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A digital microfluidic biochip (DMFB) enables the miniaturization of immunoassays, point-of-care clinical diagnostics, DNA sequencing, and other laboratory procedures in biochemistry. A recent generation of biochips uses a micro-electrode-dot-array (MEDA) architecture, which provides fine-grained control of droplets and seamlessly integrates microelectronics and microfluidics using CMOS technology and a TSMC fabrication process. To ensure that bioassays are carried out on MEDA biochips efficiently, high-level synthesis algorithms have recently been proposed. However, as in the case of conventional DMFBs, microelectrodes are likely to fail when they are heavily utilized, and previous methods fail to consider reliability issues. In this article, we first present a new microelectrode cell (MC) design such that the droplet-sensing operation can be enabled/disabled for individual MCs. Next, “partial update” and “partial sensing” operations are presented based on an IEEE Std. 1687 IJTAG network design. Finally, wear-leveling synthesis method is proposed to ensure uniform utilization of MCs on MEDA. A comprehensive set of simulation results demonstrate the effectiveness of the proposed hardware design and design automation methods. [ABSTRACT FROM AUTHOR]

New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation

Digital (droplet) microfluidics (DμF) is a powerful platform for automated lab-on-a-chip procedures, ranging from quantitative bioassays such as RT-qPCR to complete mammalian cell culturing. The simple MEMS processing protocols typically employed to fabricate DμF devices limit their functionality to two dimensions, and hence constrain the applications for which these devices can be used. This paper describes the integration of vertical functionality into a DμF platform by stacking two planar digital microfluidic devices, altering the electrode fabrication process, and incorporating channels for reversibly translating droplets between layers. Vertical droplet movement was modeled to advance the device design, and three applications that were previously unachievable using a conventional format are demonstrated: (1) solutions of calcium dichloride and sodium alginate were vertically mixed to produce a hydrogel with a radially symmetric gradient in crosslink density; (2) a calcium alginate hydrogel was formed within the through-well to create a particle sieve for filtering suspensions passed from one layer to the next; and (3) a cell spheroid formed using an on-chip hanging-drop was retrieved for use in downstream processing. The general capability of vertically delivering droplets between multiple stacked levels represents a processing innovation that increases DμF functionality and has many potential applications.

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.

Digital microfluidics based on sessile droplets has emerged as a promising technology for various applications including biochemical assays, clinical diagnostics, and drug screening. Digital microfluidic platforms provide an isolated microenvironment to prevent cross-contamination and require reduced sample volume. Despite these advantages, the droplet-based technology has the inherent limitation of the quiescent flow conditions at low Reynolds number, which causes mixing samples confined within the droplets to be challenging. Recently, solutal Marangoni flows induced by volatile liquids have been utilized for sessile droplet mixing to address the above-mentioned limitation. The volatile liquid vaporized near a sessile droplet induces a surface tension gradient throughout the droplet interface, leading to vortical flows inside a droplet. This Marangoni flow-based droplet mixing method does not require an external energy source and is easy to operate. However, this passive method requires a comparably long time of a few tens of seconds for complete mixing since it depends on the natural evaporation of the volatile liquid. Here, we propose an improved ultrasound-induced heating method based on a nature-inspired ultrasound-absorbing layer and apply it to enhance solutal Marangoni effect. The heater consists of an interdigital transducer deposited on a piezoelectric substrate and a silver nanowire-polydimethylsiloxane composite as an ultrasound-absorbing layer. When the transducer is electrically actuated, surface acoustic waves are produced and immediately absorbed in the composite layer by viscoelastic wave attenuation. The conversion from acoustic to thermal energy occurs, leading to rapid heating. The heating-mediated enhanced vaporization of a volatile liquid accelerates the solutal Marangoni flows and thus enables mixing high-viscosity droplets, which is unachievable by the passive solutal Marangoni effect. We theoretically and experimentally investigated the enhanced Marangoni flow and confirmed that rapid droplet mixing can be achieved within a few seconds. The proposed heater-embedded sessile droplet mixing platform can be fabricated in small size and easily integrated with other digital microfluidic platforms. Therefore, we expect that the proposed sample mixing method can be utilized for various applications in digital microfluidics and contribute to the advancements in the medical and biochemical fields.

Summary: Digital microfluidic biochips (DMFBs) are designed to efficiently carry out biochemical and biomedical analysis in a miniaturized way. DMFBs offer various advantages over traditional laboratory techniques and reduces cost, and increases automation and software programmability. Scheduling of microfluidic operations is the first and essential step in the fluidic‐level synthesis of DMFBs, while the other two are the module placement and droplet routing. Scheduling DMFB operations is a multiconstrained optimization problem, and the particular decision problem is NP‐complete. We propose a hybrid artificial bee colony (ABC) algorithm using generalized N‐point crossover (GNX) based scheduling of DMFB operations. Proposed ABC‐GNX perturbs through search space, evaluates various schedules possible, and returns the best schedule among the evaluated schedules. Simple list scheduling based heuristic algorithms can explore a single schedule based on the sequence generated by the priority function. Iterative improvement based search algorithms explore the search space and evaluate more schedules, but the proposed ABC‐GNX algorithm produces optimal solutions in shorter execution times. Simulation results show that the proposed ABC‐GNX produces a higher number of optimal completion times and faster execution times than existing algorithms. [ABSTRACT FROM AUTHOR]

Sensitive and rapid antigen detection is critical for the diagnosis and treatment of infectious diseases, but conventional ELISAs including chemiluminescence-based assays are limited in sensitivity and require many operation steps. Fluorescence immunoassays are fast and convenient but often show limited sensitivity and dynamic range. To address the need, an aggregation-induced emission fluorgens (AIEgens) enhanced i mmunofluorescent assay with beads-based quantification on the digital microfluidic (DMF) platform was developed. Portable DMF devices and chips with small electrodes were fabricated, capable of manipulating droplets within 100 nL and boosting the reaction efficiency. AIEgen nanoparticles (NPs) with high fluorescence and photostability were synthesized to enhance the test sensitivity and detection range. The integration of AIEgen probes, transparent DMF chip design, and the large magnetic beads (10 μm) as capture agents enabled rapid and direct image-taking and signal calculation of the test result. The performance of this platform was demonstrated by point-of-care quantification of SARS-CoV-2 nucleocapsid (N) protein. Within 25 min, a limit of detection of 5.08 pg mL−1 and a limit of quantification of 8.91 pg mL−1 can be achieved using <1 μL sample. The system showed high reproducibility across the wide dynamic range (10–105 pg mL−1), with the coefficient of variance ranging from 2.6% to 9.8%. This rapid, sensitive AIEgens-enhanced immunofluorescent assay on the DMF platform showed simplified reaction steps and improved performance, providing insight into the small-volume point-of-care testing of different biomarkers in research and clinical applications. [Display omitted] • Enhance fluorescent signal and dynamic range through AIEgens probes. • DMF chip with small electrode grid and ultra-thin gap layer for nanoliter droplet. • Transparent design of chips enables direct imaging with fluorescent microscope. • Sensitive DMF fluorescent immunoassay detects antigen at 5 pg mL−1 within 25 min. [ABSTRACT FROM AUTHOR]

As a prerequisite for extracellular vesicle (EV) -based studies and diagnosis, effective isolation, enrichment and retrieval of EV biomarkers are crucial to subsequent analyses, such as miRNA-based liquid biopsy for non-small-cell lung cancer (NSCLC). However, most conventional approaches for EV isolation suffer from lengthy procedure, high cost, and intense labor. Herein, we introduce the digital microfluidic (DMF) technology to EV pretreatment protocols and demonstrate a rapid and fully automated sample preparation platform for clinical tumor liquid biopsy. Combining a reusable DMF chip technique with a low-cost EV isolation and miRNA preparation protocol, the platform completes automated sample processing in 20–30 min, supporting immediate RT-qPCR analyses on EV-derived miRNAs (EV-miRNAs). The utility and reliability of the platform was validated via clinical sample processing for EV-miRNA detection. With 23 tumor and 20 non-tumor clinical plasma samples, we concluded that EV-miR-486-5p and miR-21-5p are effective biomarkers for NSCLC with a small sample volumn (20–40 μL). The result was consistent to that of a commercial exosome miRNA extraction kit. These results demonstrate the effectiveness of DMF in EV pretreatment for miRNA detection, providing a facile solution to EV isolation for liquid biopsy. [Display omitted] •Digital microfluidics was developed for isolation and lysis of extracellular vesicles (EVs) from plasma samples. •Isolation efficiency of EVs reached 77% in 20 min using the novel method. •A cost-effective rework method for cleanroom-based DMF chips is proposed and validated. •Full-automatic procedure was realized with evaluated qPCR compatibility. •EV-miRNAs miR-486-5p and miR-21-5p were validated for detection of non-small cell lung cancer. [ABSTRACT FROM AUTHOR]

Signal transduction mediated by epidermal growth factor receptor (EGFR) gene affects the proliferation, invasion, metastasis, and angiogenesis of tumor cells. In particular, non-small cell lung cancer (NSCLC) patients with increased in copy number of EGFR gene are often sensitive to tyrosine kinase inhibitors. Despite being the standard for detecting EGFR amplification in the clinic, fluorescence in situ hybridization (FISH) traditionally involves repetitive and complex benchtop procedures that are not only time consuming but also require well-trained personnel. To address these limitations, we develop a digital microfluidics-based FISH platform (DMF-FISH) that automatically implements FISH operations. This system mainly consists of a DMF chip for reagent operation, a heating array for temperature control and a signal processing system. With the capability of automatic droplet handling and efficient temperature control, DMF-FISH performs cell digestion, gradient elution, hybridization and DAPI staining without manual intervention. In addition to operational feasibility, DMF-FISH yields comparable performance with the benchtop FISH protocol but reducing the consumption of DNA probe by 87 % when tested with cell lines and clinical samples. These results highlight unique advantages of the fully automated DMF-FISH system and thus suggest its great potential for clinical diagnosis and personalized therapy of NSCLC. Automated fluorescence in situ hybridization experimental platform based on digital microfluidics. [Display omitted] • Automated fluorescence in situ hybridization protocol by digital microfluidics. • Similar performance of detecting EGFR gene amplification to the benchtop protocol. • Significant reduction in manual labor and reagent consumption, especially costly DNA probe with an 87 % reduction. • The platform shows great potential for clinical diagnosis and personalized therapy of NSCLC. [ABSTRACT FROM AUTHOR]

Digital microfluidics (DMF) shows great potential in the manipulation of individual droplets. However, the limited number of electrode control wirings for droplet control intrinsically restrains the massive generation of pL/nL individual droplets, fundamentally hindering the realization of high throughput applications on DMF such as digital nucleic acid amplification and analysis. This work demonstrates on-chip high throughput digital loop-mediated isothermal amplification (dLAMP) on a DMF platform based on a functional substrate with super-wettability contrast. Exploiting the passive dispensing technique as empowered by electrowetting on dielectric (EWOD) effect, a total of 1818 individual droplets with high uniformity are generated on the super-hydrophilic microarray within 1 min. Separation within the oil phase without tubes and valves can avoid evaporation, ensuring a precise total reaction volume. Absolute quantification of λDNA with a dynamic range of 1–1000 copies/μL is achieved by counting the amount of positive microdroplets after on-chip incubation, demonstrating the strong potential for rapid, low-cost, reliable, and quantitative nucleic acid analysis with high accuracy. • High-throughput digital microfluidics platform. • High uniformity among microdroplets. • No dead volume for ensuring a precise total reaction volume. • On-chip high precision absolute quantification of nucleic acid. [ABSTRACT FROM AUTHOR]

The emerging microfluidic tools bring new methods for miniaturized analytical systems and single cells manipulation. This study presents an active‐matrix digital microfluidic platform for single cells generation and manipulation. The microfluidic chip was fabricated by amorphous silicon (a‐6i) thin‐film transistor (TFT) technology. The active device contains 26,368 electrodes that can be independently addressed to perform multiple and simultaneous droplets manipulation. The electrode array consists of electrodes differing in shape and size. The combination of various electrodes can give full play of their advantages, thus improve the droplet movement stability and generation uniformity. This work paves new ways for conducting low‐cost and high‐throughput applications in single cells manipulation. [ABSTRACT FROM AUTHOR]

Abstract This study reports the first comprehensive investigation of separation of the immiscible phases of multiphase droplets in digital microfluidics (DMF) platform. Electrowetting-on-dielectric (EWOD) actuation has been used to mechanically separate the phases. Phase separation performance in terms of percentage residue of one phase into another phase has been quantified. It was conceived that the residue formation can be controlled by controlling the deformation of the phases. The larger capillary number of the neck forming phase is associated with the larger amount of deformation as well as more residue. In this study, we propose two different ways to control the deformation of the phases. In the first method, we applied different EWOD operation voltages on two phases to maintain equal capillary numbers during phase separation. In the second method, while keeping the applied voltages same on both sides, we tested the phase separation performance by varying the actuation schemes. Less than 2% of residue was achieved by both methods, which is almost 90% improvement compared to the phase separation by the conventional droplet splitting technique in EWOD DMF platform, where the residue percentage can go up to 20%.

Digital microfluidics (DMF) is a versatile platform for conducting a variety of biological and chemical assays. The most commonly used set-up for the actuation of microliter droplets is electrowetting on dielectric (EWOD), where the liquid is moved by an electrostatic force on a dielectric layer. Superhydrophobic materials are promising materials for dielectric layers, especially since the minimum contact between droplet and surface is key for low adhesion of biomolecules, as it causes droplet pinning and cross contamination. However, superhydrophobic surfaces show limitations, such as full wetting transition between Cassie and Wenzel under applied voltage, expensive and complex fabrication and difficult integration into already existing devices. Here we present Fluoropor, a superhydrophobic fluorinated polymer foam with pores on the micro/nanoscale as a dielectric layer in DMF. Fluoropor shows stable wetting properties with no significant changes in the wetting behavior, or full wetting transition, until potentials of 400 V. Furthermore, Fluoropor shows low attachment of biomolecules to the surface upon droplet movement. Due to its simple fabrication process, its resistance to adhesion of biomolecules and the fact it is capable of being integrated and exchanged as thin films into commercial DMF devices, Fluoropor is a promising material for wide application in DMF.

The development of large-scale integration based on soft lithography has ushered a new revolution in microfluidics. This technology, however, relies inherently on pneumatic control of micromechanical valves that require air pressure to operate, while digital microfluidics uses a purely electrical signal on an electrode for droplet manipulation. In this article, we discuss the prospect and current challenges of digital microfluidics to solve the problem of the tyranny of numbers in arbitrary fluidic manipulation. We distill the fundamental physics governing electrowetting and their implications for specifications of the control electronics. We survey existing control electronics in digital microfluidics and detail the improvements needed to realize a low-power, programmable digital microfluidic system. Such an instrument would attract wide interest in both professional and non-professional (hobbyist) communities.

Delicate animal experiments and microdose clinical trials using short-lived radioisotopes require rapid preparation with high accuracy and careful attention to safety within a limited timeframe. We have developed an open-style electrowetting on dielectric (EWOD) device containing dimple structures for the rapid preparation of radiolabelled reagents. The device was demonstrated by automatic preparation of a technetium diethylenetriamine pentaacetate (99mTc-DTPA) with high chelation efficiency (99.7 ± 0.13%). Additionally, we demonstrated the single-photon emission computed tomography/computed tomography imaging of mouse kidney using the 99mTc-DTPA prepared with the EWOD device. The obtained organ tomographic images were sufficient for the evaluation of mouse models for specific diseases. These results indicate that manual radiolabelling for a small amount of nuclear medicine can be replaced by a process using the proposed EWOD device as a human error reduction technique.

The dynamic immune response to various diseases and therapies has been considered a promising indicator of disease status and therapeutic effectiveness. For instance, the human peripheral blood mononuclear cell (PBMC), as a major player in the immune system, is an important index to indicate a patient’s immune function. Therefore, establishing a simple yet sensitive tool that can frequently assess the immune system during the course of disease and treatment is of great importance. This study introduced an integrated system that includes an electrochemical impedance spectroscope (EIS)-based biosensor in a digital microfluidic (DMF) device, to quantify the PBMC abundance with minimally trained hands. Moreover, we exploited the unique droplet manipulation feature of the DMF platform and conducted a dynamic cell capture assay, which enhanced the detection signal by 2.4-fold. This integrated system was able to detect as few as 104 PBMCs per mL, presenting suitable sensitivity to quantify PBMCs. This integrated system is easy-to-operate and sensitive, and therefore holds great potential as a powerful tool to profile immune-mediated therapeutic responses in a timely manner, which can be further evolved as a point-of-care diagnostic device to conduct near-patient tests from blood samples.

In this study, an “all-in-one” digital microfluidics (DMF) system was developed for automatic and rapid molecular diagnosis and integrated with magnetic bead-based nucleic acid extraction, loop-mediated isothermal amplification (LAMP), and real-time optical signal monitoring. First, we performed on- and off-chip comparison experiments for the magnetic bead nucleic acid extraction module and LAMP amplification function. The extraction efficiency for the on-chip test was comparable to that of conventional off-chip methods. The processing time for the automatic on-chip workflow was only 23 min, which was less than that of the conventional methods of 28 min 45 s. Meanwhile, the number of samples used in on-chip experiments was significantly smaller than that used in off-chip experiments; only 5 µL of E. coli samples was required for nucleic acid extraction, and 1 µL of the nucleic acid template was needed for the amplification reaction. In addition, we selected SARS-CoV-2 nucleic acid reference materials for the nucleic acid detection experiment, demonstrating a limit of detection of 10 copies/µL. The proposed “all-in-one” DMF system provides an on-site “sample to answer” time of approximately 60 min, which can be a powerful tool for point-of-care molecular diagnostics.

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.

Contact charge electrophoresis (CCEP) is an electrically controllable manipulation technique of conductive droplets and particles by charging and discharging when in contact with the electrode. Given its straightforward operation mechanism, low cost, and ease of system construction, it has gained traction as a versatile and potential strategy for the realistic establishment of lab-on-a-chip (LOC) in various engineering applications. We present a CCEP-based digital microfluidics (DMF) platform with two parallel electrode modules comprising assembled conventional pin header sockets, allowing for efficient mixing through horizontal and vertical shaking via droplet reciprocating motions. The temporal chromic change caused by the chemical reaction between the pH indicator and base solutions within the shaking droplets is quantitatively analyzed under various CCEP actuation conditions to evaluate the mixing performance in shaking droplets by vertical and horizontal reciprocating motions on the DMF platform. Furthermore, mixing flow patterns within shaking droplets are successfully visualized by a high-speed camera system. The suggested techniques can mix samples and reagents rapidly and efficiently in droplet-based microreactors for DMF applications, such as biochemical analysis and medical diagnostics.

We introduce a digital microfluidics (DMF) platform specifically designed to perform a loop-mediated isothermal amplification (LAMP) of DNA and applied it to a real-time amplification to monitor a cancer biomarker, c-Myc (associated to 40% of all human tumors), using fluorescence microscopy. We demonstrate the full manipulation of the sample and reagents on the DMF platform, resulting in the successful amplification of 90 pg of the target DNA (0.5 ng/µL) in less than one hour. Furthermore, we test the efficiency of an innovative mixing strategy in DMF by employing two mixing methodologies onto the DMF droplets—low frequency AC (alternating current) actuation as well as back-and-forth droplet motion—which allows for improved fluorescence readouts. Fluorophore bleaching effects are minimized through on-chip sample partitioning by DMF processes and sequential droplet irradiation. Finally, LAMP reactions require only 2 µL volume droplets, which represents a 10-fold volume reduction in comparison to benchtop LAMP.

This study proposed a fabrication method for thin, film-based, transparent, and flexible digital microfluidic devices. A series of characterizations were also conducted with the fabricated digital microfluidic devices. For the device fabrication, the electrodes were patterned by laser ablation of 220 nm-thick indium tin oxide (ITO) layer on a 175 μm-thick polyethylene terephthalate (PET) substrate. The electrodes were insulated with a layer of 12 μm-thick polyethylene (PE) film as the dielectric layer, and finally, a surface treatment was conducted on PE film in order to enhance the hydrophobicity. The whole digital microfluidic device has a total thickness of less than 200 μm and is nearly transparent in the visible range. The droplet manipulation with the proposed digital microfluidic device was also achieved. In addition, a series of characterization studies were conducted as follows: the contact angles under different driving voltages, the leakage current density across the patterned electrodes, and the minimum driving voltage with different control algorithms and droplet volume were measured and discussed. The UV–VIS spectrum of the proposed digital microfluidic devices was also provided in order to verify the transparency of the fabricated device. Compared with conventional methods for the fabrication of digital microfluidic devices, which usually have opaque metal/carbon electrodes, the proposed transparent and flexible digital microfluidics could have significant advantages for the observation of the droplets on the digital microfluidic device, especially for colorimetric analysis using the digital microfluidic approach.

Electro-wetting-on-dielectric (EWOD) enables the manipulation of droplets on a two-dimensional surface, which provides a versatile technique for digital microfluidics at a micro- or nano-scale. However, the deficiency of the dispensing precision has long limited its applications in micro total analysis systems (μ-TAS) where the accuracy of assays is largely determined by the volume control of the reagent dosing. This paper proposes optimum electrode designs and carries out characterization experiments to demonstrate the reproducibility of on-chip droplet generation with no extra external apparatus. The coefficient variation of the volumes of consecutively dispensed droplets from a non-refilling reservoir can be limited to below 0.3%, indicating the validity of the new electrode structure in practical applications.

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xinyu Liu reports financial support was provided by Shenzhen Science and Technology Innovation Commission. Yujuan Chai reports financial support was provided by Guangdong Province Young Innovative Talents Project. Yujuan Chai reports financial support was provided by Guangdong Province University Key Project. Yujuan Chai reports financial support was provided by Medical-Engineering Interdisciplinary Research Foundation of ShenZhen University., (Copyright © 2023. Published by Elsevier Inc.)

A digital microfluidic biochip (DMFB) is an attractive platform for immunoassays, point-of-care clinical diagnostics, DNA sequencing, and other laboratory procedures in biochemistry. A recent generation of biochips uses a micro-electrode-dot-array (MEDA) architecture, which provides fine-grained controllability of droplets and seamlessly integrates microelectronics and microfluidics using CMOS technology. In order to ensure robust fluidic operations and high confidence in the outcome of biochemical experiments, chip testing, fault diagnosis, and fault recovery are critical for MEDA biochips. In this article, we present an effective fault-recovery solution based on the homogeneous structure of MEDA. Since the microelectrode cells (MCs) in an MEDA biochip are identical, we add multiplexers for reconfigurability, whereby an MC with faulty components can use the hardware resources in a neighboring MC. In addition, we use the IEEE 1687 (also known as IJTAG) network to reduce the number of control signals needed for the multiplexers, and to provide flexible subscan chain access for the fault-recovery control flow. A comprehensive set of simulation results demonstrates the effectiveness of the proposed fault-recovery solution for MEDA biochips. [ABSTRACT FROM AUTHOR]

This paper reviews publications that have fortified our understanding of the electrowetting-on-dielectric (EWOD) actuation mechanism. Over the last decade, growing interest in EWOD has led to a wide range of scientific and technological investigations motivated by its applicability in microfluidics, especially for droplet-based optical and lab-on-a-chip systems. At this point in time, we believe that it is helpful to summarize the observations, insights, and modeling techniques that have led to the current picture showing how forces act on liquid droplets and how droplets respond in EWOD microfluidic devices. We discuss the basic physics of EWOD and explain the mechanical response of a droplet using free-body diagrams. It is our hope that this review will inspire new research approaches and help design useful devices. © 2012 Copyright Taylor and Francis Group, LLC.

Point-of-care (POC) tests capable of individual health monitoring, transmission reduction, and contact tracing are especially important in a pandemic such as the coronavirus disease 2019 (COVID-19). We develop a disposable POC cartridge that can be mass produced to detect the SARS-CoV-2 N gene through real-time quantitative polymerase chain reaction (qPCR) based on digital microfluidics (DMF). Several critical parameters are studied and improved, including droplet volume consistency, temperature uniformity, and fluorescence intensity linearity on the designed DMF cartridge. The qPCR results showed high accuracy and efficiency for two primer-probe sets of N1 and N2 target regions of the SARS-CoV-2 N gene on the DMF cartridge. Having multiple droplet tracks for qPCR, the presented DMF cartridge can perform multiple tests and controls at once.

Abstract Background Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. Results We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 μL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb− 1. Conclusion We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

Digital microfluidic (DMF) platforms have contributed immensely to the development of multifunctional lab-on-chip systems for performing complete sets of biological and analytical assays. Electrowetting-on-dielectric (EWOD) technology, due to its outstanding flexibility and integrability, has emerged as a promising candidate for such lab-on-chip applications. Triggered by an electrical stimulus, EWOD devices allow precise manipulation of single droplets along the designed electrode arrays without employing external pumps and valves, thereby enhancing the miniaturization and portability of the system towards transcending important laboratory assays in resource-limited settings. In recent years, the simple fabrication process and reprogrammable architecture of EWOD chips have led to their widespread applications in food safety analysis. Various EWOD devices have been developed for the quantitative monitoring of analytes such as food-borne pathogens, heavy metal ions, vitamins, and antioxidants, which are significant in food samples. In this paper, we reviewed the advances and developments in the design of EWOD systems for performing versatile functions starting from sample preparation to sample detection, enabling rapid and high-throughput food analysis. [ABSTRACT FROM AUTHOR]

Microfluidic sample manipulation is a key enabler in modern chemical biology research. Both discrete droplet‐based digital microfluidic (DMF) assays and continuous flow in‐channel assays are well established, each featuring unique advantages from the viewpoint of automation and parallelization. However, there are marked differences in the applicable microfabrication materials and methods, which limit the interfacing of DMF sample preparation with in‐channel separation systems, such as the gold standard microchip electrophoresis. Simultaneously, there is an increasing demand for low‐cost and user‐friendly manufacturing techniques to foster the adaptation of microfluidic technology in routine laboratory analyses. This work demonstrates integration of DMF with in‐channel separation systems using only low‐cost and accessible (non‐cleanroom) manufacturing techniques, i.e., inkjet printing of silver for patterning of the driving electrodes and UV curing of off‐stoichiometric thiol–ene (OSTE) polymers both for dielectric coating of the electrode arrays and replica molding of the microchannel network. As a dielectric, OSTE performs similar to Parylene C (a gold standard dielectric in electrowetting), whereas its tunable surface and bulk properties facilitate straightforward bonding of the microchannel with the dielectric layer. In addition, a new chip design that facilitates efficient droplet transfer from the DMF part to the microchannel inlet solely by electrowetting is showcased. [ABSTRACT FROM AUTHOR]

Liquid marble, an emerging platform for digital microfluidics, has shown its potential in biomedical applications, cosmetics, and chemical industries. Recently, the manipulation and fundamental aspects of liquid marbles have been reported and attracted attention from the microfluidics community. Insights into their physical and chemical properties allow liquid marbles to be utilised in practical applications. This review summarises and revisits the effect of capillarity on the formation of liquid marbles and how it affects the effective surface tension as well as their robustness. The paper also systematically discusses the applied aspect of capillarity of the carrier liquid for transporting floating liquid marbles. [ABSTRACT FROM AUTHOR]

In recent years, application of digital microfluidic biochips (DMFBs) in an extended range, suffers from a few limitations namely (a) flexibility on droplet size (b) requirement of special fabrication process (c) need for integrated sensor systems for real-time detection. To overcome these challenging issues, a DMFB based new advanced microelectrode dot array architecture (MEDA) with advanced characteristics such as field programmability, higher reconfigurability and scalability has been introduced. This paper provides a comprehensive survey of MEDA-based DMFBs, a review on fluidic level synthesis as well as chip-level design to solve problems such as malfunctioning microelectrodes, droplet classification, system automation, biomedical detection and surface flatness testing, followed by a brief description on fault Modeling, testing and error recovery technique on MEDA architecture. Finally, the survey concludes with suggestions for future advancement possibilities in the areas of development of MEDA-based Biochips. [ABSTRACT FROM AUTHOR]

High control voltage and low success rate limit the application of droplet cutting on digital microfluidic chip, hence, the traditional square electrode was designed to crescent electrode to solve these problems in this paper. First, the relationship between the EWOD tension of micro-droplet and the chord length of effective Triple Contact Line (TCL) was analyzed based on the theory of electrowetting-on-dielectric. Then, the droplet cutting processes of different electrodes were numerically simulated and the results were analyzed. Finally, the effect of droplet cutting on four kinds of chips were tested. The results revel that the crescent electrode can decrease the applied voltage for drop et cutting and the minimum voltage required for cutting on crescent electrode (A=1.41) was at least 13.9% lower than that of square electrode. In addition, the success rates of droplet cutting on crescent electrode at different channel heights are higher than that of square electrode. [ABSTRACT FROM AUTHOR]

In this paper, we investigated the droplet splitting using different spacers in the air. The droplets were manipulated by the EWOD force and the level set method was used for our numerical implementation. The results show that the property of spacer has the effect on the droplet splitting. Specifically, we showed that the re-merging occurred when split daughter droplets pass the sub-channels for the short spacer and unequal droplet splitting can be achieved for the off-centered obstacle. In the study of the thickness, the thicker spacer made the splitting process unstable. Furthermore, we found that the triangle spacer was optical for the separation from the consideration of pressure. Our task can provide some help for the design of the spacer size in the future. [ABSTRACT FROM AUTHOR]

The current technique for the detection of fetus genotype by in-vitro fertilization (IVF) is quite expensive and is unsuitable/ inconvenient for all patients and even affects the embryonic growth cycle. We thus propose a new method to extract small amount of cell-free DNA (cf-DNA) from embryo culture medium. We introduce a new format for DNA extraction depending on digital microfluidics (DMF), electrowetting-on-dielectric (EWOD) and magnetic forces to separate, suspend DNA-coated magnetic particles. Nano-droplets (of nano-liter size) are electrostatically controlled on an array of insulated electrodes by EWOD system. By applying voltage to the various electrodes, multiple nano-droplets can be manipulated precisely in the same device. This feature enables one to extract DNA at ultra-small concentrations (fg/μL to pg/μL) from an embryo culture medium with an EWOD system. We applied our EWOD platform to extract a small amount of cf-DNA from a clinical embryo culture medium of mouse. In the future, we will distinguish the DNA in the real embryo buffer according to the length of the telomere. This feature would be valuable for the selection of an embryo before an implantation when the gene information can be recognized. [ABSTRACT FROM AUTHOR]

Digital microfluidic biochips (DMFBs) are an emerging technology that implements bioassays through manipulation of discrete fluid droplets. Recent results have shown that DMFBs are vulnerable to actuation tampering attacks, where a malicious adversary modifies control signals for the purposes of manipulating results or causing denial-of-service. Such attacks leverage the highly programmable nature of DMFBs. However, practical DMFBs often employ a technique called pin mapping to reduce control pin count while simultaneously reducing the degrees of freedom available for droplet manipulation. Attempts to control specific electrodes as part of an attack cannot be made without inadvertently actuating other electrodes on-chip, which makes the tampering evident. This paper explores this tamper resistance property of pin mapping in detail. We derive relevant security metrics, evaluate the tamper resistance of several existing pin mapping algorithms, and propose a new security-aware pin mapper. Further, we develop integer linear programming-based methodologies for inserting indicator droplets into a DMFB in order to boost tamper resistance. Experimental results show that the proposed techniques can significantly increase the difficulty for an attacker to make stealthy changes to the execution of a bioassay. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are prone to form protein particles through aggregation, fragmentation, and oxidation under varying stress conditions during the manufacturing, shipping, and storage of parenteral drug products. According to pharmacopeia requirements, sub-visible particle levels need to be controlled throughout the shelf life of the product. Therefore, in addition to determining particle counts, it is crucial to accurately characterize particles in drug product to understand the stress condition of exposure and to implement appropriate mitigation actions for a specific formulation. In this study, we developed a new method for intelligent characterization of protein particles using micro-Raman spectroscopy on a digital microfluidic chip (DMF). Several microliters of protein particle solutions induced by stress degradation were loaded onto a DMF chip to generate multiple droplets for Raman spectroscopy testing. By training multiple machine learning classification models on the obtained Raman spectra of protein particles, eight types of protein particles were successfully characterized and predicted with high classification accuracy (93%–100%). The advantages of the novel particle characterization method proposed in this study include a closed system to prevent particle contamination, one-stop testing of morphological and chemical structure information, low sample volume consumption, reusable particle droplets, and simplified data analysis with high classification accuracy. It provides great potential to determine the probable root cause of the particle source or stress conditions by a single testing, so that an accurate particle control strategy can be developed and ultimately extend the product shelf-life. [Display omitted] • Determine probable particle source based on a single assay for particle control strategy. • Closed DMF prevents particle contamination and provides reusable sample droplets. • One-stop testing of morphological and chemical structure information by Raman spec. • Machine learning simplifies data analysis and provides high prediction accuracy. • Low sample volume consumption on Digital Microfluidic (DMF). [ABSTRACT FROM AUTHOR]

The development of a rapid and reliable polymerase chain reaction (PCR) method for point-of-care (POC) diagnosis is crucial for the timely identification of pathogens. Microfluidics, which involves the manipulation of small volumes of fluidic samples, has been shown to be an ideal approach for POC analysis. Among the various microfluidic platforms available, digital microfluidics (DMF) offers high degree of configurability in manipulating μL/nL-scale liquid and achieving automation. However, the successful implementation of ultrafast PCR on DMF platforms presents challenges due to inherent system instability. In this study, we developed a robust and ultrafast PCR in 3.7–5 min with a detection sensitivity comparable to conventional PCR. Specifically, the implementation of the pincer heating scheme homogenises the temperature within a drop. The utilization of a μm-scale porous hydrophobic membrane suppresses the formation of bubbles under high temperatures. The design of a groove around the high-temperature zone effectively mitigates the temperature interference. The integration of a soluble sensor into the droplets provides an accurate and instant in-drop temperature sensing. We envision that the fast, robust, sensitive, and automatic DMF system will empower the POC testing for infectious diseases. [ABSTRACT FROM AUTHOR]

Abstract This letter reports two novel electrode design considerations to satisfy two very important aspects of EWOD operation—(1) Highly consistent volume of generated droplets and (2) Highly improved accuracy in the generated droplet volume. Considering the design principles investigated two novel designs were proposed; L-junction electrode design to offer high throughput droplet generation and Y-junction electrode design to split a droplet very fast while maintaining equal volume of each part. Devices of novel designs were fabricated and tested, and the results are compared with those of conventional approach. It is demonstrated that inaccuracy and inconsistency of droplet volume dispensed in the device with novel electrode designs are as low as 0.17 and 0.10%, respectively, while those of conventional approach are 25 and 0.76%, respectively. The dispensing frequency is enhanced from 4 to 9 Hz by using the novel design.

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., Competing Interests: The SEEKER® instruments and reagents were loaned to the Enzyme Unit at Great Ormond Street Hospital by Baebies Inc. via Technopath Distribution Ltd., Tipperary, Ireland. The MPS Society (UK) provided an unrestricted grant to Simon Heales to cover the salary of Rohit Hirachan. Derek Burke and Alistair Horman declare that they have no conflict of interest., (© 2024 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Droplet manipulation has garnered significant attention in various fields due to its wide range of applications. Among many different methods, magnetic actuation has emerged as a promising approach for remote and instantaneous droplet manipulation. In this study, we present the bidirectional droplet manipulation on a magnetically actuated superhydrophobic ratchet surface. The surface consists of silicon strips anchored on elastomer ridges with superhydrophobic black silicon structures on the top side and magnetic layers on the bottom side. The soft magnetic properties of the strips enable their bidirectional tilting to form a ratchet surface and thus bidirectional droplet manipulation upon varying external magnetic field location and strength. Computational multiphysics models were developed to predict the tilting of the strips, demonstrating the concept of bidirectional tilting along with a tilting angle hysteresis theory. Experimental results confirmed the soft magnetic hysteresis and consequential bidirectional tilting of the strips. The superhydrophobic ratchet surface formed by the tilting strips induced the bidirectional self-propulsion of dispensed droplets through the Laplace pressure gradient, and the horizontal acceleration of the droplets was found to be positively correlated with the tilting angle of the strips. Additionally, a finite element analysis was conducted to identify the critical conditions for dispensed droplet penetration through the gaps between the strips, which hinder the droplet's self-propulsion. The models and findings here provide substantial insights into the design and optimization of magnetically actuated superhydrophobic ratchet surfaces to manipulate droplets in the context of digital microfluidic applications.

Digital microfluidics (DMF) devices enable precise manipulation of small liquid volumes in point-of-care testing. A printed circuit board (PCB) substrate is commonly utilized to build DMF devices. However, inkjet printing can be used to fabricate DMF circuits, providing a less expensive alternative to PCB-based DMF designs while enabling more rapid design iteration cycles. We demonstrate the cleanroom-free fabrication process of a low-cost inkjet-printed DMF circuit. We compare Kapton and polymethyl methacrylate (PMMA) as dielectric coatings by measuring the minimal droplet actuation voltage for a range of actuation frequencies. A minimum actuation voltage of 5.6 V was required for droplet movement with the PMMA layer thickness of 0.2 μm and a hydrophobic layer of 0.17 μm. Significant issues with PMMA dielectric breakdown were observed at actuation voltages above 10 V. In comparison, devices that utilized Kapton were found to be more robust, even at an actuation voltage up to 100 V.