Digital Microfluidic System Research Articles

Optimization of Electrode Patterns for an ITO-Based Digital Microfluidic through the Finite Element Simulation.

Indium tin oxide (ITO)-based digital microfluidics (DMF) with unique optical and electrical properties are promising in the development of integrated, automatic and portable analytical systems. The fabrication technique using laser direct etching (LDE) on ITO glass has the advantages of being rapid, low cost and convenient. However, the fabrication resolution of LDE limits the minimum line width for patterns on ITO glasses, leading to a related wider lead wire for the actuating electrodes of DMF compared with photolithography. Therefore, the lead wire of electrodes could affect the droplet motion on the digital microfluidic chip due to the increased contact line with the droplet. Herein, we developed a finite element model of a DMF with improved efficiency to investigate the effect of the lead wire. An optimized electrode pattern was then designed based on a theoretical analysis and validated by a simulation, which significantly decreased the deformation of the droplets down to 0.012 mm. The performance of the optimized electrode was also verified in an experiment. The proposed simulation method could be further extended to other DMF systems or applications to provide an efficient approach for the design and optimization of DMF chips.

All-in-One Digital Microfluidics System for Molecular Diagnosis with Loop-Mediated Isothermal Amplification.

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.

A digital microfluidic system with integrated electrochemical impedance detection arrays

Electrochemical sensing provides a new way for miniaturization and low cost of equipment. Meanwhile, digital microfluidic (DMF) technology is making lab-on-chip a reality. The two technologies are essential in the point-of-care area. Electrochemical detection on electrodes and DMF-based droplet driving electrode realize their respective functions by means of electrical signals. In terms of working mode, it seems to have certain similarity, and electrochemical sensing seems to be more suitable for integrating digital microfluidic systems. We thus integrated electrochemical sensing on an active matrix (AM) DMF system. As a common transparent conductive glass, indium tin oxide (ITO) has been widely used as the top plate of the digital microfluidic chip. In this work, we used ITO glass with patterned interdigital electrodes as the top plate of the AM-DMF chip. We achieved on-chip droplet volume detection by electrochemical impedance spectroscopy (EIS). We also proposed a novel sample dilution method on the DMF chip. It takes only 30 seconds to dilute solutions into 4 different concentrations. We then analyzed droplet samples of different concentrations with EIS method. And the correlation coefficient of the fitted curve is as high as 0.9964. In conclusion, EIS is a powerful detection technology for fast, high-integration and low-cost detection on DMF system. The integration of EIS and DMF can be used in various biochemical analyses in the future.

Volumeless reagent delivery: a liquid handling method for adding reagents to microscale droplets without increasing volume.

The addition of reagents for assays in digital microfluidic (DMF) systems is traditionally done by merging of droplets containing different analytes or reagents in solution. However, this process significantly increases droplet volume after each step, resulting in dilution of the analyte and reagents. Here, we report a new technique for performing reagent additions to aqueous droplets without significantly increasing the droplet's volume: volume-less reagent delivery (VRD). VRD is enabled by a physical phenomenon we call "exclusive liquid repellency" (ELR), which describes an aqueous/oil/solid 3-phase system where the aqueous phase can be fully repelled from a solid phase (contact angle ∼180°). When performing VRD, a reagent of interest in solution is deposited onto the ELR solid surface and allowed to dry. The ELR surface containing the dried reagent is then immersed under oil, followed by introduction of an aqueous droplet. By dragging the aqueous droplet over the spot of dried reagent using paramagnetic particles or via a physical sliding wall, the droplet can then recover and reconstitute the reagent with negligible increase in its total volume, returning the ELR surface to its initial liquid repellent state in the process. We demonstrate that VRD can be performed across a wide range of reagent types including sugars, proteins (antibodies), nucleic acids (DNA), antibiotics, and even complex enzyme/substrate/buffer "kit" mixtures. We believe VRD is a flexible and powerful technique which can further the development of self-contained, multi-step assays in DMF and other microfluidic systems.

Portable sample processing for molecular assays: application to Zika virus diagnostics.

This paper introduces a digital microfluidic (DMF) platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges offering a closed, humidified environment for sample processing at elevated temperatures, as well as programmable control instrumentation with a novel thermal cycling unit regulated using a proportional integral derivative (PID) feedback loop. The system operates on 12 V DC power, which can be supplied by rechargeable battery packs for remote testing. The DMF system was optimized for an RNA processing pipeline consisting of the following steps: 1) magnetic-bead based RNA extraction from lysed plasma samples, 2) RNA clean-up, and 3) integrated, isothermal amplification of Zika RNA. The DMF pipeline was coupled to a paper-based, colorimetric cell-free protein expression assay for amplified Zika RNA mediated by toehold switch-based sensors. Blinded laboratory evaluation of Zika RNA spiked in human plasma yielded a sensitivity and specificity of 100% and 75% respectively. The platform was then transported to Recife, Brazil for evaluation with infectious Zika viruses, which were detected at the 100 PFU mL-1 level from a 5 μL sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.

Programmable High Integration and Resolution Digital Microfluidic Device Driven by Thin Film Transistor Arrays

A digital microfluidic (DMF) system based on electrowetting on dielectrics (EWOD) is a new technology that is able to manipulate microlitre droplets on the plane. Conventional EWOD devices have difficulty achieving high integration and a large number of electrodes due to the limitation of electrical connections. To realize the precise control of multiple droplets in a large-area EWOD device, we propose a programmable high integration and resolution DMF device based on a thin film transistor array (TFTA) &#x2013; EWOD in this paper. The TFTA backplane with thousands of individually addressable electrodes used in electrowetting displays (EWDs) is integrated into this device. A source integrated circuit (IC) and a gate IC are bonded on the substrate glass of the device as the driver chip. A TFTA-EWOD device with <inline-formula> <tex-math notation="LaTeX">$\mathrm {320 \times 240}$ </tex-math></inline-formula> electrodes is designed, fabricated and tested in our experiments. Furthermore, a matching droplet control system with detection and feedback functions was developed and implemented. This system is capable of detecting and analysing individual droplet status while concurrently continuing with other droplet manipulations on the TFTA-EWOD device. The experimental results show that different droplet manipulations, including dispensing, merging and transport, can be carried out successfully on this device. We also successfully applied the proposed device and system to a colorimetric enzymatic assay to show that the proposed scheme is capable of chemical analysis experiments. There are no additional errors introduced by the proposed TFTA-EWOD device. The designed device and matching system proposes a programmable high integration, high resolution, and universal solution for complex DMF applications. We hope that the proposed device and system can provide an experimental platform to other research teams who focus on DMF applications.

Analysis of the effects of aryl hydrocarbon receptor expression on cancer cell invasion via three-dimensional microfluidic invasion assays.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds to xenobiotics and activates expression of response elements to metabolize these compounds. The AHR pathway has been associated with a long list of diseases including cancer; however, it is debated whether AHR is tumorigenic or tumour-inhibiting. In particular, there are contradictory reports in the literature regarding the effects of AHR expression level on metastatic breast cancer. Here we used a 3D invasion assay called cell invasion in digital microfluidic microgel systems (CIMMS) to study the effect of AHR expression on invasion. In this study, MDA-MB-231 cells with stable knockout of AHR (AHRko) showed enhanced invasive characteristics and reduced proliferation, and cells with transient overexpression of AHR showed reduced invasiveness. Overexpression of AHR with a mutation in the DNA binding domain showed no difference in invasiveness compared to control, which suggests that the changes in invasiveness are related to the expression of AHR. CIMMS also allowed for extraction of sub-populations of invaded cells for RNA sequencing experiments. A comparison of the transcriptomes of invaded subpopulations of wild-type and AHRko cells identified 1809 genes that were differentially expressed, with enriched pathways including cell cycle, proliferation, survival, immunoproteasome activation, and activation of matrix metalloproteases. In sum, the data reported here for MDA-MB-231 cells suggests some new interpretations of the discrepancy in the literature on the role of AHR in breast cancer. We propose that the unique combination of functional discrimination with transcriptome profiling provided by CIMMS will be valuable for a wide range of mechanistic invasion-biology studies in the future.

Concentration on demand – A microfluidic system for precise adjustment of the content of single droplets

We present a novel microfluidic system that produces the required concentration of a reagent in a single droplet or that produces a sequence of droplets with a defined periodic distribution of concentrations. We use digital algorithms that, through a series of simple operations, such as merging and splitting droplets, ensure superior precision, repeatability and flexibility in concentration setting.Unlike Digital microfluidic (DMF) systems based on electrowetting on dielectric (EWOD) commonly used to implement digital algorithms in the droplet world, our approach is based on much more available channel-based microfluidics operated by programmable syringe pumps. Furthermore, the small footprint of our system makes it easy to integrate with other structures of microfluidic networks. Thus, this technique is a comprehensive component that can be built into the microfluidic networks executing laboratory analytical tasks in chemistry or biology to enrich their performance and offer new functionalities.

Machine vision-based driving and feedback scheme for digital microfluidics system

Abstract A digital microfluidic system based on electrowetting-on-dielectric is a new technology for controlling microliter-sized droplets on a plane. By applying a voltage signal to an electrode, the droplets can be controlled to move, merge, and split. Due to device design, fabrication, and runtime uncertainties, feedback control schemes are necessary to ensure the reliability and accuracy of a digital microfluidic system for practical application. The premise of feedback is to obtain accurate droplet position information. Therefore, there is a strong need to develop a digital microfluidics system integrated with driving, position, and feedback functions for different areas of study. In this article, we propose a driving and feedback scheme based on machine vision for the digital microfluidics system. A series of experiments including droplet motion, merging, status detection, and self-adaption are performed to evaluate the feasibility and the reliability of the proposed scheme. The experimental results show that the proposed scheme can accurately locate multiple droplets and improve the success rate of different applications. Furthermore, the proposed scheme provides an experimental platform for scientists who focused on the digital microfluidics system.

Mechanowetting drives droplet and fluid transport on traveling surface waves generated by light-responsive liquid crystal polymers

In nature, capillary forces are often driving microfluidic propulsion and droplet manipulation, and technologies have been developed to utilize these forces in applications such as lab-on-a-chip biosensors and microfluidic systems. At the same time, responsive materials have been developed that can be activated by a variety of external triggers, including light, electric fields, and temperature, to locally deform and create dynamic surface structures, such as traveling waves. Here, we combine these developments into a system that enables capillary-driven droplet transport and fluid propulsion generated by light-induced surface waves in azobenzene-embedded liquid crystal polymers. We demonstrate that the traveling waves are able to efficiently propel fluids by means of mechanowetting. We couple the wave profiles to the fluid simulations using a multiphase computational fluid dynamics approach. We study three different fluid propulsion systems, i.e., peristaltic flow, liquid slug transport, and free-standing droplet transport. The first system operates on a fluid-filled single channel and achieves relative flow speeds of u/uwave&amp;lt;0.01. In contrast, the slugs and droplets are transported at two orders of magnitude higher speed equal to the wave speed (u/uwave=1) by exploiting the mechanowetting effect. We quantify the capillary forces generated by the traveling surface waves. Our method opens new avenues in light-driven (digital) microfluidic systems with enhanced control of fluid flow.

Conquering the Tyranny of Number With Digital Microfluidics.

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.

Active Femtoliter Droplet Generation in Microfluidics by Confined Interface Vibration.

The precise and effective generation of micron-sized droplets is one of the most common and important issues for droplet-based microfluidics. Active droplet generation makes use of additional energy input in promoting interfacial instabilities for droplet generation. Here, we report a new technique for the active generation of femtoliter droplets in microfluidic systems using confined interfacial vibration (CIV). The CIV is formed at the orifice of a traditional inkjet nozzle first by pushing the liquid out and then pulling it back. Droplets are pinched off during the withdrawal process, and this is different from the current active droplet generation techniques, which only monodirectionally push the liquid out. Droplets with radius ranging from ca. 1 to 28 μm can be actively generated by CIV at an orifice with radius 30 μm, distinguishing from conventional active generation techniques in which the droplets are always comparable or slightly bigger than the orifice. Experimental results showed that the droplet volume can be customized by controlling the intensity of the CIV. The inherent digital nature of the inkjet technique enables easy and precise regulating of the droplet volume, making it seamlessly compatible with the digital microfluidic systems.

Bacterial classification and antibiotic susceptibility testing on an integrated microfluidic platform.

With the prevalence of bacterial infections and increasing levels of antibiotic resistance comes the need for rapid and accurate methods for bacterial classification (BC) and antibiotic susceptibility testing (AST). Here we demonstrate the use of the fluid handling technique digital microfluidics (DMF) for automated and simultaneous BC and AST using growth metabolic markers. Custom instrumentation was developed for this application including an integrated heating module and a machine-learning-enabled low-cost colour camera for real-time absorbance and fluorescent sample monitoring on multipurpose devices. Antibiotic dilutions along with sample handling, mixing and incubation at 37 °C were all pre-programmed and processed automatically. By monitoring the metabolism of resazurin, resorufin beta-D-glucuronide and resorufin beta-D-galactopyranoside to resorufin, BC and AST were achieved in under 18 h. AST was validated in two uropathogenic E. coli strains with antibiotics ciprofloxacin and nitrofurantoin. BC was performed independently and simultaneously with ciprofloxacin AST for E. coli, K. pneumoniae, P. mirabilis and S. aureus. Finally, a proof-of-concept multiplexed system for breakpoint testing of two antibiotics, as well as E. coli and coliform classification was investigated with a multidrug-resistant E. coli strain. All bacteria were correctly identified, while AST and breakpoint test results were in essential and category agreement with reference methods. These results show the versatility and accuracy of this all-in-one microfluidic system for analysis of bacterial growth and phenotype.

Cancer drug screening with an on-chip multi-drug dispenser in digital microfluidics.

Microfluidics has been the most promising platform for drug screening with a limited number of cells. However, convenient on-chip preparation of a wide range of drug concentrations remains a large challenge and has restricted wide acceptance of microfluidics in precision medicine. In this paper, we report a digital microfluidic system with an innovative control structure and chip design for on-chip drug dispensing to generate concentrations that span three to four orders of magnitude, enabling single drug or combinatorial multi-drug screening with simple electronic control. Specifically, we utilize droplet ejection from a drug drop sitting on a special electrode, named a drug dispenser, under high-voltage pulse actuation to deliver the desired amount of drugs to be picked up by a cell suspension drop driven by low-voltage sine wave actuation. Our proof-of-principle validation for this technique as a convenient single and multi-drug screening involved testing of the drug toxicity of two chemotherapeutics, cisplatin (Cis) and epirubicin (EP), towards MDA-MB-231 breast cancer cells and MCF-10A normal breast cells. The results are consistent with those screened based on traditional 96-well plates. These findings demonstrate the reliability of the drug screening system with an on-chip drug dispenser. This system with fewer cancer cells, less drug consumption, a small footprint, and high scalability with regard to concentration could pave the way for drug screening on biopsied primary tumor cells for precision medicine or any concentration-related research.

Artificial Xylem Chip: A Three-Dimensionally Printed Vertical Digital Microfluidic Platform.

Digital microfluidics (DMF) is a promising lab-on-a-chip technology which has been applied in a wide variety of fields, including chemical sensing, biological detection, and even mechanical transportation. However, the appearance and functions of current DMF have been limited within two-dimensional planar space because of the conventional fabrication methods, such as photolithography or screen printing. In this paper, we report a DMF system which utilizes the advantage of three-dimensional (3D) printing to develop the novel form factor of electrodes and conversion of channels from planar to 3D forms. Vertical channels have been fabricated through combined 3D printing methods to facilitate stable and controlled movement of water droplets. The interfaces among liquid, gas, and solid were analyzed through Young-Lippmann law. We calculated the actuation force in a series of different configurations to enable us to optimize the system. Inspired by xylem structures in plants, the vertical movement and pumping of droplets are demonstrated by a programmable control system with a built-in boost converter for a real-time operating and portable DMF system. This work validates the promise of 3D printing to make 3D vertical DMF devices and the potential of the artificial xylem chip for micropumping applications.

Applications of electrowetting-on-dielectric (EWOD) technology for droplet digital PCR.

Digital microfluidics is an elegant technique based on single droplets for the design, composition, and manipulation of microfluidic systems. In digital microfluidics, especially in the electrowetting on dielectric (EWOD) system, each droplet acts as an independent reactor, which enables a wide range of multiple parallel biological and chemical reactions at the microscale. EWOD digital microfluidics reduces reagent and energy consumption, accelerates analysis, enables point-of-care diagnostic, simplifies integration with sensors, etc. Such a digital microfluidic system is especially relevant for droplet digital PCR (ddPCR), thanks to its nanoliter droplets and well-controlled volume distribution. At low DNA concentration, these small volumes allow less than one DNA strand per droplet on average (limited dilution) so that after a fixed number of PCR cycles (endpoint PCR), only the DNA in droplets containing the sequence of interest has been amplified and can be detected by fluorescence to yield an accurate count of the sequences of interest using statistical models. Focusing on ddPCR, this article summarizes the latest development and research on EWOD technology for droplet PCR over the last decade.

An integrated digital microfluidic bioreactor for fully automatic screening of microalgal growth and stress-induced lipid accumulation.

Algae are the promising feedstock of biofuel. The screening of competent species and proper fertilizer supply is of the most important tasks. To accelerate this rather slow and laborious step, we developed an integrated high-throughput digital microfluidic (DMF) system that uses a discrete droplet to serve as a microbioreactor, encapsulating microalgal cells. On the basis of fundamental understanding of various droplet hydrodynamics induced by the existence of different sorts of ions and biological species, incorporation of capacitance-based position estimator, electrode-saving-based compensation, and deterministic splitting approach, was performed to optimize the DMF bioreactor. Thus, it enables all processes (e.g.,nutrient gradient generation, algae culturing, and analyzing of growth and lipid accumulation) occurring automatically on-chip especially in a high-fidelity way. The ability of the system to compare different microalgal strains on-chip was investigated. Also, the Chlorella sp. were stressed by various conditions and then growth and oil accumulation were analyzed and compared, which demonstrated its potential as a powerful tool to investigate microalgal lipid accumulation at significantly lower laborites and reduced time.

Low-Cost Graphene-Based Digital Microfluidic System.

In this work, the laser-scribing technique was used as a low-cost, rapid and facile method for fabricating digital microfluidic (DMF) systems. Laser-scribed graphene (LSG) electrodes are directly synthesized on flexible substrates to pattern the DMF electrode arrays. This facilitates the DMF electrodes’ fabrication process by eliminating many microfabrication steps. An electrowetting test was performed to investigate the effectiveness of the LSG DMF electrodes in changing the contact angles of droplets. Different DMF operations were successfully performed using the proposed LSG DMF chips in both open and closed DMF systems. The quality and output resolution were examined to assess the performance of such patterned electrodes in the DMF systems. To verify the efficacy of the LSG DMF chips, a one-step direct assay for the detection of Legionella pneumophila deoxyribonucleic acid (DNA) was performed on the chip without the need for any washing step. The high specificity in distinguishing a single-nucleotide mismatch was achieved by detecting target DNA concentrations as low as 1 nM. Our findings suggest that the proposed rapid and easy fabrication method for LSG DMF electrodes offers a great platform for low-cost and easily accessible point-of-care diagnostic devices.

PortaDrop: A portable digital microfluidic platform providing versatile opportunities for Lab-On-A-Chip applications.

Electrowetting-on-dielectric is a decent technique to manipulate discrete volumes of liquid in form of droplets. In the last decade, electrowetting-on-dielectric systems, also called digital microfluidic systems, became more frequently used for a variety of applications because of their high flexibility and reconfigurability. Thus, one design can be adapted to different assays by only reprogramming. However, this flexibility can only be useful if the entire system is portable and easy to use. This paper presents the development of a portable, stand-alone digital microfluidic system based on a Linux-based operating system running on a Raspberry Pi, which is unique. We present “PortaDrop” exhibiting the following key features: (1) an “all-in-one box” approach, (2) a user-friendly, self-explaining graphical user interface and easy handling, (3) the ability of integrated electrochemical measurements, (4) the ease to implement additional lab equipment via Universal Serial Bus and the General Purpose Interface Bus as well as (5) a standardized experiment documentation. We propose that PortaDrop can be used to carry out experiments in different applications, where small sample volumes in the nanoliter to picoliter range need to be handled an analyzed automatically. As a first application, we present a protocol, where a droplet is consequently exchanged by droplets of another medium using passive dispensing. The exchange is monitored by electrical impedance spectroscopy. It is the first time, the media exchange caused by passive dispensing is characterized by electrochemical impedance spectroscopy. Summarizing, PortaDrop allows easy combination of fluid handling by means of electrowetting and additional sensing.

Stripped Electrode Based Electrowetting-on-Dielectric Digital Microfluidics for Precise and Controllable Parallel Microdrop Generation

Microdrop generation with excellent controllability and volume precision is of paramount significance for a large variety of microfluidic applications. In this work, we propose a new configuration comprising only stripped electrodes of rectangular shape for the closed electrowetting-on-dielectric digital microfluidic (EWOD DMF) system and investigate its parallel microdrop generation outcomes via a numerical approach. The microfluidic droplet motion is solved by a finite-volume scheme on a fixed computational domain. The numerical model is verified by an experimental study of microdrop production from an EWOD DMF device with three different electrode designs. After model verification, we examine the influences of the equilibrium contact angle and the spacing of the microchannel on stripped electrode based microdrop generation outcomes, and discover five different regimes including the phenomena of satellite droplet formation and separation cessation. Despite the various generation outcomes, the daughter droplet size is found to vary linearly with a dimensionless EWOD parameter κ*. More importantly, for all successful generations, the deviation of the daughter droplet size from that of the stripped electrode is smaller than 3.5%, which even reaches zero in proper conditions. This new configuration can be utilized as a convenient alternative for electrowetting-induced parallel microdrop production with excellent precision and controllability.