Ultrasonic mixing in microfluidic channels using integrated transducers

This paper presents a microfluidic mixer that uses acoustic stirring created by ultrasonic waves. The ultrasound is introduced into the channel by integrated piezoelectric transducers. The transducers are made of a zinc oxide thin film, which is deposited on the bottom surface of a quartz substrate. The poly(dimethylsiloxane) channel is aligned to the transducers on the top surface of the substrate. The transducers are designed for operation around 450 MHz. The main mechanism of the mixing is the acoustic stirring of the fluid perpendicular to the flow direction. The radiation pressure that is generated by the transducer causes the stirring inside the microfluidic channel. The performance of the mixer is characterized by mixing phenolphthalein solution and sodium hydroxide dissolved in ethyl alcohol. Flow rates on the order of 1-100 microL/min are used. The transducers are driven by 1.2 V(rms) sinusoidal voltages at 450 MHz.     

Rapid microfluidic mixing.

A preformed T-microchannel imprinted in polycarbonate was postmodified with a pulsed UV excimer laser (KrF, 248 nm) to create a series of slanted wells at the junction. The presence of the wells leads to a high degree of lateral transport within the channel and rapid mixing of two confluent streams undergoing electroosmotic flow. Several mixer designs were fabricated and investigated. All designs were relatively successful at low flow rates (0.06 cm/s, > or = 75% mixing), but had varying degrees of success at high flow rates (0.81 cm/s, 45-80% mixing). For example, one design operating at high flow rates was able to split an incoming fluorescent stream into two streams of varying concentrations depending on the number of slanted wells present. The final mixer design was able to overcome stream splitting at high flow rates, and it was shown that the two incoming streams were 80% mixed within 443 microm of the T-junction for a flow rate of 0.81 cm/s. Without the presence of the mixer and at the same high flow rate, a channel length of 2.3 cm would be required to achieve the same extent of mixing when relying upon molecular diffusion entirely, while 6.9 cm would be required for 99% mixing.     

Experimental test of scaling of mixing by chaotic advection in droplets moving through microfluidic channels

This letter describes an experimental test of a simple argument that predicts the scaling of chaotic mixing in a droplet moving through a winding microfluidic channel. Previously, scaling arguments for chaotic mixing have been described for a flow that reduces striation length by stretching, folding, and reorienting the fluid in a manner similar to that of the baker's transformation. The experimentally observed flow patterns within droplets (or plugs) resembled the baker's transformation. Therefore, the ideas described in the literature could be applied to mixing in droplets to obtain the scaling argument for the dependence of the mixing time, t~(aw/U)log(Pe), where w [m] is the cross-sectional dimension of the microchannel, a is the dimensionless length of the plug measured relative to w, U [m s(-1)] is the flow velocity, Pe is the Péclet number (Pe=wU/D), and D [m(2)s(-1)] is the diffusion coefficient of the reagent being mixed. Experiments were performed to confirm the scaling argument by varying the parameters w, U, and D. Under favorable conditions, submillisecond mixing has been demonstrated in this system.     

Controlled mixing in microfluidic systems using bacterial chemotaxis

We demonstrate the use of Escherichia coli and their chemotactic characteristics to enhance mixing in a microchannel in a controlled and bi-directional manner. The presence of a chemoattractant in one arm of a three-junction microchannel results in an asymmetric increase in the effective diffusion coefficient of extremely high molecular weight TMR-Dextran (MW 2 000 000), which rises linearly with the concentration of attractant from a baseline value of 8-42 microm(2)/s at a concentration of 0.1 M. The response to a repellent is similar, with the opposite bias.     

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.     

Hybridization enhancement using microfluidic planetary centrifugal mixing

DNA microarrays produce their greatest sensitivities when hybridized using concentrated samples and effective mixing; however, these goals have proved elusive to combine. If samples are diluted enough to fill larger chambers, then mixing works well using either pumping or gravity with rotation, although sensitivities will suffer. Various techniques for mixing concentrated samples in small thin chambers have been proposed; however, they often leave streaks or scars, and their reusable components require careful cleaning. Here we introduce a versatile new microfluidics platform, a two-axis centrifuge whose fluidic chambers rotate in a planetary relationship to a radial gravitational field. This paradigm readily overcomes surface and viscous forces even in chambers only 50 microm thin. Thin chambers obviate the need for sample dilution and increase sensitivities and dynamic ranges 10-fold. In comparisons against conventional mixing using the same 10 microg of starting total RNA on 22 000-probe arrays, 10 000 more usable signals rose above the noise. In other experiments, planetary mixing was able to produce comparable results while using only one-tenth the starting sample. The benefits of planetary mixing include sample conservation, shorter hybridizations, less reliance on amplification, and the ability to quantify many gene signals otherwise obscured by noise.     

Principles of surface-directed liquid flow in microfluidic channels

To direct liquid flow inside microchannels, surface free energies were patterned by use of self-assembled monolayers (SAMs) in combination with either multistream laminar flow or photolithography. For the photolithographic method, two photocleavable SAMs were designed and synthesized. Carboxylic acid-terminated monolayers were obtained by photodeprotection, which was confirmed by contact angle and X-ray photoelectron spectroscopy. Using either of these patterning methods, we show that aqueous liquids flow only along the hydrophilic pathways when the pressure is maintained below a critical value; the liquids are referred to as being confined by virtual walls. Several principles of liquid flow in surface-patterned channels were derived analytically and verified experimentally. These principles include the maximum pressure that virtual walls can withstand, the critical width of the hydrophilic pathway that can support spontaneous flow, the smallest width of the liquid streams under an external pressure, the critical radius of curvature of turns that can be introduced into the hydrophilic pathway without liquid crossing the hydrophilic-hydrophobic boundary, and the minimal distance for two liquid streams to remain separated under the maximum pressure. Experimental results are in good agreement with the analytical predictions.     

3D focusing of nanoparticles in microfluidic channels

Dynamic focusing of particles can be used to centre particles in a fluid stream, ensuring the passage of the particles through a specified detection volume. This paper describes a method for focusing nanoparticles using dielectrophoresis. The method differs from other focusing methods in that it manipulates the particles and not the fluid. Experimental focusing is demonstrated for a range of different particle types, and discussed in terms of the operational limits of the device. Dynamic numerical simulations of the particle motion in the device are presented and compared with the experimental results. The potential of the device for nanoparticle control and manipulation in microfluidic chips is discussed.     

Immobilization of DNA hydrogel plugs in microfluidic channels

Acrylamide-modified DNA probes are immobilized in polycarbonate microfluidic channels via photopolymerization in a polyacrylamide matrix. The resulting polymeric, hydrogel plugs are porous under electrophoretic conditions and hybridize with fluorescently tagged complementary DNA. The double-stranded DNA can be chemically denatured, and the chip may be reused with a new analytical sample. Conditions for photopolymerization, hybridization, and denaturation are discussed. We also demonstrate the photopolymerization of plugs containing different DNA probe sequences in one microfluidic channel, thereby enabling the selective detection of multiple DNA targets in one electrophoretic pathway.     

Surface-directed, graft polymerization within microfluidic channels

We demonstrate a simple procedure to coat the surfaces of enclosed PDMS microchannels by UV-mediated graft polymerization. In prior applications, only disassembled channels could be coated by this method. This limited the utility of the method to coatings that could easily and tightly seal with themselves. By preadsorbing a photoinitiator onto the surface of PDMS microchannels, the rate of polymer formation at the surface was greatly accelerated compared to that in solution. Thus, a gel did not form in the lumen of enclosed microchannels. We demonstrate that the photoinitiator benzophenone remained on the surface of PDMS even after extensive washing. After addition of a variety of monomer solutions (acrylic acid, poly(ethylene glycol) monomethoxyl acrylate, or poly(ethylene glycol) diacrylate) and illumination with UV light, a stable, covalently attached surface coating formed in the microchannels. The electroosmotic mobility was stable in response to air exposure and to repeated cycles of hydration-dehydration of the coating. These surfaces also supported the electrophoretic separation of two model analytes. Placement of an opaque mask over a portion of the channel permitted photopatterning of the microchannels with a resolution of approximately 100 microm. By using an appropriate mixture of monomers combined with masks, it should be possible to fabricate PDMS microfluidic devices with distinct surface properties in different regions or channels.     

Recirculation of nanoliter volumes within microfluidic channels

A microfluidic device is described, capable of recirculating nanoliter volumes in restricted microchannel segments. The device consists of a PDMS microfluidic structure, reversibly sealed to a glass substrate with integrated platinum electrodes. The integrated electrodes generate electroosmotic flow locally, which results in a cycling flow in the channel segment between the two electrodes in case one channel exit is closed (dead-end channel). This cycling flow is a consequence of the counterbalancing hydrodynamic pressure against the electroosmotically generated flow. Acid-base indicators were employed to study the formation of H(+) and OH(-) at both the in-channel electrodes. The formation of acid can locally change the zeta-potential of the channel wall, which will affect the flow profile. Using this method, small analyte volumes can be mixed for prolonged times within well-defined channel segments and/or exposed to in-channel sensor surfaces.     

DNA displacement assay integrated into microfluidic channels

This paper describes the development of a unique fluorescence-based DNA diagnostic microfluidic assay that does not require labeling of the target sequence prior to analysis. The assay is based on the displacement of a short sacrificial fluorescent-tagged indicator oligomer by a longer untagged target sequence as it is electrophoresed through a DNA-containing hydrogel plug immobilized in a microfluidic channel. The distinct advantages of this assay are the short sensing times, as a result of directed electrophoretic transport of target DNA to the sensing element, combined with the ability to detect nonlabeled target DNA.     

Biomolecular recognition on well-characterized beads packed in microfluidic channels

We describe a new approach for the analysis of biomolecular recognition in microfluidic channels. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer (FRET). We establish a model system that detects the FLAG epitope. The assay can potentially detect subfemtomole quantities of antibody with a high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter-to-submicroliter volumes of analyte fluid. Kinetic and equilibrium constants for the reaction of this receptor-ligand pair are obtained through modeling of kinetic responses of the affinity microcolumn and are consistent with those obtained by flow cytometry. Because of the correlation between kinetic and equilibrium data obtained for the microcolumns, quantitative analysis can be done prior to the steady-state end point of the recognition reaction. This method has the promise of combining the utility of affinity chromatography with the advantage of direct, quantitative, and real-time analysis and the cost-effectiveness of microanalytical devices. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes and molecular assembly and microsystem-based multianalyte determinations.     

Patterning enzymes inside microfluidic channels via photoattachment chemistry

We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.     

Laser-induced fluorescence detection system for microfluidic chips based on an orthogonal optical arrangement

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45 degrees in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90 degrees. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N = 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building.     

Fabrication of spiral-shaped microfluidic channels in glass by femtosecond laser

Long spiral-shaped microfluidic channels in glass have been fabricated by femtosecond laser direct writing. After hydrofluoric acid etching and post baking, the laser modified regions in glass formed hollow microstructures. The diameter size and the screw-pitch of the channels can be set freely. The experimental results showed that the etched internal surface of the microchannel by hydrofluoric acid will become smoother after the subsequent baking. The incident laser power and scanning speed can also influence the channel quality.     

Carrier medium exchange through ultrasonic particle switching in microfluidic channels

This paper describes a method, utilizing acoustic force manipulation of suspended particles, in which particles in a laminar flow microchannel are continuously translated from one medium to another with virtually no mixing of the two media. During the study, 5-microm polyamide spheres suspended in distilled water, spiked (contaminated) with Evans blue, were switched over to clean distilled water. More than 95% of the polyamide spheres could be collected in the clean medium while removing up to 95% of the contaminant. Preliminary experiments to use this method to wash blood were performed. Red blood cells were switched from blood, spiked with Evans blue, to clean blood plasma. At least 95% of the red blood cells (bovine blood) could be collected in clean blood plasma while up to 98% of the contaminant was removed. The obtained results indicate that the presented method can be used as a generic method for particle washing and, more specifically, be applied for both intraoperative and postoperative blood washing.     

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.     

Fabrication of microfluidic reactors and mixing studies for luciferase detection

We report the detection of luciferase by implementing a bioluminescent assay in microfluidic reactors. The reactors were fabricated in poly(methyl methacrylate) by hot embossing using a mold master with the reactor layouts made by high-precision micromilling. The overall fabrication process was simple to implement and had a quick turnaround time with low cost. Two reactors, one with smooth channels (called reactor I) and the other with staggered herringbone mixers (called reactor II), were studied for the bioluminescent assay. The assay was implemented by introducing a sample and an assay solution into the reactors and then mixing took place to achieve the enzymatic reactions. We found that the mixing efficiency in reactor II was 17.8 times higher than reactor I. Theoretical analysis of the experimental results indicated that the required channel length of mixing was linearly proportional to the flow rate. A calibration curve for luciferase was obtained for both reactors. We found that the detection sensitivity of reactor II was 3 times higher than reactor I. The limit of detection in reactor II was determined to be 0.14 microg/mL luciferase. The device was further exploited to determine the concentration of luciferase samples obtained from in vitro protein expression.     

Coupled electrorotation of polymer microspheres for microfluidic sensing and mixing

We show that coupled electrorotation (CER) of microscopic particles using microfabricated electrodes can be used for localized sensing and mixing. The effective use of microelectromechanical systems and micro total analysis systems requires many types of control. These include the abilityto (1) manipulate objects within microchannels by noncontact means, (2) mix fluids, and (3) sense local chemical parameters. Coupled electrorotation, in which the interactions between induced electric dipoles of adjacent particles lead to particle rotation, addresses aspects of all three challenges simultaneously. CER is a simple means of controlling the rotation of dielectric objects using homogeneous external radio frequency electric fields. CER is sensitive to several chemical and physical parameters such as the solution conductivity, pH, and viscosity. As a step toward integrating CER devices into microfluidic systems, a simple chip was designed to induce local mixing and to detect local changes in salt concentration, pH, and viscosity.