Digital Microfluidic Platform Research Articles

Shape-memory microfluidic chips for fluid and droplet manipulation.

Fluid manipulation is an important foundation of microfluidic technology. Various methods and devices have been developed for fluid control, such as electrowetting-on-dielectric-based digital microfluidic platforms, microfluidic pumps, and pneumatic valves. These devices enable precise manipulation of small volumes of fluids. However, their complexity and high cost limit the commercialization and widespread adoption of microfluidic technology. Shape memory polymers as smart materials can adjust their shape in response to external stimuli. By integrating shape memory polymers into microfluidic chips, new possibilities for expanding the application areas of microfluidic technology emerge. These shape memory polymers can serve as actuators or regulators to drive or control fluid flow in microfluidic systems, offering innovative approaches for fluid manipulation. Due to their unique properties, shape memory polymers provide a new solution for the construction of intelligent and automated microfluidic systems. Shape memory microfluidic chips are expected to be one of the future directions in the development of microfluidic technology. This article offers a summary of recent research achievements in the field of shape memory microfluidic chips for fluid and droplet manipulation and provides insights into the future development direction of shape memory microfluidic devices.

AIEgens-enhanced rapid sensitive immunofluorescent assay for SARS-CoV-2 with digital microfluidics

BackgroundSensitive 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. ResultsTo address the need, an aggregation-induced emission fluorgens (AIEgens) enhanced immunofluorescent 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%. SignificanceThis 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.

Evaluation, in a highly specialised enzyme laboratory, of a digital microfluidics platform for rapid assessment of lysosomal enzyme activity in dried blood spots.

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.

Rapid automated extracellular vesicle isolation and miRNA preparation on a cost-effective digital microfluidic platform

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.

Automated chemoenzymatic modular synthesis of human milk oligosaccharides on a digital microfluidic platform.

Glycans, along with proteins, nucleic acids, and lipids, constitute the four fundamental classes of biomacromolecules found in living organisms. Generally, glycans are attached to proteins or lipids to form glycoconjugates that perform critical roles in various biological processes. Automatic synthesis of glycans is essential for investigation into structure-function relationships of glycans. In this study, we presented a method that integrated magnetic bead-based manipulation and modular chemoenzymatic synthesis of human milk oligosaccharides (HMOs), on a DMF (Digital Microfluidics) platform. On the DMF platform, enzymatic modular reactions were conducted in solution, and purification of products or intermediates was achieved by using DEAE magnetic beads, circumventing the intricate steps required for traditional solid-phase synthesis. With this approach, we have successfully synthesized eleven HMOs with highest yields of up to >90% on the DMF platform. This study would not only lay the foundation for OPME synthesis of glycans on the DMF platform, but also set the stage for developing automated enzymatic glycan synthesizers based on the DMF platform.

Droplet Actuation Enhancement through Voltage Control and Hydrophobic Coating Selection

This paper presents a novel digital microfluidics platform that utilizes PCB substrate designed on Easy EDA software and is based on electrowetting on a dielectric (EWOD) phenomenon. For droplet actuation, the platform’s performance was examined at different DC voltages ranging from 300V to 450V. The fabrication process was made simple and inexpensive by using readily available, low-cost ingredients including silicone oil, cooking oil, and olive oil with grafting tape as a dielectric layer. Compared to other oils used for droplet actuation, cooking oil yields the highest droplet velocity. Due to its affordable and easy-to-fabricate nature, our suggested digital microfluidics platform will be feasible for droplet actuation and can also be applied to medical diagnostic applications like DNA analysis and cell culture, as well as the detection of environmental pollutants.

On-chip droplet analysis and cell spheroid screening by capillary wrapping enabled shape-adaptive ferrofluid transporters.

Non-invasive droplet manipulation with no physical damage to the sample is important for the practical value of manipulation tools in multidisciplinary applications from biochemical analysis and diagnostics to cell engineering. It is a challenge to achieve this for most existing photothermal, electric stimuli, and magnetic field-based technologies. Herein, we present a droplet handling toolbox, the ferrofluid transporter, for non-invasive droplet manipulation in an oil environment. It involves the transport of droplets with high robustness and efficiency owing to low interfacial friction. This capability caters to various scenarios including droplets with varying components and solid cargo. Moreover, we fabricated a droplet array by transporter positioning and achieved droplet gating and sorting for complex manipulation in the droplet array. Benefiting from the ease of scale-up and high biocompatibility, the transporter-based droplet array can serve as a digital microfluidic platform for on-chip droplet-based bioanalysis, cell spheroid culture, and downstream drug screening tests.

Droplet manipulation on an adjustable closed-open digital microfluidic system utilizing asymmetric EWOD.

The closed-open digital microfluidic (DMF) system offers a versatile and powerful platform for various applications by combining the advantages of both closed and open structures. The current closed-open DMF system faces challenges in scaling up due to electrode structural differences between closed and open regions. Here we developed an adjustable closed-open DMF platform by utilizing the modified slippery liquid-infused porous surfaces (SLIPS) with asymmetric electrowetting on dielectric (AEWOD) as a hydrophobic dielectric layer. The consistent electrode structures of the bottom printed circuit board (PCB) electrode array on both the closed and open regions, and the utilization of a transparent acrylic with floating potential as the top plate allow a low-cost and easily scalable closed-open DMF system to be achieved. The impacts of applied voltage, parallel plate spacing, electrode switching interval, and electrode driving strategies on various droplet manipulations were investigated. The results show that the optimal plate spacings range from 340-510 μm within the closed region. Meanwhile, we also studied the influence of the thickness, geometry, and position of the top plate on the droplet movement at the closed-open boundary. Through force analysis and experimentation, it is found that a thin top plate and a bevel of ∼4° can effectively facilitate the movement of droplets at the boundary. Finally, we successfully achieved protein staining experiments on this platform and developed a customized smartphone application for the accurate detection of protein concentration. This innovative closed-open DMF system provides new possibilities for future applications in real-time biological sample processing and detection.

Absolute quantification of nucleic acid on digital microfluidics platform based on superhydrophobic–superhydrophilic micropatterning

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.

Multi-Sample Detection of Soil Nitrate Nitrogen Using a Digital Microfluidic Platform

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.

Deep learning enhanced multiplex detection of viable foodborne pathogens in digital microfluidic chip

Culture plating is worldwide accepted as the gold standard for quantifying viable foodborne pathogens. However, it is time-consuming (1–2 days) and requires specialized laboratory and personnel. This study reported a deep learning enhanced digital microfluidic platform for multiplex detection of viable foodborne pathogens. The new method used a Time-Lapse images driven EfficientNet-Transformer Network (TLENTNet) to type and quantify the bacteria through spatiotemporal features of bacterial growth and digital enumeration of bacterial culture. First, the bacterial sample was prepared with LB medium and injected into a pre-vacuumed microfluidic chip with an array of 800 microwells to encapsulate at most one bacterium in each well. Then, a programmed sliding microscopic platform was used to scan all microwells every 15 min, capturing time-lapse images of bacterial growth within each microwell. Finally, the TLENTNet was used to facilitate bacterial typing and quantification. Under optimal conditions, this platform was able to detect four bacterial species (S.typhimurium, E. coli O157:H7, S. aureus and B. cereus) with an average accuracy of 97.72% and a detection limit of 63 CFU/mL in 7 h.

PH Regulator on Digital Microfluidics with Pico-Dosing Technique.

Real-time pH control on-chip is a crucial factor for cell-based experiments in microfluidics, yet difficult to realize. In this paper, we present a flexible pH regulator on a digital microfluidic (DMF) platform. The pico-dosing technology, which can generate and transfer satellite droplets, is presented to deliver alkali/acid into the sample solution to change the pH value of the sample. An image analysis method based on ImageJ is developed to calculate the delivered volume and an on-chip colorimetric method is proposed to determine the pH value of the sample solution containing the acid-base indicator. The calculated pH values show consistency with the measured ones. Our approach makes the real-time pH control of the on-chip biological experiment more easy to control and flexible.

A-329 Preliminary Clinical Method Comparison of a Near-Patient Platform for Low Blood Volume Testing of aFXa in Pediatric Patients on Heparin Therapy

Abstract Background Up to 15% of hospitalized children receive heparin therapy and require frequent monitoring for Anti-Factor Xa activity (aFXa) and activated partial thromboplastin time (aPTT) to achieve hemostatic balance. Current heparin tests require large blood volumes, which can lead to iatrogenic anemia with frequent sample collection. We performed a small clinical method comparison of a rapid, near-patient platform for simultaneous whole blood testing of aFXa from 50 μL of sample against the clinical standard test. The aPTT assay is in development on the same digital microfluidic (DMF) platform. Methods Four DMF instruments were placed in our laboratory. Preliminary precision of the DMF aFXa assay was obtained using commercial controls (n = 19 for aFXa at 0.98 U/mL; n = 20 for aFXa at 0.42 U/mL). We obtained whole blood or plasma from residual aFXa samples from pediatric patients. Venous samples were collected from patients for routine testing in the core laboratory, and 150 μL of whole blood was saved for aFXa method comparison testing. Patients were either on low molecular weight heparin or unfractionated heparin. We performed a method comparison study to determine assay equivalence to in-house clinical standard tests using plasma for aFXa on the Stago STA R MAX to both plasma and whole blood on the DMF platform. Results For precision, we observed an assay coefficient of variation (CV) of 4.68% and 6.6% for aFXa at 0.98 U/mL and 0.42 U/mL, respectively. The aFXa assay correlated well (R2 = 0.94 for whole blood (n = 9) and R2 = 0.97 for plasma (n = 10)); additional studies are needed to demonstrate aPTT assay equivalence. Conclusions We demonstrated good correlation of the DMF aFXa assay to the clinical standard test. The low blood volume requirement make these tests suitable for use in heparin monitoring where frequent testing is necessary. A large scale clinical method comparison is planned to ensure clinical utility.

EWOD Chip with Micro-Barrier Electrode for Simultaneous Enhanced Mixing during Transportation.

Digital microfluidic platforms have been extensively studied in biology. However, achieving efficient mixing of macromolecules in microscale, low Reynolds number fluids remains a major challenge. To address this challenge, this study presents a novel design solution based on dielectric electro-wetting (EWOD) by optimizing the geometry of the transport electrode. The new design integrates micro-barriers on the electrodes to generate vortex currents that promote mixing during droplet transport. This design solution requires only two activation signals, minimizing the number of pins required. The mixing performance of the new design was evaluated by analyzing the degree of mixing inside the droplet and quantifying the motion of the internal particles. In addition, the rapid mixing capability of the new platform was demonstrated by successfully mixing the sorbitol solution with the detection solution and detecting the resulting reaction products. The experimental results show that the transfer electrode with a micro-barrier enables rapid mixing of liquids with a six-fold increase in mixing efficiency, making it ideal for the development of EWOD devices.

Digital microfluidics-engaged automated enzymatic degradation and synthesis of oligosaccharides.

Glycans are an important group of natural biopolymers, which not only play the role of a major biological energy resource but also as signaling molecules. As a result, structural characterization or sequencing of glycans, as well as targeted synthesis of glycans, is of great interest for understanding their structure-function relationship. However, this generally involves tedious manual operations and high reagent consumptions, which are the main technical bottlenecks retarding the advances of both automatic glycan sequencing and synthesis. Until now, automated enzymatic glycan sequencers or synthesizers are still not available on the market. In this study, to promote the development of automation in glycan sequencing or synthesis, first, programmed degradation and synthesis of glycans catalyzed by enzymes were successfully conducted on a digital microfluidic (DMF) device by using microdroplets as microreactors. In order to develop automatic glycan synthesizers and sequencers, a strategy integrating enzymatic oligosaccharide degradation or synthesis and magnetic manipulation to realize the separation and purification process after enzymatic reactions was designed and performed on DMF. An automatic process for enzymatic degradation of tetra-N-acetyl chitotetraose was achieved. Furthermore, the two-step enzymatic synthesis of lacto-N-tetraose was successfully and efficiently completed on the DMF platform. This work demonstrated here would open the door to further develop automatic enzymatic glycan synthesizers or sequencers based on DMF.

Hybrid electrodes effective for both electrowetting‐ and dielectrowetting‐driven digital microfluidics

AbstractElectrowetting on dielectric (EWOD) and dielectrowetting (DEW) are two major principles to drive droplets in digital microfluidics. EWOD is effective to manipulate (create, transport, split, and merge) conductive droplets being currently used for many biological, chemical, and optical applications. DEW can also manipulate droplets but more efficiently with dielectric (nonconductive) fluids. A digital microfluidic platform efficiently operable by both EWOD and DEW would offer higher versatility in handling a wide range of fluids, regardless of their conductivities. In this regard, this article presents a new hybrid electrode design enabling EWOD and DEW to drive various kinds of droplet fluids on a single platform. In addition, a slippery liquid‐infused surface (SLIPS) is integrated with the hybrid electrodes. The SLIPS is well known to resist biofouling and repel sticky fluids, which endows the hybrid electrodes with much wider application spectra. As a result, the present SLIPS‐integrated hybrid electrodes facilitate actuating various kinds of fluids which would not be driven by conventional EWOD and/or DEW electrodes. This paper presents the successful transportation of not only conductive fluids including water, protein solution, glycerol, and honey but also nonconductive fluids including dodecane, silicone oil, and light and heavy crude oil, all driven by the SLIPS‐integrated hybrid electrodes. The performance comparisons among solid, interdigitating, and hybrid electrodes are made by testing both conductive and nonconductive droplets.

A universal model for continuous “one-to-two” high-efficient droplet generation in digital microfluidics

High-efficient droplet generation is crucial in microfluidics platforms in bio-applications. Among the microfluidic techniques, digital microfluidics, although with the capability of programmatically manipulating the droplets, is less effective in high-throughput droplets generation. In this Letter, a continuous “one-to-two” droplet generation strategy on digital microfluidics platform is achieved with an exponential generation efficiency, while overcoming the restriction on the droplet geometries (aspect ratio) in a wide range. An energy-based sub-droplet generation model is also proposed, as an expansion to the previous Laplace pressure-based geometrical model. The electrical parameters are taken into computing the sub-droplet generation on digital microfluidics from the aspect of energy. The proposed continuous “one-to-two” droplet generation strategy can generate sub-droplets with a minimized electrodes' number while with a higher energy input in each operation. The exponential efficiency will open the era for high-throughput droplets' application developments in digital microfluidics.

Digital microfluidic platform assembled into a home-made studio for sample preparation and colorimetric sensing of S-nitrosocysteine.

Digital microfluidics (DMF) is a versatile lab-on-a-chip platform that allows integration with several types of sensors and detection techniques, including colorimetric sensors. Here, we propose, for the first time, the integration of DMF chips into a mini studio containing a 3D-printed holder with previously fixed UV-LEDs to promote sample degradation on the chip surface before a complete analytical procedure involving reagent mixture, colorimetric reaction, and detection through a webcam integrated on the equipment. As a proof-of-concept, the feasibility of the integrated system was successfully through the indirect analysis of S-nitrosocysteine (CySNO) in biological samples. For this purpose, UV-LEDs were explored to perform the photolytic cleavage of CySNO, thus generating nitrite and subproducts directly on DMF chip. Nitrite was then colorimetrically detected based on a modified Griess reaction, in which reagents were prepared through a programable movement of droplets on DMF devices. The assembling and the experimental parameters were optimized, and the proposed integration exhibited a satisfactory correlation with the results acquired using a desktop scanner. Under the optimal experimental conditions, the obtained CySNO degradation to nitrite was 96%. Considering the analytical parameters, the proposed approach revealed linear behavior in the CySNO concentration range between 12.5 and 400μmolL-1 and a limit of detection equal to 2.8μmolL-1. Synthetic serum and human plasma samples were successfully analyzed, and the achieved results did not statistically differ from the data recorded by spectrophotometry at the confidence level of 95%, thus indicating the huge potential of the integration between DMF and mini studio to promote complete analysis of lowmolecular weight compounds.

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

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

Asymmetric electrodes for droplet directional actuation by a square wave on an open surface

The large-scale electrowetting-on-dielectric (EWOD) digital microfluidic platforms always require complex electrode-addressing schemes and high-cost fabrication methods of dielectric and hydrophobic layers. Hence, the voltage polarity-dependent electrowetting was used to actuate droplets with asymmetric printed circuit board (PCB) electrodes on an open slippery liquid-infused porous surface (SLIPS) chip. The V-shaped, convex-shaped, and curve-shaped coplanar asymmetric electrode arrays were designed to achieve the directional motion of a droplet by a square wave voltage signal. The operation ranges of voltage amplitude and frequency for continuously actuating a 10 µl droplet were obtained on the three asymmetric electrode arrays. Our results found that the curve-shaped electrode array has a broader voltage operation range than the other two for continuous pumping of a 10 µl droplet. It is because the droplet has a long effective width of contact line with the front curve-shaped electrode and does not overlap with the rear electrode during the movement. Meanwhile, a reconfigurable convex-shaped electrode structure was also proposed to achieve bidirectional actuation of a droplet by controlling the square wave voltage. This work provides a low-cost, straightforward electrode-addressing scheme for designing digital microfluidic devices.