多功能芯片对合成样本中肝癌细胞HepG2的测试研究

设计并制作了一种集多孔流分离(Multi-orifice flow fractionation,MOFF)技术与磁捕获技术于一体的用于特异性分离和捕获合成样本中肝癌细胞HepG2的多功能微流控细胞芯片.此芯片由玻璃基片和PDMS微通道盖片组成,PDMS盖片上含有3条进样通道、MOFF分离区和六边形腔体的细胞富集检测区.其中,MOFF分离区总长20 mm,由80组长度为0.18 mm、深度为50μm、收缩区域宽度为0.06 mm、扩张区域宽度为0.20 mm的半菱形收缩/扩张重复单元组成,每组收缩/扩张重复单元间的夹角为103.0°.实验以肝癌细胞HepG2-血细胞混悬液为样本;根据磁珠表面修饰c-Met抗体能与肝癌细胞HepG2特异性结合的原理,通过表面羧基化的磁珠、EDC(1 mg/mL)、NHS(1 mg/mL)和c-Met抗体制备了浓度为50μg/mL的免疫磁珠(Anti-MNCs)悬浮液.在样本流速为50μL/min条件下,利用外加磁场实现了血细胞合成样本中微量肝癌细胞HepG2的有效捕获;采用微波加热法以柠檬酸、硫脲为原料制备了用于荧光标记HepG2的碳量子点,在芯片上实现了血液中肝癌细胞HepG2的原位荧光可视化观测.对芯片检测区捕获到的HepG2进行了显微计数分析,对500μL血细胞(107 cell/mL)中含10个HepG2细胞的合成样本,捕获效率达到88.5%±6.7%(n=20).结果表明,所设计的多模式多功能的微流控芯片具有良好的肿瘤细胞分离和检测功能.     

Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP)

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 μL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications.     

Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid

Pure separation and sorting of microparticles from complex fluids are essential for biochemical analyses and clinical diagnostics. However, conventional techniques require highly complex and expensive labeling processes for high purity separation. In this study, we present a simple and label-free method for separating microparticles with high purity using the elasto-inertial characteristic of a non-Newtonian fluid in microchannel flow. At the inlet, particle-containing sample flow was pushed toward the side walls by introducing sheath fluid from the center inlet. Particles of 1 μm and 5 μm in diameter, which were suspended in viscoelastic fluid, were successfully separated in the outlet channels: larger particles were notably focused on the centerline of the channel at the outlet, while smaller particles continued flowing along the side walls with minimal lateral migration towards the centerline. The same technique was further applied to separate platelets from diluted whole blood. Through cytometric analysis, we obtained a purity of collected platelets of close to 99.9%. Conclusively, our microparticle separation technique using elasto-inertial forces in non-Newtonian fluid is an effective method for separating and collecting microparticles on the basis of size differences with high purity.     

Improved preparative-scale. continuous, free-flow electrophoretic separation of the enantiomers of terbutaline utilizing equal-but-opposite enantiomer mobilities.

The factors that influence yield and product purity in the continuous, preparative-scale electrophoretic separation of the enantiomers of terbutaline when using the principle of equal-but-opposite effective mobilities were studied. The sodium salt of heptakis-6-sulfato-beta-cyclodextrin was used as the resolving agent, in acidic, isopropanol-containing background electrolytes, in the continuous, free-flow, preparative electrophoretic instrument, the Octopus. By matching the linear velocity of the feed solution to that of the background electrolyte, lateral hydrodynamic dispersion was minimized resulting in a nonelectrophoresed feed band that was only three fractions (about 3 mm) wide as it exited the 0.5 m long separation channel. The multiple of residence time and applied potential was also optimized, constrained by migration of the front of heptakis-6-sulfato-beta-cyclodextrin out of the separation zone, leading to the recovery of 95% of both enantiomers in better than 99.99% purity, at a production rate of 0.1 mg/h.     

Pressure generation at the junction of two microchannels with different depths

In this study, we report the design of a microchip‐based hydraulic pump that comprises three glass conduits arranged in a T‐geometry, one of which has a 2 mm long segment shallower (0.5–3 μm in depth) than the remaining 15 μm deep microfluidic network. Upon application of an electric field across this microchannel junction, a mismatch in EOF rate is introduced due to a differential in the fluid conductivity across the deep and shallow segments. Using the reported micropump, pressure‐driven velocities up to 3.2 mm/s have been generated in a 15 μm deep separation channel for an applied voltage of 1.75 kV allowing us to operate under separation conditions that yield the minimum plate height. Moreover, we have shown that this flow velocity can be maximized by optimizing the depth in the shallow region of the T‐geometry. Interestingly however, a simple theory accounting for fluid conductivity differences across microchannels of different depths significantly underestimates the pressure‐driven velocities observed in our experiments. The Taylor dispersion coefficient in our system on the other hand compares well with the theoretical predictions reported in the literature. Finally, the functionality of our device has been demonstrated by implementing a reverse‐phase chromatographic separation that was driven by the pressure‐driven flow generated on‐chip.     

Microalgae separation by inertia-enhanced pinched flow fractionation

To improve the accuracy and efficiency of ships’ ballast water detection, the separation of microalgae according to size is significant. In this article, a method to separate microalgae based on inertia-enhanced pinched flow fractionation (iPFF) was reported. The method utilized the inertial lift force induced by flow to separate microalgae according to size continuously. The experimental results show that, as the Reynolds number increases, the separation effect becomes better at first, but then stays unchanged. The best separation effect can be obtained when the Reynolds number is 12.3. In addition, with the increase of the flow rate ratio between sheath fluid and microalgae mixture, the separation effect becomes better and the best separation effect can be obtained when the flow rate ratio reaches 10. In this case, the recovery rate of Tetraselmis sp. is about 90%, and the purity is about 86%; the recovery rate of Chlorella sp. is as high as 99%, and the purity is about 99%. After that, the separation effect keeps getting better but very slowly. In general, this study provides a simple method for the separation of microalgae with different sizes, and lays a foundation for the accurate detection of microalgae in the ballast water.     

Non-orthogonal micro-free flow electrophoresis: From theory to design concept

Micro-free flow electrophoresis (μFFE) is a technique that facilitates continuous separation of molecules in a shallow channel with a hydrodynamic flow and an electric field at an angle to the flow. We recently developed a general theory of μFFE that suggested that an electric field non-orthogonal to the flow could improve resolution. Here, we used computer modeling to study resolution as a function of the electric field strength and the angle between the electric field and the hydrodynamic flow. In addition we used our general theory of μFFE to investigate other important influences on resolution, which include the velocity of the hydrodynamic flow, the height of the separation channel, and the magnitude and direction of the electroosmotic flow. Finally, we propose four designs that could be used to generate non-orthogonal electric fields and discuss their relative merits.     

Electrophoretic chip for high-fidelity fractionation of double-stranded DNA

We report the high fidelity, on-chip fractionation of selected segments from an electrophoretic flow of separated fragments. dsDNA fragments (10-330 base pairs (bp)) were initially separated using a 6.5 cm long channel with an electric field strength of 150 V/cm. As an example of the fractionation process, a target fragment of 20 bp was selected and extracted from the separation channel. The extraction was confirmed and evaluated by fluorescence imaging. High resolution and extraction fidelity were achieved by introducing new procedures for (i) extraction channel-blocking and (ii) segment transfer with cleaning. These procedures are necessary for the development of a practical, fully automated multitarget fractionation electrophoretic chip. A kind of CCD image processing method was introduced to monitor, control, and evaluate the procedure of fractionation. The resolution limits of the separation and extraction are discussed.     

SPLITT Cell Analytical Separation of Silica Particles. Non-Specific Crossover Effects: Does the Shear-Induced Diffusion Play a Role?

The gravitational split-flow lateral transport thin fractionation is known to be a fast, simple, theoretically tractable and tunable tool for the binary separation of molecular or particulate samples into different dimensional fractions. This fractionation is performed in a so-called SPLITT cell and is due to the combined effect of the gravitational force field and the flow rates inside the separation channel. It is known that separation performance is strongly dependent on the flow rate conditions and feed flow concentration, however, to date, few studies have been conducted to investigate the effect non-specific crossover has on separation. The aim of this work is to establish whether diffusive processes stemming from hydrodynamic effects contribute in any way to the quality of separation. A silica sample of known granule size distribution was chosen for this study which has environmental applications.     

Low-capacity channel designed for particle separation with controlled electric fields and evaluation of involved forces

Electric field is one of the suitable physical fields applicable to particle separations. Although long rectangular channel is used for particle separation in usual electrical field flow fractionation (FFF), a short low-capacity channel can replace it if the field is precisely controlled. Several separation principles are proposed with this channel. The elution behavior of particles has revealed that the gravitational, diffusion, and hydrodynamic lift force (HLF) play important roles in the determination of the elution behavior of particles. The elution threshold voltage (V(th)) was defined and experimentally determined for various system configurations and particles. The electric force no longer overcomes the other forces, and particles are taken off the wall, when the applied voltage becomes lower than V(th). V(th) values have allowed us not only to estimate surface charge density of a particle but also to evaluate the hydrodynamic lift force against particle.     

Effect of flow development region and fringing magnetic force field on annular split-flow thin fractionation

Split-flow thin (SPLITT) fractionation devices have been widely used to separate macromolecules, colloids, cells and particles. Recently, the quadrupole magnetic flow sorter (QMS) has been reported in the literature as another family of SPLITT fractionation device. However, the separation performance observed in the experimental measurements is generally found to deviate from the ideal behaviour. Possible causes such as hydrodynamic lift force, high particle concentration and imperfect geometries have been extensively examined. However, the effects of flow development regions and fringing magnetic force field at the separation channel inlet and outlet, which are ignored by the theory, have not been investigated. The error introduced by ignoring these effects need to be rigorously studied so that the theory can be used to optimise operation flow rates with confidence. Indeed, we find in this paper that these ignored effects are responsible to the discrepancy between the experimental data and the theoretical predictions. A new theory has been proposed for optimisation of device operation.     

Electrophoretic chip for fractionation of selective DNA fragment

A microchannel chip has been used to fractionate selected segments from an electrophoretic flow of separated fragments. A sample, which covers the size from 35 to 670 bp, was initially separated using an 8.8-cm-long channel at the electric field strength of 100 V/cm. The target fragment of 318 bp was selected and extracted from the separation channel. High-resolution fractionation was achieved by introducing new procedures for blocking, extraction, and segment transfer. Fractionation quality with and without blocking were compared using a 310 Genetic Analyzer (Applied Biosystems). The results show that no contamination was found in the sample, which was fractionated with blocking; however, a contamination by short segments was found in the sample, which was fractionated without blocking. Furthermore, fractionation by the chip was found to be of higher fidelity than that by the polyacrylamide slab gel, which displayed a small overlapped peak after the target peak. Compared with the traditional method, our chips enable faster and high-fidelity fractionation, thus providing a new tool for bioanalysis and other applications.     

Velocity domain and volume fraction distribution of heavy microparticles in low reynolds number flow in microchannel

In this paper, a two-dimensional Stokes flow having particle size distribution ranging from 10 to 50 µm in a rectangular channel is simulated numerically with focus on the hydrodynamic forces. The results show that due to the disparity between the density of the fluid and particles, velocity domain of particles deviates from the fluid velocity domain and this phenomenon occurs significantly for the larger particles. Also, with the increase of Reynolds number, the volume fraction of dispersed phase near the bottom wall of the channel increases either. Compared to similar studies, this investigation employs numerical simulation of microparticulate flow and interparticle hydrodynamic forces with emphasis on the dispersed phase volume fraction in order to present the microchannel flow properties.     

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.     

流动聚焦型微流控体系中的液滴的图案化排列和转变模式

流体在微流通道中形成剪切流场（低雷诺数）．不同于宏观体系,由于剪切力和表面张力的竞争作用,产生的液滴在微尺度下的微流通道中形成特殊的排列现象---周期性类似“晶格”排列现象．设计了新型流动聚焦型微流控芯片,分析研究在微流体系中液滴周期性图案化排列和转变机理性,液滴排列模式受两方面因素影响：水油两相的流速比值和微通道尺寸．当微通道宽度为250或300 μm时,液滴形成单层分散,双层和单层挤压排列．当微通道宽度为350 μm 时,液滴会形成单层分散到三层排列到双层挤压最后到单层挤压排列．当出口通道宽度增加到400 μm时,甚至出现了液滴四层排列的现象．同时研究了各个液滴排列模式的“转变点”.     

On-chip fraction collection for multiple selected ssDNA fragments using isolated extraction channels

High efficiency and high-purity fraction collection is highly sought in analysis of fragments-of-interest from selective polymerase chain reaction (PCR) products generated by High Coverage Gene Expression Profiling (HiCEP) methods. Here we demonstrate a new electrophoretic chip device enabling automatic high-efficient fractionation of multiple ssDNA target fragments during a run of separation. We used thoroughly isolated extraction channels for each selected target to reduce the risk of cross-contamination between targets due to cross-talk of extraction channels. Fragments of 35, 108 and 138 b, were successfully isolated, then the recovery was PCR-amplified and assessed by capillary electrophoresis (CE) analysis. Total impurity level of the targets due to unwanted fragments of 0.7%, 2% and 6% respectively, was estimated. Difficulties in collecting multiple target factions are due to band diffusion and DNA adsorption to the walls for the fragments in the separation channel, which is generated by transferring the DNA target fraction from the extraction section to the target reservoir. Therefore, we have carefully measured band broadening and analyzed its influence on the separation resolution due to the delay.     

Improvement of size-based particle separation throughput in slanted spiral microchannel by modifying outlet geometry

The inertial microfluidic technique, as a powerful new tool for accurate cell/particle separation based on the hydrodynamic phenomenon, has drawn considerable interest in recent years. Despite numerous microfluidic techniques of particle separation, there are few articles in the literature on separation techniques addressing external outlet geometry to increase the throughput efficiency and purity. In this work, we report on a spiral inertial microfluidic device with high efficiency (>98%). Herein, we demonstrate how changing the outlet geometry can improve the particle separation throughput. We present a complete separation of 4 and 6 μm from 10 μm particles potentially applicable to separate microalgae (Tetraselmis suecica from Phaeodactylum tricornutum). Two spiral microchannels with the same cross section dimension but different outlet geometry were considered and tested to investigate the particle focusing behavior and separation efficiency. As compared with particle focusing observed in channels with a simple outlet, the particle focusing in a modified outlet geometry appears in a more successful focusing manner with complete separation. This simple approach of particle separation makes it attractive for lab-on-a-chip devices for continuous extraction and filtration of a wide range of cell/particle sizes.     

Separation of polystyrenes by means of open tubular capillary chromatography

Simulating polymer separation in flow-through channels of monolithic columns, separation of a mixture of polystyrene standards was investigated using open tubular capillary column of 2 μm inner diameter. High column efficiency was observed for polymers of molar mass ranged from few tens to few hundred kDas. Column efficiency significantly decreased for polymers with molar mass larger than 500 kDa nevertheless preserving value of few tens of thousands theoretical plates. Calibration curve observed for open capillary column is rather steep and can be well described by simple equation without quadratic term. In spite of low selectivity, capillary columns were able in separating wide range of polystyrene standards due to column high efficiency and in such a way supported an idea of hydrodynamic mechanism of polymer separation in flow-through channel of monolithic packings.     

Purification of 3-hydroxypropionitrile by wiped molecular distillation

3-hydroxypropionitrile (HPN) is widely used asmaterials in many organic reactions such as synthe-sizing medicine, pesticide and polymeric compound.Since the 1970s, HPN has mostly been synthesizedwith the reaction of acrylonitrile and H2O catalyzed byNaOH aqueous solution, and the mole yield of HPN isabove 85%. However, the HPN must be separatedfrom the acrylonitrile hydration mixture and get rid ofwater, salt and other side products. The traditional separation method is evapora-tion…     

Biocompatible channels for field-flow fractionation of biological samples: correlation between surface composition and operating performance

Biocompatible methods capable of rapid purification and fractionation of analytes from complex natural matrices are increasingly in demand, particularly at the forefront of biotechnological applications. Field-flow fractionation is a separation technique suitable for nano-sized and micro-sized analytes among which bioanalytes are an important family. The objective of this preliminary study is to start a more general approach to field-flow fractionation for bio-samples by investigation of the correlation between channel surface composition and biosample adhesion. For the first time we report on the use of X-ray photoelectron spectroscopy (XPS) to study the surface properties of channels of known performance. By XPS, a polar hydrophobic environment was found on PVC material commonly used as accumulation wall in gravitational field-flow fractionation (GrFFF), which explains the low recovery obtained when GrFFF was used to fractionate a biological sample such as Staphylococcus aureus. An increase in separation performance was obtained first by conditioning the accumulation wall with bovine serum albumin and then by using the ion-beam sputtering technique to cover the GrFFF channel surface with a controlled inert film. XPS analysis was also employed to determine the composition of membranes used in hollow-fiber flow field-flow fractionation (HF FlFFF). The results obtained revealed homogeneous composition along the HF FlFFF channel both before and after its use for fractionation of an intact protein such as ferritin.