Hydrodynamic gating valve for microfluidic fluorescence-activated cell sorting

Microfluidic cell sorter allows efficient separation of small number of cells, which is beneficial in handling cells, especially primary cells that cannot be expanded to large populations. Here, we demonstrate a microfluidic fluorescence-activated cell sorter (μFACS) with a novel sorting mechanism, in which automatic on-chip sorting is realized by turning on/off the hydrodynamic gating valve when a fluorescent target is detected. Formation of the hydrodynamic gating valve was investigated by both numerical simulation and flow visualization experiment. Separation of fluorescent polystyrene beads was then conducted to evaluate this sorting mechanism and to optimize the separation conditions. Isolation of fluorescent HeLa-DsRed cells was further demonstrated with high purity and recovery rate. Viability of the sorted cells was also examined, suggesting a survival rate of more than 90%. We expect this sorting approach to find widespread applications in bioanalysis.     

Label-free cell separation and sorting in microfluidic systems

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible.     

A micropillar-integrated smart microfluidic device for specific capture and sorting of cells

An integrated smart microfluidic device consisting of nickel micropillars, microvalves, and microchannels was developed for specific capture and sorting of cells. A regular hexagonal array of nickel micropillars was integrated on the bottom of a microchannel by standard photolithography, which can generate strong induced magnetic field gradients under an external magnetic field to efficiently trap superparamagnetic beads (SPMBs) in a flowing stream, forming a bed with sufficient magnetic beads as a capture zone. Fluids could be manipulated by programmed controlling the integrated air-pressure-actuated microvalves, based on which in situ bio-functionalization of SPMBs trapped in the capture zone was realized by covalent attachment of specific proteins directly to their surface on the integrated microfluidic device. In this case, only small volumes of protein solutions (62.5 nL in the capture zone; 375 nL in total volume needed to fill the device from inlet A to the intersection of outlet channels F and G) can meet the need for protein! The newly designed microfluidic device reduced greatly chemical and biological reagent consumption and simplified drastically tedious manual handling. Based on the specific interaction between wheat germ agglutinin (WGA) and N-acetylglucosamine on the cell membrane, A549 cancer cells were effectively captured and sorted on the microfluidic device. Capture efficiency ranged from 62 to 74%. The integrated microfluidic device provides a reliable technique for cell sorting.     

微流控芯片分选临床样品中循环肿瘤细胞的研究进展

癌症作为常见病正严重威胁着我国乃至全球居民的健康。循环肿瘤细胞(CTCs)是一类由癌变部位释放并进入血液中的癌细胞,其在癌症的早期诊断、个体化及肿瘤转移机制研究等方面的作用正逐渐被发现和认可,但由于血液中的CTCs含量极少,对其分选极具挑战。微流控芯片作为一种微型化、高通量、集成化平台,在CTCs研究中彰显了独特的优势,相关报道也越来越多。随着研究的深入,微流控芯片技术不再局限于基于模型样品的方法学开发,而是更注重于能否用于临床实际样品中CTCs的检测,但目前未见该角度的综述报道。为此,文章综述了近年来用于临床实际样品CTCs分析的微流控芯片分选技术,并探讨了微流控芯片用于CTCs分选的发展趋势。     

Particle sorting using a porous membrane in a microfluidic device

Porous membranes have been fabricated based on the development of the perforated membrane mold [Y. Luo and R. N. Zare, Lab Chip, 2008, 8, 1688-1694] to create a single filter that contains multiple pore sizes ranging from 6.4 to 16.6 μm inside a monolithic three-dimensional poly(dimethylsiloxane) microfluidic structure. By overlapping two filters we are able to achieve smaller pore size openings (2.5 to 3.3 μm). This filter operates without any detectable irreversible clogging, which is achieved using a cross-flow placed in front of each filtration section. The utility of a particle-sorting device that contains this filter is demonstrated by separating polystyrene beads of different diameters with an efficiency greater than 99.9%. Additionally, we demonstrate the effectiveness of this particle-sorting device by separating whole blood samples into white blood cells and red blood cells with platelets.     

An integrated microfluidic device for the simultaneous detection of multiple antibiotics

Residual antibiotics in food pose a serious long-term threat to human health. Therefore, an on-site visualization method for antibiotic detection is required. However, the requirements of traditional antibiotic testing methods in terms of operator proficiency and equipment cost hinder the rapid point-of-care-testing detection of suspected samples. Herein, we reported an integrated microfluidic device combining a microfluidic chip containing cruciform valves with immunochromatographic strips for the rapid detection of multiple antibiotics in milk. The rapid qualitative and quantitative analysis of four types of antibiotics (sulfonamides, β-lactams, streptomycin, and tetracyclines) was performed using mobile phone photography and mobile phone application analysis. The detection time was maintained at 10 min. The limits of detection (LODs) for the four antibiotics were 0.15, 0.12, 0.25, and 0.29 ng/mL, respectively, and the selectivity for the different antibiotics was observed even in a highly complex matrix. This device successfully integrated separation and real-time detection onto a chip and might provide a promising perspective for the detection of multiple antibiotics in milk.     

Cell sorting by endocytotic capacity in a microfluidic magnetophoresis device

Magnetically labelled cells are finding a wealth of applications for in vitro analysis as well as in vivo treatments. Sorting of cells into subpopulations based on their magnetite loading is an important step in such procedures. Here, we study the sorting of monocytes and macrophages which internalise nanoparticles to different extents based on their endocytotic capacity. Macrophages featured a high endocytotic activity and were found to internalise between 4 and 60 pg of iron per cell. They were successfully sorted into five subpopulations of narrow iron loading distributions via on-chip free-flow magnetophoresis, thus demonstrating the potential of sorting of relatively similarly loaded cells. Monocytes featured a low endocytotic capacity and took on 1 to 4 pg of iron per cell. Mixtures of monocytes and macrophages were successfully sorted within the free-flow magnetophoresis chip and good purity (>88%), efficacy (>60%) and throughput (from 10 to 100 cells s(-1)) could be achieved. The introduced method constitutes a viable tool for studies of endocytotic capacity and sorting/selection of cells based on this functionality.     

Conductivity detection for monitoring mixing reactions in microfluidic devices

A conductivity detector was coupled to poly(dimethylsiloxane)-glass capillary electrophoresis microchips to monitor microfluidic flow. Electroosmotic flow was investigated with both conductivity detection (CD) and the current monitoring method. No significant variation was observed between these methods, but CD showed a lower relative standard deviation. Gradient mixing experiments were employed to investigate the relationship between the electrolyte conductivity and the electrolyte concentration. A good linear response of conductivity to concentration was obtained for solutions whose difference in concentrations were less than 27 mM. The new system holds great promise for precision mixing in microfluidic devices using electrically driven flows.     

Polymer microfluidic chip for online monitoring of microarray hybridizations

A disposable single use polymer microfluidics chip has been developed and manufactured by micro injection molding. The chip has the same outer dimensions as a standard microscope slide (25 x 76 x 1.1 mm) and is designed to be compatible with existing microscope slide handling equipment like microarray scanners. The chip contains an inlet, a 10 microL hybridization chamber capable of holding a 1000 spot array, a waste chamber and a vent to allow air to escape when sample is injected. The hybridization chamber ensures highly homogeneous hybridization conditions across the microarray. We describe the use of this chip in a flexible setup with fluorescence based detection, temperature control and liquid handling by computer controlled syringe pumps. The chip and the setup presented in this article provide a powerful tool for highly parallel studies of kinetics and thermodynamics of duplex formation in DNA microarrays. The experimental setup presented in this article enables the on-chip microarray to be hybridized and monitored at several different stringency conditions during a single assay. The performance of the chip and the setup is demonstrated by on-line measurements of a hybridization of a DNA target solution to a microarray. A presented numerical model indicates that the hybridization process in microfluidic hybridization assays is diffusion limited, due to the low values of the diffusion coefficients D of the DNA and RNA molecules involved.     

Inertial lift enhanced phase partitioning for continuous microfluidic surface energy based sorting of particles

A new microfluidics technique that exploits the selectivity of phase partitioning and high-speed focusing capabilities of the inertial effects in flow was developed for continuous label-free sorting of particles and cells. Separations were accomplished by introducing particles at the interface of polyethylene glycol (PEG) and dextran (DEX) phases in rectangular high aspect-ratio microfluidic channels and allowing them to partition to energetically favorable locations within the PEG phase, DEX phase or interface at the center of the microchannel. Separation of partitioned particles was further enhanced via inertial lift forces that develop in high aspect-ratio microchannels that move particles to equilibrium positions close to the outer wall. Combining phase partitioning with inertial focusing ensures selectivity is possible using phase partitioning with sufficient throughput (at least an order of magnitude greater than phase partitioning alone) for application in the clinical and research setting. Using this system we accomplished separation of 15 μm polystyrene (PS) particles from 1-20 μm polymethylmethacrylate (PMMA) particles. Results confirm the feasibility of separation based on phase partitioning and enhancement of separation via inertial focusing. Approximately 86% of PS particles were isolated within the PEG phase whereas 78% of PMMA particles were isolated within the DEX phase. When a binary mixture of PS and PMMA was introduced within the device, ~83% of PS particles were isolated in the PEG phase and ~74% of PMMA particles were isolated in the DEX phase. These results confirm the feasibility of this technique for rapid and reliable separation of particles and potentially cells.     

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.     

Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes

Microfluidics has enabled new cell biology experiments. Incorporating chemical monitoring of cellular secretion into chips offers the potential to increase information content and utility of such systems. In this work, an integrated, multilayer polydimethylsiloxane microfluidic chip was developed to simultaneously measure fatty acids and glycerol secreted from cultured adipocytes on chip in near real time. Approximately 48,000 adipocytes were loaded into a cell chamber in a reversibly sealed chip. Cells were perfused at 0.75 μL/min. Cell perfusate was split and directed to separate, continuously operating fluorescent enzyme assay channel networks. The fluorescent assay products were detected simultaneously near the outlet of the chip. The fatty acid and glycerol assays had linear dynamic ranges of 150 and 110 μM and limit of detection (LOD) of 6 and 5 μM, respectively. Surface modifications including pretreatment with sodium dodecyl sulfate were utilized to prevent adsorption of fatty acids to the chip surface. Using the chip, basal fatty acid and glycerol concentrations ranged from 0.18 to 0.7 nmol?×?10⁶ cell^?1 min^?1 and from 0.23 to 0.85 nmol?×?10⁶ cell^?1 min^?1, respectively. Using valves built into the chip, the perfusion solution was switched to add 20 μM isoproterenol, a β-adrenergic agonist, which stimulates the release of glycerol and fatty acids in adipocytes. This manipulation resulted in a rapid and stable 1.5- to 6.0-fold increase of non-esterified fatty acid (NEFA) and glycerol. The ratio of NEFA:glycerol released increased with adipocyte age. These experiments illustrate the potential for performing multiple real-time assays on cells in culture using microfluidic devices.     

A glass microfluidic chip for continuous blood cell sorting by a magnetic gradient without labeling

This paper presents a microfluidic chip for highly efficient separation of red blood cells (RBCs) from whole blood on the basis of their native magnetic properties. The glass chip was fabricated by photolithography and thermal bonding. It consisted of one inlet and three outlets, and a nickel wire of 69-microm diameter was positioned in the center of a separation channel with 149-microm top width and 73-microm depth by two parallel ridges (about 10 microm high). The two ridges were formed simultaneously during the wet etching of the channels. The nickel wire for generating the magnetic gradient inside the separation channel was introduced from the side of the chip through a guide channel. The external magnetic field was applied by a permanent magnet of 0.3 T placed by the side of the chip and parallel to the main separation channel. The RBCs were separated continuously from the 1:40 (v/v) diluted blood sample at a flow rate in the range 0.12-0.92 microL/min (9-74 mm/min) with the chip, and up to 93.7% of the RBCs were collected in the middle outlet under a flow rate of 0.23 microL/min. The cell sedimentation was alleviated by adjusting the specific density of the supporting media with bovine serum albumin. Quantum dot labeling was introduced for visual fluorescence tracking of the separation process. The uneven distribution phenomenon of the blood cells around the nickel wire was reported and discussed.     

Design of microfluidic channel geometries for the control of droplet volume, chemical concentration, and sorting

Passive microfluidic channel geometries for control of droplet fission, fusion and sorting are designed, fabricated, and tested. In droplet fission, the inlet width of the bifurcating junction is used to control the range of breakable droplet sizes and the relative resistances of the daughter channels were used to control the volume of the daughter droplets. Droplet fission is shown to produce concentration differences in the daughter droplets generated from a primary drop with an incompletely mixed chemical gradient, and for droplets in each of the bifurcated channels, droplets were found to be monodispersed with a less than 2% variation in size. Droplet fusion is demonstrated using a flow rectifying design that can fuse multiple droplets of same or different sizes generated at various frequencies. Droplet sorting is achieved using a bifurcating flow design that allows droplets to be separated base on their sizes by controlling the widths of the daughter channels. Using this sorting design, submicron satellite droplets are separated from the larger droplets.     

Fish and Chips: a microfluidic perfusion platform for monitoring zebrafish development

We have developed a multi-channel microfluidic perfusion platform for culturing zebrafish embryos and capturing live images of various tissues and organs inside the embryo. The Fish and Chips was micro-fabricated in silicon and glass for reproducibility and accuracy of the microfluidic structure. The microfluidic platform consists of three parts: a microfluidic gradient generator, a row of eight fish tanks, in which the fish embryos are individually placed, and eight output channels. The fluidic gradient generator supports dose-dependent drug and chemical studies. A unique perfusion system ensures a uniform and constant flow of media to the fish tank while the wastes are efficiently removed. The fish tanks restrict the embryo movements, except rotationally, for live imaging of internal tissues and organs. The embryos showed developmental abnormalities under the influence of the drug valproic acid (VPA).     

A microfluidic device based on gravity and electric force driving for flow cytometry and fluorescence activated cell sorting

A novel method based on gravity and electric force driving of cells was developed for flow cytometry and fluorescence activated cell sorting in a microfluidic chip system. In the experiments cells flowed spontaneously under their own gravity in a upright microchip, passed through the detection region and then entered into the sorting electric field one by one at an average velocity of 0.55 mm s(-1) and were fluorescence activated cell sorted (FACS) by a switch-off activation program. In order to study the dynamical and kinematic characteristics of single cells in gravity and electric field of microchannels a physical and numerical module based on Newton's Law of motion was established and optimized. Hydroxylpropylmethyl cellulose (HPMC) was used to minimize cell assembling, sedimentation and adsorption to microchannels. This system was applied to estimate the necrotic and apoptotic effects of ultraviolet (UV) light on HeLa cells by exposing them to UV radiation for 10, 20 or 40 min and the results showed that UV radiation induced membrane damage contributed to the apoptosis and necrosis of HeLa cells.     

Recent advances in microfluidic cell sorting techniques based on both physical and biochemical principles

Microfluidic technologies for isolating cells of interest from a heterogeneous sample have attracted great attentions, due to the advantages of less sample consumption, simple operating procedure, and high separation accuracy. According to the working principles, the microfluidic cell sorting techniques can be categorized into biochemical (labeled) and physical (label‐free) methods. However, the inherent drawbacks of each type of method may somehow influence the popularization of these cell sorting techniques. Using the multiple complementary isolation principles is a promising strategy to overcome this problem, therefore there appears to be a continuing trend to integrate two or more sorting methods together. In this review, we focus on the recent advances in microfluidic cell sorting techniques relied on both physical and biochemical principles, with emphasis on the mechanisms of cell separation. The biochemical cell sorting techniques enhanced by physical principles and the physical cell sorting techniques enhanced by biochemical principles, are first introduced. Then, we highlight on‐chip magnetic‐activated cell sorting, on‐chip fluorescence‐activated cell sorting, multi‐step cell sorting and multi‐principle cell sorting techniques, which are based on both physical and biochemical separation mechanisms. Finally, the challenges and future perspectives of the integrated microfluidics for cell sorting are discussed.     

Fast microfluidic temperature control for high resolution live cell imaging

One major advantage of using genetically tractable model organisms such as the fission yeast Schizosaccharomyces pombe is the ability to construct temperature-sensitive mutations in a gene. The resulting gene product or protein behaves as wildtype at permissive temperatures. At non-permissive or restrictive temperatures the protein becomes unstable and some or all of its functions are abrogated. The protein regains its function when returning to a permissive temperature. In principle, temperature-sensitive mutation enables precise temporal control of protein activity when coupled to a fast temperature controller. Current commercial temperature control devices do not have fast switching capability over a wide range of temperatures, making repeated temperature changes impossible or impractical at the cellular timescale of seconds or minutes. Microfabrication using soft-lithography is emerging as a powerful tool for cell biological research. We present here a simple disposable polydimethylsiloxane (PDMS) based microfluidic device capable of reversibly switching between 5 °C and 45 °C in less than 10 s. This device allows high-resolution live cell imaging with an oil immersion objective lens. We demonstrate the utility of this device for studying microtubule dynamics throughout the cell cycle.     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.     

Rapid simultaneous determination of nitrate and nitrite on a centrifugal microfluidic device

A centrifugal microfluidic device was developed for the rapid sequential determination of two critical environmental species, nitrate and nitrite, in water samples. The nitrate is reduced to nitrite and the nitrite is derivatized. The analytes are determined spectrophotometrically through the disc with a 1.4 mm pathlength. The detection limits are 0.05 and 0.16 mg L⁻¹ for nitrite and nitrate respectively. The use of powdered reagents, the 100 μL sample required and the design of the device suggest that it would be suitable for field use.