Comparison of a thickness-tapered channel in flow field-flow fractionation with a conventional channel with flow rate programming.

The thickness-tapered channel structure in flow field-flow fractionation (FlFFF), recently introduced by constructing a channel with a linear decrease in thickness along its length, demonstrated effectiveness in steric/hyperlayer separation of supramicron particles with improvements in separation speed, elution recovery, and an expanded dynamic size range of separation. In this study, we conducted a comparative analysis of the performance between the impact of field (or crossflow rate) programming or outflow rate programming for the separation of polystyrene latex standards (50 ∼ 800 nm) with a conventional channel having uniform thickness and a thickness-tapered channel without programming. Outlet flow rate and crossflow rate conditions were also varied. Although the particle size resolution of the tapered channel does not surpass that of field programming in uniform thickness channel, it achieves higher-speed separation without a significant loss of resolution and without the need for a complex flow controller system even at a low outflow rate condition. Furthermore, it yielded an improved resolution for particles close to the steric transition regime (400 ∼ 600 nm) in the normal mode of separation. Due to the continuous increase in mean flow velocity down the channel, the tapered channel exhibits flexibility in separating submicron-sized particles at high crossflow rate conditions or low outflow rate conditions, of which the latter can be advantageous when coupled with mass spectrometry in a miniaturized setup.

Microfluidic chip for graduated magnetic separation of circulating tumor cells by their epithelial cell adhesion molecule expression and magnetic nanoparticle binding.

The enumeration of circulating tumor cells (CTCs) in the peripheral bloodstream of metastatic cancer patients has contributed to improvements in prognosis and therapeutics. There have been numerous approaches to capture and counting of CTCs. However, CTCs have potential information beyond simple enumeration and hold promise as a liquid biopsy for cancer and a pathway for personalized cancer therapy by detecting the subset of CTCs having the highest metastatic potential. There is evidence that epithelial cell adhesion molecule (EpCAM) expression level distinguishes these highly metastatic CTCs. The few previous approaches to selective CTC capture according to EpCAM expression level are reviewed. A new two-stage microfluidic device for separation, enrichment and release of CTCs into subpopulations sorted by EpCAM expression level is presented here. It relies upon immunospecific magnetic nanoparticle labeling of CTCs followed by their field- and flow-based separation in the first stage and capture as discrete subpopulations in the second stage. To fine tune the separation, the magnetic field profile across the first stage microfluidic channel may be modified by bonding small Vanadium Permendur strips to its outer walls. Mathematical modeling of magnetic fields and fluid flows supports the soundness of the design.

Tessellated permanent magnet circuits for flow-through, open gradient separations of weakly magnetic materials.

Emerging microfluidic-based cell assays favor label-free red blood cell (RBC) depletion. Magnetic separation of RBC is possible because of the paramagnetism of deoxygenated hemoglobin but the process is slow for open-gradient field configurations. In order to increase the throughput, periodic arrangements of the unit magnets were considered, consisting of commercially available Nd-Fe-B permanent magnets and soft steel flux return pieces. The magnet design is uniquely suitable for multiplexing by magnet tessellation, here meaning the tiling of the magnet assembly cross-sectional plane by periodic repetition of the magnet and the flow channel shapes. The periodic pattern of magnet magnetizations allows a reduction of the magnetic material per channel with minimal distortion of the field cylindrical symmetry inside the magnet apertures. A number of such magnet patterns are investigated for separator performance, size and economy with the goal of designing an open-gradient magnetic separator capable of reducing the RBC number concentration a hundred-fold in 1 mL whole blood per hour.

Fractionating power and outlet stream polydispersity in asymmetrical flow field-flow fractionation. Part II: programmed operation.

Asymmetrical flow field-flow fractionation (As-FlFFF) is a widely used technique for analyzing polydisperse nanoparticle and macromolecular samples. The programmed decay of cross flow rate is often employed. The interdependence of the cross flow rate through the membrane and the fluid flow along the channel length complicates the prediction of elution time and fractionating power. The theory for their calculation is presented. It is also confirmed for examples of exponential decay of cross flow rate with constant channel outlet flow rate that the residual sample polydispersity at the channel outlet is quite well approximated by the reciprocal of four times the fractionating power. Residual polydispersity is of importance when online MALS or DLS detection are used to extract quantitative information on particle size or molecular weight. The theory presented here provides a firm basis for the optimization of programmed flow conditions in As-FlFFF. Graphical abstract Channel outlet polydispersity remains significant following fractionation by As-FlFFF under conditions of programmed decay of cross flow rate.

Fractionating power and outlet stream polydispersity in asymmetrical flow field-flow fractionation. Part I: isocratic operation.

Asymmetrical flow field-flow fractionation (As-FlFFF) has become the most commonly used of the field-flow fractionation techniques. However, because of the interdependence of the channel flow and the cross flow through the accumulation wall, it is the most difficult of the techniques to optimize, particularly for programmed cross flow operation. For the analysis of polydisperse samples, the optimization should ideally be guided by the predicted fractionating power. Many experimentalists, however, neglect fractionating power and rely on light scattering detection simply to confirm apparent selectivity across the breadth of the eluted peak. The size information returned by the light scattering software is assumed to dispense with any reliance on theory to predict retention, and any departure of theoretical predictions from experimental observations is therefore considered of no importance. Separation depends on efficiency as well as selectivity, however, and efficiency can be a strong function of retention. The fractionation of a polydisperse sample by field-flow fractionation never provides a perfectly separated series of monodisperse fractions at the channel outlet. The outlet stream has some residual polydispersity, and it will be shown in this manuscript that the residual polydispersity is inversely related to the fractionating power. Due to the strong dependence of light scattering intensity and its angular distribution on the size of the scattering species, the outlet polydispersity must be minimized if reliable size data are to be obtained from the light scattering detector signal. It is shown that light scattering detection should be used with careful control of fractionating power to obtain optimized analysis of polydisperse samples. Part I is concerned with isocratic operation of As-FlFFF, and part II with programmed operation.

Circular Halbach array for fast magnetic separation of hyaluronan-expressing tissue progenitors.

Connective tissue progenitors (CTPs) are a promising therapeutic agent for bone repair. Hyaluronan, a high molecular mass glycosaminoglycan, has been shown by us to be a suitable biomarker for magnetic separation of CTPs from bone marrow aspirates in a canine model. For the therapy to be applicable in humans, the magnetic separation process requires scale-up without compromising the viability of the cells. The scaled-up device presented here utilizes a circular Halbach array of diametrically magnetized, cylindrical permanent magnets. This allows precise control of the magnetic field gradient driving the separation, with theoretical analysis favoring a hexapole field. The separation vessel has the external diameter of a 50 mL conical centrifuge tube and has an internal rod that excludes cells from around the central axis. The magnet and separation vessel (collectively dubbed the hexapole magnet separator or HMS) was tested on four human and four canine bone marrow aspirates. Each CTP-enriched cell product was tested using cell culture bioassays as surrogates for in vivo engraftment quality. The magnetically enriched cell fractions showed statistically significant, superior performance compared to the unenriched and depleted cell fractions for all parameters tested, including CTP prevalence (CTPs per 10(6) nucleated cells), proliferation by colony forming unit (CFU) counts, and differentiation by staining for the presence of osteogenic and chondrogenic cells. The simplicity and speed of the HMS operation could allow both CTP isolation and engraftment during a single surgical procedure, minimizing trauma to patients and lowering cost to health care providers.

Retention ratio and nonequilibrium bandspreading in asymmetrical flow field-flow fractionation.

In asymmetrical flow field-flow fractionation (As-FlFFF), only the membrane-covered accumulation wall is permeable to fluid; the opposite channel wall is impermeable. Fluid enters the channel at the inlet and exits partly through the membrane-covered accumulation wall and partly through the channel outlet. This means that not only does the volumetric channel flow rate decrease along the channel length as fluid exits through the membrane but also the cross-channel component to fluid velocity must approach zero at the impermeable wall. This dependence of cross-channel fluid velocity on distance across the channel thickness influences the equilibrium concentration profile for the sample components introduced to the channel. The concentration profile departs from the exponential profile predicted for the ideal model of field-flow fractionation. This influences both the retention ratio and the principal contribution to bandspreading--the nonequilibrium contribution. The derivation of an equation for the nonequilibrium bandspreading parameter χ in As-FlFFF is presented, and its numerical solution graphed. At high retention, it is shown that the solutions for both retention ratio R and χ converge on those for the ideal model, as expected. At lower levels of retention, the departures from the ideal model are significant, particularly for bandspreading. For example, at a level of retention corresponding to a retention parameter λ of 0.05, R is almost 4% higher than for the ideal model (0.28047 as compared to 0.27000) but the value of χ is almost 60% higher. The equations presented for both R and χ include a first-order correction for the finite size of the particles--the steric exclusion correction. These corrections are shown to be significant for particle sizes eluting well before steric inversion. For example, particles of half the inversion diameter are predicted to elute 25% slower and to show almost 40% higher bandspreading when steric effects are not accounted for. The work presented contributes to the fundamental theory of As-FlFFF and allows quantitative prediction of both retention and bandspreading at all levels of retention.

Retention in continuous two-dimensional thermal field-flow fractionation: comparison of experimental results with theory.

A theoretical and experimental study of continuous two-dimensional thermal field-flow fractionation (2D-ThFFF) is presented. Separation takes place in radial flow between two closely spaced discs, one of which is heated and the other cooled in order to maintain a temperature gradient across the channel. The cooled disc, which serves as the accumulation wall, is rotated relative to the other to create a shear component to the fluid flow. Under the influence of the thermal gradient and flow components, the sample components spiral outwards along different paths to the outer rim of the channel to be collected. The general principle of operation is described and an approximate theoretical model formulated for predicting the outlet position for the path of each sample component. The influence of the principal operational parameters, such as radial and angular flow rates and thermal gradient, on the deflection angle of the sample trajectory is investigated. Fractionation is demonstrated for polystyrene polymer standards in a binary solvent consisting of cyclohexane and ethylbenzene. Experimental results are compared with theoretical predictions.

Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow.

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures, require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation.

Effect of carrier fluid viscosity on retention time and resolution in gravitational field-flow fractionation.

Gravitational field-flow fractionation (GrFFF) is a useful technique for fast separation of micrometer-sized particles. Different sized particles are carried at different velocities by a flow of fluid along an unobstructed thin channel, resulting in a size-based separation. They are confined to thin focused layers in the channel thickness where force due to gravity is exactly opposed by hydrodynamic lift forces (HLF). It has been reported that the HLF are a function of various parameters including the flow rate (or shear rate), the size of the particles, and the density and viscosity of the liquid. The dependence of HLF on these parameters offers a means of altering the equilibrium transverse positions of the particles in GrFFF, and hence their elution times. In this study, the effect of the viscosity of the carrier fluid on the elution behavior (retention, zone broadening, and resolution) of micrometer-sized particles in GrFFF was investigated using polystyrene (PS) latex beads as model particles. In order to change the carrier liquid viscosity without affecting its density, various amounts of (hydroxypropyl) methyl cellulose (HPMC) were added to the aqueous carrier liquid. It was found that particles migrate at faster rates as the carrier viscosity is increased, which confirms the dependence of HLF on viscosity. At the same time, particle size selectivity decreased but peak shape and symmetry for the more strongly retained particles improved. As a result, separation was improved in terms of both the separation time and resolution with increase of carrier viscosity. A theoretical model for plate height in GrFFF is also presented, and its predictions are compared to experimentally measured values.

The use of a linear Halbach array combined with a step-SPLITT channel for continuous sorting of magnetic species.

The Quadrupole Magnetic Sorter (QMS), employing an annular flow channel concentric with the aperture of a quadrupole magnet, is well established for cell and particle separations. Here we propose a magnetic particle separator comprising a linear array of cylindrical magnets, analogous to the array proposed by Klaus Halbach, mated to a substantially improved form of parallel-plate SPLITT channel, known as the step-SPLITT channel. While the magnetic force and throughput are generally lower than for the QMS, the new separator has advantages in ease of fabrication and the ability to vary the magnetic force to suit the separands. Preliminary experiments yield results consistent with prediction and show promise regarding future separations of cells of biomedical interest.

Characterization of magnetic nanoparticles using programmed quadrupole magnetic field-flow fractionation.

Quadrupole magnetic field-flow fractionation is a relatively new technique for the separation and characterization of magnetic nanoparticles. Magnetic nanoparticles are often of composite nature having a magnetic component, which may be a very finely divided material, and a polymeric or other material coating that incorporates this magnetic material and stabilizes the particles in suspension. There may be other components such as antibodies on the surface for specific binding to biological cells, or chemotherapeutic drugs for magnetic drug delivery. Magnetic field-flow fractionation (MgFFF) has the potential for determining the distribution of the magnetic material among the particles in a given sample. MgFFF differs from most other forms of field-flow fractionation in that the magnetic field that brings about particle separation induces magnetic dipole moments in the nanoparticles, and these potentially can interact with one another and perturb the separation. This aspect is examined in the present work. Samples of magnetic nanoparticles were analysed under different experimental conditions to determine the sensitivity of the method to variation of conditions. The results are shown to be consistent and insensitive to conditions, although magnetite content appeared to be somewhat higher than expected.

Effect of asymmetrical flow field-flow fractionation channel geometry on separation efficiency.

The separation efficiencies of three different asymmetrical flow field-flow fractionation (AF4) channel designs were evaluated using polystyrene latex standards. Channel breadth was held constant for one channel (rectangular profile), and was reduced either linearly (trapezoidal profile) or exponentially (exponential profile) along the length for the other two. The effective void volumes of the three channel types were designed to be equivalent. Theoretically, under certain flow conditions, the mean channel flow velocity of the exponential channel could be arranged to remain constant along the channel length, thereby improving separation in AF4. Particle separation obtained with the exponential channel was compared with particle separation obtained with the trapezoidal and rectangular channels. We demonstrated that at a certain flow rate condition (outflow/inflow rate=0.2), the exponential channel design indeed provided better performance with respect to the separation of polystyrene nanoparticles in terms of reducing band broadening. While the trapezoidal channel exhibited a little poorer performance than the exponential, the strongly decreasing mean flow velocity in the rectangular channel resulted in serious band broadening, a delay in retention time, and even failure of larger particles to elute.

Magnetic Pressure as a Scalar Representation of Field Effects in Magnetic Suspensions.

Magnetic microsphere suspensions undergo complex motion when exposed to finite sources of the magnetic field, such as small permanent magnets. The computational complexity is compounded by a difficulty in choosing a suitable choice of visualization tools because this often requires using the magnetic force vector field in three dimensions. Here we present a potentially simpler approach by using the magnetic pressure. It is a scalar quantity, pm = B2/2µ0, and its usefulness has been already demonstrated in applications to magnetohydrodynamics and ferrohydrodynamics (where B is the applied field and µ0 = 4π×10-7 T.m/A). The equilibrium distribution of the magnetic bead plug in aqueous suspension is calculated as an isosurface of the magnitude of the magnetic pressure pm = const, in the field of two permanent magnet blocks calculated from closed formulas. The geometry was adapted from a publication on the magnetic bead suspensions in microsystems and the predicted bead plug distribution is shown to agree remarkably well with the experiment.

Sequential CD34 cell fractionation by magnetophoresis in a magnetic dipole flow sorter.

Cell separation and fractionation based on fluorescent and magnetic labeling procedures are common tools in contemporary research. These techniques rely on binding of fluorophores or magnetic particles conjugated to antibodies to target cells. Cell surface marker expression levels within cell populations vary with progression through the cell cycle. In an earlier work we showed the reproducible magnetic fractionation (single pass) of the Jurkat cell line based on the population distribution of CD45 surface marker expression. Here we present a study on magnetic fractionation of a stem and progenitor cell (SPC) population using the established acute myelogenous leukemia cell line KG-1a as a cell model. The cells express a CD34 cell surface marker associated with the hematopoietic progenitor cell activity and the progenitor cell lineage commitment. The CD34 expression level is approximately an order of magnitude lower than that of the CD45 marker, which required further improvements of the magnetic fractionation apparatus. The cells were immunomagnetically labeled using a sandwich of anti-CD34 antibody-phycoerythrin (PE) conjugate and anti-PE magnetic nanobead and fractionated into eight components using a continuous flow dipole magnetophoresis apparatus. The CD34 marker expression distribution between sorted fractions was measured by quantitative PE flow cytometry (using QuantiBRITE PE calibration beads), and it was shown to be correlated with the cell magnetophoretic mobility distribution. A flow outlet addressing scheme based on the concept of the transport lamina thickness was used to control cell distribution between the eight outlet ports. The fractional cell distributions showed good agreement with numerical simulations of the fractionation based on the cell magnetophoretic mobility distribution in the unsorted sample.

Magnetic nanoparticle drug carriers and their study by quadrupole magnetic field-flow fractionation.

Magnetic nanoparticle drug carriers continue to attract considerable interest for drug targeting in the treatment of cancers and other pathological conditions. The efficient delivery of therapeutic levels of drug to a target site while limiting nonspecific, systemic toxicity requires optimization of the drug delivery materials, the applied magnetic field, and the treatment protocol. The history and current state of magnetic drug targeting is reviewed. While initial studies involved micrometer-sized and larger carriers, and work with these microcarriers continues, it is the sub-micrometer carriers or nanocarriers that are of increasing interest. An aspect of magnetic drug targeting using nanoparticle carriers that has not been considered is then addressed. This aspect involves the variation in the magnetic properties of the nanocarriers. Quadrupole magnetic field-flow fractionation (QMgFFF) is a relatively new technique for characterizing magnetic nanoparticles. It is unique in its capability of determining the distribution in magnetic properties of a nanoparticle sample in suspension. The development and current state of this technique is also reviewed. Magnetic nanoparticle drug carriers have been found by QMgFFF analysis to be highly polydisperse in their magnetic properties, and the strength of response of the particles to magnetic field gradients is predicted to vary by orders of magnitude. It is expected that the least magnetic fraction of a formulation will contribute the most to systemic toxicity, and the depletion of this fraction will result in a more effective drug carrying material. A material that has a reduced systemic toxicity will allow higher doses of cytotoxic drugs to be delivered to the tumor with reduced side effects. Preliminary experiments involving a novel method of refining a magnetic nanoparticle drug carrier to achieve this result are described. QMgFFF is used to characterize the refined and unrefined material.

Development of multistage magnetic deposition microscopy.

Magnetic deposition microscropy (MDM) combines magnetic deposition and optical analysis of magnetically tagged cells into a single platform. Our multistage MDM uses enclosed microfabricated channels and a magnet assembly comprising four zones in series. The enclosed channels alleviate the problem plaguing previous versions of MDM: scouring of the cell deposition layer by the air-liquid interface as the channel is drained. The four-zone magnet assembly was designed to maximize capture efficiency, and experiments yielded total capture efficiencies of >99% of fluorescent- and magnetically-labeled Jurkat cells at reasonable throughputs (10(3) cells/min). A digital image processing protocol was developed to measure the average pixel intensities of the deposited cells in different zones, indicative of the marker expression. Preliminary findings indicate that the multistage MDM may be suitable for depositing cells and particles in successive zones according to their magnetic properties (e.g., magnetic susceptibilities or magnetophoretic mobilities). The overall goal is to allow the screening of multiple disease conditions in a single platform.

Theory for nanoparticle retention time in the helical channel of quadrupole magnetic field-flow fractionation.

Quadrupole magnetic field-flow fractionation (QMgFFF) is a separation and characterization technique for magnetic nanoparticles such as those used for cell labeling and for targeted drug therapy. A helical separation channel is used to efficiently exploit the quadrupole magnetic field. The fluid and sample components therefore have angular and longitudinal components to their motion in the thin annular space occupied by the helical channel. The retention ratio is defined as the ratio of the times for non retained and a retained material to pass through the channel. Equations are derived for the respective angular and longitudinal components to retention ratio.

Differences in magnetically induced motion of diamagnetic, paramagnetic, and superparamagnetic microparticles detected by cell tracking velocimetry.

Magnetic separation in biomedical applications is based on differential magnetophoretic mobility (MM) of microparticulate matter in viscous media. Typically, the difference in MM is obtained by selectively labeling the target cells with superparamagnetic iron oxide nanoparticles (SPIONs). We have measured the MM of monodisperse, polystyrene microspheres (PSMs), with and without attached SPIONs as a model of cell motion induced by nanoparticle magnetization, using variable H field and cell tracking velocimetry (CTV). As a model of paramagnetic microparticle motion, the MM measurements were performed on the same PSMs in paramagnetic gadolinium solutions, and on spores of a prokaryotic organism, Bacillus globigii (shown to contain paramagnetic manganese). The CTV analysis was sensitive to the type of the microparticle magnetization, producing a value of MM independent of the applied H field for the paramagnetic species, and a decreasing MM value with an increasing field for superparamagnetic species, as predicted from theory. The SPION-labeled PSMs exhibited a saturation magnetization above H approximately = 64,000 A m(-1) (or 0.08 tesla). Based on those data, the average saturation magnetizations of the SPIONs was calculated and shown to vary between different commercial sources. The results demonstrate sensitivity of the CTV analysis to different magnetization mechanisms of the microparticles.

Characterization of nonspecific crossover in split-flow thin channel fractionation.

Split-flow thin channel (SPLITT) fractionation is a technique for continuous separation of particles or macromolecules in a fluid stream into fractions according to the lateral migration induced by application of a field perpendicular to the direction of flow. Typical applications have involved isolation of different fractions from a polydisperse sample. Some specialized applications involve the separation of the fraction influenced by the transverse field from the fraction that is not. For example, immunomagnetically labeled biological cells may be separated from nonlabeled cells with the application of a transverse magnetic field gradient. In such cases, it may be critically important to minimize contamination of the labeled cells with nonlabeled cells while at the same time maximizing the throughput. Such contamination is known as nonspecific crossover (NSC) and refers to the real or apparent migration of nonmobile particles or cells across stream lines with the mobile material. The possible mechanisms for NSC are discussed, and experimental results interpreted in terms of shear-induced diffusion (SID) caused by viscous interactions between particles in a sheared flow. It is concluded that SID may contribute to NSC, but that further experiments and mathematical modeling are necessary to more fully explore the phenomenon.