Control of flow rate and concentration in microchannel branches by induced-charge electrokinetic flow

This paper presents a numerical study of controlling the flow rate and the concentration in a microchannel network by utilizing induced-charge electrokinetic flow (ICEKF). ICEKF over an electrically conducting surface in a microchannel will generate vortices, which can be used to adjust the flow rates and the concentrations in different microchannel branches. The flow field and concentration field were studied under different applied electric fields and with different sizes of the conducting surfaces. The results show that, by using appropriate size of the conducting surfaces in appropriate locations, the microfluidic system can generate not only streams of the same flow rate or linearly decreased flow rates in different channels, but also different, uniform concentrations within a short mixing length quickly.     

Microfluidic pool structure for cell docking and rapid mixing

A microfluidic pool structure for cell docking and rapid mixing is described. The pool structure is defined as a microchamber on one structural layer of a bilayer chip and connects with two or more individual microchannels on the other structural layer. In contrast to the turbulent flow in a macroscale pool, laminar streams enter and exit this microfluidic pool structure with definite and controllable direction that may be influenced by the location and geometry of the pool. A simple microfluidic model was used to validate this hypothesis. In this model, a microscale pool structure was made on the lower layer of a chip and connected with three parallel microchannels in the upper layer. Simulation and experimental results indicated that the flow profile within the pool structure was determined by its geometry and location. This could be used as a flow control method and it was simpler than designs based on microvalve, hydraulic pressure, or electrokinetic force, and has some important applications. For example, controllable streams within this structure were used to immobilize biological cells along the microchannel walls. When different solution streams flowed through the pool, rapid diffusion of analytes occurred for short diffusion distance between vertical flow laminas. Furthermore, desired dilution (mixing) ratio could be obtained by controlling the geometry of the microfluidic pool.     

Measurements of scattered light on a microchip flow cytometer with integrated polymer based optical elements

Flow cytometry is widely used for analyzing microparticles, such as cells and bacteria. In this paper, we report an innovative microsystem, in which several different optical elements (waveguides, lens and fiber-to-waveguide couplers) are integrated with microfluidic channels to form a complete microchip flow cytometer. All the optical elements, the microfluidic system, and the fiber-to-waveguide couplers were defined in one layer of polymer (SU-8, negative photoresist) by standard photolithography. With only a single mask procedure required, all the fabrication and packaging processes can be finished in one day. Polystyrene beads were measured in the microchip flow cytometer, and three signals (forward scattering, large angle scattering and extinction) were measured simultaneously for each bead. To our knowledge this is the first time forward scattered light and incident light extinction were measured in a microsystem using integrated optics. The microsystem can be applied for analyzing different kinds of particles and cells, and can easily be integrated with other microfluidic components.     

Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation

This paper describes the use of arrays of horizontally-oriented reservoirs to deliver liquids through microchannels at a constant flow rate over extended periods of time (hours to days). The horizontal orientation maintains a constant hydraulic pressure drop across microfluidic channels even as the volumes of liquids within the reservoirs change over time. For a given channel-reservoir system, the magnitude of the flow velocity depends linearly on the height difference between reservoirs. The simple structure and operation mechanism make this pumping system versatile. A one-inlet-one-outlet system was used to continuously deliver media for perfusion cell culture, and an array of inlet reservoirs coupled to an outlet reservoir via microchannels was used to drive flows of multiple laminar streams. The parallel pumping scheme conveniently generated various smooth and step concentration gradients, and allowed evaluation of the effect of colchicine on myoblasts. Since the reservoir arrays are configured to be compatible with commercialized multichannel pipettors designed for 96 well plate handling, this simple pumping scheme is envisioned to be broadly useful for medium to high throughput microfluidic perfusion cell culture assays, cell migration assays, multiple laminar flow drug tests, and any other applications needing multiple microfluidic streams.     

Velocity-independent microfluidic flow cytometry

Pressure-driven flow in microfluidic channels is characterized by a distribution of velocities. This distribution makes it difficult to implement conventional flow cytometry data analysis. We have demonstrated a method to measure velocity as an independent parameter when performing microfluidic flow cytometry. This method allows velocity-independent analysis of particles such as beads or cells, and allows flow cytometry analysis of extended objects, such as long DNA molecules. It allows accurate flow cytometry in transient and nonuniform flows. This general measurement method could be used in the future to measure the velocity of particles in a variety of existing microfluidic devices without the need for changes in their design.     

Surface enhanced Raman spectroscopy for microfluidic pillar arrayed separation chips

Numerous studies have addressed the challenges of implementing miniaturized microfluidic platforms for chemical and biological separation applications. However, the integration of real time detection schemes capable of providing valuable sample information under continuous, ultra low volume flow regimes has not fully been addressed. In this report we present a chip based chromatography system comprising of a pillar array separation column followed by a reagent channel for passive mixing of a silver colloidal solution into the eluent stream to enable surface enhanced Raman spectroscopy (SERS) detection. Our design is the first integrated chip based microfluidic device to combine pressure driven separation capability with real time SERS detection. With this approach we demonstrate the ability to collect distinctive SERS spectra with or without complete resolution of chromatographic bands. Computational fluidic dynamic (CFD) simulations are used to model the diffusive mixing behaviour and velocity profiles of the two confluent streams in the microfluidic channels. We evaluate the SERS spectral band intensity and chromatographic efficiency of model analytes with respect to kinetic factors as well as signal acquisition rates. Additionally, we discuss the use of a pluronic modified silver colloidal solution as a means of eliminating contamination generally caused by nanoparticle adhesion to channel surfaces.     

Multi-step synthesis of nanoparticles performed on millisecond time scale in a microfluidic droplet-based system

This paper reports a plug-based, microfluidic method for performing multi-step chemical reactions with millisecond time-control. It builds upon a previously reported method where aqueous reagents were injected into a flow of immiscible fluid (fluorocarbons)(H. Song et al., Angew. Chem. Int. Ed., 2003, 42, 768). The aqueous reagents formed plugs--droplets surrounded and transported by the immiscible fluid. Winding channels rapidly mixed the reagents in droplets. This paper shows that further stages of the reaction could be initiated by flowing additional reagent streams directly into the droplets of initial reaction mixture. The conditions necessary for an aqueous stream to merge with aqueous droplets were characterized. The Capillary number could be used to predict the behavior of the two-phase flow at the merging junction. By transporting solid reaction products in droplets, the products were kept from aggregating on the walls of the microchannels. To demonstrate the utility of this microfluidic method it was used to synthesize colloidal CdS and CdS/CdSe core-shell nanoparticles.     

An electrokinetic/hydrodynamic flow microfluidic CE-ESI-MS interface utilizing a hydrodynamic flow restrictor for delivery of samples under low EOF conditions

A hydrodynamic flow restrictor (HDR) that is used to combine electrokinetic and hydrodynamic flow streams has been fabricated in a microfluidic channel by laser micromachining. Combining electrokinetic and hydrodynamic flow streams is challenging in microfluidic devices, because the hydrodynamic flow often overpowers the electrokinetic flow, making it more difficult to use low electroosmotic flow in the electrokinetic portion of the system. The HDR has been incorporated into a capillary electrophoresis-mass spectrometry interface that provides continuous introduction of a make-up solution and negates the hydrodynamic backpressure in the capillary electrophoresis channel to the extent that low EOF can be utilized. Moreover, the hydrodynamic backpressure is sufficiently minimized to allow coatings that minimize EOF to be used in the electrokinetically driven channel. Such coatings are of great importance for the analysis of proteins and other biomolecules that adsorb to charged surfaces.     

Polar stimulation and constrained cell migration in microfluidic channels

Asymmetrical delivery of stimuli to moving cells for perturbing spatially-heterogeneous intracellular signaling is an experimental challenge not adequately met by existing technologies. Here, we report a robust microfluidic platform allowing localized treatment of the front and/or back of moving cells which crawl through narrow channels that they completely occlude. The enabling technical element for this study is a novel design for precise, passive balancing of flow inside the microfluidic device by contacting two fluid streams before splitting them again. The microchannels constrain cell morphology and induce qualitative and quantitative changes in neutrophil chemotaxis that mimic cells crawling through tissues.     

Microfluidic fabrication of complex-shaped microfibers by liquid template-aided multiphase microflow

This study presents a simple microfluidic approach to the rapid fabrication of complex-shaped microfibers (e.g., single hollow, double hollow, and microbelt), with highly uniform structures, based on a combination of the spontaneous formation of polymeric jet streams and in situ photopolymerization. Two laminar flows of a photocurable fluid and a liquid template (nonpolymerizing fluid) spontaneously form jet streams in equilibrium states in microfluidic channels because of the minimization of the interfacial energy between the two fluids. The formation of the jet streams strongly depends on the spreading coefficients and the evolution time along the downstream of the microfluidic system. Thus, the simple control of the spreading coefficients can guide microfibers into various shapes. The sizes of the core and shell of the hollow fibers can also be readily manipulated by the flow rates of the polymerizing fluid and the liquid template phase. Asymmetric hollow fibers can also be produced in different evolutionary states in the microfluidic system. The microfluidic approach shown here represents a significant step toward the easy fabrication of microfibers with readily controllable structures and geometries. We anticipate that this novel fabrication approach and the prediction method based on spreading coefficients presented in this work can be applied to produce a wide variety of functional microfibrous materials.     

Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing

Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic flow cytometer developed in this paper provides a practical platform that can be used for routine analysis in biological laboratories. Additionally, the 3D hydrodynamic focusing channel design can also be applied to various applications that can advance the lab on a chip research.     

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.     

A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array

We present a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 microm in height surrounded by multiple narrow perfusion channels 2 microm in height. The high aspect ratio (approximately 20) between the microchamber and the perfusion channels offers advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C and was grown to confluency. The microfluidic cell culture array could potentially offer an affordable platform for a wide range of applications in high throughput cell-based screening, bioinformatics, synthetic biology, quantitative cell biology, and systems biology.     

Generation of stable complex gradients across two-dimensional surfaces and three-dimensional gels

Many chemical and biological processes are dependent on molecular gradients. We describe a new microfluidic approach that can be used to produce spatiotemporal gradients across two-dimensional surfaces and three-dimensional gels under flow-free conditions. Free diffusion between dynamically replenished flow channels acting as a sink and source is utilized to give rise to stable steady-state gradient profiles. The gradient profile is dictated by the engineered design of the device's gradient-generating region. Different designs can yield both linear and non-linear gradients of varying profiles. More complex gradients can be made by juxtaposing different designs within a single gradient-generating region. By fabricating an array of designs along the gradient-generating region, different gradient profiles can be generated simultaneously, allowing for parallel analysis. Additionally, simple methods of localizing gels into microdevices are demonstrated. The device was characterized by experimentally obtained gradient profiles of fluorescent molecules that corroborated closely with a simulated finite element model.     

Optical tweezers applied to a microfluidic system

We will demonstrate how optical tweezers can be combined with a microfluidic system to create a versatile microlaboratory. Cells are moved between reservoirs filled with different media by means of optical tweezers. We show that the cells, on a timescale of a few seconds, can be moved from one reservoir to another without the media being dragged along with them. The system is demonstrated with an experiment where we expose E. coli bacteria to different fluorescent markers. We will also discuss how the system can be used as an advanced cell sorter. It can favorably be used to sort out a small fraction of cells from a large population, in particular when advanced microscopic techniques are required to distinguish various cells. Patterns of channels and reservoirs were generated in a computer and transferred to a mask using either a sophisticated electron beam technique or a standard laser printer. Lithographic methods were applied to create microchannels in rubber silicon (PDMS). Media were transported in the channels using electroosmotic flow. The optical system consisted of a combined confocal and epi-fluorescence microscope, dual optical tweezers and a laser scalpel.     

Relaxation of microparticles exposed to hydrodynamic forces in microfluidic conduits

The behavior of microparticles exposed to gravitational and lift forces and to the velocity gradient in flow velocity profile formed in microfluidic conduits is studied from the viewpoint of the transient period (the relaxation) between the moment at which a particle starts to be transported by the hydrodynamic flow and the time at which it reaches an equilibrium position, characterized by a balance of all active forces. The theoretical model allowing the calculation of the relaxation time is proposed. The numerical calculus based on the proposed model is compared with the experimental data obtained under different experimental conditions, namely, for different lengths of microfluidic channels, different average linear velocities of the carrier liquid, and different sizes and densities of the particles used in the study. The results are important for the optimization of microfluidic separation units such as microthermal field-flow fractionation channels in which the separation or manipulation of the microparticles of various origin, synthetic, natural, biological, etc., is performed under similar experimental conditions but by applying an additional thermodynamic force.

A novel 3D mammalian cell perfusion-culture system in microfluidic channels

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.     

Fabrication of a modular tissue construct in a microfluidic chip

By combining microfluidics and soft-lithographic molding of gels containing mammalian cells, a device for three-dimensional (3D) culture of mammalian cells in microchannels was developed. Native components of the extracellular matrix, including collagen or Matrigel, made up the matrix of each molded piece (module) of cell-containing gel. Each module had at least one dimension below approximately 300 microm; in modules of these sizes, the flux of oxygen, nutrients, and metabolic products into and out of the modules was sufficient to allow cells in the modules to proliferate to densities comparable to those of native tissue (10(8)-10(9) cells cm(-3)). Packing modules loosely into microfluidic channels and chambers yielded structures permeated with a network of pores through which cell culture medium could flow to feed the encapsulated cells. The order in the packed assemblies increased as the width of the microchannels approached the width of the modules. Multiple cell types could be spatially organized in the small microfluidic channels. Recovery and analysis of modules after 24 h under constant flow of medium (200 microL h(-1)) showed that over 99% of encapsulated cells survived this interval in the microfluidic chamber.     

Continuous flow multi-stage microfluidic reactors via hydrodynamic microparticle railing

"Multi-stage" fluidic reactions are integral to diverse biochemical assays; however, such processes typically require laborious and time-intensive fluidic mixing procedures in which distinct reagents and/or washes must be loaded sequentially and separately (i.e., one-at-a-time). Microfluidic processors that enable multi-stage fluidic reactions with suspended microparticles (e.g., microbeads and cells) to be performed autonomously could greatly extend the efficacy of lab-on-a-chip technologies. Here we present a single-layer microfluidic reactor that utilizes a microfluidic railing methodology to passively transport suspended microbeads and cells into distinct, adjacent laminar flow streams for rapid fluidic mixing and assaying. Four distinct molecular synthesis processes (i.e., consisting of 48 discrete fluidic mixing stages in total) were accomplished on polystyrene microbead substrates (15 μm in diameter) in parallel, without the need for external observation or regulation during device operation. Experimental results also revealed successful railing of suspended bovine aortic endothelial cells (approximately 13 to 17 μm in diameter). The presented railing system provides an effective continuous flow methodology to achieve bead-based and cell-based microfluidic reactors for applications including point-of-care (POC) molecular diagnostics, pharmacological screening, and quantitative cell biology.