- Email: [email protected]
EWOD-based chip characterization under AC voltage
Microelectronic Engineering 88 (2011) 1745–1748
Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
EWOD-based chip characterization under AC voltage Rachid Malk a,b, Laurent Davoust b,⇑, Yves Fouillet a a b
CEA, LETI, Minatec, 38054 Grenoble, France CNRS-LEGI, Microfluidics, Interfaces & Particles Team, BP 53, 38041 Grenoble, France
a r t i c l e
i n f o
Article history: Available online 13 December 2010 Keywords: Electrowetting on dielectric Microfluidics Droplet Cells Contamination Characterisation
a b s t r a c t This paper addresses one basic issue of EWOD chip as a consumable: how to detect the alteration of hydrophobic layers? We investigate the possibility of using droplet oscillations to finely characterize chip ability to EWOD protocols. Experiments are performed in coplanar electrodes configuration. Dedicated setup and software have been developed for a simple in situ characterization. When a low-frequency AC voltage is used, drop surface oscillations are created from the time-varying component of the normal electrostatic stress at drop surface near the contact line. As an analysis tool, our software is based on droplet contour detection and delivers dynamical contact angle and contact line motion. Careful attention is brought to surface wetting or dewetting of the droplet during long-term AC actuation and surface ageing. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Digital lab-on-chip (DLC) consists in handling discrete amounts of liquid all over a surface [1]. The droplets are considered as micro reactors inside which biological (or chemical) reactions can be individually achieved. Electrowetting-on-dielectric (EWOD) is probably one of the most appropriate drivers for activating droplets in DLCs enabling the integration of complex bio-protocols [2]. EWOD phenomenon consists in controlling apparent wettability of a droplet by generating an electrostatic force at the triple contact line (TCL). When AC voltage is used, the electrowetting stress displays a time-varying dependence which leads to droplet oscillations [3]. So far these oscillations have been studied from a fundamental point of view only. This paper aims at exploiting drop shape oscillations for a fine characterization of chip surface. After describing the experimental setup and droplet analysis software, droplet dynamics is investigated through two tests. The first one is mechanical wearing of the hydrophobic layer during long-term actuation. The second one is the detection of surface contamination along EWOD chip from a drop made with a biological sample containing cells. The possibility of using EWOD-driven droplet oscillations as a fast means of surface characterization is finally discussed.
Technological steps are made on a 200 mm silicon wafer and chips are composed of three main layers: the electrodes (200 nm thick AlCu); the dielectric layer (600 nm thick Si3N4); and, finally, the hydrophobic layer (1 lm thick SiOC [4]). These materials enable to integrate the whole fabrication process into cleanrooms with no compromise on EWOD performance and a good reproducibility during assays. More details of the technology are described in [5].
⇑ Corresponding author. Present address: SIMAP/EPM Laboratory, Domaine Universitaire, 38400 Saint Martin d’Hères, France. E-mail address: [email protected] (L. Davoust). 0167-9317/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2010.11.057
2.1. Experimental setup The simple chip design (Fig. 1) used here is made from two half disks shaped coplanar electrodes (2 mm radius) separated by a 3 lm gap. The advantage of this coplanar electrodes configuration compared with the classical needle electrode configuration is that droplet oscillations are not disturbed by any needle counterelectrode and image analysis is therefore facilitated. A 1.5 ll droplet of Phosphate Buffered Saline (PBS) is placed on the chip surface with a micropipette. Silicone oil (Paragon Scientific) is used as ambient phase. AC electrical signal is generated from a generator (Yokogawa FG120) and a home-made amplifier. A CCD camera (Pixelfly 200 XS) and a zoom lens are used for image acquisition. The camera is placed so as to visualize droplet profiles. A LED is used as a stroboscopic lighting source placed behind the droplet in order to create a backlight and to minimize light reflection. The LED is plugged to the same generator. The frequencies of AC actuation and stroboscopic lighting are same, and by
1746
R. Malk et al. / Microelectronic Engineering 88 (2011) 1745–1748
Fig. 1. Experimental setup (left) and electrodes chip design (right).
shifting phase between both signals the instantaneous shape of the droplet can be clearly imaged. Time-dependent droplet profiles are acquired and analysed using dedicated software. 2.2. Software package Software package has been developed for both EWOD and imaging experiments. The experimental setup is monitored by a Labview interface which drives the CCD camera and the function generator as well. An image of the droplet is taken while the voltage or phase are changed step by step. Once the experiments are completed, successive images are analysed using a Graphical User Interface (GUI) developed under Matlab environment. Image analysis based on edge detection allows time-dependent wetting angle and TCL position to be measured. Measurement errors for contact angle and TCL position are 3–4° and 3 lm, respectively. Special attention has been paid to simplify assays and to fasten experiments. Thus the experimental setup and dedicated software developed here enable complete automation of experimental chain including data acquisition as well as data analysis.
3. Surface stability as estimated from repeated TCL oscillations The protocol used for this long-term stability experiment is detailed here: we apply AC voltage (75 Vrms) at a frequency of 100 Hz low enough to enhance EWOD in the oscillating regime. At such a frequency, drop oscillations are axisymmetric while being large enough to be analysed from imaging [5]. Oscillations are analysed and then the droplet is left to oscillate during 3 h so that the chip surface undergoes more than 2 millions TCL displacements. Finally, the droplet is removed, and we put another droplet on the same surface to analyse its oscillations. Fig. 2 represents oscillations of the droplet before and after the surface has experienced 2 millions oscillating cycles. When the TCL is advancing, contact angle decreases until reaching its minimum value corresponding to the stop of the TCL displacement. On the contrary, when TCL is receding, contact angle increases until TCL stops. Thus, peak values are in phase for TCL and contact angle curves are in phase. Observation shows that the amplitude of oscillations of the contact angle is very close between the beginning and the end of
Fig. 2. Contact angle (blue curve) and contact line oscillations (black curve) before and after 2 millions oscillation cycles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
R. Malk et al. / Microelectronic Engineering 88 (2011) 1745–1748
experiment (27° and 29°, respectively); the same is true as for the TCL displacement (68 and 71 lm, respectively). Differences of values are comparable with the measurement error. Deviation of mean contact angle and mean TCL displacement are only 1° and 2 lm, respectively.
4. Surface contamination as estimated from repeated TCL oscillations This time, shape oscillations are analysed before and after the chip surface has been in contact with a droplet containing PBS (Phosphate Buffered Saline) and adherent cells (U373B, 1.5.106 cells/ml) as well. Silicone oil is used as ambient phase. The droplet is left at rest during 5 min enabling cell sedimentation and surface contamination. The droplet containing cells is then removed and to keep the same droplet characteristics, a new PBS droplet is deposited on the same chip surface. Its oscillations are analysed and compared to non-contaminated surface oscillations (Fig. 3). Curves show that TCL oscillates around a mean position which is 570 lm if the surface is clean against 613 lm if the surface is contaminated. The amplitude of oscillation of the TCL is 65 lm when the surface is clean while it varies from 40 to
1747
60 lm when the surface is contaminated and contact angle for a contaminated surface is found about 12° smaller than the one for a clean surface. It is also worthy to note that surface contamination yields a symmetry break-up between advancing and receding contact angles. The same experiment is reproduced but now the contaminating droplet contains cells and surfactants (pluronic F68) added at critical micellar concentration (CMC: 0.04 mM) (Fig. 4). In this case, differences between curves are found less than 3° and 3 lm for contact angle and droplet radius, respectively.
5. Discussion Hydrophobic and dielectric layers are the key point of EWOD technology and so require specific careful attention. There is a need to develop analysis tool in order to insure that good surface properties are guaranteed during the whole bio-protocol duration. A first step before starting the protocol is to ensure that no surface mechanical wearing will be caused by repeated droplet displacements. Droplet displacement induces contact line friction that may cause ageing of the hydrophobic layer due to long-term actuation. Here instead of using common static electrowetting
Fig. 3. Oscillating curves of a PBS droplet before (black curves) and after surface chip contamination by cells (blue curves). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Oscillating curves of a PBS droplet before (black curves) and after surface chip was put in contact with a droplet containing cells and pluronic F68 (blue curves). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
1748
R. Malk et al. / Microelectronic Engineering 88 (2011) 1745–1748
experiments or loop-like droplet EWOD-driven displacements, we exploit droplet EWOD-driven oscillations achieved from a lowfrequency actuation. As droplet oscillations originate from the electrical stress acting at the TCL, wetting properties are a key parameter for TCL displacement and contact angle oscillations. Consequently, the measurement of these variables enables to follow wetting stability of the chip surface: any change in wetting ability is expected to be detected from drop shape oscillations. Another advantage of using oscillations is that the droplet is at rest so the area of chip required for surface characterization is dramatically reduced. Moreover the back and forth motion of the TCL is concentrated on the same location of the surface chip which increases the test efficiency. As can been shown in the curves (Fig. 3), for each period of the input frequency, 2 periods of oscillations are observed which means that the droplet oscillates at twice the input frequency; the oscillating test is faster to be implemented than droplet displacements because the frequency of droplet oscillations (twice the input AC frequency (200 Hz)) is far higher than the maximal droplet displacement frequency (40 Hz) [1]. Here, small differences between the curves (Fig. 3) show that the chip surface has not been altered in spite of the numbers of cycles of the TCL which is far higher than what most protocols require. Neither surface wearing nor surface erosion has been detected showing that wetting properties of the SiOC surface are stable under EWOD actuation. SiOC as a hydrophobic layer proves to be a very stable material for EWOD actuation. The use of biological materials (cells, proteins) may induce surface adhesion, cross contamination and contact line pinning. Droplet oscillations can also be used to detect surface contamination. As can be shown in Fig. 3, oscillations are very different depending on if the surface is contaminated or not. The results clearly show a loss of hydrophobicity of the contaminated surface due to cells adsorption on the surface of the chip. The use of pluronic F68 proves to be very efficient to prevent cell contamination, a
finding which was also reported in [6] for another type of biological material (proteins). Further work will consist in investigating the correlation amplitude of oscillations and concentration of adsorbed cells, in order to better quantify chip surface contamination. 6. Conclusion This work focuses on the possibility of using EWOD in the oscillating regime for a fast characterization of solid supports used in digital lab on chips. A dedicated software has been developed to image and analyse EWOD-driven oscillations of a droplet and two typical experiments have been investigated: surface stability and surface contamination. The advantages of using droplet oscillations have been outlined and applications have been illustrated. The hydrophobic layer has demonstrated good stability to a longterm actuation while surface contamination can be easily detected by oscillations. The use of pluronic F68 as a means to prevent EWOD-based chips from cells adhesion has been validated. Further investigation is required in order to fully exploit our method of surface characterization but droplet oscillations seem to be a very promising way to explore. References [1] R.B. Fair, Microfluid. Nanofluid. (3) (2007) 245–281. [2] Y. Fouillet, D. Jary, C. Chabrol, P. Claustre, C. Peponnet, Microfluid. Nanofluid. (4) (2008) 159–165. [3] M. Oh, S.H. Ko, K.H. Kang, Langmuir 24 (2008) 8379. [4] J. Thery, M. Borella, S. Le Vot, D. Jary, F. Rivera, G. Castellan, A.G. Brachet, M. Plissonnier, Y. Fouillet, Sioc as a hydrophobic layer for electrowetting on dielectric applications, in: Proceedings of lTas 2007 conference, vol. 1, pp. 349– 351, ISBN:978-0-9798064-0-7. [5] R. Malk, Y. Fouillet, L. Davoust, Sens. Actuators B: Chem. (2010), doi:10.1016/ j.snb.2009.12.066. [6] V.N. Luk, G.C.H. Mo, A.R. Wheeler, Langmuir 24 (2008) 6382–6389.
Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
EWOD-based chip characterization under AC voltage Rachid Malk a,b, Laurent Davoust b,⇑, Yves Fouillet a a b
CEA, LETI, Minatec, 38054 Grenoble, France CNRS-LEGI, Microfluidics, Interfaces & Particles Team, BP 53, 38041 Grenoble, France
a r t i c l e
i n f o
Article history: Available online 13 December 2010 Keywords: Electrowetting on dielectric Microfluidics Droplet Cells Contamination Characterisation
a b s t r a c t This paper addresses one basic issue of EWOD chip as a consumable: how to detect the alteration of hydrophobic layers? We investigate the possibility of using droplet oscillations to finely characterize chip ability to EWOD protocols. Experiments are performed in coplanar electrodes configuration. Dedicated setup and software have been developed for a simple in situ characterization. When a low-frequency AC voltage is used, drop surface oscillations are created from the time-varying component of the normal electrostatic stress at drop surface near the contact line. As an analysis tool, our software is based on droplet contour detection and delivers dynamical contact angle and contact line motion. Careful attention is brought to surface wetting or dewetting of the droplet during long-term AC actuation and surface ageing. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
Digital lab-on-chip (DLC) consists in handling discrete amounts of liquid all over a surface [1]. The droplets are considered as micro reactors inside which biological (or chemical) reactions can be individually achieved. Electrowetting-on-dielectric (EWOD) is probably one of the most appropriate drivers for activating droplets in DLCs enabling the integration of complex bio-protocols [2]. EWOD phenomenon consists in controlling apparent wettability of a droplet by generating an electrostatic force at the triple contact line (TCL). When AC voltage is used, the electrowetting stress displays a time-varying dependence which leads to droplet oscillations [3]. So far these oscillations have been studied from a fundamental point of view only. This paper aims at exploiting drop shape oscillations for a fine characterization of chip surface. After describing the experimental setup and droplet analysis software, droplet dynamics is investigated through two tests. The first one is mechanical wearing of the hydrophobic layer during long-term actuation. The second one is the detection of surface contamination along EWOD chip from a drop made with a biological sample containing cells. The possibility of using EWOD-driven droplet oscillations as a fast means of surface characterization is finally discussed.
Technological steps are made on a 200 mm silicon wafer and chips are composed of three main layers: the electrodes (200 nm thick AlCu); the dielectric layer (600 nm thick Si3N4); and, finally, the hydrophobic layer (1 lm thick SiOC [4]). These materials enable to integrate the whole fabrication process into cleanrooms with no compromise on EWOD performance and a good reproducibility during assays. More details of the technology are described in [5].
⇑ Corresponding author. Present address: SIMAP/EPM Laboratory, Domaine Universitaire, 38400 Saint Martin d’Hères, France. E-mail address: [email protected] (L. Davoust). 0167-9317/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mee.2010.11.057
2.1. Experimental setup The simple chip design (Fig. 1) used here is made from two half disks shaped coplanar electrodes (2 mm radius) separated by a 3 lm gap. The advantage of this coplanar electrodes configuration compared with the classical needle electrode configuration is that droplet oscillations are not disturbed by any needle counterelectrode and image analysis is therefore facilitated. A 1.5 ll droplet of Phosphate Buffered Saline (PBS) is placed on the chip surface with a micropipette. Silicone oil (Paragon Scientific) is used as ambient phase. AC electrical signal is generated from a generator (Yokogawa FG120) and a home-made amplifier. A CCD camera (Pixelfly 200 XS) and a zoom lens are used for image acquisition. The camera is placed so as to visualize droplet profiles. A LED is used as a stroboscopic lighting source placed behind the droplet in order to create a backlight and to minimize light reflection. The LED is plugged to the same generator. The frequencies of AC actuation and stroboscopic lighting are same, and by
1746
R. Malk et al. / Microelectronic Engineering 88 (2011) 1745–1748
Fig. 1. Experimental setup (left) and electrodes chip design (right).
shifting phase between both signals the instantaneous shape of the droplet can be clearly imaged. Time-dependent droplet profiles are acquired and analysed using dedicated software. 2.2. Software package Software package has been developed for both EWOD and imaging experiments. The experimental setup is monitored by a Labview interface which drives the CCD camera and the function generator as well. An image of the droplet is taken while the voltage or phase are changed step by step. Once the experiments are completed, successive images are analysed using a Graphical User Interface (GUI) developed under Matlab environment. Image analysis based on edge detection allows time-dependent wetting angle and TCL position to be measured. Measurement errors for contact angle and TCL position are 3–4° and 3 lm, respectively. Special attention has been paid to simplify assays and to fasten experiments. Thus the experimental setup and dedicated software developed here enable complete automation of experimental chain including data acquisition as well as data analysis.
3. Surface stability as estimated from repeated TCL oscillations The protocol used for this long-term stability experiment is detailed here: we apply AC voltage (75 Vrms) at a frequency of 100 Hz low enough to enhance EWOD in the oscillating regime. At such a frequency, drop oscillations are axisymmetric while being large enough to be analysed from imaging [5]. Oscillations are analysed and then the droplet is left to oscillate during 3 h so that the chip surface undergoes more than 2 millions TCL displacements. Finally, the droplet is removed, and we put another droplet on the same surface to analyse its oscillations. Fig. 2 represents oscillations of the droplet before and after the surface has experienced 2 millions oscillating cycles. When the TCL is advancing, contact angle decreases until reaching its minimum value corresponding to the stop of the TCL displacement. On the contrary, when TCL is receding, contact angle increases until TCL stops. Thus, peak values are in phase for TCL and contact angle curves are in phase. Observation shows that the amplitude of oscillations of the contact angle is very close between the beginning and the end of
Fig. 2. Contact angle (blue curve) and contact line oscillations (black curve) before and after 2 millions oscillation cycles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
R. Malk et al. / Microelectronic Engineering 88 (2011) 1745–1748
experiment (27° and 29°, respectively); the same is true as for the TCL displacement (68 and 71 lm, respectively). Differences of values are comparable with the measurement error. Deviation of mean contact angle and mean TCL displacement are only 1° and 2 lm, respectively.
4. Surface contamination as estimated from repeated TCL oscillations This time, shape oscillations are analysed before and after the chip surface has been in contact with a droplet containing PBS (Phosphate Buffered Saline) and adherent cells (U373B, 1.5.106 cells/ml) as well. Silicone oil is used as ambient phase. The droplet is left at rest during 5 min enabling cell sedimentation and surface contamination. The droplet containing cells is then removed and to keep the same droplet characteristics, a new PBS droplet is deposited on the same chip surface. Its oscillations are analysed and compared to non-contaminated surface oscillations (Fig. 3). Curves show that TCL oscillates around a mean position which is 570 lm if the surface is clean against 613 lm if the surface is contaminated. The amplitude of oscillation of the TCL is 65 lm when the surface is clean while it varies from 40 to
1747
60 lm when the surface is contaminated and contact angle for a contaminated surface is found about 12° smaller than the one for a clean surface. It is also worthy to note that surface contamination yields a symmetry break-up between advancing and receding contact angles. The same experiment is reproduced but now the contaminating droplet contains cells and surfactants (pluronic F68) added at critical micellar concentration (CMC: 0.04 mM) (Fig. 4). In this case, differences between curves are found less than 3° and 3 lm for contact angle and droplet radius, respectively.
5. Discussion Hydrophobic and dielectric layers are the key point of EWOD technology and so require specific careful attention. There is a need to develop analysis tool in order to insure that good surface properties are guaranteed during the whole bio-protocol duration. A first step before starting the protocol is to ensure that no surface mechanical wearing will be caused by repeated droplet displacements. Droplet displacement induces contact line friction that may cause ageing of the hydrophobic layer due to long-term actuation. Here instead of using common static electrowetting
Fig. 3. Oscillating curves of a PBS droplet before (black curves) and after surface chip contamination by cells (blue curves). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Oscillating curves of a PBS droplet before (black curves) and after surface chip was put in contact with a droplet containing cells and pluronic F68 (blue curves). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
1748
R. Malk et al. / Microelectronic Engineering 88 (2011) 1745–1748
experiments or loop-like droplet EWOD-driven displacements, we exploit droplet EWOD-driven oscillations achieved from a lowfrequency actuation. As droplet oscillations originate from the electrical stress acting at the TCL, wetting properties are a key parameter for TCL displacement and contact angle oscillations. Consequently, the measurement of these variables enables to follow wetting stability of the chip surface: any change in wetting ability is expected to be detected from drop shape oscillations. Another advantage of using oscillations is that the droplet is at rest so the area of chip required for surface characterization is dramatically reduced. Moreover the back and forth motion of the TCL is concentrated on the same location of the surface chip which increases the test efficiency. As can been shown in the curves (Fig. 3), for each period of the input frequency, 2 periods of oscillations are observed which means that the droplet oscillates at twice the input frequency; the oscillating test is faster to be implemented than droplet displacements because the frequency of droplet oscillations (twice the input AC frequency (200 Hz)) is far higher than the maximal droplet displacement frequency (40 Hz) [1]. Here, small differences between the curves (Fig. 3) show that the chip surface has not been altered in spite of the numbers of cycles of the TCL which is far higher than what most protocols require. Neither surface wearing nor surface erosion has been detected showing that wetting properties of the SiOC surface are stable under EWOD actuation. SiOC as a hydrophobic layer proves to be a very stable material for EWOD actuation. The use of biological materials (cells, proteins) may induce surface adhesion, cross contamination and contact line pinning. Droplet oscillations can also be used to detect surface contamination. As can be shown in Fig. 3, oscillations are very different depending on if the surface is contaminated or not. The results clearly show a loss of hydrophobicity of the contaminated surface due to cells adsorption on the surface of the chip. The use of pluronic F68 proves to be very efficient to prevent cell contamination, a
finding which was also reported in [6] for another type of biological material (proteins). Further work will consist in investigating the correlation amplitude of oscillations and concentration of adsorbed cells, in order to better quantify chip surface contamination. 6. Conclusion This work focuses on the possibility of using EWOD in the oscillating regime for a fast characterization of solid supports used in digital lab on chips. A dedicated software has been developed to image and analyse EWOD-driven oscillations of a droplet and two typical experiments have been investigated: surface stability and surface contamination. The advantages of using droplet oscillations have been outlined and applications have been illustrated. The hydrophobic layer has demonstrated good stability to a longterm actuation while surface contamination can be easily detected by oscillations. The use of pluronic F68 as a means to prevent EWOD-based chips from cells adhesion has been validated. Further investigation is required in order to fully exploit our method of surface characterization but droplet oscillations seem to be a very promising way to explore. References [1] R.B. Fair, Microfluid. Nanofluid. (3) (2007) 245–281. [2] Y. Fouillet, D. Jary, C. Chabrol, P. Claustre, C. Peponnet, Microfluid. Nanofluid. (4) (2008) 159–165. [3] M. Oh, S.H. Ko, K.H. Kang, Langmuir 24 (2008) 8379. [4] J. Thery, M. Borella, S. Le Vot, D. Jary, F. Rivera, G. Castellan, A.G. Brachet, M. Plissonnier, Y. Fouillet, Sioc as a hydrophobic layer for electrowetting on dielectric applications, in: Proceedings of lTas 2007 conference, vol. 1, pp. 349– 351, ISBN:978-0-9798064-0-7. [5] R. Malk, Y. Fouillet, L. Davoust, Sens. Actuators B: Chem. (2010), doi:10.1016/ j.snb.2009.12.066. [6] V.N. Luk, G.C.H. Mo, A.R. Wheeler, Langmuir 24 (2008) 6382–6389.