Ultrasensitive amplification-free detection of circulating miRNA via droplet-based processing of SERS tag-miRNA-magnetic nanoparticle sandwich nanocomplexes on a paper-based electrowetting-on-dielectric platform.

MicroRNAs (miRNAs) have emerged as a promising class of biomarkers for early detection of various cancers, including ovarian cancer. However, quantifying miRNAs in human blood samples is challenging owing to the issues of sensitivity and specificity. In this study, hsa-miR-200a-3p of the miR-200a sub-family, which is a biomarker of ovarian cancer, was used as the analyte to demonstrate the analytical capability of an integrated biosensing platform using an extremely sensitive surface-enhanced Raman scattering (SERS) nanotag-nanoaggregate-embedded beads (NAEBs), magnetic nanoparticles (MNPs), a pair of highly specific locked nucleic acid (LNA) probes, and a semi-automated paper-based electrowetting-on-dielectric (pEWOD) device to provide labor-less and thorough sample cleanup and recovery. A sandwich approach where NAEBs are modified by one LNA-1 probe and MNPs are modified by another LNA-2 probe was applied. Then, the target analyte miRNA-200a-3p was introduced to form a sandwich nanocomplex through hybridization with the pair of LNA probes. The pEWOD device was used to achieve short cleanup time and good recovery of the nanocomplex, bringing the total analysis time to less than 30 min. The detection limit of this approach can reach 0.26 fM through SERS detection. The versatility of this method without the need for RNA extraction from clinical samples is expected to have good potential in detecting other miRNAs.

Transitional Electrodes in Electrowetting-Based Droplet Dispensing.

Digital microfluidic systems based on electrowetting-on-dielectric technology, particularly valuable in producing and manipulating microdroplets steadily and consistently, have experienced notable advancements in recent years. In this paper, experimental characterizations reveal that simply adding one transitional electrode between the reservoir and the splitting electrode improves the volumetric consistency and reproducibility for droplet dispensing. The volumetric coefficient variation of the consecutively dispensed droplets from a non-refilling reservoir decreases by 1% after the addition of one transitional electrode, with no extra external apparatus. This work provides a straightforward yet effective approach to the improvement of digital microfluidic systems and micro total analysis systems.

Advanced design and applications of digital microfluidics in biomedical fields: An update of recent progress.

Significant breakthroughs have been made in digital microfluidic (DMF)-based technologies over the past decades. DMF technology has attracted great interest in bioassays depending on automatic microscale liquid manipulations and complicated multi-step processing. In this review, the recent advances of DMF platforms in the biomedical field were summarized, focusing on the integrated design and applications of the DMF system. Firstly, the electrowetting-on-dielectric principle, fabrication of DMF chips, and commercialization of the DMF system were elaborated. Then, the updated droplets and magnetic beads manipulation strategies with DMF were explored. DMF-based biomedical applications were comprehensively discussed, including automated sample preparation strategies, immunoassays, molecular diagnosis, blood processing/testing, and microbe analysis. Emerging applications such as enzyme activity assessment and DNA storage were also explored. The performance of each bioassay was compared and discussed, providing insight into the novel design and applications of the DMF technology. Finally, the advantages, challenges, and future trends of DMF systems were systematically summarized, demonstrating new perspectives on the extensive applications of DMF in basic research and commercialization.

Development of a Microfluidic Chip Powered by EWOD for In Vitro Manipulation of Bovine Embryos.

Digital microfluidics (DMF) holds great potential for the alleviation of laboratory procedures in assisted reproductive technologies (ARTs). The electrowetting on dielectric (EWOD) technology provides dynamic culture conditions in vitro that may better mimic the natural embryo microenvironment. Thus far, EWOD microdevices have been proposed for in vitro gamete and embryo handling in mice and for analyzing the human embryo secretome. This article presents the development of the first microfluidic chip utilizing EWOD technology designed for the manipulation of bovine embryos in vitro. The prototype sustains the cell cycles of embryos manipulated individually on the chips during in vitro culture (IVC). Challenges related to the chip fabrication as well as to its application during bovine embryo IVC in accordance with the adapted on-chip protocol are thoroughly discussed, and future directions for DMF in ARTs are indicated.

Morphogenesis-inspired two-dimensional electrowetting in droplet networks.

Living tissues dynamically reshape their internal cellular structures through carefully regulated cell-to-cell interactions during morphogenesis. These cellular rearrangement events, such as cell sorting and mutual tissue spreading, have been explained using the differential adhesion hypothesis, which describes the sorting of cells through their adhesive interactions with their neighbors. In this manuscript we explore a simplified form of differential adhesion within a bioinspired lipid-stabilized emulsion approximating cellular tissues. The artificial cellular tissues are created as a collection of aqueous droplets adhered together in a network of lipid membranes. Since this abstraction of the tissue does not retain the ability to locally vary the adhesion of the interfaces through biological mechanisms, instead we employ electrowetting with offsets generated by spatial variations in lipid compositions to capture a simple form of bioelectric control over the tissue characteristics. This is accomplished by first conducting experiments on electrowetting in droplet networks, next creating a model for describing electrowetting in collections of adhered droplets, then validating the model against the experimental measurements. This work demonstrates how the distribution of voltage within a droplet network may be tuned through lipid composition then used to shape directional contraction of the adhered structure using two-dimensional electrowetting events. Predictions from this model were used to explore the governing mechanics for complex electrowetting events in networks, including directional contraction and the formation of new interfaces.

Combining sensors and actuators with electrowetting-on-dielectric (EWOD): advanced digital microfluidic systems for biomedical applications.

The concept of digital microfluidics (DMF) enables highly flexible and precise droplet manipulation at a picoliter scale, making DMF a promising approach to realize integrated, miniaturized "lab-on-a-chip" (LOC) systems for research and clinical purposes. Owing to its simplicity and effectiveness, electrowetting-on-dielectric (EWOD) is one of the most commonly studied and applied effects to implement DMF. However, complex biomedical assays usually require more sophisticated sample handling and detection capabilities than basic EWOD manipulation. Alternatively, combined systems integrating EWOD actuators and other fluidic handling techniques are essential for bringing DMF into practical use. In this paper, we briefly review the main approaches for the integration/combination of EWOD with other microfluidic manipulation methods or additional external fields for specified biomedical applications. The form of integration ranges from independently operating sub-systems to fully coupled hybrid actuators. The corresponding biomedical applications of these works are also summarized to illustrate the significance of these innovative combination attempts.

Digital microfluidics for biological analysis and applications.

Digital microfluidics (DMF) is an emerging liquid-handling technology based on arrays of microelectrodes for the precise manipulation of discrete droplets. DMF offers the benefits of automation, addressability, integration and dynamic configuration ability, and provides enclosed picoliter-to-microliter reaction space, making it suitable for lab-on-a-chip biological analysis and applications that require high integration and intricate processes. A review of DMF bioassays with a special emphasis on those actuated by electrowetting on dielectric (EWOD) force is presented here. Firstly, a brief introduction is presented on both the theory of EWOD actuation and the types of droplet motion. Subsequently, a comprehensive overview of DMF-based biological analysis and applications, including nucleic acid, protein, immunoreaction and cell assays, is provided. Finally, a discussion on the strengths, challenges, and potential applications and perspectives in this field is presented.

Liquid metal droplet motion transferred from an alkaline solution by a robot arm.

The excellent motion performance of gallium-based liquid metals (LMs) upon the application of a modest electric field has provided a new opportunity for the development of autonomous soft robots. However, the locomotion of LMs often appears in an alkaline solution, which hampers the application under other different conditions. In this work, a novel robot arm is designed to transfer the motion of the LM from an alkaline solution in a synchronous drive mode. The liquid metal droplet (LMD) at the bottom of the robot arm is actuated using a DC voltage to provide the driving force for the system. By introducing an end effector at the center of the robot arm, the synchronous motion of the system is replicated and can be applied to different situations. The theoretical understanding of continuous electrowetting (CEW) at the LM interface is explained, and then the motion performance of the robot arm against the function of the applied voltage and driving direction is investigated. Moreover, several applications using this robot arm, such as pattern drawing, cargo transportation, and drug concentration detection, are demonstrated. The presented robot arm has the potential to observably expand the application fields of the LM.

Electro-Microfluidic Assembly Platform for Manipulating Colloidal Structures inside Water-in-Oil Emulsion Droplets.

Colloidal assembly is a key strategy in nature and artificial device. Hereby, an electromicrofluidic assembly platform (eMAP) is proposed and validated to achieve 3D colloidal assembly and manipulation within water droplets. The water-in-oil emulsion droplets autoposition in the eMAP driven by dielectrophoresis, where the (di)electrowetting effect induces droplet deformation, facilitating quadratic growth of the electric field in water droplet to achieve "far-field" dielectrophoretic colloidal assembly. Reconfigurable 3D colloidal configurations are observed and dynamically programmed via applied electric fields, colloidal properties, and droplet size. Binary and ternary colloidal assemblies in one droplet allow designable chemical and physical anisotropies for functional materials and devices. Integration of eMAP in high throughput enables mass production of functional microcapsules, and programmable optoelectronic units for display devices. This eMAP is a valuable reference for expanding fundamental and practical exploration of colloidal systems.

A review of optoelectrowetting (OEW): from fundamentals to lab-on-a-smartphone (LOS) applications to environmental sensors.

Electrowetting-on-dielectric (EWOD) has been extensively explored as an active-type technology for small-scale liquid handling due to its several unique advantages, including no requirement of mechanical components, low power consumption, and rapid response time. However, conventional EWOD devices are often accompanied with complex fabrication processes for patterning and wiring of 2D arrayed electrodes. Furthermore, their sandwich device configuration makes integration with other microfluidic components difficult. More recently, optoelectrowetting (OEW), a light-driven mechanism for effective droplet manipulation, has been proposed as an alternative approach to overcome these issues. By utilizing optical addressing on a photoconductive surface, OEW can dynamically control an electrowetting phenomenon without the need for complex control circuitry on a chip, while providing higher functionality and flexibility. Using commercially available spatial light modulators such as LCD displays and smartphones, millions of optical pixels are readily generated to modulate virtual electrodes for large-scale droplet manipulations in parallel on low-cost OEW devices. The benefits of the OEW mechanism have seen it being variously explored in its potential biological and biochemical applications. This review article presents the fundamentals of OEW, discusses its research progress and limitations, highlights various technological advances and innovations, and finally introduces the emergence of the OEW technology as portable smartphone-integrated environmental sensors.

Electrically-driven handling of gametes and embryos: taking a step towards the future of ARTs.

Electrical stimulation of gametes and embryos and on-chip manipulation of microdroplets of culture medium serve as promising tools for assisted reproductive technologies (ARTs). Thus far, dielectrophoresis (DEP), electrorotation (ER) and electrowetting on dielectric (EWOD) proved compatible with most laboratory procedures offered by ARTs. Positioning, entrapment and selection of reproductive cells can be achieved with DEP and ER, while EWOD provides the dynamic microenvironment of a developing embryo to better mimic the functions of the oviduct. Furthermore, these techniques are applicable for the assessment of the developmental competence of a mammalian embryo in vitro. Such research paves the way towards the amelioration and full automation of the assisted reproduction methods. This article aims to provide a summary on the recent developments regarding electrically stimulated lab-on-chip devices and their application for the manipulation of gametes and embryos in vitro.

Minimization of MEDA Biochip-Size in Droplet Routing.

With the increasing demand for fast, accurate, and reliable biological sensor systems, miniaturized systems have been aimed at droplet-based sensor systems and have been promising. A micro-electrode dot array (MEDA) biochip, which is one kind of the miniaturized systems for biochemical protocols such as dispensing, dilutions, mixing, and so on, has become widespread due to enabling dynamical control of the droplets in microfluidic manipulations. In MEDA biochips, the electrowetting-on-dielectric (EWOD) technique stands out since it can actuate droplets with nano/picoliter volumes. Microelectrode cells on MEDA actuate multiple droplets simultaneously to route locations for the purpose of the biochemical operations. Taking advantage of the feature, droplets are often routed in parallel to achieve high-throughput outcomes. Regarding parallel manipulation of multiple droplets, however, the droplets are known to be initially placed at a distant position to avoid undesirable mixing. The droplets thus result in traveling a long way for a manipulation, and the required biochip size for routing is also enlarged. This paper proposes a routing method for droplets to reduce the biochip size on a MEDA biochip with the allowance of splitting during routing operations. We mathematically derive the routing problem, and the experiments demonstrate that our proposal can significantly reduce the biochip size by 70.8% on average, compared to the state-of-the-art method.

Open-channel microfluidics via resonant wireless power transfer.

Open-channel microfluidics enables precise positioning and confinement of liquid volume to interface with tightly integrated optics, sensors, and circuit elements. Active actuation via electric fields can offer a reduced footprint compared to passive microfluidic ensembles and removes the burden of intricate mechanical assembly of enclosed systems. Typical systems actuate via manipulating surface wettability (i.e., electrowetting), which can render low-voltage but forfeits open-microchannel confinement. The dielectric polarization force is an alternative which can generate open liquid microchannels (sub-100 µm) but requires large operating voltages (50-200 VRMS) and low conductivity solutions. Here we show actuation of microchannels as narrow as 1 µm using voltages as low as 0.5 VRMS for both deionized water and physiological buffer. This was achieved using resonant, nanoscale focusing of radio frequency power and an electrode geometry designed to abate surface tension. We demonstrate practical fluidic applications including open mixing, lateral-flow protein labeling, filtration, and viral transport for infrared biosensing-known to suffer strong absorption losses from enclosed channel material and water. This tube-free system is coupled with resonant wireless power transfer to remove all obstructing hardware - ideal for high-numerical-aperture microscopy. Wireless, smartphone-driven fluidics is presented to fully showcase the practical application of this technology.

A self-powered wireless motion sensor based on a high-surface area reverse electrowetting-on-dielectric energy harvester.

This paper presents a motion-sensing device with the capability of harvesting energy from low-frequency motion activities. Based on the high surface area reverse electrowetting-on-dielectric (REWOD) energy harvesting technique, mechanical modulation of the liquid generates an AC signal, which is modeled analytically and implemented in Matlab and COMSOL. A constant DC voltage is produced by using a rectifier and a DC-DC converter to power up the motion-sensing read-out circuit. A charge amplifier converts the generated charge into a proportional output voltage, which is transmitted wirelessly to a remote receiver. The harvested DC voltage after the rectifier and DC-DC converter is found to be 3.3 V, having a measured power conversion efficiency (PCE) of the rectifier as high as 40.26% at 5 Hz frequency. The energy harvester demonstrates a linear relationship between the frequency of motion and the generated output power, making it highly suitable as a self-powered wearable motion sensor.

Achromatic doublet electrowetting prism array for beam steering device in foveated display.

A foveated display is a technology that can solve the problem of insufficient angular resolution (relative to the human eye) for near-eye display. In a high-resolution foveated display, a beam steering element is required to track the human gaze. An electrowetting prism array is a transmissive non-mechanical beam steering device, that allows a light and compact optical system to be configured and a large aperture possible. However, the view is obstructed by the sidewall of the prism array. When the size of the cell prism is 7mm, the prism array has an 87% fill-factor. To push the fill-factor to 100%, the cell prisms were magnified using a lens array. Image processing was performed such that the image produced by the lens array was identical to the original. Beam steering by refraction is accompanied by chromatic dispersion, which causes chromatic aberration, making colors appear blurry. The refractive index condition to reduce chromatic dispersion was obtained using the doublet structure of the electrowetting prism. The chromatic dispersion was reduced by 70% on average.

DC electric field-driven heartbeat phenomenon of gallium-based liquid metal on a floating electrode.

The heart beating phenomenon of room temperature liquid metal (LM) mercury has attracted much attention in the past years, but its research and application are limited because of the low vapor pressure and high toxicity. Here, a fundamental scientific finding is reported that the non-toxic eutectic gallium indium (EGaIn) alloy droplets beat periodically at a certain frequency based on a floating electrode under the stimulation of the direct current (DC) field. The essential characteristics of heart beating are the displacement and the projected area change of the LM droplet. The mechanism of this phenomenon is the self-regulation of interfacial tension caused by chemical oxidation, chemical corrosion, and continuous electrowetting. In this article, a series of experiments are also carried out to examine the effects of different factors on the heartbeat, such as voltage, the volume of the droplet, the droplet immersion depth, the electrolyte solution concentration, the distance of electrodes, and the type of floating electrode. Finally, the heartbeat state and application boundary of the LM droplet under different conditions are summarized by imitating the human life process. The periodic changes of the LM droplet under an external DC electric field provide a new method to simulate the beating of the heart artificially, and can be applied to the research of organ chip fluid pumping in the future.

Permeation by Electrowetting Actuation: Revealing the Prospect of a Micro-valve Based on Ionic Liquid.

The electrowetting behavior of ionic liquid significantly promotes microfluidic technology due to the advantage of manipulation of ionic liquid without additional mechanical parts. Recently, a novel micro-valve that shows good prospects was proposed by MacArthur et al. based on the permeation of ionic liquid under electric field. Inspired by their work, the permeation process of ionic liquid (EMIM-Im) droplets actuated by electrowetting was investigated in this work using molecular dynamics simulation. The wettability of substrate, electric field strength and electric field polarity were varied to investigate their influences. On the substrate side, results showed that the hydrophilic substrates tend to stretch and adsorb the droplet and hence hinder the permeation process, whereas the hydrophobic substrates facilitate permeation due to their low attraction for liquid. Particularly, super hydrophilic substrates should be avoided in practice, because their strong adsorption effects will override the electric field effects and disable the permeation process. On the electric field side, results showed that increased electric field strength enhances the permeation, but varying electric field polarity will result in an asymmetric permeation behavior, which was found to be the result of the different evaporation rate of the ion species that ultimately caused a non-charge-neutral droplet. Our investigation then uncovered the two critical roles of the electric field: elongating the droplet and providing the driving force for the permeation.

Integrated Full-Range Droplet Actuation for Inkjet-Printed Digital Microfluidic Chip on Flexible Substrates.

Flexible printed electronic technology makes it possible to fabricate low-cost digital microfluidic (DMF) chips. Inkjet printing on flexible substrates is one of the most cost-effective fabrication processes for DMF chips. Based on inkjet printing technology and simplified coating methods of dielectric and hydrophobic layers, we fabricated low-cost flexible DMF chips (FDMFCs) on PET sheet and on matte photo paper. The surface quality, conductivity and spatial output resolution of the silver lines under different number of printings, different line widths and line gaps on the two types of FDMFCs were comprehensively analyzed. The traditional square dispensing electrodes were optimized to reduce the volume error of the droplets generated during the repeated dispensing operations. Droplets can be driven to implement all the operations on various configurations of FDMFCs by electrowetting-on-dielectric, including closed configuration, open configuration, hybrid configuration composed of closed and open regions on a single chip, and open curved configuration, which are defined as full-range droplet actuation. The droplet motion between closed and open regions in two position modes of the top plate was deeply studied. Droplet operation experiments prove that the motion performance of droplets can be comparable to that of chips processed by traditional technology, and the rapid prototyping technology of the FDMFCs can make the performance of droplet operation mainly depend on the conductivity of the electrode layer and the electrode gap and greatly weaken the influence of the substrate surface quality on the FDMFCs.

"One-to-three" droplet generation in digital microfluidics for parallel chemiluminescence immunoassays.

In digital microfluidics, droplet generation is a fundamental operation for quantitative liquid manipulation. The generation of well-defined micro-droplets on a chip with restricted device geometries has become a real obstacle for digital microfluidics platforms to be used in parallel for in vitro diagnostic applications. Here, we propose a "one-to-three" droplet splitting technique that is able to generate sub-microlitre droplets beyond the "well-known" geometry limit in electrowetting-on-dielectric digital microfluidics. Accordingly, we realized an on-chip magnetic bead chemiluminescence immunoassay for parallel detection with the "one-to-three" technique. With the help of the generated micro droplets, we were able to retain the magnetic beads by a significantly reduced magnetic force. We have shown the detection of five B-type natriuretic peptide analyte samples on a single chip for around 10 minutes. The correlation coefficient of the calibration curve was 0.9942, and the detection limit was lower than 5 pg mL-1.

Small universal mechanical module driven by a liquid metal droplet.

Gallium-based liquid metal droplets (LMDs) from micro-electromechanical systems (MEMS) have gained much attention due to their precise and sensitive controllability under an electric field. Considerable research progress has been made in the field of actuators by taking advantage of the continuous electrowetting (CEW) present within the solution. However, the motion generated is confined within the specific liquid environment and is lacking a way to transmit its motion outwardly, which undoubtedly serves as the greatest obstacle restricting any further development. Therefore, a driving module is proposed to generate rotational motion outside the solution for universality. Its performance can be easily tuned by adjusting the applied voltage. As an example of further application, the module is designed in the form of a pump that realizes the continuous/intermittent propulsion to mimic the veins/arteries of the human body without the problem in the previous LMD-based pumps. The feasibility of this pump in the on-chip in vitro analysis is proved by preparing a dynamic cell culture to simulate the movement of biofluids within human bodies. This study proposes an optional solution with an LMD-based motor for generating rotational motion and to expand current research on soft materials in actuators.