Designing for chaos: applications of chaotic advection at the microscale

Chaotic advection can play an important role in efficient microfluidic mixers. We discuss a design paradigm that exploits chaotic advection and illustrate by two recent examples, namely enhancing gene expression profiling and constructing an in-line microfluidic mixing channel, how application of this paradigm has led to successful micromixers. We suggest that 'designing for chaos', that is, basing practical mixer design on chaotic advection analysis, is a promising approach to adopt in this developing field which otherwise has little to guide it and is constrained by issues of scale and manufacturability.     

Using bioinspired thermally triggered liposomes for high-efficiency mixing and reagent delivery in microfluidic devices

High-efficiency mixing is of fundamental importance for the successful development and application of lab-on-a-chip devices. In this report, we present the use of bioinspired thermally triggered liposomes for the controlled delivery and subsequent rapid mixing of reagents in a microfluidic device. In this technique, reagents are encapsulated inside the aqueous interior of liposomes that are dispersed evenly throughout a microfluidic system. Mixing of the encapsulated reagent and reaction do not occur until the reagent is released by a thermal trigger. This approach takes advantage of the dramatically increased lipid membrane permeability of liposomes near the gel-to-liquid phase transition temperature (T(m)) to deliver reagents at a precise location in the microfluidic device through the modulation of temperature. Implementation of this technique requires the encapsulation of the desired reagent in a liposome whose formulation has an appropriate T(m), as well as accurate spatial control of the temperature in the microfluidic device. As the liposomes are uniformly dispersed through the microfluidic channel, mixing occurs quite rapidly upon the release of the reagent. We demonstrate this technique by using several formulations of thermally triggered liposomes to release the hydrophilic fluorescent dyes at controlled locations in a polycarbonate microfluidic device. Additionally, we demonstrate a DNA labeling reaction using liposomes in a capillary-based microfluidic device. Under the conditions studied here, mixing and reaction are complete in approximately 200 microm of channel length. We believe this approach holds great promise for the performance of rapid high-throughput assays and in particular for biological analytes whose native environment is mimicked by the liposome.     

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.     

Monitoring a reaction at submillisecond resolution in picoliter volumes

Well-established rapid mixing techniques such as stopped-flow have been used to push the dead time for kinetic experiments down to a few milliseconds. However, very fast reactions are difficult to resolve below this limit. We now outline an approach that provides access to ultrafast kinetics but does not rely on active mixing at all. Here, the reagents are compartmentalized into water-in-oil emulsion microdroplets (diameter ～50 μm) that are statically arrayed in pairs, resting side-by-side in a well feature of a poly(dimethylsiloxane) (PDMS) device. A reaction between the contents of two droplets arrayed in such a holding trap is initiated by droplet fusion that is brought about by electrocoalescence and known to occur on a time scale of about 100 μs. A reaction between the reactants (Fe(3+) and SCN(-)) is monitored by image analysis measuring the product formation in the newly merged drop in both space and time, by use of a fast camera. A comparison of the concentration field of the reaction product with the output of a reaction-diffusion system of equations yields a rate constant k ～ 3 × 10(4) M(-3)·s(-1). Since reaction and diffusion are formally included in the mathematical model, measurements can proceed immediately after the drop fusion, removing the need to allow time for mixing. This approach is different from existing methodologies, for example, experiments in a conventional stopped-flow apparatus but also electrofusion of moving droplets where contents are mixed by chaotic advection before a reaction is monitored. Our analysis makes kinetics accessible that are several times faster than techniques that have to allow time for mixing.     

Transverse diffusion of laminar flow profiles to produce capillary nanoreactors

We introduce transverse diffusion of laminar flow profiles (TDLFP), the first generic method for mixing two or more reactants inside capillaries. Conceptually, solutions of reactants are injected inside the capillary by pressure as a series of consecutive plugs. Due to the laminar nature of flow inside the capillary, the nondiffused plugs have parabolic profiles with predominantly longitudinal interfaces between them. After injection, the plugs are mixed by transverse diffusion; longitudinal diffusion does not contribute to mixing. To prove the principle, we used TDLFP to mix two reactants-an enzyme and its substrate. After mixing the reactants by TDLFP, we incubated reaction mixtures for different periods of time and measured the reaction kinetics. We found that the reaction proceeded in time- and concentration-dependent fashion, thus confirming that the reactants were mixed by TDLFP. Remarkably, the experimental reaction kinetics were not only in qualitative agreement but also in good quantitative agreement with theoretically predicted ones. TDLFP has a number of enabling features. By facilitating the preparation of reaction mixtures inside the capillary, TDLFP lowers reagent consumption to nanoliters (microliters are required for conventionally mixing reagents in a vial). The reaction products can be then analyzed "on-line" by capillary separation coupled with optical, electrochemical, or mass spectrometric detection. The combination of TDLFP with capillary separation will be an indispensable tool in screening large combinatorial libraries for affinity probes and drug candidates: a few microliters of a target protein will be sufficient to screen thousands of compounds. The new method paves the road to a wide use of capillary nanoreactors in different areas of physical and life sciences.     

Mixing crowded biological solutions in milliseconds

In vitro studies of biological reactions are rarely performed in conditions that reflect their native intracellular environments where macromolecular crowding can drastically change reaction rates. Kinetics experiments require reactants to be mixed on a time scale faster than that of the reaction. Unfortunately, highly concentrated solutions of crowding agents such as bovine serum albumin and hemoglobin that are viscous and sticky are extremely difficult to mix rapidly. We demonstrate a new droplet-based microfluidic mixer that induces chaotic mixing of crowded solutions in milliseconds due to protrusions of the microchannel walls that generate oscillating interfacial shear within the droplets. Mixing in the microfluidic mixer is characterized, mechanisms underlying mixing are discussed, and evidence of biocompatibility is presented. This microfluidic platform will allow for the first kinetic studies of biological reactions with millisecond time resolution under conditions of macromolecular crowding similar to those within cells.     

Parameters influencing pulsed flow mixing in microchannels

The rapid mixing of reagents is a crucial step for on-chip chemical and biological analysis. It has been recently suggested that microfluidic mixing can be greatly enhanced by simply using time pulsing of the incoming flow rates of the two fluids to be mixed (Glasgow, I.; Aubry, N. Lab Chip 2003, 3, 114-120). This paper elaborates on the latter technique, showing through computational fluid dynamics how the mixing efficiency strongly depends on certain dimensionless parameters of the system, while remaining nearly insensitive to others. In particular, it is demonstrated that higher Strouhal numbers (ratio of flow characteristic time scale to the pulsing time period) and pulse volume ratios (ratio of the volume of fluid pulsed to the volume of inlet/outlet intersection) lead to better mixing. This paper also presents a physical device capable of mixing two reagents using pulsing, which shows improved mixing with greater values of the Strouhal number.     

Reagent-loaded cartridges for valveless and automated fluid delivery in microfluidic devices

An important problem in the life sciences and in health care is simple and rapid detection of biomarkers. Although microfluidic devices are potentially useful in addressing this problem, current techniques for automating fluid delivery--which include valves and electroosmosis--require sophisticated microfabrication of the chip, bulky instrumentation, or both. In this paper, we describe a simple and reliable technique for storing and delivering a sequence of reagents to a microfluidic device. The technique is low-cost, requires minimal user intervention, and can be performed in resource-poor settings (e.g., outside of a laboratory) in the absence of electricity and computer-controlled equipment. In this method, cartridges made of commercially available tubing are filled by sequentially injecting plugs of reagents separated by air spacers. The air spacers prevent the reagents from mixing with each other during cartridge preparation, storage, and usage. As an example, we used this "plug-in cartridge" technology to complete a solid-phase immunoassay in a microchannel in 2 min with low-nanomolar sensitivity and demonstrate the diagnosis of HIV in 13 min.     

A chaotic electroosmotic stirrer

Two-dimensional, time-independent, and time-dependent electroosmotic flows driven by a uniform electric field in a conduit with nonuniform zeta potential distributions along its walls are investigated theoretically. The time-independent flow fields are computed with the aid of Fourier series. The series' convergence is accelerated so that highly accurate solutions are obtained with just a few terms in the series. The analytic solution is used to compute flow patterns for various distributions of the zeta potential along the conduit's boundaries. Subsequently, it is demonstrated that by time-wise periodic alternations of the zeta potentials, one can induce chaotic advection. This chaotic flow can be used to efficiently stir and mix fluids in microfluidic devices.     

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.     

Processing of nanolitre liquid plugs for microfluidic cell-based assays

Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.     

Optimization of a Microfluidic Mixing Process for Gene Expression-Based Bio-dosimetry

In recent decades advances in radiation imaging and radiation therapy have led to a dramatic increase in the number of people exposed to radiation. Consequently, there is a clear need for personalized biodosimetry diagnostics in order to monitor the dose of radiation received and adapt it to each patient depending on their sensitivity to radiation exposure (Hall E.J. and Brenner D. J., 2008). Similarly, after a large-scale radiological event such as a dirty bomb attack, there will be a major need to assess, within a few days the radiation doses received by tens of thousands of individuals. Current high throughput devices can handle only a few hundred individuals per day. Hence there is a great need for a very fast self-contained non-invasive biodosimetric device based on a rapid blood test.This paper presents a case study where regression methods and designed experiments are used to arrive at the optimal settings for various factors that impact the kinetics in a biodosimetric device. We use ridge regression to initially identify a set of potentially important variables in the mixing process which is one of the critical sub systems of the device. This was followed by a series of designed experiments to arrive at the optimal setting of the significant microfluidic cartridge and piezoelectric disk (PZT) (D. Sadler, F. Zenhausern, U.S. Patent 6,986,601; Lee, S. Y., Ko, B., Yang, W., 2005) related factors. This statistical approach has been utilized to study the microfluidic mixing to mix water and dye mixtures of 70 μl volume. The outcome of the statistical design, experimentation and analysis was then exploited for optimizing the design, fabrication and assembly of the microfluidic devices. As a result of the experiments that were performed, the system was fine tuned and the mixing time was reduced from 5.5 minutes to 2 minutes.     

Processing of nanolitre liquid plugs for microfluidic cell-based assays

Abstract

Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.     

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.     

Using a multijunction microfluidic device to inject substrate into an array of preformed plugs without cross-contamination: comparing theory and experiments

In this paper we describe a multijunction microfluidic device for the injection of a substrate into an array of preformed plugs carried by an immiscible fluid in a microchannel. The device uses multiple junctions to inject substrate into preformed plugs without synchronization of the flow of substrate and the array of preformed plugs of reagent, which reduces cross-contamination of the plugs, eliminates formation of small droplets of substrate, and allows a greater range of injection ratios compared to that of a single T-junction. The device was based on a previously developed physical model for transport that was here adapted to describe injection and experimentally verified. After characterization, the device was applied to two biochemical assays, including evaluation of the enzymatic activity of thrombin and determination of the coagulation time of human blood plasma, which both provided reliable results. The reduction of cross-contamination and greater range of injection ratios achieved by this device may improve the processes that involve addition and titration of reagents into plugs, such as high-throughput screening of protein crystallization conditions.     

Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing

Rapid detection and identification of influenza virus is becoming increasingly important in the face of concerns over an influenza pandemic. A fully integrated and self-contained microfluidic device has been developed to rapidly identify influenza A hemagglutinin and neuraminidase subtypes and sequence portions of both genes. The device consists of a DNA microarray with 12 000 features and a microfluidic cartridge that automates the fluidic handling steps required to carry out a genotyping assay for pathogen identification and sequencing. The fully integrated microfluidic device consists of microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross talk of the stored reagents. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows the detection and identification of influenza virus in a rapid and automated fashion.     

On-chip titration of an anticoagulant argatroban and determination of the clotting time within whole blood or plasma using a plug-based microfluidic system

This paper describes extending plug-based microfluidics to handling complex biological fluids such as blood, solving the problem of injecting additional reagents into plugs, and applying this system to measuring of clotting time in small volumes of whole blood and plasma. Plugs are droplets transported through microchannels by fluorocarbon fluids. A plug-based microfluidic system was developed to titrate an anticoagulant (argatroban) into blood samples and to measure the clotting time using the activated partial thromboplastin time (APTT) test. To carry out these experiments, the following techniques were developed for a plug-based system: (i) using Teflon AF coating on the microchannel wall to enable formation of plugs containing blood and transport of the solid fibrin clots within plugs, (ii) using a hydrophilic glass capillary to enable reliable merging of a reagent from an aqueous stream into plugs, (iii) using bright-field microscopy to detect the formation of a fibrin clot within plugs and using fluorescent microscopy to detect the production of thrombin using a fluorogenic substrate, and (iv) titration of argatroban (0-1.5 microg/mL) into plugs and measurement of the resulting APTTs at room temperature (23 degrees C) and physiological temperature (37 degrees C). APTT measurements were conducted with normal pooled plasma (platelet-poor plasma) and with donor's blood samples (both whole blood and platelet-rich plasma). APTT values and APTT ratios measured by the plug-based microfluidic device were compared to the results from a clinical laboratory at 37 degrees C. APTT obtained from the on-chip assay were about double those from the clinical laboratory but the APTT ratios from these two methods agreed well with each other.     

Automated microfluidic screening assay platform based on DropLab

This paper describes DropLab, an automated microfluidic platform for programming droplet-based reactions and screening in the nanoliter range. DropLab can meter liquids with picoliter-scale precision, mix multiple components sequentially to assemble composite droplets, and perform screening reactions and assays in linear or two-dimensional droplet array with extremely low sample and reagent consumptions. A novel droplet generation approach based on the droplet assembling strategy was developed to produce multicomponent droplets in the nanoliter to picoliter range with high controllability on the size and composition of each droplet. The DropLab system was built using a short capillary with a tapered tip, a syringe pump with picoliter precision, and an automated liquid presenting system. The tapered capillary was used for precise liquid metering and mixing, droplet assembling, and droplet array storage. Two different liquid presenting systems were developed based on the slotted-vial array design and multiwell plate design to automatically present various samples, reagents, and oil to the capillary. Using the tapered-tip capillary and the picoliter-scale precision syringe pump, the minimum unit of the droplet volume in the present system reached ~20 pL. Without the need of complex microchannel networks, various droplets with different size (20 pL-25 nL), composition, and sequence were automatically assembled, aiming to multiple screening targets by simply adjusting the types, volumes, and mixing ratios of aspirated liquids on demand. The utility of DropLab was demonstrated in enzyme inhibition assays, protein crystallization screening, and identification of trace reducible carbohydrates.     

Sequential Coalescence Enabled Two‐Step Microreactions in Triple‐Core Double‐Emulsion Droplets Triggered by an Electric Field

Advances in microfluidic emulsification have enabled the generation of exquisite multiple‐core droplets, which are promising structures to accommodate microreactions. An essential requirement for conducting reactions is the sequential coalescence of the multiple cores encapsulated within these droplets, therefore, mixing the reagents together in a controlled sequence. Here, a microfluidic approach is reported for the conduction of two‐step microreactions by electrically fusing three cores inside double‐emulsion droplets. Using a microcapillary glass device, monodisperse water‐in‐oil‐in‐water droplets are fabricated with three compartmented reagents encapsulated inside. An AC electric field is then applied through a polydimethylsiloxane chip to trigger the sequential mixing of the reagents, where the precise sequence is guaranteed by the discrepancy of the volume or conductivity of the inner cores. A two‐step reaction in each droplet is ensured by two times of core coalescence, which totally takes 20–40 s depending on varying conditions. The optimal parameters of the AC signal for the sequential fusion of the inner droplets are identified. Moreover, the capability of this technique is demonstrated by conducting an enzyme‐catalyzed reaction used for glucose detection with the double‐emulsion droplets. This technique should benefit a wide range of applications that require multistep reactions in micrometer scale.     

A digital microfluidic system for the investigation of pre-steady-state enzyme kinetics using rapid quenching with MALDI-TOF mass spectrometry

A digital microfluidic system based on electrowetting has been developed to facilitate the investigation of pre-steady-state reaction kinetics using rapid quenching and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The device consists of individually addressable electrodes arranged to allow the combination of liquid droplets at well-defined time intervals and an integrated, electrohydrodynamically driven mixer. The device combines two droplets to initiate a reaction, then, with precise timing, combines a third droplet to quench the reaction, and finally combines a fourth droplet to form a matrix. Improvements to throughput when compared to traditional laboratory-scale methods, and previous MALDI-TOF MS digital microfluidic systems, were made. The device was tested against a model protein tyrosine phosphatase system, and results agreed well with published data. The system therefore allows for the analysis of reaction kinetics that were previously too rapid to analyze using MALDI-TOF MS.