Continuous separation of particles using a microfluidic device equipped with flow rate control valves

We propose herein an improved microfluidic system for continuous and precise particle separation. We have previously proposed a method for particle separation called "pinched flow fractionation." Using the previously reported method, particles can be continuously separated according to differences in their diameters, simply by introducing liquid flows with and without particles into a specific microchannel structure. In this study, we incorporated PDMS membrane microvalves for flow rate control into the microfluidic device to improve the separation accuracy. By adjusting the flow rates distributed to each outlet, target particles could be precisely collected from the desired outlet. We succeeded in separating micron and submicron-size polymer particles. This method can be used widely for continuous and precise separation of various kinds of particles, and can function as an important part of microfluidic systems.     

DC dielectrophoresis separation of marine algae and particles in a microfluidic chip

This paper reports a microfluidic method of continuous separation of marine algae and particles by DC dielectrophoresis. The locally non-uniform electric field is generated by an insulating PDMS triangle hurdle fabricated within a PDMS microchannel. Both the particles and algae are subject to negative DEP forces at the hurdle where the gradient of local electric-field strength is the strongest. The DEP force acting on the particle or the algae depends on particles’ or algae’s volume, shape and dielectric properties. Thus the moving particles and algae will be repelled to different streamlines when passing the hurdle. In this way, combined with the electroosmotic flow, continuous separation of algae of two different sizes, and continuous separation of polystyrene particles and algae with similar volume but different shape were achieved. This first demonstration of DC DEP separation of polystyrene particles and algae with similar sizes illustrates the great influence of dielectric properties on particle separation and potentials for sample pretreatment.     

Steady flow generation in microcirculatory systems

In this paper we explore the mechanical generation of steady-non pulsatile-flow in microfluidic systems. The rationale of the paper is inspired in the example of cardiovascular systems where at the microscale (i.e. capillaries) the flow is steady rather than pulsatile to optimize performance. We present a solution to the generation of steady flow in engineered microfluidic systems either in open or closed loop configurations via the use of disc pumps. The disc pump consists of a flat rotating disc and utilizes both viscous drag and centrifugal force to achieve pumping. Experiments using single loop and double loop microfluidic systems are presented to characterize the disc pump. Continuous flow generated by the disc pumps can be used to separate particles based on size using recirculating loops and for extraction of small particles without disturbing the concentration of bigger particles. The potential impact of this technology includes sample separation and extraction techniques into portable microfluidic labs-on-a-chip, and long term culture systems for cells in suspension.     

Continuous flow separations in microfluidic devices

Biochemical sample mixtures are commonly separated in batch processes, such as filtration, centrifugation, chromatography or electrophoresis. In recent years, however, many research groups have demonstrated continuous flow separation methods in microfluidic devices. Such separation methods are characterised by continuous injection, real-time monitoring, as well as continuous collection, which makes them ideal for combination with upstream and downstream applications. Importantly, in continuous flow separation the sample components are deflected from the main direction of flow, either by means of a force field (electric, magnetic, acoustic, optical etc.), or by intelligent positioning of obstacles in combination with laminar flow profiles. Sample components susceptible to deflection can be spatially separated. A large variety of methods has been reported, some of these are miniaturised versions of larger scale methods, others are only possible in microfluidic regimes. Researchers now have a diverse toolbox to choose from and it is likely that continuous flow methods will play an important role in future point-of-care or in-the-field analysis devices.     

Droplet microfluidics based microseparation systems

Lab on a chip (LOC) technology is a promising miniaturization approach. The feature that it significantly reduced sample consumption makes great sense in analytical and bioanalytical chemistry. Since the start of LOC technology, much attention has been focused on continuous flow microfluidic systems. At the turn of the century, droplet microfluidics, which was also termed segmented flow microfluidics, was introduced. Droplet microfluidics employs two immiscible phases to form discrete droplets, which are ideal vessels with confined volume, restricted dispersion, limited cross-contamination, and high surface area. Due to these unique features, droplet microfluidics proves to be a versatile tool in microscale sample handling. This article reviews the utility of droplet microfluidics in microanalytical systems with an emphasize on separation science, including sample encapsulation at ultra-small volume, compartmentalization of separation bands, isolation of droplet contents, and related detection techniques.     

Continuous and precise particle separation by electroosmotic flow control in microfluidic devices

A new scheme has been described for continuous particle separation using EOF in microfluidic devices. We have previously reported a method for particle separation, called "pinched flow fractionation (PFF)", in which size-dependent and continuous particle separation can be achieved by introducing pressure-driven flows with and without particles into a pinched microchannel. In this study, EOF was employed to transport fluid flows inside a microchannel. By controlling the applied voltage to electrodes inserted in each inlet/outlet port, the flow rates from both inlets, and flow rates distributed to each outlet could be accurately tuned, thus enabling more effective separation compared to the pressure-driven scheme. In the experiment, the particle behaviors were compared between EOF and pressure-driven flow schemes. In addition, micrometer- and submicrometer-sized particles were accurately separated and individually collected using a microchannel with multiple outlet branch channels, demonstrating the high efficiency of the presented scheme.     

Chip integrated strategies for acoustic separation and manipulation of cells and particles

Acoustic standing wave technology combined with microtechnology opens up new areas for the development of advanced particle and cell separating microfluidic systems. This tutorial review outlines the fundamental work performed on continuous flow acoustic standing wave separation of particles in macro scale systems. The transition to the microchip format is further surveyed, where both fabrication and design issues are discussed. The acoustic technology offers attractive features, such as reasonable throughput and ability to separate particles in a size domain of about tenths of micrometers to tens of micrometers. Examples of different particle separation modes enabled in microfluidic chips, utilizing standing wave technology, are described along a discussion of several potential applications in life science research and in the medical clinic. Chip integrated acoustic standing wave separation technology is still in its infancy and it can be anticipated that new laboratory standards very well may emerge from the current research.     

High-resolution DNA separation in microcapillary electrophoresis chips utilizing double-L injection techniques

An experimental and numerical investigation into the use of high-resolution injection techniques to separate DNA fragments within electrophoresis microchips is presented. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered, and several variable-volume injection methods are discussed. A detailed analysis is provided of a double-L injection technique, which employs appropriate electrokinetic manipulations to reduce sample leakage within the microchip. The leakage effect in electroosmotic flow (EOF) is investigated using a sample composed of rhodamine B and Cy3 dye. Meanwhile, the effects of sample leakage in capillary electrophoresis (CE) separation are studied by considering the separation of 100-base pairs (bp) DNA ladders and HaeIII-digested PhiX-174 DNA samples. The present experimental and simulation results indicate that the unique injection system employed in the current microfluidic chip has the ability to replicate the functions of both the conventional cross-channel and the shift-channel injection systems. Furthermore, applying the double-L injection method to these two injection systems is shown to reduce sample leakage significantly. The proposed microfluidic chip and double-L injection technique developed in this study have an exciting potential for use in high-resolution, high-throughput biochemical analysis applications and in many other applications throughout the micrototal analysis systems field.     

Continuous sorting of magnetic cells via on-chip free-flow magnetophoresis

The ability to separate living cells is an essential aspect of cell research. Magnetic cell separation methods are among some of the most efficient methods for bulk cell separation. With the development of microfluidic platforms within the biotechnology sector, the design of miniaturised magnetic cell sorters is desirable. Here, we report the continuous sorting of cells loaded with magnetic nanoparticles in a microfluidic magnetic separation device. Cells were passed through a microfluidic chamber and were deflected from the direction of flow by means of a magnetic field. Two types of cells were studied, mouse macrophages and human ovarian cancer cells (HeLa cells). The deflection was dependent on the magnetic moment and size of the cells as well as on the applied flow rate. The experimentally observed deflection matched well with calculations. Furthermore, the separation of magnetic and non-magnetic cells was demonstrated using the same microfluidic device.     

外加场分离技术与微流控技术联用在微纳尺度物质分离中的研究进展

微纳尺度物质的分离和分选在精准医学、材料科学和单细胞分析等研究中至关重要。精准、高效和快速的分离微纳尺度物质能够为癌症的早期诊断、生物样品检测和细胞筛选提供重要帮助,其中基于外加场分离技术的分离微纳尺度物质因可以对微纳尺度物质高效在线分离和分选,被广泛应用于微纳米颗粒、外泌体以及生物细胞的分离工作中,而目前多数外加场分离技术存在装备繁琐和样品消耗大等问题。微流控技术是一种通过制作微通道和微流控芯片操纵微小流体对微纳尺度样品组分进行分离的技术,因具有快速检测、高通量、在线分离、集成性高、成本低等优势现被应用于微纳尺度物质分离分析中,是一种微纳尺度物质分离的有效方法,通过在微流控芯片上设计不同的通道及外部配件提高主动场对微纳尺度物质分离效率。外加场分离技术与微流控技术联用可以实现微纳尺度物质的无损、高效、在线分离。该综述主要概述了近年来在微流控芯片上依托流动场、电场、磁场及声场等外加场分离技术来提高对微纳尺度物质分离效率的研究现状,并将各个外力场对单细胞、微颗粒等微纳尺度物质的分离进行分类介绍,总结各自的优缺点及发展应用,最后展望了外加场分离技术与微流控技术联用在应用于癌细胞的早期筛查、精确分离微尺度物质领域的未来发展前景,并提出联用技术的优势和未来应用等。     

Miniaturizing free-flow electrophoresis - a critical review

Free-flow electrophoresis (FFE) separation methods have been developed and investigated for around 50 years and have been applied not only to many types of analytes for various biomedical applications, but also for the separation of inorganic and organic substances. Its continuous sample preparation and mild separation conditions make it also interesting for online monitoring and detection applications. Since 1994 several microfluidic, miniaturized FFE devices were developed and experimentally characterized. In contrast to their large-scale counterparts microfluidic FFE (mu-FFE) devices offer new possibilities due to the very rapid separations within several seconds or below and the requirement for sample volumes in the microliter range. Eventually, these mu-FFE systems might find application in so-called lab-on-a-chip devices for real-time monitoring and separation applications. This review gives detailed information on the results so far published on mu-FFE chips, comprising its four main modes, namely free-flow zone electrophoresis (FFZE), free-flow IEF (FFIEF), free-flow ITP (FFITP), and free-flow field-step electrophoresis (FFFSE). The principles of the different FFE modes and the basic underlying theory are given and discussed with special emphasis on miniaturization. Different designs as well as fabrication methods and applied materials are discussed and evaluated. Furthermore, the separation results shown indicate that similar separation quality with respect to conventional FFE systems, as defined by the resolution and peak capacity, can be achieved with mu-FFE separations when applying much lower electrical voltages. Furthermore, innovations still occur and several approaches for hyphenated, more integrated systems have been proposed so far, some of which are discussed here. This review is intended as an introduction and early compendium for research and development within this field.     

Orthogonal optical force separation simulation of particle and molecular species mixtures under direct current electroosmotic driven flow for applications in biological sample preparation

Presented here are the results from numerical simulations applying optical forces orthogonally to electroosmotically induced flow containing both molecular species and particles. Simulations were conducted using COMSOL v4.2a Multiphysics® software including the particle tracking module. The study addresses the application of optical forces to selectively remove particulates from a mixed sample stream that also includes molecular species in a pinched flow microfluidic device. This study explores the optimization of microfluidic cell geometry, magnitude of the applied direct current electric field, EOF rate, diffusion, and magnitude of the applied optical forces. The optimized equilibrium of these various contributing factors aids in the development of experimental conditions and geometry for future experimentation as well as directing experimental expectations, such as diffusional losses, separation resolution, and percent yield. The result of this work generated an optimized geometry with flow conditions leading to negligible diffusional losses of the molecular species while also being able to produce particle removal at near 100% levels. An analytical device, such as the one described herein with the capability to separate particulate and molecular species in a continuous, high‐throughput fashion would be valuable by minimizing sample preparation and integrating gross sample collection seamlessly into traditional analytical detection methods.     

A microfluidic device for continuous capture and concentration of microorganisms from potable water

A microfluidic device based on electrophoretic transport and electrostatic trapping of charged particles has been developed for continuous capture and concentration of microorganisms from water. Reclaimed and bottled water samples at pH values ranging from 5.2-6.5 were seeded with bacteria (E. coli, Salmonella, and Pseudomonas) and viruses (MS-2 and Echovirus). Negative control and capture experiments were performed simultaneously using two identical devices. Culture based methods were utilized to characterize the capture efficiency as a function of the species type, time, flow rate, and applied electric field. Based on differences between the capture and negative control data, capture efficiencies of 90% to 99% are reported for E. coli, Salmonella, Pseudomonas, and MS-2, while the capture efficiency for Echovirus was between 70% and 80%. Overall, the device exhibits a 16.67 fold sample volume reduction within an hour at 6 mL h(-1) flow rate, resulting in a concentration factor of 14.2 at 85.2% capture efficiency. The device can function either as a filter or a sample concentrator without using any chemical additives. It can function as an integral component of a continuous, microbial capture and concentration system from large volumes of potable water.     

Integrated continuous microfluidic liquid-liquid extraction

We describe continuous flow liquid-liquid phase separation in microfluidic devices based on capillary forces and selective wetting surfaces. Effective liquid-liquid phase separation is achieved by using a thin porous fluoropolymer membrane that selectively wets non-aqueous solvents, has average pore sizes in the 0.1-1 microm range, and has a high pore density for high separation throughput. Pressure drops throughout the microfluidic network are modelled and operating regimes for the membrane phase separator are determined based on hydrodynamic pressure drops and capillary forces. A microfluidic extraction device integrating mixing and phase separation is realized by using silicon micromachining. Modeling of the phase separator establishes the operating limits. The device is capable of completely separating several organic-aqueous and fluorous-aqueous liquid-liquid systems, even with high fractions of partially miscible compounds. In each case, extraction is equivalent to one equilibrium extraction stage.     

Continuous on-line concentration based on dynamic pH junction for trimethoprim and sulfamethoxazole by microfluidic capillary electrophoresis combined with flow injection analysis system

A novel, rapid and continuous on-line concentration approach based on dynamic pH junction for the analysis of trimethoprim (TMP) and sulfamethoxazole (SMZ) by microfluidic capillary electrophoresis (CE) combined with flow injection analysis is developed in this paper. Stacking is due to decreases in the velocity of analytes when migrating from the low-pH sample zone (sample was dissolved in 50 mM HCl) to a relatively high-pH buffer (30 mM phosphate buffer, pH 8.5) filled in the capillary. This results in 2.9-4.7-fold improvement in concentration sensitivity relative to conventional capillary electrophoresis methods. The separation could be achieved within 2 min and sample throughput rate can reach up to 38 h(-1).     

用于细胞破裂的微流控生物芯片的研制

基于微电子机械系统(MEMS)技术,研制成一种夹流式血细胞破裂微流控生物芯片。细胞样品在破胞试剂夹流作用下导入芯片并在微沟道中流动,两种液体在流动过程中充分混合,导致细胞破裂。采用抗凝全血为细胞样品,比较胍盐和曲拉通的破胞效果;并分析在胍盐破裂细胞条件下,细胞浓度和流速对破胞效果的影响。控制破胞试剂流速远大于样品流速,可在几秒钟内完成细胞的破裂;保持破胞试剂与样品流速的比例,同时提高流速可在芯片上实现细胞的快速破裂。夹流式细胞破裂芯片具有与细胞分离芯片和脱氧核糖核酸(DNA)提取芯片相集成的潜力,可实现对复杂生物样品预处理操作,为实现微全分析系统打下良好基础。     

Continuous particle separation in spiral microchannels using Dean flows and differential migration

Microparticle separation and concentration based on size has become indispensable in many biomedical and environmental applications. In this paper we describe a passive microfluidic device with spiral microchannel geometry for complete separation of particles. The design takes advantage of the inertial lift and viscous drag forces acting on particles of various sizes to achieve differential migration, and hence separation, of microparticles. The dominant inertial forces and the Dean rotation force due to the spiral microchannel geometry cause the larger particles to occupy a single equilibrium position near the inner microchannel wall. The smaller particles migrate to the outer half of the channel under the influence of Dean forces resulting in the formation of two distinct particle streams which are collected in two separate outputs. This is the first demonstration that takes advantage of the dual role of Dean forces for focusing larger particles in a single equilibrium position and transposing the smaller particles from the inner half to the outer half of the microchannel cross-section. The 5-loop spiral microchannel 100 microm wide and 50 microm high was used to successfully demonstrate a complete separation of 7.32 microm and 1.9 microm particles at Dean number De = 0.47. Analytical analysis supporting the experiments and models is also presented. The simple planar structure of the separator offers simple fabrication and makes it ideal for integration with on-chip microfluidic systems, such as micro total analysis systems (muTAS) or lab-on-a-chip (LOC) for continuous filtration and separation applications.     

Continuous dielectrophoretic separation of particles in a spiral microchannel

Particle separation is a fundamental operation in the areas of biology and physical chemistry. A variety of force fields have been used to separate particles in microfluidic devices, among which electric field may be the most popular one due to its general applicability and adaptability. So far, however, electrophoresis‐based separations have been limited primarily to batchwise processes. Dielectrophoresis (DEP)‐based separations require in‐channel micro‐electrodes or micro‐insulators to produce electric field gradients. This article introduces a novel particle separation technique in DC electrokinetic flow through a planar double‐spiral microchannel. The continuous separation arises from the cross‐stream dielectrophoretic motion of particles induced by the non‐uniform electric field inherent to curved channels. Specifically, particles are focused by DEP to one sidewall of the first spiral, and then dielectrophoretically deflected toward the other sidewall of the second spiral at a particle‐dependent rate, leading to focused particle streams along different flow paths. This DEP‐based particle separation technique is demonstrated in an asymmetric double‐spiral microchannel by continuously separating a mixture of 5/10 μm particles and 3/5 μm particles.     

Continuous molecular enrichment in microfluidic systems

Highly efficient molecular extractions in continuous flow microfluidic systems are demonstrated utilising the rapid mixing properties of biphasic segmented flow in conjunction with suspended micro-particulate adsorbents. A continuous flow technique providing potential for continual on-line sample enrichment, purification and clean-up in chemical synthesis, and sample preparation.     

Continuous blood cell separation by hydrophoretic filtration

We propose a new hydrophoretic method for continuous blood cell separation using a microfluidic device composed of slanted obstacles and filtration obstacles. The slanted obstacles have a larger height and gap than the particles in order to focus them to a sidewall by hydrophoresis. In the successive structure, the height and gap of the filtration obstacles with a filtration pore are set between the diameters of small and large particles, which defines the critical separation diameter. Accordingly, the particles smaller than the criterion freely pass through the gap and keep their focused position. In contrast, the particles larger than the criterion collide against the filtration obstacle and move into the filtration pore. The microfluidic device was characterized with polystyrene beads with a minimum diameter difference of 7.3%. We completely separated polystyrene microbeads of 9 and 12 microm diameter with a separation resolution of approximately 6.2. This resolution is increased by 6.4-fold compared with our previous separation method based on hydrophoresis (S. Choi and J.-K. Park, Lab Chip, 2007, 7, 890, ref. 1). In the isolation of white blood cells (WBCs) from red blood cells (RBCs), the microfluidic device isolated WBCs with 210-fold enrichment within a short filtration time of approximately 0.3 s. These results show that the device can be useful for the binary separation of a wide range of biological particles by size. The hydrophoretic filtration as a sample preparation unit offers potential for a power-free cell sorter to be integrated into disposable lab-on-a-chip devices.