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A microfabricated reservoir-type oxygen sensor for measuring the real-time cellular oxygen consumption rate at various conditions
Sensors and Actuators B 147 (2010) 263–269
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A microfabricated reservoir-type oxygen sensor for measuring the real-time cellular oxygen consumption rate at various conditions Jungil Park a , Youngmi Kim Pak b,c,∗∗ , James Jungho Pak a,∗ a b c
School of Electrical Engineering, Korea University, Seoul 136-713, Republic of Korea Department of Nanopharmaceutical and Life Science, Age-Related and Brain Disease Research Center, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Physiology, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 December 2009 Received in revised form 17 March 2010 Accepted 23 March 2010 Available online 30 March 2010 Keywords: Electrochemistry Reservoir-type Oxygen sensor Cellular respiration Oxygen consumption rate
a b s t r a c t This paper presents a microfabricated reservoir-type oxygen sensor, which can accurately measure the solubilized oxygen concentration in real time, in order to measure the cellular oxygen consumption rate (OCR) in a solution containing cells. The fabricated oxygen sensor is composed of three-parts: electrochemical sensing electrodes, an oxygen-permeable membrane, and a reservoir for storing the solution. The oxygen transport rate through the membrane and the oxygen reaction rate at the working electrode (WE) surface are the two dominant parameters in determining the sensitivity of the oxygen sensor. The fabricated sensor showed a sensitivity of 2.84 A/cm2 M and a 90% response time of 4.9 s in an average of 5 sensors when a 25,000 m2 WE and a 20 m polydimethylsiloxane membrane were used. This is the first report in which the fastest response time has been achieved for the oxygen sensor. The fabricated sensor showed the repeatability with 154.05 ± 1.87 nA at the full-oxygen state and 2.77 ± 1.0 nA at the zero-oxygen state. The fabricated sensor was used to measure the uncoupled OCR of the L6 cells, and its result of 3.69 ± 0.30 was almost identical to the result of 3.70 ± 0.26 obtained from a commercial system. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Mitochondrial dysfunction has been generally accepted as one of the causes of age-related degenerative diseases including metabolic syndrome, insulin resistance, and diabetes [1–3]. The tissues or cells affected with the diseases exhibit a decrease in mitochondrial functions which may be monitored by the mitochondrial membrane potential, mitochondrial DNA contents, intracellular ATP contents, mitochondrial gene expressions and the cellular oxygen consumption rate (OCR) [4]. Among those parameters, the oxygen consumption rate, also known as the respiration rate as related to the cells or the isolated mitochondria, is an important physiologic profile of mitochondrial respiratory function [1,2,5,6]. Large-scale screening of cell-based mitochondrial activities using a 96-well or a 384-well has been applied to investigate problems in mitochondrial biology, drug toxicity, and therapeutics [4]. In this screening compendium, however, the OCR was not included solely because the current OCR measurement instruments do not allow for a high-throughput analysis and require a large number
∗ Corresponding author. Tel.: +82 2 3290 3238; fax: +82 2 921 0544. ∗∗ Co-corresponding author. Tel.: +82 2 969 0958; fax: +82 2 969 6343. E-mail addresses: [email protected] (J. Park), [email protected] (Y.K. Pak), [email protected] (J.J. Pak). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.03.069
of cells in a time-consuming process. Therefore, it was valuable to develop a small oxygen biosensor to measure the OCR precisely and rapidly using a small number of cells in a small volume of solution. Recently, Seahorse Bioscience has produced XF24 Analyzer which uses the small volume of solution and can measure over 24 samples, simultaneously [7,8]. The cellular OCR is determined by measuring the oxygen concentration dissolved in a solution containing cells with time. This is accomplished by two different techniques, an optical or an electrochemical measurement method [7,9–15]. Both optical and electrochemical oxygen sensors have the advantages of continuous measurement, simple operation, and use of a small volume of solution. XF 24 Analyzer using the optical method and 782 Oxygen meter (Strathkelvin Instruments, Germany) using the electrochemical method are known to use about 10 l and 200 l solution in the experiment of measuring the cellular consumption rate, respectively [7,15]. While XF24 Analyzer is the opened system because it is not completely isolated from the outside environment, 782 Oxygen meter and Oxygraph-2k (Oroboros Instruments, Austria) have the closed systems that do not allow ambient oxygen to access the measurement solution. The closed system can provide more accurate output results because the environmental effect would be lower [8]. The electrochemical oxygen sensor is more suitable in measuring the cellular respiration level because it responds to a sudden change in the oxygen concentration more rapidly than the optical oxygen
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sensor does [11]. Tengberg et al. and Wu et al. reported that 90% response time of the optical oxygen sensor and the electrochemical oxygen sensor were approximately 10 s and 6.8 s, respectively [16,17]. Also, the electrochemical oxygen sensor is known to show a higher sensitivity than the optical oxygen sensor [11]. However Clark and Whalen first invented the different electrochemical oxygen sensors, known as a Clark-type sensor and Whalen-type sensor [13,18,19], Clark-type sensor has been widely used to measure the dissolved oxygen concentrations because the Whalen-type senor has a complicated structure to fabricate, very low response current (pA range) and resulting in limited applications. The Clark-type sensors have been used to determine the biochemical oxygen demand (BOD) in the environmental field as well as the pO2 in a blood vessel in the medical field [20–22]. Currently, the Clark-type sensor is employed by commercial measurement systems, such as the 782 Oxygen meter and the Oxygraph-2k [14,15]. These two systems are widely used because they can make relatively precise measurements and carry out data analysis easily. However, since they are expensive and can only measure the oxygen levels of one or two samples at a time, researchers have desired inexpensive and precise sensors that can take simultaneous measurements of more than two samples. For the fabrication of inexpensive and precise sensors or systems, micromachining technology was used because it offers precise control of volume, excellent uniformity, and reproducibility [23]. For the past two decades, many researchers have developed several types of the Clark-type sensors, which are fabricated as the pencil-type, the needle-type, and the plane-type, by using microfabrication technology. These sensors can measure the pO2 in a blood vessel, the oxygen respiration level of cells, and the dissolved oxygen level in water [17,24–27]. This paper presents a microfabricated reservoir-type oxygen sensor, which can accurately measure the solubilized oxygen concentration in real time, in order to measure the cellular OCR in a small volume of solution containing cells. The sensing principle, fabrication procedure, and characteristics of the microfabricated reservoir-type oxygen sensor were first presented. Then, real-time OCRs of the harvested L6 cells in 200 l were measured with the fabricated sensors and their results were compared with the result obtained from a commercially available Oxygraph-2k in 2 ml, in order to confirm their compatibility. 2. Design of experiments The fabricated reservoir-type oxygen sensor consists of three main parts: the electrodes with an electrolyte, which are used for the electrochemical detection, a membrane for oxygen permeation, and a reservoir for storing a sample solution. This sensor measures the reduction current of the dissolved oxygen, which permeates through the membrane from the solution containing cells to the inner electrolyte, at the WE (working electrode) as shown in Fig. 1. When a negative potential is applied to the WE relative to the CE (counter electrode), the dissolved oxygen is reduced at the WE surface and an oxidation reaction against the WE occurs at the CE according to the following Eqs. (1) and (2) [28,29]: WE : CE :
−
O2 + 2H2 O + 4e → 4OH −
4OH → O2 + 2H2 O + 4e
−
−
(1) (2)
The reduction current value of the dissolved oxygen can be related to the partial concentration of oxygen, C, as follows [29]:
I(t) = nFAPm
∂C ∂x
(3) x=0
Fig. 1. The schematic diagram for the reaction mechanism of a general Clark-type sensor.
The derivative term of (∂C/∂x)x=0 can be obtained by the differentiation of Eq. (4) and rearranged to Eq. (5) [29]:
C x = + C0 dm ∞
n=0
nFAPm I(t) = dm C0
2 n
(−12 ) sin
∞
1+2
nx dm
2
(−1) exp
n=1
exp
−n2 2 Dm t
−n2 2 Dm t 2 dm
2 dm
(4)
(5)
where n is the number of electrons, F is Faraday’s constant, A is the surface area of the working electrode, Pm is the permeability of the membrane, dm is the membrane thickness, Dm is the oxygen diffusivity of the membrane, and C0 is the oxygen concentration of the bulk solution. Therefore, the reduction current value of the dissolved oxygen can be determined with the factors of the exponential function. The sensitivity and response time of the fabricated reservoirtype oxygen sensor is related to the oxygen transport rate through the membrane and the oxygen reaction rate at the WE surface as shown in Eq. (5). Therefore, an experiment was carried out to determine the oxygen transport rate through the membrane and the oxygen reaction rate at the WE surface. When the oxygen permeability of the membrane is higher and the WE surface area is large enough for oxygen reduction, the reduction current of the oxygen will be higher because of the higher oxygen concentration gradient at the WE surface (Fig. 2a). On the other hand, when the WE surface area is small enough and the oxygen permeability of the membrane is higher, the reduction current of oxygen will be smaller because of the smaller oxygen concentration gradient at the WE surface (Fig. 2b). Since the oxygen concentration gradient at the WE surface varied with both the oxygen transport rate and the oxygen reaction rate, it can affect both the sensitivity and the response time. 3. Experimental procedure 3.1. Materials and apparatus A 500 m thick 4 7740 glass wafer (JMC Glass, manufactured in Korea) was used as the substrate for realizing the reservoirtype oxygen sensor. Polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) and silicone adhesive (3145 silicone adhesive, Dow Corning) were used to form both a reservoir and a membrane for the oxygen sensor structure. Gas-permeable polypropylene (PP) and fluorinated ethylene propylene (FEP) polymer membranes are commercially available from IWOO Scientific Corporation in Korea. A potentiostat (PC4/750, Gamry Instruments, United States) and a computer installed with the software Framework ver. 5.1 (Gamry
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cation since they are of electronic grade. Potassium chloride (KCl), Tris–HCl, and sodium sulfite (Na2 SO3 ) were purchased from Sigma–Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin (PS) and the other materials required for cell culturing were purchased from Gibco BRL, Life Technologies. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) was purchased from Sigma Co. 3.3. Cell culture and OCR assays L6 rat skeletal muscle cells were cultured in 100 mm petridishes in a complete growth medium (DMEM) supplemented with 10% FBS + 100 g/ml PS in 5% CO2 incubator at 37 ◦ C. The cells were harvested by 0.25% trypsin–EDTA, washed with DMEM by centrifugation at 1000 rpm for 5 min at 24 ◦ C, and re-suspended in DMEM media. The viable cells were counted by using hemocytometer after a Trypan blue exclusion staining. Endogenous OCR was measured as described before [3] with some modifications. Briefly, the isolated L6 cells (1 × 106 cells) in the 100 l DMEM were transferred to the chambers of an Oxygraph-2k apparatus (chamber size: 2 ml) or the fabricated oxygen sensor (chamber size: 200 l) containing 1.9 ml or 0.1 ml DMEM, respectively. The coupled and uncoupled OCRs were measured before and after adding FCCP, an uncoupler, at the final concentration of 25 M. 3.4. Fabrication of reservoir-type oxygen sensor
Fig. 2. The schematic of a one-dimension model of the oxygen flux to the WE; (a) (1) high gas permeability and (2) low gas permeability and (b) (1) a small WE surface area, (2) a large WE surface area.
Instruments) were used to measure the electrochemical redox current of the oxygen sensor. An Oxygraph-2k and a computer installed with software Oroboros Datlab (Oroboros Instruments in Austria) were used as a reference commercial oxygen sensor in order to compare the results of the fabricated oxygen sensor to that of a commercial oxygen sensor. 3.2. Chemicals All the chemicals used in the sensor fabrication process are commercially available and they were used without further purifi-
The designed reservoir-type oxygen sensor has three layers and a substrate area of 1.2 cm × 2 cm. The first layer, where the potential is applied and the current is measured, consists of the WE, the CE, and the RE (reference electrode) on a glass substrate. The second layer is the chamber for the inner electrolyte and for the passivation of the electrode leads. The inner electrolyte chamber is divided into two parts, and microchannels between them are designed in such a way that the inner electrolyte can move between the two parts. The third layer is used to store the sample solution. The designed oxygen sensor was fabricated on a glass substrate. After cleaning the glass wafer in piranha solution (H2 SO4 :H2 O2 = 4:1) for 5 min, Ti/Au (500 Å/2500 Å) was deposited and patterned for the WE and the CE. Ti/Ag (500 Å/2500 Å) was deposited and patterned on the Ti/Au patterned substrate for the RE. Then, SU-8 2100 was patterned to make the inner electrolyte chamber and for the passivation of the electrode leads. The RE, Ag/AgCl, was formed electrochemically by using a potentiostat in 0.1 M KCl/Tris–HCl. For creating AgCl on the Ag surface, a current of 50 nA was applied to the Ag electrode for 3 h [30,31]. The PDMS reservoir was made by molding, and an oxygen-
Fig. 3. Photographs of the patterned electrode substrate and the fabricated reservoir-type oxygen sensor.
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permeable membrane was either purchased or made by spin coating. The membrane was bonded onto the bottom of the PDMS reservoir opening. The patterned electrode substrate was bonded to the PDMS reservoir by using a silicone adhesive. Fig. 3 shows the photographs of the fabricated reservoir-type oxygen sensor. 4. Results and discussions 4.1. Oxygen transport rate through the membrane In the oxygen sensor, the most important property of the membrane is its oxygen permeability because it is closely related to the oxygen transport rate and the oxygen concentration gradient. In the oxygen transport rate experiment, PP, FEP, and PDMS were employed as the polymer membranes of the fabricated reservoirtype oxygen sensor with the WE area of 25,000 m2 . The oxygen permeability of PP, FEP, and PDMS are 1.6, 5.9, and 600 Barrer, respectively [32,33]. Fig. 4a shows the results of the reduction currents of the fabricated oxygen sensors with PP, FEP, and PDMS membranes when the oxygen sensors were applied at −0.8 V vs. Ag/AgCl by the amperometry method as the sensors were changed from the full-oxygen state (air-saturated) to the zero-oxygen state (0.1 M Na2 SO3 ). O2 concentration at air-saturated pure water at
37 ◦ C is reported to be 207.3 mol/l, or approximately 0.2 M [34]. Therefore, the injected 0.1 M Na2 SO3 200 l solution into the reservoir of the fabricated oxygen sensor should be enough to remove all the oxygen in the inner electrolyte and to change the sensor state from the full-oxygen state to the zero-oxygen state. When the membrane with high oxygen permeability was employed by the sensor, the fabricated oxygen sensor showed a fast response time due to the high oxygen concentration gradient. The 90% response time of an oxygen sensor is defined as the time difference between the 100% oxygen level electrical current and the 10% oxygen level electrical current when the oxygen level changes from the fulloxygen state to the zero-oxygen state. The 90% response times of the oxygen sensors with the PDMS, the FEP, and the PP membrane were 4.9 ± 0.1 s, 41.4 ± 4.7 s, and 155 ± 22.9 s. The oxygen sensor with the PDMS membrane showed the shortest response time. The sensitivities of the fabricated oxygen sensors with the PDMS, the FEP, and the PP membrane were 2.84 A/cm2 M, 1.82 A/cm2 M, and 1.29 A/cm2 M, respectively. 4.2. Oxygen reaction rate at the WE surface If the oxygen transport rate through the membrane is high enough, then the sensitivity of the sensor will change according to the WE surface areas. In order to examine the effect of the oxygen reaction rate at the WE surface, the WE surface area of the fabricated reservoir-type oxygen sensor was varied to be 5000 m2 , 10,000 m2 , 15,000 m2 , 20,000 m2 , and 25,000 m2 . At −0.8 V vs. Ag/AgCl by the amperometry, the reduction current was measured as the sensor was changed from the full-oxygen state to the zero-oxygen state. Fig. 4b shows the response times results of the fabricated oxygen sensors with different WE surface areas. For the sensor with the largest WE surface area, the response time was the shortest as shown in Fig. 4b due to the fastest oxygen reaction rate at the WE surface. Based on the response time of approximately 5 s for the WE surface areas at 15,000–25,000 m2 , it can be concluded that these larger WE surface areas are the most effective for measuring the cellular oxygen consumption rate. The largest WE area gave the fastest response time of 4.9 ± 0.1 s. The response time of the fabricated oxygen sensor is compared with the previously reported oxygen sensor based on the utilization of various materials as the membrane and the WE in Table 1. The results obtained in the present study are summarized in Table 1 along with those reported in the literature. Table 1 indicates the WE surface area and the membrane which is related to the 90% response time. It was confirmed that the fabricated Clark-type sensor exhibited the fastest response time except Whalen-type sensor. 4.3. Repeatability and lifetime
Fig. 4. The output current results of the fabricated reservoir-type oxygen sensor (n = 5); (a) PP, FEP, and PDMS membrane at −0.8 V vs. Ag/AgCl. When the sensor changes from the full-oxygen state to the zero-oxygen state, 0.1 M Na2 SO3 solution was injected in the reservoir of the sensor. (b) Various WE surface areas during change from the full-oxygen state to the zero-oxygen state. PDMS membrane was used, and −0.8 V vs. Ag/AgCl was applied.
A repeatability test was performed by repeating the injection and then the removal of the Na2 SO3 solution in the PDMS reservoir periodically for 1 h. The reduction current was measured at −0.8 V vs. Ag/AgCl. Fig. 5 shows the reduction current results with the periodic injection and then suction of the Na2 SO3 solution seven times from the reservoir of the oxygen sensor with the PDMS membrane. Without the Na2 SO3 solution in the PDMS reservoir, the mean and the standard deviation of the measured current were 154.05 nA and 1.87 nA, respectively. With the Na2 SO3 solution in the reservoir, the mean and the standard deviation of the measured current were 2.77 nA and 1.0 nA, respectively. Although the reduction current in each state changed slightly, it was considered that it may not affect the results owing to the small standard deviation in each state and the extremely high current difference between the full-oxygen state and the zero-oxygen state. The measured current response showed a good repeatability for 1 h, and it was maintained at the same level.
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Table 1 Comparison of the 90% response time of the reported oxygen sensors based on the different materials. W.E. material
W.E. area
Pt Pt Pt Ag Pt Pt Au Au
<3.14 m 12.56 mm2 1520 m2 0.4 mm2 2.83 × 10−5 cm2 400 m2 – 25,000 m2 2
Membrane/thickness
90% response time
References
Cellulose acetate/3 m FEP/25 m Polypropylene FEP/12 m RTV silicone PDMS/15 m – PDMS/20 m
0.95 s 25 s 18 s 30 s <1 min 6.8 s 20 s <5 s
[13] [14] [15] [24] [25] [17] [36,37] Current work
shows the calibration curve of the fabricated oxygen sensors equipped with the PDMS membrane. The curve shows a good linearity with the correlation coefficient of 0.998. 4.5. Measurement of oxygen consumption rate
Fig. 5. The repeatability results of the fabricated reservoir-type oxygen sensor with the PDMS membrane and a 25,000 m2 WE; measuring the reduction currents at both a full-oxygen state and a zero-oxygen state seven times sequentially.
Fig. 6 shows the result of the lifetime test. The fabricated oxygen sensor continuously operated for 22 h at −0.8 V in the air-filled state. The oxygen reduction current value was stable at about 130 nA from 0 h to 10.6 h. After 10.6 h, the reduction current abruptly changed over 20%. Although the lifetime was about 10 h, it was enough time to measure the oxygen consumption rate. 4.4. Linearity Solutions of various oxygen concentrations were made by mixing deionized water and Na2 SO3 at different levels and the reduction currents from those solutions were measured. Fig 7
Fig. 6. The lifetime result of the fabricated reservoir-type oxygen sensor with the PDMS membrane and a 25,000 m2 WE.
To measure the cellular respiration rate, we injected the harvested L6 cells (1 × 106 /100 l DMEM) into the reservoirs of the fabricated oxygen sensor or Oxygraph-2k containing 100 l or 1.9 ml DMEM at 37 ◦ C, respectively. We converted the current changes with time into the dissolved oxygen concentrations by using linear regression curve shown in Fig. 7. Fig. 8 shows the time-dependent changes of the oxygen concentrations dissolved in DMEM containing cells before and after the addition of 0.5 l and 5 l of 10 mM FCCP uncoupler in the reservoirs of the fabricated oxygen sensor and Oxygraph-2k, respectively. Since the cells were stored at 4 ◦ C before injection to maintain mitochondrial activity, we observed a membrane permeability shift for 100 s, reflecting a temperature adjustment period to 37 ◦ C. The shift was not observed in Oxygraph-2k since its chamber volume was 2 ml while that of the fabricated sensor was 200 l. However, the temperature change did not affect the decrease of oxygen in the closed chamber. The slope of oxygen concentration with time is defined as the oxygen consumption rate. Coupled and uncoupled OCRs are the slopes before and after adding FCCP, respectively. If the cells are intact and not damaged, the uncoupled OCR is greater than the coupled OCR because the FCCP uncouples the electron transport chain from the ATP synthase, resulting in the maximum level of electron flow to the oxygen adaptor, which means the maximum cellular respiration [35]. The measured coupled and uncoupled OCRs of our sensor were approximately 10-fold because the chamber volume of our sensor was 1/10 of the Oxygraph-2k (Table 2). However,
Fig. 7. The calibration curve for the fabricated reservoir-type oxygen sensor (n = 5) with the PDMS membrane at 25,000 m2 .
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The electrodes and the passivation layer of the reservoir-type oxygen sensor were easily fabricated using MEMS technology, and a PDMS reservoir was made by molding. When a 25,000 m2 WE and an 20 m PDMS membrane were applied, the fabricated oxygen sensor showed a sensitivity of 2.84 A/cm2 M and a 90% response time of 4.9 s. The fastest response time has been achieved for the oxygen sensor compared with the previously reported. With the periodic injection and removal of the Na2 SO3 solution in the reservoir, the current response showed a good repeatability for 1 h. Also, the fabricated oxygen sensor showed good lifetime for 10 h when the fabricated oxygen sensor was in the full-oxygen state. The linearity was excellent with the correlation coefficient of 0.998. The fabricated oxygen sensor was evaluated by taking the ratio of the uncoupled OCR to the coupled OCR within 10 min. The result (ratio = 3.70 ± 0.26) of the fabricated oxygen sensor was similar to that (ratio = 3.68 ± 0.30) of the commercial oxygen measurement system. The fabricated reservoir-type oxygen sensor showed the result of the cellular OCR with a high speed with a great reliability. The reservoir-type oxygen sensor later could become a part of an array-type oxygen sensor for multiple simultaneous measurements. Acknowledgements This research was supported by Seoul R&BD Program (No. 10920) and National Research Foundation (NRF) through a grant provided by Ministry of Education, Science and Technology (MEST) (Nos. R01-2006-000-11371-0, K20601000002-09E010000200, and 20090063278). References
Fig. 8. The measurement results of the oxygen respiration of cells containing the solution by (a) the fabricated reservoir-type oxygen sensor and (b) Oxygraph-2k. The oxygen concentration was monitored before and after addition of FCCP solution (2.5 M) in the reservoir of the fabricated reservoir-type oxygen sensor and Oxygraph-2k, respectively. (1) and (2) are the regions utilized for slope calculations for the coupled OCRs and the FCCP-uncoupled OCRs region, respectively.
both sensors showed nearly the same maximum respiration ratio which is the ratio of the uncoupled OCR to the coupled OCR. Table 2 summarized the characteristics of two oxygen sensors. It should be noted that we achieved the measurements of OCRs within 10 min when using the fabricated oxygen sensor while it took 40 min when using the Oxygraph-2k. The fabricated oxygen sensors may allow us to assay OCR at a high speed with a great reliability and sensitivity. Also, this reservoir-type oxygen sensor can be a part of an oxygen sensor array for multiple simultaneous measurements. 5. Conclusions A miniaturized reservoir-type oxygen sensor which can utilize a solution containing cells and measure the oxygen respiration level of that solution accurately was successfully demonstrated. Table 2 Comparison between the fabricated oxygen sensor and Oxygraph-2k.
Chamber volume (ml) Coupled OCR (pmol/s/ml) Uncoupled OCR (pmol/s/ml) Maximum respiration ratio (uncoulped/coupled) Duration for assay (min)
Fabricated O2 sensor
Oxygraph-2k
0.2 283.3 ± 27.7 1046.7 ± 186.5 3.69
2 21.3 ± 0.4 78.5 ± 4.1 3.68
10
40
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Biographies Jungil Park is currently an integrated MS and PhD student in the School of Electrical Engineering at Korea University. He received his bachelor’s degree in Division of Electronics and Information Engineering in 2004 at Korea University. Youngmi Kim Pak received her BS and MS degrees in Pharmacy in 1983 and 1985 at Seoul National University, Korea. In 1991, she received her PhD degree in Biochemistry at Purdue University. After 5 year of post-doctoral training in Stanford University School of Medicine, she returned to Korea. She was a section leader of Division of Metabolic Disease, Korean NIH during 1996–2002 and a professor in School of Medicine, University of Ulsan during 2002–2007. Currently she is a professor of Kyung Hee University College of Medicine. Her research interests are mitochondrial pathogenesis of human diseases and biosensor. James Jungho Pak received his BS, MS and PhD degrees in Electrical Engineering respectively in 1985, 1988 and 1992 at Purdue University. He had worked in Intel Corporation in Santa Clara, CA, USA from 1992 to 1995 as a senior device physicist. Since 1995, he has been a professor in the School of Electrical Engineering at Korea University. His research interests include the Microsystems including bio-MEMS, biosensor, applications of polymer in microsensors and microactuators, flexible electronics and novel semiconductor devices and processing.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A microfabricated reservoir-type oxygen sensor for measuring the real-time cellular oxygen consumption rate at various conditions Jungil Park a , Youngmi Kim Pak b,c,∗∗ , James Jungho Pak a,∗ a b c
School of Electrical Engineering, Korea University, Seoul 136-713, Republic of Korea Department of Nanopharmaceutical and Life Science, Age-Related and Brain Disease Research Center, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Physiology, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 2 December 2009 Received in revised form 17 March 2010 Accepted 23 March 2010 Available online 30 March 2010 Keywords: Electrochemistry Reservoir-type Oxygen sensor Cellular respiration Oxygen consumption rate
a b s t r a c t This paper presents a microfabricated reservoir-type oxygen sensor, which can accurately measure the solubilized oxygen concentration in real time, in order to measure the cellular oxygen consumption rate (OCR) in a solution containing cells. The fabricated oxygen sensor is composed of three-parts: electrochemical sensing electrodes, an oxygen-permeable membrane, and a reservoir for storing the solution. The oxygen transport rate through the membrane and the oxygen reaction rate at the working electrode (WE) surface are the two dominant parameters in determining the sensitivity of the oxygen sensor. The fabricated sensor showed a sensitivity of 2.84 A/cm2 M and a 90% response time of 4.9 s in an average of 5 sensors when a 25,000 m2 WE and a 20 m polydimethylsiloxane membrane were used. This is the first report in which the fastest response time has been achieved for the oxygen sensor. The fabricated sensor showed the repeatability with 154.05 ± 1.87 nA at the full-oxygen state and 2.77 ± 1.0 nA at the zero-oxygen state. The fabricated sensor was used to measure the uncoupled OCR of the L6 cells, and its result of 3.69 ± 0.30 was almost identical to the result of 3.70 ± 0.26 obtained from a commercial system. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Mitochondrial dysfunction has been generally accepted as one of the causes of age-related degenerative diseases including metabolic syndrome, insulin resistance, and diabetes [1–3]. The tissues or cells affected with the diseases exhibit a decrease in mitochondrial functions which may be monitored by the mitochondrial membrane potential, mitochondrial DNA contents, intracellular ATP contents, mitochondrial gene expressions and the cellular oxygen consumption rate (OCR) [4]. Among those parameters, the oxygen consumption rate, also known as the respiration rate as related to the cells or the isolated mitochondria, is an important physiologic profile of mitochondrial respiratory function [1,2,5,6]. Large-scale screening of cell-based mitochondrial activities using a 96-well or a 384-well has been applied to investigate problems in mitochondrial biology, drug toxicity, and therapeutics [4]. In this screening compendium, however, the OCR was not included solely because the current OCR measurement instruments do not allow for a high-throughput analysis and require a large number
∗ Corresponding author. Tel.: +82 2 3290 3238; fax: +82 2 921 0544. ∗∗ Co-corresponding author. Tel.: +82 2 969 0958; fax: +82 2 969 6343. E-mail addresses: [email protected] (J. Park), [email protected] (Y.K. Pak), [email protected] (J.J. Pak). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.03.069
of cells in a time-consuming process. Therefore, it was valuable to develop a small oxygen biosensor to measure the OCR precisely and rapidly using a small number of cells in a small volume of solution. Recently, Seahorse Bioscience has produced XF24 Analyzer which uses the small volume of solution and can measure over 24 samples, simultaneously [7,8]. The cellular OCR is determined by measuring the oxygen concentration dissolved in a solution containing cells with time. This is accomplished by two different techniques, an optical or an electrochemical measurement method [7,9–15]. Both optical and electrochemical oxygen sensors have the advantages of continuous measurement, simple operation, and use of a small volume of solution. XF 24 Analyzer using the optical method and 782 Oxygen meter (Strathkelvin Instruments, Germany) using the electrochemical method are known to use about 10 l and 200 l solution in the experiment of measuring the cellular consumption rate, respectively [7,15]. While XF24 Analyzer is the opened system because it is not completely isolated from the outside environment, 782 Oxygen meter and Oxygraph-2k (Oroboros Instruments, Austria) have the closed systems that do not allow ambient oxygen to access the measurement solution. The closed system can provide more accurate output results because the environmental effect would be lower [8]. The electrochemical oxygen sensor is more suitable in measuring the cellular respiration level because it responds to a sudden change in the oxygen concentration more rapidly than the optical oxygen
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sensor does [11]. Tengberg et al. and Wu et al. reported that 90% response time of the optical oxygen sensor and the electrochemical oxygen sensor were approximately 10 s and 6.8 s, respectively [16,17]. Also, the electrochemical oxygen sensor is known to show a higher sensitivity than the optical oxygen sensor [11]. However Clark and Whalen first invented the different electrochemical oxygen sensors, known as a Clark-type sensor and Whalen-type sensor [13,18,19], Clark-type sensor has been widely used to measure the dissolved oxygen concentrations because the Whalen-type senor has a complicated structure to fabricate, very low response current (pA range) and resulting in limited applications. The Clark-type sensors have been used to determine the biochemical oxygen demand (BOD) in the environmental field as well as the pO2 in a blood vessel in the medical field [20–22]. Currently, the Clark-type sensor is employed by commercial measurement systems, such as the 782 Oxygen meter and the Oxygraph-2k [14,15]. These two systems are widely used because they can make relatively precise measurements and carry out data analysis easily. However, since they are expensive and can only measure the oxygen levels of one or two samples at a time, researchers have desired inexpensive and precise sensors that can take simultaneous measurements of more than two samples. For the fabrication of inexpensive and precise sensors or systems, micromachining technology was used because it offers precise control of volume, excellent uniformity, and reproducibility [23]. For the past two decades, many researchers have developed several types of the Clark-type sensors, which are fabricated as the pencil-type, the needle-type, and the plane-type, by using microfabrication technology. These sensors can measure the pO2 in a blood vessel, the oxygen respiration level of cells, and the dissolved oxygen level in water [17,24–27]. This paper presents a microfabricated reservoir-type oxygen sensor, which can accurately measure the solubilized oxygen concentration in real time, in order to measure the cellular OCR in a small volume of solution containing cells. The sensing principle, fabrication procedure, and characteristics of the microfabricated reservoir-type oxygen sensor were first presented. Then, real-time OCRs of the harvested L6 cells in 200 l were measured with the fabricated sensors and their results were compared with the result obtained from a commercially available Oxygraph-2k in 2 ml, in order to confirm their compatibility. 2. Design of experiments The fabricated reservoir-type oxygen sensor consists of three main parts: the electrodes with an electrolyte, which are used for the electrochemical detection, a membrane for oxygen permeation, and a reservoir for storing a sample solution. This sensor measures the reduction current of the dissolved oxygen, which permeates through the membrane from the solution containing cells to the inner electrolyte, at the WE (working electrode) as shown in Fig. 1. When a negative potential is applied to the WE relative to the CE (counter electrode), the dissolved oxygen is reduced at the WE surface and an oxidation reaction against the WE occurs at the CE according to the following Eqs. (1) and (2) [28,29]: WE : CE :
−
O2 + 2H2 O + 4e → 4OH −
4OH → O2 + 2H2 O + 4e
−
−
(1) (2)
The reduction current value of the dissolved oxygen can be related to the partial concentration of oxygen, C, as follows [29]:
I(t) = nFAPm
∂C ∂x
(3) x=0
Fig. 1. The schematic diagram for the reaction mechanism of a general Clark-type sensor.
The derivative term of (∂C/∂x)x=0 can be obtained by the differentiation of Eq. (4) and rearranged to Eq. (5) [29]:
C x = + C0 dm ∞
n=0
nFAPm I(t) = dm C0
2 n
(−12 ) sin
∞
1+2
nx dm
2
(−1) exp
n=1
exp
−n2 2 Dm t
−n2 2 Dm t 2 dm
2 dm
(4)
(5)
where n is the number of electrons, F is Faraday’s constant, A is the surface area of the working electrode, Pm is the permeability of the membrane, dm is the membrane thickness, Dm is the oxygen diffusivity of the membrane, and C0 is the oxygen concentration of the bulk solution. Therefore, the reduction current value of the dissolved oxygen can be determined with the factors of the exponential function. The sensitivity and response time of the fabricated reservoirtype oxygen sensor is related to the oxygen transport rate through the membrane and the oxygen reaction rate at the WE surface as shown in Eq. (5). Therefore, an experiment was carried out to determine the oxygen transport rate through the membrane and the oxygen reaction rate at the WE surface. When the oxygen permeability of the membrane is higher and the WE surface area is large enough for oxygen reduction, the reduction current of the oxygen will be higher because of the higher oxygen concentration gradient at the WE surface (Fig. 2a). On the other hand, when the WE surface area is small enough and the oxygen permeability of the membrane is higher, the reduction current of oxygen will be smaller because of the smaller oxygen concentration gradient at the WE surface (Fig. 2b). Since the oxygen concentration gradient at the WE surface varied with both the oxygen transport rate and the oxygen reaction rate, it can affect both the sensitivity and the response time. 3. Experimental procedure 3.1. Materials and apparatus A 500 m thick 4 7740 glass wafer (JMC Glass, manufactured in Korea) was used as the substrate for realizing the reservoirtype oxygen sensor. Polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) and silicone adhesive (3145 silicone adhesive, Dow Corning) were used to form both a reservoir and a membrane for the oxygen sensor structure. Gas-permeable polypropylene (PP) and fluorinated ethylene propylene (FEP) polymer membranes are commercially available from IWOO Scientific Corporation in Korea. A potentiostat (PC4/750, Gamry Instruments, United States) and a computer installed with the software Framework ver. 5.1 (Gamry
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cation since they are of electronic grade. Potassium chloride (KCl), Tris–HCl, and sodium sulfite (Na2 SO3 ) were purchased from Sigma–Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin (PS) and the other materials required for cell culturing were purchased from Gibco BRL, Life Technologies. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) was purchased from Sigma Co. 3.3. Cell culture and OCR assays L6 rat skeletal muscle cells were cultured in 100 mm petridishes in a complete growth medium (DMEM) supplemented with 10% FBS + 100 g/ml PS in 5% CO2 incubator at 37 ◦ C. The cells were harvested by 0.25% trypsin–EDTA, washed with DMEM by centrifugation at 1000 rpm for 5 min at 24 ◦ C, and re-suspended in DMEM media. The viable cells were counted by using hemocytometer after a Trypan blue exclusion staining. Endogenous OCR was measured as described before [3] with some modifications. Briefly, the isolated L6 cells (1 × 106 cells) in the 100 l DMEM were transferred to the chambers of an Oxygraph-2k apparatus (chamber size: 2 ml) or the fabricated oxygen sensor (chamber size: 200 l) containing 1.9 ml or 0.1 ml DMEM, respectively. The coupled and uncoupled OCRs were measured before and after adding FCCP, an uncoupler, at the final concentration of 25 M. 3.4. Fabrication of reservoir-type oxygen sensor
Fig. 2. The schematic of a one-dimension model of the oxygen flux to the WE; (a) (1) high gas permeability and (2) low gas permeability and (b) (1) a small WE surface area, (2) a large WE surface area.
Instruments) were used to measure the electrochemical redox current of the oxygen sensor. An Oxygraph-2k and a computer installed with software Oroboros Datlab (Oroboros Instruments in Austria) were used as a reference commercial oxygen sensor in order to compare the results of the fabricated oxygen sensor to that of a commercial oxygen sensor. 3.2. Chemicals All the chemicals used in the sensor fabrication process are commercially available and they were used without further purifi-
The designed reservoir-type oxygen sensor has three layers and a substrate area of 1.2 cm × 2 cm. The first layer, where the potential is applied and the current is measured, consists of the WE, the CE, and the RE (reference electrode) on a glass substrate. The second layer is the chamber for the inner electrolyte and for the passivation of the electrode leads. The inner electrolyte chamber is divided into two parts, and microchannels between them are designed in such a way that the inner electrolyte can move between the two parts. The third layer is used to store the sample solution. The designed oxygen sensor was fabricated on a glass substrate. After cleaning the glass wafer in piranha solution (H2 SO4 :H2 O2 = 4:1) for 5 min, Ti/Au (500 Å/2500 Å) was deposited and patterned for the WE and the CE. Ti/Ag (500 Å/2500 Å) was deposited and patterned on the Ti/Au patterned substrate for the RE. Then, SU-8 2100 was patterned to make the inner electrolyte chamber and for the passivation of the electrode leads. The RE, Ag/AgCl, was formed electrochemically by using a potentiostat in 0.1 M KCl/Tris–HCl. For creating AgCl on the Ag surface, a current of 50 nA was applied to the Ag electrode for 3 h [30,31]. The PDMS reservoir was made by molding, and an oxygen-
Fig. 3. Photographs of the patterned electrode substrate and the fabricated reservoir-type oxygen sensor.
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permeable membrane was either purchased or made by spin coating. The membrane was bonded onto the bottom of the PDMS reservoir opening. The patterned electrode substrate was bonded to the PDMS reservoir by using a silicone adhesive. Fig. 3 shows the photographs of the fabricated reservoir-type oxygen sensor. 4. Results and discussions 4.1. Oxygen transport rate through the membrane In the oxygen sensor, the most important property of the membrane is its oxygen permeability because it is closely related to the oxygen transport rate and the oxygen concentration gradient. In the oxygen transport rate experiment, PP, FEP, and PDMS were employed as the polymer membranes of the fabricated reservoirtype oxygen sensor with the WE area of 25,000 m2 . The oxygen permeability of PP, FEP, and PDMS are 1.6, 5.9, and 600 Barrer, respectively [32,33]. Fig. 4a shows the results of the reduction currents of the fabricated oxygen sensors with PP, FEP, and PDMS membranes when the oxygen sensors were applied at −0.8 V vs. Ag/AgCl by the amperometry method as the sensors were changed from the full-oxygen state (air-saturated) to the zero-oxygen state (0.1 M Na2 SO3 ). O2 concentration at air-saturated pure water at
37 ◦ C is reported to be 207.3 mol/l, or approximately 0.2 M [34]. Therefore, the injected 0.1 M Na2 SO3 200 l solution into the reservoir of the fabricated oxygen sensor should be enough to remove all the oxygen in the inner electrolyte and to change the sensor state from the full-oxygen state to the zero-oxygen state. When the membrane with high oxygen permeability was employed by the sensor, the fabricated oxygen sensor showed a fast response time due to the high oxygen concentration gradient. The 90% response time of an oxygen sensor is defined as the time difference between the 100% oxygen level electrical current and the 10% oxygen level electrical current when the oxygen level changes from the fulloxygen state to the zero-oxygen state. The 90% response times of the oxygen sensors with the PDMS, the FEP, and the PP membrane were 4.9 ± 0.1 s, 41.4 ± 4.7 s, and 155 ± 22.9 s. The oxygen sensor with the PDMS membrane showed the shortest response time. The sensitivities of the fabricated oxygen sensors with the PDMS, the FEP, and the PP membrane were 2.84 A/cm2 M, 1.82 A/cm2 M, and 1.29 A/cm2 M, respectively. 4.2. Oxygen reaction rate at the WE surface If the oxygen transport rate through the membrane is high enough, then the sensitivity of the sensor will change according to the WE surface areas. In order to examine the effect of the oxygen reaction rate at the WE surface, the WE surface area of the fabricated reservoir-type oxygen sensor was varied to be 5000 m2 , 10,000 m2 , 15,000 m2 , 20,000 m2 , and 25,000 m2 . At −0.8 V vs. Ag/AgCl by the amperometry, the reduction current was measured as the sensor was changed from the full-oxygen state to the zero-oxygen state. Fig. 4b shows the response times results of the fabricated oxygen sensors with different WE surface areas. For the sensor with the largest WE surface area, the response time was the shortest as shown in Fig. 4b due to the fastest oxygen reaction rate at the WE surface. Based on the response time of approximately 5 s for the WE surface areas at 15,000–25,000 m2 , it can be concluded that these larger WE surface areas are the most effective for measuring the cellular oxygen consumption rate. The largest WE area gave the fastest response time of 4.9 ± 0.1 s. The response time of the fabricated oxygen sensor is compared with the previously reported oxygen sensor based on the utilization of various materials as the membrane and the WE in Table 1. The results obtained in the present study are summarized in Table 1 along with those reported in the literature. Table 1 indicates the WE surface area and the membrane which is related to the 90% response time. It was confirmed that the fabricated Clark-type sensor exhibited the fastest response time except Whalen-type sensor. 4.3. Repeatability and lifetime
Fig. 4. The output current results of the fabricated reservoir-type oxygen sensor (n = 5); (a) PP, FEP, and PDMS membrane at −0.8 V vs. Ag/AgCl. When the sensor changes from the full-oxygen state to the zero-oxygen state, 0.1 M Na2 SO3 solution was injected in the reservoir of the sensor. (b) Various WE surface areas during change from the full-oxygen state to the zero-oxygen state. PDMS membrane was used, and −0.8 V vs. Ag/AgCl was applied.
A repeatability test was performed by repeating the injection and then the removal of the Na2 SO3 solution in the PDMS reservoir periodically for 1 h. The reduction current was measured at −0.8 V vs. Ag/AgCl. Fig. 5 shows the reduction current results with the periodic injection and then suction of the Na2 SO3 solution seven times from the reservoir of the oxygen sensor with the PDMS membrane. Without the Na2 SO3 solution in the PDMS reservoir, the mean and the standard deviation of the measured current were 154.05 nA and 1.87 nA, respectively. With the Na2 SO3 solution in the reservoir, the mean and the standard deviation of the measured current were 2.77 nA and 1.0 nA, respectively. Although the reduction current in each state changed slightly, it was considered that it may not affect the results owing to the small standard deviation in each state and the extremely high current difference between the full-oxygen state and the zero-oxygen state. The measured current response showed a good repeatability for 1 h, and it was maintained at the same level.
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Table 1 Comparison of the 90% response time of the reported oxygen sensors based on the different materials. W.E. material
W.E. area
Pt Pt Pt Ag Pt Pt Au Au
<3.14 m 12.56 mm2 1520 m2 0.4 mm2 2.83 × 10−5 cm2 400 m2 – 25,000 m2 2
Membrane/thickness
90% response time
References
Cellulose acetate/3 m FEP/25 m Polypropylene FEP/12 m RTV silicone PDMS/15 m – PDMS/20 m
0.95 s 25 s 18 s 30 s <1 min 6.8 s 20 s <5 s
[13] [14] [15] [24] [25] [17] [36,37] Current work
shows the calibration curve of the fabricated oxygen sensors equipped with the PDMS membrane. The curve shows a good linearity with the correlation coefficient of 0.998. 4.5. Measurement of oxygen consumption rate
Fig. 5. The repeatability results of the fabricated reservoir-type oxygen sensor with the PDMS membrane and a 25,000 m2 WE; measuring the reduction currents at both a full-oxygen state and a zero-oxygen state seven times sequentially.
Fig. 6 shows the result of the lifetime test. The fabricated oxygen sensor continuously operated for 22 h at −0.8 V in the air-filled state. The oxygen reduction current value was stable at about 130 nA from 0 h to 10.6 h. After 10.6 h, the reduction current abruptly changed over 20%. Although the lifetime was about 10 h, it was enough time to measure the oxygen consumption rate. 4.4. Linearity Solutions of various oxygen concentrations were made by mixing deionized water and Na2 SO3 at different levels and the reduction currents from those solutions were measured. Fig 7
Fig. 6. The lifetime result of the fabricated reservoir-type oxygen sensor with the PDMS membrane and a 25,000 m2 WE.
To measure the cellular respiration rate, we injected the harvested L6 cells (1 × 106 /100 l DMEM) into the reservoirs of the fabricated oxygen sensor or Oxygraph-2k containing 100 l or 1.9 ml DMEM at 37 ◦ C, respectively. We converted the current changes with time into the dissolved oxygen concentrations by using linear regression curve shown in Fig. 7. Fig. 8 shows the time-dependent changes of the oxygen concentrations dissolved in DMEM containing cells before and after the addition of 0.5 l and 5 l of 10 mM FCCP uncoupler in the reservoirs of the fabricated oxygen sensor and Oxygraph-2k, respectively. Since the cells were stored at 4 ◦ C before injection to maintain mitochondrial activity, we observed a membrane permeability shift for 100 s, reflecting a temperature adjustment period to 37 ◦ C. The shift was not observed in Oxygraph-2k since its chamber volume was 2 ml while that of the fabricated sensor was 200 l. However, the temperature change did not affect the decrease of oxygen in the closed chamber. The slope of oxygen concentration with time is defined as the oxygen consumption rate. Coupled and uncoupled OCRs are the slopes before and after adding FCCP, respectively. If the cells are intact and not damaged, the uncoupled OCR is greater than the coupled OCR because the FCCP uncouples the electron transport chain from the ATP synthase, resulting in the maximum level of electron flow to the oxygen adaptor, which means the maximum cellular respiration [35]. The measured coupled and uncoupled OCRs of our sensor were approximately 10-fold because the chamber volume of our sensor was 1/10 of the Oxygraph-2k (Table 2). However,
Fig. 7. The calibration curve for the fabricated reservoir-type oxygen sensor (n = 5) with the PDMS membrane at 25,000 m2 .
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The electrodes and the passivation layer of the reservoir-type oxygen sensor were easily fabricated using MEMS technology, and a PDMS reservoir was made by molding. When a 25,000 m2 WE and an 20 m PDMS membrane were applied, the fabricated oxygen sensor showed a sensitivity of 2.84 A/cm2 M and a 90% response time of 4.9 s. The fastest response time has been achieved for the oxygen sensor compared with the previously reported. With the periodic injection and removal of the Na2 SO3 solution in the reservoir, the current response showed a good repeatability for 1 h. Also, the fabricated oxygen sensor showed good lifetime for 10 h when the fabricated oxygen sensor was in the full-oxygen state. The linearity was excellent with the correlation coefficient of 0.998. The fabricated oxygen sensor was evaluated by taking the ratio of the uncoupled OCR to the coupled OCR within 10 min. The result (ratio = 3.70 ± 0.26) of the fabricated oxygen sensor was similar to that (ratio = 3.68 ± 0.30) of the commercial oxygen measurement system. The fabricated reservoir-type oxygen sensor showed the result of the cellular OCR with a high speed with a great reliability. The reservoir-type oxygen sensor later could become a part of an array-type oxygen sensor for multiple simultaneous measurements. Acknowledgements This research was supported by Seoul R&BD Program (No. 10920) and National Research Foundation (NRF) through a grant provided by Ministry of Education, Science and Technology (MEST) (Nos. R01-2006-000-11371-0, K20601000002-09E010000200, and 20090063278). References
Fig. 8. The measurement results of the oxygen respiration of cells containing the solution by (a) the fabricated reservoir-type oxygen sensor and (b) Oxygraph-2k. The oxygen concentration was monitored before and after addition of FCCP solution (2.5 M) in the reservoir of the fabricated reservoir-type oxygen sensor and Oxygraph-2k, respectively. (1) and (2) are the regions utilized for slope calculations for the coupled OCRs and the FCCP-uncoupled OCRs region, respectively.
both sensors showed nearly the same maximum respiration ratio which is the ratio of the uncoupled OCR to the coupled OCR. Table 2 summarized the characteristics of two oxygen sensors. It should be noted that we achieved the measurements of OCRs within 10 min when using the fabricated oxygen sensor while it took 40 min when using the Oxygraph-2k. The fabricated oxygen sensors may allow us to assay OCR at a high speed with a great reliability and sensitivity. Also, this reservoir-type oxygen sensor can be a part of an oxygen sensor array for multiple simultaneous measurements. 5. Conclusions A miniaturized reservoir-type oxygen sensor which can utilize a solution containing cells and measure the oxygen respiration level of that solution accurately was successfully demonstrated. Table 2 Comparison between the fabricated oxygen sensor and Oxygraph-2k.
Chamber volume (ml) Coupled OCR (pmol/s/ml) Uncoupled OCR (pmol/s/ml) Maximum respiration ratio (uncoulped/coupled) Duration for assay (min)
Fabricated O2 sensor
Oxygraph-2k
0.2 283.3 ± 27.7 1046.7 ± 186.5 3.69
2 21.3 ± 0.4 78.5 ± 4.1 3.68
10
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Biographies Jungil Park is currently an integrated MS and PhD student in the School of Electrical Engineering at Korea University. He received his bachelor’s degree in Division of Electronics and Information Engineering in 2004 at Korea University. Youngmi Kim Pak received her BS and MS degrees in Pharmacy in 1983 and 1985 at Seoul National University, Korea. In 1991, she received her PhD degree in Biochemistry at Purdue University. After 5 year of post-doctoral training in Stanford University School of Medicine, she returned to Korea. She was a section leader of Division of Metabolic Disease, Korean NIH during 1996–2002 and a professor in School of Medicine, University of Ulsan during 2002–2007. Currently she is a professor of Kyung Hee University College of Medicine. Her research interests are mitochondrial pathogenesis of human diseases and biosensor. James Jungho Pak received his BS, MS and PhD degrees in Electrical Engineering respectively in 1985, 1988 and 1992 at Purdue University. He had worked in Intel Corporation in Santa Clara, CA, USA from 1992 to 1995 as a senior device physicist. Since 1995, he has been a professor in the School of Electrical Engineering at Korea University. His research interests include the Microsystems including bio-MEMS, biosensor, applications of polymer in microsensors and microactuators, flexible electronics and novel semiconductor devices and processing.