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Applications of electrowetting-on-dielectric (EWOD) technology for droplet digital PCR
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Abstract
Digital microfluidics is an elegant technique based on single droplets for the design, composition, and manipulation of microfluidic systems. In digital microfluidics, especially in the electrowetting on dielectric (EWOD) system, each droplet acts as an independent reactor, which enables a wide range of multiple parallel biological and chemical reactions at the microscale. EWOD digital microfluidics reduces reagent and energy consumption, accelerates analysis, enables point-of-care diagnostic, simplifies integration with sensors, etc. Such a digital microfluidic system is especially relevant for droplet digital PCR (ddPCR), thanks to its nanoliter droplets and well-controlled volume distribution. At low DNA concentration, these small volumes allow less than one DNA strand per droplet on average (limited dilution) so that after a fixed number of PCR cycles (endpoint PCR), only the DNA in droplets containing the sequence of interest has been amplified and can be detected by fluorescence to yield an accurate count of the sequences of interest using statistical models. Focusing on ddPCR, this article summarizes the latest development and research on EWOD technology for droplet PCR over the last decade.
I. INTRODUCTION
Digital microfluidic technology is a novel microscale liquid processing technology in which ultra-small droplets can be operated.1 Different droplet driving methods have been studied, including thermal capillary forces,2–4 surface acoustic wave (SAW),5,6 dielectrophoresis,7–10 photoelectric wetting,11–14 mechanical drive,15,16 and EWOD.17–28 Among them, EWOD-based device has been used for electronic cooling,25,26 optical display and lens systems,29 micro viscometers,30 micro conveyor systems,31 particle sampling and separation,32–35 chemical synthesis,36 and microbubbles manipulation.26 Nevertheless, the most popular applications exist in the field of biochemical medical research, where EWOD versatility and reconfigurability have addressed some of the key challenges in developing lab on chip (LOC) technology. Fast droplet transportation and easy sensor integration are also cited as key advantages of EWOD-based microfluidic technology. Therefore, EWOD devices have been widely used to perform on-chip creation and distribution of droplets.20,27,37,38 However, much work so far has focused on the electrowetting theory39,40 and operating characteristics of EWOD devices,41 while the actual applications of EWOD are still at a very early stage.
PCR is an in vitro technique that amplifies DNA and generates millions of copies of specific fragments of DNA from a very small amount of starting materials.42 With the development of quantitative real-time PCR (qPCR) technology, this technology enables quantitative analysis by using fluorescent probes to monitor the progress of amplification after each cycle, which is the gold standard for diagnosing infectious diseases in nucleic acid amplification.43–45 However, the final detection sensitivity and accuracy of qPCR is significantly affected by differences in primer efficiency and template concentration. Droplet digital PCR addresses these issues by amplifying each fragment of DNA independently, at the cost of increased complexity. Indeed, on top of all the steps needed for qPCR, ddPCR additionally requires generating a highly monodisperse population of nanoliter droplets. Among the possible automation methods of ddPCR, electrowetting microfluidic technology combined with resistive microheater and temperature sensors have demonstrated precise droplet volume distribution and microliter sample processing capabilities, which makes it a promising method for PCR automation and miniaturization.43
Digital PCR (dPCR) is a method for absolute measurement of nucleic acid concentration. The difference with PCR is that dPCR does not rely on calibration curves for sample quantification, which avoids the trap from reaction efficiency.46 Instead, the DNA sample is highly diluted such that, on average, less than one fragment of DNA is found per droplet (limiting dilution). Each droplet behaves as an independent micro-reactor, such that, after amplification, the fraction of positive droplets directly indicates the amount of targeted DNA in the original sample, which allows for the computing of the concentration of the target sequence using Poisson statistics.47,48 dPCR changes quantization to a series of enumerations of positive and negative results with continuous or analog signals converted into binary, i.e., digital signals. In 1999, Vogelstein and Kinzler used microplates for dPCRs for the first time, which resulted in a high-throughput strategy.49–51 Efforts on dPCR are currently under way to be applied to gene mutation analysis,52,53 prenatal diagnosis of chromosomal abnormalities,54 DNA copy number determination,53,55 pathogen detection,56,57 genetic testing,58 etc. The application of the innovative method in nucleic acid detection and quantitative analysis has been promoted by the huge improvements of dPCR chip, which provides high throughput, high sensitivity, and high precision microfluidic technology.59
In dPCR, the sample is divided into many independent PCR sub-reactions in microdroplets (nL to pL range) so that each droplet contains exactly one or no target sequence. Sample partitioning can effectively concentrate the target sequence in the separate microreactors. With the development of droplet PCR and EWOD, dPCR will provide more and smaller droplets, more precise volume, which points to a great potential for droplet digital PCR (ddPCR) using EWOD technology. However, the combination of droplet PCR and EWOD into EWOD based ddPCR will pose additional challenges as well. Aiming to ddPCR, this article aims to summarize the latest research and development of EWOD technology for droplet digital PCR over the past ten years.
In recent years, miniaturization and automation of PCR systems have made tremendous progress, especially benefitting from microfluidic technology and advanced micromachining technology.31 In particular, the combination of PCR and EWOD microfluidics has received significant attention because of the opportunity for fully integrated automation. This review discusses the current progress on this top from the aspects of (1) surface chemistry, including reliability, feasibility, biocompatibility, and cross-contamination; (2) electrowetting challenges associated with ddPCR, such as droplet generation and splitting, driving voltage reduction, surface chemical modification of the EWOD device to improve the wettability, and droplet evaporation management during the PCR process; and (3) the existing EWOD–ddPCR platforms and their applications.
II. EWOD TECHNOLOGY FOR DROPLET DIGITAL PCR
A lot of research has focused on the development and improvement of the EWOD devices in order to apply it to droplet digital PCR. In addition to the traditional droplet splitting method, at present, several alternative approaches have been proposed to achieve efficient and precise droplet generation. EWOD devices also face the problem of excessive driving voltage and susceptibility to electrical breakdown. Improvement of dielectric performance and modification of the surface chemistry of EWOD devices are essential for droplet PCR.
A. Droplet generation by electrowetting
Lippmann found that the externally added net charge may change the capillary force at the solid–liquid interface. The concept of MEMS (Micro-Electro-Mechanical System) devices using electrical signals to control surface tension, namely, electrocapillarity or electrowetting, was introduced by Matsumoto and Colgate.60 Its high effectiveness and ease of implementation on the microscale make it very attractive for device miniaturization.
As shown in Fig. 1, when a voltage is applied, the charge decreases the free energy of the typically hydrophobic dielectric surface, forcing it to become more hydrophilic, and the contact angle of the droplet decreases. Unlike conventional electrowetting where droplets directly contact a conductive surface, this technique is called EWOD which has excellent reversibility in multilayer dielectric layers.61
Principle of EWOD. (a) Schematic configuration, (b) EWOD demonstration pictures. Reprinted with permission from Sung Kwon et al., J. Microelectromech. Syst. 12, 70 (2003). Copyright 2003 IEEE.
1. Traditional droplet splitting/generation method
In the traditional EWOD device, the contact angle on the electrode can be controlled by the potential according to the Lippman–Young (L–Y) equation,
where and are the contact angles at the electric potential V and 0, respectively. is the interfacial tension between the liquid and its surrounding phase (typically gas). , , and t are the permittivity of vacuum, the relative dielectric constant, and the thickness of the dielectric layer, respectively.
Based on the working principle above, the splitting of droplets using EWOD usually involves at least three consecutive electrodes, as shown in Fig. 2. The two side electrodes stretch the droplet by using the electrowetting effect to reduce the contact angle of the droplet along the direction of motion (A-A′) so that the radius of curvature r2 of the droplet increases. At the same time, the middle electrode is floated or grounded, which will not increase its contact angle. As a result, the meniscus of the middle electrode contracts to keep the total volume of the droplet constant, i.e., a continuous decrease of R1 and r1 along with the increase of r2 under driving voltage on the two end electrodes, which ultimately splits the droplet.61,62 This configuration is only effective if the droplet spans at least three electrodes. Besides the size of the droplet, the success of the split depends on the size of the electrode, the excitation voltage, and the gap between the bottom and top plates of the EWOD device.61
2. Novel electrode design for manipulating droplets
Considering precise droplet generation, Wang et al. introduced an electrode design for droplet dispensing with the function of stabilizing the shape of droplets before splitting. As shown in Fig. 3,28 two outer arcs and a central dumbbell are designed on the electrodes to mimic the natural shape of the cutting process. To make the batching process more controllable, Wang et al. add a necking process between the typical extrusion and breaking process, by which the typical two-step batching process (extrusion, breaking) is expanded to a three-step process (extrusion, necking, fracture). The volume of the droplet is determined by the dispensing process that more than 95% droplets fall within the range of 324 nl–326 nl. This attempt is considered to be an all-electric, simple, and effective method, which improves the uniformity of droplet volume and has good practical application prospects.
Schematics and photos of the EWOD dispensing process with our electrode design (top view). (a) Extrusion step. (b) Necking step. (c) Breaking step. [(d)–(f)] Photos of extrusion, necking, and breaking step, respectively. Reprinted with permission from Wang et al., Appl. Phys. Lett. 108, 243701 (2016). Copyright 2016 AIP Publishing LLC.
Ehsan et al.63 have studied asymmetric droplet splitting by geometrically modified conventional electrodes in an EWOD platform. One or more sub-electrodes are activated based on the desired volume of droplets, a portion of the droplet is pulled onto the activated sub-electrode, and then the adjacent electrode is turned off. Finally, the droplet is split into the furthest electrode. The activation time varies according to the voltage applied to the system. Using a design with 0.5 mm sub-electrodes and the gap height of h = 25 μm, droplets as small as 7 nl were generated from a mother droplet with a size of 150 nl. The results show that the applied voltage must be close to the minimum voltage for shunting (called the threshold shunt voltage), which will produce split droplets with precise volumes.
dPCR requires thousands of discrete droplets with possibly continuous addition of reagents, which means that one electrode has to deal with droplets of different volumes during PCR. Figure 438 shows such multi-volume droplet manipulation driven by a variable size interdigitated EWOD electrode, which can produce/manipulate droplet volumes from 15 nl to 180 nl. Chen et al. have achieved automatic control of droplets of different sizes under the same electrode array. According to theoretical calculations and experiments, the triangular finger electrode enables to drive droplets up to 36 times smaller than when using traditional square electrode arrays. It enables more efficient large-scale droplet driving than typical rectangular fingers do. This work provides a way for the realization of multifunctional EWOD devices in the future applications requiring a broad range of droplet volumes, such as dPCR.
Different volumes of droplets transported from left to right under the same driving signal in a parallel-plate electrowetting device with triangle interdigitated fingers: (a) 15 nL, (b) 90 nL, and (c) 180 nL. Reprinted with permission from Chen et al., Appl. Phys. Lett. 101, 234102 (2012). Copyright 2012 AIP Publishing LLC.
3. Multi-droplet generation through microstructure on chip
Dong et al.64 proposed an EWOD chip with a 3D micro-blade structure to enhance droplet separation performance, as shown in Fig. 5. The chip is composed of a set of electrodes and on-chip 3D micro-blades, which can perform a variety of droplet splitting functions. According to the required final division, the blade is placed in different positions on the path. During the splitting process, the mother drops are cut off by the tip of the blade, and the daughter drops are separated by the body of the blade. The position of the blade depends on the desired application and the tips of the blades are equally spaced on the electrode E2 to achieve equal volume division. The droplets of different volumes are split by placing the blades in an appropriate position. Just four electrodes are needed to generate up to five sub-droplets simultaneously, which greatly simplifies the electronic control system.
EWOD chip made with an on-chip microstructure (embedded 3D blade). (a) The EWOD chip with a single on-chip blade for 50/50 splitting. (b) The EWOD chip fabricated with blades for quarter-splitting and fences to prevent drifting. Reprinted with permission from Dong et al., Lab Chip 17, 896 (2017). Copyright 2017 Royal Society of Chemistry.
4. Gradually ramping down a voltage to split the droplet volumes precisely
Ananda et al. studied the accurate generation and measurement of the sample volume in electrowetting equipment. The device consists of three 500 μm wide microchannel-shaped electrodes, the shortest of which is a separate electrode with a length of 3 mm in the middle [Fig. 6(a)]. The method is to reduce the potential of the split electrode slowly (>1 s), which can minimize the hydrodynamic instability in the split region, thus replacing the electrode suddenly to accurately divide the sample volume (minimum: 0.5 μL). This method can potentially improve the performance of applications that require precise metering of samples, such as immunoassays65 or DNA amplification.66,67
Electrowetting-based equipment for precise droplet segmentation. (a) Electrowetting based device for demonstrating deterministic splitting and volume generation. (b) Cross section of an electrowetting based device. (c) Formation of 500 μm wide, 25 mm long microfluidic channel. (d) Magnified image of the inlet/outlet reservoir patterned in the spacer layer. Reprinted with permission from Banerjee et al., Lab Chip 12, 5138 (2012). Copyright 2012 Royal Society of Chemistry.
B. Improvement of dielectric performance
Dielectric breakdown, that is the catastrophic failure of EWOD devices when an excessive voltage is applied, is one of the critical issues that hinders further integration of EWOD with IC and limits the safety of potentially portable use of EWOD for POCT (point of care technology). Therefore, lowering the driving voltage and increasing the dielectric reliability are existential challenges for the EWOD device and systems. On a typical Teflon-coated hydrophobic surface, the contact angle needs to be changed from 120° to 80°68 by electrical voltage as high as 150 V. According to the L–Y equation,69 there are two methods to reduce this driving voltage: (1) reducing the surface tension of droplets (), for instance, using air instead of oil as the outer phase, (2) increasing the relative dielectric constant (), and (3) reducing the dielectric thickness (t). Combined approaches by building multiple dielectric layers and modification of dielectric properties have also been proposed to address to lower the driving voltage and improve the reliability of EWOD devices.
1. Encapsulating droplets with the oil film
Fan et al.70 found that encapsulated droplets have a lower viscosity and a larger volume of oil shell than bare and immersed droplets, which can then be driven at lower voltage or larger speed. By applying a voltage of 48 V, a 1 × 1 mm2 EWOD electrode is used to generate 25 nl DI water droplets and 2.5 nl oil droplets in a 25 μm high gap between the parallel plates, as shown in Fig. 7. The electrowetting force of encapsulated droplets is lower than that of the exposed droplets in the air. The oil viscosity reduces the driving voltage of the encapsulated droplets. The driving speed of the encapsulated droplets is greater than for immersed droplets.
Configuration of the designed parallel-plate device having water, oil, and rinsing reservoirs. (a) A parallel plate with three reservoirs, to perform the four main stages of droplet. (b) Generation and encapsulation. (c) Rinsing. (d) Emersion. Reprinted with permission from Fan et al., Lab Chip 11, 2500 (2011). Copyright 2011 Royal Society of Chemistry.
2. Dielectrics with high relative permittivity
Chen et al. studied the electrowetting properties of cyanoethyl pullulan (CEP), which was used as an insulator for EWOD devices in our research. As shown in Fig. 8, Chen et al. found that CEP had the best dielectric properties when annealed at 100 °C atmospheric pressure in air, with a dielectric constant of about 18 (100 kHz). When CEP is used to manufacture the parallel-plate EWOD device, it successfully drives water droplets at DC voltage as low as 20 V. The voltage-dependent speed indicates that the DC signal has a greater electrowetting driving force than the AC signal. When using AC driving signals, the driving force will be reduced as the frequency increases. This annealed CEP is a promising insulator for EWOD devices.71
Schematics of sample preparation and structure setup for (a) CA measurement; (b) dielectric characterization; and (c) EWOD manipulation and (d) the molecular structure of CEP. Reprinted with permission from Dong et al., Sens. Actuators B Chem. 199, 183 (2014). Copyright 2014 Elsevier.
Chang et al.23 studied the use of alumina as a high-κ material for reducing the EWOD driving voltage. The 1, 270 Å aluminum oxide in the coplanar EWOD device is deposited by atomic layer deposition (ALD) to obtain a control electrode array of 11 × 1 mm2 with a pitch of 50 μm. They demonstrated the movement of 2 μl water droplets on EWOD in air and observed that the droplet velocity increases exponentially with the voltage applied below 15 V. The threshold voltage is reduced to 3 V.
Barium strontium titanate (BST) was studied by Moon et al. with a layer of fluoropolymer, which has a very high dielectric constant, can achieve a 40° contact angle decrease with driving voltage as low as 15 V.72 High-κ materials such as BST and alumina usually require a higher annealing temperature (about 700 °C), which may soften or melt the typical glass substrate of the EWOD device, i.e., possibly incompatible with glass-based micromachining.
3. Building multiple dielectric layers
Lin et al.73 designed a multi-layer insulator EWOD device that can dispense 300 pl droplets from a 140 nl closed on-chip liquid reservoir with EWOD excitation as low as 11.4 V for a voltage switching frequency between the electrodes of 1 Hz. The driving threshold voltage is 7.2 V. A multilayer insulator device with 135 nm Ta2O5 and 180 nm parylene C is used to minimize the applied voltage. The electrode size of the EWOD device is 35 μm and can dispense 30 pl droplets from the container on the chip. Then, Lin et al.74 developed a low-voltage picoliter EWOD device with a new multi-layer insulator structure and interleaved electrode design to increase electrowetting force. It can achieve droplet distribution, while the improved gasket design allows for better sealing and sufficient flow concentration to dispense droplets from the on-chip container. A 12 pl droplet can be split into two 6 pl daughter droplets at 18.7 Vrms with 33 μm electrode devices.
Gao et al.69 introduced methacryloxypropyltrimethoxysilane (A-174) as an adhesion promoter in the deposition process of multiple dielectric layers Ta2O5 and parylene C, thereby forming chemical bonds at the interface.75–78 In this way, as shown in Fig. 9, the conversion results in a stronger and more stable chemical bond than before, which is different from the typical single or double dielectric layer. The device lifetime is extended over 100 times compared with the original device. The EWOD excitation voltage can be as low as 5 V. The chip has high strength and can withstand more than 10 000 droplets reciprocating between two adjacent electrodes without showing the signs of dielectric breakdown.
Setup of the control-engaged EWOD device. (a) The EWOD module consists of four operation parts: the chip holder, the control electronics layer, the field-programmable gate array (FPGA) board, and the software control program. (b) The fabricated EWOD chip. (c) The control electronics for real-time droplet actuation and sensing. Reprinted with permission from Gao et al., RSC Adv. 5, 48626 (2015). Copyright 2015 Royal Society of Chemistry.
C. Surface chemical modification of EWOD devices
During biochemical experiments, the surface energy of the device will increase considerably due to the very large surface/volume ratio, thus may interfere with the PCRs. Therefore, microdevices require proper surface chemistry to achieve the required loading and PCR functions, especially surface wettability. PCR-friendly coatings, chemical coating control for manufacturing purposes, and evaluation of coating chemical and physical stability are critical fluid handling techniques.
1. Double-layered deposition of hydrophobic films
One of the key structural features of EWOD devices is a surface coating to modify the surface wettability because it directly affects the performance and reliability of the device. On the one hand, a multilayer polymer composite coating can reduce the electrowetting actuation voltage and, on the other hand, it simplifies the EWOD fabrication process, thanks to conformal deposition, thickness uniformity, and better adhesion to the substrate.
Papageorgiou et al.79 proposed a multi-layer fluoropolymer surface coating that exhibited high dielectric breakdown resistance under low-voltage AC or DC driving and confirmed its dielectric properties in subsequent driving experiments. The performance of a more sophisticated composite hydrophobic coating is comparable to the spin-coated Teflon surface. Special attention should be given to such a surface preparation. To achieve a composite coating, as shown in Fig. 10, a short span of plasma is used to clean and etch the surface of the chip, followed by plasma deposition of fluorocarbon (FC), which is then coated with spin-on Teflon.80 The results show that for low-voltage and high-voltage surface microfluidic actuation schemes, the composite coating is far superior to the simple spin-coated hydrophobic surface.
2. BSA-based electrostatic passivation to improve wettability
Hydrophilic surfaces may inhibit enzymatic reactions, which causes direct interaction between polar substances and enzymes or DNA templates. To avoid this inhibition, a direct passivation step based on biological or chemical coatings is introduced, or a small amount of PCR accelerator is added to the biological reagent, for example, protein, metal-terminated nanoparticles, and traditional PCR additives.81
Petralia et al.82 proposed a novel method for measuring the wettability of silicon embedded microchannels. Tetraethyl orthosilicate (TEOS) treated microchannel surfaces are modified with different chemical processes: wet cleaning, plasma cleaning, wet and gas-phase epoxy silanization, and finally bovine serum albumin (BSA) coating. Experimental results show that BSA-based electrostatic passivation can improve wettability as the contact angle is increased, which is useful for biological experiments such as dPCR. Petralia et al. also found that BSA-based electrostatic passivation is a key factor for the successful integration of sample loading and amplification processes. The chip is composed of two silicon components: (1) the bottom contains an integrated temperature sensor and (2) the top is composed of 96 square silicon micro-cavities (dimension 800 × 800 × 400 μm3). The final volume of each cavity is 200 nl, and the spacing (micro-cavity distance and distance) is between 200 and 500 μm. The passivation coating is performed by immersing the device in a BSA 1% aqueous solution (w/v) for 15 h. After the voltage is applied according to the scheme shown in Fig. 11, it can ensure the conversion from the hydrophobic surface to the hydrophilic surface to load the sample, thereby ensuring complete biocompatibility and DNA amplification reaction.83
BSA-based electrostatic passivation chip. (a) Chip structure and its components. (b) EWOD scheme. (c) System for EWOD electro-loading and PCR. Reprinted with permission from Petralia et al., BioNanoScience 6, 139 (2016). Copyright 2016 Springer Nature.
The throughput of droplet of EWOD devices is indeed an important parameter for realizing dPCR. EWOD intrinsically shows no superiority to typical microchannel devices in high throughput of droplet generation, which could hinder its application in PCR. However, EWOD possesses many other competitive and even overwhelming advantages that drive research in the field and have already yielded some exciting results. One is the 3D micro-blade design, which is used to realize the rapid segmentation of droplets on the EWOD device, and droplets of uniform or unequal volume are obtained to realize the PCR process and fluorescence analysis of the droplets. This design enables the generation and manipulation of tens of thousands of droplets for dPCR.64 Another is to use the EWOD ability device to produce droplets of well-controlled volume across a range of sizes. These droplets are used for droplet PCR and then imaged so that the measured nucleic acid concentration can be obtained. No special equipment is required to manufacture, and the droplet size range increases the dynamic range. The simulation is used to estimate the error of the best fit concentration and its dependence on the droplet number/droplet size distribution and sample concentration.59
III. EVAPORATION MANAGEMENT OF DROPLETS OF EWOD DEVICES FOR DROPLET PCR
In many cases, it is desirable to implement EWOD on open surfaces so that the medium surrounding the droplets is ambient air. However, the use of air medium EWOD devices is severely limited by the problem of droplet evaporation, especially when droplet-based biochemical reactions (such as PCR) require long periods of high temperature. Therefore, there is an urgent need for a solution to manage evaporation in EWOD devices.
A. Replenish solvent droplets in the PCR process
During operations that require high temperatures and/or long incubation times (≥65 °C, ≥1 min for water droplets) to counteract evaporation, solvent droplets are used to replenish the reaction volume promptly. Jebrail et al.84 developed an evaporation management solution. In this way, preheated solvent droplets can be replenished regularly in a temperature-controlled biochemical reaction. The device is designed with a through-hole to the bottom EWOD substrate,85 where the 8 mm through-hole is used to deliver additional solvent droplets from the external syringe pump reservoir to the surface of the device. As the reacting droplet begins to shrink due to evaporation, the solvent droplets will be generated from the through-hole and transported to the edge of the hot zone, and the solvent droplets will be preheated for 5 s before they merge (Fig. 12). The preheating of these replenishing droplets minimizes the effect on the temperature of the reaction droplets during the merger, thereby improving the consistency of the reaction kinetics. Similarly, when a droplet loses 15%–20% of its volume, the solvent should be replenished to minimize changes in solute concentration that may adversely affect the reaction kinetics. Finally, 2 μl of reaction droplets can be kept at a constant volume over a wide temperature range (35–95 °C).
Evaporation management solution: Solvent droplets are used to replenish the reaction volume promptly. (a) Schematic of the digital microfluidics device. (b) Time-lapse series of stills from a movie capturing demonstration of evaporation management solution. Reprinted with permission from Jebrail et al., Lab Chip 15, 151 (2015). Copyright 2015 Royal Society of Chemistry.
B. Water–oil core–shell droplets on EWOD devices
In addition to the use of electrowetting force to manipulate water droplets in air, it has also been used in non-conductive and immiscible fluids.62 For example, silicone oil (polydimethylsiloxane) is widely used as a transmission medium due to its low surface tension and low viscosity (as low as 0.65 CST).34 A sizeable fraction of EWOD research is conducted in low viscosity silicone oil.86–90 In general, the operation of EWOD-based equipment is greatly simplified in silicone oil. The oil medium not only reduces the contact angle hysteresis of the droplet34,61 and significantly reduces the threshold voltage required for driving, but also reduces its interfacial tension.34,88 That facilitates various fluid operations, especially the transportation in silicone oil, because it also can prevent droplets from evaporating, thus fitting for high temperature operation and minimizing equipment contamination.34,90,91
Fan et al.70 proposed water and oil droplets pre-created by EWOD to form encapsulated droplets and thereby reduce evaporation. As shown in Fig. 13, a parallel-plate device is designed for encapsulated droplet experiments. It has three containers: a water container, an oil container, and a rinsing container. Water droplets encapsulated in oil are obtained by merging water and oil droplets. The oil shell can be removed by driving the encapsulated droplet into a rinse reservoir containing liquid hexane to dissolve and remove the oil shell. Figure 7 shows these four operations: the generation of water (W) and oil (O) droplets (1); encapsulation to form W/O droplets (2); rinsing of the oil shell in the rinsing container (3); and emergence of clean water droplets from the rinsing tank (4). The bottom plate contains multiple drive electrodes covered by a dielectric and a hydrophobic layer, while the common electrode is deposited on the top plate and coated with a hydrophobic material. The device behavior is highly similar to standard EWOD devices and most electrowetting functions can be realized. In the absence of droplet breakage, the oil shell can effectively suppress water evaporation and enables long-term operation.
Encapsulated droplet formation by splitting. (a) Splitting. (b) Splitting from a larger encapsulated droplet. (c) An encapsulated droplet generated by emersion and splitting. The procedure of emersion and splitting of a water droplet from an oil reservoir is shown in (d)–(i). Reprinted with permission from Fan et al., Lab Chip 11, 2500 (2011). Copyright 2011 Royal Society of Chemistry.
Brassard et al.92 proposed another mode of operation of the EWOD-based microfluidic device, while water droplets were encapsulated in a thin shell of silicone oil. Compared with operation in air, evaporation and equipment contamination can be reduced by the water–oil core–shell droplets. In the standard configuration [Fig. 14(a)], the space between the two plates is filled with air or oil. In the core–shell structure, as shown in Fig. 14(b), a small amount of oil is then distributed with the aqueous solution to form a thin shell around the droplets. The oil phase and aqueous solution are then immediately transported in the air. Therefore, the presence of the silicone oil shell in contact with the surface of the device can ensure that the water core is operated under conditions similar to those obtained in the oil medium.
C. Waterproof layer and water channel are designed to prevent evaporation
Oil-based EWOD may result in undesirable liquid–liquid reactant extraction into the surrounding oil,93 incompatibility with oil-miscible liquids (for example, organic solvents such as alcohol), and unnecessary heat dissipation, which diffuses the local heating and often disrupts temperature-sensitive reactions. Therefore, great efforts of more effective design that does not affect the experimental process to prevent evaporation are required.
As shown in Fig. 15, Song et al.94 proposed a series of methods to improve evaporation. In the new dPCR chip, first, a glass coverslip is used as a waterproof layer to prevent the vapor of the test solution from evaporation during the dPCR process. The use of coverslips compared to the use of parylene C deposited on PDMS to act as a vapor barrier greatly simplifies the micromachining steps.50,95 Second, the water channel is designed around each sample plate. Before the dPCR, the water channel is full of water, so it can be greatly reduced the effect of reagent evaporation. Third, on the same sample plate, all the reaction chambers are connected to only one channel, and the phenomenon of selective flow seen on the branch channel chip can be avoided. Fourth, the mode of the channel and the chamber are designed on the same layer, which makes it easier to produce and reduces the operation time.96 It is a method that combines soft lithography (only one layer) and the ability to bond multiple layers of PDMS. Finally, the reactions of three different samples can be performed simultaneously on the same chip, and each sample solution is separated into 1040 reaction chambers with a volume of only 2.08 nl.
Schematic diagram of dPCR chip, showing the functional layer to prevent evaporation. (a) Photograph of the dPCR chip, with dyes in it. (b) Schematic diagram of the dPCR chip, showing different layers with different functions. Reprinted with permission from Song et al., Biomed. Microdevices 17, 64 (2015). Copyright 2015 Springer Nature.
IV. CONSTRUCTION OF AN INTEGRATED EWOD–PCR PLATFORM
At present, the integrated platform of PCR and EWOD has received a lot of attention. The EWOD device is integrated with an on-chip PCR including a microheater and a micro-temperature sensor, an optical sensor. Some automated and fully functional digital microfluidic platforms have even been developed, which will undoubtedly contribute to POCT of the future.
A. EWOD integrated with heating and temperature sensors
Droplet PCR system with a digital microfluidic and PCR system can be integrated with heating and temperature control equipment. Guttenberg et al.97 used equipment of surface acoustic wave (SAW) to transport and mix sample/reagent droplets, which can be used in PCR and hybridization processes.
Chang et al.90 proposed an innovative concept, namely, the hydrophobic/hydrophilic structure, to overcome the integration problem of EWOD-based chips and on-chip PCR. As shown in Fig. 16, the system can realize the creation, combination, mixing, and transportation of samples/reagents based on EWOD. The micro-PCR chamber on the chip is used to amplify DNA samples. The transportation and mixing of samples and reagents are realized on the EWOD platform. Then, the micro-temperature control chip, combining a microheater and a micro-temperature sensor, is used for PCR. The device includes three main areas: a container of primer reagent and cDNA (complementary DNA) solution, a mixing area, and a micro-PCR chamber. A PWM (pulse width modulator) controller and an ASIC (application-specific integrated circuit) chip are used to control the movement of sample/reagent droplets and the temperature field in the PCR chamber.98 In addition, the developed chip uses only 12 V operating voltage for a digital microfluidic drive and 9 V operating voltage for temperature sensing and heating.
A schematic diagram of the EWOD/PCR device. (a) Schematic representation of modules for mixing and DNA amplification. (b) Photograph of digital EWOD/PCR chip. (c) Close-up view of temperature sensor and heaters inside the microchamber of PCR. Reprinted with permission from Chang et al., Biomed. Microdevices 8, 215 (2006). Copyright 2006 Springer Nature.
B. EWOD integrated with channels microfluidic chip and/or optical inspection equipment
Brennan et al.99 integrated three devices with specific system functions: (1) electrowetting on silicon dielectric device for moving and mixing sample and reagent droplets in the oil phase; (2) microfluidic channels embedded in a polymer chip; and (3) aqueous glass microarrays for fluorescent microarray hybridization detection. As shown in Fig. 17, during the biological sample preparation and microarray imaging process, fluid and electrical interconnections are realized from the driving electronics and external reservoir to the EWOD device. The EWOD device provides the possibility to fully integrate on-chip sample preparation using nanoliter samples and reagent volumes. As shown on the right, the EWOD electrodes are multiplexed by a high-voltage (Vpp = 150 V) relay controlled independently by a Labview™ program. The prepared sample is sucked from the EWOD oil phase into the aqueous phase microarray for hybridization. A thermoelectric heater is fixed on the translation stage, which allows to separate the heater from the device after the hybridization washing step, leaving a clear optical path for microarray imaging.
Illustration of the Arrayed Primer Extension (APEX) system. The fluidic network is used to transfer the APEX sample from the EWOD device to microarray. It also facilitates post-hybridization wash and drying before fluorescence detection. (Left) Stacked view and (right) top view of structure. Arrows indicate the flow direction generated by the external pump/valve configuration. Reprinted with permission from Brennan et al., Meas. Sci. Technol. 23, 105704 (2012). Copyright 2012 IOP Publishing.
An integrated EWOD system based on the single-cell genetic analysis was described by Rival et al. The closed EWOD micro-system consists of two parts: silicon active device and glass cover. This benchtop instrument includes a chip holder for electrical connection and physical fixation, a magnet holder for operating the magnet on the top of the chip, and a detection module for droplet observation and fluorescence detection. The electrodes designed on the chip are developed and optimized100 using the general digital microfluidic design framework proposed by Fouillet et al.33 The entire process of transcriptome analysis on the same chip is realized by the EWOD-based microfluidic chip. Starting from reducing the number of cells, the chip can separate single or several cells in droplets, extract mRNA from the cells, catalyze RT reactions, and perform PCR. The overall performance of the system described in the study is comparable to bench-top PCR instruments, but the volume is reduced by at least 20 times. The initial sample is also much smaller. The specific design of the microfluidic chip and its automated functions make it possible to manipulate and isolate single cells from a larger population and to analyze gene expression at the single-cell level. It is even conceivable to couple the chip101 to the equipment described in the study to provide sorted single cells and obtain a complete system to enable cell sorting and gene expression. Such a system is particularly useful for oncology applications, such as monitoring circulating tumor cells.102,103
Noria et al.104 used CMOS technology to perform all the functions required for PCR, including temperature control, heating, microfluidics, and fluorescence detection. The PCR chip is implemented using a 0.35 μm high-voltage CMOS process. As shown in Fig. 18, the 4 × 4 mm2 chip contains a 7 × 8 array consisting of 200 × 200 μm2 electrodes, which enables the droplet transportation. A custom-developed SU-8 packaging solution is used to simultaneously package wire bonds and pattern the active electrowetting array. The droplet array is covered with indium tin oxide (ITO) coated polyethylene naphthalate (PEN) cover glass, which is 100 μm away from the surface of the chip and is supported by SU-8 sealant. To perform PCR on the surface of the chip, the primers are placed in one or two containers, the DNA target is placed in another container, and the DNA polymerase and the dye-embedded PCR reagent are placed in the remaining containers. Electrostatically draw droplets from each of these constituent containers and mix them. The entire surface of the chip is thermally cycled uniformly according to a prescribed heating curve. After each PCR cycle, each test droplet is brought to the fluorescence measurement area of the chip to determine the progress of the reaction. At the end of each extension phase, take a fluorescence reading of the diode.
CMOS-based PCR using EWOD. Drops of test sequences and PCR reagents are delivered to one of the four reservoirs on the chip. The entire surface is heated with three heaters in columns 14 and 7, and all test droplets are thermally cycled simultaneously. Use integrated single-photon avalanche diodes at pixels (3, 7) to monitor PCR progress. Reprinted with permission from Norian et al., Lab Chip 14, 4076 (2014). Copyright 2014 Royal Society of Chemistry.
All the high-voltage generation which is required to perform EWOD droplet control is integrated on the chip, and the droplet size is about nanoliters (droplet diameter is about 200 μm). In addition, the ability to move droplets through multiplexed detectors on the chip allows 40 PCRs in parallel. Since each electrowetting pixel is independent and isolated from its neighboring pixels, each droplet can contain a unique combination of primers extracted from two fluid containers. Sensitivity and dynamic range can achieve 1–104 quantitative copies of target DNA per 1.2 μl droplets.104
C. Construction of an automated multi-channel digital microfluidic platform
Sista et al.105 proposed the design of immunoassay and DNA amplification on a digital microfluidic platform based on electrowetting and developed an on-chip incubation scheme for immunoassay (shown in Fig. 19), which reduces the reaction time from 600 s (incubation on the magnet) to 240 s (incubation outside the magnet). It optimizes the washing program to achieve nearly 100% washing in ten washing cycles. By using optimized incubation and washing protocols, rapid immunoassay of cardiac troponin 1 can be performed in less than 8 min. A digital microfluidic box based on electrowetting consists of a “chip” with electrodes to control droplets and a conductive “cover” that provides a reference potential for the droplets. The chip and the cover plate are arranged in a parallel-plate configuration with droplets sandwiched between them. The droplets are surrounded by immiscible fluids to prevent evaporation and facilitate droplet operation. Both immunoassay and DNA amplification experiments are performed on a digital microfluidic multi-well plate box. The kit has 12 sample containers and 8 reagent containers. The sample container pitch is 4.5 mm, and the reagent vessel pitch is 9 mm. The off-chip reservoir can hold a large amount of liquid for washing buffer, waste and large volumes of reagents (chemiluminescent substrate) required to connect to the digital microfluidic chip. Immunoassay of troponin I is performed in whole blood using the same platform. The study further demonstrates the ability to perform using magnetic beads on the same microfluidic chip to extract samples of infectious disease pathogens and human genomic DNA from whole blood.
Automated multi-channel digital microfluidic platform: (a) fully assembled digital microfluidic porous plate filter element (8.55 × 12.78 cm2); (b) control instrument (20.32 × 33.02 × 53.34 cm3). Reprinted with permission from Sista et al., Lab Chip 8, 2091 (2008). Copyright 2008 Royal Society of Chemistry.
On this basis, the Pollack group has also proposed an automated and fully functional multi-channel digital microfluidic platform for real-time multiplex PCR detection. The system showed an excellent amplification efficiency of 94.7% and detected a methicillin-resistant equivalence model test system for a single genome of Staphylococcus aureus. The digital microfluidic PCR system includes instruments that combine all required control and detection capabilities, as well as disposable microfluidic cartridges in which sample processing and PCR are performed. The PCR box is inserted into the instrument platform containing the heater and magnet and communicates with the instrument through the electrical interface. The microfluidic control on the PCR box is realized by an electronic controller with a microprocessor and a switching circuit, which can transfer the electrowetting actuation voltage (0–300 V) to 64 individually addressed channels. Real-time PCR fluorescence detection is achieved using a custom-designed micro-fluorometer module.
Electrowetting avoids the need for bulky mechanical pumps and valves. Since the thermal circulation of the heater is avoided in the flow-through mode, and because electrowetting is inherently low-power, power consumption is minimized. Therefore, the digital microfluidic PCR system is very suitable as a portable device platform for further miniaturization for fast real-time inspection and on-site application, which cannot be achieved by conventional PCR instruments.31 We have summarized the relevant parameters of the current EWOD technology for droplet PCR in Table I.
TABLE I.
EWOD technology for droplet PCR: Targets, sensitivity, time, minimum volume.
| Source | Targets | Sensitivity | Time | Minimum volume |
|---|---|---|---|---|
| 64 | S. aureus, coag. negative, L. lactis, and K. pneumoniae | N/A | N/A | 900 nl |
| 83 | Human β-globin | N/A | N/A | 200 nl |
| 84 | Bacteriophage m13mp18 | N/A | 33 cycles in 52 min | 1.5 μl |
| 94 | Human β-actin | 10 human β-action molecules/μl | N/A | 2.08 nl |
| 103 | Human HaCaT adherent cells | N/A | N/A | 128 nl |
| 104 | Staphylococcus aureus | More than four orders of magnitude | N/A | 1.2 nl |
| 105 | Cardiac troponin I/candida | N/A | 40 cycles in 12 min | 300 nl |
V. CONCLUSION
Some droplet PCR systems have been successfully developed by using EWOD devices. It is likely that future research will focus on POCT applications by achieving better integration and automation and lowering driving voltages. Complete electric fluid operation avoids the need for bulky mechanical pumps and valves. Since the thermal circulation of the heater is avoided, electrowetting is inherently low power. The power consumption of droplet PCR based on EWOD is minimized. Therefore, it is very suitable as a portable platform for further miniaturization. In order to carry out rapid real-time inspection and on-site applications, which is not possible for any conventional PCR instruments. The digital microfluidic platform can provide a high degree of analytical configurability. A single general-purpose chip can be constructed by using the software so that various measurements and protocols will be implemented using a common basic droplet operation library. As the volume of droplets decreases, the precise of the droplet volume and the number of droplet increase, problems existing in droplet PCR including manipulation, serious evaporation, and integration will be solved gradually, which would be greatly helpful for ddPCR with EWOD in the future.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (NNSFC) with Grant Nos. 61874033 and 61674043, the Natural Science Foundation of Shanghai Municipal Government with Grant No. 18ZR1402600, and the State Key Laboratory of ASIC and System, Fudan University with Grant No. 2018MS003.
We want to express our heartfelt thanks to Dr. Antoine Riaud who helped on article modification. There are no conflicts of interest to declare.
DATA AVAILABILITY
The data that support the finding of this study are available from the corresponding author upon reasonable request.