Cell motion and recovery in a two-stream microfluidic device

The motion of cells in a two-stream microfluidic device designed to extract cryoprotective agents from cell suspensions was tested under a range of conditions. Jurkat cells (lymphoblasts) in a 10% dimethylsulfoxide solution were driven in parallel with phosphate-buffered saline solution wash streams through single rectangular channel sections and multiple sections in series. The influence of cell-stream flow rate and cell volume fraction (CVF) on cell viability and recovery were examined. The channel depth was 500 μm, and average cell stream velocity within the channels was varied from 3.6 to 8.5 mm/s corresponding with cell stream Reynolds numbers of 2.6–6.0. Cell viability measured at device outlets was high for all cases examined indicating no significant cell damage within the device. Downstream of a single stage, cell recoveries measured 90–100% for average cell stream velocities ≥6 mm/s and for CVFs up to 20%. Cell recovery downstream of multistage devices also measured 90–100% after a critical device population time. This time was found to be five times the average cell residence time within the device. The measured recovery values were significantly larger than those typically obtained using conventional cell washing methods.     

A two-stage electrophoretic microfluidic device for nucleic acid collection and enrichment

Sample purification and enrichment is an important and usually time-consuming step for on-chip nucleic acid detection and analysis. This paper presents an electrophoretic DNA focusing method in microfluidic devices to enrich nucleic acid concentration by around 2700-fold. The electrical waveforms applied to five individual electrodes are such designed that DNAs move successively to the collection electrodes at high speed, while the interferences from bubbles due to electrohydrolysis are minimized. In a spiral channel with a total length of 48 cm, 1 ml DNA sample is purified and enriched by 57 times at a flow rate of 30 μl/min at first. The captured DNAs are then released and transported to the second microfluidic chamber where DNAs are collected and concentrated by 49 times. Thus, in about 40 min, the two-stage device can extract DNAs from 1 ml sample volume and enrich its concentration by 2790-fold. A trade-off exists between the process throughput and the DNA collection efficiency. A DNA capture efficiency of 99.7 % is reached when the flow rate is 1 μl/min, and the maximum DNA capture throughput is achieved at a flow rate of 30 μl/min. As a platform technology, the device can be integrated into bio-sensing and genetic analysis assays for DNA extraction and pre-concentration.     

A portable,hand-powered microfluidic device for sorting of biological particles

Manually hand-powered portable microfluidic devices are cheap alternatives for point-of-care diagnostics. Currently, on-field tests are limited by the use of bulky syringe pumps, pressure controller and equipment. In this work, we present a manually operated microfluidic device incorporated with a groove-based channel. We show that the device is capable to effectively sort particles/cells by manual hand powering. First, the grooved-based channel with differently sized polystyrene particles was characterized using syringe pumps to study their distributions under various flow rate conditions. Afterward, the particle mixtures were sorted manually using hand power to verify the capability of this device. Finally, the manually operated device was used to sort platelets from peripheral blood mononuclear cells (PBMCs). The platelets were collected with a purity of ~ 100%. The purity of PBMCs was enhanced from 0.8 to 10.4% after multiple processes which results in an enrichment ratio of 13.8. During the process of manual hand pumping, the flow fluctuation caused by unstable injection will not influence the sorting performance. Due to its simplicity, this manually operated microfluidic chip is suitable for outfield settings.     

Microfluidic fluorescence-activated cell sorting (μFACS) chip with integrated piezoelectric actuators for low-cost mammalian cell enrichment

A low-cost, microfluidic fluorescence-activated cell sorting (μFACS) microchip integrated with two piezoelectric lead–zirconate–titanate actuators was demonstrated for automated, high-performance mammalian cell analysis and enrichment. In this PDMS–glass device, cells were hydrodynamically focused into a single file line in the lateral direction by two sheath flows, and then interrogated with a forward scattering and confocal fluorescent detection system. The selected cells were displaced transversely into a collection channel by two piezoelectric actuators that worked in a pull–push relay manner with a minimal switching time of ~0.8 ms. High detection throughput (~2500 cells/s), high sorting rate (~1250 cells/s), and high sorting efficiency (~98%) were successfully achieved on the μFACS system. Six cell mixture samples containing 22.87% of GFP-expressing HeLa cells were consecutively analyzed and sorted on the chip, revealing a stable sorting efficiency of 97.7 ± 0.93%. In addition, cell mixtures containing 37.65 and 3.36% GFP HeLa cells were effectively enriched up to 83.82 and 78.51%, respectively, on the microchip, and an enrichment factor of 105 for the low-purity (3.36%) sample was successfully obtained. This fully enclosed, disposable microfluidic chip provides an automated platform for low-cost fluorescence-based cell detection and enrichment, and is attractive to applications where cross-contamination between runs and aerosol hazard are the primary concerns.     

A clip-on electroosmotic pump for oscillating flow in microfluidic cell culture devices

Recent advances in microfluidic devices put a high demand on small, robust and reliable pumps suitable for high-throughput applications. Here we demonstrate a compact, low-cost, directly attachable (clip-on) electroosmotic pump that couples with standard Luer connectors on a microfluidic device. The pump is easy to make and consists of a porous polycarbonate membrane and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) electrodes. The soft electrode and membrane materials make it possible to incorporate the pump into a standard syringe filter holder, which in turn can be attached to commercial chips. The pump is less than half the size of the microscope slide used for many commercial lab-on-a-chip devices, meaning that these pumps can be used to control fluid flow in individual reactors in highly parallelized chemistry and biology experiments. Flow rates at various electric current and device dimensions are reported. We demonstrate the feasibility and safety of the pump for biological experiments by exposing endothelial cells to oscillating shear stress (up to 5 dyn/cm²) and by controlling the movement of both micro- and macroparticles, generating steady or oscillatory flow rates up to ± 400 μL/min.     

An Aptameric Microfluidic System for Specific Purification, Enrichment, and Mass Spectrometric Detection of Biomolecules

We present an innovative microfluidic system that accomplishes specific capture, enrichment, and isocratic elution of biomolecular analytes with coupling to label-free mass spectrometric detection. Analytes in a liquid phase are specifically captured and enriched via their affinity binding to aptamers, which are immobilized on microbeads packed inside a microchamber. Exploiting thermally induced reversible disruption of aptamer–analyte binding via on-chip temperature control with an integrated heater and temperature sensor, the captured analytes are released into the liquid phase and then isocratically eluted and transferred via a microfluidic flow gate for detection by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The utility of the device is demonstrated using adenosine monophosphate (AMP) as a model analyte. Experimental results indicate that the device is capable of purifying and enriching the analyte from a sample mixed with nonspecific analytes and contaminated with salts. In addition, thermally induced analyte release is performed at modest temperatures (45 $^{circ}hbox{C}$), and mass spectra obtained from MALDI-MS demonstrate successful detection of AMP at concentrations as low as 10 nM following enrichment by consecutive infusion of a diluted sample.$hfill$[2009-0176]      

Automating fluid delivery in a capillary microfluidic device using low-voltage electrowetting valves

A multilayer capillary polymeric microfluidic device integrated with three normally closed electrowetting valves for timed fluidic delivery was developed. The microfluidic channel consisted two flexible layers of poly (ethylene terephthalate) bonded by a pressure-sensitive adhesive spacer tape. Channels were patterned in the spacer tape using laser ablation. Each valve contained two inkjet-printed silver electrodes in series. Capillary flow within the microchannel was stopped at the second electrode which was modified with a hydrophobic monolayer (valve closed). When a potential was applied across the electrodes, the hydrophobic monolayer became hydrophilic and allowed flow to continue (valve opened). The relationship between the actuation voltage, the actuation time, and the distance between two electrodes was performed using a microfluidic chip containing a single microchannel design. The results showed that a low voltage (4.5 V) was able to open the valve within 1 s when the distance between two electrodes was 1 mm. Increased voltages were needed to open the valves when the distance between two electrodes was increased. Additionally, the actuation time required to open the valve increased when voltage was decreased. A multichannel device was fabricated to demonstrate timed fluid delivery between three solutions. Our electrowetting valve system was fabricated using low-cost materials and techniques, can be actuated by a battery, and can be integrated into portable microfluidic devices suitable for point-of-care analysis in resource-limited settings.     

Scale-up and control of droplet production in coupled microfluidic flow-focusing geometries

A single microfluidic chip consisting of six microfluidic flow-focusing devices operating in parallel was developed to investigate the feasibility of scaling microfluidic droplet generation up to production rates of hundreds of milliliters per hour. The design utilizes a single inlet channel for both the dispersed aqueous phase and the continuous oil phase from which the fluids were distributed to all six flow-focusing devices. The exit tubing for each of the six flow-focusing devices is separate and individually plumbed to each device. Within each flow-focusing device, the droplet size was monodisperse, but some droplet size variations were observed across devices. We show that by modifying the flow resistance in the outlet channel of an individual flow-focusing device it is possible to control both the droplet size and frequency of droplet production. This can be achieved through the use of valves or, as is done in this study, by changing the length of the exit tubing plumbed to the outlet of the each device. Longer exit tubing and larger flow resistance is found to lead to larger droplets and higher production frequencies. The devices can thus be individually tuned to create a monodisperse emulsion or an emulsion with a specific drop size distribution.     

Microfluidic devices containing thin rock sections for oil recovery studies

While there has been a shift towards renewable energy sources, oil remains an important source of not only energy but also raw materials. Oil recovery is currently an inefficient process with as much as 50% of the original oil remaining in a field. Improvement of oil recovery techniques requires a model system that is both chemically and physically representative to achieve accurate results. Current large laboratory scale systems use large cores drilled from target rock and large, high-pressure systems to recreate oil recovery systems. The cores and associated equipment required to accurately model oil recovery are expensive and time consuming to obtain and operate. As a result, there has been a continual quest to develop alternative solutions that are faster, less complicated, and less expensive while still providing accurate representation of reservoirs. An alternative to large-scale models are optically transparent two or three-dimensional microfluidic devices. Several examples of microfluidic devices used to study oil recovery processes have been published. Unfortunately, most microfluidic devices require complicated fabrication techniques, inaccurately replicate the reservoir rock surface chemistry and geometry, and are made from materials not representative of surfaces found in oil reservoirs. Herein, the Flow On Rock Device is described as an easy to fabricate microfluidic device that acts as a bridge between fully synthetic microfluidics and large laboratory models due to incorporation of reservoir rock samples directly into the microfluidic device. Results of flooding studies are presented on shale and sandstone models as an example of the potential for this system in studying oil recovery.     

An on-chip cell culturing and combinatorial drug screening system

A low-cost, convenient and precise drug combination screening microfluidic platform is developed, in which cell culture chambers designed with micropillars integrate with three laminar flow diffusion channels. This platform has several distinct features, including minimum shear stress on cells, biocompatibility, optimum concentration distribution and automatic combinatorial gradient generation, which can potentially speed up the discovery of an effective drug combination for cancer ablations. The presented device can generate two-drug combination gradients at the optimum flow rate of 90 μL/h and can be applied to identify the optimal combination of two clinically relevant chemotherapy drugs. For demonstration, paclitaxel at 0.77 × 10^?3 mg/mL and cisplatin at 0.23 × 10^?4 mg/mL were studied against lung cancer cells (A549). This microfluidic device has the potential to provide a precise and robust screening for anticancer combinational drugs practiced in clinics.     

Microfluidic devices easily created using an office inkjet printer

Although various processes have been used for producing microfluidic devices, many of them are not so simple that ordinary end-users can produce the devices by themselves. However, in this study, microfluidic devices were easily produced using an office inkjet printer. As the components of the device, channels, manifolds, and mixers were created by printing their shapes on glass slides using the printer. A syringe pump could control the flow of fluid through the manifolds and mixers. In addition, resistivity of the device to acidic and basic solutions was tested.     

Highly integrated polymeric microliquid flow controller for droplet microfluidics

Microfluidic applications demand accurate control and measurement of small fluid flows and volumes, and the majority of approaches found in the literature involve materials and fabrication methods not suitable for a monolithic integration of different microcomponents needed to make a complex Lab-on-a-Chip (LoC) system. The present work leads to a design and manufacturing approach for problem-free monolithic integration of components on thermoplastics, allowing the production of excellent quality devices either as stand-alone components or combined in a complex structures. In particular, a polymeric liquid flow controlling system (LFCS) at microscale is presented, which is composed of a pneumatic microvalve and an on-chip microflow sensor. It enables flow regulation between 30 and 230 μl/min with excellent reproducibility and accuracy (error lower than 5%). The device is made of a single Cyclic Olefin Polymer (COP) piece, where the channels and cavities are hot-embossed, sealed with a single COP membrane by solvent bonding and metalized, after sealing, to render a fully functional microfluidic control system that features on-chip flow sensing. In contrast with commercially available flow control systems, the device can be used for high-quality flow modulation in disposable LoC devices, since the microfluidic chip is low cost and replaceable from the external electronic and pneumatic actuators box. Functionality of the LFCS is tested by connecting it to a microfluidic droplet generator, rendering highly stable flow rates and allowing generation of monodisperse droplets over a wide range of flow rates. The results indicate the successful performance of the LFCS with significant improvements over existing LFCS devices, facing the possibility of using the system for biological applications such as generating distinct perfusion modes in cell culture, novel digital microfluidics. Moreover, the integration capabilities and the reproducible fabrication method enable straightforward transition from prototype to product in a way that is lean, cost-effective and with reduced risk.     

A novel filtration method integrated on centrifugal microfluidic devices

A method has been developed that integrates filters directly into centrifugal microfluidic devices. This technique is suitable for both rapid prototyping and commercial applications. Commercially available filter paper was sealed into the centrifugal microfluidic device with a simple manual fabrication procedure. The method was validated using soil slurry in water and a variety of filter papers with pore sizes ranging from 0.7 to 11 μm. Filtration times of 4 s to several minutes were obtained for 100 μL samples depending on the type of filter paper and rotation rate utilized. The validity of the method was demonstrated by assessing the amount of light lost due to the scatter or absorption caused by particles in the filtered sample while the device was in motion. Filtration and sedimentation were compared and after 30 min of centrifugation, sedimentation had not removed particles as well as filtration. This technique opens up centrifugal microfluidic devices to a wide range of samples.     

Modular membrane valves for universal integration within thermoplastic devices

While much research has been conducted on elastomeric valves within PDMS microfluidic devices, we rarely see scalable manufacturing processes for integrating such valves into rigid thermoplastic devices. Most thermoplastic materials do not share intrinsic bonding compatibility to flexible elastomer membranes, making it difficult to ensure leak-proof operation of such valves within thermoplastic devices. In order to overcome bonding compatibility issues, we propose decoupling the valve architecture from the fluidic routing device layers. This can be achieved by prefabricating modular valves via molding processes and subsequently inserting them into thermoplastic layers containing valve seats. Thermoplastic layers containing modular valves are then thermally bonded to thermoplastic layers containing the fluidic routing channels, resulting in leak-proof valve integration. At valve actuation pressures of approximately 60 kPa, the modular membrane valves seal fluidic channels operating at a flow rate of 100 µl min^?1. Modular valves that were incorporated into a concentration gradient generator demonstrated dynamically configurable fluid routing at a response frequency of 5 Hz. The integration of modular membrane valves is an effective solution to streamline and cost-down the manufacturing of hybrid elastomer–thermoplastic devices. As this solution does not rely on bonding compatibility between the elastomeric membranes and the thermoplastic device, it can be applied universally to solve integration issues for low-cost thermoplastic device fabrication.     

Fabrication of circular microfluidic channels through grayscale dual-projection lithography

Circular microfluidic channels are in great demand since they are more realistic in mimicking physiological flow systems, generating axis-symmetrical flow, and achieving uniform shear stress. A typical microchannel with rectangular cross section can induce non-physiological gradients of shear rate, pressure, and velocity. This paper presents a novel method of fabricating microfluidic channels with circular and elliptical cross sections through grayscale dual-projection lithography. Our method utilizes two projecting systems to expose grayscale image face-to-face and simultaneously polymerize the photocurable material. The cross-sectional profiles of the fabricated microchannels are consistent with mathematical predictions and, therefore, demonstrate the capability of controlling the channel shapes precisely. Customized circular microchannels can be generated with complex features such as junctions, bifurcations, hierarchies, and gradually changed diameters. This method is capable of fabricating circular channels with a wide range of diameters (39 μm–2 mm) as well as elliptical channels with a major-to-minor axis ratio up to 600%. Microfluidic devices with circular cross sections suitable for particle analysis were made as a demonstrative application in nanoparticle binding and distribution within a mimetic blood vessel. A ready-to-use microfluidic device with customized circular channels can be fabricated within 1 h without the need of clean room or expensive photolithography devices.     

Microfluidic reactor array device for massively parallel in situ synthesis of oligonucleotides

We have designed and fabricated a microfluidic reactor array device for massively parallel in situ synthesis of oligonucleotides (oDNA). The device is made of glass anodically bonded to silicon consisting of three level features: microreactors, microchannels and through inlet/outlet holes. Main challenges in the design of this device include preventing diffusion of photogenerated reagents upon activation and achieving uniform reagent flow through thousands of parallel reactors. The device embodies a simple and effective dynamic isolation mechanism which prevents the intermixing of active reagents between discrete microreactors. Depending on the design parameters, it is possible to achieve uniform flow and synthesis reaction in all of the reactors by proper design of the microreactors and the microchannels. We demonstrated the use of this device on a solution-based, light-directed parallel in situ oDNA synthesis. We were able to synthesize long oDNA, up to 120 mers at stepwise yield of 98%. The quality of our microfluidic oDNA microarray including sensitivity, signal noise, specificity, spot variation and accuracy was characterized. Our microfluidic reactor array devices show a great potential for genomics and proteomics researches.     

Investigation of wax and paper materials for the fabrication of paper-based microfluidic devices

Paper-based microfluidic devices hold great potential in today’s microfluidic applications. They offer low costs, simple and quick fabrication processes, ease of uses, etc. In this work, several wax and paper materials are investigated for the fabrication of paper-based microfluidic devices. A novel method of using wax as a suitable backing to a paper-based analytical device has been demonstrated. Governing equations for the mechanics of the fluid flow in paper-based channels with constant widths have been experimentally validated. Experimental results showing deviations from the governing equations have been verified using fluidic channels with varying widths. There lies the possibility of manipulation of the fluid flow in paper-based microfluidic devices solely using geometric factors. This opens up many potential applications that may require sequential delivery of reagents or samples. Lastly, properties of paper such as the average pore diameter and permeability can be deduced from experimental results.     

Aqueous two-phase extraction for bovine serum albumin (BSA) with co-laminar flow in a simple coaxial capillary microfluidic device

Microfluidic extraction based on a co-laminar flow of aqueous two-phase system is used to separate bovine serum albumin (BSA). Mass transfer between the continuous two-phase flows is demonstrated by the extraction of BSA in a microfluidic device. The protein concentrations of the BSA samples were determined using the Bradford method. Polyethylene glycol 4000 and ammonium sulfate ((NH₄)₂SO₄) served as model aqueous two-phase solutions. The appropriate flow rates of the aqueous two phases were thus determined. We can flexibly control the mass transfer area and time by simply adjusting the flow rate. It takes only 3.6 s for three extraction cycles in a coaxial microfluidic device to achieve a BSA recovery yield of 71.1 %, which is superior to the traditional beaker aqueous two-phase extraction process. In this study, co-laminar flow-based continuous microextraction is demonstrated and its mass transfer is analyzed by solving the diffusion model, based on a large specific interfacial area and surface renewal.     

Deterministic lateral displacement (DLD) in the high Reynolds number regime: high-throughput and dynamic separation characteristics

Recent progress in the development of biosensors has created a demand for high-throughput sample preparation techniques that can be easily integrated into microfluidic or lab-on-a-chip platforms. One mechanism that may satisfy this demand is deterministic lateral displacement (DLD), which uses hydrodynamic forces to separate particles based on size. Numerous medically relevant cellular organisms, such as circulating tumor cells (10–15 µm) and red blood cells (6–8 µm), can be manipulated using microscale DLD devices. In general, these often-viscous samples require some form of dilution or other treatment prior to microfluidic transport, further increasing the need for high-throughput operation to compensate for the increased sample volume. However, high-throughput DLD devices will require a high flow rate, leading to an increase in Reynolds numbers (Re) much higher than those covered by existing studies for microscale (≤?100 µm) DLD devices. This study characterizes the separation performance for microscale DLD devices in the high-Re regime (10?<?Re?<?60) through numerical simulation and experimental validation. As Re increases, streamlines evolve and microvortices emerge in the wake of the pillars, resulting in a particle trajectory shift within the DLD array. This differs from previous DLD works, in that traditional models only account for streamlines that are characteristic of low-Re flow, with no consideration for the transformation of these streamlines with increasing Re. We have established a trend through numerical modeling, which agrees with our experimental findings, to serve as a guideline for microscale DLD performance in the high-Re regime. Finally, this new phenomenon could be exploited to design passive DLD devices with a dynamic separation range, controlled simply by adjusting the device flow rate.     

Microfluidic aptameric affinity sensing of vasopressin for clinical diagnostic and therapeutic applications

We present a microfluidic aptameric biosensor, or aptasensor, for selective detection of clinically relevant analytes with integrated analyte enrichment, isocratic elution and label-free detection by mass spectrometry. Using a microfluidic platform that is coupled to matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), we demonstrate specific purification, enrichment, and label-free detection of trace amounts of arginine vasopressin (AVP), a peptide hormone that is responsible for arterial vasoconstriction. During extreme physical trauma, in particular immunological shock or congestive heart failure, AVP is excreted abnormally and is hence a biomarker for such conditions. The device uses an aptamer, i.e., an oligonucleotide that binds specifically to an analyte via affinity interactions, to achieve highly selective analyte capture and enrichment. In addition, via thermally induced reversible disruption of the aptamer-analyte binding, the device can be easily regenerated for reuse and allows isocratic analyte elution, i.e., release and collection of analytes using a single aqueous solution. Furthermore, the device is coupled to MALDI-MS using a microfluidic flow gate, which directs the eluted analyte onto a MALDI sample plate for mass spectrometry. We first perform systematic characterization of kinetic and thermal release properties, as well as the overall timescale of the assay, using fluorescently labeled AVP. We then demonstrate MALDI-MS detection of unlabeled AVP at clinically relevant concentrations approaching 1 pM.