A magnetophoresis-based microfluidic detection platform under a static-fluid environment

Microfluidic cell separations and immunoassays exploit a dynamic flow environment by electrical pumps to manipulate fluids containing biomolecules and microbeads. In particular, the magnetophoresis-based microfluidics requires a delicate flow control of pumps because the flow rate affects the result sensitively. Consequently, the dynamic flow environment requiring pumps prevents the magnetophoresis-based microfluidics from popularization and miniaturization. Herein, we present a magnetophoresis-based microfluidic platform under a static-fluid environment for the detection of microbeads labeled with magnetic nanoparticles (MNPs) by simple manual operation of fluids. To overcome the residual flow caused by the manual operation, we designed a microfluidic device having a pair of microchannels; one for detecting the target and the other for a reference. The deviations due to the residual flow were corrected by comparing the difference between the mean velocities of microbeads in each microchannel where microbeads labeled with five different concentrations of MNPs could be classified. On the basis of the convenience and portability of magnetophoresis under a static-fluidic environment, this new microfluidic platform enabled semiquantitative detection of labeled particles without any complex electrical devices and could thus be used as a portable detection platform.     

Modular component design for portable microfluidic devices

A new modular design concept for microfluidic devices is proposed and demonstrated in this study. We designed three key modular microfluidic components: pumps, valves, and reservoirs, and demonstrated that a microfluidic device with specific functions can be easily assembled with those key modular components. Our pumps are man-powerable so that the assembled microfluidic devices require no any other power sources like expensive syringe pumps or air compressors. This feature makes the assembled microfluidic devices completely portable. We also combined our assembled device with other existing mixing microchannels to serve as the mixing and loading system in polymerase chain reaction experiment to amplify DNA successfully. This result shows that those modular components can be integrated into other microchannels, implying great potential applications of the modular design.     

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.     

Localized substance delivery to single cell and 4D imaging of its uptake using a flow channel with a lateral aperture

We have developed a novel microfluidic device for space- and time-resolved (4D) visualization of intracellular events when a cell surface is partially exposed to external stimuli. The device, fabricated using 3D rotational inclined UV lithography of photoresist SU-8, consists of a cell-containing chamber and a flow channel separated by a thin vertical wall having a lateral micrometer aperture smaller than a cell. A cell is first immobilized on the aperture by suction from the flow channel using a syringe pump, and a chemical stimulant is then fed to the channel so that only the cell surface bounded by the aperture is subjected to the stimulus without leakage to other part of the cell surface. The subsequent lateral signal propagation inside the cell can be visualized using high-speed fluorescence confocal microscopy. As an experimental demonstration of the device, 2-NBDG (fluorescence glucose analog) intake into a mouse insulinoma cell, MIN6m9, was visualized in 4D resolution.     

The microscopy cell (MicCell), a versatile modular flowthrough system for cell biology, biomaterial research, and nanotechnology

We describe a novel microfluidic perfusion system for high-resolution microscopes. Its modular design allows pre-coating of the coverslip surface with reagents, biomolecules, or cells. A poly(dimethylsiloxane) (PDMS) layer is cast in a special molding station, using masters made by photolithography and dry etching of silicon or by photoresist patterning on glass or silicon. This channel system can be reused while the coverslip is exchanged between experiments. As normal fluidic connectors are used, the link to external, computer-programmable syringe pumps is standardized and various fluidic channel networks can be used in the same setup. The system can house hydrogel microvalves and microelectrodes close to the imaging area to control the influx of reaction partners. We present a range of applications, including single-molecule analysis by fluorescence correlation spectroscopy (FCS), manipulation of single molecules for nanostructuring by hydrodynamic flow fields or the action of motor proteins, generation of concentration gradients, trapping and stretching of live cells using optical fibers precisely mounted in the PDMS layer, and the integration of microelectrodes for actuation and sensing.     

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.     

集成惯性聚焦结构的介电泳微流控芯片的粒子连续分离

设计并制造了一种带有惯性聚焦结构的介电泳微流控芯片,以实现不同介电性质的粒子连续分离.采用MEMS工艺制作了介电泳微流控芯片:通道入口侧壁设置一对梯形结构使经过的粒子受惯性升力的作用聚焦到通道两侧;通道底部光刻一组夹角为90°的倾斜叉指电极产生非均匀电场,利用介电泳力和流体曳力的合力使通道两侧不同的粒子发生角度不同的偏转进入不同通道,从而实现分离.将酵母菌细胞和聚苯乙烯小球作为实验样本,分析了流速和交流电压对分离的影响,确定了二者分离的最优条件并进行分离.实验结果表明,将电导率为20μS/cm的样本溶液以5μL/min的流速注入到通道中,施加6 Vp-p、10 kHz的正弦信号,酵母菌细胞沿电极运动至夹角处后沿通道中心排出,聚苯乙烯小球沿通道两侧排出,成功实现分离,平均分离效率达92.8％、平均分离纯度达90.7％.     

Exploitation of a microfluidic device capable of generating size-tunable droplets for gene delivery

This study presents a new microfluidic chip that generates micro-scale emulsion droplets for gene delivery applications. Compared with conventional methods of droplet formation, the proposed chip can create uniform droplets (size variation <7.1%) and hence enhance the efficiency of the subsequent gene delivery. A new microfluidic chip was developed in this study, which used a new design with a pneumatic membrane chamber integrated into a T-junction microchannel. Traditionally, the size of droplets was controlled by the flow rate ratio of the continuous and disperse phase flows, which can be controlled by syringe pumps. In this study, a pneumatic chamber near the intersection of the T-junction channel was designed to locally change the flow velocity and the shear force. When the upper air chamber was filled with compressed air, the membrane was deflected and then the droplet size could be fine-tuned accordingly. Experimental data showed that using the new design, the higher the air pressure applied to the active tunable membrane, the smaller the droplet size. Finally, droplets were used as carriers for DNA to be transfected into the Cos-7 cells. It was also experimentally found that the size of the emulsion droplets plays an important role on the efficiency of the gene delivery. The preliminary results of this paper have been presented at the 2007 IEEE International Conference of Nano/Molecular Medicine and Engineering (IEEE NANOMED 2007), Macau, China, 6–9 August, 2007.     

Viscosity-difference-induced asymmetric selective focusing for large stroke particle separation

We developed a new approach for particle separation by introducing viscosity difference of the sheath flows to form an asymmetric focusing of sample particle flow. This approach relies on the high-velocity gradient in the asymmetric focusing of the particle flow to generate a lift force, which plays a dominated role in the particle separation. The larger particles migrate away from the original streamline to the side of the higher relative velocity, while the smaller particles remain close to the streamline. Under high-viscosity (glycerol–water solution) and low-viscosity (PBS) sheath flows, a significant large stroke separation between the smaller (1.0 μm) and larger (9.9 μm) particles was achieved in a sample microfluidic device. We demonstrate that the flow rate and the viscosity difference of the sheath flows have an impact on the interval distance of the particle separation that affects the collected purity and on the focusing distribution of the smaller particles that affects the collected concentration. The interval distance of 293 μm (relative to the channel width: 0.281) and the focusing distribution of 112 μm (relative to the channel width: 0.107) were obtained in the 1042-μm-width separation area of the device. This separation method proposed in our work can potentially be applied to biological and medical applications due to the wide interval distance and the narrow focusing distribution of the particle separation, by easy manufacturing in a simple device.     

Characterization of DNA hybridization kinetics in a microfluidic flow channel

In this investigation we report on the influence of volumetric flow rate, flow velocity, complementary DNA concentration, height of a microfluidic flow channel and time on DNA hybridization kinetics. A syringe pump was used to drive Cy3-labeled target DNA through a polydimethylsiloxane (PDMS) microfluidic flow channel to hybridize with immobilized DNA from the West Nile Virus. We demonstrate that a reduction of channel height, while keeping a fixed volumetric flow rate or a fixed flow velocity, enhances mass transport of target DNA to the capture probes. Compared to a passive hybridization, the DNA hybridization in the microfluidic flow channel generates higher fluorescence intensities for lower concentration of target DNA during the same fixed period of time. Within a fixed 2 min time period the fastest DNA hybridization at a 50 pM concentration of target DNA is achieved with a continuous flow of target DNA at the highest flow rate and the lowest channel height.     

Circuit model for microfluidic bubble generation under controlled pressure

We explore the microfluidic generation of bubbles in a flow-focusing junction using a pressure-controlled device rather than the more common flow rate-controlled devices. This device is a prototype for extending microfluidic drop generation methods to molten polymers. We show that the bubble generation process is highly sensitive to pressure: small changes in pressure induce large changes in bubble size and bubble formation frequency. A simple resistance circuit model can explain this pressure dependence. Briefly, we show that bubble generation is possible only within a finite pressure range. Near the ends of this pressure range, the ratio of the flow rates of the dispersed to continuous phase is highly sensitive to pressure, and therefore so also is the bubble generation process. The circuit model offers a way to use existing models of drop generation (which are based on flow rate-controlled operation) to predict pressure-controlled operation. We also examine drop formation using a highly viscous polymer as the dispersed phase. Drops are formed far downstream of the flow-focusing junction, and they are far smaller than the microfluidic channel dimensions. These results suggest that existing microfluidic drop generation methods may be exploited to make complex particles from thermoplastic polymers.     

Computational and performance analysis of a continuous magnetophoretic bioseparation chip with alternating magnetic fields

This paper presents the modeling and optimization of a magnetophoretic bioseparation chip for isolating cells, such as circulating tumor cells from the peripheral blood. The chip consists of a continuous-flow microfluidic platform that contains locally engineered magnetic field gradients. The high-gradient magnetic field produced by the magnets is spatially non-uniform and gives rise to an attractive force on magnetic particles flowing through a fluidic channel. Simulations of the particle–fluid transport and the magnetic force are performed to predict the trajectories and capture lengths of the particles within the fluidic channel. The computational model takes into account key forces, such as the magnetic and fluidic forces and their effect on design parameters for an effective separation. The results show that the microfluidic device has the capability of separating various cells from their native environment. An experimental study is also conducted to verify and validate the simulation results. Finally, to improve the performance of the separation device, a parametric study is performed to investigate the effects of the magnetic bead size, cell size, number of beads per cell, and flow rate on the cell separation performance.     

Microfluidic counterflow centrifugal elutriation system for sedimentation-based cell separation

We present a centrifugal microfluidic system for precise cell/particle sorting using the concept of counterflow centrifugal elutriation (CCE). A conventional CCE system uses a rotor device incorporating a flow-through separation chamber, in which the balance of centrifugal and counterflow drag forces exerted on particles is gradually shifted by changing the flow rate and/or the rotation speed. In the present system, both the centrifugal and the fluid forces are generated through microdevice rotation in order to significantly simplify the setup of the conventional CCE. In addition, the density gradient of the medium is employed to elute particles/cells of different sedimentation velocities stepwise from the separation chamber instead of changing the rotation speed. We successfully separated polymer particles with diameters of 1.0–5.0 μm using a branched loading channel for focusing particles to the center of the separation chamber. We also demonstrated the sorting of blood cells for biological applications. This system may provide a versatile means for cell/particle sorting in a general biological laboratory and function as a unit operation in various centrifugal microfluidic platforms for biochemical experiments and clinical diagnosis.     

Highly efficient dual-channel cytometric-detection of micron-sized particles in microfluidic device

To demonstrate the ability to efficiently count and identify suspended micron-sized particles by simultaneously detecting their fluorescence emission and light scattering in microfabricated channel, a compact configuration that used a polydimethylsiloxane (PDMS) microfabricated channel as interrogation component, hydrodynamic focusing for particle control, and a simple free-space optical setup for signal detection, was accordingly developed. Subsequently, a quantitative count of 1.013 μm diameter fluorescently labeled beads in suspension was implemented in a microfluidic device employing both fluorescence emission and light scattering at average particle throughput ranging from 83 to 416 particles/s. As a result, the detection efficiencies above 88% for both signals and correlation percentages above 97% between them were routinely achieved. In addition, it was shown that effective differentiation of 1.013 μm fluorescently labeled beads from various unlabeled beads in mixed populations of high mixing ratios had been successfully realized in this microfluidic-device-based instrumentation. Finally, the demonstrated system was used to detect fluorescein isothiocyanate (FITC) labeled nonpathogenic bacteria of Escherichia coli (E. coli) DH5α. The results showed the detection efficiencies above 89.7% for fluorescence emission and 94.5% for light scattering signals, and a correlation of 94.9% between the two signals at an average throughput of 350 cells/s have been obtained. As a comparison, the detection accuracies of the dual-channel cytometric detection of the FITC-labeled E. coli DH5α cells in the microfluidic device are approximately 84.3% and 88.8% for fluorescence emission and light scattering respectively when compared against a manual cell count using a haemocytometer as a standard.     

Microfluidic separation of magnetic particles with soft magnetic microstructures

This paper demonstrates simple and cost-effective microfluidic devices for enhanced separation of magnetic particles by using soft magnetic microstructures. By injecting a mixture of iron powder and polydimethylsiloxane (PDMS) into a prefabricated channel, an iron–PDMS microstructure was fabricated next to a microfluidic channel. Placed between two external permanent magnets, the magnetized iron–PDMS microstructure induces localized and strong forces on the magnetic particles in the direction perpendicular to the fluid flow. Due to the small distance between the microstructure and the fluid channel, the localized large magnetic field gradients result a vertical force on the magnetic particles, leading to enhanced separation of the particles. Numerical simulations were developed to compute the particle trajectories and agreed well with experimental data. Systematic experiments and numerical simulation were conducted to study the effect of relevant factors on the transport of superparamagnetic particles, including the shape of iron–PDMS microstructure, mass ratio of iron–PDMS composite, width of the microfluidic channel, and average flow velocity.     

Thin-film electrode based droplet detection for microfluidic systems

We report on a droplet-producing microfluidic system with electrical impedance-based detection. The microfluidic devices are made of polydimethylsiloxane (PDMS) and glass with thin film electrodes connected to an impedance-monitoring circuit. Immiscible fluids containing the hydrophobic and hydrophilic phases are injected with syringe pumps and spontaneously break into water-in-oil droplet trains. When a droplet passes between a pair of electrodes in a medium having different electrical conductivity, the resulting impedance change signals the presence of the particle for closed-loop feedback during processing. The circuit produces a digital pulse for input into a computer control system. The droplet detector allows estimation of a droplet's arrival time at the microfluidic chip outlet for dispensing applications. Droplet detection is required in applications that count, sort, and direct microfluidic droplets. Because of their low cost and simplicity, microelectrode-based droplet detection techniques should find applications in digital microfluidics and in three-dimensional printing technology for rapid prototyping and biotechnology.     

Orbiting magnetic microbeads enable rapid microfluidic mixing

We use three-dimensional numerical simulations and experiments to examine microfluidic mixing induced by orbiting magnetic microbeads in a microfluidic channel. We show that orbiting microbeads can lead to rapid fluid mixing in low Reynolds number flow, and identify two distinct mixing mechanisms. Bulk advection of fluid across the channel occurs due to the flow pattern that is developed when the ratio of flow velocity to bead velocity is low, and leads to rapid mixing. At higher velocity ratios, dispersion of small amounts of fluid across the channel occurs and results in increased mixing. We use simulations to investigate the effect of system parameters on the distance required to achieve a desired mixing level. We develop an experimental continuous-flow device and use it to validate our simulations and to demonstrate rapid microfluidic mixing. This device has the flexibility to also be applied to a mixing chamber or to stop-flow applications for rapid and controllable mixing. In addition to rapid mixing, the use of orbiting magnetic microbeads has the added benefit that functionalized microbeads can be used to capture particles from the fluid solution during mixing, and that they can be extracted from the device for analysis, thus serving multiple functionalities in a single device.     

Miniaturized DNA amplification platform with soft-lithographically fabricated continuous-flow PCR microfluidic device on a portable temperature controller

Fabrication of 3D microfluidic devices is normally quite expensive and tedious. A strategy was established to rapidly and effectively produce multilayer 3D microfluidic chips which are made of two layers of poly(methyl methacrylate) (PMMA) sheets and three layers of double-sided pressure sensitive adhesive (PSA) tapes. The channel structures were cut in each layer by cutting plotter before assembly. The structured channels were covered by a PMMA sheet on top and a PMMA carrier which contained threads to connect with tubing. A large variety of PMMA slides and PSA tapes can easily be designed and cut with the help of a cutting plotter. The microfluidic chip was manually assembled by a simple lamination process.The complete fabrication process from device design concept to working device can be completed in minutes without the need of expensive equipment such as laser, thermal lamination, and cleanroom. This rapid frabrication method was applied for design of a 3D hydrodynamic focusing device for synthesis of gold nanoparticles (AuNPs) as proof-of-concept. The fouling of AuNPs was prevented by means of a sheath flow. Different parameters such as flow rate and concentration of reagents were controlled to achieve AuNPs of various sizes. The sheet-based fabrication method offers a possibility to create complex microfluidic devices in a rapid, cheap and easy way.

     

Development of a biological detection platform utilizing a modular microfluidic stack

The goal of this project is to build a miniaturized, user-friendly cytometry setup (Datta et al. in Microfluidic platform for education and research. COMS, Baton Rouge, 2008; Frische et al. in Development of an miniaturized flow cytometry setup for visual cell inspection and sorting. Baton Rouge, Project Report, 2008) by combining a customized, microfluidic device with visual microscope inspection to detect and extract specific cells from a continuous sample flow. We developed a cytological tool, based on the Coulter particle counter principle, using a microelectrode array patterned on a borosilicate glass chip as electrical detection set-up which is fully embedded into a polymeric multi-layer microfluidic stack. The detection takes place between pairs of coplanar Cr/Au microelectrodes by sensing an impedance change caused by particles continuously carried within a microfluidic channel across the detection area under laminar flow conditions. A wide frequency range available for counting provides information on cell size, membrane capacitance, cytoplasm conductivity and is potentially of interest for in-depth cell diagnostic e.g. to detect damaged or cancerous cells and select them for extraction and further in-depth analysis.     

A microfluidic device for rapid concentration of particles in continuous flow by DC dielectrophoresis

This article presents a microfluidic device (so called concentrator) for rapid and efficient concentration of micro/nanoparticles using direct current dielectrophoresis (DC DEP) in continuous fluid flow. The concentrator is composed of a series of microchannels constructed with PDMS-insulating microstructures to focus efficiently the electric field in the flow direction to provide high field strength and gradient. Multiple trapping regions are formed within the concentrator. The location of particle trapping depends on the strength of the electric field applied. Under the experimental conditions, both streaming movement and DEP trapping of particles simultaneously take place within the concentrator at different regions. The former occurs upstream and is responsible for continuous transport of the particles, whereas the latter occurs downstream and rapidly traps the particles delivered from upstream. The observation agrees with the distribution of the simulated electric field and DEP force. The performance of the device is demonstrated by successfully and effectively concentrating fluorescent nanoparticles. At the sufficiently high electric field, the device demonstrates a trapping efficiency of 100%, which means downstream DEP traps and concentrates all (100%) the incoming particles from upstream. The trapping efficiency of the device is further studied by measuring the fluorescence intensity of concentrated particles in the channel. Typically, the fluorescence intensity becomes saturated in Trap 1 by applying the voltage (400 V) for >2 min, demonstrating that rapid concentration of the nanoparticles (10⁷ particles/ml) is achieved in the device. The microfluidic concentrator described can be implemented in applications where rapid concentration of targets is needed such as concentrating cells for sample preparation and concentrating molecular biomarkers for detection.