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With the continuous expansion of pixel arrays in digital microfluidics (DMF) chips, precise droplet control has emerged as a critical issue requiring detailed consideration. This paper proposes a novel capacitance-based droplet sensing system for thin-film transistor DMF. The proposed circuit features a distinctive inner and outer dual-pixel electrode structure, integrating droplet driving and sensing functionalities. Discharge occurs exclusively at the inner electrode during droplet sensing, effectively addressing droplet perturbation in existing sensing circuits. The circuit employs a novel fan-shaped structure of thin-film transistors. Simulation results show that it can provide a 48 V pixel voltage and demonstrate a sensing voltage difference of over 10 V between deionized water and silicone oil, illustrating its proficiency in droplet driving and accurate sensing. The stability of threshold voltage drift and temperature was also verified for the circuit. The design is tailored for integration into active matrix electrowetting-on-dielectric (AM-EWOD) chips, offering a novel approach to achieve precise closed-loop control of droplets. [ABSTRACT FROM AUTHOR]

Digital microfluidics is a novel technique for manipulating discrete droplets with the advantages of programmability, small device size, low cost, and easy integration. The development of droplet sensing methods advances the automation control of digital microfluidics. Impedance measurement emerges as a promising technique for droplet localization and characterization due to its non-invasive nature, high sensitivity, simplicity, and cost-effectiveness. However, traditional impedance measurement approaches in digital microfluidics based on the high-voltage actuating signal are limited in sensing accuracy in practical applications. In this paper, we propose a novel droplet impedance sensing system for digital microfluidics by introducing a low-voltage and addressable measurement circuit, which enables impedance measurement over a wide frequency range. The proposed measurement system has also been used for detecting the droplet composition, size, and position in a digital microfluidic chip. The improved impedance sensing method can also promote the applications of the digital microfluidic, which requires high accuracy, real-time, and contactless sensing with automatic sample pretreatment. [ABSTRACT FROM AUTHOR]

Single-cell joint analysis of methylome and transcriptome reveals how the methylation regulates the transcriptional activity. However, traditional bench-top protocols for single-cell DNA methylation and RNA transcription co-detection are labor-intensive, cost-ineffective and contaminant-prone. Herein, we establish the DMF-scMT-seq, a highly-efficient and cost-effective method to simultaneously analyze single-cell DNA methylation and transcriptional activity based on digital microfluidics. DMF-scMT-seq automates the workflow of single-cell isolation, cellular hypotonic lysis, nucleic acid separation and methylome/transcriptome library construction in a contactless and addressable way. The system ensures high accuracy (R>0.85), high gene detection ability (14,697 genes per cell at 4 million sequencing depth), and high CpG coverage (677,198 CpG sites per cell at 1 million sequencing depth). By using DMF-scMT-seq, the relationship of DNA methylation and RNA transcription under different genomic contexts is resolved. We further apply DMF-scMT-seq to study the dynamics of transcription regulation with methylation-inhibiting anti-tumor Decitabine, and identify the methylated promoter/gene body driven genes in response to Decitabine treatment. DMF-scMT-seq facilitates the construction of the correlation of DNA methylation and transcriptional activity at the single-cell level in a flexible, sensitive and accurate way, which is anticipated to be a powerful tool in studying single-cell biological systems. [ABSTRACT FROM AUTHOR]

Mobility manipulation of liquid droplets is an important task of digital imicrofluidics. Acoustic levitation has revolutionised the contactless manipulation of liquid droplets for various applications. Acoustic levitation technique can be effectively used to manipulate droplets to obtain their coalescence. This paper reports a unique, versatile, and material-independent approach for the vertical coalescence of the droplets suspended in an acoustic levitator. The acoustic power of the levitator is carefully engineered to obtain vertical coalescence of two liquid droplets. Water, 20% and 40% glycerol–water solutions are used as the working liquids. The results of the experiments revealed three outcomes during the coalescence. The outcomes are analysed and discussed. [ABSTRACT FROM AUTHOR]

Proteins as crucial components of cells are responsible for the majority of cellular processes. Sensitive and efficient protein detection enables a more accurate and comprehensive investigation of cellular phenotypes and life activities. Here, a protein sequencing method with high multiplexing, high throughput, high cell utilization, and integration based on digital microfluidics (DMF‐Protein‐seq) is proposed, which transforms protein information into DNA sequencing readout via DNA‐tagged antibodies and labels single cells with unique cell barcodes. In a 184‐electrode DMF‐Protein‐seq system, ≈1800 cells are simultaneously detected per experimental run. The digital microfluidics device harnessing low‐adsorbed hydrophobic surface and contaminants‐isolated reaction space supports high cell utilization (>90%) and high mapping reads (>90%) with the input cells ranging from 140 to 2000. This system leverages split&pool strategy on the DMF chip for the first time to overcome DMF platform restriction in cell analysis throughput and replace the traditionally tedious bench‐top combinatorial barcoding. With the benefits of high efficiency and sensitivity in protein analysis, the system offers great potential for cell classification and drug monitoring based on protein expression at the single‐cell level. [ABSTRACT FROM AUTHOR]

PD1/PD-L1 checkpoint inhibitors are at the forefront of cancer immunotherapies. However, the overall response rate remains only 10–30%. Even among initial responders, drug resistance often occurs, which can lead to prolonged use of a futile therapy in the race with the fatal disease. It would be ideal to closely monitor key indicators of patients’ immune responsiveness, such as circulating PD-L1 levels. Traditional PD-L1 detection methods, such as ELISA, are limited in sensitivity and rely on core lab facilities, preventing their use for the regular monitoring. Electrochemical sensors exist as an attractive candidate for point-of-care tool, yet, streamlining multiple processes in a single platform remains a challenge. To overcome this challenge, this work integrated electrochemical sensor arrays into a digital microfluidic device to combine their distinct merits, so that soluble PD-L1 (sPD-L1) molecules can be rapidly detected in a programmed and automated manner. This new platform featured microscale electrochemical sensor arrays modified with electrically conductive 3D matrix, and can detect as low as 1 pg/mL sPD-L1 with high specificity. The sensors also have desired repeatability and can obtain reproducible results on different days. To demonstrate the functionality of the device to process more complex biofluids, we used the device to detect sPD-L1 molecules secreted by human breast cancer cell line in culture media directly and observed 2X increase in signal compared with control experiment. This novel platform holds promise for the close monitoring of sPD-L1 level in human physiological fluids to evaluate the efficacy of PD-1/PD-L1 immunotherapy.

Abstract Lysosomal storage disorders (LSDs) are predominantly enzyme deficiencies leading to substrate accumulation, causing progressive damage to multiple organs. To date, a crucial part of diagnosing LSDs is measuring enzymatic activity in leucocytes, plasma, or dried blood spots (DBS). Here, we present results from a proof‐of‐principle study, evaluating an innovative digital microfluidics (DMF) platform, referred to as SEEKER®, that can measure the activity of the following four lysosomal enzymes from DBS: α‐L‐iduronidase (IDUA) for mucopolysaccharidosis I (MPS I), acid α‐glucosidase (GAA) for Pompe disease, β‐glucosidase (GBA) for Gaucher disease, and α‐galactosidase A (GLA) for Fabry disease. Over 900 DBS were analysed from newborns, children, and adults. DMF successfully detected known patients with MPS I, Pompe disease, and Gaucher disease, and known males with Fabry disease. This is the first demonstration of this multiplexed DMF platform for identification of patients with LSDs in a specialised diagnostic enzyme laboratory environment. We conclude that this DMF platform is relatively simple, high‐throughput, and could be readily accommodated into a specialised laboratory as a first‐tier test for MPS I, Pompe disease, and Gaucher disease for all patients, and Fabry disease for male patients only.

Single‐cell microRNA (miRNA) sequencing has allowed for comprehensively studying the abundance and complex networks of miRNAs, which provides insights beyond single‐cell heterogeneity into the dynamic regulation of cellular events. Current benchtop‐based technologies for single‐cell miRNA sequencing are low throughput, limited reaction efficiency, tedious manual operations, and high reagent costs. Here, a highly multiplexed, efficient, integrated, and automated sample preparation platform is introduced for single‐cell miRNA sequencing based on digital microfluidics (DMF), named Hiper‐seq. The platform integrates major steps and automates the iterative operations of miRNA sequencing library construction by digital control of addressable droplets on the DMF chip. Based on the design of hydrophilic micro‐structures and the capability of handling droplets of DMF, multiple single cells can be selectively isolated and subject to sample processing in a highly parallel way, thus increasing the throughput and efficiency for single‐cell miRNA measurement. The nanoliter reaction volume of this platform enables a much higher miRNA detection ability and lower reagent cost compared to benchtop methods. It is further applied Hiper‐seq to explore miRNAs involved in the ossification of mouse skeletal stem cells after bone fracture and discovered unreported miRNAs that regulate bone repairing. [ABSTRACT FROM AUTHOR]

Simultaneous profiling of DNA methylation and gene expression within single cells is a powerful technology to dissect complex gene regulatory network of cells. However, existing methods are based on picking a single‐cell in a tube and split single‐cell lysate into two parts for transcriptome and methylome library construction, respectively, which is costly and cumbersome. Here, DIRECT is proposed, a digital microfluidics‐based method for high‐efficiency single‐cell isolation and simultaneous analysis of the methylome and transcriptome in a single library construction. The accuracy of DIRECT is demonstrated in comparison with bulk and single‐omics data, and the high CpG site coverage of DIRECT allows for precise analysis of copy number variation information, enabling expansion of single cell analysis from two‐ to three‐omics. By applying DIRECT to monitor the dynamics of mouse embryonic stem cell differentiation, the relationship between DNA methylation and changes in gene expression during differentiation is revealed. DIRECT enables accurate, robust, and reproducible single‐cell DNA methylation and gene expression co‐analysis in a more cost‐effective, simpler library preparation and automated manner, broadening the application scenarios of single‐cell multi‐omics analysis and revealing a more comprehensive and fine‐grained map of cellular regulatory landscapes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Digital microfluidics (DMF) has found great applications in vitro diagnostics (IVD). Compared to the microfabrication-based DMF, printed circuit board (PCB)-based DMF is more economical and compatible with existing IVD instruments. Despite that, current PCB-based DMF is oftentimes limited by the available droplets that can be controlled simultaneously, compromising their throughput and applications as point-of-care tools. In this work, a platform that simultaneously controls multiple PCB-based DMF plates was constructed. The software and hardware were first developed, followed by the reliability tests. Colorimetric analysis of glucose was applied to the PCB-based DMF, demonstrating the capability of this platform. With the high throughput enabled by simultaneous operations of multiple plates, this PCB-based DMF can potentially allow point-of-care testing with low cost for resource-limited settings.

Digital microfluidics (DMF) is a technique for actively dispensing and manipulating discrete microdroplets on hydrophobic surfaces. DMF devices based on electrowetting on medium (EWOD) effect are widely used due to their advantages of fast response speed, simple system and high accuracy. Microfluidics is important for sweat sensing because it minimizes sweat pollution and evaporation of sweat on the skin. However, the current microfluidic channel has some problems, such as the old and new sweat is easy to mix in the channel, the self‐driving efficiency is too low, and the small sweat volume cannot be detected in time. Digital microfluidics can accurately transmit small sweats, avoid sweat residue in the channel, and have high voltage drive efficiency. Therefore, in this paper, we will use DMF digital microfluidic array technology to realize the collection of trace sweat, and realize the collection and detection of micro‐nano fabrication technology—photolithography. In this way, it is a microfluidic sweat sensor that realizes efficient transmission, trace detection, and intelligent monitoring, and is expected to be applied in wearable sweat sensing. [ABSTRACT FROM AUTHOR]

With the continuous expansion of pixel arrays in digital microfluidics (DMF) chips, precise droplet control has emerged as a critical issue requiring detailed consideration. This paper proposes a novel capacitance-based droplet sensing system for thin-film transistor DMF. The proposed circuit features a distinctive inner and outer dual-pixel electrode structure, integrating droplet driving and sensing functionalities. Discharge occurs exclusively at the inner electrode during droplet sensing, effectively addressing droplet perturbation in existing sensing circuits. The circuit employs a novel fan-shaped structure of thin-film transistors. Simulation results show that it can provide a 48 V pixel voltage and demonstrate a sensing voltage difference of over 10 V between deionized water and silicone oil, illustrating its proficiency in droplet driving and accurate sensing. The stability of threshold voltage drift and temperature was also verified for the circuit. The design is tailored for integration into active matrix electrowetting-on-dielectric (AM-EWOD) chips, offering a novel approach to achieve precise closed-loop control of droplets.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in the African American population. We identified eighteen G6PD-deficient samples (9%) in a study of residual, de-identified whole blood specimens from 200 African American pediatric patients using a point-of-care instrument. This highlights the possibility of a rapid time to result for G6PD testing, which can be valuable in some clinical scenarios. [ABSTRACT FROM AUTHOR]

The rapid quantification of nitrate nitrogen concentration plays a pivotal role in monitoring soil nutrient content. Nevertheless, the low detection efficiency limits the application of traditional methods in rapid testing. For this investigation, we utilized a digital microfluidic platform and 3D-printed microfluidics to accomplish automated detection of soil nitrate nitrogen with high sensitivity across numerous samples. The system combines digital microfluidics (DMF), 3D-printed microfluidics, a peristaltic pump, and a spectrometer. The soil solution, obtained after extraction, was dispensed onto the digital microfluidic platform using a micropipette. The digital microfluidic platform regulated the movement of droplets until they reached the injection area, where they were then aspirated into the 3D-printed microfluidic device for absorbance detection. Implementing this approach allows for the convenient sequential testing of multi-samples, thereby enhancing the efficiency of nitrate nitrogen detection. The results demonstrate that the device exhibits rapid detection (200 s for three samples), low reagent consumption (40 µL per sample), and low detection limit (95 µg/L). In addition, the relative error between the detected concentration and the concentration measured by ultraviolet spectrophotometry is kept within 20%, and the relative standard deviation (RSD) of the measured soil samples is between 0.9% and 4.7%. In the foreseeable future, this device will play a significant role in improving the efficiency of soil nutrient detection and guiding fertilization practices. [ABSTRACT FROM AUTHOR]

Automated techniques for mammalian cell engineering are needed to examine a wide range of unique genetic perturbations especially when working with precious patient samples. An automated and miniaturized technique making use of digital microfluidics to electroporate a minimal number of mammalian cells (≈40 000) at a time on a scalable platform is introduced. This system functions by merging three droplets into a continuous droplet chain, which is called a triDrop. In the triDrop configuration, the outer droplets are comprised of high‐conductive liquid while an inner or middle droplet comprising of low‐conductivity liquid that contains the cells and biological payloads. In this work, it is shown that applying a voltage to the outer droplets generates an effective electric field throughout the tri‐droplet structure allowing for insertion of the biological payload into the cells without sacrificing long‐term cell health. This technique is shown for a range of biological payloads including plasmids, mRNA, and fully formed proteins being inserted into adherent and suspension cells which include primary T‐cells. The unique features of flexibility and versatility of triDrop show that the platform can be used for the automation of multiplexed gene edits with the benefits of low reagent consumption and minimal cell numbers. [ABSTRACT FROM AUTHOR]

Digital microfluidics allows for controlled droplet movement by applying AC or DC voltages. In this research, we investigated the dynamic contact angle variation of droplets at different voltages and droplet volumes in a digital microfluidics platform. Volumes of 10 µL, 14 µL, and 18 µL were investigated with voltages ranging from 250 V to 400 V. The goal was to investigate how variations in voltage and droplet volume affected the contact angle, specifically by tracking variations in the advancing and receding contact angles. The findings showed that as voltage rises, the contact angle decreases more noticeably in terms of both advancing and receding angles. This shows that higher voltages boost the electrowetting effect and lead to better droplet dispersal and substrate wetting. Furthermore, it was found that across the studied voltage range, the effect of volume on the contact angle was largely consistent. The relation between voltage, volume, and contact angle in electrowetting is better understood from our analysis. [ABSTRACT FROM AUTHOR]

In digital microfluidics, discrete liquid droplets move on a dielectric surface with the help of AC or DC voltage. Digital microfluidics system has extended the scope of microfluidics by providing automated, precise flow control for point-of-care devices capable of medical diagnostics, environmental, and chemical sensing platforms. In this paper, we introduce a digital microfluidics platform based on electrowetting on a dielectric using PCB substrate designed on Easy EDA software. The platform's performance was analyzed at various DC voltages between 200 V and 400 V for droplet actuation. PCB fabrication, the utilization of easily accessible and cheap materials such as cooking oil and grafting tape as a dielectric layer, allowed the fabrication to be affordable and simple. The results indicate that droplet actuation takes place at 220 V. The droplet velocity obtained was 2.6 mm/s and 1.51 mm/s using silicon oil and cooking oil, respectively, at 400 V. Our proposed digital microfluidics platform will play a vital role in droplet actuation and can be used for diagnostic applications such as DNA analysis and cell culture due to its simple fabrication and cost-effective technique. [ABSTRACT FROM AUTHOR]

The spread of infectious diseases poses a global threat to human health and the economy. Conventional laboratory-based pathogen detection analytical techniques are reliable, but are labour and time consuming. Decentralized, rapid pathogen detection and classification devices are essential to boost biosecurity efforts and can aid in the advancement of modern medicine. Here, we describe the development of an integrated digital microfluidic (DMF) electrochemical impedimetric sensor for rapid and on-site detection of lipopolysaccharide (LPS), a molecular signature of Gram-negative bacteria. The sensor was fabricated by immobilizing toll-like receptor protein (TLR4) onto a gold sensing electrode that was fabricated on an indium tin oxide (ITO) DMF top plate. The top plate also housed lithographically patterned ITO pseudo-reference and auxiliary electrodes for a three-electrode electrochemical impedance (EIS) detection system. We exploited the unique feature of DMF to manipulate droplets consisting of samples, buffers, wash solutions and reagents to perform automated EIS measurements due to the interaction of TLR4 with LPS. The integrated sensor platform showed a detection limit of 35 ng/mL LPS and a linear range of up to 400 ng/mL. The small size and ease of operation of the integrated system holds great prospect for the development of portable, and automated generic pathogen detection and classification platform for point-of-need applications.

Continuous mutations have occurred in the genome of the SARS-CoV-2 virus since the onset of the COVID-19 pandemic. The increased transmissibility of the mutated viruses has not only imposed medical burdens but also prolonged the duration of the pandemic. A point-of-care (POC) platform that provides multitarget detection will help to track and reduce disease transmissions. Here we detected and discriminated three genotypes of SARS-CoV-2, including the wildtype and two variants of concern (VOCs), the Delta variant and Omicron variant, through reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a digital microfluidics (DMF)-based cartridge. Upon evaluating with the RNA samples of Omicron variant, the DMF RT-qPCR presented a sensitivity of 10 copies/μL and an amplification efficiency of 96.1%, capable for clinical diagnosis. When spiking with SARS-CoV-2 RNA (wildtype, Delta variant, or Omicron variant) and 18S rDNA, the clinical analog samples demonstrated accurate detection and discrimination of different SARS-CoV-2 strains in 49 min. [ABSTRACT FROM AUTHOR]

We successfully developed a platform for the magnetic manipulation of droplets containing magnetic beads and examined the washing behaviors of the droplets, including droplet transportation, magnetic bead agitation inside droplets, and separation from parent droplets. Magnetic field gradients were produced with two layers of 6 × 1 planar coils fabricated by using printed circuit board technology. We performed theoretical analyses to understand the characteristics of the coils and successfully predicted the magnetic field and thermal temperature of a single coil. We then investigated experimentally the agitation and splitting kinetics of the magnetic beads inside droplets and experimentally observed the washing performance in different neck-shaped gaps. The performance of the washing process was evaluated by measuring both the particle loss ratio and the optical density. The findings of this work will be used to design a magnetic-actuated droplet platform, which will separate magnetic beads from their parent droplets and enhance washing performance. We hope that this study will provide digital microfluidics for application in point-of-care testing. The developed microchip will be of great benefit for genetic analysis and infectious disease detection in the future. [ABSTRACT FROM AUTHOR]

Digital microfluidics, which relies on the movement of drops, is relatively immune to clogging problems, making it suited for micro-reactor applications. Here, graphene oxide paper of 100 μm thickness, fabricated by blade coating sedimented dispersions onto roughened substrates, followed by drying and mechanical exfoliation, was found to be relatively free of cracks and curling. It also exhibited high wettability and elasto-capillary characteristics. Possessing low enough stiffness, it could rapidly and totally self-wrap water drops of 20 μL volume placed 2 mm from its edge when oriented between 0 and 60° to the horizontal. This complete wrapping behavior allowed drops to be translated via movement of the paper over long distances without dislodgement notwithstanding accelerations and decelerations. An amount of 2 drops that were wrapped with separate papers, when collided with each other at speeds up to 0.64 m/s, were found to eschew coalescence. This portends the development of robust digital microfluidic approaches for micro-reactors. [ABSTRACT FROM AUTHOR]

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.

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.

Rapid diagnosis of acute myocardial infarction (AMI) is critical in the clinical setting. An automated and rapid chemiluminescence immunoassay system based on digital microfluidics (DMF) is suggested for cardiac troponin I (cTnI). This method involves a double antibody sandwich structure. The cTnI antigen in the sample to be analyzed was captured by magnetic beads encapsulated with the cTnI antibody, which was subsequently recognized by the biotin-labeled secondary antibody. Horseradish peroxidase (HRP)-labeled streptavidin may be conjugated to biotin and used to mark the cTnI antigen because of the unique way that biotin binds to avidin. The simultaneous recognition of the cTnI antigen by primary and secondary antibodies in sandwich form, as well as the high specificity of the reaction between streptavidin and biotin, considerably improves the sensitivity and specificity of this technique for cTnI detection. This approach has decent stability. For cTnI samples, the full process took 40 min, with a limit of detection (LOD) of 0.34 ng mL−1. Additionally, there was a significant correlation range from 0.39 to 25 ng mL−1, with a correlation value of 0.994. The CV (Coefficient of variation) value was 2.26%. This system is anticipated to be used for the clinical measurement of cTnI in human serum. [ABSTRACT FROM AUTHOR]

Over the past two decades, digital microfluidic biochips have been in much demand for safety-critical and biomedical applications and increasingly important in point-of-care analysis, drug discovery, and immunoassays, among other areas. However, for complex bioassays, finding routes for the transportation of droplets in an electrowetting-on-dielectric digital biochip while maintaining their discreteness is a challenging task. In this study, we propose a deep reinforcement learning-based droplet routing technique for digital microfluidic biochips. The technique is implemented on a distributed architecture to optimize the possible paths for predefined source–target pairs of droplets. The actors of the technique calculate the possible routes of the source–target pairs and store the experience in a replay buffer, and the learner fetches the experiences and updates the routing paths. The proposed algorithm was applied to benchmark suites I and III as two different test benches, and it achieved significant improvements over state-of-the-art techniques. HIGHLIGHTS: • Deep-reinforcement learning approach in a distributed framework for droplet transportation on digital microfluidic biochips. • Optimizing the droplet transportation time for homogeneous multiple droplets by sharing the common electrodes. • A sequence of intelligent trade-offs to handle the fluidic constraints for parallel droplet transportation. [ABSTRACT FROM AUTHOR]

Microfluidic-based platforms have become a hallmark for chemical and biological assays, empowering micro- and nano-reaction vessels. The fusion of microfluidic technologies (digital microfluidics, continuous-flow microfluidics, and droplet microfluidics, just to name a few) presents great potential for overcoming the inherent limitations of each approach, while also elevating their respective strengths. This work exploits the combination of digital microfluidics (DMF) and droplet microfluidics (DrMF) on a single substrate, where DMF enables droplet mixing and further acts as a controlled liquid supplier for a high-throughput nano-liter droplet generator. Droplet generation is performed at a flow-focusing region, operating on dual pressure: negative pressure applied to the aqueous phase and positive pressure applied to the oil phase. We evaluate the droplets produced with our hybrid DMF–DrMF devices in terms of droplet volume, speed, and production frequency and further compare them with standalone DrMF devices. Both types of devices enable customizable droplet production (various volumes and circulation speeds), yet hybrid DMF–DrMF devices yield more controlled droplet production while achieving throughputs that are similar to standalone DrMF devices. These hybrid devices enable the production of up to four droplets per second, which reach a maximum circulation speed close to 1540 µm/s and volumes as low as 0.5 nL. [ABSTRACT FROM AUTHOR]

In this paper, we present different microelectromechanical systems based on electrostatic actuators, and demonstrate their capacity to achieve large and stepwise displacements using a cooperative function of the actuators themselves. To explore this, we introduced micro-stepper actuators to our experimental systems, both with and without a guiding spring mechanism; mechanisms with such guiding springs can be applied to comb-drive and parallel-plate actuators. Our focus was on comparing various guiding spring designs, so as to increase the actuator displacement. In addition, we present systems based on cascaded actuators; these are converted to micromechanical digital-to-analog converters (DAC). With DACs, the number of actuators (and thus the complexity of the digital control) are significantly reduced in comparison to analog stepper-actuators. We also discuss systems that can achieve even larger displacements by using droplet-based bearings placed on an array of aluminum electrodes, rather than guiding springs. By commutating the voltages within these electrode arrays, the droplets follow the activated electrodes, carrying platforms atop themselves as they do so. This process thus introduces new applications for springless large displacement stepper-actuators. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Electrowetting on dielectric-based digital microfluidic platforms (EWOD-DMF) have a potential to impact point-of-care diagnostics. Conventionally, EWOD-DMF platforms are manufactured in cleanrooms by expert technicians using costly and time consuming micro-nanofabrication processes such as optical lithography, depositions and etching. However, such high-end microfabrication facilities are extremely challenging to establish in resource-poor and low-income countries, due to their high capital investment and operating costs. This makes the fabrication of EWOD-DMF platforms extremely challenging in low-income countries, where such platforms are most needed for many applications such as point-of-care testing applications. To address this challenge, we present a low-cost and simple fabrication procedure for EWOD-DMF electrode arrays, which can be performed anywhere with a commercial office inkjet printer without the need of expensive cleanroom facilities. We demonstrate the utility of our platform to move and mix droplets of different reagents and physiologically conductive buffers, thereby showing its capability to potentially perform a variety of biochemical assays. By combining our low-cost, inkjet-printed EWOD-DMF platform with smartphone imaging technology and a compact control system for droplet manipulation, we also demonstrate a portable and hand-held device which can be programmed to potentially perform a variety of biochemical assays.

Core–shell particles are micro- or nanoparticles with solid, liquid, or gas cores encapsulated by protective solid shells. The unique composition of core and shell materials imparts smart properties on the particles. Core–shell particles are gaining increasing attention as tuneable and versatile carriers for pharmaceutical and biomedical applications including targeted drug delivery, controlled drug release, and biosensing. This review provides an overview of fabrication methods for core–shell particles followed by a brief discussion of their application and a detailed analysis of their manipulation including assembly, sorting, and triggered release. We compile current methodologies employed for manipulation of core–shell particles and demonstrate how existing methods of assembly and sorting micro/nanospheres can be adopted or modified for core–shell particles. Various triggered release approaches for diagnostics and drug delivery are also discussed in detail. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Electrowetting‐on‐dielectric (EWOD) devices have proven to be effective tools for precise microfluidic manipulation or in liquid lenses that surpass conventional solid lenses in versatility. However, the fabrication of these devices presents many challenges, such as their scalability or the growing concern on their environmental impact due to materials used in their fabrication. This review provides a comprehensive analysis of the materials currently used in the fabrication of EWOD devices and the characteristics they must meet. In addition, a discussion of future challenges in the fabrication of EWOD devices is presented, in particular the environmental problems presented by some of the materials currently in use. [ABSTRACT FROM AUTHOR]

Liquid marbles are droplets encapsulated by a layer of hydrophobic nanoparticles and have been extensively employed in digital microfluidics and lab-on-a-chip systems in recent years. In this study, magnetic liquid marbles were used to manipulate nonmagnetic liquid marbles. To achieve this purpose, a ferrofluid liquid marble (FLM) was employed and attracted toward an electromagnet, resulting in an impulse to a water liquid marble (WLM) on its way to the electromagnet. It was observed that the manipulation of the WLM by the FLM was similar to the collision of billiard balls except that the liquid marbles exhibited an inelastic collision. Taking the FLM as the projectile ball and the WLM as the other target balls, one can adjust the displacement and direction of the WLM precisely, similar to an expert billiard player. Firstly, the WLM displacement can be adjusted by altering the liquid marble volumes, the initial distances from the electromagnet, and the coil current. Secondly, the WLM direction can be adjusted by changing the position of the WLM relative to the connecting line between the FLM center and the electromagnet. Results show that when the FLM or WLM volume increases by five times, the WLM shooting distance approximately increases by 200% and decreases by 75%, respectively. [ABSTRACT FROM AUTHOR]

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in the African American population. We identified eighteen G6PD-deficient samples (9%) in a study of residual, de-identified whole blood specimens from 200 African American pediatric patients using a point-of-care instrument. This highlights the possibility of a rapid time to result for G6PD testing, which can be valuable in some clinical scenarios.

The rapid quantification of nitrate nitrogen concentration plays a pivotal role in monitoring soil nutrient content. Nevertheless, the low detection efficiency limits the application of traditional methods in rapid testing. For this investigation, we utilized a digital microfluidic platform and 3D-printed microfluidics to accomplish automated detection of soil nitrate nitrogen with high sensitivity across numerous samples. The system combines digital microfluidics (DMF), 3D-printed microfluidics, a peristaltic pump, and a spectrometer. The soil solution, obtained after extraction, was dispensed onto the digital microfluidic platform using a micropipette. The digital microfluidic platform regulated the movement of droplets until they reached the injection area, where they were then aspirated into the 3D-printed microfluidic device for absorbance detection. Implementing this approach allows for the convenient sequential testing of multi-samples, thereby enhancing the efficiency of nitrate nitrogen detection. The results demonstrate that the device exhibits rapid detection (200 s for three samples), low reagent consumption (40 µL per sample), and low detection limit (95 µg/L). In addition, the relative error between the detected concentration and the concentration measured by ultraviolet spectrophotometry is kept within 20%, and the relative standard deviation (RSD) of the measured soil samples is between 0.9% and 4.7%. In the foreseeable future, this device will play a significant role in improving the efficiency of soil nutrient detection and guiding fertilization practices.

Abstract Electrowetting‐on‐dielectric (EWOD) devices have proven to be effective tools for precise microfluidic manipulation or in liquid lenses that surpass conventional solid lenses in versatility. However, the fabrication of these devices presents many challenges, such as their scalability or the growing concern on their environmental impact due to materials used in their fabrication. This review provides a comprehensive analysis of the materials currently used in the fabrication of EWOD devices and the characteristics they must meet. In addition, a discussion of future challenges in the fabrication of EWOD devices is presented, in particular the environmental problems presented by some of the materials currently in use.

Antibiotic detection in dairy is crucial, as introduction of antibiotics in food can lead to antibiotic resistance and cause allergic reactions in consumers. Dairy farmers risk monetary fines for shipping contaminated milk. Microfluidics is an appealing technology, as it is portable, can be implemented on-site, and is highly automated. This work presents a digital microfluidic dairy device for antibiotic detection. The digital microfluidic dairy device explores integration of a lock-in amplifier with droplet-based microfluidic techniques, being electrowetting actuation and droplet control. The lock-in amplifier setup is initially demonstrated for the fluorescent dye rhodamine B, to establish baseline operation. Ultimately, the lock-in amplifier setup is demonstrated for detection of ciprofloxacin in milk. The limit of detection is significantly lowered through the integration of the lock-in amplifier technology. Actuation of milk and water samples is shown, demonstrating full two dimensional control over the position of water and milk droplets, to enable a complete lab-on-a-chip system. The results show promise for modern dairy testing of antibiotic signatures. [2022-0120] [ABSTRACT FROM AUTHOR]

An accurate description of contact angle hysteresis in AC electrowetting is important for a wide range of practical electrowetting applications. This work demonstrates that electrowetting actuated advancing contact angles and the reduction of contact angle hysteresis in AC electrowetting are better predicted when characterizing the advancing electrowetting force by its root mean square (rms) value as opposed to its time-averaged or maximum values. Characterizing the electrowetting force by its rms value allows the transient electrowetting force to exceed the pinning force for some amount of time overcoming the inertial effects at the contact line before advancement. This is opposed to the maximum force characterization which implies that inertial effects can be neglected at the contact line resulting in immediate advancement when the forces are unbalanced. [ABSTRACT FROM AUTHOR]

A magnetically actuated chip was successfully developed in this study to perform the purpose of transportation for a droplet containing clustered magnetic beads. The magnetic field gradient is generated by the chip of the two-layer 4 × 4 array micro-coils, which was commercially fabricated by printing circuit board (PCB) technology. A numerical model was first established to investigate the magnetic field and thermal field for such a micro-coil. Consequently, the numerical simulations were in reasonable agreement with the experimental results. Moreover, a theoretical analysis was derived to predict the dynamic behaviors of the droplets. This analysis will offer the optimal operation for such a magnetically actuated chip. This study aims to successfully implement the concept of "digital microfluidics" in "point-of-care testing" (POCT). In the future, the micro-coil chip will be of substantial benefit to genetic analysis and infectious disease detection. [ABSTRACT FROM AUTHOR]

Digital microfluidic biochip (DMFB) has attracted attention in the biochemical and medical industries. In particular, a microelectrode dot array (MEDA) biochip, which is composed of a two-dimensional microelectrode array, enables to realize fine-grained manipulation such as dilution, mixing, sensing, and so on in real-time. Unlike existing DMFB biochips, a MEDA architecture allows microelectrodes to control a certain volume of droplet in a fine-grained manner and can vary droplet volume and shape in such a way that it efficiently conducts synthesis and manipulation of droplets. There have been many works in order to improve the efficiency of synthesis of MEDA biochips; however, the synthesis, especially droplet routing, has never considered the shape-dependent velocity of droplets. In this paper, we propose the droplet routing techniques for MEDA biochips with shape-dependent velocity of droplets. The proposed techniques take the advantage of variant velocities of droplets dependent on the shapes and aim to reduce the overall routing time of a droplet from a source to a destination. Simulation results confirm that the proposed techniques can shorten the routing time by 80% compared to the state-of-the-art techniques.

Helicobacter pylori (H. pylori) infection correlates closely with gastric diseases such as gastritis, ulcers, and cancer, influencing more than half of the world's population. Establishing a rapid, precise, and automated platform for H. pylori diagnosis is an urgent clinical need and would significantly benefit therapeutic intervention. Recombinase polymerase amplification (RPA)-CRISPR recently emerged as a promising molecular diagnostic assay due to its rapid detection capability, high specificity, and mild reaction conditions. In this work, we adapted the RPA-CRISPR assay on a digital microfluidics (DMF) system for automated H. pylori detection and genotyping. The system can achieve multi-target parallel detection of H. pylori nucleotide conservative genes (ureB) and virulence genes (cagA and vacA) across different samples within 30 min, exhibiting a detection limit of 10 copies/rxn and no false positives. We further conducted tests on 80 clinical saliva samples and compared the results with those derived from real-time quantitative polymerase chain reaction, demonstrating 100% diagnostic sensitivity and specificity for the RPA-CRISPR/DMF method. By automating the assay process on a single chip, the DMF system can significantly reduce the usage of reagents and samples, minimize the cross-contamination effect, and shorten the reaction time, with the additional benefit of losing the chance of experiment failure/inconsistency due to manual operations. The DMF system together with the RPA-CRISPR assay can be used for early detection and genotyping of H. pylori with high sensitivity and specificity, and has the potential to become a universal molecular diagnostic platform. [ABSTRACT FROM AUTHOR]

In digital microfluidics, discrete liquid droplets move on a dielectric surface with the help of AC or DC voltage. Digital microfluidics system has extended the scope of microfluidics by providing automated, precise flow control for point-of-care devices capable of medical diagnostics, environmental, and chemical sensing platforms. In this paper, we introduce a digital microfluidics platform based on electrowetting on a dielectric using PCB substrate designed on Easy EDA software. The platform’s performance was analyzed at various DC voltages between 200 V and 400 V for droplet actuation. PCB fabrication, the utilization of easily accessible and cheap materials such as cooking oil and grafting tape as a dielectric layer, allowed the fabrication to be affordable and simple. The results indicate that droplet actuation takes place at 220 V. The droplet velocity obtained was 2.6 mm/s and 1.51 mm/s using silicon oil and cooking oil, respectively, at 400 V. Our proposed digital microfluidics platform will play a vital role in droplet actuation and can be used for diagnostic applications such as DNA analysis and cell culture due to its simple fabrication and cost-effective technique.