On-demand patterning of protein matrixes inside a microfluidic device

On-demand immobilization of proteins at specific locations in a microfluidic device would advance many types of bioassays. We describe a strategy to create a patterned surface within a microfluidic channel by electrochemical means, which enables site-specific immobilization of protein matrixes and cells under physiological conditions, even after the device is fully assembled. By locally generating hypobromous acid at a microelectrode in the microchannel, the heparin-coated channel surface rapidly switches from antibiofouling to protein-adhering. Since this transformation allows compartmentalizing of multiple types of antibodies into distinct regions throughout the single microchannel, simultaneous assay of two kinds of complementary proteins was possible. This patterning procedure can be applied to conventional microfluidic devices since it requires only some electrodes and a voltage source (1.7 V, DC).     

Microfluidic particle sorter employing flow splitting and recombining

This paper describes an improved microfluidic device that enables hydrodynamic particle concentration and size-dependent separation to be carried out in a continuous manner. In our previous study, a method for hydrodynamic filtration and sorting of particles was proposed using a microchannel having multiple branch points and side channels, and it was applied for continuous concentration and separation of polymer particles and cells. In the current study, the efficiency of particle sorting was dramatically improved by geometrically splitting fluid flow from a main stream and recombining. With these operations, particles with diameters larger than a specific value move toward one sidewall in the mainstream. This control of particle positions is followed by the perfect particle alignment onto the sidewall, which increases the selectivity and recovery rates without using a liquid that does not contain particles. In this study, a microchannel having one inlet and five outlets was designed and fabricated. By simply introducing particle suspension into the device, concentrations of 2.1-3.0-microm particles were increased 60-80-fold, and they were collected independently from each outlet. In addition, it was demonstrated that the measured flow rates distributed into each side channel corresponded well to the theoretical values when regarding the microchannel network as a resistive circuit.     

Sheathless hydrophoretic particle focusing in a microchannel with exponentially increasing obstacle arrays

We present a novel microfluidic device with exponentially increasing obstacle arrays to enable sheathless particle focusing. The anisotropic fluidic resistance of slant obstacles generates transverse flows, along which particles are focused to one sidewall. In the successive channel with exponentially increasing widths, bent obstacles extended from the slant obstacles increase the focusing efficiency of the particles. With the device, we achieved the focusing efficiency of 76%, 94%, and 98% for 6, 10, and 15 microm beads, respectively. The focusing efficiency of the particles can be further improved in the devices with more extension steps. In addition, using the microfluidic devices with the symmetric structure of the slant and bent obstacles, we achieved complete focusing of biological cells to the centerline of a channel within 1.7% coefficient of variation. The results demonstrated the sheathless hydrophoretic focusing of microparticles and cells with the advantages of a sheathless method, passive operation, single channel, and flow rate independence.     

Generating nonlinear concentration gradients in microfluidic devices for cell studies

We describe a microfluidic device for generating nonlinear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for coflowing multiple aqueous solutions, a main coflow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the coflow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H(2)O(2)) concentrations.     

Microfluidic Design of Magnetoresponsive Photonic Microcylinders with Multicompartments

Colloidal photonic structures have been designed to have granular format to use them for paint pigments, encoded carriers, and display pixels. However, conventional approaches only provide spherical or discoid shapes, restricting their applications. Cylindrical granules with fan‐shaped compartments in the cross section are appealing for microcarriers with abundant optical codes and active display pigments for color switching. In this work, a stratified laminar flow of concentrated silica particles is employed, formed in a cylindrical microchannel, to produce cylindrical photonic microparticles with multiple compartments. To accomplish this, a microfluidic device is designed to have one cylindrical main channel connected with four branch channels. Four different photocurable suspensions are independently injected through the branches to form quarter‐cylindrically compartmentalized streams in the main channel. Local ultraviolet irradiation on the main channel polymerizes the suspension, thereby forming cylindrical microparticles with four compartments. In each compartment, silica particles form ordered array which develops particle size–dependent structural color. Therefore, different colors can be incorporated into single microcylinder by employing different sizes of silica particles. Moreover, one of the compartments can be rendered to be magnetoresponsive by embedding aligned magnetic particles, which enables the remote control of microcylinder orientation and therefore the switching of structural colors.     

Control of flow direction in microfluidic devices with polyelectrolyte multilayers

Electroosmotic flow (EOF) is commonly utilized in microfluidics. Because the direction of the EOF can be determined by the substrate surface charge, control of the surface chemical state offers the potential, in addition to voltage control, to direct the flow in microfluidic devices. We report the use of polyelectrolyte multilayers (PEMs) to alter the surface charge and control the direction of flow in polystyrene and acrylic microfluidic devices. Relatively complex flow patterns with simple arrangements of applied voltages are realized by derivatization of different arms of a single device with oppositely charged polyelectrolytes. In addition, flow in opposite directions in the same channel is possible. A positively derivatized plastic substrate with a negatively charged lid was used to achieve top-bottom opposite flows. Derivatization of the two sides of a plastic microchannel with oppositely charged polyelectrolytes was used to achieve side-by-side opposite flows. The flow is characterized using fluorescence imaging and particle velocimetry.     

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.     

Dynamics of capturing process of multiple magnetic nanoparticles in a flow through microfluidic bioseparation system

A mathematical model based on finite-element technique is developed for predicting the transport and capture of multiple magnetic nanoparticles in a microfluidic system that consists of a microfluidic channel enclosed by a permanent magnet. The trajectories and trapping efficiencies are calculated for multiple magnetic nanoparticles when released in the microsystem. It is demonstrated that not only the size but also the point of release of nanoparticles within the microchannel affects the capturing process. Influence of three important parameters, inlet velocities of fluid containing magnetic nanoparticles, diameter of magnetic nanoparticles and magnetic field strength on the trapping efficiency are investigated and optimised values of inlet velocity and magnetic field strength for completely trapping 50 nm magnetic nanoparticles are predicted. It is further demonstrated that the angular position of magnet around the microchannel is also critical in dictating the resulting bioseparation performance. Furthermore, combination of these analyses using the mathematical model will be very useful in the design and development of novel microfluidic bioseparation microsystems.     

Perfusion in microfluidic cross-flow: separation of white blood cells from whole blood and exchange of medium in a continuous flow

We describe a microfluidic technique for separation of particles and cells and a device that employs this technique to separate white blood cells (WBC) from whole human blood. The separation is performed in cross-flow in an array of microchannels with a deep main channel and large number of orthogonal, shallow side channels. As a suspension of particles advances through the main channel, a perfusion flow through the side channels gradually exchanges the medium of the suspension and washes away particles that are sufficiently small to enter the shallow side channels. The microfluidic device is tested with a suspension of polystyrene beads and is shown to efficaciously exchange the carrier medium while retaining all beads. In tests with whole human blood, the device is shown to reduce the content of red blood cells (RBC) by a factor of approximately 4000 with retention of 98% of WBCs. The ratio between WBCs and RBCs reached at an outlet of the device is 2.4 on average. The device is made of a single cast of poly(dimethylsiloxane) sealed with a cover glass and is simple to fabricate. The proposed technique of separation by perfusion in continuous cross-flow could be used to enrich rare populations of cells based on differences in size, shape, and deformability.     

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.     

Temperature Gradients Drive Bulk Flow Within Microchannel Lined by Fluid–Fluid Interfaces

Surface tension gradients induce Marangoni flow, which may be exploited for fluid transport. At the micrometer scale, these surface‐driven flows can be quite significant. By introducing fluid–fluid interfaces along the walls of microfluidic channels, bulk fluid flows driven by temperature gradients are observed. The temperature dependence of the fluid–fluid interfacial tension appears responsible for these flows. In this report, the design concept for a biocompatible microchannel capable of being powered by solar irradiation is provided. Using microscale particle image velocimetry, a bulk flow generated by apparent surface tension gradients along the walls is observed. The direction of flow relative to the imposed temperature gradient agrees with the expected surface tension gradient. The phenomenon's ability to replace bulky peripherals, like traditional syringe pumps, on a diagnostic microfluidic device that captures and detects leukocyte subpopulations within blood is demonstrated. Such microfluidic devices may be implemented for clinical assays at the point of care without the use of electricity.     

Microfluidic device for electric field-driven single-cell capture and activation

A microchip that performs directed capture and chemical activation of surface-modified single cells has been developed. The cell capture system is comprised of interdigitated gold electrodes microfabricated on a glass substrate within PDMS channels. The cell surface is labeled with thiol functional groups using endogenous RGD receptors, and adhesion to exposed gold pads on the electrodes is directed by applying a driving electric potential. Multiple cell types can thus be sequentially and selectively captured on desired electrodes. Single-cell capture efficiency is optimized by varying the duration of field application. Maximum single-cell capture is attained for the 10-min trial, with 63 +/- 9% (n = 30) of the electrode pad rows having a single cell. In activation studies, single M1WT3 CHO cells loaded with the calcium-sensitive dye fluo-4 AM were captured; exposure to the muscarinic agonist carbachol increased the fluorescence to 220 +/- 74% (n = 79) of the original intensity. These results demonstrate the ability to direct the adhesion of selected living single cells on electrodes in a microfluidic device and to analyze their response to chemical stimuli.     

Formation of stable stacking zones in a flow stream for sample immobilization in microfluidic systems

Electrocapture is a multifunctional microfluidic tool that can be used for concentration, sample cleanup, multistep reactions, and separation of biomolecules. Herein, we investigate the mechanisms underlying the electrocapture principle. A microfluidic electrocapture device was found to be capable of generating regions of different electric field, which are maintained in the flow by electric and hydrodynamic forces, with the zones of lower electric field strength upstream of those with higher strength. In addition to detection of the local electric fields by direct measurements, the existence of the zones was observed by the capture of a solution containing Coomassie and myoglobin. The two molecules were captured at different spots in a steady-state manner and were released (separated) at different electric fields. Considering these observations and the experimental values for the electric field strengths, flow velocities, and electrophoretic mobilities of DNA, proteins, and peptides, it is concluded that the macromolecules are captured between the field zones by a stacking mechanism.     

Optimization of microfluidic fuel cells using transport principles

Microfluidic fuel cells exploit the lack of convective mixing at low Reynolds number to eliminate the need for a physical membrane to separate the fuel from the oxidant. Slow transport of reactants in combination with high catalytic surface-to-volume ratios often inhibit the efficiency of a microfluidic fuel cell. The performance of microfluidic devices that rely on surface electrochemical reactions is controlled by the interplay between reaction kinetics and the rate of mass transfer to the reactive surfaces. This paper presents theoretical and experimental work to describe the role of flow rate, microchannel geometry, and location of electrodes within a microfluidic fuel cell on its performance. A transport model, based on the convective-diffusive flux of reactants, is developed that describes the optimal conditions for maximizing both the average current density and the percentage of fuel utilized. The results show that the performance can be improved when the design of the device includes electrodes smaller than a critical length. The results of this study advance current approaches to the design of microfluidic fuel cells and other electrochemically-coupled microfluidic devices.     

Electrokinetic fluid control in two-dimensional planar microfluidic devices

We present microfluidic device designs with a two-dimensional planar format and methods to facilitate efficient sample transport along both dimensions. The basic device design consisted of a single channel for the first dimension which orthogonally intersected a high-aspect ratio second-dimension channel. To minimize dispersion of sample moving into and through the sample transfer region, control channels were placed on both sides of the first-dimension channel, and the electrokinetic flow from these control channels was used to confine the sample stream. We used SIMION and COMSOL simulations of the electric fields and fluid flow to guide device design. First, devices with one, two, and four control channels were fabricated and tested, and four control channels provided the most effective sample confinement. The designs were evaluated by measuring the sample stream widths and concentration to width ratios as a function of the electric field strength ratio in the control channels and first-dimension (1D) channel (EC/E1D). Next, both a single open channel and an array of parallel channels were tested for the second dimension, and improved performance was observed for the parallel channel design, with stream widths as narrow as 120 microm. The ease with which fluids could be introduced into both the first and second dimensions was also illustrated. Sample plugs injected into the planar region were confined as effectively as sample streams and were easily routed into the planar region by reconfiguring the applied potentials.     

Electrokinetic injection techniques in microfluidic chips

The separation efficiency of a microfluidic chip is influenced to a significant degree by the flow field conditions within the injection microchannel. Therefore, an understanding of the physics of the flow within this channel is beneficial in the design and operation of such a system. The configuration of an injection system is determined by the volume of the sample plug that is to be delivered to the separation process. Accordingly, this paper addresses the design and testing of injection systems with a variety of configurations, including a simple cross, a double-T, and a triple-T configuration. This paper also presents the design of a unique multi-T injection configuration. Each injection system cycles through a predetermined series of steps, in which the electric field magnitude and distribution within the various channels is strictly manipulated, to effectuate a virtual valve. The uniquemulti-T configuration injection system presented within this paper has the ability to simulate the functions of the cross, double-T, and triple-T systems through appropriate manipulations of the electric field within its various channels. In other words, the proposed design successfully combines several conventional injection systems within a single microfluidic chip.     

On‐Demand Droplet Collection for Capturing Single Cells

Droplet‐based microfluidic techniques are extensively used in efficient manipulation and genome‐wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single‐droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on‐demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on‐demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell‐to‐cell variations.     

Aptamer-enabled efficient isolation of cancer cells from whole blood using a microfluidic device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

Liquid membrane operations in a microfluidic device for selective separation of metal ions

A three-phase flow, water/n-heptane/water, was constructed in a microchannel (100-microm width, 25-microm depth) on a glass microchip (3 cm x 7 cm) and was used as a liquid membrane for separation of metal ions. Surface modification of the microchannel by octadecylsilane groups induced spontaneous phase separation of the three-phase flow in the microfluidic device, which allows control of interfacial contact time and off-chip analysis using conventional analytical apparatus. Prior to the selective transport of a metal ion through the liquid membrane in the microchannel, the forward and backward extraction of yttrium and zinc ions was investigated in a two-phase flow on a microfluidic device using 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester (commercial name, PC-88A) as an extractant. The extraction conditions (contact time of the two phases, pH, extractant concentration) in the microfluidic device were examined. These investigations demonstrated that the conventional methodology for solvent extraction of metal ions is applicable to solvent extraction in a microchannel. Finally, we employed the three-phase flow in the microchannel as a liquid membrane and observed the selective transport of Y ion through the liquid membrane. In the present study, we succeeded, for the first time, in the selective separation of a targeted metal ion from an aqueous feed solution to a receiving phase within a few seconds by employing a liquid membrane formed in a microfluidic device.