Laser-induced mixing in microfluidic channels

We demonstrate a novel strategy for mixing solutions and initiating chemical reactions in microfluidic systems. This method utilizes highly focused nanosecond laser pulses from a Q-switched Nd:YAG laser at lambda = 532 nm to generate cavitation bubbles within 100- and 200-microm-wide microfluidic channels containing the parallel laminar flow of two fluids. The bubble expansion and subsequent collapse within the channel disrupts the laminar flow of the parallel fluid streams and produces a localized region of mixed fluid. We use time-resolved imaging and fluorescence detection methods to visualize the mixing process and to estimate both the volume of mixed fluid and the time scale for the re-establishment of laminar flow. The results show that mixing is initiated by liquid jets that form upon cavitation bubble collapse and occurs approximately 20 micros following the delivery of the laser pulse. The images also reveal that mixing occurs on the millisecond time scale and that laminar flow is re-established on a 50-ms time scale. This process results in a locally mixed fluid volume in the range of 0.5-1.5 nL that is convected downstream with the main flow in the microchannel. We demonstrate the use of this mixing technique by initiating the horseradish peroxidase-catalyzed reaction between hydrogen peroxide and nonfluorescent N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to yield fluorescent resorufin. This approach to generate the mixing of adjacent fluids may prove advantageous in many microfluidic applications as it requires neither tailored channel geometries nor the fabrication of specialized on-chip instrumentation.     

Improvements in mixing time and mixing uniformity in devices designed for studies of protein folding kinetics

Using a microfluidic laminar flow mixer designed for studies of protein folding kinetics, we demonstrate a mixing time of 1 +/- 1 micros with sample consumption on the order of femtomoles. We recognize two limitations of previously proposed designs: (1) size and shape of the mixing region, which limits mixing uniformity and (2) the formation of Dean vortices at high flow rates, which limits the mixing time. We address these limitations by using a narrow shape-optimized nozzle and by reducing the bend of the side channel streamlines. The final design, which combines both of these features, achieves the best performance. We quantified the mixing performance of the different designs by numerical simulation of coupled Navier-Stokes and convection-diffusion equations and experiments using fluorescence resonance energy-transfer (FRET)-labeled DNA.     

Quantitative analysis of methyl parathion pesticides in a polydimethylsiloxane microfluidic channel using confocal surface-enhanced Raman spectroscopy

A fast and ultra-sensitive trace analysis of methyl parathion pesticides in a polydimethylsiloxane (PDMS) microfluidic channel was investigated using confocal surface-enhanced Raman spectroscopy (SERS). A three-dimensional PDMS-based passive micromixer was fabricated for this purpose. This PDMS micromixer showed a high mixing efficiency because a strong chaotic advection was developed by the simultaneous vertical and transverse dispersion of the confluent streams. The confocal SERS signal was measured after methyl parathion pesticides were effectively adsorbed onto silver nanoparticles while flowing along the upper and lower alligator-teeth-shaped PDMS channel. A quantitative analysis of the methyl parathion pesticides was performed based on the measured peak height at 1246 cm-1. Our method has a detection limit of 0.1 ppm. This value satisfies the requirement recommended by the Collaborative International Pesticides Analytical Council (CIPAC) for the determination of methyl parathion in pesticide formulations. This study demonstrates the feasibility of using confocal SERS for the highly sensitive detection of methyl parathion pesticides in a PDMS microfluidic channel.     

Deconvolution microscopy for flow visualization in microchannels

Quantitative visualization of microflows is often needed to evaluate the efficiency of fluid mixing, study flow properties, investigate unusual flow behavior, and verify computational fluid dynamic simulations. In this work, we explore the technique of coupling a conventional optical microscope with a computational deconvolution algorithm to produce images of three-dimensional flows in plastic microfluidic channels. The approach, called deconvolution microscopy, is achieved by (1) optically sectioning the flow in the microchannel by collecting a series of fluorescence images at different focal planes along the optical axis and (2) removing the out-of-focus fluorescence signal by a deconvolution method to reconstruct the corrected three-dimensional concentration image. We compare three different classes of deconvolution algorithms for a uniform concentration test case and then demonstrate how deconvolution microscopy is useful for flow visualization and analysis of mixing in microfluidic channels. In particular, we employ the method to confirm the presence of twisting flows in a microchannel containing microfabricated ridges.     

A soft lithographic approach to fabricate patterned microfluidic channels

The control of surface properties and spatial presentation of functional molecules within a microfluidic channel is important for the development of diagnostic assays and microreactors and for performing fundamental studies of cell biology and fluid mechanics. Here, we present a simple technique, applicable to many soft lithographic methods, to fabricate robust microchannels with precise control over the spatial properties of the substrate. In this approach, the patterned regions were protected from oxygen plasma by controlling the dimensions of the poly(dimethylsiloxane) (PDMS) stamp and by leaving the stamp in place during the plasma treatment process. The PDMS stamp was then removed, and the microfluidic mold was irreversibly bonded to the substrate. The approach was used to pattern a nonbiofouling poly(ethylene glycol)-based copolymer or the polysaccharide hyaluronic acid within microfluidic channels. These nonbiofouling patterns were then used to fabricate arrays of fibronectin and bovine serum albumin as well as mammalian cells. In addition, further control over the deposition of multiple proteins onto multiple or individual patterns was achieved using laminar flow. Also, cells that were patterned within channels remained viable and capable of performing intracellular reactions and could be potentially lysed for analysis.     

On-chip microfluidic transport and mixing using electrowetting and incorporation of sensing functions

An integrated system was developed that performs microfluidic transport, mixing, and sensing on a single chip. The operation principle for the microfluidic transport was based on electrowetting. A solution to be transported was confined in a space between a row of gold working electrodes and a protruding poly(dimethylsiloxane) (PDMS) structure. When a negative potential was applied to one of the gold working electrodes, it became hydrophilic, and the solution was transported through the flow channel. The solution could be transported in any desired direction in a network of flow channels by switching on necessary electrodes one by one. Furthermore, two solutions transported through two flow channels could be mixed using a mixing electrode based on the same principle. To demonstrate the applicability of a lab-on-a-chip, an air gap ammonia electrode was integrated by taking advantage of the open structure of the flow channel. Gaseous ammonia that was produced after pH adjustment and diffused through an air gap caused a pH change in the electrolyte layer, which was measured with an iridium oxide pH indicator electrode. The 90% response time was less than 1 min for the millimolar order of ammonia. The calibration curve was linear down to 10 microM. The ammonia-sensing system was also applied to construct biosensing systems for urea and creatinine. A linear relationship was observed between the potential and the logarithm of the concentration of the analytes down to 50 microM for both urea and creatinine. The developed microfluidic system can be a basic building block for future systems.     

Torque-actuated valves for microfluidics

This paper describes torque-actuated valves for controlling the flow of fluids in microfluidic channels. The valves consist of small machine screws (> or =500 microm) embedded in a layer of polyurethane cast above microfluidic channels fabricated in poly(dimethylsiloxane) (PDMS). The polyurethane is cured photochemically with the screws in place; on curing, it bonds to the surrounding layer of PDMS and forms a stiff layer that retains an impression of the threads of the screws. The valves were separated from the ceiling of microfluidic channels by a layer of PDMS and were integrated into channels using a simple procedure compatible with soft lithography and rapid prototyping. Turning the screws actuated the valves by collapsing the PDMS layer between the valve and channel, controlling the flow of fluids in the underlying channels. These valves have the useful characteristic that they do not require power to retain their setting (on/off). They also allow settings between "on" and "off" and can be integrated into portable, disposable microfluidic devices for carrying out sandwich immunoassays.     

Capillary-assembled microchip for universal integration of various chemical functions onto a single microfluidic device

A novel concept for assembling various chemical functions onto a single microfluidic device is proposed. The concept, called a capillary-assembled microchip, involves embedding chemically functionalized capillaries into a lattice microchannel network fabricated on poly(dimethylsiloxane) (PDMS). The network has the same channel dimensions as the outer dimensions of the capillaries. In this paper, we focus on square capillaries to be embedded into a PDMS microchannel network having a square cross section. The combination of hard glass square capillary and soft square PDMS channel allows successful fabrication of a microfluidic device without any solution leakage, and which can use diffusion-based two-solution mixing. Two different types of chemically modified capillaries, an ion-sensing capillary and a pH-sensing capillary, are prepared by coating a hydrophobic plasticized poly(vinyl chloride) membrane and a hydrophilic poly(ethyleneglycol) membrane containing functional molecules onto the inner surface of capillaries. Then, they are cut into appropriate lengths and arranged on a single microchip to prepare a dual-analyte sensing system. The concept proposed here offers advantages inherent to using a planar microfluidic device and of chemical functionality of immobilized molecules. Therefore, we expect to fabricate various types of chemically functionalized microfluidic devices soon.     

Enhanced bio-molecular interactions through recirculating microflows

A recirculating microfluidic platform has been developed for carrying out optical bio-detection. The present device can be used for passive mixing of the biological species with the microfluidic channel without immobilisation, through appropriate design and flow control. The feasibility of bio-detection using the present setup has been demonstrated through the method of fluorescence and the experiments were carried out with Antisheep Antibody (AB) tagged with Alexafluor 647 (AF647) fluorophore particles. By controlling the fluid flow, it was possible to isolate AB separately into a recirculation zone within the microfluidic channel, thereby enabling qualitative and quantitative bio-detection. Finite element modelling of the flow behaviour has been carried out and the results were similar to the results of flow visualisation obtained with tagged antibody particles. The present work thus provides confidence in using the hybrid integrated device for in situ rapid biomedical detection of biological pairs or individual specimen in fluorescence-based chemical and biological sensing.     

Microfluidic characterization and continuous separation of cells and particles using conducting poly(dimethyl siloxane) electrode induced alternating current-dielectrophoresis

This paper presents a poly(dimethyl siloxane) (PDMS) polymer microfluidic device using alternating current (ac) dielectrophoresis (DEP) for separating live cells from interfering particles of similar sizes by their polarizabilities under continuous flow and for characterizing DEP behaviors of cells in stagnant flow. The ac-DEP force is generated by three-dimensional (3D) conducting PDMS composite electrodes fabricated on a sidewall of the device main channel. Such 3D PDMS composite electrodes are made by dispersing microsized silver (Ag) fillers into PDMS gel. The sidewall AgPDMS electrodes can generate a 3D electric field that uniformly distributes throughout the channel height and varies along the channel lateral direction, thereby producing stronger lateral DEP effects over the entire channel. This allows not only easy observation of cell/particle lateral motion but also using the lateral DEP force for manipulation of cells/particles. The former feature is used to characterize the frequency-dependent DEP behaviors of Saccharomyces cerevisiae (yeast) and Escherichia coli (bacteria). The latter is utilized for continuous separation of live yeast and bacterial cells from similar-size latex particles as well as live yeast cells from dead yeast cells. The separation efficiency of 97% is achieved in all cases. The demonstration of these functions shows promising applications of the microfluidic device.     

Enzyme kinetic measurements using a droplet-based microfluidic system with a concentration gradient

In this paper, we propose a microfluidic device that is capable of generating a concentration gradient followed by parallel droplet formation within channels with a simple T-junction geometry. Linear concentration gradient profiles can be obtained based on fluid diffusion under laminar flow. Optimized conditions for generating a linear concentration gradient and parallel droplet formation were investigated using fluorescent dye. The concentration gradient profile under diffusive mixing was dominated by the flow rate at sample inlets, while parallel droplet formation was affected by the channel geometry at both the inlet and outlet. The microfluidic device was experimentally characterized using optimal layout and operating conditions selected through a design process. Furthermore, in situ enzyme kinetic measurements of the β-galactosidase-catalyzed hydrolysis of resorufin-β-d-galactopyranoside were performed to demonstrate the application potential of our simple, time-effective, and low sample volume microfluidic device. We expect that, in addition to enzyme kinetics, drug screening and clinical diagnostic tests can be rapidly and accurately performed using this droplet-based microfluidic system.     

On-chip transduction of nucleic acid hybridization using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1.     

Controlling nonspecific protein adsorption in a plug-based microfluidic system by controlling interfacial chemistry using fluorous-phase surfactants

Control of surface chemistry and protein adsorption is important for using microfluidic devices for biochemical analysis and high-throughput screening assays. This paper describes the control of protein adsorption at the liquid-liquid interface in a plug-based microfluidic system. The microfluidic system uses multiphase flows of immiscible fluorous and aqueous fluids to form plugs, which are aqueous droplets that are completely surrounded by fluorocarbon oil and do not come into direct contact with the hydrophobic surface of the microchannel. Protein adsorption at the aqueous-fluorous interface was controlled by using surfactants that were soluble in fluorocarbon oil but insoluble in aqueous solutions. Three perfluorinated alkane surfactants capped with different functional groups were used: a carboxylic acid, an alcohol, and a triethylene glycol group that was synthesized from commercially available materials. Using complementary methods of analysis, adsorption was characterized for several proteins (bovine serum albumin (BSA) and fibrinogen), including enzymes (ribonuclease A (RNase A) and alkaline phosphatase). These complementary methods involved characterizing adsorption in microliter-sized droplets by drop tensiometry and in nanoliter plugs by fluorescence microscopy and kinetic measurements of enzyme catalysis. The oligoethylene glycol-capped surfactant prevented protein adsorption in all cases. Adsorption of proteins to the carboxylic acid-capped surfactant in nanoliter plugs could be described by using the Langmuir model and tensiometry results for microliter drops. The microfluidic system was fabricated using rapid prototyping in poly(dimethylsiloxane) (PDMS). Black PDMS microfluidic devices, fabricated by curing a suspension of charcoal in PDMS, were used to measure the changes in fluorescence intensity more sensitively. This system will be useful for microfluidic bioassays, enzymatic kinetics, and protein crystallization, because it does not require surface modification during fabrication to control surface chemistry and protein adsorption.     

Investigation of the mixing efficiency of a chaotic micromixer using thermal lens spectrometry

This work investigates the efficiency of a chaotic micromixer using thermal lens spectrometry. The outlet of the mixing device was connected to a thermal lens detection head integrating the probe beam optical fibers and the sample capillary. The chaotic micromixer consisted of a Y-shaped poly(dimethylsiloxane) (PDMS) microchip in which ribbed herringbone microstructures were etched on the floor of the main channel. Due to the solvent composition dependence of the thermal lens response, the photothermal method was shown to be highly sensitive to nonhomogeneous mixing compared to fluorescence detection. The apparatus was applied to the determination of Fe2+ with 1,10-phenanthroline using flow injection analysis; a limit of detection of 11 microg L(-1) of iron was obtained.     

Patterning multiplex protein microarrays in a single microfluidic channel

The development of versatile biofunctional surfaces is a fundamental prerequisite in designing Lab on a Chip (LOC) devices for applications in biosensing interfaces and microbioreactors. The current paper presents a rapid combinatorial approach to create multiplex protein patterns in a single microfluidic channel. This approach consists of coupling microcontact printing with microfluidic patterning, where microcontact printing is employed for silanization using (3-Aminopropyl) triethoxysilane (APTES), followed by microfluidic patterning of multiple antibodies. As a result, the biomolecules of choice could be covalently attached to the microchannel surface, thus creating a durable and highly resistant functional interface. Moreover, the experimental procedure was designed to create a microfluidic platform that maintains functionality at high flow rates. The functionalized surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and monitored with fluorescence microscopy at each step of functionalization. To illustrate the possibility of patterning multiple biomolecules along the cross section of a single microfluidic channel, microarrays of five different primary antibodies were patterned onto a single channel and their functionality was evaluated accordingly through a multiplex immunoassay using secondary antibodies specific to each patterned primary antibody. The resulting patterns remained stable at shear stresses of up to 50 dyn/cm(2). The overall findings suggest that the developed multiplex functional interface on a single channel can successfully lead to highly resistant multiplex functional surfaces for high throughput biological assays.     

The detection of p53 gene via fluorescence quenching of quantum dot in microfluidic chip

Recently, quantum dot (QD) has been used widely in the field of bio assay including cell imaging, biomarker, and fluorescence resonance energy transfer (FRET) sensor. The DNA assay without labeling process has several advantages including low cost, short time, and simplicity. Microbeads of agarose, glass, and polystyrene have been used as a solid support in microfluidic devices to trace molecules. The main advantages of microfluidics include high throughput, short analysis time, small sample volume, and high sensitivity. PDMS based microfluidic chips were prepared for the detection of p53 gene by using QD-DNA conjugate. The microfluidic chip has a weir in the channel to trap microbeads to which QD-DNA probes bind. Carboxylated CdSe/ZnS QDs (wavelength of emission: 605 nm) could bind to microbeads of polystyrene/divinyl benzene via EDC/NHS crosslinking reaction. The target gene and DNA intercalating dye (TOTO-3) were loaded into the micro-channel. Fluorescence quenching from QDs by intercalating dye was observed after hybridization of DNA at the weir in the channel of microfluidic chip. The fluorescence quenching from QDs by TOTO-3 was dependent on the concentration of target gene. This experiment shows the possibility of rapid detection of DNA via bead-QD complex on microfluidic chip.     

Design of PDMS microlenses bonded to a lab-CD chip for ELISA applications

A novel device comprising polydimethyl-siloxane (PDMS) microlenses bonded to a microfluidic compact disk (CD) is proposed for enzyme-linked immunosorbent assay (ELISA) applications. The PDMS microlenses were fabricated using a simple soft replica molding method and were bonded to the microfluidic CD using oxygen plasma treatment. A commercial software tool (ZEMAX) has been used to analyze the focal length of the microlens. A laser-induced fluorescence bio-detection system, consisting of the integrated microfluidic CD/PDMS microlenses and an optical detection module, was constructed and used to examine the enzymatic reaction of 3-(4-hydroxy) phenly propionic acid. The experimental results show that the PDMS microlens focusing effect yields a significant improvement in the intensity of the detected fluorescence signals. As a result, the proposed device represents an ideal solution for ELISAs and other high-sensitivity bio-detection applications.     

Pressure-driven laminar flow in tangential microchannels: an elastomeric microfluidic switch

This paper describes laminar fluid flow through a three-dimensional elastomeric microstructure formed by two microfluidic channels, fabricated in layers that contact one another face-to-face (typically at a 90 degree angle), with the fluid flows in tangential contact. There are two ways to control fluid flow through these tangentially connected microchannels. First, the flow profiles through the crossings are sensitive to the aspect ratio of the channels; the flow can be controlled by applying external pressure and changing this aspect ratio. Second, the flow direction of an individual laminar stream in multiphase laminar flow depends on the lateral position of the stream within the channel; this position can be controlled by injecting additional streams of fluid into the channel. We describe two microfluidic switches based on these two ways for controlling fluid flow through tangential microchannels and present theoretical arguments that explain the observed dependence of the flow profiles on the aspect ratio of the channels.     

Achieving uniform mixing in a microfluidic device: hydrodynamic focusing prior to mixing

We describe a microfluidic mixer that is well-suited for kinetic studies of macromolecular conformational change under a broad range of experimental conditions. The mixer exploits hydrodynamic focusing to create a thin jet containing the macromolecules of interest. Kinetic reactions are triggered by molecular diffusion into the jet from adjacent flow layers. The ultimate time resolution of these devices can be restricted by premature contact between co-flowing solutions during the focusing process. Here, we describe the design and characterization of a mixer in which hydrodynamic focusing is decoupled from the diffusion of reactants, so that the focusing region is free from undesirable contact between the reactants. Uniform mixing on the microsecond time scale is demonstrated using a device fabricated by imprinting optical-grade plastic. Device characterization is carried out using fluorescence correlation spectroscopy (FCS) and two-photon microscopy to measure flow speeds and to quantify diffusive mixing by monitoring the collisional fluorescence quenching, respectively. Criteria for achieving microsecond time resolution are described and modeled.     

FT-IR spectroscopic imaging of reactions in multiphase flow in microfluidic channels

Rapid, in situ, and label-free chemical analysis in microfluidic devices is highly desirable. FT-IR spectroscopic imaging has previously been shown to be a powerful tool to visualize the distribution of different chemicals in flows in a microfluidic device at near video rate imaging speed without tracers or dyes. This paper demonstrates the possibility of using this imaging technology to capture the chemical information of all reactants and products at different points in time and space in a two-phase system. Differences in the rates of chemical reactions in laminar flow and segmented flow systems are also compared. Neutralization of benzoic acid in decanol with disodium phosphate in water has been used as the model reaction. Quantitative information, such as concentration profiles of reactant and products, can be extracted from the imaging data. The same feed flow rate was used in both the laminar flow and segmented flow systems. The laminar flow pattern was achieved using a plain wide T-junction, whereas the segmented flow was achieved by introducing a narrowed section and a nozzle at the T-junction. The results show that the reaction rate is limited by diffusion and is much slower with the laminar flow pattern, whereas the reaction is completed more quickly in the segmented flow due to better mixing.