Continuous Flow Microfluidic Device Research Articles

Continuous-flow multi-pulse electroporation at low DC voltages by microfluidic flipping of the voltage space topology.

Concerns over biosafety, cost, and carrying capacity of viral vectors have accelerated research into physical techniques for gene delivery such as electroporation and mechanoporation. Advances in microfabrication have made it possible to create high electric fields over microscales, resulting in more efficient DNA delivery and higher cell viability. Continuous-flow microfluidic methods are typically more suitable for cellular therapies where a large number of cells need to be transfected under sterile conditions. However, the existing continuous-flow designs used to generate multiple pulses either require expensive peripherals such as high-voltage (>400 V) sources or function generators, or result in reduced cell viability due to the proximity of the cells to the electrodes. In this paper, we report a continuous-flow microfluidic device whose channel geometry reduces instrumentation demands and minimizes cellular toxicity. Our design can generate multiple pulses of high DC electric field strength using significantly lower voltages (15-60 V) than previous designs. The cells flow along a serpentine channel that repeatedly flips the cells between a cathode and an anode at high throughput. The cells must flow through a constriction each time they pass from an anode to a cathode, exposing them to high electric field strength for short durations of time (the "pulse-width"). A conductive biocompatible poly-aniline hydrogel network formed in situ is used to apply the DC voltage without bringing the metal electrodes close to the cells, further sheltering cells from the already low voltage electrodes. The device was used to electroporate multiple cell lines using electric field strengths between 700 and 800 V/cm with transfection efficiencies superior than previous flow-through designs.

Dielectrophoresis based cell switching in continuous flow microfluidic devices

This article presents a novel negative-dielectrophoresis based approach for switching of a focused stream of micro-sized particles, including cells, to desired locations inside a continuous flow microfluidic device. The first section, of the device, focuses the incoming stream of micro-sized particles while the second section switches this focused stream of micro-sized particles. The microfluidic device consists of a glass substrate and a PDMS layer. The microfluidic device is realized using standard microfabrication. Tests are carried out using blood cells to demonstrate the efficacy of the approach in switching a stream of micro-sized particles to multiple locations inside the microchannel.

Formal Verification of a Microfluidic Device for Blood Cell Separation

Blood cell separation microdevices are designed in biomedical engineering for separation of cancer cells from blood. The movement of cancer cells particles in a continuous flow microfluidic device is a challenging problem since there are several forces incorporated. For instance, forces due to inertia, gravity, buoyancy, dielectrophoresis and virtual mass are accounted for in this system. Understanding the cell particle movement and behavior at high level of abstraction is necessary in order to avoid fundamental errors in the design of systems that can make use of this behavior. In this paper we use formal analysis in order to formalize and validate the movement of microparticles under DEP forces for blood cell separation microdevice. This is achieved by modeling the dynamic behavior that can predict the trajectory of microparticles as a transition state based system. The model is used to validate the correctness of the microdevice at early stages of the design process.

Model for tracing the path of microparticles in continuous flow microfluidic devices for 2D focusing via standing acoustic waves

An, experimentally validated, two-dimensional dynamic model for tracing the path of microparticles in a microfluidic layered transducer is developed. The model is based on Newton’s 2nd law and considers forces due to inertia, gravity, buoyancy, virtual mass and acoustics; it is solved using finite difference method. Microparticles’ trajectory consists of transient and steady state phases. All operating and geometric parameters are influential during the transient phase. The final levitation height is independent of the radius and initial vertical location of the microparticle as well as volumetric flow rate; however, dependent on the acoustic energy density and wavelength. There exists a threshold acoustic energy density for levitating microparticles from a specific initial vertical displacement; analytical equation for determining this acoustic energy density is provided.

Modeling the trajectory of microparticles subjected to dielectrophoresis in a microfluidic device for field flow fractionation

This article details the development of an experimentally validated model for tracking the movement of microparticles in a continuous flow microfluidic device employing dielectrophoresis for purposes of field-flow fractionation. This device employs interdigitated transducer electrodes on the bottom surface of the microchannel. The electric potential inside the microchannel is defined by Laplace equation while the trajectory of the microparticles is described by governing equations based on Newton’s second law. Forces due to inertia, gravity, buoyancy, dielectrophoresis and virtual mass are accounted for in this model. The governing equations are solved using finite difference method. The model is subsequently used for parametric study; the parameters analyzed include microparticle radius, applied voltage, volumetric flow rate, microchannel height and electrode/gap length. As per the model the levitation height, under steady state conditions, of the microparticles is independent of the microparticle radius, volumetric flow rate and microchannel height, it is dependent on the applied voltage and electrode/gap length. The levitation height, under transient conditions, is dependent on all these parameters.

Experimental validation of a simple, low-cost, T-junction droplet generator fabricated through 3D printing

Three-dimensional printing has been recently proposed and assessed for continuous flow microfluidic devices. In this paper the focus is on a new application of this rapid and low cost method for microfluidic device prototyping: droplets production through a T-junction generator. The feasibility of this new methodology is assessed by means of an experimental study in which the statistical parameters which characterize the production of droplets are analyzed. Furthermore, this study assesses the validity of previous theoretical and experimental results, obtained for a PDMS T-junction droplet generator, also in the case of a 3D printed Acrylonitrile microfluidic chip. Finally, the feasibility of producing monodisperse droplets by analyzing the polydispersity index of the prepared droplets is demonstrated.

Magnetophoretic-based microfluidic device for DNA isolation.

This paper presents a continuous flow microfluidic device for the separation of DNA from blood using magnetophoresis for biological applications and analysis. This microfluidic bio-separation device has several benefits, including decreased sample handling, smaller sample and reagent volumes, faster isolation time, and decreased cost to perform DNA isolation. One of the key features of this device is the use of short-range magnetic field gradients, generated by a micro-patterned nickel array on the bottom surface of the separation channel. In addition, the device utilizes an array of oppositely oriented, external permanent magnets to produce strong long-range field gradients at the interfaces between magnets, further increasing the effectiveness of the device. A comprehensive simulation is performed using COMSOL Multiphysics to study the effect of various parameters on the magnetic flux within the separation channel. Additionally, a microfluidic device is designed, fabricated, and tested to isolate DNA from blood. The results show that the device has the capability of separating DNA from a blood sample with a purity of 1.8 or higher, a yield of up to 33 μg of polymerase chain reaction ready DNA per milliliter of blood, and a volumetric throughput of up to 50 ml/h.

All-plastic, low-power, disposable, continuous-flow PCR chip with integrated microheaters for rapid DNA amplification

The design, fabrication and evaluation of a low-cost and low-power, continuous-flow microfluidic device for DNA amplification by polymerase chain reaction (PCR) with integrated heating elements, on a commercially available thin polymeric substrate (Pyralux® Polyimide), is presented. The small thermal mass of the chip, in combination with the low thermal diffusivity of the polymeric substrate on which the heating elements reside, yields a low power consumption PCR chip with fast amplification rates. A flow-through μPCR device is designed and fabricated using flexible printed circuit (FPC) technology on a foot-print area of 8cm×6cm with a meandering microchannel realized at a very small distance (50μm) above 3 independently operating resistive (copper) serpentine microheaters, each one defining one of the three PCR temperature zones. The 145cm-long microchannel is appropriately designed to cross the alternating temperature zones as many times as necessary for the DNA sample to perform 30 PCR cycles. Numerical computations lead the design so that there is no thermal crosstalk between the 3 zones of our chip and indicate excellent temperature uniformity in each zone. In addition, the total power consumption during the chip operation is calculated to be in the order of a few Watts, verified experimentally by means of thermal characterization of our heaters. Thermal camera measurements also verified the excellent temperature uniformity in the three thermal zones. An external, home-made temperature control system was utilized to maintain the heater temperatures in the designated values (±0.2°C). The PCR chip was validated by a successful amplification of a 90 base-pairs DNA template of the mouse GAPDH housekeeping gene within 5min.

A continuous-flow high-throughput microfluidic device for airborne bacteria PCR detection

Rapid analysis of airborne pathogens plays a critical role in early warning of spreading infectious diseases, which is essential for public health and disease prevention. Herein we report an integrated microfluidic device that can perform airborne pathogen capture, enrichment and continuous-flow high-throughput gene analysis. The device was validated by six frequently encountered bacteria and shows great potential in environmental analysis and for public health protection.

120 Deglycerolization and separation of human red blood cells in a microfluidic focusing channel

In current practice, removal of glycerol from frozen-thawed red blood cells (RBCs) is completed through a batch process that requires approximately 45 min per unit of blood. This process demands that the need for frozen RBCs be anticipated, and any unused deglycerolized units of blood be disposed of after a short post-thaw shelf life. Previous research in our lab demonstrated that rapid glycerol removal is possible using a multistep process involving exposure to solutions with decreasing concentrations of non-permeating solutes. This process maintains RBCs within their osmotic tolerance limits to minimize cell damage while enhancing the speed of glycerol removal. The ultimate goal is to perform the aforementioned rapid deglycerolization procedure in a continuous flow microfluidic device. The current research focuses on the first step of a proposed three-step deglycerolization process to demonstrate the ability to remove glycerol from a 40% w/v glycerolized human RBCs in a microfocusing device and to separate wash solution from cells at the outlet. The glycerolized cell stream is flowed between two 3270 mOsm saline wash streams allowing for a high contact area between the wash and cells for rapid diffusion of glycerol. The device separates RBCs from the wash solution at the exit of the device by taking advantage of the Fåhræus–Lindqvist effect in which blood cells tend to occupy the center of a microchannel creating a cell-free boundary layer. The device is able to skim off the cell-free layer with minimal collection of RBCs, creating an all in one passive deglycerolization device. In order to facilitate rapid prototyping and allow for cleaning and sterilization between runs, a device was fabricated by compressing a polyimide shim between two pieces of 21 cm× 5 cm× 0.95 cm acrylic with CNC milled inlet holes and through holes for compression screws. The polyimide shim was micromachined with a 200 μ m W × 127 μ m H × 14 cm L main channel from a CAD drawing, using a UV laser cutting tool. The optically transparent device allows for visualization of the flow for the entire length of the microchannel via microscopy and high speed imaging. Blood and wash solutions are flowed through the device and collected from their respective outlets for analysis. Syringe pumps feed the device with 3:1 ratio of wash to glycerolized blood. At the outlet, approximately 92% of the red blood cells exited the device in the expected blood collection channel, with the remaining 8% leaving with the wash streams. The wash stream represented 46% of the total volume out of the device. The difference in glycerol mass percent between the deglycerolized blood and wash output was approximately 30%, indicating substantial mass transfer into the wash solution during the short (∼12 s) transit time through the device. Further work is being done to improve the separation of wash solution from deglycerolized cells with additional separation branches as well as running multiple devices in series to carry out a complete multistep RBC deglycerolization process. Source of funding: National Science Foundation Grant#1150861. Conflict of interest: None declared. adam.higgins@oregonstate.edu

Continuous-Flow Microfluidic Electrochemical Synthesis: Investigating a New Tool for Oxidative Chemistry

A continuous-flow microfluidic electrochemical device (Flux Module) has been designed and evaluated as a practical new laboratory tool to facilitate electrochemical synthetic transformations. Four- and six-electron benzylic oxidations are reported to illustrate the utility afforded by a unique route of synthesis using this technology. Through the utilization of an electron-rich substrate (p-methoxytoluene), a continuous-flow electrochemical oxidation process was optimized. Using a general continuous-flow protocol, a series of diverse tolyl-based substrates were evaluated and the resulting data are reported. The Flux Module results were correlated with the oxidation potential of each substrate as measured by cyclic voltammetry. This established a trend regarding the nature of available oxidation product profiles using this synthesis platform.

Rapid dielectrophoretic characterization of single cells using the dielectrophoretic spring

Dielectrophoresis-based cell separation has significant promise for separation of cells from heterogeneous mixtures based on their electrical properties and is used in diverse areas ranging from hematopoietic stem cell purification to cancer cell isolation. The electrical properties of cells in heterogeneous populations determine if and how well cell subpopulations are separable, and therefore the utility of dielectrophoretic separation is fundamentally determined by our ability to measure electrical properties of cell populations on a cell-by-cell basis. We developed an automated system for electrical characterization of cells that can characterize 1000's of individual cells across a range of conditions (>30 conditions/h). The system uses a continuous-flow microfluidic device and a method termed the dielectrophoretic spring that uses the force balance between dielectrophoresis and fluid drag to measure electrical properties of cells independent of size. We present characterization of the method with beads and cells as well as its application to rapidly find conditions that can discriminate neutrophils with different activation states.

Kinetics screening of the N-alkylation of organic superbases using a continuous flow microfluidic device: basicity versus nucleophilicity

We describe here the determination of the alkylation rate of a set of organic superbases by iodomethane in DMF using a microfluidic continuous flow reactor. Surprisingly, log k(Alkylation) follows the inverse trend of pK(BH+) of the base. Mayr's equation allows a more quantitative approach. From a synthetic point of view, TMGN and BEMP are demonstrated to be the best choices.

Single-step radiofluorination of peptides using continuous flow microreactor

18F radiolabelling of peptides bearing two different prosthetic groups was successfully conducted in a continuous flow microfluidic device for the first time. Radiochemical yields were dependent on precursor concentration, reaction temperature and flow rate. The choice of leaving group had a dramatic influence on the reaction outcome. Rapid reaction optimization was possible.

A continuous flow μPCR device with integrated microheaters on a flexible polyimide substrate

The purpose of this work is to develop a simple and low cost microfluidic device with integrated microheaters on a thin flexible polymeric substrate, in order to perform DNA amplification on chip, at faster amplification rates due to the reduced thermal capacity and large heat transfer rate between the sample and the temperature-controlled heating zones. We fabricate a continuous-flow PCR microfluidic device, developed on a commercially available substrate (PyraluxTM) and we focus on the electrical and thermal characterization of the integrated microheaters. Each microheater defines one of the three temperature zones through which the DNA sample will be cycled for 25 times as it flows continuously through the microchannel. Electrical measurements confirm the functionality of the microheaters both as temperature sensors and thermal elements. Heat transfer and fluid dynamics computations are also performed to ascertain that the temperature of each microheater is almost identical to that in the microchannel crossing the corresponding thermal zone.

Optimal Conditions for Protein Array Deposition Using Continuous Flow

Optimal conditions for depositing protein microarrays using a continuous-flow microfluidic device, the continuous-flow microspotter (CFM), have been determined using a design of experiments approach. The amount of protein deposited on the surface depends on the rates of convective and diffusive transport to the surface and binding at the surface. These rates depend on parameters such as the flow rate, time, and capture mechanism at the surface. The process parameters were optimized, and uniform protein spots were obtained at a protein concentration of 10 microg/mL and even at 0.4 microg/mL. A 150-fold dilution in protein concentration in the sample solution decreased surface concentration by a factor of only 16. If the capture mechanism of the protein on the substrate is nonspecific, optimal deposition is obtained at higher flow rates for short periods of time. If the capture mechanism is specific, such as biotin-avidin, deposition is optimal at medium flow rates with little advantage beyond 30 min. The CFM can be used to deposit protein arrays with good spot morphology, spot-to-spot uniformity and enhanced surface concentration. The CFM was used to deposit an array of various antibodies, and their interactions with an antigen were studied using surface plasmon resonance (SPR). Affinity values were obtained at low antibody concentrations (5 microg/mL) with low coefficients of variation. Thus, the CFM can be used to effectively capture proteins and antibodies from dilute samples while depositing multiple spots, thereby increasing the quality of spots in protein microarrays and especially improving screening throughput of SPR.

From Microdroplets to Microfluidics: Selective Emulsion Separation in Microfluidic Devices

Microdroplets show great promise as a new high-throughput technology in chemistry, biochemistry, and molecular biology. Microdroplets can be generated at rates in excess of several thousands per second and accurately formulated using minute amounts of small molecules, DNA, proteins, or cells. Furthermore, integrated active elements can be used to control individual droplets. With the technology for creating, dividing, fusing, interrogating, and even sorting microdroplets already developed, one of the main problems to be resolved is how to access their contents. Droplets are naturally self-contained microreactors that prevent sample loss, diffusion, and cross-contamination, general issues that afflict traditional microfluidics. However, the isolated nature of droplets prevents physical access of their contents on-chip. Even though this does not represent a problem for many of the applications that have already been demonstrated, it limits the integration of microdroplets with other platforms. Analytical techniques such as mass spectrometry, capillary electrophoresis, and liquid chromatography have been successfully integrated with continuous-flow microfluidic devices, but their integration with microdroplets remains challenging. If the contents of microdroplets could be readily extracted on demand, the carrier fluid discarded, and the microdroplets converted into a continuous stream, microfluidic functionality could be combined with the advantages of microdroplets. In this paper we present a technology that bridges the fields of microdroplets and continuous-flow microfluidics by extracting on-chip the contents of microdroplets and incorporating them into a continuous stream. The extraction is achieved through electrocoalescence: droplets are forced to coalesce with an aqueous stream by application of an electric field across the channel. The extraction is controlled through the voltage applied at microfabricated electrodes on each side of the channel and can be performed in a continuous or discrete fashion. The discrete collection of droplets can be controlled by an external electrical signal related to the contents of the droplets. As a proof of principle, we have implemented a fluorescence intensity based detection system to control the collection of the droplets, resulting in a device capable of selectively incorporating the contents of droplets of interest to a continuous microfluidic stream. We used flow-focusing to generate microdroplets. An aqueous stream was focused between two oil streams as they pass through a junction. Shear forces make the aqueous thread break up into monodisperse droplets. Droplet size and frequency were controlled by a combination of channel dimensions and flow rates. We used a mixture of fluorous oil (FC-77) and 1H,1H,2H,2H-perfluorooctanol (70:30 by weight) as the carrier phase. The oil and aqueous flows at the flow-focusing device were adjusted to generate the desired droplet frequency, typically ranging from 10–250 Hz. The flow of the lateral aqueous phase was adjusted so an interface was held in the region between the electrodes without overflow in either direction. Figure 1a shows a scheme of a typical device where droplets flow parallel to a stream of water between two electrodes. In the absence of an electric field, the droplets are not perturbed by the presence of the aqueous stream and follow the geometrically determined flow lines. Figure 1b and c show micrographs of such a device in operation. Droplets of a dye generated at the flow-focusing device flow past the

The influence of magnetic carrier size on the performance of microfluidic integrated micro-electromagnetic traps

Efficient manipulation and capture of magnetic carriers in fluid stream require appropriate magnetic confinement devices whose performances are strongly dependent on the nature of the magnetic carriers. In this sense, we have performed a systematic investigation of the magnetic capture efficiencies for five commercially available superparamagnetic particles pumped along rectangular microfluidic channels using microelectromagnetic traps composed of planar circular current-carrying microwires and cylindrical ferromagnetic posts. In addition, in order to obtain a quantitative description of particle movement, we have implemented a numerical model for the dynamics of magnetic objects subjected to magnetic field gradients in conventional continuous-flow microfluidic devices. Fully 3D trajectories of the particles, effective cross-sectional areas of the microchannel as well as micro-electromagnet trapping efficiencies are compared to experimental measurements and a very good agreement is obtained. Finally, a simple and effective analytical model to determine the critical velocity, i.e. when the magnetic trapping device is no longer able to capture and hold 100% of the magnetic superparamagnetic particles, is also presented.

Magnetic nanocarriers: from material design to magnetic manipulation

Magnetic nanocarriers play an increasing role in various biomedical applications such as the separation of magnetically 'tagged' DNA, drug delivery or identification of biological species. Recent developments in nanotechnology allow the fabrication of both artificial nanocarriers and magnetic separation devices which may achieve great performances at an incredibly small-volume sample handling. However, as the sizes of magnetic carriers and separation devices diminish, important theoretical and experimental challenges may occur mainly due to the important variations of the surface-to-volume ratio. Under these circumstances, intrinsic properties of individual carriers and their influence on functionality and performances of the magnetic manipulation have to be investigated with a higher degree of accuracy. In this paper, we present the state-of-the-art techniques for extracting accurate information about individual magnetic entities from magnetic measurements performed on ordered arrays or clusters of magnetic nano-objects of various dimensionalities and geometries. As the mutual magnetic interactions may be responsible for collective effects, both analytical and numerical techniques for evaluating the mutual interactions between magnetic nanoparticles and nanowires are reviewed, special emphasis being put on the dimensionality of their assemblages. As the efficiency of the manipulation and capture of individual magnetic carriers in magnetic confinement devices depend strongly on their size, the theoretical description of the motion of magnetic particles under various conditions of flow and field configurations become very important especially with the transition to the nanoscale regime. The equations of motion of magnetic nanocarriers in continuous-flow microfluidic devices are reviewed and some solutions for superparamagnetic interacting clusters, non-interacting beads and ferromagnetic contiguous or codebar nanowires are presented. The influence of both carrier size and transition toward the nanoscale regime on the trappability of some magnetic nanocarriers is evaluated in terms of finite-length Navier boundary conditions and applied in order to compare the motion of superparamagnetic beads and ferromagnetic nanowires in conventional continuous-flow magnetic confinement devices.

Product differentiation during continuous-flow thermal gradient PCR

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.