Additive-Free Digital Microfluidics - Langmuir (ACS Publications)

Jun 11, 2013 - Here, a new strategy is introduced to move droplets containing cells and other analytes on solid substrates, without extra moieties; in...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/Langmuir

Additive-Free Digital Microfluidics Sergio L. S. Freire* and Brendan Tanner Department of Mathematics, Physics and Statistics, University of the Sciences, 600 South 43rd Street, Philadelphia, Pennsylvania 19104, United States S Supporting Information *

ABSTRACT: Digital microfluidics, a technique for manipulation of droplets, is becoming increasingly important for the development of miniaturized platforms for laboratory processes. Despite the enthusiasm, droplet motion is frequently hindered by the desorption of proteins or other analytes to surfaces. Current approaches to minimize this unwanted surface fouling involve the addition of extra species to the droplet or its surroundings, which might be problematic depending on the droplet content. Here, a new strategy is introduced to move droplets containing cells and other analytes on solid substrates, without extra moieties; in particular, droplets with bovine serum albumin could be moved at a concentration 2000 times higher than previously reported (without additives). This capability is achieved by using a soot-based superamphiphobic surface combined with a new device geometry, which favors droplet rolling. Contrasting with electrowetting, wetting forces are not required for droplet motion.

■

Here, for the first time (to the best of our knowledge), a strategy is introduced to allow the manipulation of droplets containing the protein BSA, or cells suspended in media, directly on solid substrates without any extra additives. This is achieved by combining the soot-based superamphiphobic surface with a new device geometry, which allows an optimized force distribution that favors droplet rolling. Also, an additional force component, acting upward on the droplet, further decreases droplet−surface interaction; no wetting is required for device operation. These devices are termed field dewetting (field-DW) devices. At frequencies below 18 kHz, the mechanism of droplet motion in DMF is mainly associated with electrowetting (EW) on a dielectric (see ref 13, particularly eqs 30a and 30b). Apparently, dielectrophoresis (DEP) is the main mechanism in field-DW, generally associated with smaller forces. Note, however, that the distinction between DEP and EW is not necessary for the force calculations done here. The model organism Tetrahymena thermophila was used in the experiments. It is a 50 μm long motile cell, widely used for studies on motility14 and genetics.15 Cilia (each cilium is ∼5 μm long), distributed along the cell body, are the structures responsible for cell motion. These cells are easy to culture, nonpathogenic, and suitable for our future studies on ciliary motility and molecular motors. Note that our previous work showed that Tetrahymena cells suspended in media cannot be transported by EW without additives;16 the application of voltage spreads the droplet on surfaces, preventing motion. The choice of cells in suspension

INTRODUCTION Digital microfluidics (DMF) is a technique for manipulation of droplets on surfaces by an electric potential applied to an array of electrodes.1,2 DMF allows automation and control of droplets unparalleled by any other technique of fluid transport, which makes it particularly attractive for low-volume applications involving a variety of bioanalytes, including enzyme assays,3 protein analysis,4 and cell-based assays.5 One technological limitation preventing an even broader usage of DMF is surface fouling (or biofouling). This unwanted desorption of proteins or other analytes to surfaces hinders droplet transport. Current approaches to reduce biofouling include the control of droplet pH,6 which is complicated for samples with a variety of chemical species, Pluronic additives,7,8 the use of oil,9,10 which is the most common alternative, or the use of graphene oxide.11 Altogether, these approaches involve the addition of extra species to the droplet or its surroundings, which might be problematic depending on the droplet constituents; for example, some Pluronic solutions are cytotoxic,7 and nonpolar solutes can migrate into the oil. Also, the stability of the oil film is dependent on the droplet protein content.9 With the inclusion of graphene oxide, the maximum concentration of bovine serum albumin (BSA) for droplet motion is about 0.3 g/L (30 times less than here; see the Experimental Results and Discussion); also, graphene oxide adsorbs to proteins. Altogether, alternatives that do not alter droplet chemistry would be preferable. Recently, candle soot has been proposed as a template to fabricate superamphiphobic surfaces, i.e., surfaces that are simultaneously superhydrophobic and superoleophobic, which are not wetted by liquids with relatively low surface tension.12 We expand the application of soot to improve the performance of digital microfluidic devices. © XXXX American Chemical Society

Received: October 28, 2012 Revised: June 11, 2013

A

dx.doi.org/10.1021/la401616j | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

the nine components of T⃡ has the average ⟨Tij⟩ = 1/2 Re[εẼ iẼ j* − 0.5εδijẼ ·Ẽ *], where δij is the Kronecker delta and the asterisk refers to the complex conjugate of the complex ac electric field Ẽ .20

does not necessarily limit the results presented here; in fact, Tcells (human leukemia lymphocites) also need additives (Pluronics, in this case) to diminish surface fouling in EW.5 As mentioned above, it is well-known that when appreciable biofouling occurs, droplets become stuck, rendering DMF devices inoperable; although yet with limited studies, the investigation suggests that the converse is also true in the devices developed here: droplet motion is correlated with reduced desorption of proteins to surfaces. Below, theory and simulations are presented to allow design optimization and performance comparison between EW devices and the proposed architecture. The experimental results are then discussed, including the actuation of T. thermophila cells and BSA in field-DW devices.

■

Recently, candle soot has been proposed as a template to fabricate superamphiphobic surfaces.12 Here, the procedure was modified to expedite fabrication, while allowing at least partial protection of the soot network texture. Copper laminates (35 μm thick, LF9110, from DuPont Flexible Circuit Materials, Research Triangle Park, NC) were attached to glass slides (75 × 25 mm) using double-sided tape, coated with candle soot (typically for 30 s for a ∼30 μm thick layer), and protected with a gold layer (150−200 nm thick) using the sputter coater Desk V HP equipped with a Au target (Denton Vacuum, Moorestown, NJ). After the coating, the surface was made amphiphobic by dip-coating (10 min, inside a chemical hood) in a 1-dodecanethiol solution (1%, v/v, in ethanol). The surface was then gently washed with a few droplets of ethanol and dried in air. These devices were termed gold-coated soot, GS, devices. In other cases, for even faster fabrication of several surfaces required for multiple experiments, after coating with candle soot (∼45 s), copper substrates (75 × 43 mm, 0.5 mm thick) were moved into a chemical hood, coated only with Teflon-AF (Dupont, Wilmington, DE) (Teflon-AF resin in Fluorinert FC-40, 1:100 (w/w)), and then baked on a hot plate (160 °C, 15 min) (these devices were termed Teflon-coated soot, TS, devices). For this method to work, droplets of Teflon-AF were deposited on the edges of the soot-coated substrate while warm from the candle flame; the substrate was then immediately inclined at an angle close to 90°, and more droplets of Teflon-AF were carefully dispensed until the entire surface was covered (these procedures were done inside a chemical hood). Note that the best approach was to let the droplet spread as much as possible on the soot surface; when the droplet fell directly on a spot, soot was washed away from that area and dipcoating in Teflon-AF readily damaged the soot surface. The choice of copper as the substrate in the two methods above was a matter of convenience; other conductive materials can be used as well. Top electrodes (see the section “Simulations”) of field-DW devices (2 mm long, 0.3 mm wide) were fabricated by etching a patterned copper laminate (35 μm thick).21 The distance between electrodes was 0.3 mm, and they required no coating since they did not contact the droplets. However, to prevent accidental contact of the droplet and top electrodes, which could produce a short circuit and electrode damage, a 30 μm film of PFA (perfluoroalkoxy, from McMaster-Carr, Robbinsville, NJ) was attached to the top electrodes using a piece of tape on the top of the device. Droplets were actuated by applying voltages (8−18 kHz, 500−660 VRMS (root mean squared voltage)) to the top electrodes and were obtained by a signal generator (33220A, Agilent Technologies, Santa Clara, CA), connected to a high-voltage amplifier (PZD700, Trek, Inc., Medina, NY). Operations were monitored by a Hitachi charge-coupled device (CCD) camera and an imaging system (VZM 200i, Edmund Optics, Barrington, NJ). 1-Dodecanethiol, dibucaine, and Triton X-100 were from SigmaAldrich (St. Louis, MO). T. thermophila cells (strain SB 255), from the Tetrahymena stock center at Cornell University (Ithaca, NY), were cultured in media (0.25% proteose peptone, 0.55% glucose, 0.25% yeast extract, 33 μM FeCl3)22 at room temperature. The cell concentration was about 105 cells/mL. Often, the cell droplet volume was larger than 2 μL for optimal off-chip manipulation since smaller volumes tended to stick to the pipet tips during handling. BSA was from New England Biolabs (Ipswich, MA), and BSA Alexa Fluor 488 conjugate (fluorescently tagged BSA) was from Invitrogen (Grand Island, NY). Indium tin oxide (ITO)-coated glass slides were from Delta Technologies, Ltd. (Stillwater, MN). They were coated with Teflon-AF to provide a reference surface for studies of protein attachment.

■

THEORY To obtain the electric potential and electric field distribution in digital microfluidic devices, the Gauss law (eq 1) and the equation for charge conservation (eq 2) were used:17 ρ ∇·E = free (1) ε ∂ρfree ∂t

+ ∇·J = 0

(2)

where E is the electric field, ρfree is the volumetric free charge density, ε = εrε0 (εr is the relative permittivity of the medium, ε0 is the vacuum permittivity, and ε is the permittivity of the medium), and J is the current density. It is assumed that magnetic fields are negligible and that all materials are ohmic, i.e., J = σE, where σ is the electrical conductivity. Taking the time derivative on both sides of eq 1, and including eq 2, one obtains ⎛ ∂E ⎞ ∇·⎜ε + σ E⎟ = 0 ⎝ ∂t ⎠

(3)

The electric potential Φ can be defined, being associated with the electric field by the equation E = −∇Φ. When a sinusoidal voltage (ac) is applied to the devices, the electric potential Φ can be described by Φ = Re{φejωt},where j is (−1)1/2, ω = 2πf is the angular frequency, and φ is the complex electric potential, or the phasor of the electric potential, which is time-independent. The associated ac electric field is given by E = Re{Ẽ ejωt}, where Ẽ = −∇φ. Substituting Ẽ into eq 3, and keeping in mind that in a harmonic field the operator ∂/∂t ⇒ jω,18 one obtains ∇·[(σ + jωε)∇φ] = 0

(4)

This equation was solved numerically using the commercial software COMSOL Multiphysics (version 4.3). At the interfaces between materials A and B, current continuity was considered, i.e., (JÃ − JB̃ )·n = 0, where the complex current density is given by J ̃ = (σ + jωε)Ẽ . Also using COMSOL, the average force ⟨F⟩ on a droplet was obtained by integrating the average of Maxwell’s stress tensor (T⃡ ) over the exterior droplet surface,17,19 according to ⟨Fi ⟩ =

∮s ⟨Tij⟩nj da

MATERIALS AND METHODS

(5)

where the indices i and j refer to the coordinates x, y, and z, the Einstein convention is considered (summation over repeated indices), and n is the unity vector normal to the area. Each of B

dx.doi.org/10.1021/la401616j | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

Confocal microscopy was used for the analysis of BSA adsorbed to surfaces. The system was a Nikon TiE (Eclipse) confocal microscope with a CSU-X spinning disk confocal scan head (Yokogawa). An electronic filter wheel was used for selection of the Alexa Fluor fluorescence emission filter (centered at 525 nm, Chroma Corp., ET525/36m); excitation (488 nm) was produced by a 50 mW laser (MLC400, Agilent Technologies). Images were acquired using the Clara Interline CCD camera (Andor Technology) in conjunction with a 40× objective (numerical aperture (NA) 0.6). Experimental parameters were optimized to prevent saturation of the CCD (including exposure time, 500 ms) with signal from the ITO-coated substrates (the most intense emissions). In each region of interest (ROI) in the various samples, corresponding to the signal and background, the fluorescence was quantified by integrating the brightness values over all pixels. All measurements were background corrected by subtracting the background ROI from the corresponding integrated value. Fluorescence signals of the field-DW substrates were compared with signals from ITO slides coated with Teflon-AF. A two-tailed Wilcoxon−Mann−Whitney test was used to determine if the difference between the two groups of samples was statistically significant at α = 0.05. A nonparametric method was deemed necessary due to the small sample size and the lack of normality of the data; however, there is a significant difference in normalized fluorescence between the two groups of samples (p = 0.0167). For the confocal studies above, stock solutions (10 mg/mL) of BSA Alexa Fluor 488 conjugate were prepared in 10 mM Tris−HCl (pH 7.8) buffer containing 1.8 mM sodium azide. These solutions were stored in the dark at −20 °C until ready to use. The buffer without proteins was stored at room temperature and used to wash BSA droplets from ITO-coated substrates. In the procedures described in this paper, laboratory safety guidelines should always be followed. Table 1 shows the physical properties of the materials considered in the simulations. The top electrodes in field-DW devices had the same

Figure 1. Simulations (∼2 μL DI water droplet) indicating the electric field distribution (only along the plane, x direction in simulations, for clarity) for weaker (gray) and stronger (blue) fields for EW (A) and (B) proposed devices. The arrows indicate the force along the plane and the predicted vertical forces, pushing the droplet against the surface (A) or pulling it upward (B). Voltages Φ (500 VRMS, 18 kHz) were applied to the bottom right electrode or to top electrodes 1−3.

gap between electrodes was 40 μm; the top electrode (top− bottom distance 0.4 mm) enclosed the device. Electrodes were modeled as made of copper. Hydrophobic layers on the top electrode and on Parylene were disregarded since they are much thinner (∼100 nm) than the Parylene layer (2−10 μm) in EW devices.13 A voltage Φ (500 VRMS, 18 kHz) was considered applied as indicated. The force along the plane, responsible for droplet motion, was ∼150 μN, compatible with the range of values measured by other authors (100−300 μN).27 They also reported downward forces on droplets in the millinewton range, in agreement with our results, which indicated a large vertical force (∼5 mN) pushing the droplet against the dielectric layer. For further validation, an open EW device (instead of enclosed) was also modeled. Once again, a downward net force on the droplet was obtained, as previously observed (see Figure 3 of Abdelgawad et al.25). Different designs were simulated to study force distribution in DMF. The one adopted here uses top electrodes with ac voltages, located slightly (0.1−0.5 mm) above the droplet but not touching it. This geometry has the advantages of (1) creating an upward force, which pulls the droplet away from the substrate, and (2) favoring droplet rolling. A sequential application of voltages to closely spaced top electrodes created an imbalance in the electric field (Figure 1B), enabling motion along the plane. This structure was modeled as shown in Figure 1B. A DI water droplet (∼2 μL) was on top of Parylene-C (10 μm thick), 0.3 mm below a set of top electrodes. The voltages were also 500 VRMS at 18 kHz. The results indicated an upward force of ∼4 μN, with a weak force component along the plane (∼3 μN). Note that this force is approximately 50 times lower than in EW, indicating that droplet motion along the plane would be possible only under very low friction between the droplet and substrate (close to the friction required for rolling only, i.e., without dissipative forces). For water droplets, this could be accomplished by using superhydrophobic surfaces;28−31 for droplets containing cells in media or other moieties used here, the soot-based superamphiphobic substrate resulted in an effective choice. To obtain forces in the devices on soot-based surfaces (termed field dewetting, field-DW, devices) presented below, simulations disregarded layers of Teflon-AF and 1dodecanethiol (see the Materials and Methods). The simulations indicated that the force along the plane on the droplet (2 μL) with cells is ∼3 μN, contrasting with a larger force in EW (∼60 μN), considering the same applied voltage

Table 1. Properties of the Materials Used in the Simulations σ (S/m) air soot (see the text) DI water25

0 1000 5.5 × 10−6

εr 1 2

Parylene-C26 PBS5

σ (S/m)

εr

1 × 10−15 4.7

3.1 70

80

dimensions as the fabricated ones (see above). To validate the experimental results, droplets with cells in media were modeled as composed by PBS (phosphate-buffered saline). Some considerations about candle soot are required. Soot is a byproduct of the combustion of carbonaceous materials, and its composition can vary significantly. This implies variations in the conductivity and dielectric constant, which are difficult to track. By design, however, the air layer between the top electrodes and droplet (0.1−0.5 mm thick; see below) in the field-DW devices is responsible for the largest voltage drop in the device because of its low conductivity (close to zero20) compared to those of the water (or PBS) and soot layers. In fact, the admittance (the ac equivalent of the dc conductance) of the air layer is small compared to that of other elements; the air layer is almost an open circuit. This implies a large insensitivity to the parameters of the soot layer, and to run the simulations, the electrical conductivity σ and relative dielectric constant εr of candle soot were assumed to be 1000 S/m (a guess for diesel soot23) and 2 (assumed as close to that of carbon black24), respectively.

■

SIMULATIONS A typical enclosed EW device1 was modeled as shown in Figure 1A. A deionized (DI) water droplet (∼2 μL), surrounded by air, was on top of a Parylene-C layer (10 μm thick). The bottom electrodes were 1.9 mm long and 2.7 mm wide, and the C

dx.doi.org/10.1021/la401616j | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

predicted by simulations, the electric field responsible for the horizontal motion also created an upward, AC dewetting force (∼6 μN) on the droplet. This vertical force, associated with the weak interaction with the substrate, could almost entirely lift a DI water droplet (Figure 2C). For the tests described here, droplets with cells could be moved on at least 3 GS and 3 TS devices, with a minimum of 10 transfer operations. Note that the results above contrast with their EW counterparts, in which motion occurred only with the introduction of oil; without it, our experiments in EW16 showed that droplets with cells in media spread following the application of voltage and could not be moved. In fact, here, simulations indicated a net force of electric origin in EW (∼2 mN), acting downward on a 2 μL droplet with cells on top of Parylene-C (30 μm thick); this force correlates with the decrease in the contact angle.32 Also, note that this wetting force is strong (∼30 times stronger than the component responsible for horizontal motion), favoring surface fouling. Altogether, Tetrahymena (and other cells5) in their media cannot be transported by EW without additives. Previously, dc electric fields have been shown to produce an upward force on water droplets,30 which was used to reverse wetting. Here, droplets do not touch the top surface, and besides the easier fabrication, displacement and upward (AC dewetting) forces occurred simultaneously in field-DW devices: wetting forces were not required for operation. Recombination (merging) of droplets of different volumes was immediate. In particular, Figure 2D shows the deciliation of Tetrahymena cells by the anesthetic dibucaine33 (17 mM final concentration). In about 10−15 min, cells were concentrated at the bottom of the droplet (not shown) after becoming immotile due to loss of cilia (note that cell lysis could have happened as well33). However, experiments showed that droplets of similar volumes (with cells or DI water only) tend not to recombine. Simulations indicated that, as two droplets get closer, they are subjected to smaller, or even repulsive, forces, preventing recombination; therefore, concentrations should be tailored to have droplets of different volumes for immediate recombination. One limitation, however, is that splitting cannot be performed, which limits the number of mixing operations. Droplets with BSA, a sticky protein that adsorbs to solid surfaces, were successfully transported in the field-DW devices, without any extra additives (Figure 3A; for these tests, 4 μL droplets of BSA were moved across at least 10 positions on 4 different devices). The concentration (10 mg/mL) was 2000 times higher than previously reported (0.005 mg/mL8,34). In EW devices, it has been shown that BSA droplets in the milligram per milliliter concentration range could only be transported by using oil1 or Pluronics.8 Altogether, the reduced wetting/adhesive force between the droplet and substrate is key for device operation, also allowing the successful transport of droplets containing a 0.01% concentration of the detergent Triton X-100 in DI water, also without extra additives. Although preliminary (the droplet could be displaced a few times only), we believe that this has been accomplished for the first time in DMF. TS substrates supported approximately 2000 droplet transfers (cells or BSA) for a maximum of 20 min. Approximately 40% (10 out of 25) of the substrates worked. The main problem was that the Teflon solution partially/completely washed out the soot pattern during the coating; with practice,

(500 VRMS, 18 kHz) and same soot and Parylene-C thickness (30 μm), respectively. Although forces change with droplet position, this number is representative of an average force in EW;25 therefore, considering the same droplet displacement, the estimated work (force times displacement) performed, and the associated energy to move a droplet along the plane, is 20 times less in the field-DW devices (see the Supporting Information for further comments). The distance between the droplet and top plate was adjusted for optimal droplet displacement during each experiment (also, the experiments have shown that the vertical force could be fine-tuned by changing the frequency between 8 and 18 kHz). Note that, if the top plate was too close to the droplet, and depending on the adhesion force between the droplet and substrate, the droplet could be attracted to the top electrodes, hindering motion. Here, approximately three top electrodes (0.3 mm each) with a 0.3 mm gap between two electrodes equaled the diameter of the droplet. These dimensions were compatible with the resolution of the printer used to fabricate the electrodes and were not unique; narrower and closer electrodes would also work. What matters is subjecting the droplet to a nonuniform electric field, which will make it move toward the region where the field is more intense.

■

EXPERIMENTAL RESULTS AND DISCUSSION The field-DW devices (Figure 2A) allowed successful manipulation of droplets with T. thermophila cells in media

Figure 2. (A) Schematic view of a droplet with cells in a field-DW device (not to scale). Top coatings were formed by 1-dodecanethiol on gold or by Teflon-AF (see Materials and Methods). The inset shows a picture of a Tetrahymena cell. (B) Motion of a 2 μL droplet with cells along seven positions. (C) Note the reduced interaction of a DI water droplet (∼0.4 μL) with the surface, particularly in the picture on the right. (D) Merging of two droplets, one with cells (10 μL) and another with dibucaine (2 μL). Dibucaine droplets could also be moved (not shown).

(2 μL), without oil or any extra additives (Figure 2B). Note the high contact angle (CA) (∼160°) [experiments have shown that if the CA is below ∼150°, the droplets will not be movable; the forces of electrical origin could not overcome the adhesion forces between the droplet and surface], approximately ∼5− 10° larger than before the voltage was applied, corresponding to a small deviation from the droplet spherical profile: as D

dx.doi.org/10.1021/la401616j | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

tilted to allow droplet roll-off. Substrates were stored in the dark. Currently, ITO slides coated with Teflon-AF are widely employed in DMF and were used here as references for BSA adhesion and the associated fluorescence intensity. Droplets were directly pipetted onto the ITO-coated substrates (two samples, three different droplets) and left sitting in the same spot for 20 min; they were then washed off with droplets of Tris−HCl. Subtrates were stored in the dark. Note that the normalized intensity indicated a larger amount of protein adhesion to the ITO surfaces coated with Teflon-AF when compared to the field-DW counterparts (Figure 4). Figure 3. (A) Motion sequence of a BSA droplet (4 μL) on a TS substrate and (B) for a fluorescently tagged BSA droplet (4 μL) on another TS substrate. The BSA concentration is 10 g/L in both cases. The inset shows the droplet under white light illumination. As the droplet moves to the right (C) or left (D), note the liquid menisci (indicated by the arrows) working against motion. As the surface degraded, further motion was allowed by decreasing the distance between the top electrodes and droplet, which increased the upward and along the plane force components on the droplet; note that a stretched up liquid bridge was formed (see arrow), a consequence of the applied vertical force (E). At 25 min, the surface degraded to a point in which increasing the vertical force caused the permanent attachment of the droplet to the top electrodes, leaving a fraction (indicated by the arrow) attached to the substrate (F).

we were able to achieve a 50% (19 out of 37) success rate; it was, however, an easy surface to fabricate, required minimal resources, and could be reused. GS substrates supported approximately 700 droplet transfers for a maximum of 9 min. All of the fabricated substrates supported droplet motion for gold layers 150−200 nm thick (five substrates). In any substrate, the maximum transfer speed was ∼6 cm/s, limited, most possibly, by the minimum actuation time of the relays in the automated setup.16 Fluorescently tagged BSA allowed for a closer inspection of the interaction between the droplet and surface in a typical experiment. A blue LED (light-emitting diode; λ ∼ 466 nm) was used to illuminate the droplet, which appeared in a green color due to the Alexa fluor emission (centered at 519 nm). When the droplet moved (Figure 3B; also see movie S1 in the Supporting Information, showing the motion sequence of a droplet), note the presence of liquid menisci, acting against rolling (Figure 3C,D). As the surface deteriorated, the distance between the top electrodes and substrate was lowered, increasing forces on the droplet and diminishing the interaction between the droplet and surface (Figure 3E). This allowed for an additional 2 min of continuous droplet motion, after which the surface degraded to a point that prevented further droplet motion. Now, decreasing the distance between the top electrodes and droplet produced such a high vertical force that the droplet was attracted to the top electrodes, leaving a fraction stuck to the substrate (Figure 3F); so a certain fraction of BSA was left behind (surface fouling). Confocal microscopy was used to evaluate surface desorption of BSA. After confirmation of droplet mobility on each device (one back-and-forth motion), droplets (4 μL) containing BSA Alexa Fluor 488 conjugate (10 mg/mL) were left on the same position for a period of 20 min (chosen to be equal to the maximum operation time of the TS substrates, as mentioned above) on each substrate (seven field-DW devices). After this time, the electric field was deactivated and the substrates were

Figure 4. Fluorescence signal of the field-DW substrates compared with ITO slides coated with Teflon-AF. On the basis of the Wilcoxon−Mann−Whitney test, there is a significant difference in normalized fluorescence between the two groups of samples (p = 0.0167). The insets show the droplets after 20 min sitting on the same spot on a field-DW device (left) and immediately after being pipetted onto a coated ITO slide (right) when illuminated with blue light (see the text).

A blue LED was used to illuminate the droplets for the pictures to be taken (Figure 4, insets). When the droplets were washed away from the coated ITOs, a green emission, corresponding to the emission of the dye, could easily be seen by the naked eye; this did not happen with the field-DW devices: no fluorescence could be seen. In any case, the blue light was kept on just for a few seconds to prevent photobleaching. In summary, the experiments indicated a correlation between the device lifetime and the time during which BSA desorption was negligible (when compared to an ITO-coated substrate). After this time period (∼20 min), droplets would increasingly stick to the surfaces until motion ceased completely (note that Figure 3 C−F shows one particular case, but similar degradation effects happened to other devices as well in a similar timeline). Attempts to increase the forces led to the detachment of droplets from the bottom substrate, leaving an amount stuck on the substrate. Since contact line pinning, contact line friction, and wall shear30 are reduced in field-DW, it is reasonable to believe that droplet motion was associated with reduced biofouling in the field-DW devices. However, more experiments are under design to further characterize surface desorption of bioanalytes, which is a complex matter, also dependent on the protein/bioanalyte being transported. E

dx.doi.org/10.1021/la401616j | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

droplet motion. This material is available free of charge via the Internet at http://pubs.acs.org.

Finally, simulations indicated that forces increase by only 7% if the soot thickness increases from 30 to 300 μm for a fixed distance between the top electrodes and a 2 μL droplet with cells. Furthermore, the experiments showed that the top− droplet distances could vary along the droplet path by ∼100 μm without hindering droplet transport. Therefore, field-DW devices tolerate variations in thickness and uniformity of the soot layer, convenient for candle soot deposition.

■

Corresponding Author

*E-mail: [email protected]. Phone: 215-596-8958. Fax: 215-895-1112. Notes

■

The authors declare no competing financial interest.

■

SUMMARY AND CONCLUSIONS Here an experimental design is introduced to allow the actuation of droplets with cells and the protein BSA, without any extra additives. This contrasts with EW, where previous experiments have shown the collapse of droplets containing cells on surfaces, with catastrophic failure;16 in fact, Tetrahymena (and other cells5) in their media cannot be transported by EW without additives. The transport of BSA droplets also requires oil,1 Pluronics,8 or graphene oxide;11 otherwise, only very small concentrations (0.005 mg/mL8) can be moved by EW. Apparently, there is a correlation between continued motion and reduced surface desorption of BSA. Notwithstanding, biofouling is a complex matter, and some authors35 even suggest that it might be impossible to completely suppress the effect; in theory, if only a single protein attaches to a surface, more will be attracted to this site. In fact, the maximum operation time reported for DMF devices was about 40 min, with the addition of Pluronics.7 No wetting and changes in contact angle are required for droplet motion in the field-DW devices. Also, droplets deviate very little from the spherical profile, and there is no need to solve the Navier−Stokes equation to include droplet deformation, allowing a reasonable prediction of experimental trends/results with simpler and faster computer simulations. This agrees with previous results showing that changes in the contact angle and center-of-mass motion are distinct phenomena;36 although this is also true in EW, forces and changes in the contact angle are often related because of the strong electric forces in the contact line, which are balanced by surface tension.37 Note that the successful performance of field-DW devices stems from the geometry (which favors rolling and enables an upward force) combined with a superamphiphobic surface. The use of candle soot is an easy, cheap, and possibly an effective alternative to produce superamphiphobic surfaces for DMF. The characteristics of the field-DW devices are largely independent of the thickness, uniformity, and electrical properties of the soot layer (see the Materials and Methods). Furthermore, soot can be made transparent if necessary.12 Current efforts are under way to increase device longevity, improve the quality of the soot layer coating, and further address biofouling. Finally, since droplets can almost roll freely, field-DW devices might allow faster droplet motion when compared to EW, useful for the development of droplet displays.

■

AUTHOR INFORMATION

ACKNOWLEDGMENTS We thank the Lindback Foundation for financial support, Dr. Alexander Sidorenko and Elza Chu for fruitful discussions and technical assistance, Dr. Kenneth Myers for assistance with confocal microscopy, and Dr. Laura Pontiggia for assistance with statistical analysis of the data.

■

REFERENCES

(1) Fair, R. B. Digital microfluidics: is a true lab-on-a-chip possible? Microfluid. Nanofluid. 2007, 3 (3), 245−281. (2) Washizu, M. Electrostatic actuation of liquid droplets for microreactor applications. IEEE Trans. Ind. Appl. 1998, 34, 732−737. (3) Miller, E. M.; Wheeler, A. R. A digital microfluidic approach to homogeneous enzyme assays. Anal. Chem. 2008, 80 (5), 1614−1619. (4) Nelson, W. C.; Peng, I.; Lee, G.-A.; Loo, J. A.; Garrell, R. L.; Kim, C.-J. Incubated protein reduction and digestion on an electrowettingon-dielectric digital microfluidic chip for MALDI-MS. Anal. Chem. 2010, 82 (23), 9932−9937. (5) Barbulovic-Nad, I.; Yang, H.; Park, P. S.; Wheeler, A. R. Digital microfluidics for cell-based assays. Lab Chip 2008, 8 (4), 519−526. (6) Yoon, J.-Y.; Garrell, R. L. Preventing biomolecular adsorption in electrowetting-based microchips. Anal. Chem. 2003, 75, 5097−5102. (7) Au, S. H.; Kumar, P.; Wheeler, A. R. A new angle on Pluronic additives: advancing droplets and understanding in digital microfluidics. Langmuir 2011, 27 (13), 8586−8594. (8) Luk, V. N.; Mo, G. C. H.; Wheeler, A. R. Pluronic additives: a solution to sticky problems in digital microfluidics. Langmuir 2008, 24 (12), 6382−6389. (9) Srinivasan, V.; Pamula, V. K.; Fair, R. B. An integrated digital mirofluidic lab-on-a-chip for clinical diagnostics on human physiological fluids. Lab Chip 2004, 4, 310−315. (10) Velev, O. D.; Prevo, B. G.; Bhatt, K. H. On-chip manipulation of free droplets. Nature 2003, 426 (6966), 515−516. (11) Perry, G.; Thomy, V.; Das, M. R.; Coffinier, Y.; Boukherroub, R. Inhibiting protein biofouling using graphene oxide in droplet-based microfluidic microsystems. Lab Chip 2012, 12 (9), 1601−1604. (12) Deng, X.; Mammen, L.; Butt, H.-J.; Vollmer, D. Candle soot as a template for a transparent robust superamphiphobic coating. Science 2011, 335, 67−70. (13) Jones, T. B. More about the electromechanics of electrowetting. Mech. Res. Commun. 2009, 36 (1), 2−9. (14) Seetharam, R. N.; Satir, P. Coordination of outer arm dynein activity along axonemal doublet microtubules. Cell Motil. Cytoskeleton 2008, 65 (7), 572−80. (15) Aldag, I.; Bockau, U.; Rossdorf, J.; Laarmann, S.; Raaben, W.; Herrmann, L.; Weide, T.; Hartmann, M. Expression, secretion and surface display of a human alkaline phosphatase by the ciliate Tetrahymena thermophila. BMC Biotechnol. 2011, 11 (1), 11. (16) Thorne, N.; Lamberto, M.; Mazza, A.; Freire, S. L. S. An alternative strategy for fabrication of robust and flexible digital microfluidic devices. Pacific Northwest J. Undergrad. Res. Creat. Act. 2011, 2 (1), No. 2 (17) Griffiths, D. J. Introduction to Electrodynamics; Prentice Hall: Upper Saddle River, NJ, 1999. (18) Morgan, H.; Green, N. G. AC Electrokinetics: Colloids and Nanoparticles; Research Studies Press Ltd.: Baldock, Hertfordshire, England, 2003.

ASSOCIATED CONTENT

S Supporting Information *

Movie showing a motion sequence of a fluorescently tagged BSA droplet (4 μL), movie showing motion between two positions of a droplet containing cells (2 μL) on a field-DW device, and a brief additional discussion on the characteristics of F

dx.doi.org/10.1021/la401616j | Langmuir XXXX, XXX, XXX−XXX

Langmuir

Article

(19) Berthier, J. Microdrops and Digital Microfluidics; William Andrew: Norwich, NY, 2008. (20) Hong, J.; Ko, S.; Kang, K.; Kang, I. A numerical investigation on AC electrowetting of a droplet. Microfluid. Nanofluid. 2008, 5 (2), 263−271. (21) Abdelgawad, M.; Watson, M. W. L.; Young, E. W. K.; Mudrik, J. M.; Ungrin, M. D.; Wheeler, A. R. Soft lithography: masters on demand. Lab Chip 2008, 8 (8), 1379−1385. (22) Cassidy-Hanley, D.; Bowen, J.; Lee, J. H.; Cole, E.; VerPlank, L. A.; Gaertig, J.; Gorovsky, M. A.; Bruns, P. J. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics 1997, 146, 135−147. (23) Grob, B.; Schmid, J.; Ivleva, N. P.; Niessner, R. Conductivity for soot sensing: possibilities and limitations. Anal. Chem. 2012, 84 (8), 3586−3592. (24) Speight, J. G. Perry’s Standard Tables and Formulas for Chemical Engineers. McGraw-Hill: New York, 2003; p 66. (25) Abdelgawad, M.; Park, P.; Wheeler, A. R. Optimization of device geometry in single-plate digital microfluidics. J. Appl. Phys. 2009, 105 (9), 094506-7. (26) Parylene conformal coating specifications & properties. http:// www.nbtc.cornell.edu/facilities/downloads/ Parylene%20Information%20Sheets.pdf (extracted from the Specialty Coating Systems Web site); Accessed on June 23, 2013. (27) Crane, N. B.; Mishra, P.; Volinsky, A. A. Characterization of electrowetting processes through force measurements. Rev. Sci. Instrum. 2010, 81 (4), 043902−7. (28) Dash, S.; Alt, M. T.; Garimella, S. V. Hybrid surface design for robust superhydrophobicity. Langmuir 2012, 28 (25), 9606−9615. (29) Jonsson-Niedziolka, M.; Lapierre, F.; Coffinier, Y.; Parry, S. J.; Zoueshtiagh, F.; Foat, T.; Thomy, V.; Boukherroub, R. EWOD driven cleaning of bioparticles on hydrophobic and superhydrophobic surfaces. Lab Chip 2011, 11 (3), 490−496. (30) Kumari, N.; Garimella, S. V. Electrowetting-induced dewetting transitions on superhydrophobic surfaces. Langmuir 2011, 27 (17), 10342−6. (31) Manukyan, G.; Oh, J. M.; van den Ende, D.; Lammertink, R. G. H.; Mugele, F. Electrical switching of wetting states on superhydrophobic surfaces: a route towards reversible Cassie-to-Wenzel transitions. Phys. Rev. Lett. 2011, 106 (1), 014501. (32) Lee, J.; Moon, H.; Fowler, J.; Schoellhammer, T.; Kim, C.-J. Electrowetting and electrowetting-on-dielectric for microscale liquid handling. Sens. Actuators, A 2002, 95, 259−268. (33) Thompson, G. A., Jr.; Baugh, L. C.; Walker, L. F. Nonlethal deciliation of Tetrahymena by a local anesthetic and its utility as a tool for studying cilia regeneration. J. Cell Biol. 1974, 61 (1), 253−7. (34) Yoon, J. Y.; Garrell, R. L. Preventing biomolecular adsorption in electrowetting-based biofluidic chips. Anal. Chem. 2003, 75 (19), 5097−5102. (35) Koc, Y.; de Mello, A. J.; McHale, G.; Newton, M. I.; Roach, P.; Shirtcliffe, N. J. Nano-scale superhydrophobicity: suppression of protein adsorption and promotion of flow-induced detachment. Lab Chip 2008, 8 (4), 582−586. (36) Jones, T. B. On the relationship of dielectrophoresis and electrowetting. Langmuir 2002, 18 (11), 4437−4443. (37) Kang, K. H. How electrostatic fields change contact angle in electrowetting. Langmuir 2002, 18 (26), 10318−22.

G

dx.doi.org/10.1021/la401616j | Langmuir XXXX, XXX, XXX−XXX