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Proceedings of 2006 International Conference on Microtechnologies in Medicine and Biology Okinawa, Japan 9-12 May 2006


Development of an integrated microfluidic device for sensing dynamic response of cells and tissues N. Pereira Rodrigues, H. Kimura, Y. Sakai, T. Fujii LIMMS/CNRS-IIS (UMI 2820) Institute of Industrial Science, University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8505, Japan Tel/Fax: +81-3-5452-6213, E-mail: [email protected]

Abstract The aim of this study is to develop a novel cell culture device with an integrated array of electrochemical sensors that will allow the monitoring of cell activity. This device “on a chip” will therefore be dedicated and realize a complete, precise and real-time monitoring of the composition and modulation of cell medium. The first experiments presented here are dedicated to oxygen electrochemical detection by using an integrated array of microsensors inserted in a PDMS microfluidic device. The configuration of the sensors is fabricated by thin film technology and is composed of an array of gold working microelectrodes, a gold counter electrode and an Ag/AgCl reference electrode. The array of gold microelectrodes is covered by a layer of Nafion®, which is a gas permeable membrane that enables a long lifetime of the electrodes and a good sensitivity of detection of oxygen reduction. First results dedicated to perform O2 calibration showed good performances of the O2 sensor in terms of linearity of response and in terms of sensitivity of O2 monitoring in a physiological buffer medium. Keywords: integrated electrochemical sensors, oxygen monitoring, microelectrodes array, PDMS, microfluidic device

of in vivo electrochemical techniques have been reported. Nafion®, a perfluorinated cation-exchange polymer, has been widely used in the construction of in vivo sensors because the electrostatic and/or discriminating properties of the film give protection against fouling. This study is aimed to develop a novel cell culture device with an integrated array of electrochemical sensors. Local concentration of oxygen in a polydimethylsiloxane (PDMS) microfluidic device is monitored by integrating an array of specific sensors at the inlet and outlet of the device. Fabrication process of the integrated sensors in the PDMS microchannel is detailed, and calibration curves as well as linearity of response of the Nafion® modified sensors are put in evidence. The performances of the present sensor are compared to a commercial oxygen sensor located outside the device.

1. INTRODUCTION Recently the micro total analysis system (µ-TAS) has attracted widespread attention due to its excellent potential for offering highly efficient and on-line analysis of biological molecules or metabolisms by integrating the sensing system in a microfluidic device [1]. A controlled oxygen supply and accurate sensing of dissolved oxygen levels within a miniaturized environment are necessary in biological assay and tissue engineering applications. In cellular assays, rapid determination of cell viability is frequently accomplished by the monitoring of cellular metabolic activity by monitoring oxygen concentrations in the medium [2]. A controllable source of oxygen is therefore an important parameter for the study of cellular growth and development in tissue engineering applications. For this purpose, refinement of techniques of integrated sensors in microfluidic systems that allow real-time measurements of oxygen concentration during cell culture is a subject of growing interest. Electrochemical microsensors have received extensive attention in life sciences and electroanalytical chemistry and many applications 1-4244-0338-3/06/$20.00 ©2006 IEEE

2. MATERIALS AND METHODS 2.a. Reagents and Materials Nafion® alcoholic solution (5%) was purchased from Aldrich. Slide glasses with a thickness of 1.8


microscopy by using, when it is necessary, a small droplet of methanol deposited on the PDMS.

mm purchased from Matsunami were used as the electrode substrate. A negative photoresist, SU82025 MicroChem, was used for the microfabricated structure. A positive photoresist, S1813 (AZ Photoresist), was used to define the design of the electrodes. Silpot 184W/C poly(dimethylsiloxane) (PDMS) purchased from Dow Corning was used for the oxygen-permeable membrane and for the microfluidic device. All other chemicals were analytical grade and used without further purification.

2.d. Final configuration of the device A scheme of the final microfluidic device containing the electrodes at the inlet and at the outlet of the cell chamber is showed in Figure 1. FLOW INLET

2.b. Fabrication and configuration of the microelectrodes


Microfluidic structure (PDMS) Glass substrate

The structure of the sensor consists of a glass substrate and a PDMS container. The glasses were first cleaned by immersing them in Piranha solution (25% H2SO4, 75% H2O2) during 15 min. The liftoff technique was used to pattern the electrode geometry on the glass substrate, and 5 nm Cr and 200 nm Au were sequentially deposited by evaporation to obtain an array of 5 working microelectrodes (200 X 50 µm), and a reference and counter electrodes (50 X 2000 µm). Reference electrode was obtained by evaporating 200 µm Ag after another lift-off process. AgCl layer was obtained by dipping the Ag electrode in a solution of FeCl3 during a few seconds. During each step of the thin-film electrodes fabrication process, a 35 µm thick insulating layer fabricated using a S1813 positive photoresist with 3000 rpm for 30 s was used to define the configuration of the array of electrodes.

Cell culture Integrated electrochemical microsensors (Au, L =50X50 to 50X200 µm)

Figure 1: Scheme of the cell-based microfluidic device comprising arrays of microsensors at the inlet and the outlet The configuration and the positioning of the sensors in the inlet and the outlet of the microchannel can be aimed at giving information about local oxygen concentration variations between the inlet and the outlet of the cell chamber. In that case, measurements have to be done in parallel by independently connecting one or many O2 sensors belonging to each array of microelectrodes. Therefore, this configuration of sensor gives many possibilities concerning series of experiments that are possible to perform. In this study, preliminary experiments are done in way to validate the O2 sensor performances.

2.c. Fabrication of the PDMS container and its bonding to glass substrate The microfluidic structure is fabricated through replica moulding processes with a master which contains the negative pattern of the structure. First, a negative master is made with a SU8 photoresist put on a Si wafer using a conventional photolithography process [3]. CHF3 plasma is applied in way to deposit a fluorocarbon layer onto the obtained SU8 mould for future easy release of the PDMS layers [4]. Then, the liquid-state PDMS is poured after removing all bubbles by putting the master and PDMS in a vacuum chamber. The solidification step consists in baking the PDMS at 70°C during 1h30. The PDMS is then peeled off from the master and inlets and outlets of each PDMS layer are formed with 1 mm diameter punch. After this step, the PDMS layers and glass substrate containing the integrated array of microelectrodes are aligned and stacked onto each other after being treated by O2 plasma. Alignment between microfluidic channels and the array of microelectrodes, which is the most important step of the process, is performed under optical

2.e. Electrochemical measurements All chronoamperometric measurements were performed with a Electrochemical anlyser Model 600B potentiostat (BAS, Japan). No special pretreatments of the working electrodes were used before measurements. All amperometric measurements were collected at E = -0.6 V vs Ag/AgCl. The experimental protocol comprised a peristaltic pump (Perista pump, bioinstrument ATTO) (flow rate: 100 µL/min) and a commercial oxygen meter (Strathkelvin Instruments, Model 782) directly connected to the Au/Nafion® sensor. The function of the commercial O2 meter was to allow an exact determination of O2 concentrations during experiment. For that purpose, gases (nitrogen and oxygen) were purged through a Plexiglas bottle at T = 25°C to ensure modulation of oxygen composition of the electrolyte solution, which was in our case a phosphate buffer solution (PBS, pH = 7.4).


concentration measured (S = 100 pA/s for 0.02 µM/s in these experimental conditions). Therefore, the results obtained show that the developed sensor is able to monitor oxygen during a long time period without degradation in these experimental conditions.

3. RESULTS AND DISCUSSION 3.a. Fabrication of the oxygen sensor

Reference electrode (Ag/AgCl)

125 400 120

-i (nA)

380 115



340 320






Working electrode (Au) (200X50 µm)


oxygen concentration (µM)

The integrated array of electrodes was composed of 5 Au working electrodes (200X50 µm) which could be independently connected. First, a droplet of diluted Nafion® alcoholic solution layer was deposed onto the surface of the electrodes and was allowed to dry at 70°C during 30 min. In way to perform dynamic on-line measurements of oxygen, a microchannel made of PDMS (width = 400µm) was bonded to the glass substrate containing the array of electrodes. This first configuration of the O2 sensor showed Figure 2 was aimed to validate its performances about sensitivity and linearity of response during continuous measurements.

100 200 300 400 500 600 700 800 900 1000 t (s)

Figure 3: Simultaneous oxygen monitoring by using Au/Nafion® working electrode (E = -0.6 V vs Ag/AgCl) (continuous line) and oxygen concentration measured with a commercial O2 meter (⋅ marks). PDMS microchannel (w = 400 µm)

Counter electrode (Au)

Figure 4 shows the calibration curve obtained for a larger O2 concentration range. Good linearity, with a correlation coefficient of 0.997 and a sensitivity of 5 nA/µM were obtained due to no electrochemical crosstalk between the working, the counter and the reference electrodes. Further experiments are now in progress to enlarge oxygen concentration range.

500 µm

Figure 2: Image of the integrated O2 sensors in a microfluidic channel (Nafion® recovers Au electrodes) 3.b. Performances of the O2 sensor Figure 3 shows the linearity of response obtained for the Au/Nafion® sensor during on-line monitoring of the dissolved oxygen that flows through the microfluidic channel (T= 25°C). The results obtained showed perfect concordance between the amperometric signal and the oxygen concentration measured with a commercial oxygen meter during a long period (experiments were done up to 1h). Indeed, a correlation coefficient of 0.995 was observed and no degradation of the electrodes during time was observed due to protection induced by the Nafion® layer. Thus, same experiments performed without Nafion® layer showed colour modification and then degradation of the counter electrode due to the potential applied (E = -0.6 vs Ag/AgCl). By the other way, the periodical small variations of current observed during measurements are due to periodical small variations of the flow rate induced by the peristaltic pump, but, as we can see Figure 3, the variation of amperometric signal observed are not significant comparing to the variations of

400 350

-i (nA)

300 250 200 150 60








oxygen concentration (µM)

Figure 4: Linear relation between amperometric oxygen measurements performed by Au/Nafion® working electrode (E = -0.6 V vs Ag/AgCl) and oxygen concentration measured with a commercial O2 meter.


4. CONCLUSION In this study, we presented a novel oxygen sensor with a fast time of response and a good sensitivity and linearity of detection. The results obtained are very promising and further experiments will be done with cell cultures that will be introduced to a chamber directly connected to the microchannel containing the electrodes. Therefore, kinetics of oxygen response in biological samples will be done. By the other side, as the array of electrode is composed of many electrodes, experiments implying a glucose sensor are now in progress in way to make simultaneous measurements of oxygen and glucose levels in the presence of the same cell culture.


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K. Hosokawa, T. Fujii, I. Endo. ”Handling picoliter samples in a poly (dimethylsiloxane) based microfluidic device”. Anal. Chem., vol. 71, pp. 47814785, 1999

6. ACKNOWLEDGEMENTS We would like to acknowledge CNRS, JSPS and MEXT for their financial support.


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