A MINIATURIZED CLARK OXYGEN SENSOR FOR ORGAN-ON-CHIP
DEVICES
Elsbeth G.B.M. Bossink1,*, Olivier Y.F. Henry2, Maximilian A. Benz2, Loes I. Segerink1, Donald
E. Ingber2,3,4, and Mathieu Odijk1
1BIOS Lab on a Chip group, MESA+ Institute of Nanotechnology, MRA Institute for Biomedical Technology and Technical Medicine, Max Planck Center for Complex Fluid Dynamics, University of
Twente, NETHERLANDS and
2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115; 3Harvard John A. Paulson School of Engineering and Applied Sciences, Cambridge, MA 02138; 4Vascular Biology Program and Department of Surgery, Boston Children’s Hospital and Harvard
Medical School, Boston, MA 02115.
ABSTRACT
Oxygen concentration is an essential regulatory parameter in cell culture and organ-on-chip devices. Measuring variations in oxygen concentration near cells during cell culture can provide a wealth of information on cell viabil-ity, cellular activviabil-ity, cell differentiation and response to external stimuli.
Here, we present a miniaturized three-electrode Clark oxygen sensor, which is shown to be biocompatible, pro-tected from biofouling and has a 7.5s response time. This miniaturized sensor allows to measure the oxygen con-centration in a microfluidic channel of an organ-on-chip, whereby this chemical microenvironment can be moni-tored.
KEYWORDS: Clark oxygen sensor, miniaturization, oxygen concentration, organ-on-a-chip INTRODUCTION
In organ-on-chip devices, and specifically gut-on-a-chip devices, oxygen concentration is a tremendously im-portant regulatory parameter [1]. In the lumen of the human intestine, the commensal microbiome resides under anaerobic conditions, resulting in a steep oxygen gradient along the radial axis of the intestine (Figure 1). This oxygen gradient is essential to be monitored as it affects the organization, development and dynamic nature of the gut bacterial microbiome and intestinal physiology [2].
Clark-type oxygen sensors are well known oxygen sensors, based on the reduction of oxygen at the working electrode. In this work, we integrate such oxygen sensor in an existing organ-on-chip device [3]. For this, we need a miniaturized sensor, which is biocompatible, protected from biofouling and has a stable reference electrode.
Figure 1: Gut-on-a-chip oxygen gradient concept. Schematic illus-tration of the steep oxygen gradient present in the small intestine, along the radial axis from the submucosa to the lumen of the small intestine.
Figure 2: Top view of the three electrode Clark oxy-gen sensor on glass, with Pt working electrode (WE) and counter electrode (CE), and Ag/AgCl reference electrode (RE). Purple illustrates a layer of pHEMA.
EXPERIMENTAL
The Clark oxygen sensor consists of platinum (Pt) de-posited on a glass chip (Figure 2). Electrode conduction paths were covered with SU8 to insulate all platinum, ex-cept the electrodes. A silver/silver chloride (Ag/AgCl) quasi reference electrode was fabricated by electroplat-ing silver atop one platinum electrode and subsequent electrochemical chlorination. The stability of the Ag/AgCl electrode was validated by measuring the open circuit potential between the fabricated reference elec-trode and a commercially available Ag/AgCl elecelec-trode (BASi®).
A thin protective layer consisting of poly(hydroxy-ethyl methacrylate) (pHEMA) was formed by UV curing, partly atop the electrodes in order to protect the sensor from biofouling and conserve sensitivity. A method was also developed to covalently attach the pHEMA layer to the sensor by surface treatment of the chip prior to UV curing (Figure 3). Briefly, the method consisted of treat-ment with APTES and VTMS consecutively.
The oxygen sensor was tested by adding 0.1M Na2SO3 to the 0.1M KCl electrolyte. The Na2SO3
de-pletes oxygen, resulting in a 0% oxygen solution. The re-sponse curve of the sensor was determined by adding dif-ferent amounts of Na2SO3.
RESULTS AND DISCUSSION
The oxygen sensor is shown to have a stable Ag/AgCl quasi reference electrode, with a potential drop of ap-proximately 0.1 mV/hour (Figure 4). This is low, compared to a calculated potential difference of 0.6 mV caused by a temperature change of 1˚C (21˚C-22°C) [4]. The pHEMA is in direct contact with cells in the microfluidic channel above and is therefore required to be biocompatible. To test the biocompatibility, the pHEMA was exposed to a culture of Caco2 cells for 48h (Figure 5). No significant difference in cell death in the presence or absence of pHEMA was observed.
Figure 3: Surface chemistry of the chip to ensure a tight adherence of the pHEMA layer to the chip. The chip is im-mersed in 1% APTES ((3-Aminopropyl)-triethoxysilane) and hydrolyzed overnight. Subsequently, the chip is im-mersed in 1% VTMS (vinyltrimethoxysilane). After drying, a HEMA mixture is pipetted on the surface and cross-linked by UV curing.
Figure 4: Open circuit potential of the fabricated Ag/AgCl quasi reference electrode over 2 hours in blue. A potential drop of approximately 0.1 mV/hour was observed. A temper-ature change of 1 ˚C (21˚C-22°C) causes a theoretical poten-tial difference of 0.6 mV, indicated with the two red dashed horizontal lines.
Figure 5: Testing biocompatibility of pHEMA. Caco2 cells P58 after 48 hours of culture. Brightfield images: A) Con-trol, well plate, n=3. B) Microscopic glass slide with pHEMA, n=4. Fluorescent microscope images: C) Control, well plate, n=3. D) Microscopic glass slide with pHEMA, n=4. Dead cells are stained red, live cells green. Scale bars are 400 µm.
Figure 6 shows the result of the chronoamperometry measurement at -0.7 V in 0.1M KCl. The 90% response time of the oxygen sensor from 21% oxygen to 0% oxygen is approximately 7.5 seconds. The response curve of the oxygen sensor (Figure 7) has a linear fit with a correlation coefficient of R2=0.9315. Part of the uncertainty in the
correlation between current and the concentration of Na2SO3 is due to the inherent lack of control of this chemical
method of changing the oxygen concentration. We plan to improve the determination of the sensor response by controlling the oxygen concentration more precisely using mass flow controllers and bubbling predefined concen-trations of oxygen/nitrogen gas through the liquid.
Figure 6: Chronoamperometry at -0.7 V in 0.1 M KCl of the oxygen sensor covered with pHEMA. At approximately t=50 seconds, Na2SO3 was added to obtain a 0% oxygen
solution. 90% Response time is 7.5 seconds.
Figure 7: Response curve of the oxygen sensor. Chronoam-perometry at -0.7 V is performed, in 0.1 M KCl with various amounts of Na2SO3 added. The linear fit has a correlation
CONCLUSION AND OUTLOOK
In conclusion, the developed three-electrode electrochemical oxygen sensor can measure oxygen, with a re-sponse time of 7.5 seconds. The sensor is protected from biofouling with pHEMA, which is proven to be biocom-patible. Due to its small dimensions, the sensor can be integrated into an existing organ-on-chip device with a 1 mm wide microfluidic channel [3]. Future experiments will focus on characterizing the oxygen sensor in an organ-on-chip-device during cell culture.
ACKNOWLEDGEMENTS
Funding source: This project has been funded by a Building Blocks of Life grant from the Netherlands Organ-ization for Scientific Research (NWO), grant no. 737.016.003.
REFERENCES
[1] P.E. Oomen, M.D. Skolimowski, E. Verpoorte, Implementing oxygen control in chip-based cell and tissue culture systems, Lab Chip. 16 (2016) 3394–3414. doi:10.1039/C6LC00772D.
[2] M.G. Espey, Role of oxygen gradients in shaping redox relationships between the human intestine and its microbiota, Free Radic. Biol. Med. 55 (2013) 130–140. doi:10.1016/j.freeradbiomed.2012.10.554.
[3] O.Y.F. Henry, R. Villenave, M.J. Cronce, W.D. Leineweber, M.A. Benz, D.E. Ingber, Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function, Lab Chip. 17 (2017) 2264–2271. doi:10.1039/C7LC00155J.
[4] R.G. Bates, V.E. Bower, Standard Potential of the Silver-Silver-Chloride Electrode from 0° to 95° C and the Thermodynamic Properties of Dilute Hydrochloric Acid Solutions, J. Res. Natl. Bur. Stand. (1934). 53 (1954).
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