Continuous hydrophoretic separation and sizing of microparticles using slanted obstacles in a microchannel

We report a microfluidic separation and sizing method of microparticles with hydrophoresis--the movement of suspended particles under the influence of a microstructure-induced pressure field. By exploiting slanted obstacles in a microchannel, we can generate a lateral pressure gradient so that microparticles can be deflected and arranged along the lateral flows induced by the gradient. Using such movements of particles, we completely separated polystyrene microbeads with 9 and 12 microm diameters. Also, we discriminated polystyrene microbeads with diameter differences of approximately 7.3%. Additionally, we measured the diameter of 10.4 microm beads with high coefficient of variation and compared the result with a conventional laser diffraction method. The slanted obstacle as a microfluidic control element in a microchannel is analogous to the electric, magnetic, optical, or acoustic counterparts in that their function is to generate a field gradient. Since our method is based on intrinsic pressure fields, we could eliminate the need for external potential fields to induce the movement of particles. Therefore, our hydrophoretic method will offer a new opportunity for power-free and biocompatible particle control within integrated microfluidic devices.     

Hydrophoretic high-throughput selection of platelets in physiological shear-stress range

A gentle, but fast means for low-stress, high-throughput platelet purification is of significant clinical and biotechnological utility. Current implementations to sort platelets, however, require an external physical field, specialized buffer, or the harsh separation condition of high shear stress that tends to cause platelet stimulation. Here we report the use of hydrophoretic size separation in a wider channel and its parallelization to augment its throughput capability, maintaining physiological shear-stress range. We demonstrate a parallelized device comprising 10 stacks of the wide-channel hydrophoresis device, yielding a throughput of 2.9 million cells s(-1) and a platelet purity of 76.8%. The use of the wide channel for hydrophoresis also facilitates clogging-free separation by sorting blood clots and plaques. The wide-channel hydrophoresis offers the potential for gentle, fast, clogging-free sorting of rare blood cells with extreme throughput capabilities.     

Investigation of viscoelastic focusing of particles and cells in a zigzag microchannel

Microfluidic particle focusing has been a vital prerequisite step in sample preparation for downstream particle separation, counting, detection, or analysis, and has attracted broad applications in biomedical and chemical areas. Besides all the active and passive focusing methods in Newtonian fluids, particle focusing in viscoelastic fluids has been attracting increasing interest because of its advantages induced by intrinsic fluid property. However, to achieve a well-defined focusing position, there is a need to extend channel lengths when focusing micrometer-sized or sub-microsized particles, which would result in the size increase of the microfluidic devices. This work investigated the sheathless viscoelastic focusing of particles and cells in a zigzag microfluidic channel. Benefit from the zigzag structure of the channel, the channel length and the footprint of the device can be reduced without sacrificing the focusing performance. In this work, the viscoelastic focusing, including the focusing of 10 μm polystyrene particles, 5 μm polystyrene particles, 5 μm magnetic particles, white blood cells (WBCs), red blood cells (RBCs), and cancer cells, were all demonstrated. Moreover, magnetophoretic separation of magnetic and nonmagnetic particles after viscoelastic pre-focusing was shown. This focusing technique has the potential to be used in a range of biomedical applications.     

A hybrid dielectrophoretic and hydrophoretic microchip for particle sorting using integrated prefocusing and sorting steps

This work explores dielectrophoresis (DEP)‐active hydrophoresis in sorting particles and cells. The device consists of prefocusing region and sorting region with great potential to be integrated into advanced lab‐on‐a‐chip bioanalysis devices. Particles or cells can be focused in the prefocusing region and then sorted in the sorting region. The DEP‐active hydrophoretic sorting is not only based on size but also on dielectric properties of the particles or cells of interest without any labelling. A mixture of 3 and 10 μm particles were sorted and collected from corresponding outlets with high separation efficiency. According to the different dielectric properties of viable and nonviable Chinese Hamster Ovary (CHO) cells at the medium conductivity of 0.03 S/m, the viable CHO cells were focused well and sorted from cell sample with a high purity.     

A low-cost and high-throughput benchtop cell sorter for isolating white blood cells from whole blood

The ability to isolate and purify white blood cells (WBCs) from mixed ensembles such as blood would benefit autologous cell-based therapeutics as well as diagnosis of WBC disorders. Current WBCs isolation methods have the limitations of low purity or requiring complex and expensive equipment. In addition, due to the overlap in size distribution between lymphocytes (i.e., a sub-population of WBCs) and red blood cells (RBCs), it is challenging to achieve isolation of entire WBCs populations. In this work, we developed an inertial microfluidics-based cell sorter, which enables size-based, high-throughput isolation, and enrichment of WBCs from RBC-lysed whole blood. Using the developed inertial microfluidic chip, the sorting resolution is sharpened within 2 μm, which achieved separation between 3 and 5 μm diameter particles. Thus, with the present cell sorter, a full population of WBCs can be isolated from RBC-lysed blood samples with recovery ratio of 92%, and merely 5% difference in the composition percentage of the three subpopulations of granulocytes, monocytes, and lymphocytes compared to the original sample. Furthermore, our cell sorter is designed to enable broad application of size-based inertial cell sorting by supplying a series of microchips with different sorting cutoff size. This strategy allows us to further enrich the lymphocytes population by twofold using another microchip with a cutoff size between 10 and 15 μm. With simplicity and efficiency, our cell sorter provides a powerful platform for isolating and sorting of WBCs and also envisions broad potential sorting applications for other cell types.     

Elastic-inertial separation of microparticle in a gradually contracted microchannel

Separation of microparticle in viscoelastic fluid is highly required in the field of biology and clinical medicine. For instance, the separation of the target cell from blood is an important prerequisite step for the drug screening and design. The microfluidic device is an efficient way to achieve the separation of the microparticle in the viscoelastic fluid. However, the existing microfluidic methods often have some limitations, including the requirement of the long channel length, the labeling process, and the low throughput. In this work, based on the elastic-inertial effect in the viscoelastic fluid, a new separation method is proposed where a gradually contracted microchannel is designed to efficiently adjust the forces exerted on the particle, eventually achieving the high-efficiency separation of different sized particles in a short channel length and at a high throughput. In addition, the separation of WBCs and RBCs is also validated in the present device. The effect of the flow rate, the fluid property, and the channel geometry on the particle separation is systematically investigated by the experiment. With the advantage of small footprint, simple structure, high throughput, and high efficiency, the present microfluidic device could be utilized in the biological and clinical fields, such as the cell analysis and disease diagnosis.     

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.     

Study on the discrete dielectrophoresis for particle–cell separation

This paper presents the application of the discrete dielectrophoretic force to separate polystyrene particles from red blood cells. The separation process employs a simple microfluidic device that is composed of interdigitated electrodes and a microchannel. The discrete dielectrophoretic force is generated by adjusting the duty cycle of the applied voltage. The electrodes make a tilt angle with the microchannel to change the moving direction of the red blood cells. By adjusting the voltage magnitude and duty cycle, we investigate the deflection of red blood cells and the variation of cell velocity along electrode edge under positive dielectrophoresis. The experiments with polystyrene particles show that the enrichment of the particles is greater than 150 times. The maximum separation efficiency is 97% for particle-to-cell number ratio equal to 1:2000 in the sample having high cell concentration. Using the appropriate applied voltage magnitude and duty cycle, the discrete dielectrophoretic force can prevent the clogging of microchannel while successfully separating the particles from the cells with high enrichment and efficiency. The proposed principle can be readily applied to dielectrophoresis-based devices for biomedical sample preparation or diagnosis such as the separation of rare or infected cells from a blood sample.     

Inertial separation in a contraction-expansion array microchannel

We report a contraction-expansion array (CEA) microchannel that allows inertial size separation by a force balance between inertial lift and Dean drag forces in fluid regimes in which inertial fluid effects become significant. An abrupt change of the cross-sectional area of the channel curves fluid streams and produces a similar effect compared to Dean flows in a curved microchannel of constant cross-section, thereby inducing Dean drag forces acting on particles. In addition, the particles are influenced by inertial lift forces throughout the contraction regions. These two forces act in opposite directions each other throughout the CEA microchannel, and their force balancing determines whether the particles cross the channel, following Dean flows. Here we describe the physics and design of the CEA microfluidic device, and demonstrate complete separation of microparticles (polystyrene beads of 4 and 10 μm in diameter) and efficient exchange of the carrier medium while retaining 10 μm beads.     

Low-cost polymer-film spiral inertial microfluidic device for label-free separation of malignant tumor cells

We developed a low-cost polymer-film spiral inertial microfluidic device for the effective size-dependent separation of malignant tumor cells. The device was fabricated in polymer films by rapid laser cutting and chemical bonding. After fabricating the prototype device, the separation performance of our device was evaluated using particles and cells. The effects of operational flow rate, cell diameter, and cell concentration on the separation performance were explored. Our device successfully separated tumor cells from polydisperse white blood cells according to their different migration modes and lateral positions. Then, the separation of rare cells was carried out using the high-concentration lysed blood spiked with 200 tumor cells. Experimental results showed that 83.90% of the tumor cells could be recovered, while 99.87% of white blood cells could be removed. We successfully employed our device for processing clinical pleural effusion samples from patients with advanced metastatic breast cancer. Malignant tumor cells with an average purity of 2.37% could be effectively enriched, improving downstream diagnostic accuracy. Our device offers the advantages of label-free operation, low cost, and fast fabrication, thus being a potential tool for effective cell separation.     

基于错流过滤原理的微流控细胞分离芯片的研制

首次提出并制备了一种错流过滤式细胞分离微流控芯片.     

Controllable Synthesis of Multicompartmental Particles Using 3D Microfluidics

A microfluidic assembly method based on a microfluidic chip and capillary device was developed to create multicompartmental particles. The microfluidic chip design endows the particles with regulable internal structure. By adjusting the microstructure of the chip, the diameter of the capillary, the gap length between the two microfluidic components, and the flow rates, the size of the particles and the number or the ratio of different regions within the particle could be widely varied. As a proof of concept, we have produced some complicated particles that even contain 20 compartments. Furthermore, the potential applications of the anisotropic particles are explored by encapsulating magnetic beads, fluorescent nanoparticles, and the cells into different compartments of the microparticles. We believe that this method will open new avenues for the design and application of multicompartmental particles.     

Pinched flow fractionation devices for detection of single nucleotide polymorphisms

We demonstrate a new and flexible microfluidic based method for genotyping single nucleotide polymorphisms (SNPs). The method relies on size separation of selectively hybridized polystyrene microspheres in a microfluidic pinched flow fractionation (PFF) device. The microfluidic PFF devices with 13 mum deep channels were fabricated by thermal nanoimprint lithography (NIL) in a thin film of cyclic-olefin copolymer (mr-I T85) on a silicon wafer substrate, and the channels were sealed by thermal polymer bonding. Streptavidin coated polystyrene microspheres with a mean diameter of 3.09 microm and 5.6 microm were functionalized with biotin-labeled oligonucleotides for the detection of a mutant (Mt) or wild-type (Wt) DNA sequence in the HBB gene, respectively. Hybridization to functionalized beads was performed with fluorescent targets comprising synthetic DNA oligonucleotides or amplified RNA, synthesized using human DNA samples from individuals with point mutations in the HBB gene. Following a stringent wash, the beads were separated in a PFF device and the fluorescent signal from the beads was analyzed. Patients being wildtypes, heterozygotes or mutated respectively for the investigated mutation could reliably be diagnosed in the PFF device. This indicates that the PFF technique can be used for accurate and fast genotyping of SNPs.     

Theoretical and experimental analysis of negative dielectrophoresis-induced particle trajectories

Many biomedical analysis applications require trapping and manipulating single cells and cell clusters within microfluidic devices. Dielectrophoresis (DEP) is a label-free technique that can achieve flexible cell trapping, without physical barriers, using electric field gradients created in the device by an electrode microarray. Little is known about how fluid flow forces created by the electrodes, such as thermally driven convection and electroosmosis, affect DEP-based cell capture under high conductance media conditions that simulate physiologically relevant fluids such as blood or plasma. Here, we compare theoretical trajectories of particles under the influence of negative DEP (nDEP) with observed trajectories of real particles in a high conductance buffer. We used 10-µm diameter polystyrene beads as model cells and tracked their trajectories in the DEP microfluidic chip. The theoretical nDEP trajectories were in close agreement with the observed particle behavior. This agreement indicates that the movement of the particles was highly dominated by the DEP force and that contributions from thermal- and electroosmotic-driven flows were negligible under these experimental conditions. The analysis protocol developed here offers a strategy that can be applied to future studies with different applied voltages, frequencies, conductivities, and polarization properties of the targeted particles and surrounding medium. These findings motivate further DEP device development to manipulate particle trajectories for trapping applications.     

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.     

Numerical study of dielectrophoresis-modified inertial migration for overlapping sized cell separation

Circulating tumor cells (CTCs) have been proven to have significant prognostic, diagnostic, and clinical values in early-stage cancer detection and treatment. The efficient separation of CTCs from peripheral blood can ensure intact and viable CTCs and can, thus, give proper genetic characterization and drug innovation. In this study, continuous and high-throughput separation of MDA-231 CTCs from overlapping sized white blood cells (WBCs) is achieved by modifying inertial cell focusing with dielectrophoresis (DEP) in a single-stage microfluidic platform by numeric simulation. The DEP is enabled by embedding interdigitated electrodes with alternating field control on a serpentine microchannel to avoid creating two-stage separation. Rather than using the electrokinetic migration of cells which slows down the throughput, the system leverages the inertial microfluidic flow to achieve high-speed continuous separation. The cell migration and cell positioning characteristics are quantified through coupled physics analyses to evaluate the effects of the applied voltages and Reynolds numbers (Re) on the separation performance. The results indicate that the introduction of DEP successfully migrates WBCs away from CTCs and that separation of MDA-231 CTCs from similar sized WBCs at a high Re of 100 can be achieved with a low voltage of magnitude 4 ×10⁶ V/m. Additionally, the viability of MDA-231 CTCs is expected to be sustained after separation due to the short-term DEP exposure. The developed technique could be exploited to design active microchips for high-throughput separation of mixed cell beads despite their significant size overlap, using DEP-modified inertial focusing controlled simply by adjusting the applied external field.     

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.     

Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

Continuous flow microfluidic device for cell separation, cell lysis and DNA purification

A novel integrated microfluidic device that consisted of microfilter, micromixer, micropillar array, microweir, microchannel, microchamber, and porous matrix was developed to perform sample pre-treatment of whole blood. Cell separation, cell lysis and DNA purification were performed in this miniaturized device during a continuous flow process. Crossflow filtration was proposed to separate blood cells, which could successfully avoid clogging or jamming. After blood cells were lyzed in guanidine buffer, genomic DNA in white blood cells was released and adsorbed on porous matrix fabricated by anodizing silicon in HF/ethanol electrolyte. The flow process of solutions was simulated and optimized. The anodization process of porous matrix was also studied. Using the continuous flow procedure of cell separation, cell lysis and DNA adsorption, average 35.7 ng genomic DNA was purified on the integrated microfluidic device from 1 μL rat whole blood. Comparison with a commercial centrifuge method, the miniaturized device can extract comparable amounts of PCR-amplifiable DNA in 50 min. The greatest potential of this integrated miniaturized device was illustrated by pre-treating whole blood sample, where eventual integration of sample preparation, PCR, and separation on a single device could potentially enable complete detection in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.