Droplet-based DEP microfluidics - High speed liquid actuation on planar substrates and factors influencing picolitre droplet formation

Transcription

1 Droplet-based DEP microfluidics - High speed liquid actuation on planar substrates and factors influencing picolitre droplet formation Thirukumaran T.K., Thomas B. Jones *, Karan V.I.S. Kaler Department of Electrical and Computer Engineering University of Calgary, Calgary, Alberta, Canada T2N 1N4 * University of Rochester, Rochester, NY, United States Abstract Successful lab-on-a-chip (LOC) devices resort to some sort of microfluidic system to dispense, transport, and mix small volumes of liquid analyte. We propose a new LOC scheme that integrates liquid dielectrophoretic (DEP) actuation for analyte distribution and droplet dispensing, and then particulate DEP, for subsequent, frequency-selective manipulation of bio-particles contained within the droplets. Multiple picolitre(pl) sized droplets have been formed simultaneously from a parent microliter droplet pipetted onto a coplanar electrode structure patterned and microcoated with dielectrics to control surface wetting. The volumes of the dispensed droplets are controlled by the width of the electrode structure and their number by semicircular bumps patterned into the coplanar strips. We have furthermore shown that integration of particle DEP electrodes within the droplet forming structures can be effectively utilized to sort cell based on their frequency dependent polarization response. KEYWORDS Dielectrophoresis, Liquid actuation, particle DEP, droplet dispensing, microfluidics, Lab-on-chip INTRODUCTION Voltage-controlled formation, conveyance and subsequent manipulation of submicroliter sized fluidic droplet containing cells, macromolecules and/or reagents, without the need for walled microchannels, external pumps and/or valves opens up a new arena of surface fluidic based lab-on-a-chip devices. Such wall-less, openchannel surface fluidic systems, are particularly suited to handling biological cells, DNA and other molecular components as they are immune to the clogging and sample contamination common in closed microfluidic systems.

2 Furthermore, open channel systems, unlike their closed channel counterparts, are immune to leakage problems often encountered at high pumping and channel pressures. The work by Pellat, Melcher, and others [1-3] provided a sound foundation for exploiting electrostatic fields to siphon, transport, and manipulate liquids. Similarly, we leverage the phenomena of dielectrophoresis (DEP) on a microscopic scale, using nonuniform electric fields, to actuate liquids and dispense sub-nanoliter sized droplets. Jones and co-workers [4] have demonstrated the actuation and conveyance of liquids on the surface of insulating substrates. The basic mechanism of electric field induced liquid actuation achieved by employing dielectrically coated coplanar microelectrodes is reasonably well understood [5]. The driving force behind this liquid actuation is DEP, where the non-uniform field required for DEP is generated by appropriate ac excitation of the electrode pair. Electrode widths typically range from 30 µm to 100 µm and are coated by a thin (2 10 µm) insulating dielectric layer [1]. In this paper, we report the development of a DEP actuated surface microfluidic scheme that integrates fluidic transport and droplet formation with DEP of particles contained within the liquid. This capability facilitates and provides for rapid sampling and parallel processing of multiple pl sized droplets containing cells and fractionating them based on their polarization response. BACKGROUND Liquid Dielectrophoresis (L-DEP) To gain insight concerning DEP actuation of liquids, consider a simple arrangement of two coplanar electrodes, patterned on an insulating substrate and coated with a thin insulating dielectric (see Fig. 1). Now consider a drop of liquid (water) placed on the coated coplanar electrode pair, so that it covers a short section of the electrodes, as shown in Fig. 1. When sufficient AC voltage typically ~150 V at a suitable frequency (typically 60 khz or above) is applied, a liquid finger projects from the droplet and travels along the electrodes until it reaches the end of the structure. For water, this DEP actuation is rapid, with transient "meniscus" speeds exceeding 5 cm/s and flow rates of up to ~1 µl/s [4]. The basis for liquid actuation in this case is the interaction of the nonuniform electric field, generated by the electrodes, and the polar liquid molecules. The field gives rise to a net body force acting on the liquid surface to draw out the liquid finger and move it along the electrodes to establish a new hydrostatic equilibrium, with the entire length of the electrodes covered by the finger. The dynamics of the liquid finger formation has been discussed at length by Jones et al [6]. A reduced-order lumped parameter model that takes into account liquid surface tension viscous drag, and the DEP force gives the following [6]: 2 2 ( εliq 1) cc d airv FLiq DEP = (1.1) 2( c + 2 ε c )( c + 2 c ) d liq air d air

3 Under the above actuation force the liquid finger displacement (Z(t) ) is 1/2 proportional to the square root of time (t), namely zt () t, for viscosity dominates flow. The liquid finger once drawn from the parent drop due to liquid DEP actuation is stably sustained between the electrodes as long as the voltage is on. But once the voltage is removed, the liquid finger rapidly breaks up due to capillary stability into multiple droplets, each far smaller than the original parent drop. Droplet formation can be achieved more reliably using semicircular droplet forming bumps patterned on to the electrodes [7]. The bumps must be spaced evenly at the so-called most unstable wavelength according to Rayleigh s instability theory for liquid jets. Particle dielectrophoresis In the present work, we have integrated DEP liquid actuation and subsequent droplet formation with particulate DEP to enable frequency selective manipulation of cells and other microscopic particles suspended in the actuated liquid. Microscopic sized particles such a cells suspended in liquid media and subjected to a inhomogenous electric field ( E 0 ) exhibit a frequency dependent DEP response. For spherically shaped particles of radius a, the time average DEP force exerted on the particle is as follows: uuuur 3 2 ) F = 2πa ε Re[ K ( ω)] E r (1.2) DEP m e 0( rms) where the polarization factor K e ε ε ( ω) =, ε + 2ε p p m m * ε is the complex permittivity, ω is angular velocity of the electric field, is the gradient operator and the subscripts p and m represent particle and medium respectively. It important to note that the DEP force is sensitive to the particle size, the gradient of the electric field squared and furthermore the magnitude and sign of the frequency dependent Clausis Mossotti polarization factor ( Ke( ω )). If Ke( ω ) >0, the DEP interaction force impels particle into region of field intensity maxima (positive DEP), conversely if K ( ) e ω < 0, the particle experience a negative DEP force and are translated to region of field intensity minima. Past work in biological DEP teaches us that the different cells and/or particles exhibit unique frequency dependences in their polarization response arising for a variety of reasons, related to size, shape, composition, surface character and in cells on their physiochemical makeup. We propose to leverage this difference in polarization between different cell types to affect their separation, utilizing a surface fluidic microsystem, as described below.

4 Integrated liquid and Particle Dielectrophoresis The goal of this work is to leverage liquid DEP and particle DEP phenomena to affect the physical separation of a heterogeneous mixture of cells suspended in fluidic media. To achieve in a practical way, we have integrated the finer microelectrode structures that will facilitate positive DEP collection and/or negative DEP repulsion of cells with the fluidic actuation and droplet forming structures. A schematic diagram of this integrated liquid and particle DEP structure is shown in Fig. 2. The coplanar electrodes marked as 1 and 2 in Fig. 2 is used for synthesizing the L-DEP force responsible for liquid actuation while the interdigitated electrodes in the bump region (marked as 3 in Fig. 2) facilitates particle separation based on their polarization behavior. The lower bumps (marked as 4) are used for collecting purer cell population. EXPERIMENTS Methods and Materials - Patterning microelectrodes on Glass The microelectrode structure, shown in Fig. 2, was realized using standard photolithography techniques to pattern the electrodes on a thin layer of metal evaporated on an insulating glass substrate. Details of the fabrication protocol are presented elsewhere [8]. Successful liquid actuation depends heavily on proper dielectric coating of the metal electrode. Dielectric materials of high dielectric constant (K) reduce the required actuation voltage, thus reducing the likelihood of electrical breakdown. We use SU-8 photoresist (K=4) spin-coated to a thickness of ~2 µm which is then covered by a 1.3µm layer of positive photoresist HPR-504, which renders the surface hydrophilic and hence improve rivulet formation. Results and Discussion When an AC voltage is applied across the co-planar electrodes, the non-uniform field acting along the periphery of the droplet tends to lower the droplet contact angle with the surface. The change in contact angle is quiet significant with an increasing voltage and upon a threshold voltage, liquid rivulet projects to balance the hydrodynamic equilibrium caused by the non-uniform electric field. The liquid rivulet moves along the electrode length to maintain a stable hydrodynamic equilibrium. When the voltage across the electrodes is removed, the liquid looses its stability and breaks-up to form sessile droplet based on Rayleigh s instability criteria as discussed before. The time sequence response of the parent droplet to an applied AC voltage of 220 V RMS is shown in Fig. 3. The change in contact angle followed by the liquid rivulet projecting along the electrode length is quite significant from Fig. 3A. Fig. 3B & 3C shows the movement of liquid rivulet along the electrode, while disintegration of rivulet into droplets upon the removal of voltage is as shown in 3D. With the flexibility to form precise droplets at regions of interest [7], thrust was to transport microscopic particles including biological cells along the liquid rivulet and to disperse them to the subsequent daughter droplets. Particles thus dispersed can be manipulated dielectrophoretically, based on their polarization response.

5 For a successful particulate DEP separation, the maximum surface area of the droplet needs to be exposed to the field synthesized by interdigitated electrodes in the bump region. In this work, analytical estimation of the ratio of bump radius to the rivulet width was estimated as in Fig. 4. Experiments with structures based on this estimation show the formation of equal volume droplets as in Fig. 5. Further, it was observed that upon removal of voltage, a portion of the liquid is pulled back into the parent droplet resulting in volume loss in the daughter droplets. To avoid this, three electrode structures [9] were used for the experimental verification of this analytical estimation and the results gave a satisfactory evidence of the bump radius to rivulet width relationship. The electrode structures based on the relation as in eq. (1.3), aids in forming equal volume droplets covering the entire surface area of the bump. x y λ * R bump =(x+y) Fig. 4 Bump ratio calculation; with the volume of liquid trapped between the bumps during actuation being equal to the volume of droplet formed upon voltage removal. The ratio of bump radius (BR) to rivulet radius is estimated as: Fig. 5 Droplets of uniform volume covering the entire surface of the bump 1.92 (rivulet radius) BR = ( Bump radius + Rivulet radius) 2 (1.3) Particulate DEP To verify the particulate DEP effects on open droplets, initial experiments were performed with sample media containing yeast cells (Saccharomyces cerevisiae) placed directly on the bump region. When a sinusoidal AC voltage of 2.47 V RMS is applied at a frequency of 600 khz, yeast cells were observed to be pulled towards the electrodes and also forms pearl chain along the periphery of the bump region due to +ve DEP effect (Fig. 6A). However, at frequencies above 10 MHz, the same cells were observed to be repelled from the electrode periphery (-ve DEP) as in Fig. 6B. Under ve DEP conditions, some cells are trapped within the interdigitated electrodes, this phenomenon though not quantified can be attributed to the smaller electrode gaps resulting in very high particle-particle interaction and thereby forming cell-traps. Cells repelled under ve DEP conditions were actuated along the

6 electrodes below the bumps, forming picolitre droplets on the lower bumps. Experiments are underway to verify the purity of the cell population in these picolitre droplets. CONCLUSION A novel technique for integrating liquid and particulate DEP has been introduced as a new prototype for a laboratory on a chip. The scheme avoids certain difficulties of closed channel devices. With the capability to transport and distribute biological samples to specific locations, this integrated system can be further extended to downstream sample processing including cell lysing, cell cycle monitoring, DNA amplification (PCR reactions) and other detection systems (ex. Hybridization, fluorescent labeling, etc.,). Currently, experiments with modified electrode patterns are in progress for efficient particle transport and particle trapping. Further, utilization of this unique technique for on-chip droplet mixing and integration with droplet-pcr is explored. ACKNOWLEDGEMENTS The authors acknowledge the support provided by the University of Calgary in the form of International fellowship abroad grant and the National Science and Engineering Research Council of Canada (NSERC). The authors also acknowledge the Nanofab staff (Univ. of Alberta, Edmonton) for their guidance in fabrication. REFERENCES 1. Pellat, H., Mesure de la force agissant sur les dielectriques liquides non electrises places dans un champ elitrique. C. R. Acad. Sci. Paris, 1895(119): p Jones, T.B. and J.R. Melcher, Dynamics of electromechanical flow structures. Phys. Fluids, 1973(16): p Melcher, J.R., M. Hurwitz, and R. Fax, Dielectric liquid expulsion. J. Spacecr. Rockets, 1969(6): p Jones, T.B., et al., Dielectrophoretic liquid actuation and nanodroplet formation. Journal of Applied Physics, (2): p Jones, T.B., K.L. Wang, and D.J. Yao, Frequency-dependent electromechanics of aqueous liquids: Electrowetting and dielectrophoresis. Langmuir, (7): p Jones, T.B., Dynamics of Dielectrophoretic Liquid Microactuation. Proceedings of 4th Int'l Conference on Applied Electrostatics, Dalin, China, Ahmed, R., et al., Dispensing picoliter droplets using dielectrophoretic (DEP) microactuation. Microscale Thermophysical Engineering, (3): p

7 8. Kanagasabathi, T., C.J. Backhouse, and K. Kaler. Dielectrophoresis (DEP) of cells and microparticles in PDMS microfluidic channels. in Nanotech Boston, MA. 9. Private communication with Rajib Ahmed, Univ of Rochester, 2004 Parent liquid droplet ( ε liq ) liquid finger dielectric film ( ε d ) Insulating substrate Fig. 1A Fig. 1 Dynamics of liquid actuation. Fig. 1A Schematic side-view of actuation; Fig. 1B Cross-sectional view of the finger. w g Fig. 1B Parent droplet with heterogeneous mixture of cells 4 Fig. 2 Schematics scheme for integrating L-DEP and P-DEP structures

8 3(A) 3(B) 3(C) 3(D) Fig. 3 Time sequence response of liquid actuation at an applied AC voltage of 220 V RMS and frequency 100 khz; 3(A) Initial parent droplet before applying voltage; 3(B) Liquid rivulet projecting from the parent droplet; 3(C) Rivulet traveling to the end of the structure to balance the hydrodynamic equilibrium; 3(D) Breaking-up to form a droplet at the second bump Droplets missing the first bump were considered due to the fairly larger bump size compared to the rivulet radius. Fig. 6A Fig. 6B Fig. 6 +ve and ve DEP response of yeast cells; Fig. 6A +ve DEP of yeast cells at an applied voltage of 2.47 V RMS and frequency 500 khz; Fig. 6B - ve DEP of yeast cells under the same experimental conditions with the AC field frequency changed to 10 MHz