Phaseguide-assisted blood separation microfluidic device for point-of-care applications

We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 μl of whole blood but yielding approximately 0.38 μl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.     

Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml⁻¹), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.     

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.     

Rapid and cost-effective fabrication of selectively permeable calcium-alginate microfluidic device using "modified" embedded template method

In this paper, we have presented a non-lithographic embedded template method for rapid and cost-effective fabrication of a selectively permeable calcium-alginate (Ca-alginate) based microfluidic device with long serpentine delay channel. To demonstrate the versatility of the presented method, we have demonstrated two different strategies to fabricate serpentine long delay channels without using any sophisticated microfabrication techniques, in formal lab atmosphere. The procedure presented here, also, enables the preparation of a multilayered microfluidic device with channels of varying dimensions, in a single device without using any sophisticated micromachining instrumentation. In addition, we have also qualitatively studied the diffusion of small and large molecules from a Ca-alginate based microfluidic device and proposed a method to effectively control the out-flow of macro biomolecules from the crosslinked Ca-alginate matrix to create a selectively permeable matrix required for various biological and biomimetic applications, as mentioned in the Introduction section of this work.     

Hybrid capillary-inserted microfluidic device for sheathless particle focusing and separation in viscoelastic flow

A novel microfluidic device which consists of two stages for particle focusing and separation using a viscoelastic fluid has been developed. A circular capillary tube was used for three-dimensional particle pre-alignment before the separation process, which was inserted in a polydimethylsiloxane microchannel. Particles with diameters of 5 and 10 μm were focused at the centerline in the capillary tube, and the location of particles was initialized at the first bifurcation. Then, 5 and 10 μm particles were successfully separated in the expansion region based on size-dependent lateral migration, with ∼99% separation efficiency. The proposed device was further applied to separation of MCF-7 cells from leukocytes. Based on the cell size distribution, an approximate size cutoff for separation was determined to be 16 μm. At 200 μl/min, 94% of MCF-7 cells were separated with the purity of ∼97%. According to the trypan blue exclusion assay, high viability (∼90%) could be achieved for the separated MCF-7 cells. The use of a commercially available capillary tube enables the device to be highly versatile in dealing with particles in a wide size range by using capillary tubes with different inner diameters.     

Modulating chemotaxis of lung cancer cells by using electric fields in a microfluidic device

We employed direct-current electric fields (dcEFs) to modulate the chemotaxis of lung cancer cells in a microfluidic cell culture device that incorporates both stable concentration gradients and dcEFs. We found that the chemotaxis induced by a 0.5 μM/mm concentration gradient of epidermal growth factor can be nearly compensated by a 360 mV/mm dcEF. When the effect of chemical stimulation was balanced by the electrical drive, the cells migrated randomly, and the path lengths were largely reduced. We also demonstrated electrically modulated chemotaxis of two types of lung cancer cells with opposite directions of electrotaxis in this device.     

Density-dependent separation of encapsulated cells in a microfluidic channel by using a standing surface acoustic wave

This study presents a method for density-based separation of monodisperse encapsulated cells using a standing surface acoustic wave (SSAW) in a microchannel. Even though monodisperse polymer beads can be generated by the state-of-the-art technology in microfluidics, the quantity of encapsulated cells cannot be controlled precisely. In the present study, mono-disperse alginate beads in a laminar flow can be separated based on their density using acoustophoresis. A mixture of beads of equal sizes but dissimilar densities was hydrodynamically focused at the entrance and then actively driven toward the sidewalls by a SSAW. The lateral displacement of a bead is proportional to the density of the bead, i.e., the number of encapsulated cells in an alginate bead. Under optimized conditions, the recovery rate of a target bead group (large-cell-quantity alginate beads) reached up to 97% at a rate of 2300 beads per minute. A cell viability test also confirmed that the encapsulated cells were hardly damaged by the acoustic force. Moreover, cell-encapsulating beads that were cultured for 1 day were separated in a similar manner. In conclusion, this study demonstrated that a SSAW can successfully separate monodisperse particles by their density. With the present technique for separating cell-encapsulating beads, the current cell engineering technology can be significantly advanced.     

Trapping single human osteoblast-like cells from a heterogeneous population using a dielectrophoretic microfluidic device

We describe a system for the isolation, concentration, separation, and recovery of human osteoblast-like cells from a heterogeneous population using dielectrophoretic ring traps. Cells flowing in a microfluidic channel are immobilized inside an electric field cage using negative dielectrophoresis. A planar ring electrode creates a closed trap while repelling surrounding cells. Target cells are identified by fluorescent labeling, and are trapped as they pass across a ring electrode by an automated system. We demonstrate recovery of small populations of human osteoblast-like cells with a purity of 100%, which in turn demonstrates the potential of such a device for cell selection from a heterogeneous population.     

Probing the mechanical properties of brain cancer cells using a microfluidic cell squeezer device

Despite being invasive within surrounding brain tissues and the central nervous system, little is known about the mechanical properties of brain tumor cells in comparison with benign cells. Here, we present the first measurements of the peak pressure drop due to the passage of benign and cancerous brain cells through confined microchannels in a “microfluidic cell squeezer” device, as well as the elongation, speed, and entry time of the cells in confined channels. We find that cancerous and benign brain cells cannot be differentiated based on speeds or elongation. We have found that the entry time into a narrow constriction is a more sensitive indicator of the differences between malignant and healthy glial cells than pressure drops. Importantly, we also find that brain tumor cells take a longer time to squeeze through a constriction and migrate more slowly than benign cells in two dimensional wound healing assays. Based on these observations, we arrive at the surprising conclusion that the prevailing notion of extraneural cancer cells being more mechanically compliant than benign cells may not apply to brain cancer cells.     

Hydrodynamic self-focusing in a parallel microfluidic device through cross-filtration

The flow focusing is a fundamental prior step in order to sort, analyze, and detect particles or cells. The standard hydrodynamic approach requires two fluids to be injected into the microfluidic device: one containing the sample and the other one, called the sheath fluid, allows squeezing the sample fluid into a narrow stream. The major drawback of this approach is the high complexity of the layout for microfluidic devices when parallel streams are required. In this work, we present a novel parallelized microfluidic device that enables hydrodynamic focusing in each microchannel using a single feed flow. At each of the parallel channels, a cross-filter region is present that allows removing fluid from the sample fluid. This fluid is used to create local sheath fluids that hydrodynamically pinch the sample fluid. The great advantage of the proposed device is that, since only one inlet is needed, multiple parallel micro-channels can be easily introduced into the design. In the paper, the design method is described and the numerical simulations performed to define the optimal design are summarized. Moreover, the operational functionality of devices tested by using both polystyrene beads and Acute Lymphoid Leukemia cells are shown.     

Effects of hydraulic pressure on cardiomyoblasts in a microfluidic device

We employed a microfluidic device to study the effects of hydraulic pressure on cardiomyoblast H9c2. The 170 mm Hg pressure increased the cellular area and the expression of atrial natriuretic peptide. With the same device, we demonstrated that the effects of hydraulic pressure on the cardiomyoblast could be reduced by the inhibitor of focal adhesion kinase. This mechanical–chemical antagonism could lead to a potential therapeutic strategy of hypertension-induced cardiac hypertrophy.     

Cascaded spiral microfluidic device for deterministic and high purity continuous separation of circulating tumor cells

Inertial microfluidics is an emerging class of technologies developed to separate circulating tumor cells (CTCs). However, defining design parameters and flow conditions for optimal operation remains nondeterministic due to incomplete understanding of the mechanics, which has led to challenges in designing efficient systems. Here, we perform a parametric study of the inertial focusing effects observed in low aspect ratio curvilinear microchannels and utilize the results to demonstrate the isolation of CTCs with high purity. First, we systematically vary parameters including the channel height, width, and radius of curvature over a wide range of flow velocities to analyze its effect on size dependent differential focusing and migration behaviors of binary (10 μm and 20 μm) particles. Second, we use these results to identify optimal flow regimes to achieve maximum separation in various channel configurations and establish design guidelines to readily provide information for developing spiral channels tailored to potentially arbitrary flow conditions that yield a desired equilibrium position for optimal size based CTC separation. Finally, we describe a fully integrated, sheath-less cascaded spiral microfluidic device to continuously isolate CTCs. Human breast cancer epithelial cells were successfully extracted from leukocytes, achieving 86.76% recovery, 97.91% depletion rate, and sustaining high viability upon collection to demonstrate the versatility of the device. Importantly, this device was designed without the cumbersome trail-and-error optimization process that has hindered the development of designing such inertial microfluidic systems.     

Handling of artificial membranes using electrowetting-actuated droplets on a microfluidic device combined with integrated pA-measurements

Artificial membranes, as a controllable environment, are an essential tool to study membrane proteins. Electrophysiology provides information about the ion transport mechanism across a membrane at the single-protein level. Unfortunately, high-throughput studies and screening are not accessible to electrophysiology because it is a set of not automated and technically delicate methods. Therefore, it is necessary to automate and parallelize electrophysiology measurement in artificial membranes. Here, we present a first step toward this goal: the fabrication and characterization of a microfluidic device integrating electrophysiology measurements and the handling of an artificial membrane which includes its formation, its displacement and the separation of its leaflets using electrowetting actuation of sub-μL droplets. To validate this device, we recorded the insertion of a model porin, α-hemolysin.     

Single-cell attachment and culture method using a photochemical reaction in a closed microfluidic system

Recently, interest in single cell analysis has increased because of its potential for improving our understanding of cellular processes. Single cell operation and attachment is indispensable to realize this task. In this paper, we employed a simple and direct method for single-cell attachment and culture in a closed microchannel. The microchannel surface was modified by applying a nonbiofouling polymer, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, and a nitrobenzyl photocleavable linker. Using ultraviolet (UV) light irradiation, the MPC polymer was selectively removed by a photochemical reaction that adjusted the cell adherence inside the microchannel. To obtain the desired single endothelial cell patterning in the microchannel, cell-adhesive regions were controlled by use of round photomasks with diameters of 10, 20, 30, or 50 μm. Single-cell adherence patterns were formed after 12 h of incubation, only when 20 and 30 μm photomasks were used, and the proportions of adherent and nonadherent cells among the entire UV-illuminated areas were 21.3%±0.3% and 7.9%±0.3%, respectively. The frequency of single-cell adherence in the case of the 20 μm photomask was 2.7 times greater than that in the case of the 30 μm photomask. We found that the 20 μm photomask was optimal for the formation of single-cell adherence patterns in the microchannel. This technique can be a powerful tool for analyzing environmental factors like cell-surface and cell-extracellular matrix contact.     

Rapid,one-step fabrication and loading of nanoscale 1,2-distearoyl-sn-glycero-3-phosphocholine liposomes in a simple,double flow-focusing microfluidic device

Liposomes are currently well-established as biocompatible delivery vehicles for numerous compounds. However, conventional manufacturing tends to rely on time-consuming processes, costly equipment, unstable reaction parameters, and numerous pre- and post-processing steps. Herein, we demonstrate a microscope-slide-sized alternative: a double flow-focusing microfluidic geometry capable of sub-hour synthesis and controlled loading of tunable liposomes. Using phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine as the bilayer constituent, the effect of varying the dissolved lipid concentration and flow rate ratio on synthesized liposome diameters was investigated and the encapsulation of fluorescent hydrophobic drug model ergost-5,7,9(11),22-tetraen-3β-ol was performed to ascertain the potential of this device as a loading platform.     

Vortex-aided inertial microfluidic device for continuous particle separation with high size-selectivity,efficiency, and purity

In this paper, we report an inertial microfluidic device with simple geometry for continuous extraction of large particles with high size-selectivity (<2 μm), high efficiency (∼90%), and high purity (>90%). The design takes advantage of a high-aspect-ratio microchannel to inertially equilibrate cells and symmetric chambers for microvortex-aided cell extraction. A side outlet in each chamber continuously siphons larger particles, while the smaller particles or cells exit through the main outlet. The design has several advantages, including simple design, small footprint, ease of paralleling and cascading, one-step operation, and continuous separation with ultra-selectivity, high efficiency and purity. The described approach is applied to manipulating cells and particles for ultra-selective separation, quickly and effectively extracting larger sizes from the main flow, with broad applications in cell separations.     

Cell-enclosing gelatin-based microcapsule production for tissue engineering using a microfluidic flow-focusing system

Gelatin-based microcapsule production using a microfluidic system and the feasibility of the resultant microcapsules for constructing spherical tissues surrounded by heterogeneous cells were studied. The first cell-encapsulation and subsequent cell-enclosing microparticle encapsulation were achieved using a microfluidic flow-focusing droplet production system. A hollow-core structure of about 150 μm in diameter was developed by incubating the resultant microparticles at 37 °C, which induced thermal melting of the enclosed unmodified gelatin microparticles. Mammalian cells filled the hollow-cores after 4 days of incubation. A cell layer on the cell-enclosing microcapsules was developed by simply suspending the microcapsules in medium containing adherent fibroblast cells. This method may prove useful for the generation of gelatin microcapsules using a microfluidic system for formation of artificial tissue constructs.     

Quantification of the specific membrane capacitance of single cells using a microfluidic device and impedance spectroscopy measurement

The specific membrane capacitance (SMC) is an electrical parameter that correlates with both the electrical activity and morphology of the plasma membrane, which are physiological markers for cellular phenotype and health. We have developed a microfluidic device that enables impedance spectroscopy measurements of the SMC of single biological cells. Impedance spectra induced by single cells aspirated into the device are captured over a moderate frequency range (5 kHz–1 MHz). Maximum impedance sensitivity is achieved using a tapered microfluidic channel, which effectively routes electric fields across the cell membranes. The SMC is extracted by curve-fitting impedance spectra to an equivalent circuit model. From our measurement, acute myeloid leukemia (AML) cells are found to exhibit larger SMC values in hypertonic solutions as compared with those in isotonic solutions. In addition, AML cell phenotypes (AML2 and NB4) exhibiting varying metastatic potential yield distinct SMC values (AML2: 16.9 ± 1.9 mF/m² (n = 23); NB4: 22.5 ± 4.7 mF/m² (n = 23)). Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach.     

A modular cell culture device for generating arrays of gradients using stacked microfluidic flows

Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.     

Transport and shear in a microfluidic membrane bilayer device for cell culture

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.