A “twisted” microfluidic mixer suitable for a wide range of flow rate applications

This paper proposes a new “twisted” 3D microfluidic mixer fabricated by a laser writing/microfabrication technique. Effective and efficient mixing using the twisted micromixers can be obtained by combining two general chaotic mixing mechanisms: splitting/recombining and chaotic advection. The lamination of mixer units provides the splitting and recombination mechanism when the quadrant of circles is arranged in a two-layered serial arrangement of mixing units. The overall 3D path of the microchannel introduces the advection. An experimental investigation using chemical solutions revealed that these novel 3D passive microfluidic mixers were stable and could be operated at a wide range of flow rates. This micromixer finds application in the manipulation of tiny volumes of liquids that are crucial in diagnostics. The mixing performance was evaluated by dye visualization, and using a pH test that determined the chemical reaction of the solutions. A comparison of the tornado-mixer with this twisted micromixer was made to evaluate the efficiency of mixing. The efficiency of mixing was calculated within the channel by acquiring intensities using ImageJ software. Results suggested that efficient mixing can be obtained when more than 3 units were consecutively placed. The geometry of the device, which has a length of 30 mm, enables the device to be integrated with micro total analysis systems and other lab-on-chip devices.     

Effect of the crossing-structure sequence on mixing performance within three-dimensional micromixers

The geometry of crossing structure formed by two-layer microchannels determines the axial and transverse movements of contact interface between two liquid streams, which gives us a new method for promoting the micromixers. Hence, we designed four different three-dimensional micromixers by selecting two different crossing structures as basic units (one unit is a crossing structure called “X” and the other is a reversed crossing structure called “rX”). In order to find out how the crossing-structure sequence affects the mixing performance within three-dimensional micromixers, we organized these four mixers in different ways, i.e., the first combination is X-rX-X-rX-…, the second is X-rX-rX-X-…, the third is X-X-rX-X-…, and the last one is X-X-X-X…. Consequently, quite distinct mixing phenomena are engendered. Furthermore, experiments were also conducted using the first and the last models to verify the simulation results. We infer that the last mixer is more likely to trigger chaos and convection by rotating the contact surface than the first one that merely swings the surface even when the flow rates and viscosities of the two liquid streams are increased.     

Novel index for micromixing characterization and comparative analysis

The most basic micromixer is a T- or Y-mixer, where two confluent streams mix due to transverse diffusion. To enhance micromixing, various modifications of T-mixers are reported such as heterogeneously charged walls, grooves on the channel base, geometric variations by introducing physical constrictions, etc. The performance of these reported designs is evaluated against the T-mixer in terms of the deviation from perfectly mixed state and mixing length (device length required to achieve perfect mixing). Although many studies have noticed the reduced flow rates for improved mixer designs, the residence time is not taken into consideration for micromixing performance evaluation. In this work, we propose a novel index, based on residence time, for micromixing characterization and comparative analysis. For any given mixer, the proposed index identifies the nondiffusive mixing enhancement with respect to the T-mixer. Various micromixers are evaluated using the proposed index to demonstrate the usefulness of the index. It is also shown that physical constriction mixer types are equivalent to T-mixers. The proposed index is found to be insightful and could be used as a benchmark for comparing different mixing strategies.     

Focusing-enhanced mixing in microfluidic channels

A focusing-based microfluidic mixer was studied. The micromixer utilizes the focusing process required for cytometry to reduce the diffusion distance of molecules to be mixed in order to facilitate the passive diffusion-controlled mixing process. It was found that both the high flow rate ratio of the sheath flow to the flows to be mixed and the low flow rate of the mixing fluids resulted in the short mixing length required within the microfluidic channel. It was shown that a complete mixing was achieved within a distance of 4 mm in the micromixer for the focused mixing fluids at a flow rate of 2 μl∕min and a flow rate ratio of the sheath flow to the flows to be mixed at 4:1. The mixer described here is simple and can be easily fabricated and controlled.     

Micromixer based on viscoelastic flow instability at low Reynolds number

We exploited the viscoelasticity of biocompatible dilute polymeric solutions, namely, dilute poly(ethylene oxide) solutions, to significantly enhance mixing in microfluidic devices at a very small Reynolds number, i.e., Re≈0.023, but large Peclet and elasticity numbers. With an abrupt contraction microgeometry (8:1 contraction ratio), two different dilute poly(ethylene oxide) solutions were successfully mixed with a short flow length at a relatively fast mixing time of <10 μs. Microparticle image velocimetry was employed in our investigations to characterize the flow fields. The increase in velocity fluctuation with an increase in flow rate and Deborah number indicates the increase in viscoelastic flow instability. Mixing efficiency was characterized by fluorescent concentration measurements. Our results showed that enhanced mixing can be achieved through viscoelastic flow instability under situations where molecular-diffusion and inertia effects are negligible. This approach bypasses the laminar flow limitation, usually associated with a low Reynolds number, which is not conducive to mixing.     

On-chip recalcification of citrated whole blood using a microfluidic herringbone mixer

In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.     

Quantitative characterization of micromixing simulation

Micromixers with floor-grooved microfluidic channels have been successfully demonstrated in experiment. In this work, we numerically simulated the mixing within the devices and used the obtained concentration versus channel length profiles to quantitatively characterize the process. It was found that the concentration at any given cross-section location of the microfluidic channel periodically oscillates along the channel length, in coordination with the groove-caused helical flow during the mixing, and eventually converges to the neutral concentration value of two the mixing fluids. With these data, the specific channel length required for each helical flow to complete, the mixing efficiency of the devices, and the total channel length required to complete a mixing were easily defined and quantified, and were used to directly and comprehensively characterize the micromixing. This concentration versus channel length profile-based characterization method was also demonstrated in quantitatively analyzing the micromixing within a classic T mixer. It has clear advantages over the traditional concentration image-based characterization method that is only able to provide qualitative or semiquantitative information about a micromixing, and is expected to find an increasing use in studying mixing and optimizing device structure through numerical simulations.     

Linear conversion of pressure into concentration, rapid switching of concentration, and generation of linear ramps of concentration in a microfluidic device

Mixing of liquids to produce solutions with different concentrations is one of the basic functionalities of microfluidic devices. Generation of specific temporal patterns of concentration in microfluidic devices is an important technique to study responses of cells and model organisms to variations in the chemical composition of their environment. Here, we present a simple microfluidic network that linearly converts pressure at an inlet into concentration of a soluble reagent in an observation region and also enables independent concurrent linear control of concentrations of two reagents. The microfluidic device has an integrated mixer channel with chaotic three-dimensional flow that facilitates rapid switching of concentrations in a continuous range. A simple pneumatic setup generating linear ramps of pressure is used to produce smooth linear ramps and triangular waves of concentration with different slopes. The use of chaotic vs. laminar mixers is discussed in the context of microfluidic devices providing rapid switching and generating temporal waves of concentration.     

An efficient micromixer based on multidirectional vortices due to baffles and channel curvature

An efficient planar micromixer based on multidirectional vortices in a curved channel with radial baffles is proposed and examined in this work. The curvature of the microchannel and the radial baffles induce vortices in different directions. The multidirectional vortices and the converging-diverging flow caused by the baffles contribute together to the enhancement of mixing. The micromixer is fabricated with polydimethylsiloxane by a single planar microlithography process and the mixing behaviors are observed by a confocal spectral microscope imaging system to validate the simulation obtained by a commercial code. The simulation and experimental results are in reasonable agreement. The concentration distributions and flow patterns obtained reveal the following trends. (i) The mixing efficiency of the basic C-shaped micromixer with the first baffle attached to the internal cylinder and the second attached to the external cylinder is better than that of the C-shaped micromixer with inverted arrangement of baffles. (ii) When the radius of the curved channel and the width of the passage between the baffle and the cylindrical wall are small enough and the Reynolds number (Re) is large enough, an extra separation vortex develops in the downstream of the second baffle. This phenomenon is one of the reasons of trend (i). (iii) A micromixer consisting of a few basic C-shaped micromixers connected by straight channels may generate a high degree of mixing for the case with a large Re.     

Sorting of circulating tumor cells (MV3-melanoma) and red blood cells using non-inertial lift

We demonstrate the method of non-inertial lift induced cell sorting (NILICS), a continuous, passive, and label-free cell sorting approach in a simple single layer microfluidic device at low Reynolds number flow conditions. In the experiments, we exploit the non-inertial lift effect to sort circulating MV3-melanoma cells from red blood cell suspensions at different hematocrits as high as 9%. We analyze the separation process and the influence of hematocrit and volume flow rates. We achieve sorting efficiencies for MV3-cells up to E_MV3 = 100% at Hct = 9% and demonstrate cell viability by recultivation of the sorted cells.     

Analysis of a laminar-flow diffusional mixer for directed self-assembly of liposomes

The present work describes the operation and simulation of a microfluidic laminar-flow mixer. Diffusive mixing takes place between a core solution containing lipids in ethanol and a sheath solution containing aqueous buffer, leading to self assembly of liposomes. Present device architecture hydrodynamically focuses the lipid solution into a cylindrical core positioned at the center of a microfluidic channel of 125 × 125-μm² cross-section. Use of the device produces liposomes in the size range of 100–300 nm, with larger liposomes forming at greater ionic strength in the sheath solution and at lower lipid concentration in the core solution. Finite element simulations compute the concentration distributions of solutes at axial distances of greater than 100 channel widths. These simulations reduce computation time and enable computation at long axial distances by utilizing long hexahedral elements in the axial flow region and fine tetrahedral elements in the hydrodynamic focusing region. Present meshing technique is generally useful for simulation of long microfluidic channels and is fully implementable using comsol Multiphysics. Confocal microscopy provides experimental validation of the simulations using fluorescent solutions containing fluorescein or enhanced green fluorescent protein.     

Studying enzymatic bioreactions in a millisecond microfluidic flow mixer

In this study, the pre-steady state development of enzymatic bioreactions using a microfluidic mixer is presented. To follow such reactions fast mixing of reagents (enzyme and substrate) is crucial. By using a highly efficient passive micromixer based on multilaminar flow, mixing times in the low millisecond range are reached. Four lamination layers in a shallow channel reduce the diffusion lengths to a few micrometers only, enabling very fast mixing. This was proven by confocal fluorescence measurements in the channel’s cross sectional area. Adjusting the overall flow rate in the 200 μm wide and 900 μm long mixing and observation channel makes it possible to investigate enzyme reactions over several seconds. Further, the device enables changing the enzyme/substrate ratio from 1:1 up to 3:1, while still providing high mixing efficiency, as shown for the enzymatic hydrolysis using β-galactosidase. This way, the early kinetics of the enzyme reaction at multiple enzyme/substrate concentrations can be collected in a very short time (minutes). The fast and easy handling of the mixing device makes it a very powerful and convenient instrument for millisecond temporal analysis of bioreactions.     

Effect of a dual inlet channel on cell loading in microfluidics

Unwanted sedimentation and attachment of a number of cells onto the bottom channel often occur on relatively large-scale inlets of conventional microfluidic channels as a result of gravity and fluid shear. Phenomena such as sedimentation have become recognized problems that can be overcome by performing microfluidic experiments properly, such as by calculating a meaningful output efficiency with respect to real input. Here, we present a dual-inlet design method for reducing cell loss at the inlet of channels by adding a new “ upstream inlet ” to a single main inlet design. The simple addition of an upstream inlet can create a vertically layered sheath flow prior to the main inlet for cell loading. The bottom layer flow plays a critical role in preventing the cells from attaching to the bottom of the channel entrance, resulting in a low possibility of cell sedimentation at the main channel entrance. To provide proof-of-concept validation, we applied our design to a microfabricated flow cytometer system (μFCS) and compared the cell counting efficiency of the proposed μFCS with that of the previous single-inlet μFCS and conventional FCS. We used human white blood cells and fluorescent microspheres to quantitatively evaluate the rate of cell sedimentation in the main inlet and to measure fluorescence sensitivity at the detection zone of the flow cytometer microchip. Generating a sheath flow as the bottom layer was meaningfully used to reduce the depth of field as well as the relative deviation of targets in the z-direction (compared to the x-y flow plane), leading to an increased counting sensitivity of fluorescent detection signals. Counting results using fluorescent microspheres showed both a 40% reduction in the rate of sedimentation and a 2-fold higher sensitivity in comparison with the single-inlet μFCS. The results of CD4⁺ T-cell counting also showed that the proposed design results in a 25% decrease in the rate of cell sedimentation and a 28% increase in sensitivity when compared to the single-inlet μFCS. This method is simple and easy to use in design, yet requires no additional time or cost in fabrication. Furthermore, we expect that this approach could potentially be helpful for calculating exact cell loading and counting efficiency for a small input number of cells, such as primary cells and rare cells, in microfluidic channel applications.     

Micromixing visualization and quantification in a microscale multi-inlet vortex nanoprecipitation reactor using confocal-based reactive micro laser-induced fluorescence

A technique for visualizing and quantifying reactive mixing for laminar and turbulent flow in a microscale chemical reactor using confocal-based microscopic laser induced fluorescence (confocal μ-LIF) was demonstrated in a microscale multi-inlet vortex nanoprecipitation reactor. Unlike passive scalar μ-LIF, the reactive μ-LIF technique is able to visualize and quantify micromixing effects. The confocal imaging results indicated that the flow in the reactor was laminar and steady for inlet Reynolds numbers of 10, 53, and 93. Mixing and reaction were incomplete at each of these Reynolds numbers. The results also suggested that although mixing by diffusion was enhanced near the midplane of the reactor at Re_j = 53 and 93 due to very thin bands of acidic and basic fluid forming as the fluid spiraled towards the center of the reactor, near the top, and bottom walls of the reactor, the lower velocities due to fluid friction with the walls hindered the formation of these thin bands, and, thus, resulted in large regions of unmixed and unreacted fluid. At Re_j = 240, the flow was turbulent and unsteady. The mixing and reaction processes were still found to be incomplete even at this highest Reynolds number. At the reactor midplane, the flow images at Re_j = 240 showed unmixed base fluid near the center of the reactor, suggesting that just as in the Re_j = 53 and 93 cases, lower velocities near the top and bottom walls of the reactor hinder the mixing and rection of the acidic and basic streams. Ensemble averages of line-scan profiles for the Re_j = 240 were then calculated to provide statistical quantification of the microscale mixing in the reactor. These results further demonstrate that even at this highest Reynolds number investigated, mixing and reaction are incomplete. Visualization and quantification of micromixing using this reactive μ-LIF technique can prove useful in the validation of computational fluid dynamics models of micromixing within microscale chemical reactors.     

Lab-on-a-chip workshop activities for secondary school students

The ability to engage and inspire younger generations in novel areas of science is important for bringing new researchers into a burgeoning field, such as lab-on-a-chip. We recently held a lab-on-a-chip workshop for secondary school students, for which we developed a number of hands-on activities that explained various aspects of microfluidic technology, including fabrication (milling and moulding of microfluidic devices, and wax printing of microfluidic paper-based analytical devices, so-called μPADs), flow regimes (gradient formation via diffusive mixing), and applications (tissue analysis and μPADs). Questionnaires completed by the students indicated that they found the workshop both interesting and informative, with all activities proving successful, while providing feedback that could be incorporated into later iterations of the event.     

Mixing enhancement in microfluidic channel with a constriction under periodic electro-osmotic flow

We present a new approach to enhance mixing in T-type micromixers by introducing a constriction in the microchannel under periodic electro-osmotic flow. Two sinusoidal ac electric fields with 180° phase difference and similar dc bias are applied at the two inlets. The out of phase ac electric field induces oscillation of fluid interface at the junction of the two inlet channels and the constriction. Due to the constriction introduced at the junction, fluids from these two inlets form alternative plugs at the constricted channel. These plugs of fluids radiate downstream from the constriction into the large channel and form alternate thin crescent-shaped layers of fluids. These crescent-shaped layers of fluids increase tremendously the contact surface area between the two streams of fluid and thus enhance significantly the mixing efficiency. Experimental results and mixing mechanism analysis show that amplitude and frequency of the ac electric field and the length of the constriction govern the mixing efficiency.     

Visualizing millisecond chaotic mixing dynamics in microdroplets: A direct comparison of experiment and simulation

In order to fully explore and utilize the advantages of droplet-based microfluidics, fast, sensitive, and quantitative measurements are indispensable for the diagnosis of biochemical reactions in microdroplets. Here, we report an optical detection technique using two-photon fluorescence lifetime imaging microscopy, with an aligning-summing and non-fitting division method, to depict two-dimensional (2D) maps of mixing dynamics by chaotic advection in microdroplets with high temporal and spatial resolution. The mixing patterns of two dye solutions inside droplets were quantitatively and accurately measured. The mixing efficiency in a serpentine droplet mixer was also quantified and compared with the simulation data. The mapped chaotic mixing dynamics agree well with the numerical simulation and theoretical prediction. This quantitative characterization is potentially applicable to the real-time kinetic study of biological and chemical reactions in droplet-based microfluidic systems.     

A microfluidic chip for direct and rapid trapping of white blood cells from whole blood

Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 10³ from 3.3 × 10³ WBCs.     

Study on the use of dielectrophoresis and electrothermal forces to produce on-chip micromixers and microconcentrators

The present study uses the dielectrophoresis (DEP) and electrothermal (ET) forces to develop on-chip micromixers and microconcentrators. A microchannel with rectangular array of microelectrodes, patterned either on its bottom surface only or on both the top and the bottom surfaces, is considered for the analysis. A mathematical model to compute electrical field, temperature field, the fluid velocity, and the concentration distributions is developed. Both analytical and numerical solutions of standing wave DEP (SWDEP), traveling wave DEP (TWDEP), standing wave ET (SWET), and traveling wave ET (TWET) forces along the length and the height of the channel are compared. The effects of electrode size and their placement in the microsystem on micromixing and microconcentrating performance are studied and compared to velocity and concentration profiles. SWDEP forces can be used to collect the particles at different locations in the microchannel. Under positive and negative DEP effect, the particles are collected at electrode edges and away from the electrodes, respectively, irrespective of the position, size, and number of electrodes. The location of the concentration region can be shifted by changing the electrode position. SWET and TWET forces are used for mixing and producing concentration regions by circulating the fluid at a given location. The effect of forces can be changed with the applied voltage. The TWDEP method is the better method for mixing along the length of the channels among the four options explored in the present work. If two layers of particle suspension are placed side by side in the channel, triangular electrode configuration can be used to mix the suspensions. Triangular and rectangular electrode configurations can efficiently mix two layers of particle suspension placed side-by-side and one-atop-the-other, respectively. Hence, SWDEP forces can be successfully used to create microconcentrators, whereas TWDEP, SWET, and TWET can be used to produce efficient micromixers in a microfluidic chip.     

Enhanced H-filter based on F?hr?us-Lindqvist effect for efficient and robust dialysis without membrane

A novel microfluidic device for highly efficient and robust dialysis without membrane is highly desired for the development of portable or wearable microdialyzer. Here we report an enhanced H-filter with pillar array based on Fåhræus-Lindqvist effect (F-L effect) for highly efficient and robust membraneless dialysis of simplified blood for the first time. The H-filter employs two fluids laminarly flowing in the microchannel for continuously membraneless dialysis. With pillar array in the microchannel, the two laminar flows, with one containing blood cells and small molecules and another containing dialyzate solution, can form a cell-free layer at the interface as selective zones for separation. This provides enhanced mixing yet extremely low shear for extraction of small molecules from the blood-cell-containing flow into the dialyzate flow, resulting in robust separation with reduced cell loss and improved efficiency. We demonstrate this by first using Chlorella pyrenoidosa as model cells to quantitatively study the separation performances, and then using simplified human blood for dialysis. The advanced H-filter, with highly efficient and robust performance for membraneless dialysis, shows great potential as promising candidate for rapid blood analysis/separation, and as fundamental structure for portable dialyzer.