Droplet generating with accurate volume for EWOD digital microfluidics

Liquid-liquid extraction techniques are one of the major tools in chemical engineering, analytical chemistry, and biology, especially in a system where two immiscible liquids have an interface solutes exchange between the two liquid phases along the interface up to a point where the concentration ratios in the two liquids reach their equilibrium values [1]. Solutes including nucleic acids and proteins of interests can be extracted from one liquid phase to the other immiscible liquid phase as a preparation step for many analytical processes. There are several advantages in miniaturizing the liquid-liquid extraction methods to on-chip level extraction. Usual advantages of miniaturization are the reduction in the sample size and portability. In addition, transport phenomena is faster in Micro-systems than in ordinary size systems, and therefore, one may expect that liquid-liquid extraction takes less time to achieve in miniaturized devices. It is due to shorter diffusion time in micro scale as well as high surface to volume ratio of Microsystems. Electrowetting on dielectric (EWOD) digital microfluidics is an efficient platform to process droplet based analytical processes [2]. Nanoliter (nL) or smaller volume of aqueous liquid droplets can be generated and transported on a chip by EWOD process. In addition to the high surface to volume ratio, high chemical potential can be expected in droplet based extraction when the droplets are in motion. In this paper, we propose to use room temperature ionic liquid (RTIL) as a second liquid phase for extraction, which forms immiscible interface with aqueous solutions. Properties of RTIL can be tailored by choice of cation, anion and substituents. RTIL has been investigated as replacements for the organic solvents and various “task-specific” ionic liquid are being developed which exhibit many attractive properties such as very low vapor pressure, high thermal stability [3]. We recently published EWOD properties of various RTILs toward microfluidic applications [4]. To demonstrate liquid-liquid micro extraction on chip, we fabricated and tested EWOD digital microfluidic devices. Fig. 1 shows (a) top and (b) cross sectional views of EWOD device. Two model extraction systems were tested. One is organic dye extracted from RTIL (1-butyl-3-methylimidazolium bis(trifluoromethanesulfonylimide or BMIMNTf2) to water and the other is iodine (I2) extracted from water to BMIMNTf2. The later model experiment is demonstrated in Fig. 2. Droplets of aqueous solution and BMIMNTf2 solution were generated on chip reservoir then transported for extraction and separated by EWOD actuation. When an aqueous solution and BMIMNTf2 solution join together, they created an interface, since water and BMIMNTf2 are immiscible. Extraction of I2 was done along the interface. After successful extraction, two immiscible liquid phases were separated by EWOD actuation and formed two separate droplets. From the result shown in Fig 2 (g), it is expected that extraction performance at the interface of moving droplet would be enhanced compared to the stationary droplet, because a moving interface prevent the chemical equilibrium, thus more chemical extraction potential can be provided with a moving interface than at a stationary interface. This demonstration is the first step toward total analysis system. The presented result opens the way to on-chip micro extraction, which will be readily integrated with other sample preparation microfluidic components and detection components. Currently, micro extraction systems for larger molecules such as nucleic acids, proteins and biological cells are being developed for further analytical applications.

To meet the increasing demand of efficient cooling performance in small scale, this paper presents a digital microfluidics (DMF) microscale liquid cooling system which works on the principle of electrowetting on dielectric (EWOD). In EWOD DMF, fluids are handled drop-wise by external electric field. When the dispensed liquid droplet arrives at the hotspot by EWOD DMF operation, it picks up heat and removes heat when it leaves. This process can be repeated for a series of droplets by using a completely automated LabVIEW controlled system connected to the PCB package. With the help of indium tin oxide (ITO) thin film resistance temperature detectors (RTD) and pre-calibrated temperature coefficient of resistance (TCR) data, the temperatures of the hotspot before and after the residence of liquid droplet (i.e., cooling) can be recorded for different frequencies (dwelling time period of droplet on the hotspot) of the drop motion and varying heater power. Future work will involve RTD resistance data collection to plot the heat flux and the temperature difference (before and after cooling) for different frequencies of drop motion. Although the primary focus is to study single phase cooling, the DI water drop will experience considerable evaporation resulting in higher cooling performance. The single phase cooling studies will help in establishing a robust platform for future two-phase cooling analysis in which evaporation effects will be considered.

It has been more than 20 years since the first micro total analysis systems (μTAS) papers were published. Initial reports of these devices, which are also commonly referred to as Labs-on-a-Chip (LOC), LabChips, microchips or microfluidic devices, generally focused on separations and the development of a variety of functional elements for sample manipulation and handling. One of the greatest potentials of μTAS, however, has always been in the integration of multiple functional elements to produce truly sample-in/answer-out systems. In the last decade, the march toward developing such integrated devices has accelerated significantly. Many μTAS reported now are quite sophisticated with multiple sample handling and processing steps that are highly integrated and often automated. While most of these devices are not yet strictly sample-in/answer-out several come quite close. There are, however, some significant hurdles still facing the development of true sample-in/answer-out systems especially in the areas of sample preparation, chip-to-real-world interfacing and detection. Additionally, further progress is needed in the miniaturization or elimination of external fluidic control elements. μTAS have found a major niche in the areas of biological and biomedical analyses, especially cellular and nucleic acid analysis. This focus on biological applications reflects the capabilities of these devices to precisely and accurately handle picoliter volumes of materials and to integrate cell transport, culturing or trapping with reagent delivery, and on-chip detection. Significant progress has been made in the development of a variety of cellular analysis systems; this field, however, is still rapidly growing and many papers focused on the expansion of such capabilities continue to be seen. Areas of focus remain the development of substrate materials and culturing conditions that do not unnaturally perturb or stress cells and that allow for extended culturing so that changes in cell physiology over time can be monitored. In addition, a significant amount of work has been directed to developing cell co-cultures on μTAS to mimic tissues, organs, and organ systems. μTAS can create unique, controlled environments to study cell-cell interactions that can not be replicated in any other way. For cellular assays substantial increases in throughput are also a focus. While significant development toward completely integrated cell assays has occurred and even some clinical demonstrations of such assays have been reported, the availability of commercial, fully integrated devices, however, has lagged. In addition to biological assays, the creative expansion of the basic μTAS toolkit with centrifugal platforms, digital microfluidics, and paper-based devices has substantially expanded its potential application base. Interest in these devices is generally more clinical in nature and again focused on generating sample-in/answer-out analyses. Significant work, however, is still needed for most of these platforms in terms of substrate materials, fluid control, sample handling, integration and throughput. Finally, the development of label-free detection technologies remains of interest. This review focuses on recent advances in μTAS technology in the areas of integrated biological assays and diagnostics with an analytical focus. We have also tried to highlight some material, fabrication, coating, separation, and detection advances with more general applicability. We have not included, for the most part, papers on synthesis, biosensors, theory, simulations or reviews. The papers included in this review were published between September 2012 and September 2013. The material was compiled using several strategies including extensive searches using Scifinder, Web of Science, PubMed, and Google Scholar. The contents of high impact journals were also scanned, including Analytical Chemistry, Lab-on-a-Chip, Nature, PNS, Appl. Phys., Letters, and Langmuir. Almost 2000 papers relating in some way to microfluidics were examined. We have done our best to try to identify some of the most interesting and promising papers and to report on them in this review. Without a doubt we have missed a few excellent papers and had to eliminate others based on space constraints and readability. For those papers that we have failed to include, we apologize in advance and welcome comments regarding any oversight that we have made.

We report the development of a microfluidic device that is the first of its kind to generate uniform arrays of individually addressable cell seeded calcium alginate gels for 3D cell culture using electrowetting on dielectric (EWOD) digital microfluidics (DMF). This is combined with EWOD DMF's multiplexing abilities to demonstrate how a single device is capable of forming cell seeded alginate hydrogels, generating different concentrations of chemicals and delivering these generated chemical concentrations to different alginate gels to observe the effect of chemicals on 3D cultured cells. This lays the foundation for a more efficient and versatile 3D cell culture and chemical screening platform.

Sensing of inorganic ions in microfluidic devices

This paper reviews the principles, operations, and applications of bubble-based electrowetting-on-dielectric (EWOD). EWOD has proved to be an efficient tool in digital microfluidics that employs discrete droplets, and various applications that use the principles of EWOD have been developed from lab-on-a-chip to optical systems. Similar to its use with droplets, EWOD can also be applied to gaseous bubbles. This review begins with a discussion of the principles of EWOD for a bubble on an electrode covered with a hydrophobic dielectric layer. It then addresses EWOD actuation and the transportation of a bubble in an aqueous medium, along with a physical explanation of bubble motion. The operation of EWOD is then extended to the on-chip creation/elimination and splitting of bubbles. In particular, micro-mixers and pumps are discussed as potential applications of these operations. Unlike droplets, bubbles can be easily oscillated by external excitation, which provides additional functionalities. By integrating EWOD with external excitation, a number of new advanced applications are introduced, including the capture/separation of particles and the propulsion of objects. In these advanced operations, cavitational microstreaming flows and acoustic radiation forces are mainly responsible for the physical mechanisms. This paper also discusses these advanced operations along with their underlying physics. It is expected that in addition to bubble oscillation, other bubble actuation modes will create new functionalities and new potential applications.

This paper describes highly efficient in-droplet particle concentrations/separations (over 91% in efficiency), where magnetic particles are concentrated/separated within a droplet by applying a magnetic field and a subsequent EWOD (electro wetting-on-dielectric) actuation physically splits a magnetic particle-rich region into an individual droplet. This in-droplet method will provide a new functionality of separation/concentration for digital (droplet-based) microfluidics, possibly allowing use of many magnetic-particles-based bio assays in digital microfluidics.

Electrowetting-on-dielectric (EWOD) technology has been considered as a promising candidate for digital microfluidic (DMF) applications due to its outstanding flexibility and integrability. The dielectric layer with a hydrophobic surface is the key element of an EWOD device, determining its driving voltage, reliability, and lifetime. Hereby, inspired by the ionic-liquid-filled structuring polymer with high capacitance independent on thickness, namely ion gel (IG), we develop a polymer (P)-ion gel-amorphous fluoropolymer, namely, PIGAF, composite film as a replaceable hydrophobic dielectric layer for fabrication of a high-efficiency and stable EWOD-DMF device at relatively low voltage. The results show that the proposed EWOD devices using the PIGAF-based dielectric layer can achieve a large contact angle (θ) change of ∼50° and excellent reversibility with a contact angle hysteresis of ≤5° at a relatively low voltage of 30 Vrms. More importantly, the EWOD actuation voltage did not change obviously with the PIGAF film thickness in the range of several to tens of microns, enabling the thickness of the film to be adjusted according to the demand within a certain range while keeping the actuation voltage low. An EWOD-DMF device can be prepared by simply stacking a PIGAF film onto a PCB board, demonstrating stable droplet actuation (motion) at 30 Vrms and 1 kHz as well as a maximum moving velocity of 69 mm/s at 140 Vrms and 1 kHz. The PIGAF film was highly stable and reliable, maintaining excellent EWOD performance after multiple droplet manipulations (≥50 cycles) or long-term storage of 1 year. The proposed EWOD-DMF device has been demonstrated for digital chemical reactions and biomedical sensing applications.

A new sample introduction/analysis approach was developed by combining a digital microfluidic (DMF) device with a portable capillary electrophoresis (CE) analyzer based on short separation capillary and contactless conductivity detection. The DMF sample injection was performed by transporting sample and buffer droplets in succession under the CE capillary inlet end allowing the capillary to be immersed into the sample/buffer droplet, and CE separation was performed by applying a high voltage between the (grounded) buffer droplet and CE outlet reservoir. Electrowetting on dielectric (EWOD) phenomenon was used for droplets actuation. With the use of the DMF sampler, CE separation of a mixture of vitamins was achieved. A droplet evaporation process with simultaneous concentration of sample in the droplet was monitored. It was found that the concentration process closely followed the theoretically predicted function.

Digital microfluidics, by the electrowetting-on-dielectric (EWOD), is a promising way to manipulate biological targets like DNA, proteins, or cells in very small liquid volumes. Conventionally, digital microfluidic operations are performed on a two-dimensional electrode array. This research expands the two-dimensional conventional digital microfluidic architecture into a three-dimensional architecture. The concept relies on the possibility that back and forth motions between open and covered regions are possible under electrowetting actuation. Two face-to-face plates based on “microelectrode dot array” (MEDA) architecture form the foundation of the two-layer microfluidic operations and a dual open/covered hybrid design adds the inter-layer microfluidic connection to complete the three-dimensional system. By combining the advantages of the open/covered systems, a three-dimensional digital microfluidics may achieve better droplet routing capabilities and fit more functions into a given footprint.

Miniaturization and automation are highly important issues for the development of high-throughput processes. The area of micro total analysis systems (muTAS) is growing rapidly and the design of new schemes which are suitable for miniaturized analytical devices is of great importance. In this paper we report the immobilization of self-assembled monolayers (SAMs) with metal ion sensing properties, on the walls of glass microchannels. The parallel combinatorial synthesis of sensing SAMs in individually addressable microchannels towards the generation of optical sensor arrays and sensing chips has been developed. [figure: see text] The advantages of microfluidic devices, surface chemistry, parallel synthesis, and combinatorial approaches have been merged to integrate a fluorescent chemical sensor array in a microfluidic chip. Specifically, five different fluorescent self-assembled monolayers have been created on the internal walls of glass microchannels confined in a microfluidic chip.

Here, we report an approach to realising the first IR-transparent digital microfluidic (DMF) chip. This enables spatially and temporally resolved infrared spectroscopy analysis directly within a DMF platform. To achieve IR compatibility, we have developed a manufacturing process that integrates calcium fluoride substrates with sputtered thin-film indium tin oxide (ITO) electrodes, creating fully functional DMF components that are transparent in the mid-IR range. This design enables real-time droplet imaging and reaction monitoring in transmission mode using infrared microscopy with a quantum cascade laser (QCL). The device incorporates a novel hexagonal electrode design that encircles droplets, forming a ring-shaped liquid boundary. This design mitigates evaporation, facilitating extended reaction times and enabling long term IR-reaction monitoring in volatile organic solvents. The device and its components are reusable, exhibit long-term stability, are robust, easy to assemble, and can operate without oil. To demonstrate the feasibility and analytical performance, we performed chemical transformations in acetonitrile on the DMF platform and monitored their progress spectroscopically for more than 100 minutes. These combined advances establish a new class of IR-integrated DMF devices, thereby enabling on-chip reaction monitoring, spatially resolved chemical imaging, and real-time mechanistic studies in microfluidic environments. • We report the first successful implementation of infrared (IR) absorption analysis within a digital microfluidics platform. • The developed digital microfluidics chip utilises calcium fluoride as an IR-transparent substrate and indium tin oxide (ITO) as the electrode material and establishes a reliable photolithographic fabrication workflow. • The IR properties of the chip materials were characterised and the chips analytical capabilities were demonstrated by repeated reaction monitoring of a model reaction, limit of detection experiments and extensive control experiments. • This work extends the analytical toolkit for digital microfluidics, utilising the advantages of IR analysis while maintaining the advantages of digital microfluidics.

We demonstrated a novel electrowetting-on-dielectric (EWOD) device for digital microfluidic using a flexible, transparent, and conducting graphene electrode. We showed that a graphene electrode had a lower leakage current and a higher breakdown voltage compared to a gold electrode when they were under the same dielectric layer such as Teflon. The droplet movement was manipulated on graphene-based EWOD using a sequential switching voltage controlled by a programmable logic controller (PLC) relay array.

Biosensing at Drop Scale Assisted by Electrowetting at Moderate Frequency

Electrowetting-on-dielectric (EWD) digital microfluidic laboratory-on-a-chip platforms demonstrate excellent performance in automating labor-intensive protocols. When coupled with an on-chip electroporation capability, these systems hold promise for streamlining cumbersome processes such as multiplex automated genome engineering (MAGE). We integrated a single Ti:Au electroporation electrode into an otherwise standard parallel-plate EWD geometry to enable high-efficiency transformation of Escherichia coli with reporter plasmid DNA in a 200 nL droplet. Test devices exhibited robust operation with more than 10 transformation experiments performed per device without cross-contamination or failure. Despite intrinsic electric-field nonuniformity present in the EP/EWD device, the peak on-chip transformation efficiency was measured to be 8.6 ± 1.0 × 108 cfu·μg-1 for an average applied electric field strength of 2.25 ± 0.50 kV·mm-1. Cell survival and transformation fractions at this electroporation pulse strength were found to be 1.5 ± 0.3 and 2.3 ± 0.1%, respectively. Our work expands the EWD toolkit to include on-chip microbial electroporation and opens the possibility of scaling advanced genome engineering methods, like MAGE, into the submicroliter regime.