Digital Microfluidic Research Articles

Large-Area Electronics-Enabled High-Resolution Digital Microfluidics for Parallel Single-Cell Manipulation.

Large-area electronics as switching elements are an ideal option for electrode-array-based digital microfluidics. With support of highly scalable thin-film semiconductor technology, high-resolution digital droplets (diameter around 100 μm) containing single-cell samples can be manipulated freely on a two-dimensional plane with programmable addressing logic. In addition, single-cell generation and manipulation as foundations for single-cell research demand ease of operation, multifunctionality, and accurate tools. In this work, we reported an active-matrix digital microfluidic platform for single-cell generation and manipulation. The active device contained 26,368 electrodes that could be independently addressed to perform parallel and simultaneous droplet generation and achieved single-cell manipulation. We demonstrate a high-resolution digital droplet generation with a droplet volume limit of 500 pL and show the continuous and stable movement of droplet-contained cells for over 1 h. Furthermore, the success rate of single droplet formation was higher than 98%, generating tens of single cells within 10 s. In addition, a pristine single-cell generation rate of 29% was achieved without further selection procedures, and the droplets containing single cells could then be tested for on-chip cell culturing. After 20 h of culturing, about 12.5% of the single cells showed cell proliferation.

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

The Long and Winding Road: On-Demand DNA Synthesis in High Demand

Integrated Digital Microfluidics NMR Spectroscopy: A Key Step toward Automated In Vivo Metabolomics.

Toxicity testing is currently undergoing a paradigm shift from examining apical end points such as death, to monitoring sub-lethal toxicity in vivo. In vivo nuclear magnetic resonance (NMR) spectroscopy is a key platform in this endeavor. A proof-of-principle study is presented which directly interfaces NMR with digital microfluidics (DMF). DMF is a "lab on a chip" method allowing for the movement, mixing, splitting, and dispensing of μL-sized droplets. The goal is for DMF to supply oxygenated water to keep the organisms alive while NMR detects metabolomic changes. Here, both vertical and horizontal NMR coil configurations are compared. While a horizontal configuration is ideal for DMF, NMR performance was found to be sub-par and instead, a vertical-optimized single-sided stripline showed most promise. In this configuration, three organisms were monitored in vivo using 1H-13C 2D NMR. Without support from DMF droplet exchange, the organisms quickly showed signs of anoxic stress; however, with droplet exchange, this was completely suppressed. The results demonstrate that DMF can be used to maintain living organisms and holds potential for automated exposures in future. However, due to numerous limitations of vertically orientated DMF, along with space limitations in standard bore NMR spectrometers, we recommend future development be performed using a horizontal (MRI style) magnet which would eliminate practically all the drawbacks identified here.

Electrowetting-Induced Coalescence of Sessile Droplets in Viscous Medium

Manipulating the coalescence of microdroplets has recently gained enormous attention in digital microfluidics and biological and chemical industries. Here, coalescence between two sessile droplets is induced by spreading them due to electrowetting. The electrocoalescence dynamics is investigated for a wide range of operating parameters such as electrowetting number, Ohnesorge number, driving frequency, and drop to surrounding medium viscosity ratio. Here, the characteristic time scale from the classical lubrication theory is modified with an additional driving and resisting force due to the electrostatic pressure force and liquid-liquid viscous dissipation, respectively. With the revised characteristic time scale, a universal bridge growth is shown between the two merging droplets following a 1/3 power law during early coalescence followed by a long-range linear variation. To ensure precise control on droplet coalescence, a geometric analysis is also performed to define the initial separation distance.

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.

Utilizing an Automated SERS-Digital Microfluidic System for High-Throughput Detection of Explosives.

The surface-enhanced Raman scattering (SERS) technique is a promising method for the detection of explosives such as 2,4,6-trinitrotoluene (TNT) and 3-nitro-1,2,4-triazol-5-one (NTO) because of its high sensitivity to trace substances. However, most SERS detection processes are often nonautomated as well as exhibit low efficiency and toxic exposure, which often poses potential danger to operators. Herein, we propose the integration of SERS with digital microfluidics (SERS-DMF) for automated, high-throughput, and high-sensitivity detection of explosives. First, we carefully designed a DMF chip comprising 40 drive electrodes and 8 storage electrodes to achieve a high-throughput process. And different concentrations of target molecules, silver nanoparticles (Ag NPs), and salts were loaded into the DMF chip. Then, the droplet aggregation, incubation, and detection processes were automatically controlled using the SERS-DMF platform. In addition, Ag NPs were efficiently aggregated by screening different types and concentrations of salts, resulting in "hotspots" and the SERS effect. With the help of the SERS-DMF platform, two explosive samples were automatically detected with high throughput and high sensitivity. The detection limits of TNT and NTO were 10-7 and 10-8 M, respectively. In addition, compared with nonautomatic operations, the SERS-DMF platform exhibited better reproducibility and higher efficiency for the detection of explosives. The proposed SERS-DMF thus has considerable potential as an analytical technique for detecting hazardous substances.

Self-powered digital microfluidics driven by rotational triboelectric nanogenerator

Digital microfluidic (DMF) has emerged as one of the most popular microfluidic platforms for sample-preparation in biochemical analysis and lab-on-a-chip applications. Operated with electrowetting on dielectric (EWOD) mechanism, DMF conventionally requires an external power source to provide the actuation voltage, which limited its portability and broader applications in point-of-care testing (POCT) environment. Herein, a DMF device, self-powered by triboelectric nanogenerator (TENG) is presented. TENG possesses a number of unique characteristics, and is very attractive to be integrated with DMF. It only requires a simple configuration with low-cost fabrication that can improve the DMF portability, but it also provides high voltage, low current output characteristics that are consistent with the EWOD actuation requirements. Basic droplet manipulations, including transportation, split, merge, dispense, and even elongate to follow the electrode patterns of alphabets, on a DMF device powered with manually-rotated Disk-TENG are demonstrated for the first time. Further, droplets containing samples and reagents are transported and mixed on the programmed electrode patterns on the chip to conduct chemical reactions, including nucleic acid amplification and phenol red test, showing that Disk-TENG can serve as the power source for DMF chips in POCT applications.

Current State and Innovations in Newborn Screening: Continuing to Do Good and Avoid Harm.

In 1963, Robert Guthrie's pioneering work developing a bacterial inhibition assay to measure phenylalanine in dried blood spots, provided the means for whole-population screening to detect phenylketonuria in the USA. In the following decades, NBS became firmly established as a part of public health in developed countries. Technological advances allowed for the addition of new disorders into routine programmes and thereby resulted in a paradigm shift. Today, technological advances in immunological methods, tandem mass spectrometry, PCR techniques, DNA sequencing for mutational variant analysis, ultra-high performance liquid chromatography (UPLC), iso-electric focusing, and digital microfluidics are employed in the NBS laboratory to detect more than 60 disorders. In this review, we will provide the current state of methodological advances that have been introduced into NBS. Particularly, 'second-tier' methods have significantly improved both the specificity and sensitivity of testing. We will also present how proteomic and metabolomic techniques can potentially improve screening strategies to reduce the number of false-positive results and improve the prediction of pathogenicity. Additionally, we discuss the application of complex, multiparameter statistical procedures that use large datasets and statistical algorithms to improve the predictive outcomes of tests. Future developments, utilizing genomic techniques, are also likely to play an increasingly important role, possibly combined with artificial intelligence (AI)-driven software. We will consider the balance required to harness the potential of these new advances whilst maintaining the benefits and reducing the risks for harm associated with all screening.

Harmonization of Newborn Screening Results for Pompe Disease and Mucopolysaccharidosis Type I.

In newborn screening, false-negative results can be disastrous, leading to disability and death, while false-positive results contribute to parental anxiety and unnecessary follow-ups. Cutoffs are set conservatively to prevent missed cases for Pompe and MPS I, resulting in increased falsepositive results and lower positive predictive values. Harmonization has been proposed as a way to minimize false-negative and false-positive results and correct for method differences, so we harmonized enzyme activities for Pompe and MPS I across laboratories and testing methods (Tandem Mass Spectrometry (MS/MS) or Digital Microfluidics (DMF)). Participating states analyzed proofof- concept calibrators, blanks, and contrived specimens and reported enzyme activities, cutoffs, and other testing parameters to Tennessee. Regression and multiples of the median were used to harmonize the data. We observed varied cutoffs and results. Six of seven MS/MS labs reported enzyme activities for one specimen for MPS I marginally above their respective cutoffs with results classified as negative, whereas all DMF labs reported this specimen's enzyme activity below their respective cutoffs with results classified as positive. Reasonable agreement in enzyme activities and cutoffs was achieved with harmonization; however, harmonization does not change how a value would be reported as this is dependent on the placement of cutoffs.

Janus liquid marbles: Fabrication techniques, recent developments, and applications

AbstractLiquid marbles (LMs) are nonsticky droplets stabilized by hydrophobic solid particles that are adsorbed at the liquid–air interface. LMs are emerging as a potential platform in digital microfluidics, sensing applications, storage unit, biological incubation, and cosmetic applications. Incorporating multifunctional particles to form Janus liquid marble (JLM) could enhance the capabilities of the pristine LM. JLMs are multifunctional next‐generation LMs comprising two hemispherical domains of distinct physicochemical properties such as size, hydrophobicity, electrical conductivity, surface functionalization, stimuli‐responsivity, and so on. The JLMs offer precise control on the manipulability and enhanced performance over pristine LMs. Though the properties, applications, and progress of LMs are detailed in the recent literature, a focused review encompassing the fabrication, recent developments, potential applications of JLMs, and the challenges regarding its reliable fabrication remains a gap in the literature. The review provides insights into the importance of JLMs, systematically discussing the fabrication strategies, applications, challenges, and future directions.

A bidirectional droplet routing in digital microfluidics biochip

Droplet routing in digital microfluidic biochip is very challenging. It involves moving droplets from its source cell to destination cell. The challenge is to maintain fluidic constraints for every droplet movement. In this article, we have proposed a greedy bidirectional droplet routing based on divide and conquerer approach. The biochip is explored using two waves i.e. one from source cell and other from destination cell. Then, the biochip is converted into a bipartite graph by identifying strongly connected components. The route selection is done using our four greedy selection heuristics. The final route is generated by appending multiple sub-routes. The simulation result show an improvement of 7.32% and 18.05% in latest arrival time and number of shared cells respectively in free topology. Similarly we register an enhancement of 8.43% and 19.70% latest arrival time and number of shared cells respectively in virtual topology.

Digital microfluidics as an emerging tool for bacterial protocols.

Bacteria are widely studied in various research areas, including synthetic biology, sequencing and diagnostic testing. Protocols involving bacteria are often multistep, cumbersome and require access to a long list of instruments to perform experiments. In order to streamline these processes, the fluid handling technique digital microfluidics (DMF) has provided a miniaturized platform to perform various steps of bacterial protocols from sample preparation to analysis. DMF devices can be paired/interfaced with instrumentation such as microscopes, plate readers, and incubators, demonstrating their versatility with existing research tools. Alternatively, DMF instruments can be integrated into all-in-one packages with on-chip magnetic separation for sample preparation, heating/cooling modules to perform assay steps and cameras for absorbance and/or fluorescence measurements. This perspective outlines the beneficial features DMF offers to bacterial protocols, highlights limitations of current work and proposes future directions for this tool's expansion in the field.

A review of droplet bouncing behaviors on superhydrophobic surfaces: Theory, methods, and applications

The phenomenon of droplet bouncing on superhydrophobic surfaces has received extensive attention in the academic and industrial fields, as it is critical for various engineering applications, such as anti-icing, spray cooling, and metal quenching. In this review, the research of droplet bouncing behaviors is comprehensively introduced from the bouncing mechanism, research methods, and potential applications. The bouncing mechanism is related to three aspects: droplet properties, surface characteristics, and ambient conditions. Among them, droplet size, impact velocity, gas film, surface morphology, surface temperature, and applied electric field are frequently concerned. Surface wettability is critical for droplet bouncing behaviors, which affects the droplet movement on the surface. Momentum and mass distribution are essential to reduce contact time, which can be achieved through the surface morphology design. The manipulation of solid–liquid contact lines and surface tension can achieve directional droplet transportation. In addition, typical droplet bouncing experiments are presented, and experimental studies of single and successive droplets in recent decades are collated. Volume of fluid, the lattice Boltzmann method, and molecular dynamics are described, which are typical simulation methods for droplet bouncing dynamics at different scales. Potential engineering applications such as surface self-cleaning, digital microfluidics, and enhanced heat transfer have been developed through theoretical foundations and research methods. Finally, the conclusions and the possible future research directions are outlined. The durability of superhydrophobic surfaces becomes a bottleneck for engineering applications. The life cycle research perspective may be applied to future studies.

Experimental and Theoretical Investigation on the Dynamic Response of Ferrofluid Liquid Marbles to Steady and Pulsating Magnetic Fields.

Liquid marbles are droplets enwrapped by a layer of hydrophobic micro/nanoparticles. Due to the isolation of fluid from its environment, reduction in evaporation rate, low friction with the surfaces, and capability of manipulation even on hydrophilic surfaces, liquid marbles have attracted the attention of researchers in digital microfluidics. This study investigates the manipulation of ferrofluid liquid marbles (FLMs) under DC and pulse width-modulated (PWM) magnetic fields generated by an electromagnet for the first time. At first, the threshold of the magnetic field for manipulating these FLMs is studied. Afterward, the dynamic response of the FLMs to the DC magnetic field for different FLM volumes, coil currents, and initial distances of FLM from the coil is studied, and a theoretical model is proposed. By applying the PWM magnetic field, it is possible to gain more control over the manipulation of the FLMs on the surface and adjust their position more accurately. Results indicate that with a decrease in FLM volume, coil current, and duty cycle, the FLM step length decreases; hence, FLM manipulation is more precise. Under the PWM magnetic field, it is observed that FLM movement is not synchronous with the generated pulse, and even after the coil is turned off, FLMs keep their motion. In the end, with proper adjustment of the electromagnet pulse width, launching of FLMs at a distance farther than the coil is observed.

Dissolution-Enhanced Luminescence Enhanced Digital Microfluidics Immunoassay for Sensitive and Automated Detection of H5N1.

Protein bioassay is a critical tool for the screening and detection of protein biomarkers in disease diagnostics and biological applications. However, the detection sensitivity and system automation of current immunoassays do not meet the emerging demands of clinical applications. Here, we developed a dissolution-enhanced luminescence-enhanced digital microfluidics immunoassay (DEL-DMF), which significantly improves the sensitivity and automation of the protein bioassay. In DEL-DMF, the sample and reagent droplets are controlled to complete the processes of sample transport, immunoreaction, and buffer washing, which not only minimizes sample consumption to 2 μL and enhances the binding efficiency of immunoreaction but also streamlines all the procedures and simplifies the process of immunoassay. Moreover, dissolution-enhanced luminescence using NaEuF4 NPs as nanoprobes boosts the fluorescence and increases the sensitivity of the bioassay. We demonstrate the enhanced analytical performance of our DEL-DMF immunoassay to detect H5N1 hemagglutinin in human serum and saliva. A limit of detection of 1.16 pM was achieved in less than 0.5 h with only 2 μL sample consumption. Overall, our DEL-DMF immunoassay combines the merits of the microfluidics platform and dissolution-enhanced luminescence, thus affording superior detection sensitivity and system automation for protein biomarkers. This novel immunoassay microsystem holds great potential in clinical and biological applications.

Antifouling Properties of Pluronic and Tetronic Surfactants in Digital Microfluidics.

Fouling at liquid-solid interfaces is a pernicious problem for a wide range of applications, including those that are implemented by digital microfluidics (DMF). There are several strategies that have been used to combat surface fouling in DMF, the most common being inclusion of amphiphilic surfactant additives in the droplets to be manipulated. Initial studies relied on Pluronic additives, and more recently, Tetronic additives have been used, which has allowed manipulation of complex samples like serum and whole blood. Here, we report our evaluation of 19 different Pluronic and Tetronic additives, with attempts to determine (1) the difference in antifouling performance between the two families, (2) the structural similarities that predict exceptional antifouling performance, and (3) the mechanism of the antifouling behavior. Our analysis shows that both Pluronic and Tetronic additives with modest molar mass, poly(propylene oxide) (PPO) ≥50 units, poly(ethylene oxide) (PEO) mass percentage ≤50%, and hydrophilic-lipophilic balance (HLB) ca. 13-15 allow for exceptional antifouling performance in DMF. The most promising candidates, P104, P105, and T904, were able to support continuous movement of droplets of serum for more than 2 h, a result (for devices operating in air) previously thought to be out of reach for this technique. Additional results generated using device longevity assays, intrinsic fluorescence measurements, dynamic light scattering, asymmetric flow field flow fractionation, supercritical angle fluorescence microscopy, atomic force microscopy, and quartz crystal microbalance measurements suggest that the best-performing surfactants are more likely to operate by forming a protective layer at the liquid-solid interface than by complexation with proteins. We propose that these results and their implications are an important step forward for the growing community of users of this technique, which may provide guidance in selecting surfactants for manipulating biological matrices for a wide range of applications.

A digital microfluidic single-cell manipulation system optimized by extending-depth-of-field device

Microfluidic systems have been widely utilized in high-throughput biology analysis, but the difficulties in liquid manipulation and cell cultivation limit its application. This work has developed a new digital microfluidic (DMF) system for on-demand droplet control. By adopting an extending-depth-of-field (EDoF) phase modulator to the optical system, the entire depth of the microfluidic channel can be covered in one image without any refocusing process, ensuring that 95% of the particles in the droplet are captured within three shots together with shaking processes. With this system, suspension droplets are generated and droplets containing only one yeast cell can be recognized, then each single cell is cultured in the array of the chip. By observing their growth in cell numbers and the green fluorescence protein (GFP) production via fluorescence imaging, the single cell with the highest production can be identified. The results have proved the heterogeneity of yeast cells, and showed that the combined system can be applied for rapid single-cell sorting, cultivation, and analysis.

Sensitive and automated detection of bacteria by CRISPR/Cas12a-assisted amplification with digital microfluidics

The development of new diagnostic platforms for sensitive and rapid detection of bacterial pathogens is of great significance for improving precision medication for infectious disease and food safety control. Currently, many bacterial detection methods are limited by low detection sensitivity, sophisticated protocols, long processing times and labor-intensive procedures. Here, we report a DMF-Cas12a method for rapid, sensitive and automated analysis of bacterial pathogens by integrating the CRISPR/Cas12a system with a digital microfluidic chip (DMF). In the assay, Staphylococcus aureus could be detected with high sensitivity and specificity, and the detection limit of the assay was found to be 32 CFU/mL. With the tiny volume and automation capabilities of DMF, this assay was accomplished in 55 min and consumed only 6.6 μL reagent. This platform was successfully applied to urine and milk samples with excellent sensitivity and reliability. The DMF-Cas12a method shows good potential in automated and highly sensitive pathogen detection for a wide variety of biological applications.

Induced flow inside a droplet by static electrical charge

Introducing controlled fluid motion in a droplet turns out to be of outstanding scientific importance, hallmarked by a plethora of applications ranging from engineering to biology. While internal mechanisms such as interfacial tension or buoyancy-driven dynamics may trigger fascinating flow structures inside a droplet, controllability of the same without external forcing remains questionable. On the other hand, in an electrically forced environment, complex fabrication steps and special choices of the ionic liquid are often demanded. Circumventing these limits, here we bring out a new method of flow manipulation inside a sessile droplet by simply deploying a static charge produced by the triboelectric effect. This is physically actuated by charge transfer between the two lateral electrodes within which the droplet is entrained, triggering a strong ionized air current. The flow inside the droplet is generated due to the shear exerted at the interface by the charge-induced ionized airflow around the droplet, a paradigm that has hitherto remained unexplored. The strength of the fluid flow can be controlled by adjusting the supplied static charge. Such unique controllability without sacrificing the physical simplicity opens up new possibilities for flow manipulation in a multitude of applications ranging from droplet microreactors to digital microfluidics.

Low-cost Polyurethane Coating as Dielectric Component in Digital Microfluidics

Digital microfluidics (DMF) as a platform for precise handling of liquid droplets is a powerful tool but the affordability of the device has been one of the hindrances to its wide implementation. This paper reports the development of DMF devices using low-cost materials and simple deposition techniques specifically for the device dielectric component. Three commercial polyurethane coatings were investigated for their feasibility as the dielectric layer. The electrowetting behaviour of these materials was investigated by evaluating the change in contact angle with applied voltage of a water droplet sitting on the dielectric samples prepared using easy deposition methods such as spraying and spin coating. Devices were then fabricated using these materials to evaluate their capability to actuate droplets. Five types of polyurethane dielectric sample exhibited contact angle reversibility with hysteresis ranging between 4° to 25° after 250 VDC application. Droplet transportation back and forth across 8 electrodes at 180 VRMS has been demonstrated in a device made of one of the polyurethane coatings using a spraying technique. This result implies the potential of using polyurethane for future development of low-cost and disposable DMF devices.