Measuring magnetic force field distributions in microfluidic devices: Experimental and numerical approaches.

Precisely and accurately determining the magnetic force and its spatial distribution in microfluidic devices is challenging. Typically, magnetic microfluidic devices are designed in a way to both maximize the force within the separation region and to minimize the necessity for knowing such details-such as designing magnetic geometries that create regions of nearly constant magnetic force or that dictate the behavior of the magnetic force to be highly predictable in a specified region. In this work, we present a method to determine the spatial distribution of the magnetic force field in a magnetic microfluidic device by particle tracking magnetophoresis. Polystyrene microparticles were suspended in a paramagnetic fluid, gadolinium, and this suspension was exposed to various magnetic field geometries. Polystyrene particle motion was tracked using a microscope and images processed using Fiji (ImageJ). From a sample with a large spatial distribution of particle tracks, the magnetic force field distribution was calculated. The force field distribution was fitted to nonlinear spatial distribution models. These experimental models are compared to and supported by 3D simulations of the magnetic force field in COMSOL.

SPIONs self-assembly and magnetic sedimentation in quadrupole magnets: Gaining insight into the separation mechanisms.

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently popular materials experiencing rapid development with potential application value, especially in biomedical and chemical engineering fields. Examples include wastewater management, bio-detection, biological imaging, targeted drug delivery and biosensing. While not exclusive, magnetically driven isolation methods are typically required to separate the desired entity from the media in specific applications and in their manufacture and/or quality control. However, due to the nano-size of SPIONs, their magnetic manipulation is affected by Brownian motion, adding considerable complexities. The two most common methods for SPION magnetic separation are high and low gradient magnetic separation (HGMS and LGMS, respectively). Nevertheless, the effect of specific magnetic energy fields on SPIONs, such as horizontal (perpendicular to gravity), high fields and gradients (higher than LGMS) on the horizontal magnetophoresis and vertical sedimentation of SPIONs has only recently been suggested as a way to separate very small particles (5 nm). In this work, we continue those studies on the magnetic separation of 5-30 nm SPIONs by applying fields and gradients perpendicular to gravity. The magnetic field was generated by permanent magnets arranged in quadrupolar configurations (QMS). Different conditions were studied, and multiple variables were evaluated, including the particle size, the initial SPIONs concentration, the temperature, the magnetic field gradient and the magnetic exposure time. Our experimental data show that particles are subjected to horizontal magnetic forces, to particle agglomeration due to dipole-dipole interactions, and to vertical sedimentation due to gravity. The particle size and the type of separator employed (i.e. different gradient and field distribution acting on the particle suspension) have significant effects on the phenomena involved in the separation, whereas the temperature and particle concentration affect the separation to a lesser extent. Finally, the separation process was observed to occur in less than 3 mins for our experimental conditions, which is encouraging considering the long operation time (up to days) necessary to separate particles of similar sizes in LGMS columns that also employ permanent magnets.

Quantification of the Mean and Distribution of Hemoglobin Content in Normal Human Blood Using Cell Tracking Velocimetry.

The current clinical method for detecting anemia focuses on measuring the concentration of hemoglobin (Hb) in blood. However, recent developments in particle tracking algorithms and the understanding of the relationship between Hb and magnetism has enabled the quantitative measurement of the Hb content in a single red blood cell, RBC, based on magnetophoretic mobility. To further explore this relationship, 22 human blood samples obtained from 17 healthy volunteers were analyzed by the cell tracking velocimetry system, and the calculated Hb concentration from these measurements was compared to the values measured by UV-visible spectrophotometry, the standard method for measuring Hb in clinical laboratories. The results show close correlations between the mean of the spectrophotometric and magnetophoretic methods; however, single cell analysis with the magnetophoretic mobility method allows further elucidation of the distribution of Hb concentration within RBCs from a donor sample to be determined. Histograms of these magnetophoretic mobility distributions indicate that the fraction of RBCs that are below the bulk Hb concentration that defines anemia varies not only from donor to donor but also in the same donor over time. Consistent with a variable fraction below the anemic Hb concentration, the distribution around the mean has a large range. Previous studies have indicated that RBCs lose Hb during ex vivo storage; however, it is not known if this variability in the distribution of Hb content is a function of the age of the RBCs in a donor, suggesting a variable rate in RBC production between donors, or variability in available iron at the time of RBC formation. We suggest our cell tracking velocimetry system can reveal more information regarding this matter.

Self-Assembly and sedimentation of 5 nm SPIONs using horizontal, high magnetic fields and gradients.

Superparamagnetic iron oxide nanoparticles (SPIONs) are employed in multiple applications, especially within medical and chemical engineering fields. However, their magnetic separation is very challenging as the magnetophoretic motion is hindered by thermal energy and viscous drag. Recent studies have addressed the recovery of SPIONs by a combination of cooperative magnetophoresis and sedimentation. Nevertheless, the effect of horizontal, high fields and gradients on the vertical sedimentation of SPIONs has not been described. In this work, we report, for the first time, the magnetically facilitated sedimentation of 5 nm particles by applying fields and gradients perpendicular to gravity. The magnetic field was generated by quadrupole magnetic sorters and the process was measured with time by tracking the concentration along the length of a channel contacting the 5 nm SPIONs within the quadrupole field. Our experimental data suggest that aggregates of 60-90 particles are formed in the system; thus, particle agglomeration by dipole-dipole interactions was promoted, and these clusters settled down as a result of gravitational forces. Multiple variables and parameters were evaluated, including the initial SPION concentration, the temperature, the magnetic field and gradient and operation time. It was found that the process was improved by decreasing the initial concentration and the temperature, but the magnitude of the magnetic field and gradient did not significantly affect the sedimentation. Finally, the separation process was rapid, with the systems reaching the equilibrium in approximately 20 minutes, which is a significant advantage in comparison to other systems that require longer times and larger particle sizes.

A Subpopulation of Monocytes in Normal Human Blood Has Significant Magnetic Susceptibility: Quantification and Potential Implications.

The presence of iron in circulating monocytes is well known as they play essential roles in iron recycling. Also, the storage of this metal as well as its incorrect uptake and/or release are important data to diagnose different pathologies. It has been demonstrated that iron storage in human blood cells can be measured through their magnetic behavior with high accuracy; however, the magnetic characteristics of monocytes have not been reported so far to the best of our knowledge. Therefore, in this work, we report, for the first time, the physical and magnetic properties of human monocytes, along with plasma platelets, oxyhemoglobin red blood cells (oxyHb-RBCs), and methemoglobin red blood cells (metHb-RBCs). The different cell populations were separated by Ficoll-density gradient centrifugation, followed by a flow sorting step to isolate monocytes from peripheral blood mononuclear cells. The different fractions were analyzed by Coulter Counter (for determining the size distribution and concentration) and the sorted monocytes were qualitatively analyzed on ImageStream, a state-of-the-art imaging cytometer. The analysis of the Coulter Counter and ImageStream data suggests that although there exists contamination in the monocyte fraction, the integrity of the sorted monocytes appears to be intact and the concentration was high enough to precisely measure their magnetic velocity by Cell Tracking Velocimetry. Surprisingly, monocytes reported the highest magnetic mobility from the four fractions under analysis, with an average magnetic velocity 7.8 times higher than MetHb-RBCs, which is the only type of cells with positive magnetic velocities. This value is equivalent to a susceptibility 2.5 times higher than the value reported by fresh MetHb-RBCs. It should be noted that this is the first study that reports that a subpopulation of human monocytes is much more magnetic than MetHb-RBCs, opening the door to the possible isolation of human monocytes by label-free magnetic techniques. Further, it is suggested that these magnetic monocytes could "contaminate" positively selected, immunomagnetically labeled blood cells (i.e., during a process using magnetically conjugated antibodies targeting cells, such as CD34 positive cells). Conversely, these magnetic monocytes could be inadvertently removed from a desired blood population when one is using a negative magnetic isolation technique to target cells for removal. © 2019 International Society for Advancement of Cytometry.

Quantitative characterization of the regulation of iron metabolism in glioblastoma stem-like cells using magnetophoresis.

This study focuses on different iron regulation mechanisms of glioblastoma (GBM) cancer stem-like cells (CSCs) and non-stem tumor cells (NSTCs) using multiple approaches: cell viability, density, and magnetophoresis. GBM CSCs and NSTCs were exposed to elevated iron concentration, and their magnetic susceptibility was measured using single cell magnetophoresis (SCM), which tracks the magnetic and settling velocities of thousands of individual cells passing through the magnetic field with a constant energy gradient. Our results consistently demonstrate that GBM NSTCs have higher magnetic susceptibility distribution at increased iron concentration compared with CSCs, and we speculate that it is because CSCs have the ability to store a high amount of iron in ferritin, whereas the free iron ions inside the NSTCs lead to higher magnetic susceptibility and reduced cell viability and growth. Further, their difference in magnetic susceptibility has led us to pursue a separate experiment using a quadrupole magnetic separator (QMS), a novel microfluidic device that uses a concentric channel and permanent magnets in a special configuration to separate samples based on their magnetic susceptibilities. GBM CSCs and NSTCs were exposed to elevated iron concentration, stained with two different trackers, mixed and introduced into QMS; subsequently, the separated fractions were analyzed by fluorescent microscopy. The separation results portray a successful label-less magnetic separation of the two populations.

Single cell analysis of aged RBCs: quantitative analysis of the aged cells and byproducts.

This study initially focused on characterizing the aging process of red blood cells by correlating the loss of hemoglobin and the translocation of phosphatidylserine (PS) in expired human red blood cells, hRBCs. Five pre-storage, leukoreduced hRBC units in AS-5 solution were stored between 1 and 6 °C for 42 days. Aliquots from each of these units were stained with Annexin-V FLUOS, which binds to externalized PS, and the hemoglobin within the cells was placed in a methemoglobin state with sodium nitrite, metHb. These aliquots were subsequently sorted into four sub-populations, ranging from no PS expression to high PS expression using a BD FACS ARIAIII. Each of these sub-fractions were introduced into the cell tracking velocimetry apparatus which measured both the magnetically-induced and the gravity-induced velocity. Subsequently, the samples were removed from the cell tracking velocimetry instrument and characterized using the Multisizer 4e Coulter Counter. From the magnetically-induced velocity, the amount of hemoglobin, in pg Hb per cell can be determined, and using an average value of the density of RBCs, the size can be determined. For the PS negative sub-fraction of RBCs, the size of the RBC was as expected but the average hemoglobin, Hb, content was below the threshold which defines anemia. In contrast, unexpected results were observed with the various levels of expression of PS. First, virtually all of the PS expressing cells were significantly smaller, on the order of 1 micron, than a normal RBC after 42 days of storage; yet the density of these small cells/microvesicles was such that they had settling velocities similar to normal-sized RBCs. Further, while the total amount of Hb per small cell/microvesicle was only approximately 25% of the full-sized RBCs, the volume of these small cells/microvesicles is only 1/200 of the PS negative RBCs. This suggests that these PS expressing cells are shrunken RBCs, or shrunken microvesicles from RBCs that concentrated the Hb internally. These results suggest not only a relationship between the loss of hemoglobin and the amount of PS exposed on the cellular outer wall, but also a mechanism by which these aged RBCs break down. It is not known at this time whether this is an artifact of storage or similar mechanisms occur in circulation within the human body.

Single cell magnetometry by magnetophoresis vs. bulk cell suspension magnetometry by SQUID-MPMS - a comparison.

Paramagnetic constituents of a cell have strong effect on cell's volume magnetic susceptibility even at low volume fraction because of their high susceptibility relative to that of the diamagnetic cell constituents. The effect can be measured at a single cell level by measuring cell terminal velocity in viscous media using a microscope equipped with a well-defined field and gradient magnet configuration (referred to as magnetophoretic analysis by cell tracking velocimetry, CTV). The sensitivity of such a microscopic-scale magnetometry was compared to that of a reference method of superconducting quantum interference-magnetic properties measurement system (SQUID-MPMS) using a red blood cell (RBC) suspension model. The RBC hemoglobin oxygen saturation determines the hemoglobin molecular magnetic susceptibility (diamagnetic when fully oxygenated, paramagnetic when fully deoxygenated or converted to methemoglobin). The SQUID-MPMS measurements were performed on an average of 5,000 RBCs in 20 µL physiological phosphate buffer at room temperature, those by CTV on a single cell track in a mean magnetic field of 1.6 T and mean gradient of 240 T/m, repeated for an average of 1,000 tracks per sample. This suggests 5,000× higher sensitivity of cell susceptometry by magnetophoretic analysis than by SQUID-MPMS. The magnetophoretic mean RBC magnetic susceptibilities were in the range determined by SQUID-MPMS (lower limit) and theory (upper limit). The ability of magnetophoretic analysis to resolve susceptibility peaks in a mixed cell populations was confirmed for an oxy RBC and met RBC mixture. Magnetophoretic analysis by CTV provides new tool for studies of emergence of paramagnetic reaction products in the cell.

Correlation of simulation/finite element analysis to the separation of intrinsically magnetic spores and red blood cells using a microfluidic magnetic deposition system.

Magnetic separation of cells has been, and continues to be, widely used in a variety of applications, ranging from healthcare diagnostics to detection of food contamination. Typically, these technologies require cells labeled with antibody magnetic particle conjugate and a high magnetic energy gradient created in the flow containing the labeled cells (i.e., a column packed with magnetically inducible material), or dense packing of magnetic particles next to the flow cell. Such designs, while creating high magnetic energy gradients, are not amenable to easy, highly detailed, mathematic characterization. Our laboratories have been characterizing and developing analysis and separation technology that can be used on intrinsically magnetic cells or spores which are typically orders of magnitude weaker than typically immunomagnetically labeled cells. One such separation system is magnetic deposition microscopy (MDM) which not only separates cells, but deposits them in specific locations on slides for further microscopic analysis. In this study, the MDM system has been further characterized, using finite element and computational fluid mechanics software, and separation performance predicted, using a model which combines: 1) the distribution of the intrinsic magnetophoretic mobility of the cells (spores); 2) the fluid flow within the separation device; and 3) accurate maps of the values of the magnetic field (max 2.27 T), and magnetic energy gradient (max of 4.41 T2 /mm) within the system. Guided by this model, experimental studies indicated that greater than 95% of the intrinsically magnetic Bacillus spores can be separated with the MDM system. Further, this model allows analysis of cell trajectories which can assist in the design of higher throughput systems.

Continuous, intrinsic magnetic depletion of erythrocytes from whole blood with a quadrupole magnet and annular flow channel; pilot scale study.

The ability to separate RBCs from the other components of whole blood has a number of useful clinical and research applications ranging from removing RBCs from typical clinical blood draw, bone marrow transplants to transfusions of these RBCs to patients after significant blood loss. Viewed from a mechanistic/process perspective, there are three routine methodologies to remove RBCs: 1) RBCs lysis, 2) separation of the RBCs from the nucleated cells (i.e., stem cells) based on density differences typically facilitated through centrifugation or sedimentation agents, and 3) antibody based separation in which a targeted RBC is bound with an affinity ligand that facilitates its removal. More recently, several microfluidic based techniques have also been reported. In this report, we describe the performance of continuous RBC separation achieved by the deflection of intrinsically magnetic, deoxygenated RBCs as they flow through a magnetic energy gradient created by quadrupole magnet. This quadrupole magnetic, with aperture of 9.65 mm, has a maximum field of B0 = 1.36 T at the pole tips and a constant field gradient of B0 /r0 = 286 T/m. The annular flow channel, contained within this quadrupole magnet, is 203 mm long, has an inner radius of 3.98 mm, and an inner, outer radius of 4.36 mm, which corresponds to an annulus radius of 380 micrometer. At the entrance and exit to this annular channel, a manifold was designed which allows a cell suspension and sheath fluid to be injected, and a RBC enriched exit flow (containing the magnetically deflected RBCs) and a RBC depleted exit flow to be collected. Guided by theoretical models previously published, a limited number of operating parameters; total flow rate, flow rate ratios of flows in and flow out, and ratios of RBC to polystyrene control beads was tested. The overall performance of this system is consistent with our previously presented, theoretical models and our intuition. As expected, the normalized recovery of RBCs in the RBC exit fraction ranged from approximately 95% down to 60%, as the total flow rate through the system increased from 0.1 to 0.6 ml/min. At the cell concentrations studied, this corresponds to a flow rate of 1.5 × 106 -9 × 106 cells/min. While the throughput of these pilot scale studies are slow for practical applications, the general agreement with theory, and the small cross-sectional area in which the actual separation is achieved, 77 mm2 (annulus radius times the length), and corresponding volume of approximately 2 mls, suggests the potential to scale-up a system for practical applications exists and is actively being pursued.