s, were utilized. In comparison, the C/C* value in the absence of embryos exhibited no dependence on r/R (Fig. S3). As mentioned above, since the amperogram for O2 reduction depends on the device configuration, solution composition, and electrode contamination, the O2 reduction current did not show as same trace as the simulation. The change in the current may restrict the precise detection of the O2 consumption of embryos. To solve this problem, we adopted the normalization process using the values in the absence of the embryos (f = 0 fmol/s) and eliminated the effects of O2 consumption on the electrodes.



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For the measurement protocol, we combined the reference state measurement before an embryo was introduced into the pit, so that the current at f = 0 fmol/s can be derived in one measurement. Since the simulation results were not used for the normalization of real samples, the method can be utilized for non-COMSOL users. We verified the O2 consumption rates of mouse embryos afforded from the proposed system by comparing the data obtained from an existing system based on SECM. Fig. 8 illustrates the O2 consumption rates of the three embryo stages. The O2 consumption rates measured using the device were as follows: two-cell for 2.1±0.3 fmol/s, four-cell for 2.3±0.5 fmol/s, and morula for 3.2±0.5 fmol/s. On the other hand, the O2 consumption rates afforded by SECM were 2.3±0.3 fmol/s, 2.2±0.3 fmol/s, and 3.5±0.3 fmol/s, respectively. In this study, more embryos were measured by the device (n = 32) than SECM (n=8), but the results demonstrated no significant difference between the two measurements. This strongly indicates that the proposed system can be applied to the detection of the O2 consumption rates of embryos. The respiration activity of embryos has been considered to reflect the developmental potential of embryos, which is one of the desired pieces of information for infertile treatment to increase the pregnancy rate. The information may help embryologists in eliciting the viable embryo for transplant. While SECM is a reliable tool for cell analysis, it is difficult to be applied for clinical use because of the complicated manipulation. The proposed system is advantageous because of its ease of handling and may serve as an alternative to SECM in such a scenario.


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Conclusions An on-chip device for the electrochemical measurement of the O2 consumption rate of embryos was developed. The results in the simulation demonstrated that the semi-spherical diffusion theory was applicable to the calculation of the O2 concentration profile in the device. The results also suggested that the O2 consumption of the electrode arrays significantly affected the analysis. Therefore, we developed a normalization method to remove these effects and to calculate the precise O2 concentration profile derived from the sample. The simulation analysis studied herein, was a simplified version. Some issues remain to be resolved for simulating continuous changes in the O2 reduction current on the device. Nonetheless, the importance of estimating the effects of O2 consumption on array electrodes has been demonstrated. By considering other chemical and physical effects such as measurement solutions, temperature, and the device components, further optimization of the measurement system can be performed. By utilizing the device and the measurement protocol, the O2 consumption rates of mouse embryos were successfully measured. The results obtained with the device were similar to those obtained using the conventional method, SECM. We predict that the device will play a strong role in promoting metabolic research on embryos and cells.



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Acknowledgements The authors would like to thank T. Hisamoto, M. Yamamoto, and T. Hiratsuka for their aid in the development of the measurement system. We would also like to thank Prof. Hiroyuki Abe in Yamagata University for device development and Dr. N. Yaegashi and his team in Tohoku University Graduate School of Medicine for their assistance in the clinical study. This work was also supported in part by a Grant-in-Aid for Scientific Research (A) (No. 16H02280) and a Grant-in-Aid for Young Scientists (No. 15H05415) from the Japan Society for the Promotion of Science (JSPS).


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Chem. 2001, 2001 73, 3751-3758. (9) Yasukawa, T.; Kondo, Y.; Uchida, I.; Matsue, T. Chem. Lett. 1998, 1998 767-768. (10) Sunkara, S. K.; Rittenberg, V.; Raine-Fenning, N.; Bhattacharya, S.; Zamora, J.; Coomarasamy, A. Hum Reprod 2011, 2011 26, 1768-1774. (11) Date, Y.; Takano, S.; Shiku, H.; Ino, K.; Ito-Sasaki, T.; Yokoo, M.; Abe, H.; Matsue, T.

Biosens. Bioelectron. 2011, 2011 30, 100-106. (12) Ghindilis, A. L.; Smith, M. W.; Schwarzkopf, K. R.; Roth, K. M.; Peyvan, K.; Munro, S. B.; Lodes, M. J.; Stover, A. G.; Bernards, K.; Dill, K.; McShea, A. Biosens. Bioelectron. 2007, 2007 22, 1853-1860. (13) Ino, K.; Nishijo, T.; Arai, T.; Kanno, Y.; Takahashi, Y.; Shiku, H.; Matsue, T. Angew.

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Figures

Fig. 1 General outline of the detection procedure using the device. (A) Detection outline for respiratory activity of an embryo on the working electrode chip. (B) Top image of the working electrode chip. (C) Top image of the reference/counter electrode chip. (D) Illustration of the device assembly.



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Fig. 2 Detection system consisting of a multi-channel potentiostat, a jig for electrical connection, a stereomicroscope for embryo manipulation, and a laptop.


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Fig. 3 (A) Device image. (B) Scanning electron microscope (SEM) image for the working electrode chip. An outermost ring electrode as a reference electrode was not used in this study.



Gamete P+HEPES HEPES

55

10 15 15 20 20 10 Time (s) Time (sec)

25 25

30 30

k = 1× ×10-5 k = 1× ×10-4

(B) 0 0 Current (nA)

00 -1 -2-2 -3 -4-4 -5 -6-6 -7 -8-8 -9 -10 -10 -11 -12 -12 -13 -14 -14 00

-1 -2-2 -3 -4-4 -5 -6-6 -7 -8-8 -9 -10 -10 -11 -12 -12 -13 -14 -14 00

Current (nA)

(A)

Current (nA) Current (nA)

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k = 1× ×10-3

k = 1× ×10-2 55

10 15 15 20 20 10 Time (s) Time (sec)

25 25

30 30

Fig. 4 Device characterization. Amperograms of O2 reduction from (A) the experiment and (B) simulation results. Both graphs display the currents at the very inner ring electrode (R = 120). In the experiments, -0.6 V was applied for 30 s to collect O2 reduction currents in Gamete, P+HEPES, and HEPES buffers. In the simulation, reaction constant k was set to 1.0 × 10-2, 1.0 × 10-3, 1.0 × 10-4, and 1.0 × 10-5 m/s.


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Fig. 5 Simulation analysis for O2 consumption rate of an embryo with no electrode O2 consumption. (A) Cross-sectional image of the O2 concentration profile at 120 s after setting the embryo (fset = 3 fmol/s). (B) C vs. R. (C) C/C* vs. r / R. (D) fcalc vs. fset; fcalc was calculated from eq.(8) using ΔC acquired from the slope of C/C* vs. r / R plot. C = O2 concentration, R = distance from the center of the sample, C* = O2 concentration of the bulk, r = radius of the sample, f = flux of the embryo O2 consumption.



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Fig. 6 Simulation analysis for O2 consumption rate of an embryo with electrode O2 consumption. (A) Cross sectional image of the O2 concentration profile with an embryo (fset = 3 fmol/s) and the O2 consumption on the electrodes. (B) Amperograms of the electrodes at distance R = 120, 170, 200, 300, 400, and 500 µm when O2 consumption of the embryo was set to 0 and 3 fmol/s. (C) i/i* vs. r/R. The values for i were acquired at 3 s from the amperograms (D) Normalized C/C* vs. r/R. (E) fcalc vs. fset; Non-normalized fcalc was calculated from eq.(8) using ΔC acquired from the slope of i/i* vs. r/R plot. On the other hand, normalized fcalc was calculated usingΔC acquired from normalized C/C* vs.

r/R plot.


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Fig. 7 Electrochemical detection of the respiratory activities of the embryos at various developmental stages. (A) Two-cell, (B) four-cell, (C) morula, and (D) blastocyst for optical images, i/i* vs. r/R and normalized C/C* vs. r/R. i/i* The i values were acquired from the current at 3 s of the last pulse waves of the reference state measurement (open circles) and O2 consumption measurements (filled circles), respectively.



5.0 device (n=32) 4.0

f [fmol/s]

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SECM (n=8)

3.0 2.0 1.0 0.0 2 cell

4 cell

morula

Fig. 8 Electrochemical detection of the respiratory activities of two-cell, four-cell and morula embryos using the device and scanning electrochemical microscopy (SECM).


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For Table of Contents graphic only


Development of Oxygen Consumption Analysis with an on-Chip

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