Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.     

Magnetic force-based multiplexed immunoassay using superparamagnetic nanoparticles in microfluidic channel

This paper describes a novel microfluidic immunoassay utilizing binding of superparamagnetic nanoparticles to beads and deflection of these beads in a magnetic field as the signal for measuring the presence of analyte. The superparamagnetic 50 nm nanoparticles and fluorescent 1 microm polystyrene beads are immobilized with specific antibodies. When target analytes react with the polystyrene beads and superparamagnetic nanoparticles simultaneously, the superparamagnetic nanoparticles can be attached onto the microbeads by the antigen-antibody complex. In the poly(dimethylsiloxane)(PDMS) microfluidic channel, only the microbeads conjugated with superparamagnetic nanoparticles by analytes consequently move to the high gradient magnetic fields under the specific applied magnetic field. In this study, the magnetic force-based microfluidic immunoassay is successfully applied to detect the rabbit IgG and mouse IgG as model analytes. The lowest concentration of rabbit IgG and mouse IgG measured over the background is 244 pg mL(-1) and 15.6 ng mL(-1), respectively. The velocities of microbeads conjugated with superparamagnetic nanoparticles are demonstrated by magnetic field gradients in microfluidic channels and compared with the calculated magnetic field gradients. Moreover, dual analyte detection in a single reaction is also performed by the fluorescent encoded microbeads in the microfluidic device. Detection range and lower detection limit can be controlled by the microbeads concentration and the higher magnetic field gradient.     

Simultaneous sample washing and concentration using a "trapping-and-releasing" mechanism of magnetic beads on a microfluidic chip

Simultaneous washing and concentration of functionalized magnetic beads in a complex sample solution were demonstrated by applying a rotational magnetic actuation system to a microfluidic chip under continuous flow conditions. The rotation of periodically arranged small permanent magnets close to the fluidic channel carrying a magnetic bead suspension allows trapping and releasing of the beads along the fluidic channel in a periodical manner. Each trapping and releasing event resembles one washing cycle. A purification efficiency of magnetic beads out of a mixed magnetic and non-magnetic bead sample solution of 83±4% at a flow rate of 0.5 μL min(-1), and a magnetic bead recovery or concentration efficiency of 91±5% were achieved using a flow rate of 0.2 μL min(-1). The detection performance of the device was experimentally evaluated with two different bioassays, using either streptavidin-coated magnetic beads in combination with biotinylated fluorescent isothiocyanate (FITC), or a mouse antigen (Ag)-antibody (Ab) system.     

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.     

Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies

We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.     

Preparation of the capillary-based microchips for solid phase extraction by using the monolithic frits prepared by UV-initiated polymerization.

A microfluidic solid phase extraction (SPE) array for sample enrichment was prepared by a simple method, a hot embossing technique. Five fused-silica capillaries (250 microm i.d., 380 microm o.d.) were partly embedded parallel in a polymethyl methacrylate (PMMA) microchip to serve as the extraction channels. Within each of the channels, a 2-mm-long monolithic porous polymer was prepared by in-situ photoinitiated polymerization. This then acted as the frit for packing of the extraction materials (octadecylsilica beads, ODS). By defining the light-exposure window on the channels, one can easily control the length and location of the polymer frits and the ODS beads can be packed at the desired location. With this method, solid phase extraction channels for microfluidic use can be easily prepared without complex fabrication of microstructures. Several SPE channels can be conveniently made in one microchip since the frits can be prepared in different channels through one polymerization; packing of the different channels can also be performed simultaneously. With the use of dilute ephedrine solutions, the sample loading capacity, linearity, and reproducibility were characterized. Coupled with the fast capillary electrophoresis separation, this microchip SPE array was applied for the detection of ephedrines in human urine.     

Direct-writing colloidal photonic crystal microfluidic chips by inkjet printing for label-free protein detection

Integrating photonic crystals (PC) into microfluidic systems has attracted immense interest for its novel functions. However, it is still a great challenge to fabricate PC microfluidic chips rapidly with complex functions. In this work, a direct-writing colloidal PC microchannel was firstly achieved by inkjet printing and was used for the surface-tension-confined microfluidic immune assay. PC channels with different structure colors have been successfully integrated on one chip. The fabricated chip has the advantages of rapid fabrication, quick fluidic transport and can monitor the fluidic fluxion using the naked eye. Utilizing this PC microfluidic chip, a colorimetric label-free immune assay was realized without nonspecific adsorption interference of the target.     

Development of a microfluidic biosensor module for pathogen detection

The development of a microfluidic biosensor module with fluorescence detection for the identification of pathogenic organisms and viruses is presented in this article. The microfluidic biosensor consists of a network of microchannels fabricated in polydimethylsiloxane (PDMS) substrate. The microchannels are sealed with a glass substrate and packed in a Plexiglas housing to provide connection to the macro-world and ensure leakage-free flow operation. Reversible sealing permits easy disassembly for cleaning and replacing the microfluidic channels. The fluidic flow is generated by an applied positive pressure gradient, and the module can be operated under continuous solution flow of up to 80 microL min(-1). The biosensor recognition principle is based on DNA/RNA hybridization and liposome signal amplification. Superparamagnetic beads are incorporated into the system as a mobile solid support and are an essential part of the analysis scheme. In this study, the design, fabrication and the optimization of concentrations and amounts of the different biosensor components are carried out. The total time required for an assay is only 15 min including sample incubation time. The biosensor module is designed so that it can be easily integrated with a micro total analysis system, which will combine sample preparation and detection steps onto a single chip.     

Microfluidic waves

The propagation of pressure waves in fluidic channels with elastic covers is discussed in view of applications to flow control in microfluidic devices. A theory is presented which describes pressure waves in the fluid that are coupled to bending waves in the elastic cover. At low frequencies, the lateral bending of the cover dominates over longitudinal bending, leading to propagating, non-dispersive longitudinal pressure waves in the channel. The theory addresses effects due to both the finite viscosity and compressibility of the fluid. The coupled waves propagate without dispersion, as long as the wave length is larger than the channel width. It is shown that in channels of typical microfluidic dimensions, wave velocities in the range of a few 10 m s(-1) result if the channels are covered by films of a compliant material such as PDMS. The application of this principle to design microfluidic band pass filters based on standing waves is discussed. Characteristic frequencies in the range of a few kHz are readily achieved with quality factors above 30.     

Polyimide-based microfluidic devices

This paper describes the development of polyimide-based microfluidic devices. A layer transfer and lamination technique is used to fabricate flexible microfluidic channels in various shapes and with a wide range of dimensions. High bond strengths can be achieved by cure cycle adaptation and surface treatment of the polyimide layers prior to bonding. The polyimide microchannels can be combined with metallization layers to fabricate electrodes inside and outside channels. The resulting devices can be used for flexible fluidic and electrical connectors, implantable fluid delivery devices, microelectrodes with embedded fluidic channels, chip-based flow cytometry and for a great variety of other applications in medical, chemical or biological research.     

Construction of microscale structures in enclosed microfluidic networks by using a magnetic beads based method

A large number of microscale structures have been used to elaborate flowing control or complex biological and chemical reaction on microfluidic chips. However, it is still inconvenient to fabricate microstructures with different heights (or depths) on the same substrate. These kinds of microstructures can be fabricated by using the photolithography and wet-etching method step by step, but involves time-consuming design and fabrication process, as well as complicated alignment of different masters. In addition, few existing methods can be used to perform fabrication within enclosed microfluidic networks. It is also difficult to change or remove existing microstructures within these networks. In this study, a magnetic-beads-based approach is presented to build microstructures in enclosed microfluidic networks. Electromagnetic field generated by microfabricated conducting wires (coils) is used to manipulate and trap magnetic beads on the bottom surface of a microchannel. These trapped beads are accumulated to form a microscale pile with desired shape, which can adjust liquid flow, dock cells, modify surface, and do some other things as those fabricated microstructures. Once the electromagnetic field is changed, trapped beads may form new shapes or be removed by a liquid flow. Besides being used in microfabrication, this magnetic-beads-based method can be used for novel microfluidic manipulation. It has been validated by forming microscale dam structure for cell docking and modified surface for cell patterning, as well as guiding the growth of neurons.     

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.     

Chitosan-mediated in situ biomolecule assembly in completely packaged microfluidic devices

We report facile in situ biomolecule assembly at readily addressable sites in microfluidic channels after complete fabrication and packaging of the microfluidic device. Aminopolysaccharide chitosan's pH responsive and chemically reactive properties allow electric signal-guided biomolecule assembly onto conductive inorganic surfaces from the aqueous environment, preserving the activity of the biomolecules. A transparent and nonpermanently packaged device allows consistently leak-free sealing, simple in situ and ex situ examination of the assembly procedures, fluidic input/outputs for transport of aqueous solutions, and electrical ports to guide the assembly onto the patterned gold electrode sites within the channel. Both in situ fluorescence and ex situ profilometer results confirm chitosan-mediated in situ biomolecule assembly, demonstrating a simple approach to direct the assembly of biological components into a completely fabricated device. We believe that this strategy holds significant potential as a simple and generic biomolecule assembly approach for future applications in complex biomolecular or biosensing analyses as well as in sophisticated microfluidic networks as anticipated for future lab-on-a-chip devices.     

Fluidic low pass filter for hydrodynamic flow stabilization in microfluidic environments

Fluctuations in flow rate invariably occur in microfluidic devices. This fluidic instability results in a deteriorating performance and the suspension of their unique functions occasionally. In this study, a fluidic-LPF (low pass filter), which is composed of an ACU (air compliance unit) and a FCSP (fluidic channel with high fluidic resistance for sufficient preload), has been proposed for providing the stabilization of hydrodynamic flow in microfluidic devices. To investigate the characteristics of various fluidic networks including our fluidic-LPF, we used a parametric identification method to estimate the time constants via a transient response that was based on a discrete parameter model. In addition, we propose the use of a pulsation index (PI) to quantify the fluctuations in flow rate. We verified the formula for PI derived herein by varying individually both the periods and the air compliance volumes in the ACU, both theoretically and experimentally. We found that the PI depended strongly on either the time constants or the periods of the flow rates at the inlet. Additionally, the normalized differences between the experimental results and the theoretical estimations were less than 6%, which shows that the proposed formula for PI can provide an accurate quantification of the fluctuations in flow, and estimate the parametric effects. Finally, we have successfully demonstrated that our fluidic-LPF can regulate fluctuations in the flow at extremely low flow rates (~ 10 μL h(-1)) and can also control severe fluidic fluctuations (PI = 0.67) with excessively long periods (100 s) via a microfluidic viscometer. We therefore believe that the stabilization of hydrodynamic flow using a fluidic-LPF could be used easily and extensively with a range of microfluidic platforms that require constant flow rates.     

Reconfigurable virtual electrowetting channels

Lab-on-a-chip systems rely on several microfluidic paradigms. The first uses a fixed layout of continuous microfluidic channels. Such lab-on-a-chip systems are almost always application specific and far from a true "laboratory." The second involves electrowetting droplet movement (digital microfluidics), and allows two-dimensional computer control of fluidic transport and mixing. The merging of the two paradigms in the form of programmable electrowetting channels takes advantage of both the "continuous" functionality of rigid channels based on which a large number of applications have been developed to date and the "programmable" functionality of digital microfluidics that permits electrical control of on-chip functions. In this work, we demonstrate for the first time programmable formation of virtual microfluidic channels and their continuous operation with pressure driven flows using an electrowetting platform. Experimental, theoretical, and numerical analyses of virtual channel formation with biologically relevant electrolyte solutions and electrically-programmable reconfiguration are presented. We demonstrate that the "wall-less" virtual channels can be formed reliably and rapidly, with propagation rates of 3.5-3.8 mm s(-1). Pressure driven transport in these virtual channels at flow rates up to 100 μL min(-1) is achievable without distortion of the channel shape. We further demonstrate that these virtual channels can be switched on-demand between multiple inputs and outputs. Ultimately, we envision a platform that would provide rapid prototyping of microfluidic concepts and would be capable of a vast library of functions and benefitting applications from clinical diagnostics in resource-limited environments to rapid system prototyping to high throughput pharmaceutical applications.     

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.     

Patterned self-assembled beads in silicon channels.

A novel technique enabling selective bead trapping in microfluidic devices without the use of physical barriers is presented in this paper. It is a fast, convenient and simple method, involving microcontact printing and self-assembly, that can be applied to silicon, quartz or plastic substrates. In the first step, channels are etched in the substrate. The surface chemistry of the internal walls of the channels is then modified by microcontact printing. The chip is submerged in a bead slurry where beads self-assemble based on surface chemistry and immobilize on the internal walls of the channels. Silicon channels (100 microm wide and 50 microm deep) have been covered with monolayers of streptavidin-, amino- and hydroxy-functionalized microspheres and resulted in good surface coverage of beads on the channel walls. A high-resolution pattern of lines of self-assembled streptavidin beads, as narrow as 5 microm, has also been generated on the bottom of a 500 microm wide and 50 microm deep channel. Flow tests were performed in sealed channels with the different immobilized beads to confirm that the immobilized beads could withstand the forces generated by water flowing in the channels. The presented results indicate that single beads can be precisely positioned within microfluidic devices based on self-assembly which is useful as screening and analysis tools within the field of biochemistry and organic chemistry.     

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.     

Logic digital fluidic in miniaturized functional devices: Perspective to the next generation of microfluidic lab‐on‐chips

Microfluidics has emerged following the quest for scale reduction inherent to micro‐ and nanotechnologies. By definition, microfluidics manipulates fluids in small channels with dimensions of tens to hundreds of micrometers. Recently, microfluidics has been greatly developed and its influence extends not only the domains of chemical synthesis, bioanalysis, and medical researches but also optics and information technology. In this review article, we will shortly discuss an enlightening analogy between electrons transport in electronics and fluids transport in microfluidic channels. This analogy helps to master transport and sorting. We will present some complex microfluidic devices showing that the analogy is going a long way off toward more complex components with impressive similarities between electronics and microfluidics. We will in particular explore the vast manifold of fluidic operations with passive and active fluidic components, respectively, as well as the associated mechanisms and corresponding applications. Finally, some relevant applications and an outlook will be cited and presented.     

Interconnected ordered nanoporous networks of colloidal crystals integrated on a microfluidic chip for highly efficient protein concentration

We report a controllable method to fabricate silica colloidal crystals at defined position in microchannel of microuidic devices using simple surface modification. The formed PCs (photonic crystals) in microfluidic channels were stabilized by chemical cross-linking of Si-O-Si bond between neighboring silica beads. The voids among colloids in PCs integrated on microfluidic devices form interconnected nanoporous networks, which show special electroosmotic properties. Due to the "surface-charge induced ion depletion effect" mechanism, FITC-labeled proteins can be efficiently and selectively concentrated in the anodic boundary of the ion depletion zone. Using this device, about 10(3) - to 10(5)-fold protein concentration was achieved within 10 min. The present simple on chip protein concentration device could be a potential sample preparation component in microfluidic systems for practical biochemical assays.