Continuous microfluidic DNA extraction using phase-transfer magnetophoresis

This paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 μl s(-1) and an eluate flow rate of 0.35 μl s(-1). DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.     

Microfluidic magnetophoretic separations of immunomagnetically labeled rare mammalian cells

Immunomagnetic isolation and magnetophoresis in microfluidics have emerged as viable techniques for the separation, fractionation, and enrichment of rare cells. Here we present the development and characterization of a microfluidic system that incorporates an angled permanent magnet for the lateral magnetophoresis of superparamagnetic beads and labeled cell-bead complexes. A numerical model, based on the relevant transport processes, is developed as a design tool for the demonstration and prediction of magnetophoretic displacement. We employ a dimensionless magnetophoresis parameter to efficiently investigate the design space, gain insight into the physics of the system, and compare results across the vast spectrum of magnetophoretic microfluidic systems. The numerical model and theoretical analysis are experimentally validated by the lateral magnetophoretic deflection of superparamagnetic beads and magnetically labeled breast adenocarcinoma MCF-7 cells in a microfluidic device that incorporates a permanent magnet angled relative to the flow. Through the dimensionless magnetophoresis parameter, the transition between regimes of magnetophoretic action, from hydrodynamically dominated (magnetic deflection) to magnetically dominated (magnetic capture), is experimentally identified. This powerful tool and theoretical framework enables efficient device and experiment design of biologically relevant systems, taking into account their inherent variability and labeling distributions. This analysis identifies the necessary beads, magnet configuration (orientation), magnet type (permanent, ferromagnetic, electromagnet), flow rate, channel geometry, and buffer to achieve the desired level of magnetophoretic deflection or capture.     

Kinetics of the phase separation of a low-viscosity liquid supersaturated with gas at the intermediate and later stages

The separation kinetics of a low-viscosity liquid supersaturated with gas is studied. It is shown that the number of bubbles per unit volume at the intermediate stage of the process remains virtually constant, whereas the amount of the gas per unit volume of the solution significantly decreases, almost attaining the equilibrium value. At the later stage, small subcritical bubbles disappear due to gas transfer to large supercritical bubbles; as a result, the number of bubbles in the virtually equilibrium (as to the dissolved gas) liquid decreases. For all the stages, the kinetics of the changes in the bubble distribution function and in the amount of gas per unit volume of the solution is determined.Translated from Kolloidnyi Zhurnal, Vol. 67, No. 1, 2005, pp. 94–105.Original Russian Text Copyright © 2005 by Slezov, Abyzov, Slezova.     

Mixing enhancement for high viscous fluids in a microfluidic chamber

Due to small channel dimensions and laminar flows, mixing in microfluidic systems is always a challenging task, especially for high viscous fluids. Here we report a method of enhancing microfluidic mixing for high viscous fluids using acoustically induced bubbles. The bubbles can be generated in an acoustically profiled microfluidic structure by using a piezoelectric disk activated at a working frequency range between 1.5 kHz and 2 kHz. The mixing enhancement is achieved through interactions between the oscillating bubbles and fluids. Both experimental studies and numerical simulations are conducted. In the experiments, DI water-glycerol mixture solutions with various viscosities were used. The results, based on the mixing efficiency calculated from experimentally acquired fluorescent images, showed that good mixing can occur in the DI water-glycerol solutions with their maximum viscosity up to 44.75 mPa s, which to our best knowledge is the highest viscosity of fluids in microfluidic mixing experiments. To explain the mechanisms of bubble generation, the numerical simulation results show that, corresponding to the actuations at the working frequency range used in the experiment, there exists a low pressure region where the pressure is lower than the water vapor pressure in the DI water-glycerol solutions, resulting in the generation of bubbles.     

Aeration of emulsions by whipping

During aeration of food emulsions such as dairy cream and ice cream, small gas bubbles are introduced, which are often stabilized by a layer of adsorbed emulsion droplets. It is shown that the maximum achievable volume of gas bubbles that can be incorporated by whipping depends on the effectiveness of the introduction of gas during the first stage of whipping and is furthermore limited by packing constraints. The main factors relevant for the latter limitation are the thickness of the coating of emulsion droplets at the bubble surface, the ratio between the droplet and bubble radii, and the fat content of the emulsion. It is hypothesized that, during whipping, a dynamic process of bubble break-up and coalescence adjusts the average bubble size and the volume of gas incorporated in the foam to the constraint of close packing of the bubbles. The consequences of this mechanism for whipping of emulsions are discussed.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

Bubbles navigating through networks of microchannels

This paper describes the behavior of bubbles suspended in a carrier liquid and moving within microfluidic networks of different connectivities. A single-phase continuum fluid, when flowing in a network of channels, partitions itself among all possible paths connecting the inlet and outlet. The flow rates along different paths are determined by the interaction between the fluid and the global structure of the network. That is, the distribution of flows depends on the fluidic resistances of all channels of the network. The movement of bubbles of gas, or droplets of liquid, suspended in a liquid can be quite different from the movement of a single-phase liquid, especially when they have sizes slightly larger than the channels, so that the bubbles (or droplets) contribute to the fluidic resistance of a channel when they are transiting it. This paper examines bubbles in this size range; in the size range examined, the bubbles are discrete and do not divide at junctions. As a consequence, a single bubble traverses only one of the possible paths through the network, and makes a sequence of binary choices ("left" or "right") at each branching intersection it encounters. We designed networks so that, at each junction, a bubble enters the channel into which the volumetric flow rate of the carrier liquid is highest. When there is only a single bubble inside a network at a time, the path taken by the bubble is, counter-intuitively, not necessarily the shortest or the fastest connecting the inlet and outlet. When a small number of bubbles move simultaneously through a network, they interact with one another by modifying fluidic resistances and flows in a time dependent manner; such groups of bubbles show very complex behaviors. When a large number of bubbles (sufficiently large that the volume of the bubbles occupies a significant fraction of the volume of the network) flow simultaneously through a network, however, the collective behavior of bubbles-the fluxes of bubbles through different paths of the network-can resemble the distribution of flows of a single-phase fluid.     

A membrane-based, high-efficiency, microfluidic debubbler

In many lab-on-chip applications, it is necessary to remove bubbles from the flow stream. Existing bubble removal strategies have various drawbacks such as low degassing efficiency, long degassing time, large dead volumes, sensitivity to surfactants, and the need for an external vacuum or pressure source. We report on a novel, simple, robust, passive, nozzle-type, membrane-based debubbler that can be readily incorporated into microfluidic devices for rapid degassing. The debubbler is particularly suitable to operate with microfluidic systems made with plastic. The debubbler consists of a hydrophobic, porous membrane that resembles a normally closed valve, which is forced open by the working fluid's pressure. To illustrate the operation of the debubbler, we describe its use in the context of a chip containing a bead array for immunoassays. Our debubbler was able to completely filter gas bubbles out of a segmented flow at rates up to 60 μl s(-1) mm(-2) of membrane area.     

Enzymatically-generated fluorescent detection in micro-channels with internal magnetic mixing for the development of parallel microfluidic ELISA

The Enzyme-Linked Immuno-Sorbent Assay, or ELISA, is commonly utilized to quantify small concentrations of specific proteins for a large variety of purposes, ranging from medical diagnosis to environmental analysis and food safety. However, this technique requires large volumes of costly reagents and long incubation periods. The use of microfluidics permits one to specifically address these drawbacks by decreasing both the volume and the distance of diffusion inside the micro-channels. Existing microfluidic systems are limited by the necessary control of extremely low flow rates to provide sufficient time for the molecules to interact with each other by diffusion only. In this paper, we describe a new microfluidic design for the realization of parallel ELISA in stop-flow conditions. Magnetic beads were used both as a solid phase to support the formation of the reactive immune complex and to achieve a magnetic mixing inside the channels. In order to test the detection procedure, the formation of the immune complex was performed off-chip before the reactive beads were injected into the reaction chamber. Anti-streptavidin antibodies were quantified with low picomolar sensitivity (0.1-6.7 pM), a linear range of 2 orders of magnitude and good reproducibility. This work represents the first step toward a new platform for simple, highly effective and parallel microfluidic ELISA.     

Analysis of pressure-driven air bubble elimination in a microfluidic device

We report an analysis of pressure-driven bubble elimination for a gas-permeable microfluidic device. In this study, we described bubble elimination in a microfluidic device employing a gas permeation model and calculated the removal efficiency of bubbles. The correction factor for the simplified model was estimated with respect to the applied pressure. Based on the established model, the required time to remove a trapped bubble with a certain area was shown to be within an error of 11.58% by comparison with experimental results. Exploiting the model equation, we were able to completely remove the air bubbles appearing during the process of filling a microfluidic device with an aqueous solution.     

Bead magnetorelaxometry with an on-chip magnetoresistive sensor

Magnetorelaxometry measurements on suspensions of magnetic beads are demonstrated using a planar Hall effect sensor chip embedded in a microfluidic system. The alternating magnetic field used for magnetizing the beads is provided by the sensor bias current and the complex magnetic susceptibility spectra are recorded as the 2nd harmonic of the sensor response. The complex magnetic susceptibility signal appears when a magnetic bead suspension is injected, it scales with the bead concentration, and it follows the Cole-Cole expression for Brownian relaxation. The complex magnetic susceptibility signal resembles that from conventional magnetorelaxometry done on the same samples apart from an offset in Brownian relaxation frequency. The time dependence of the signal can be rationalized as originating from sedimented beads.     

Gas Holdups of Small and Large Bubbles in a Large-scale Bubble Column with Elevated Pressure

Gas holdups of large bubbles and small bubbles were measured by means of dynamic gas disengagement approach in the pressured bubble column with a diameter of 0. 3 m and a height of 6.6 m. The effects of su-perficial gas velocity, liquid surface tension, liquid viscosity and system pressure on gas holdups of small bub-bles and large bubbles were investigated. The holdup of large bubbles increases and the holdup of small bub-bles decreases with an increase of liquid viscosity. Meanwhile, the holdup of large bubbles decreases with in-creasing the system pressure. A correlation for the holdup of small bubbles was obtained from the experimen-tal data.     

Coated gas bubbles for the continuous synthesis of hollow inorganic particles

We present a microfluidic approach for the controlled encapsulation of individual gas bubbles in micrometer-diameter aqueous droplets with high gas volume fractions and demonstrate this approach to making a liquid shell, which serves as a template for the synthesis of hollow inorganic particles. In particular, we find that an increase in the viscosity of the aqueous phase facilitates the encapsulation of individual gas bubbles in an aqueous droplet and allows control of the thickness of a thin aqueous shell. Furthermore, because such droplets contain a finite amount of water, uncontrolled hydrolysis reactions between reactive inorganic precursors and bulk water can be avoided. We demonstrate this approach by introducing reactive inorganic precursors, such as silane and titanium butoxide, for sol-gel reactions downstream from the formation of the bubble in a droplet and consequently fabricate hollow particles of silica or titania in one continuous flow process. These approaches provide a route to controlling double-emulsion-type gas-liquid microstructures and offer a new fabrication method for thin-shell-covered microbubbles and hollow microparticles.     

Rapid purification of cell encapsulated hydrogel beads from oil phase to aqueous phase in a microfluidic device

In this paper, we demonstrate a new type of microfluidic chip that can realize continuous-flow purification of hydrogel beads from a carrier oil into aqueous solution by using a laminar-like oil/water interface. The microfluidic chip is composed by two functional components: (1) a flow-focusing bead generation module that can control size and shape of beads, (2) a bead extraction module capable of purifying hydrogel beads from oil into aqueous solution. This module is featured with large branch channels on one side and small ones on the opposite side. Water is continuously infused into the bead extraction module through the large branch channels, resulting in a laminar-like oil/water interface between the branch junctions. Simulation and experimental data show that the efficiency of oil depletion is determined by the relative flow rates between infused water and carrier oil. By using such a microfluidic device, viable cells (HCT116, colon cancer cell line) can be encapsulated in the hydrogel beads and purified into a cell culture media. Significantly improved cell viability was achieved compared to that observed by conventional bead purification approaches. This facile microfluidic chip could be a promising candidate for sample treatment in lab-on-a-chip applications.     

Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems

The present study reports a new three-dimensional (3D) microfluidic platform capable of rapid isolation and detection of cancer cells from a large sample volume (e.g. ~1 mL) by utilizing magnetic microbead-based technologies. Several modules, including a 3D microfluidic incubator for the magnetic beads to capture cancer cells, a microfluidic control module for sample transportation and a nucleic acid amplification module for genetic identification, are integrated into this microsystem. With the incorporation of surface-modified magnetic beads, target cancer cells can be specifically recognized and conjugated onto the surface of the antibody-coated magnetic microbeads by utilizing a swirling effect generated by the new 3D microfluidic incubator, followed by isolating and purifying the magnetic complexes via the incorporation of an external magnet and a microfluidic control module, which washes away any unbound waste solution. Experimental results show that over 90% of the target cancer cells can be isolated from a large volume of bio-samples within 10 min in the 3D microfluidic incubator. In addition, the expressed genes associated with ovarian and lung cancer cells can also be successfully amplified by using the on-chip nucleic acid amplification module. More importantly, the detection limit of the developed system is found to be 5 × 10(1) cells mL(-1) for the target cancer cells, indicating that this proposed microfluidic system may be adapted for clinical use for the early detection of cancer cells. Consequently, the proposed 3D microfluidic system incorporated with immunomagnetic beads may provide a promising automated platform for the rapid isolation and detection of cancer cells with a high sensitivity.     

Dissolution of carbon dioxide bubbles and microfluidic multiphase flows

We experimentally study the dissolution of carbon dioxide bubbles into common liquids (water, ethanol, and methanol) using microfluidic devices. Elongated bubbles are individually produced using a hydrodynamic focusing section into a compact microchannel. The initial bubble size is determined based on the fluid volumetric flow rates of injection and the channel geometry. By contrast, the bubble dissolution rate is found to depend on the inlet gas pressure and the fluid pair composition. For short periods of time after the fluids initial contact, the bubble length decreases linearly with time. We show that the initial rate of bubble shrinkage is proportional to the ratio of the diffusion coefficient and the Henry's law constant associated with each fluid pair. Our study shows the possibility to rapidly impregnate liquids with CO(2) over short distances using microfluidic technology.     

Current status of membraneless water electrolysis cells

Commercial water electrolysis cells require a resistive, ion-permeable, gas-impermeable separator membrane between the electrodes to stop the hydrogen bubbles from mixing with the oxygen bubbles and vice versa. This work reviews the current status of ‘membraneless’ water electrolysis cells that safely avoid need for such a separator membrane. Three different approaches have been used to realize such cells. In the first approach, laminar flow within a microfluidic reaction chamber has been used to entrain the hydrogen and oxygen gas bubbles in separate, parallel streams that do not mix. In the second approach, closely-spaced porous electrodes have had liquid electrolyte divergently pumped through them to sweep the produced hydrogen and oxygen bubbles to different locations. In the most recent, promising approach, gas diffusion electrodes have been used to directly extract gas as it is produced, thereby avoiding discernible bubble formation and eliminating the need for a separator membrane to keep the gases separate.     

Digital droplet immunoassay based on a microfluidic chip with magnetic beads for the detection of prostate-specific antigen

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL.     

An optimal design method for preventing air bubbles in high-temperature microfluidic devices

DNA analysis with the polymerase chain reaction (PCR) has become a routine part of medical diagnostics, environmental inspections, food evaluations, and biological studies. Furthermore, the development of a microscale PCR chip is an essential component of studies aimed at integrating PCR into a micro total analysis system (μ-TAS). However, the occurrence of air bubbles in microchannels complicates this process. In this study, we investigated a new technique based on the fluid dynamics of laminar flow that utilizes a small amount of mineral oil at the beginning of sample injection to prevent air bubbles from occurring in microchannels. We also further optimized the pressure, the length of the pressurizing channel and the volume of oil, thus making our microfluidic device more useful for high-temperature PCR. Additionally, quantitative continuous-flow PCR was performed using the optimized PCR chip in order to detect genetically modified (GM) maize. DNA was extracted from GM maize, MON 810, and non-GM maize at several concentrations from 0% (w/v) to 100% (w/v). The DNA amplification signals were then analyzed on the PCR chip using a laser-based system. The signal from our microfluidic PCR chip was found to increase in direct proportion to the initial GM maize concentration.     

Bubbles no more: in-plane trapping and removal of bubbles in microfluidic devices

Gas bubbles present a frequent challenge to the on-chip investigation and culture of biological cells and small organs. The presence of a single bubble can adversely impair biological function and often viability as it increases the wall shear stress in a liquid-perfused microchannel by at least one order of magnitude. We present a microfluidic strategy for in-plane trapping and removal of gas bubbles with volumes of 0.1-500 nL. The presented bubble trap is compatible with single-layer soft lithography and requires a footprint of less than ten square millimetres. Nitrogen bubbles were consistently removed at a rate of 0.14 μL min(-1). Experiments were complemented with analytical and numerical models to comprehensively characterize bubble removal for liquids with different wetting behaviour. Consistent long-term operation of the bubble trap was demonstrated by removing approximately 4000 bubbles during one day. In a case study, we successfully applied the bubble trap to the on-chip investigation of intact small blood vessels. Scalability of the design was demonstrated by realizing eight parallel traps at a total removal rate of 0.9 μL min(-1) (measured for nitrogen).