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.     

Continuous dielectrophoretic size-based particle sorting

Continuous-flow dielectrophoretic (DEP) particle separation based on size is demonstrated in a microfluidic device. Polystyrene microspheres suspended in a neutrally buoyant aqueous solution are used as model particles to study DEP induced by an array of slanted, planar, interdigitated electrodes inside of a soft-lithography microchannel. The E-field gradients from the slanted electrodes impart a net transverse force component on the particles that causes them to "ratchet" across the channel. Over the length of the device, larger particles are deflected more than smaller particles according to the balance of hydrodynamic drag and DEP forces. Consequently, a flow-focused particle suspension containing different-sized particles is fractionated as the beads flow and separate down the length of the device. The flow behavior of spherical particles is modeled, and the total transverse particle displacement in the microfluidic device predicts fourth-order size and voltage and second-order inverse flow rate dependences. The model is verified experimentally for a range of flow rates, particle sizes, and E-field strengths.     

Continuous sorting of microparticles using dielectrophoresis

Sorting of particles such as cells is a critical process for many biomedical applications, and it is challenging to integrate it into an analytical microdevice. We report an effective and flexible dielectrophoresis (DEP)-based microfluidic device for continuous sorting of multiple particles in a microchannel. The particle sorter is composed of two components-a DEP focusing unit and a Movable DEP Trap (MDT). The trap is formed by an array of microelectrodes at the bottom of the channel and a transparent electrode plate placed at the top. The location of the trap is dependent on the configuration of voltages on the array and therefore is addressable. Flowing particles are first directed and focused into a single particle stream by the focusing unit. The streamed particles are then sorted into different fractions using the movable trap by rapidly switching the applied voltage. The performance of the sorter is demonstrated by successfully sorting microparticles in a continuous flow. The proposed DEP-based microfluidic sorter can be implemented in applications such as sample preparation and cell sorting for subsequent analytical processing, where sorting of particles is needed.     

Integrated microfluidic system enabling (bio)chemical reactions with on-line MALDI-TOF mass spectrometry

A continuous flow micro total analysis system (micro-TAS) consisting of an on-chip microfluidic device connected to a matrix assisted laser desorption ionization [MALDI] time-of-flight [TOF] mass spectrometer (MS) as an analytical screening system is presented. Reaction microchannels and inlet/outlet reservoirs were fabricated by powderblasting on glass wafers that were then bonded to silicon substrates. The novel lab-on-a-chip was realized by integrating the microdevice with a MALDI-TOFMS standard sample plate used as carrier to get the microfluidic device in the MALDI instrument. A novel pressure-driven pumping mechanism using the vacuum of the instrument as a driving force induces flow in the reaction microchannel in a self-activating way. Organic syntheses as well as biochemical reactions are carried out entirely inside the MALDI-MS ionization vacuum chamber and analyzed on-line by MALDI-TOFMS in real time. The effectiveness of the micro-TAS system has been successfully demonstrated with several examples of (bio)chemical reactions.     

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.     

High‐Density Microfluidic Particle‐Cluster‐Array Device for Parallel and Dynamic Study of Interaction between Engineered Particles

A high‐density and high‐performance microfluidic particle‐cluster‐array device utilizing a novel hydrodynamically tunable pneumatic valve (HTPV) is reported for parallel and dynamic monitoring of the interactions taking place in particle clusters. The key concept involves passive operation of the HTPV through elastic deformation of a thin membrane using only the hydrodynamic force inherent in microchannel flows. This unique feature allows the discrete and high‐density (≈30 HTPVs mm^?2) arrangement of numerous HTPVs in a microfluidic channel without any pneumatic connection. In addition, the HTPV achieves high‐performance clustering (≈92%) of three different particles in an array format through the optimization of key design and operating parameters. Finally, a contamination‐free, parallel, and dynamic biochemical analysis strategy is proposed, which employs a simple one‐inlet–one‐outlet device operated by the effective combination of several techniques, including particle clustering, the interactions between engineered particles, two‐phase partitioning and dehydration control of aqueous plugs, and shape/color‐based particle identification.     

Acoustic particle filter with adjustable effective pore size for automated sample preparation

This article presents analysis and optimization of a microfluidic particle filter that uses acoustic radiation forces to remove particles larger than a selected size by adjusting the driving conditions of the piezoelectric transducer (PZT). Operationally, the acoustic filter concentrates microparticles to the center of the microchannel, minimizing undesirable particle adsorption to the microchannel walls. Finite element models predict the complex two-dimensional acoustic radiation force field perpendicular to the flow direction in microfluidic devices. We compare these results with experimental parametric studies including variations of the PZT driving frequencies and voltages as well as various particle sizes (0.5-5.0 microm in diameter). These results provide insight into the optimal operating conditions and show the efficacy of our device as a filter with an adjustable effective pore size. We demonstrate the separation of Saccharomyces cerevisiae from MS2 bacteriophage using our acoustic device. With optimized design of our microfluidic flow system, we achieved yields of greater than 90% for the MS2 with greater than 80% removal of the S. cerevisiae in this continuous-flow sample preparation device.     

Surface modification method of microchannels for gas-liquid two-phase flow in microchips

A capillarity restricted modification method for microchannel surfaces was developed for gas--liquid microchemical operations in microchips. In this method, a microstructure combining shallow and deep microchannels and the principle of capillarity were utilized for chemical modification of a restricted area of a microchannel. A hydrophobic--hydrophilic patterning in microchannels was prepared as an example for guiding gas and liquid flows along the respective microchannels. Validity of the patterning was confirmed by measuring aqueous flow leak pressure from the hydrophilic microchannel to the hydrophobic one. The leak pressure of 7.7-1.1 kPa agreed well with that predicted theoretically from the Young-Laplace equation for the microchannel depth of 8.6-39 microm. In an experiment to demonstrate usefulness and effectiveness of the method, an air bubble was first introduced into the hydrophilic microchannel and purged from the hydrophobic-hydrophilic patterned microchannels. Next, the patterning structure was applied to remove dissolved oxygen by contacting the aqueous flow with a nitrogen flow. The concentration of dissolved oxygen decreased with contact time, and its time course agreed well with numerical simulation. These demonstrations showed that the proposed patterning method can be used in general microfluidic gas-liquid operations.     

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.     

Design and analysis of single-cell capture microfluidic device

The aim of our study was to develop microfluidic devices using microchannel technology with the capability of capturing single cells. We analyzed and compared the cell-capturing efficiencies of series-loop microchannel and parallel-loop microchannel devices that were produced using polydimethylsiloxane (PDMS). Each set of microchannels was composed of a main flow channel and several branch channels with capturing zones. The microfluidic devices were designed to use the differences in flow rates between the main flow channel and the branch channels as a means of capturing single cells based on size and sequestering them within the microstructure of multiple capture zones. The data indicated that the flow medium encountered significant resistance in the series-loop microchannel device, which resulted in an inability to hold the captured cells within any of the capture zones. Flow resistance was, however, greatly reduced in the parallel-loop microchannel device compared to the series-loop device, and single cells were captured in all the capturing zones of the device. Our data suggest that the parallel-loop microchannel technology has significant potential for development toward high-throughput platforms capable of capturing single cells for physiological analyses at the single-cell level.     

DNA mutation detection in a polymer microfluidic network using temperature gradient gel electrophoresis

A miniaturized system for DNA mutation analysis, utilizing temperature gradient gel electrophoresis (TGGE) in a polycarbonate (PC) microfluidic device, is reported. TGGE reveals the presence of sequence heterogeneity in a given heteroduplex sample by introducing a thermal denaturing gradient that results in differences between the average electrophoretic mobilities of DNA sequence variants. Bulk heater assemblies are designed and employed to externally generate temperature gradients in spatial and temporal formats along the separation channels. TGGE analyses of model mutant DNA fragments, each containing a single base substitution, are achieved using both single- and 10-channel parallel measurements in a microfluidic platform. Additionally, a comprehensive polymer microfluidic device containing an integrated microheater and sensor array is developed and demonstrated for performing spatial TGGE for DNA mutation analysis. The device consists of two PC modular substrates mechanically bonded together. One substrate is embossed with microchannels, and the other contains a tapered microheater, lithographically patterned along with an array of temperature sensors. Compared with the external heating approaches, the integrated platform provides significant reduction in power requirement and thermal response time while establishing more accurate and highly effective control of the temperature gradient for achieving improved separation resolution.     

Performance of an integrated microoptical system for fluorescence detection in microfluidic systems

This article presents a new integrated microfluidic/microoptic device designed for basic biochemical analysis. The microfluidic network is wet-etched in a Borofloat 33 (Pyrex) glass wafer and sealed by means of a second wafer. Unlike other similar microfluidic systems, elements of the detection system are realized with the help of microfabrication techniques and directly deposited on both sides of the microchemical chip. The detection system is composed of the combination of refractive circular or elliptical microlens arrays and chromium aperture arrays. The microfluidic channels are 60 microm wide and 25 microm deep. The elliptical microlenses have a major axis of 400 microm and a minor axis of 350 microm. The circular microlens diameters range from 280 microm to 350 microm. The apertures deposited on the outer chip surfaces are etched in a 3000-A-thick chromium layer. The overall thickness of this microchemical system is < 1.6 mm. A limit of detection of 3.3 nM for a Cy5 solution in phosphate buffer (pH 7.4) was demonstrated. The cross-talk signal measured between two adjacent microchannels with 1 mm pitch was < 1:5600, meaning that < or = 1.8 x 10(-4)% of the fluorescence light power emitted from one microchannel filled with a 50 microM Cy5 solution reaches the photodetector at the adjacent microchannel. This performance compares very well with that obtainable in microchemical chips using confocal fluorescence systems, taking differences in parameters, such as excitation power into microchannels, data acquisition rates, and signal filtering into account.     

On-chip diamagnetic repulsion in continuous flow

We explore the potential of a microfluidic continuous flow particle separation system based on the repulsion of diamagnetic materials from a high magnetic field. Diamagnetic polystyrene particles in paramagnetic manganese (II) chloride solution were pumped into a microfluidic chamber and their deflection behaviour in a high magnetic field applied by a superconducting magnet was investigated. Two particle sizes (5 and 10 μm) were examined in two concentrations of MnCl₂ (6 and 10%). The larger particles were repelled to a greater extent than the smaller ones, and the effect was greatly enhanced when the particles were suspended in a higher concentration of MnCl₂. These findings indicate that the system could be viable for the separation of materials of differing size and/or diamagnetic susceptibility, and as such could be suitable for the separation and sorting of small biological species for subsequent studies.     

Multifunctional picoliter droplet manipulation platform and its application in single cell analysis

We developed an automated and multifunctional microfluidic platform based on DropLab to perform flexible generation and complex manipulations of picoliter-scale droplets. Multiple manipulations including precise droplet generation, sequential reagent merging, and multistep solid-phase extraction for picoliter-scale droplets could be achieved in the present platform. The system precision in generating picoliter-scale droplets was significantly improved by minimizing the thermo-induced fluctuation of flow rate. A novel droplet fusion technique based on the difference of droplet interfacial tensions was developed without the need of special microchannel networks or external devices. It enabled sequential addition of reagents to droplets on demand for multistep reactions. We also developed an effective picoliter-scale droplet splitting technique with magnetic actuation. The difficulty in phase separation of magnetic beads from picoliter-scale droplets due to the high interfacial tension was overcome using ferromagnetic particles to carry the magnetic beads to pass through the phase interface. With this technique, multistep solid-phase extraction was achieved among picoliter-scale droplets. The present platform had the ability to perform complex multistep manipulations to picoliter-scale droplets, which is particularly required for single cell analysis. Its utility and potentials in single cell analysis were preliminarily demonstrated in achieving high-efficiency single-cell encapsulation, enzyme activity assay at the single cell level, and especially, single cell DNA purification based on solid-phase extraction.     

Ultrasound Manipulation and Extrusion of Active Nanorods

Synthetic self-propelled nano and microparticles have a growing appeal for targeted drug delivery, collective functionality, and manipulation at the nanoscale. However, it is challenging to control their positions and orientations under confinement, e.g., in microchannels, nozzles, and microcapillaries. This study reports on the synergistic effect of acoustic and flow-induced focusing in microfluidic nozzles. In a microchannel with a nozzle, the balance between the acoustophoretic forces and the fluid drag due to streaming flows generated by the acoustic field controls the microparticle's dynamics. This study manipulates the positions and orientations of dispersed particles and dense clusters inside the channel at a fixed frequency by tuning the acoustic intensity. The main findings are: first, this study successfully manipulates the positions and orientations of individual particles and dense clusters inside the channel at a fixed frequency by tuning the acoustic intensity. Second, when an external flow is applied, the acoustic field separates and selectively extrudes shape-anisotropic passive particles and self-propelled active nanorods. Finally, the observed phenomena are explained by multiphysics finite-element modeling. The results shed light on the control and extrusion of active particles in confined geometries and enable applications for acoustic cargo (e.g., drug) delivery, particle injection, and additive manufacturing via printed self-propelled active particles.     

Electrohydrodynamic generation and delivery of monodisperse picoliter droplets using a poly(dimethylsiloxane) microchip

We developed a drop-on-demand microdroplet generator for the discrete dispensing of biosamples into a bioanalytical unit. This disposable PDMS microfluidic device can generate monodisperse droplets of picoliter volume directly out of a plane sidewall of the microfluidic chip by an electrohydrodynamic mechanism. The droplet generation was accomplished without using either an inserted capillary or a monolithically built-in tip. The minimum droplet volume was approximately 4 pL, and the droplet generation was repeatable and stable for at least 30 min, with a typical variation of less than 2.0% of drop size. The Taylor cone, which is usually observed in electrospray, was suppressed by controlling the surface wetting property of the PDMS device as well as the surface tension of the sample liquids. A modification of the channel geometry right before the opening of the microchannel also enhanced the continuous droplet generation without applying any external pumping. A simple numerical simulation of the droplet generation verified the importance of controlling the surface wetting conditions for the droplet formation. Our microdroplet generator can be effectively applied to a direct interface of a microfluidic chip to a biosensing unit, such as AMS, MALDI-MS or protein microarray-type biochips.     

A ferrofluid guided system for the rapid separation of the non-magnetic particles in a microfluidic device

A microfluidic device was fabricated via UV lithography technique to separate non-magnetic fluoresbrite carboxy microspheres (approximately 4.5 microm) in the pH 7 ferrofluids made of magnetite nanoparticles (approximately 10 nm). A mixture of microspheres and ferrofluid was injected to a lithographically developed Y shape microfluidic device, and then by applying the external magnet fields (0.45 T), the microspheres were clearly separated into different channels because of the magnetic force acting on those non-magnetic particles. During this study, various pumping speeds and particle concentrations associated with the various distances between the magnet and the microfluidic device were investigated for an efficient separation. This study may be useful for the separation of biological particles, which are very sensitive to pH value of the solutions.     

Design and numerical investigation of a circular microchannel for particle/cell separation using dielectrophoresis

Precisely separating particles/cells with different sizes and physical properties has been an interest for point-of-care diagnostics and personalized treatment. Dielectrophoresis (DEP) is widely known as a powerful and non-invasive technique to separate particles and cells. This paper presents a comprehensive numerical investigation of particle/cell separation in circular microchannels using DEP. First, the geometrical parameters of the circular microchannel affecting DEP force are determined by performing an analytical solution. Then, by developing a solver in OpenFOAM, the effect of these parameters on particles deflection is investigated. According to the results, two different circular microchannels are presented to investigate the continuous separation of bio-particles (based on their physical properties) and polystyrene particles (based on their size). The results showed that a minimum voltage of 7, 9, and 12 V is required to achieve 100 % purity and separation efficiency for separating red blood cells from MDA-MB-231 cancer cells at the flow rate of 0.5, 1.0, and 1.5 µl/min, respectively. Also, the efficient separation of 5 and 10 µm polystyrene particles at the flow rate of 0.1 µl/min is possible only at the voltage of 9 V. The results of this numerical study can be useful for the fabrication of an optimal microdevice for the continuous DEP separation of particles and cells.     

On-chip diamagnetic repulsion in continuous flow

Abstract

We explore the potential of a microfluidic continuous flow particle separation system based on the repulsion of diamagnetic materials from a high magnetic field. Diamagnetic polystyrene particles in paramagnetic manganese (II) chloride solution were pumped into a microfluidic chamber and their deflection behaviour in a high magnetic field applied by a superconducting magnet was investigated. Two particle sizes (5 and 10 μm) were examined in two concentrations of MnCl₂ (6 and 10%). The larger particles were repelled to a greater extent than the smaller ones, and the effect was greatly enhanced when the particles were suspended in a higher concentration of MnCl₂. These findings indicate that the system could be viable for the separation of materials of differing size and/or diamagnetic susceptibility, and as such could be suitable for the separation and sorting of small biological species for subsequent studies.     

微流控技术制备吡柔比星聚乳酸微球及其体外释放研究

微流控液滴技术是制备单分散性药物载体的理想方法,对精确考察药物的释放行为、释放机理及动力学模型具有重要意义。采用亲水性修饰T型通道产生单分散性吡柔比星聚乳酸液滴,考察了通道性质对液滴生成的影响以及连续相和分散相流速与液滴尺寸的关系,制备了两种粒径的吡柔比星聚乳酸微球并考察了其体外释放行为。结果表明,经修饰的通道可产生稳定均一的液滴,通过调节两相流速可以有效控制液滴和微球的尺寸,制备的微球粒径均一,载药量和包封率高,吡柔比星体外释放缓慢且无明显突释,尺寸较大微球的释放速率较慢。