Metabolic Labeling and Digital Microfluidic Single-Cell Sequencing for Single Bacterial Genotypic-Phenotypic Analysis.

Accurate assessment of phenotypic and genotypic characteristics of bacteria can facilitate comprehensive cataloguing of all the resistance factors for better understanding of antibiotic resistance. However, current methods primarily focus on individual phenotypic or genotypic profiles across different colonies. Here, a Digital microfluidic-based automated assay for whole-genome sequencing of single-antibiotic-resistant bacteria is reported, enabling Genotypic and Phenotypic Analysis of antibiotic-resistant strains (Digital-GPA). Digital-GPA can efficiently isolate and sequence antibiotic-resistant bacteria illuminated by fluorescent D-amino acid (FDAA)-labeling, producing high-quality single-cell amplified genomes (SAGs). This enables identifications of both minor and major mutations, pinpointing substrains with distinctive resistance mechanisms. Digital-GPA can directly process clinical samples to detect and sequence resistant pathogens without bacterial culture, subsequently provide genetic profiles of antibiotic susceptibility, promising to expedite the analysis of hard-to-culture or slow-growing bacteria. Overall, Digital-GPA opens a new avenue for antibiotic resistance analysis by providing accurate and comprehensive molecular profiles of antibiotic resistance at single-cell resolution.

Investigation into the optoelectrowetting droplet transport mechanism.

To explore the optoelectronic wetting droplet transport mechanism, a transient numerical model of optoelectrowetting (OEW) under the coupling of flow and electric fields is established. The study investigates the impact of externally applied voltage, dielectric constant of the dielectric layer, and interfacial tension between the two phases on the dynamic behavior of droplets during transport. The proposed model employs an improved Young's equation to calculate the instantaneous voltage and contact angle of the droplet on the dielectric layer. Results indicate that, under the influence of OEW, significant variations in the interface contact angle of droplets occur in bright and dark regions, inducing droplet movement. Moreover, the dynamic behavior of droplet transport is closely associated with various parameters, including externally applied voltage, dielectric layer material, and interfacial tension between the two phases, all of which impact the contact angle and, consequently, the transport process. By summarizing the influence patterns of the three key parameters studied, the optimization of droplet transport performance is achieved. The study employs two-dimensional simulation models to emulate the droplet motion under the influence of the electric field, investigating the OEW droplet transport mechanism. The continuous movement of droplets involves three stages: initial wetting, continuous transport, and reaching a steady position. The findings contribute theoretical support for the efficient design of digital microfluidic devices for OEW droplet movement and the selection of key parameters for droplet manipulation.

Digital Microfluidics for Microproteomic Analysis of Minute Mammalian Tissue Samples Enabled by a Photocleavable Surfactant.

Proteome profiles of precious tissue samples have great clinical potential for accelerating disease biomarker discovery and promoting novel strategies for early diagnosis and treatment. However, tiny clinical tissue samples are often difficult to handle and analyze with conventional proteomic methods. Automated digital microfluidic (DMF) workflows facilitate the manipulation of size-limited tissue samples. Here, we report the assessment of a DMF microproteomics workflow enabled by a photocleavable surfactant for proteomic analysis of minute tissue samples. The surfactant 4-hexylphenylazosulfonate (Azo) was found to facilitate fast droplet movement on DMF and enhance the proteomics analysis. Comparisons of Azo and n-Dodecyl ß-d-maltoside (DDM) using small samples of HeLa digest standards and MCF-7 cell digests revealed distinct differences at the peptide level despite similar results at the protein level. The DMF microproteomics workflow was applied for the sample preparation of ∼3 µg biopsies from murine brain tissue. A total of 1969 proteins were identified in three samples, including established neural biomarkers and proteins related to synaptic signaling. Going forward, we propose that the Azo-enabled DMF workflow has the potential to advance the practical clinical application of DMF for the analysis of size-limited tissue samples.

Hybrid Digital-Droplet Microfluidic Chip for Applications in Droplet Digital Nucleic Acid Amplification: Design, Fabrication and Characterization.

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

Digital Microfluidics Supported Microproteomics for Quantitative Proteome Analysis of Single Caenorhabditis elegans Nematodes.

Miniaturization of sample preparation, including omissible manual sample handling steps, is key for reproducible nanoproteomics, as material is often restricted to only hundreds of cells or single model organisms. Here, we demonstrate a highly sensitive digital microfluidics (DMF)-based sample preparation workflow making use of single-pot solid-phase enhanced sample preparation (SP3) in combination with high-field asymmetric-waveform ion mobility spectrometry (FAIMS), and fast and sensitive ion trap detection on an Orbitrap tribrid MS system. Compared to a manual in-tube SP3-supported sample preparation, the numbers of identified peptides and proteins were markedly increased, while lower standard deviations between replicates were observed. We repeatedly identified up to 5000 proteins from single nematodes. Moreover, label-free quantification of protein changes in single Caenorhabditis elegans treated with a heat stimulus yielded 45 differentially abundant proteins when compared to the untreated control, highlighting the potential of this technology for low-input proteomics studies. LC-MS data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD033143.

Integrating DNA Encapsulates and Digital Microfluidics for Automated Data Storage in DNA.

Using DNA as a durable, high-density storage medium with eternal format relevance can address a future data storage deficiency. The proposed storage format incorporates dehydrated particle spots on glass, at a theoretical capacity of more than 20 TB per spot, which can be efficiently retrieved without significant loss of DNA. The authors measure the rapid decay of dried DNA at room temperature and present the synthesis of encapsulated DNA in silica nanoparticles as a possible solution. In this form, the protected DNA can be readily applied to digital microfluidics (DMF) used to handle retrieval operations amenable to full automation. A storage architecture is demonstrated, which can increase the storage capacity of today's archival storage systems by more than three orders of magnitude: A DNA library containing 7373 unique sequences is encapsulated and stored under accelerated aging conditions (4 days at 70 °C, 50% RH) corresponding to 116 years at room temperature and the stored information is successfully recovered.

Electrical actuation of dielectric droplets by negative liquid dielectrophoresis.

This paper presents an electrical actuation scheme of dielectric droplet by negative liquid dielectrophoresis. A general model of lumped parameter electromechanics for evaluating the electromechanical force acting on the droplets is established. The model reveals the influence of actuation voltage, device geometry, and dielectric parameter on the actuation force for both conductive and dielectric medium. Using this model, we compare the actuation forces for four liquid combinations in the parallel-plate geometry and predict the low voltage actuation of dielectric droplets by negative dielectrophoresis. Parallel experimental results demonstrate such electric actuation of dielectric droplets, including droplet transport, splitting, merging, and dispending. All these dielectric droplet manipulations are achieved at voltages < 100 Vrms . The frequency dependence of droplet actuation velocity in aqueous solution is discussed and the existence of surfactant molecules is believed to play an important role by realigning with the AC electric field. Finally, we present coplanar manipulation of oil and water droplets and formation of oil-in-water emulsion droplet by applying the same low voltage.

Characterization of Inkjet-Printed Digital Microfluidics Devices.

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.

One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation.

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in-house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knockout transfections.

Digital Microfluidics for the Detection of Selected Inorganic Ions in Aerosols.

A prototype aerosol detection system is presented that is designed to accurately and quickly measure the concentration of selected inorganic ions in the atmosphere. The aerosol detection system combines digital microfluidics technology, aerosol impaction and chemical detection integrated on the same chip. Target compounds are the major inorganic aerosol constituents: sulfate, nitrate and ammonium. The digital microfluidic system consists of top and bottom plates that sandwich a fluid layer. Nozzles for an inertial impactor are built into the top plate according to known, scaling principles. The deposited air particles are densely concentrated in well-defined deposits on the bottom plate containing droplet actuation electrodes of the chip in fixed areas. The aerosol collection efficiency for particles larger than 100 nm in diameter was higher than 95%. After a collection phase, deposits are dissolved into a scanning droplet. Due to a sub-microliter droplet size, the obtained extract is highly concentrated. Droplets then pass through an air/oil interface on chip for colorimetric analysis by spectrophotometry using optical fibers placed between the two plates of the chip. To create a standard curve for each analyte, six different concentrations of liquid standards were chosen for each assay and dispensed from on-chip reservoirs. The droplet mixing was completed in a few seconds and the final droplet was transported to the detection position as soon as the mixing was finished. Limits of detection (LOD) in the final droplet were determined to be 11 ppm for sulfate and 0.26 ppm for ammonium. For nitrate, it was impossible to get stable measurements. The LOD of the on-chip measurements for sulfate was close to that obtained by an off-chip method using a Tecan spectrometer. LOD of the on-chip method for ammonium was about five times larger than what was obtained with the off-chip method. For the current impactor collection air flow (1 L/min) and 1 hour collection time, the converted LODs in air were: 0.275 for sulfate, 6.5 for ammonium, sufficient for most ambient air monitoring applications.

A Low-Cost, Disposable and Portable Inkjet-Printed Biochip for the Developing World.

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

Magnetic digital microfluidics on a bioinspired surface for point-of-care diagnostics of infectious disease.

Magnetic digital microfluidics uses magnetic force to manipulate droplets on a Teflon-coated substrate through the added magnetic particles. To achieve a wide range of droplet manipulation, hydrophilic patterns, known as surface energy traps, are introduced onto the Teflon-coated hydrophobic substrate. However, the Teflon-coated substrate is difficult to modify because it is nonwettable, and existing techniques for patterning surface energy traps have many limitations. Inspired by the mussel adhesion mechanism, we use polydopamine, a bioinspired substance that adheres strongly to almost any materials, to pattern surface energy traps on the Teflon-coated substrate with a great ease. We have optimized the polydopamine coating protocol and characterized the surface properties of the polydopamine surface energy traps. Droplet operations including particle extraction, liquid dispensing, liquid shaping, and cross-platform transfer have been demonstrated on the polydopamine surface energy trap-enabled magnetic digital microfluidic platform in both single-plate and two-plate configurations. Furthermore, the detection of hepatitis B surface antigen using ELISA has been demonstrated on the new magnetic dgitial microfluidic platform. This new bioinspired magnetic digital microfluidic platform is easy to fabricate and operate, showing a great potential for point-of-care applications.

Rapid Chemical Reaction Monitoring by Digital Microfluidics-NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform.

Microcoil nuclear magnetic resonance (NMR) has been interfaced with digital microfluidics (DMF) and is applied to monitor organic reactions in organic solvents as a proof of concept. DMF permits droplets to be moved and mixed inside the NMR spectrometer to initiate reactions while using sub-microliter volumes of reagent, opening up the potential to follow the reactions of scarce or expensive reagents. By setting up the spectrometer shims on a reagent droplet, data acquisition can be started immediately upon droplet mixing and is only limited by the rate at which NMR data can be collected, allowing the monitoring of fast reactions. Here we report a cyclohexene carbonate hydrolysis in dimethylformamide and a Knoevenagel condensation in methanol/water. This is to our knowledge the first time rapid organic reactions in organic solvents have been monitored by high field DMF-NMR. The study represents a key first step towards larger DMF-NMR arrays that could in future serve as discovery platforms, where computer controlled DMF automates mixing/titration of chemical libraries and NMR is used to study the structures formed and kinetics in real time.

DNA assembly with error correction on a droplet digital microfluidics platform.

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 immobilized cytochrome P450 reactors with integrated inkjet-printed microheaters for droplet-based drug metabolism research.

We report the development and characterization of digital microfluidic (DMF) immobilized enzyme reactors (IMERs) for studying cytochrome P450 (CYP)-mediated drug metabolism on droplet scale. The on-chip IMERs consist of porous polymer (thiol-ene) monolith plugs prepared in situ by photopolymerization and functionalized with recombinant CYP1A1 isoforms (an important detoxification route for many drugs and other xenobiotics). The DMF devices also incorporate inexpensive, inkjet-printed microheaters for on-demand regio-specific heating of the IMERs to physiological temperature, which is crucial for maintaining the activity of the temperature-sensitive CYP reaction. For on-chip monitoring of the CYP activity, the DMF devices were combined with a commercial well-plate reader, and a custom fluorescence quantification method was developed for detection of the chosen CYP1A1 model activity (ethoxyresorufin-O-deethylation). The reproducibility of the developed assay was examined with the help of ten parallel CYP-IMERs. All CYP-IMERs provided statistically significant difference (in fluorescence response) compared to any of the negative controls (including room-temperature reactions). The average (n = 10) turnover rate was 20.3 ± 9.0 fmol resorufin per minute. Via parallelization, the concept of the droplet-based CYP-IMER developed in this study provides a viable approach to rapid and low-cost prediction of the metabolic clearance of new chemical entities in vitro.

Liquid marbles as bioreactors for the study of three-dimensional cell interactions.

Liquid marble as a bioreactor platform for cell-based studies has received significant attention, especially for developing 3D cell-based assays. This platform is particularly suitable for 3D in-vitro modeling of cell-cell interactions. For the first time, we demonstrated the interaction of olfactory ensheathing cells (OECs) with nerve debris and meningeal fibroblast using liquid marbles. As the transplantation of OECs can be used for repairing nerve injury, degenerating cell debris within the transplantation site can adversely affect the survival of transplanted OECs. In this paper, we used liquid marbles to mimic the hostile 3D environment to analyze the functional behavior of the cells and to form the basis for cell-based therapy. We show that OECs interact with debris and enhanced cellular aggregation to form a larger 3D spheroidal tissue. However, these spheroids indicated limitation in biological functions such as the inability of cells within the spheroids to migrate out and adherence to neighboring tissue by fusion. The coalescence of two liquid marbles allows for analyzing the interaction between two distinct cell types and their respective environment. We created a microenvironment consisting of 3D fibroblast spheroids and nerve debris and let it interact with OECs. We found that OECs initiate adherence with nerve debris in this 3D environment. The results suggest that liquid marbles are ideal for developing bioassays that could substantially contribute to therapeutic applications. Especially, insights for improving the survival and adherence of transplanted cells.

Advances in Testing Techniques for Digital Microfluidic Biochips.

With the advancement of digital microfluidics technology, applications such as on-chip DNA analysis, point of care diagnosis and automated drug discovery are common nowadays. The use of Digital Microfluidics Biochips (DMFBs) in disease assessment and recognition of target molecules had become popular during the past few years. The reliability of these DMFBs is crucial when they are used in various medical applications. Errors found in these biochips are mainly due to the defects developed during droplet manipulation, chip degradation and inaccuracies in the bio-assay experiments. The recently proposed Micro-electrode-dot Array (MEDA)-based DMFBs involve both fluidic and electronic domains in the micro-electrode cell. Thus, the testing techniques for these biochips should be revised in order to ensure proper functionality. This paper describes recent advances in the testing technologies for digital microfluidics biochips, which would serve as a useful platform for developing revised/new testing techniques for MEDA-based biochips. Therefore, the relevancy of these techniques with respect to testing of MEDA-based biochips is analyzed in order to exploit the full potential of these biochips.

Digital Microfluidics for Nucleic Acid Amplification.

Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification Detection.

Digital microfluidics (DMF) arises as the next step in the fast-evolving field of operation platforms for molecular diagnostics. Moreover, isothermal schemes, such as loop-mediated isothermal amplification (LAMP), allow for further simplification of amplification protocols. Integrating DMF with LAMP will be at the core of a new generation of detection devices for effective molecular diagnostics at point-of-care (POC), providing simple, fast, and automated nucleic acid amplification with exceptional integration capabilities. Here, we demonstrate for the first time the role of coupling DMF and LAMP, in a dedicated device that allows straightforward mixing of LAMP reagents and target DNA, as well as optimum temperature control (reaction droplets undergo a temperature variation of just 0.3 °C, for 65 °C at the bottom plate). This device is produced using low-temperature and low-cost production processes, adaptable to disposable and flexible substrates. DMF-LAMP is performed with enhanced sensitivity without compromising reaction efficacy or losing reliability and efficiency, by LAMP-amplifying 0.5 ng/µL of target DNA in just 45 min. Moreover, on-chip LAMP was performed in 1.5 µL, a considerably lower volume than standard bench-top reactions.

Open-atmosphere sustenance of highly volatile attoliter-size droplets on surfaces.

The controlled formation and handling of minute liquid volumes on surfaces is essential to the success of microfluidics in biology, chemistry, and materials applications. Even though current methods have demonstrated their potential in a variety of experimental assays, there remain significant difficulties concerning breadth of applicability, standardization, throughput, and economics. Here we introduce a unique microfluidic paradigm in which microscopic volatile droplets are formed, sustained, and manipulated in size and content at any desired spot on unpatterned substrates. Their sustainability is warranted by continuous replacement of the rapidly vaporizing sessile fluid through controlled equivalent volume deposition of smaller discrete liquid entities by an electrohydrodynamic nanodripping process. Using nanoparticle inks we show that the concentration of solutes in so-stabilized droplets can be linearly increased at isochoric conditions and user-defined rates. An intriguing insensitivity of the droplet shape toward surface heterogeneities ensures robustness and experimental reproducibility, even when handling attoliter quantities. The unique capabilities and technical simplicity of the presented method introduce a high degree of flexibility and make it pertinent to a diverse range of applications.