Computational and performance analysis of a continuous magnetophoretic bioseparation chip with alternating magnetic fields

This paper presents the modeling and optimization of a magnetophoretic bioseparation chip for isolating cells, such as circulating tumor cells from the peripheral blood. The chip consists of a continuous-flow microfluidic platform that contains locally engineered magnetic field gradients. The high-gradient magnetic field produced by the magnets is spatially non-uniform and gives rise to an attractive force on magnetic particles flowing through a fluidic channel. Simulations of the particle–fluid transport and the magnetic force are performed to predict the trajectories and capture lengths of the particles within the fluidic channel. The computational model takes into account key forces, such as the magnetic and fluidic forces and their effect on design parameters for an effective separation. The results show that the microfluidic device has the capability of separating various cells from their native environment. An experimental study is also conducted to verify and validate the simulation results. Finally, to improve the performance of the separation device, a parametric study is performed to investigate the effects of the magnetic bead size, cell size, number of beads per cell, and flow rate on the cell separation performance.     

Magnetophoretic mixing for in situ immunochemical binding on magnetic beads in a microfluidic channel

Functionalized magnetic beads offer promising solutions to a host of micro-total analysis systems ranging from immunomagnetic biosensors to cell separators. Immunochemical binding of functional biochemical agents or target biomolecules serves as a key step in such applications. Here we show how magnetophoretic motion of magnetic microspheres in a microchannel is harnessed to promote in situ immunochemical binding of short DNA strands (probe oligonucleotide) on the bead surface via streptavidin–biotin bonds. Using a transverse magnetic field gradient, the particles are transported across a co-flowing analyte stream containing biotinylated probe oligonucleotides that are labeled with a Cy3-fluorophore. Quantification of the resulting biotin–streptavidin promoted binding has been achieved through fluorescence imaging of the magnetophoretically separated magnetic particles in a third stream of phosphate buffered saline. Both the experimental and numerical data indicate that for a given flow rate, the analyte binding per bead depends on the flow fraction of the co-flowing analyte stream through the microchannel, but not on the fluid viscosity. Parametric studies of the effects of fluid viscosity, analyte flow fraction, and total flow rate on the extent of binding and the overall analyte separation rate are also conducted numerically to identify favorable operating regimes of a flow-through immunomagnetic separator for biosensing, cell separation, or high-throughput applications.     

Magnetic particle dosing and size separation in a microfluidic channel

Separation of functional magnetic particles or magnetically labeled entities is a key feature for bioanalytical or biomedical applications and therefore also an important component of lab-on-a-chip devices for biological applications. We present a novel integrated microfluidic magnetic bead manipulation device, comprising dosing of magnetic particles, controlled release and subsequent magnetophoretic size separation with high resolution. The system is designed to meet the requirements of specific bioassays, in particular of on-chip agglutination assays for the detection of rare analytes by particle coupling as doublets. Integrated soft-magnetic microtips with different shapes provide the magnetic driving force of the bead manipulation protocol. The magnetic tips that serve as field concentrators of an external electromagnetic field, are positioned in close contact to a microfluidic channel in order to generate high magnetic actuation forces. Mixtures of 1.0 μm and 2.8 μm superparamagnetic beads have been used to characterize the system. Magnetophoretic size separation with high resolution was performed in static conditions and in continuous flow mode. In particular, we could demonstrate the separation of 1.0 μm single beads and doublets in a sample flow.     

Operating regimes of a magnetic split-flow thin (SPLITT) fractionation microfluidic device for immunomagnetic separation

Magnetic bead-based immunoassays in the microfluidic format have attracted particular interest as it has several advantages over other microfluidic separation techniques. Magnetic split-flow thin fractionation (SPLITT) is a compact version of microfluidic sorting where a bidispersed or polydispersed suspension of magnetic particle–analyte conjugates can be selectively isolated into co-flowing streams of nearly monodispersed particles. Although the device offers capability of identifying and separating more than one target analytes simultaneously, its performance is sensitive to the slightest variation of the operating condition. Herein, we have numerically investigated the performance of a microscale magnetic SPLITT device. Using a coupled Eulerian–Lagrangian approach, we have evaluated the capture efficiency (CE) and separation index (SI) for each particle type collected at their designated outlet of the SPLITT device and identified the best regimes of operating parameters. While the CE figures are found to be best represented by a group variable Π, the SI values are better represented as function of the product of the group variables γ and β; the SI versus Π plots clearly separate into two basic trends: one for constant β (i.e., varying γ) and the other for constant γ (i.e., varying β). Our study prescribes the desired operating regimes of a microfluidic magnetophoretic SPLITT device in a practical immunomagnetic separation application.     

Low-cost credit card-based microfluidic devices for magnetic bead immobilisation

We report a simple low-cost magnetic microfluidic device for magnetic bead separation and immobilisation. One dimensional arrays of localised high magnetic field gradients are constructed at the interfaces between regions magnetised with opposing polarities on the magnetic Fe₂O₃ composite stripes of credit cards. The localised high magnetic field gradients are employed to trap magnetic beads on the surface of the magnetic stripe, without the need for external magnetic components. A magnetic card writer was used to deterministically pattern the magnetic stripes of credit cards to define arrays of magnetic reversals. The fabrication of the device is based on PDMS to credit card bonding of simple flow channels. Experimental results demonstrate that magnetic beads can be captured with efficiencies of 85, 67 and 27 % at flow rates of 25, 50 and 100 μL min^?1, respectively. The results show that the credit card-based magnetic separator might offer an efficient, simple, low-cost alternative to traditional microfluidic magnetic separators for applications such as immunomagnetic cell separation.     

Microfluidic device for continuous magnetophoretic separation of white blood cells

This paper presents a microfluidic device for magnetophoretic separation of red blood cells from blood under continuous flow. The separation method consists of continuous flow of a blood sample (diluted in PBS) through a microfluidic channel which presents on the bottom “dots” of ferromagnetic layer. By applying a magnetic field perpendicular on the flowing direction, the ferromagnetic “dots” generate a gradient of magnetic field which amplifies the magnetic force. As a result, the red blood cells are captured on the bottom of the microfluidic channel while the rest of the blood is collected at the outlet. Experimental results show that an average of 95% of red blood cells is trapped in the device.     

Isolation of magnetically tagged cancer cells through an integrated magnetofluidic device

Circulating tumour cells (CTC) in the bloodstream has been implicated in cancer metastasis. Efficient removal of CTC could potentially be an effective therapeutic measure against cancer metastasis. In this study, the hydrodynamic focusing flow in microfluidic channels (R _e  ? 1) was considered together with the magnetophoretic force. The localised magnetic field was achieved through a passivated current-carrying multilayered microstripline, where the generated field gradient was used to attract the magnetic beads to the desired outlet. The experimental results show that the device is capable of isolating purely magnetic beads with an efficiency of 91 % while isolation efficiency of the magnetically tagged HeLa cervical cancer cells from cell suspension yielded an isolation efficiency of 79 %.     

The synergistic effect of periodic immunomagnetics and microfluidics on universally capturing circulating tumor cells

Efficient capture data on circulating tumor cells (CTCs) determines early-stage cancer diagnosis and contributes to timely clinical treatment. We present an enhanced method of capturing CTCs accomplished by a microfluidic device integrated with magnetic field to activate the kinetic motion of in-device magnetic beads. The device, consisting of a microfluidic chamber and two electrode chips applied with pulsatile alternating current in both, is designed based on simulations on the periodicity characteristic of magnetic beads as well as the effect of heat dissipation on cell culture medium manifest. Using MEMS technologies, the prototype is fabricated and assembled. The cell capture experiments based on active magnetic beads are achieved in separation of rare cancer cells (MCF7 cells) with low concentration. The capture rate is estimated up to 88 %, with great potential of dramatically improving detection efficiency in disease diagnostics.     

Design of microfluidic channels for magnetic separation of malaria-infected red blood cells

This study is motivated by the development of a blood cell filtration device for removal of malaria-infected, parasitized red blood cells (pRBCs). The blood was modeled as a multi-component fluid using the computational fluid dynamics discrete element method (CFD-DEM), wherein plasma was treated as a Newtonian fluid and the red blood cells (RBCs) were modeled as soft-sphere solid particles which move under the influence of drag, collisions with other RBCs, and a magnetic force. The CFD-DEM model was first validated by a comparison with experimental data from Han and Frazier (Lab Chip 6:265–273, 2006) involving a microfluidic magnetophoretic separator for paramagnetic deoxygenated blood cells. The computational model was then applied to a parametric study of a parallel-plate separator having hematocrit of 40 % with 10 % of the RBCs as pRBCs. Specifically, we investigated the hypothesis of introducing an upstream constriction to the channel to divert the magnetic cells within the near-wall layer where the magnetic force is greatest. Simulations compared the efficacy of various geometries upon the stratification efficiency of the pRBCs. For a channel with nominal height of 100 µm, the addition of an upstream constriction of 80 % improved the proportion of pRBCs retained adjacent to the magnetic wall (separation efficiency) by almost twofold, from 26 to 49 %. Further addition of a downstream diffuser reduced remixing and hence improved separation efficiency to 72 %. The constriction introduced a greater pressure drop (from 17 to 495 Pa), which should be considered when scaling up this design for a clinical-sized system. Overall, the advantages of this design include its ability to accommodate physiological hematocrit and high throughput, which is critical for clinical implementation as a blood-filtration system.     

Generation of disk-like hydrogel beads for cell encapsulation and manipulation using a droplet-based microfluidic device

We report a droplet-based microfluidic synthetic technique to generate disk-like hydrogel beads for cell encapsulation and manipulation. Utilizing this microfluidic synthetic technique, the size of the disk-like calcium alginate (CA) hydrogel beads and the number of cells encapsulated in the disk-like CA hydrogel beads could be well controlled by individually adjusting the flow rates of reagents. As a proof-of-concept, we demonstrated that single cell (yeast cell or mammalian cell) could be successfully encapsulated into disk-like CA hydrogel beads with high cell viability. Taking advantage of the flat top/bottom surfaces of disk-like CA hydrogel beads, cell division processes in culture media were clearly observed and recorded at a desired position without rolling and moving. This facile microfluidic chip provides a feasible method for size-controlled disk-like hydrogel beads generation and cell encapsulation. It could be a promising candidate for cell division observation and quantitative biological study in lab-on-a-chip applications.     

集成惯性聚焦结构的介电泳微流控芯片的粒子连续分离

设计并制造了一种带有惯性聚焦结构的介电泳微流控芯片,以实现不同介电性质的粒子连续分离.采用MEMS工艺制作了介电泳微流控芯片:通道入口侧壁设置一对梯形结构使经过的粒子受惯性升力的作用聚焦到通道两侧;通道底部光刻一组夹角为90°的倾斜叉指电极产生非均匀电场,利用介电泳力和流体曳力的合力使通道两侧不同的粒子发生角度不同的偏转进入不同通道,从而实现分离.将酵母菌细胞和聚苯乙烯小球作为实验样本,分析了流速和交流电压对分离的影响,确定了二者分离的最优条件并进行分离.实验结果表明,将电导率为20μS/cm的样本溶液以5μL/min的流速注入到通道中,施加6 Vp-p、10 kHz的正弦信号,酵母菌细胞沿电极运动至夹角处后沿通道中心排出,聚苯乙烯小球沿通道两侧排出,成功实现分离,平均分离效率达92.8％、平均分离纯度达90.7％.     

Development of a capillary flow microfluidic Escherichia coli biosensor with on-chip reagent delivery using water-soluble nanofibers

Although the potential role of microfluidics in point of care diagnostics is widely acknowledged, the practical limitations to their use still limit deployment. Here, we developed a capillary flow microfluidic with on-chip reagent delivery which combines a lateral flow assay with microfluidic technology. The horseradish peroxidase tagged antibody was electrospun in a water-soluble polyvinylpyrrolidone nanofibers and stored in a microfluidic poly(methyl methacrylate) chip. During the assay, the sample containing Escherichia coli on immunomagnetic beads came in contact with the nanofibers causing them to dissolve and release the reagents for binding. Following hybridization, the solution moved by capillary flow toward a detection zone where the analyte was quantified using chemiluminescence. The limit of detection was found to be approximately 10⁶ CFU/mL of E. coli O157. More importantly, the ability to store sensitive reagents within a microfluidic as nanofibers was demonstrated. The fibers showed almost instant hydration and dissemination within the sample solution.     

Disk-like hydrogel bead-based immunofluorescence staining toward identification and observation of circulating tumor cells

Assays toward analysis of rare heterogeneous cells among identical specimen raise a significant challenge in many cell biological studies and clinical diagnosis applications. In this work, we report a disk-like hydrogel bead-based stratagem for rare cell researches at single cell level after a facile microfluidic-based particle synthesis approach. Cells of interested can be encapsulated into alginate droplets which are subsequently solidified into disk-like calcium alginate hydrogel beads and the bead size and cell number inside can be precisely controlled. Due to stability, permeability and disk-like shape of calcium alginate beads, cells immobilized in the disk-like beads can be treated with different chemicals with limited mechanical or fluidic operation influences and observed without distortion comparing with conventional methods or droplet microfluidic methods. Identification of circulating tumor cells, related to early-stage cancer diagnosis, is targeted to demonstrate the potential of our technique in rare cell analysis. This hydrogel bead-based stratagem is performed in immunofluorescence staining treatment and observation of cancer cells from normal hematological cells in blood sample. This method would have a great potential in single cell immobilization, manipulations and observation for biochemical cellular assays of rare cells.     

Cell separation in a microfluidic channel using magnetic microspheres

Magnetophoretic isolation of biological cells in a microfluidic environment has strong relevance in biomedicine and biotechnology. A numerical analysis of magnetophoretic cell separation using magnetic microspheres in a straight and a T-shaped microfluidic channel under the influence of a line dipole is presented. The effect of coupled particle–fluid interactions on the fluid flow and particle trajectories are investigated under different particle loading and dipole strengths. Microchannel flow and particle trajectories are simulated for different values of dipole strength and position, particle diameter and magnetic susceptibility, fluid viscosity and flow velocity in both the microchannel configurations. Residence times of the captured particles within the channel are also computed. The capture efficiency is found to be a function of two nondimensional parameters, α and β. The first parameter denotes the ratio of magnetic to viscous forces, while the second one represents the ratio of channel height to the distance of the dipole from the channel wall. Two additional nondimensional parameters γ (representing the inverse of normalized offset distance of the dipole from the line of symmetry) and σ (representing the inverse of normalized width of the outlet limbs) are found to influence the capture efficiency in the T-channel. Results of this investigation can be applied for the selection of a wide range of operating and design parameters for practical microfluidic cell separators.     

Enhancement of separation efficiency on continuous magnetophoresis by utilizing L/T-shaped microchannels

In this article a novel design of on-chip continuous magnetophoretic separator was proposed by utilizing the magnetic field and L-turning/T-junction effect of the flow field for high throughput applications. The motion of the magnetic bead was simulated based on Lagrangian tracking method and the separation efficiency was calculated according to the trajectories. Impact parameters including geometrical configuration, fluid velocity, magnetic flux density, magnetic bead size, and temperature on separation efficiency were discussed. The results show that both the L- and T-microchannel separators have higher separation efficiency as compared with the conventional straight-microchannel separator because of the L-turning/T-junction effect of the flow field. The separation efficiencies for L- and T-microchannel separators are 63.4 and 100%, respectively, while it is only 43.7% for straight-microchannel separator at the same conditions. Above a critical flow rate the separation efficiency drops drastically from nearly 100% to zero while this decrease is much slower for T-shaped configurations. The separation efficiency increases initially with the increase of the external magnetic flux density and keeps nearly constant at high magnetic flux density owing to saturated magnetization of the beads. It is also found that both the magnetic bead diameter and fluid temperature have great effect on the separation efficiency. The L/T-microchannel separators presented in this article are simple and efficient for magnetophoretic separation at high flow rates and thus useful for the high-efficiency on-chip enrichment of analytes with very low concentrations.     

Diamagnetic capture mode magnetophoretic microseparator for blood cells

This paper presents the characterization of a continuous diamagnetic capture (DMC) mode magnetophoretic microseparator for separating red and white blood cells from diluted whole blood based on their native magnetic properties. The DMC microseparator separated the blood cells using a high-gradient magnetic separation (HGMS) method without the use of additives such as magnetic beads. The microseparator was fabricated using microfabrication technology, enabling the integration of microscale magnetic flux concentrators in an aqueous microenvironment. Experimental results show that the DMC microseparator can continuously separate out 89.7% of red blood cells (RBCs) from diluted whole blood within 5 min using an external magnetic flux of 0.2 T from a permanent magnet. Monitoring white blood cells (WBCs) probed with a fluorescence dye show that 72.7% of WBCs were separated out within 10 min in the DMC microseparator using a 0.2 T external applied magnetic flux. Consequently, the DMC microseparator may facilitate the separation of WBCs from whole blood in applications such as a genetic sample preparation and blood borne disease detection. [1574].     

Geometry design of herringbone structures for cancer cell capture in a microfluidic device

Cancer cell detection with high capture efficiency is important for its extensive clinical applications. Herringbone structures in microfluidic devices have been widely adopted to increase the cell capture performance due to its chaotic effect. Given the fact of laminar flow in microfluidic devices, geometry-based optimization acting as a design strategy is effective and can help researchers reduce repetitive trial experiments. In this work, we presented a computational model to track the cell motion and used normalized capture efficiency to evaluate the tumor cell capture performance under various geometry settings. Cell adhesion probability was implemented in the model to consider the nature of ligand–receptor formation and breakage during cell–surface interactions. A facile approach was introduced to determine the two lumped coefficients of cell adhesion probability through two microfluidic experiments. A comprehensive geometric study was then performed by using this model, and results were explained from the fluid dynamics. Although most of the geometric guides agree with the general criterion concluded in the literature, we found herringbone structures with symmetric arms rather than a short arm–long arm ratio of 1/3 are optimal. This difference mainly comes from the fact that our model considers the particulate nature of cells while most studies in the literature optimize the geometry merely relying on mixing effects. Thus, our computational model implemented with cell adhesion probability can serve as a more accurate and reliable approach to optimize microfluidic devices for cancer cell capture.     

Microfluidic continuous magnetophoretic protein separation using nanoparticle aggregates

We demonstrate a microfluidic continuous-flow protein separation process in which silica-coated superparamagnetic nanoparticles interact preferentially with hemoglobin in a mixture with bovine serum albumin, and the resulting hemoglobin-nanoparticle aggregates are recovered online using magnetophoresis. We present detailed modeling and analysis of this process yielding quantitative estimates of the recovery of both proteins, validated by experiments. While several previous studies utilize an average particle size in modeling magnetophoretic particle trajectories or process design, in this study we emphasize the importance of accounting for particle size distributions in calculating particle recovery, and therefore in estimating separation efficiency. We combine experimentally measured size distributions of protein-nanoparticle aggregates with simulations of particle trajectories and provide a simple analytical method to calculate the efficiency of separation at various flow speeds, which fully accounts for heterogeneity in particle sizes. Our method can potentially be used for affinity based biomolecular separations at both analytical and preparative scales by exploiting well-established techniques to functionalize nanoparticle surfaces with selective ligands. Further, the modeling methodology presented here may be applied to provide better estimates of particle recovery in a broad range of magnetophoretic separation processes involving heterogeneity in particle sizes.     

Centrifugo-magnetophoretic particle separation

There has been a recent surge of research output on magnetophoretic lab-on-a-chip systems due to their prospective use in a range of applications in the life sciences and clinical diagnostics. Manifold applications for batch-mode or continuous-flow magnetophoretic separations of cells, proteins, and nucleic acids are found in bioanalytics, cell biology, and clinical diagnostics. To ensure stable hydrodynamic conditions and thus reproducible separation, state-of-the-art magnetophoretic lab-on-a-chip systems have been based on pressure-driven flow (Gijs in Microfluid Nanofluid 1:22–40, 2004; Pamme and Manz in Anal Chem 76:7250–7256, 2004; Pamme in Lab Chip 7:1644–1659, 2007; Karle et al. in Lab Chip 10:3284–3290, 2010), which involves rather bulky and costly instrumentation. In a flow-based system, suspended particles are following the liquid phase as a result of the Stokes drag, thus being fully exposed to divergent flow lines around obstacles and pump-induced pressure fluctuations. To eventually achieve more stable hydrodynamic conditions, improved control of magnetic particles, a more compact instrumentation footprint, and integration of high-performance upstream sample preparation, this work introduces a novel two-dimensional particle separation principle by combining magnetic deflection with centrifugal sedimentation in a stopped-flow mode (i.e., mere particle sedimentation). The experimental parameters governing our centrifugo-magnetophoretic system are the strength and orientation of the co-rotating magnetic field, the rotationally induced centrifugal field, and the size-dependent Stokes drag of the various particles with respect to the (residual) liquid phase. In this work, the following set of basic functional modes is demonstrated as proof-of-concept: separation of magnetic from non-magnetic particles, routing of magnetic particles based on control of the spin speed, and size separation of various magnetic particles. Finally, a biomimetic application involving the separation of particles representing healthy cells from a very small concentration of magnetic particles of a similar size, mass and magnetization as a immuno-magnetically tagged target cell, for instance mimicking a circulating tumor cell.     

Screening of peptide specific to cholangiocarcinoma cancer cells using an integrated microfluidic system and phage display technology

Cholangiocarcinoma (CCA) is a cancer of the bile duct with high mortality rate and poor prognosis, owing to the difficulty in the early diagnosis and prognosis. The specific biomarkers or affinity reagents toward CCA cells could be great tools to assist in detection of CCA. However, screening of biomarkers/affinity reagents are generally labor-intensive, time-consuming and requiring large volume of samples and reagents. Therefore, we developed an integrated microfluidic system which could automatically perform selections of biomarkers and affinity reagents using phage display techniques. The experimental results showed that the selection of phage-displayed peptides bound to CCA cells was successfully demonstrated on the integrated microfluidic system using fewer reagents, samples and less time (5.25 h per biopanning round, and continuously performed for only 4 panning rounds). Three oligopeptides were screened, and their specificity and affinity toward CCA cells were characterized. Furthermore, comparing to conventional EpiEnrich beads for cancer cell capture, the screened CCA-specific peptides showed relatively low capture rate over control normal cells. It is envisioned that this microfluidic system may be a powerful tool for screening of biomarkers/affinity reagents in clinical diagnosis and target therapy for CCA.