Versatile on-demand droplet generation for controlled encapsulation

We present a droplet-based microfluidic system for performing bioassays requiring controlled analyte encapsulation by employing highly flexible on-demand droplet generation. On-demand droplet generation and encapsulation are achieved pneumatically using a microdispensing pump connected to a constant pressure source. The system generates single droplets to the collection route only when the pump is actuated with a designated pressure level and produces two-phase parallel flow to the waste route during the stand-by state. We analyzed the effect of actuation pressure on the stability and size of droplets and optimized conditions for generation of stable droplets over a wide pressure range. By increasing the duration of pump actuation, we could either trigger a short train of identical size droplets or generate a single larger droplet. We also investigated the methodology to control droplet contents by fine-tuning flow rates or implementing a resistance bridge between the pump and main channels. We demonstrated the integrated chip for on-demand mixing between two aqueous phases in droplets and on-demand encapsulation of Escherichia coli cells. Our unique on-demand feature for selective encapsulation is particularly appropriate for bioassays with extremely dilute samples, such as pathogens in a clinical sample, since it can significantly reduce the number of empty droplets that impede droplet collection and subsequent data analysis.     

On demand nanoliter-scale microfluidic droplet generation,injection, and mixing using a passive microfluidic device

We here present and characterize a programmable nanoliter scale droplet-on-demand device that can be used separately or readily integrated into low cost single layer rapid prototyping microfluidic systems for a wide range of user applications. The passive microfluidic device allows external (off-the-shelf) electronically controlled pinch valves to program the delivery of nanoliter scale aqueous droplets from up to 9 different inputs to a central outlet channel. The inputs can be either continuous aqueous fluid streams or microliter scale aqueous plugs embedded in a carrier fluid, in which case the number of effective input solutions that can be employed in an experiment is no longer strongly constrained (100 s–1000 s). Both nanoliter droplet sequencing output and nanoliter-scale droplet mixing are reported with this device. Optimization of the geometry and pressure relationships in the device was achieved in several hardware iterations with the support of open source microfluidic simulation software and equivalent circuit models. The requisite modular control of pressure relationships within the device is accomplished using hydrodynamic barriers and matched resistance channels with three different channel heights, custom parallel reversible microfluidic I/O connections, low dead-volume pinch valves, and a simply adjustable array of external screw valves. Programmable sequences of droplet mixes or chains of droplets can be achieved with the device at low Hz frequencies, limited by device elasticity, and could be further enhanced by valve integration. The chip has already found use in the characterization of droplet bunching during export and the synthesis of a DNA library.     

A microfluidic platform for size-dependent generation of droplet interface bilayer networks on rails

Droplet interface bilayer (DIB) networks are emerging as a cornerstone technology for the bottom up construction of cell-like and tissue-like structures and bio-devices. They are an exciting and versatile model-membrane platform, seeing increasing use in the disciplines of synthetic biology, chemical biology, and membrane biophysics. DIBs are formed when lipid-coated water-in-oil droplets are brought together—oil is excluded from the interface, resulting in a bilayer. Perhaps the greatest feature of the DIB platform is the ability to generate bilayer networks by connecting multiple droplets together, which can in turn be used in applications ranging from tissue mimics, multicellular models, and bio-devices. For such applications, the construction and release of DIB networks of defined size and composition on-demand is crucial. We have developed a droplet-based microfluidic method for the generation of different sized DIB networks (300–1500 pl droplets) on-chip. We do this by employing a droplet-on-rails strategy where droplets are guided down designated paths of a chip with the aid of microfabricated grooves or “rails,” and droplets of set sizes are selectively directed to specific rails using auxiliary flows. In this way we can uniquely produce parallel bilayer networks of defined sizes. By trapping several droplets in a rail, extended DIB networks containing up to 20 sequential bilayers could be constructed. The trapped DIB arrays can be composed of different lipid types and can be released on-demand and regenerated within seconds. We show that chemical signals can be propagated across the bio-network by transplanting enzymatic reaction cascades for inter-droplet communication.     

SAW-driven droplet jetting technology in microfluidic: A review

The introduction of surface acoustic wave (SAW) technology on microfluidics has shown its powerfully controlling and actuating fluid and particle capability in a micro-nano scale, such as fluid mixing, fluid translation, microfluidic pumping, microfluidic rotational motor, microfluidic atomization, particle or cell concentration, droplet or cell sorting, reorientation of nano-objects, focusing and separation of particles, and droplet jetting. The SAW-driven droplet jetting technology enjoys the advantages of simple structure to fabricate with little hindrance, compact size to integrate with other components, high biocompatibility with biological cells or other molecule samples, large force in realizing fast fluidic actuation, and contact-free manipulation with fluid. The realization of this technology can effectively overcome some bottleneck problems in the current micro-injection technology, such as mechanical swear, complicated and bulky structure, and strict limitation of requirements on fluidic characteristics. This article reviews and reorganizes SAW-microfluidic jetting technology from decades of years, referring to the interaction mechanism theory of SAW and fluid, experimental methods of SAW-microfluidic jetting, effects of related parameters on objected pinch-off droplets, and applications of individual structures. Finally, we made a summary of the research results of the current literature and look forward and appraise where this discipline of SAW-microfluidic jetting could go in the future.     

The dynamics and stability of lubricating oil films during droplet transport by electrowetting in microfluidic devices

The operation of digital microfluidic devices with water droplets manipulated by electrowetting is critically dependent on the static and dynamic stability and lubrication properties of the oil films that separate the droplets from the solid surfaces. The factors determining the stability of the films and preventing surface fouling in such systems are not yet thoroughly understood and were experimentally investigated in this study. The experiments were performed using a standard digital microfluidic cartridge in which water droplets enclosed in a thin, oil-filled gap were transported over an array of electrodes. Stable, continuous oil films separated the droplets from the surfaces when the droplets were stationary. During droplet transport, capillary waves formed in the films on the electrode surfaces as the oil menisci receded. The waves evolved into dome-shaped oil lenses. Droplet deformation and oil displacement caused the films at the surface opposite the electrode array to transform into dimples of oil trapped over the centers of the droplets. Lower actuation voltages were associated with slower film thinning and formation of fewer, but larger, oil lenses. Lower ac frequencies induced oscillations in the droplets that caused the films to rupture. Films were also destabilized by addition of surfactants to the oil or droplet phases. Such a comprehensive understanding of the oil film behavior will enable more robust electrowetting-actuated lab-on-a-chip devices through prevention of loss of species from droplets and contamination of surfaces at points where films may break.     

Osteocyte culture in microfluidic devices

This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes.     

Bistability in droplet traffic at asymmetric microfluidic junctions

We present the first experimental demonstration of confined microfluidic droplets acting as discrete negative resistors, wherein the effective hydrodynamic resistance to flow in a microchannel is reduced by the presence of a droplet. The implications of this hitherto unexplored regime in the traffic of droplets in microfluidic networks are highlighted by demonstrating bistable filtering into either arm of symmetric and asymmetric microfluidic loops, and programming oscillatory droplet routing therein.     

A simple method of fabricating mask-free microfluidic devices for biological analysis

We report a simple, low-cost, rapid, and mask-free method to fabricate two-dimensional (2D) and three-dimensional (3D) microfluidic chip for biological analysis researches. In this fabrication process, a laser system is used to cut through paper to form intricate patterns and differently configured channels for specific purposes. Bonded with cyanoacrylate-based resin, the prepared paper sheet is sandwiched between glass slides (hydrophilic) or polymer-based plates (hydrophobic) to obtain a multilayer structure. In order to examine the chip’s biocompatibility and applicability, protein concentration was measured while DNA capillary electrophoresis was carried out, and both of them show positive results. With the utilization of direct laser cutting and one-step gas-sacrificing techniques, the whole fabrication processes for complicated 2D and 3D microfluidic devices are shorten into several minutes which make it a good alternative of poly(dimethylsiloxane) microfluidic chips used in biological analysis researches.     

Controlled electroporation of the plasma membrane in microfluidic devices for single cell analysis

Chemical cytometry on a single cell level is of interest to various biological fields ranging from cancer to stem cell research. The impact chemical cytometry can exert in these fields depends on the dimensionality of the retrievable analytes content. To this point, the number of different analytes identifiable and additionally their subcellular localization is of interest. To address this, we present an electroporation based approach for selective lysis of only the plasma membrane, which permits analysis of the dissolved cytoplasm, while reducing contributions from the nucleus and membrane bound fractions of the cell analytes. The use of 100 μs long pulse and a well defined DC electric field gradient of ∼4.5 kV·cm⁻¹ generated by 3D electrodes initiates release of a cytoplasm marker in ≪1 s, while retaining nuclear fluorescence markers.     

Monodisperse alginate microgel formation in a three-dimensional microfluidic droplet generator

Droplet based microfluidic systems provide an ideal platform for partitioning and manipulating aqueous samples for analysis. Identifying stable operating conditions under which droplets are generated is challenging yet crucial for real-world applications. A novel three-dimensional microfluidic platform that facilitates the consistent generation and gelation of alginate-calcium hydrogel microbeads for microbial encapsulation, over a broad range of input pressures, in the absence of surfactants is described. The unique three-dimensional design of the fluidic network utilizes a height difference at the junction between the aqueous sample injection and organic carrier channels to induce droplet formation via a surface tension enhanced self-shearing mechanism. Combined within a flow-focusing geometry, under constant pressure control, this arrangement facilitates predictable generation of droplets over a much broader range of operating conditions than that of conventional two-dimensional systems. The impact of operating pressures and geometry on droplet gelation, aqueous and organic material flow rates, microbead size, and bead generation frequency are described. The system presented provides a robust platform for encapsulating single microbes in complex mixtures into individual hydrogel beads, and provides the foundation for the development of a complete system for sorting and analyzing microbes at the single cell level.     

Shrink-film microfluidic education modules: Complete devices within minutes

As advances in microfluidics continue to make contributions to diagnostics and life sciences, broader awareness of this expanding field becomes necessary. By leveraging low-cost microfabrication techniques that require no capital equipment or infrastructure, simple, accessible, and effective educational modules can be made available for a broad range of educational needs from middle school demonstrations to college laboratory classes. These modules demonstrate key microfluidic concepts such as diffusion and separation as well as laboratory on-chip applications including chemical reactions and biological assays. These modules are intended to provide an interdisciplinary hands-on experience, including chip design, fabrication of functional devices, and experiments at the microscale. Consequently, students will be able to conceptualize physics at small scales, gain experience in computer-aided design and microfabrication, and perform experiments-all in the context of addressing real-world challenges by making their own lab-on-chip devices.     

Droplet dispensing in digital microfluidic devices: Assessment of long-term reproducibility

We report an in-depth study of the long-term reproducibility and reliability of droplet dispensing in digital microfluidic devices (DMF). This involved dispensing droplets from a reservoir, measuring the volume of both the droplet and the reservoir droplet and then returning the daughter droplet to the original reservoir. The repetition of this process over the course of several hundred iterations offers, for the first time, a long-term view of droplet dispensing in DMF devices. Results indicate that the ratio between the spacer thickness and the electrode size influences the reliability of droplet dispensing. In addition, when the separation between the plates is large, the volume of the reservoir greatly affects the reproducibility in the volume of the dispensed droplets, creating "reliability regimes." We conclude that droplet dispensing exhibits superior reliability as inter-plate device spacing is decreased, and the daughter droplet volume is most consistent when the reservoir volume matches that of the reservoir electrode.     

Vascular smooth muscle cell culture in microfluidic devices

This paper presents a microfluidic device enabling culture of vascular smooth muscle cells (VSMCs) where extracellular matrix coating, VSMC seeding, culture, and immunostaining are demonstrated in a tubing-free manner. By optimizing droplet volume differences between inlets and outlets of micro channels, VSMCs were evenly seeded into microfluidic devices. Furthermore, the effects of extracellular matrix (e.g., collagen, poly-l-Lysine (PLL), and fibronectin) on VSMC proliferation and phenotype expression were explored. As a platform technology, this microfluidic device may function as a new VSMC culture model enabling VSMC studies.     

Diffusion phenomena of cells and biomolecules in microfluidic devices

Biomicrofluidics is an emerging field at the cross roads of microfluidics and life sciences which requires intensive research efforts in terms of introducing appropriate designs, production techniques, and analysis. The ultimate goal is to deliver innovative and cost-effective microfluidic devices to biotech, biomedical, and pharmaceutical industries. Therefore, creating an in-depth understanding of the transport phenomena of cells and biomolecules becomes vital and concurrently poses significant challenges. The present article outlines the recent advancements in diffusion phenomena of cells and biomolecules by highlighting transport principles from an engineering perspective, cell responses in microfluidic devices with emphases on diffusion- and flow-based microfluidic gradient platforms, macroscopic and microscopic approaches for investigating the diffusion phenomena of biomolecules, microfluidic platforms for the delivery of these molecules, as well as the state of the art in biological applications of mammalian cell responses and diffusion of biomolecules.     

Stretching DNA by electric field and flow field in microfluidic devices: An experimental validation to the devices designed with computer simulations

We examined the performance of three microfluidic devices for stretching DNA. The first device is a microchannel with a contraction, and the remaining two are the modifications to the first. The modified designs were made with the help of computer simulations [C. C. Hsieh and T. H. Lin, Biomicrofluidics 5(4), 044106 (2011) and C. C. Hsieh, T. H. Lin, and C. D. Huang, Biomicrofluidics 6, 044105 (2012)] and they were optimized for operating with electric field. In our experiments, we first used DC electric field to stretch DNA. However, the experimental results were not even in qualitative agreement with our simulations. More detailed investigation revealed that DNA molecules adopt a globular conformation in high DC field and therefore become more difficult to stretch. Owing to the similarity between flow field and electric field, we turned to use flow field to stretch DNA with the same devices. The evolution patterns of DNA conformation in flow field were found qualitatively the same as our prediction based on electric field. We analyzed the maximum values, the evolution and the distributions of DNA extension at different Deborah number in each device. We found that the shear and the hydrodynamic interaction have significant influence on the performance of the devices.     

Extensional flow of blood analog solutions in microfluidic devices

In this study, we show the importance of extensional rheology, in addition to the shear rheology, in the choice of blood analog solutions intended to be used in vitro for mimicking the microcirculatory system. For this purpose, we compare the flow of a Newtonian fluid and two well-established viscoelastic blood analog polymer solutions through microfluidic channels containing both hyperbolic and abrupt contractions∕expansions. The hyperbolic shape was selected in order to impose a nearly constant strain rate at the centerline of the microchannels and achieve a quasihomogeneous and strong extensional flow often found in features of the human microcirculatory system such as stenoses. The two blood analog fluids used are aqueous solutions of a polyacrylamide (125 ppm w∕w) and of a xanthan gum (500 ppm w∕w), which were characterized rheologically in steady-shear flow using a rotational rheometer and in extension using a capillary breakup extensional rheometer (CaBER). Both blood analogs exhibit a shear-thinning behavior similar to that of whole human blood, but their relaxation times, obtained from CaBER experiments, are substantially different (by one order of magnitude). Visualizations of the flow patterns using streak photography, measurements of the velocity field using microparticle image velocimetry, and pressure-drop measurements were carried out experimentally for a wide range of flow rates. The experimental results were also compared with the numerical simulations of the flow of a Newtonian fluid and a generalized Newtonian fluid with shear-thinning behavior. Our results show that the flow patterns of the two blood analog solutions are considerably different, despite their similar shear rheology. Furthermore, we demonstrate that the elastic properties of the fluid have a major impact on the flow characteristics, with the polyacrylamide solution exhibiting a much stronger elastic character. As such, these properties must be taken into account in the choice or development of analog fluids that are adequate to replicate blood behavior at the microscale.     

Continuous separation of blood cells in spiral microfluidic devices

Blood cell sorting is critical to sample preparation for both clinical diagnosis and therapeutic research. The spiral inertial microfluidic devices can achieve label-free, continuous separation of cell mixtures with high throughput and efficiency. The devices utilize hydrodynamic forces acting on cells within laminar flow, coupled with rotational Dean drag due to curvilinear microchannel geometry. Here, we report on optimized Archimedean spiral devices to achieve cell separation in less than 8 cm of downstream focusing length. These improved devices are small in size (<1 in.²), exhibit high separation efficiency (∼95%), and high throughput with rates up to 1 × 10⁶ cells per minute. These device concepts offer a path towards possible development of a lab-on-chip for point-of-care blood analysis with high efficiency, low cost, and reduced analysis time.     

Applying an optical space-time coding method to enhance light scattering signals in microfluidic devices

An "optical space-time coding method" was applied to microfluidic devices to detect the forward and large angle light scattering signals for unlabelled bead and cell detection. Because of the enhanced sensitivity by this method, silicon pin photoreceivers can be used to detect both forward scattering (FS) and large angle (45-60°) scattering (LAS) signals, the latter of which has been traditionally detected by a photomultiplier tube. This method yields significant improvements in coefficients of variation (CV), producing CVs of 3.95% to 10.05% for FS and 7.97% to 26.12% for LAS with 15 μm, 10 μm, and 5 μm beads. These are among the best values ever demonstrated with microfluidic devices. The optical space-time coding method also enables us to measure the speed and position of each particle, producing valuable information for the design and assessment of microfluidic lab-on-a-chip devices such as flow cytometers and complete blood count devices.     

Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices

We have developed a coaxial flow focusing geometry that can be fabricated using soft lithography in poly(dimethylsiloxane) (PDMS). Like coaxial flow focusing in glass capillary microfluidics, our geometry can form double emulsions in channels with uniform wettability and of a size much smaller than the channel dimensions. However, In contrast to glass capillary coaxial flow focusing, our geometry can be fabricated using lithographic techniques, allowing it to be integrated as the drop making unit in parallel drop maker arrays. Our geometry enables scalable formation of emulsions down 7 μm in diameter, in large channels that are robust against fouling and clogging.     

Molecular diffusion analysis of dynamic blood flow and plasma separation driven by self-powered microfluidic devices

Integration of microfluidic devices with pressure-driven, self-powered fluid flow propulsion methods has provided a very effective solution for on-chip, droplet blood testing applications. However, precise understanding of the physical process governing fluid dynamics in polydimethylsiloxane (PDMS)-based microfluidic devices remains unclear. Here, we propose a pressure-driven diffusion model using Fick''s law and the ideal gas law, the results of which agree well with the experimental fluid dynamics observed in our vacuum pocket-assisted, self-powered microfluidic devices. Notably, this model enables us to precisely tune the flow rate by adjusting two geometrical parameters of the vacuum pocket. By linking the self-powered fluid flow propulsion method to the sedimentation, we also show that direct plasma separation from a drop of whole blood can be achieved using only a simple construction without the need for external power sources, connectors, or a complex operational procedure. Finally, the potential of the vacuum pocket, along with a removable vacuum battery to be integrated with non-PDMS microfluidic devices to drive and control the fluid flow, is demonstrated.