Bead Transport Research Articles

Quantification of cell shape, intracellular flows and transport based on DIC object detection and tracking.

Computational image analysis combined with label-free imaging has helped maintain its relevance for cell biology, despite the rapid technical improvements in fluorescence microscopy with the molecular specificity of tags. Here, we discuss some computational tools developed in our lab and their application to quantify cell shape, intracellular organelle movement and bead transport in vitro, using differential interference contrast (DIC) microscopy data as inputs. The focus of these methods is image filtering to enhance image gradients, and combining them with segmentation and single particle tracking (SPT). We demonstrate the application of these methods to Escherichia coli cell length estimation and tracking of densely packed lipid granules in Caenorhabditis elegans one-celled embryos, diffusing beads in solutions of different viscosities and kinesin-driven transport on microtubules. These approaches demonstrate how improvements to low-level image analysis methods can help obtain insights through quantitative cellular and subcellularmicroscopy.

Combined Funnel, Concentrator, and Particle Valve Functional Element for Magnetophoretic Bead Transport Based on Engineered Magnetic Domain Patterns.

Controlled actuation of superparamagnetic beads (SPBs) within a microfluidic environment using tailored dynamic magnetic field landscapes (MFLs) is a potent approach for the realization of point-of-care diagnostics within Lab-on-a-chip (LOC) systems. Making use of an engineered magnetic domain pattern as the MFL source, a functional LOC-element with combined magnetophoretic "funnel", concentrator, and "valve" functions for micron-sized SPBs is presented. A parallel-stripe domain pattern design with periodically decreasing/increasing stripe lengths is fabricated in a topographically flat continuous exchange biased (EB) thin film system by ion bombardment induced magnetic patterning (IBMP). It is demonstrated that, upon application of external magnetic field pulses, a fully reversible concentration of SPBs at the domain pattern's focal point occurs. In addition, it is shown that this functionality may be used as an SPB "funnel", allowing only a maximum number of particles to pass through the focal point. Adjusting the pulse time length, the focal point can be clogged up for incoming SPBs, resembling an on/off switchable particle "valve". The observations are supported by quantitative theoretical force considerations.

An investigation into simplifying total RNA extraction with minimal equipment using a low volume, electrokinetically driven microfluidic protocol.

Current methods for total RNA extraction are time-consuming and require several hands-on steps and specialized equipment. Microfluidic devices can offer the opportunity to reduce the number of hands-on steps, decrease the volumes of reagents required for purification, and make extraction high throughput. Here, we investigated the translation of a high volume magnetic bead-based total RNA extraction method (from human whole blood) onto a low input volume microfluidic device. Our results first show that RNA integrity is maintained when the reagent volumes are scaled down by a factor of 22 and the wash buffers are combined 1:1. With our microfluidic method, the number of wash steps can be reduced from four to one. Thus, the time to complete RNA extraction can be reduced from 2 h to 40 min. These manipulations to the conventional protocol yielded RNA amplifiable within 40 cycles of reverse transcription quantitative PCR (RT-qPCR) when using the microfluidic device to simplify the wash steps. To improve the purification of the RNA during the bead transport through the microchannel, we also investigated the effect of a synergetic application of the electrokinetic flow. Our results show that DNase I and other contaminants surrounding the beads get washed away more effectively via electrophoretic transport. Most notably, RNA adsorption on the beads is strong enough to counter electrophoretically-driven desorption. In all, our work opens new ways to extract high-quality total RNA rapidly and simply from a small quantity of blood, making the process of RNA extraction more accessible.

Trench field-effect transistors integrated in a microfluidic channel and design considerations for charge detection.

Field-effect transistors (FETs) combined with a microfluidic system allow for the electrical detection of charged materials moving in a microfluidic channel. Here, we demonstrate trench-shaped silicon FETs with the combination of a microfluidic channel that can be used for simultaneous electrical and optical detection of charged fluorescent beads. The n-channel silicon trench FETs have a maximum transconductance of 1.83 × 10-5 S at near-zero gate bias voltage, which is beneficial for the high sensitivity of electrical detection. The optical transparency and physical robustness of the integrated microfluidic channel are achieved by a polydimethylsiloxane (PDMS)/glass hybrid cover combining the good sealing characteristics of PDMS, and the thin and flat properties of glass. Device evaluation methodologies and measurement approaches are also presented demonstrating a synchronized time-lapse imaging and electronic detection of bead transport. The proposed device and design consideration could advance the promise of electronic sensing to measure potential differences induced by charged analytes.

Anisotropic Rod-Shaped Particles Influence Injectable Granular Hydrogel Properties and Cell Invasion.

Granular hydrogels have emerged as a new class of injectable and porous biomaterials that improve integration with host tissue when compared to solid hydrogels. Granular hydrogels are typically prepared using spherical particles and this study considers whether particle shape (i.e., isotropic spheres vs anisotropic rods) influences granular hydrogel properties and cellular invasion. Simulations predict that anisotropic rods influence pore shape and interconnectivity, as well as bead transport through granular assemblies. Photo-cross-linkable norbornene-modified hyaluronic acid is used to produce spherical and rod-shaped particles using microfluidic droplet generators and formed into shear-thinning and self-healing granular hydrogels, with particle shape influencing mechanics and injectability. Rod-shaped particles form granular hydrogels that have anisotropic and interconnected pores, with pore size and number influenced by particle shape and degree of packing. Robust invitro sprouting of endothelial cells from embedded cellular spheroids is observed with rod-shaped particles, including higher sprouting densities and sprout lengths when compared to hydrogels with spherical particles. Cell and vessel invasion into granular hydrogels when injected subcutaneously in vivo are significantly greater with rod-shaped particles, whereas a gradient of cellularity is observed with spherical particles. Overall, this work demonstrates potentially superior functional properties of granular hydrogels with rod-shaped particles for tissue repair.

Isolation of target DNA using synergistic magnetic bead transport and electrokinetic flow.

The advent and dissemination of next-generation sequencing (NGS) technologies such as Illumina's sequencing platforms has brought forth vast reductions in the cost, time, and technical difficulties associated with DNA and RNA sequencing. Despite this trend, the workflow required to generate nucleic acid libraries for sequencing remains time-consuming and laborious. The following research proposes a method for simplifying and streamlining this process by replacing the manual washing steps of the common magnetic bead-based cleanup with a novel microfluidic method by integrating magnetic separation and electrokinetic purification (MSEP). Requiring no pumps, pipette mixing, vortexing, or centrifugation, MSEP relies on selective adsorption of target DNA onto the magnetic beads with subsequent transport of beads through a microchannel undergoing an antiparallel electroosmotic flow. The synergetic flow conditions were optimized using a simple electrohydrodynamic flow model. This work demonstrates that MSEP is as effective in eliminating adapter-dimers from the post-ligation library mix as the manual method while also greatly reducing the hands-on time and amount of pipetting required. Although MSEP has been applied specifically toward NGS library preparation at this time, it has the potential to be adapted and employed for any bead-based separation scheme, namely, solid phase extraction, sequence-specific hybridization, and immunoprecipitation on a microscale.

Experimental assessment of grinding bead velocity distributions and stressing conditions in stirred media mills

Fine grinding in stirred media mills is an important process in mineral, chemical and pharmaceutical industry. The stressing mechanism in these mills is grinding through compression and shearing by grinding beads. A few studies have examined grinding bead transport in stirred media mills. Radiometric methods detect the local filling degree, but little is known about the experimental motion of the grinding beads in the mill. This study aims to investigate the position, velocity and radial circulation of individual grinding beads at different operation parameters (viscosity and tip speed) by a fast and inexpensive measurement technique. Additional information about stresses in the mill were derived, such as a relation between axial grinding media distribution and grinding bead velocity. As a result, the two investigated operation parameter (viscosity and tip speed) effect the grinding bead velocity and grinding bead transport in the mill. The mentioned results can be used to validate DEM simulation results.

Altered N-glycan composition impacts flagella-mediated adhesion in Chlamydomonas reinhardtii.

For the unicellular alga Chlamydomonas reinhardtii, the presence of N-glycosylated proteins on the surface of two flagella is crucial for both cell-cell interaction during mating and flagellar surface adhesion. However, it is not known whether only the presence or also the composition of N-glycans attached to respective proteins is important for these processes. To this end, we tested several C. reinhardtii insertional mutants and a CRISPR/Cas9 knockout mutant of xylosyltransferase 1A, all possessing altered N-glycan compositions. Taking advantage of atomic force microscopy and micropipette force measurements, our data revealed that reduction in N-glycan complexity impedes the adhesion force required for binding the flagella to surfaces. This results in impaired polystyrene bead binding and transport but not gliding of cells on solid surfaces. Notably, assembly, intraflagellar transport, and protein import into flagella are not affected by altered N-glycosylation. Thus, we conclude that proper N-glycosylation of flagellar proteins is crucial for adhering C. reinhardtii cells onto surfaces, indicating that N-glycans mediate surface adhesion via direct surface contact.

Modelling of magnetic bead transport in a microvascular network

In in vivo applications of magnetic particles one crucial aspect is the control of their transport in the tissue microvasculature and their release to target areas. These mechanisms can be driven and influenced by external magnetic fields with large gradients. Experimental results can be elucidated with the aid of in silico models that can also be employed to design optimized multifunctional particles and magnetic field sources. In this framework, we have developed a numerical model that enables us to calculate the trajectory of an ensemble of magnetic nano/microbeads injected in a blood vessel and manipulated by an applied magnetic field. The model combines Navier-Stokes equations for the fluid dynamics simulation of blood flow with classical Newtonian mechanics for bead motion, performing the analysis in a 3D vessel segment reconstructed from a computed tomography scan. The numerical model is applied to study the influence of bead properties (size and magnetic moment) on both transport and adhesion rate, taking into account the interplay of magnetic and viscous drag forces, as well as magnetic dipole–dipole and steric interactions. We also consider different configurations of the external magnetic field source, in order to optimize bead accumulation and adhesion to the vessel wall in a specific target region, processes that can be driven by magnetic forces.

Parallel DNA Extraction From Whole Blood for Rapid Sample Generation in Genetic Epidemiological Studies.

Large-scale genetic epidemiological studies require high-quality analysis of samples such as blood or saliva from multiple patients, which is challenging at the point of care. To expand these studies’ impact, minimal sample storage time and less complex extraction of a substantial quantity and good purity of DNA or RNA for downstream applications are necessary. Here, a simple microfluidics-based system that performs genomic DNA (gDNA) extraction from whole blood was developed. In this system, a mixture of blood lysate, paramagnetic beads, and binding buffer are first placed into the input well. Then, the gDNA-bound paramagnetic beads are pulled using a magnet through a central channel containing a wash buffer to the output well, which contains elution buffer. The gDNA is eluted at 55°C off the chip. The 40-minute microfluidic protocol extracts gDNA from six samples simultaneously and requires an input of 4 μL of diluted blood and a total reagent volume of 75 μL per reaction. Techniques including quantitative PCR (qPCR) and spectrofluorimetry were used to test the purity and quantity of gDNA eluted from the chip following extraction. Bead transport and molecular diffusional analysis showed that an input of less than 4 ng of gDNA (∼667 white blood cells) is optimal for on-chip extraction. There was no observable transport of inhibitors into the eluate that would greatly affect qPCR, and a sample was successfully prepared for next-generation sequencing (NGS). The microfluidics-based extraction of DNA from whole blood described here is paramount for future work in DNA-based point-of-care diagnostics and NGS library workflows.

The FOXJ1 target Cfap206 is required for sperm motility, mucociliary clearance of the airways and brain development.

Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206, orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus, Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects.

Mucociliary Clearance in Mice Measured by Tracking Trans-tracheal Fluorescence of Nasally Aerosolized Beads

Mucociliary clearance (MCC) is the first line of defense in clearing airways. In genetically engineered mice, each component of this system (ciliary beat, mucus, airway surface hydration) can be studied separately to determine its contribution to MCC. Because MCC is difficult to measure in mice, MCC measurements are often omitted from these studies. We report a simple method to measure MCC in mice involving nasal inhalation of aerosolized fluorescent beads and trans-tracheal bead tracking. This method has a number of advantages over existing methods: (1) a small volume of liquid is deposited thus minimally disturbing the airway surface; (2) bead behavior on airways can be visualized; (3) useful for adult or neonatal mice; (4) the equipment is relatively inexpensive and easily obtainable. The type of anesthetic had no significant effect on the rate of MCC, but overloading the airways with beads significantly decreased MCC. In addition, the rate of bead transport was not different in alive (3.11 mm/min) vs recently euthanized mice (3.10 mm/min). A 5-min aerosolization of beads in a solution containing UTP significantly increased the rate of MCC, demonstrating that our method would be of value in testing the role of various pharmacological agents on MCC.

Microfluidic communicating vessel chip for expedited and automated immunomagnetic assays.

Rapid, sensitive analysis of protein biomarkers is of tremendous biological and clinical significance. Immunoassays are workhorse tools for protein analysis and have been under continuous investigation to develop new methods and to improve the analytical performance. Herein we report a pneumatically gated microfluidic communicating vessel (μCOVE) chip for rapid and sensitive immunomagnetic ELISA. A distinct feature of our device is that it employs the communicating vessel principle as a simple means to generate a fast transient hydrodynamic flow to enable effective flow washing without the need for excessive incubation, which greatly simplifies and expedites the assay workflow, compared to conventional microfluidic flow-based immunoassays. Stationary multi-phase microfluidic techniques have been developed for fast bead washing. However, they have some limitations, such as the need for careful control of interfacial properties, large bead quantity required for reliable interphase bead transport, and relatively high bead loss during surface tension-gated traverse. Our single-phase μCOVE chip can overcome such limitations and facilitate the manipulation of magnetic beads to streamline the assay workflow. We showed that the μCOVE device affords highly sensitive quantification of the CEA and EGFR proteins with a LOD down to the sub-picogram per mL level. Direct detection of the EGFR in the crude A431 cell lysate was also demonstrated to further validate the ability of our device for rapid and quantitative analysis of complex biological samples. Overall, our work presents a unique platform that combines the merits of the stationary multi-phase systems and the flow-based microfluidics. This novel immunoassay microsystem has promising potential for a broad range of biological and clinical applications, owing to its simplicity and high performance.

Adrenomedullin regulates the speed of oviductal fluid flow in cattle.

Unidirectional flow of oviductal fluid from the ovarian to uterine side of the ampulla plays a significant role in successful pregnancy, and is produced by ciliary beating. Various systems regulate ciliary beating, such as paracrine, autocrine, and endocrine. We hypothesized that Adrenomedullin (ADM)-a peptide hormone that acts via its receptors, which are complexes of Calcitonin receptor-like receptor (CRLR) and Receptor activity-modifying protein (RAMP) 2 or 3 - promotes oviductal fluid flow in the ampulla of bovine oviducts. First, we examined the expression of ADM, CRLR, RAMP2, and RAMP3 mRNAs in isolated epithelial cells throughout the estrous cycle, and the localization of ADM receptor protein constituents in the ampulla. RAMP2 expression was significantly higher in the follicular phase. Furthermore, RAMP2 protein was detected only in ciliated cells, whereas CRLR and RAMP3 were detected in all epithelial cells. The effects of ADM and an ADM antagonist on fluid-flow speed were examined using microbeads in ampullary tissue. ADM antagonist decreased bead transport speed, and this decrease was reversed by ADM. In addition, ADM recovered the bead transport speed that decreased in the absence of calcium. Overall, our results suggest that ADM contributes to the regulation of oviductal fluid flow in ampulla.

Active Cargo Positioning from Actin-Polarity Sensing by Small Myosin Assemblies

Molecular motors navigate the cytoskeleton to position vesicles and organelles at specific locations in the cell. Cytoskeletal filaments assemble into parallel, antiparallel or disordered networks, providing a complex environment that constrains active transport properties. Using surface micro-patterns of nucleation-promoting factors to control the geometry of actin polymerization, we studied in vitro the interplay between the actin-network architecture and cargo transport by small myosin assemblies. With two parallel nucleation lines, we produced an antiparallel network of overlapping filaments. We found that 200-nm beads coated with processive myosin-5 motors displayed directed movements towards the midline of the pattern, where the net polarity of the actin network was null, and accumulated there. The bead distribution was dictated by the spatial profiles of bead velocity and diffusion coefficient, indicating that a diffusion-drift process was at work. Interestingly, beads coated with skeletal heavy mero-myosin-2 motors showed a similar behavior. However, although velocity gradients were sharper with myosin 2, the much larger bead diffusion observed with this non-processive motor resulted in less precise positioning. Strikingly, bead positioning did not depend on the spacing between the nucleation lines. Our observations are well described by a three-state model of bead transport, in which active beads locally sense the net polarity of the filament network by frequently detaching from and reattaching to the filaments. A stochastic sequence of processive runs and diffusive searches results in a biased random walk with an effective drift velocity and diffusion coefficient. Positioning relies on spatial gradients of the net actin polarity, as well as on the run length of the cargo in the attached state. Altogether, our results on a minimal acto-myosin system demonstrate the key role played by the actin-network architecture on motor transport.

Quantitative Determination of the Probability of Multiple-Motor Transport in Bead-Based Assays

With their longest dimension typically being less than 100 nm, molecular motors are significantly below the optical-resolution limit. Despite substantial advances in fluorescence-based imaging methodologies, labeling with beads remains critical for optical-trapping-based investigations of molecular motors. A key experimental challenge in bead-based assays is that the number of motors on a bead is not well defined. Particularly for single-molecule investigations, the probability of single- versus multiple-motor events has not been experimentally investigated. Here, we used bead travel distance as an indicator of multiple-motor transport and determined the lower-bound probability of bead transport by two or more motors. We limited the ATP concentration to increase our detection sensitivity for multiple- versus single-kinesin transport. Surprisingly, for all but the lowest motor number examined, our measurements exceeded estimations of a previous model by ≥2-fold. To bridge this apparent gap between theory and experiment, we derived a closed-form expression for the probability of bead transport by multiple motors, and constrained the only free parameter in this model using our experimental measurements. Our data indicate that kinesin extends to ∼57 nm during bead transport, suggesting that kinesin exploits its conformational flexibility to interact with microtubules at highly curved interfaces such as those present for vesicle transport in cells. To our knowledge, our findings provide the first experimentally constrained guide for estimating the probability of multiple-motor transport in optical trapping studies. The experimental approach utilized here (limiting ATP concentration) may be generally applicable to studies in which molecular motors are labeled with cargos that are artificial or are purified from cellular extracts.

Finite element simulations of hydrodynamic trapping in microfluidic particle-trap array systems

Computational fluid dynamic (CFD) simulation is a powerful tool in the design and implementation of microfluidic systems, especially for systems that involve hydrodynamic behavior of objects such as functionalized microspheres, biological cells, or biopolymers in complex structures. In this work, we investigate hydrodynamic trapping of microspheres in a novel microfluidic particle-trap array device by finite element simulations. The accuracy of the time-dependent simulation of a microsphere's motion towards the traps is validated by our experimental results. Based on the simulation, we study the fluid velocity field, pressure field, and force and stress on the microsphere in the device. We further explore the trap array's geometric parameters and critical fluid velocity, which affect the microsphere's hydrodynamic trapping. The information is valuable for designing microfluidic devices and guiding experimental operation. Besides, we provide guidelines on the simulation set-up and release an openly available implementation of our simulation in one of the popular FEM softwares, COMSOL Multiphysics. Researchers may tailor the model to simulate similar microfluidic systems that may accommodate a variety of structured particles. Therefore, the simulation will be of particular interest to biomedical research involving cell or bead transport and migration, blood flow within microvessels, and drug delivery.

Transport dynamics of superparamagnetic microbeads trapped by mobile magnetic domain walls

The dynamics of fluid-borne superparamagnetic bead transport by field-driven domain walls (DWs) in submicrometer ferromagnetic tracks is studied experimentally together with numerical and analytical modeling. A combination of micromagnetic modeling and numerical calculation is used to determine the strength of bead-DW interaction for a range of track geometries and bead sizes. The maximum DW velocity for continuous bead transport is predicted from these results and shown to be supported by experimental measurements. Enhancement of the maximum velocity by appropriate material selection or field application is demonstrated, and an analysis of the source of statistical variation is presented. Finally, the dynamics of bead-DW interaction and bead transport above the maximum DW velocity for continuous DW-mediated bead transport is characterized.

Design criteria for developing low-resource magnetic bead assays using surface tension valves

Many assays for biological sample processing and diagnostics are not suitable for use in settings that lack laboratory resources. We have recently described a simple, self-contained format based on magnetic beads for extracting infectious disease biomarkers from complex biological samples, which significantly reduces the time, expertise, and infrastructure required. This self-contained format has the potential to facilitate the application of other laboratory-based sample processing assays in low-resource settings. The technology is enabled by immiscible fluid barriers, or surface tension valves, which stably separate adjacent processing solutions within millimeter-diameter tubing and simultaneously permit the transit of magnetic beads across the interfaces. In this report, we identify the physical parameters of the materials that maximize fluid stability and bead transport and minimize solution carryover. We found that fluid stability is maximized with ≤0.8 mm i.d. tubing, valve fluids of similar density to the adjacent solutions, and tubing with ≤20 dyn/cm surface energy. Maximizing bead transport was achieved using ≥2.4 mm i.d. tubing, mineral oil valve fluid, and a mass of 1-3 mg beads. The amount of solution carryover across a surface tension valve was minimized using ≤0.2 mg of beads, tubing with ≤20 dyn/cm surface energy, and air separators. The most favorable parameter space for valve stability and bead transport was identified by combining our experimental results into a single plot using two dimensionless numbers. A strategy is presented for developing additional self-contained assays based on magnetic beads and surface tension valves for low-resource diagnostic applications.

Cilia-driven particle and fluid transport over mucus-free mice tracheae

To date, there is only a fragmentary understanding of the fundamental mechanisms of airway mucociliary transport. Application of the latest measurement techniques can aid in deciphering the complex interplay between ciliary beat and airway surface liquid (ASL) transport. In the present study, direct, quasi-simultaneous measurements of the cilia-induced fluid and bead transport were performed to gain a better insight into both transport mechanisms. In this study cilia-induced periciliary liquid (PCL) transport is measured by means of micro Particle Image Velocimetry (μPIV) with neutrally buoyant tracers. Particle Tracking Velocimetry (PTV) with heavier polystyrene-ferrite beads is performed to simulate particle transport. Contrary to recent literature, in which the presence of mucus was deemed necessary to maintain periciliary liquid (PCL) transport, effective particle and fluid transport was measured in our experiments in the absence of mucus. In response to muscarine or ATP stimulation, maximum fluid transport rates of 250μm/s at 15μm distance to the tracheal epithelia were measured while bead transport rates over the epithelia surfaces reached 200μm/s. We estimated that the mean bead transport is dominated by viscous drag compared to inertial fluid forces. Furthermore, mean bead transport velocities appear to be two orders of magnitude larger compared to bead sedimentation velocities. Therefore, beads are expected to closely follow the mean PCL flow in non-ciliated epithelium regions. Based on our results, we have shown that PCL transport can be directly driven by the cilia beat and that the PCL motion may be capable of driving bead transport by fluid drag.