Now showing 1 - 10 of 18
  • Publication
    Affinity Separation: M13 Bacteriophage-Activated Superparamagnetic Beads for Affinity Separation
    The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemically cross-linking the pVIII proteins to a carboxyl functionalized bead produced highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His6 peptide sequence allowed reversible assembly of the bacteriophage on a nitrilotriacetic acid functionalized core in an end-on configuration. These phage-magnetic particles were successfully used to separate antibodies from high-protein concentration solutions in a single step with a > 90 % purity. The dense magnetic core of these particles makes themfive times more responsive to magnetic fields than commercial materialscomposed of polymer-iron oxide compositesand a monolayer of phage could produced a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage
      254
  • Publication
    Advances in Affinity Ligand-Functionalized Nanomaterials for Biomagnetic Separation
    The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future.
      673Scopus© Citations 29
  • Publication
    Neuron Sub-Populations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment
    (American Chemical Society, 2015) ; ;
    Correct wiring of the nervous system requires guidance cues, diffusible or substrate-bound proteins that steer elongating axons to their target tissues. Netrin-1, the best characterized member of the Netrins family of guidance molecules, is known to induce axon turning and modulate axon elongation rate; however, the factors regulating the axonal response to Netrin-1 are not fully understood. Using microfluidics, we treated fluidically isolated axons of mouse primary cortical neurons with Netrin-1 and characterized axon elongation rates, as well as the membrane localization of deleted in colorectal cancer (DCC), a well-established receptor of Netrin-1. The capacity to stimulate and observe a large number of individual axons allowed us to conduct distribution analyses, through which we identified two distinct neuron subpopulations based on different elongation behavior and different DCC membrane dynamics. Netrin-1 reduced the elongation rates in both subpopulations, where the effect was more pronounced in the slow growing subpopulation. Both the source of Ca2+ influx and the basal cytosolic Ca2+ levels regulated the effect of Netrin-1, for example, Ca2+ efflux from the endoplasmic reticulum due to the activation of Ryanodine channels blocked Netrin-1-induced axon slowdown. Netrin-1 treatment resulted in a rapid membrane insertion of DCC, followed by a gradual internalization. DCC membrane dynamics were different in the central regions of the growth cones compared to filopodia and axon shafts, highlighting the temporal and spatial heterogeneity in the signaling events downstream of Netrin-1. Cumulatively, these results demonstrate the power of microfluidic compartmentalization and distribution analysis in describing the complex axonal Netrin-1 response.
      581Scopus© Citations 11
  • Publication
    Resistive pulse sensing of magnetic beads and supraparticle structures using tunable pores
    Tunable pores (TPs) have been used for resistive pulse sensing of 1 μm superparamagnetic beads, both dispersed and within a magnetic field. Upon application of this field, magnetic supraparticle structures (SPSs) were observed. Onset of aggregation was most effectively indicated by an increase in the mean event magnitude, with data collected using an automated thresholding method. Simulations enabled discrimination between resistive pulses caused by dimers and individual particles. Distinct but time-correlated peaks were often observed, suggesting that SPSs became separated in pressure-driven flow focused at the pore constriction. The distinct properties of magnetophoretic and pressure-driven transport mechanisms can explain variations in the event rate when particles move through an asymmetric pore in either direction, with or without a magnetic field applied. Use of TPs for resistive pulse sensing holds potential for efficient, versatile analysis and measurement of nano- and microparticles, while magnetic beads and particle aggregation play important roles in many prospective biosensing applications.
      412Scopus© Citations 32
  • Publication
    Characterization of carboxylate nanoparticle adhesion with the fungal pathogen Candida albicans
    Candida albicans is the lead fungal pathogen of nosocomial bloodstream infections worldwide and has mortality rates of 43%. Nanoparticles have been identified as a means to improve medical outcomes for Candida infections, enabling sample concentration, serving as contrast agents for in vivo imaging, and delivering therapeutics. However, little is known about how nanoparticles interact with the fungal cell wall. In this report we used laser scanning confocal microscopy to examine the interaction of fluorescent polystyrene nanoparticles of specific surface chemistry and diameter with C. albicans and mutant strains deficient in various C. albicans surface proteins. Carboxylate-functionalized nanoparticles adsorbed mainly to the hyphae of wild-type C. albicans. The dissociative binding constant of the nanoparticles was ∼150, ∼30 and ∼2.5 pM for 40, 100 nm and 200 nm diameter particles, respectively. A significant reduction in particle binding was observed with a Δals3 strain compared to wild-type strains, identifying the Als3 adhesin as the main mediator of this nanoparticle adhesion. In the absence of Als3, nanoparticles bound to germ tubes and yeast cells in a pattern resembling the localization of Als1, indicating Als1 also plays a role. Nanoparticle surface charge was shown to influence binding – positively charged amine-functionalized nanoparticles failed to bind to the hyphal cell wall. Binding of carboxylate-functionalized nanoparticles was observed in the presence of serum, though interactions were reduced. These observations show that Als3 and Als1 are important targets for nanoparticle-mediated diagnostics and therapeutics, and provide direction for optimal diameter and surface characteristics of nanoparticles that bind to the fungal cell wall.
      367Scopus© Citations 14
  • Publication
    Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and single cells
    Nonlinear magnetophoresis (NLM) is a powerful approach for on-chip transport and separation of superparamagnetic (SPM) beads, based on a travelling magnetic field wave generated by the combination of a micromagnet array (MMA) and an applied rotating magnetic field. Here, we present two novel MMA designs that allow SPM beads to be focused, sorted, and separated on-chip. Converging MMAs were used to rapidly collect the SPM beads from a large region of the chip and focus them into synchronized lines. We characterise the collection efficiency of the devices and demonstrate that they can facilitate on-chip analysis of populations of SPM beads using a single-point optical detector. The diverging MMAs were used to control the transport of the beads and to separate them based on their size. The separation efficiency of these devices was determined by the orientation of the magnetisation of the micromagnets relative to the external magnetic field and the size of the beads relative to that of micromagnets. By controlling these parameters and the rotation of the external magnetic field we demonstrated the controlled transport of SPM bead-labelled single MDA-MB-231 cells. The use of these novel MMAs promises to allow magnetically-labelled cells to be efficiently isolated and then manipulated on-chip for analysis with high-resolution chemical and physical techniques.
      376Scopus© Citations 9
  • Publication
    Charge and topography patterned lithium niobate provides physical cues to fluidically isolated cortical axons
    In vitro devices that combine chemotactic and physical cues are needed for understanding how cells integrate different stimuli. We explored the suitability of lithium niobate (LiNbO3), a transparent ferroelectric material that can be patterned with electrical charge domains and micro/nanotopography, as a neural substrate. On flat LiNbO3 z-surfaces with periodically alternating charge domains, cortical axons are partially aligned with domain boundaries. On submicron-deep etched trenches, neurites are aligned with the edges of the topographical features. Finally, we bonded a bicompartmental microfluidic chip to LiNbO3 surfaces patterned by etching, to create isolated axon microenvironments with predefined topographical cues. LiNbO3 is shown to be an emerging neuron culture substrate with tunable electrical and topographical properties that can be integrated with microfluidic devices, suitable for studying axon growth and guidance mechanisms under combined topographical/chemical stimuli.
      394Scopus© Citations 16
  • Publication
    Magnetic Tweezers-Based Force Clamp Reveals Mechanically Distinct apCAM Domain Interactions
    Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) play a crucial role in cell-cell interactions during nervous system development and function. The Aplysia CAM (apCAM), an invertebrate IgCAM, shares structural and functional similarities with vertebrate NCAM and therefore has been considered as the Aplysia homolog of NCAM. Despite these similarities, the binding properties of apCAM have not been investigated thus far. Using magnetic tweezers, we applied physiologically relevant, constant forces to apCAM-coated magnetic particles interacting with apCAM-coated model surfaces and characterized the kinetics of bond rupture. The average bond lifetime decreased with increasing external force, as predicted by theoretical considerations. Mathematical simulations suggest that the apCAM homophilic interaction is mediated by two distinct bonds, one involving all five immunoglobulin (Ig)-like domains in an antiparallel alignment and the other involving only two Ig domains. In summary, this study provides biophysical evidence that apCAM undergoes homophilic interactions, and that magnetic tweezers-based, force-clamp measurements provide a rapid and reliable method for characterizing relatively weak CAM interactions.
      397Scopus© Citations 14
  • Publication
    A microfluidic dual gradient generator for conducting cell-based drug combination assays
    We present a microfluidic chip that generates linear concentration gradients of multiple solutes that are orthogonally-aligned to each other. The kinetics of gradient formation was characterized using a fluorescent tracer matching the molecular weight of small inhibitory drugs. Live-cell signalling and motility experiments were conducted to demonstrate the potential uses and advantages of the device. A431 epidermoid carcinoma cells, where EGF induces apoptosis in a concentration-dependent manner, were simultaneously exposed to gradients of MEK inhibitor and EGF receptor (EGFR) inhibitor. By monitoring live caspase activation in the entire chip, we were able to quickly assess the combinatorial interaction between MEK and EGFR pathways, which otherwise would require costly and time consuming titration experiments. We also characterized the motility and morphology of MDA-MB-231 breast cancer cells exposed to orthogonal gradients of EGF and EGFR inhibitor. The microfluidic chip not only permitted the quantitative analysis of a population of cells exposed to drug combinations, but also enabled the morphological characterization of individual cells. In summary, our microfluidic device, capable of establishing concentration gradients of multiple compounds over a group of cells, facilitates and accelerates in vitro cell biology experiments, such as those required for cell-based drug combination assays.
      433Scopus© Citations 20
  • Publication
    Flow-Enhanced Nonlinear Magnetophoresis for High-Resolution Bioseparation
    (American Chemical Society, 2011-04-20) ; ;
    A new mode of transport is described that was capable of high-resolution separation of superparamagnetic materials from complex mixtures based on their size. Laminar flow and a rotating external magnetic field were applied to superparamagnetic beads assembled on a semiperiodic micromagnet array. Beads at the edge of the micromagnet array oscillated in-phase with the external magnetic field with an amplitude that decreased with increasing frequency, omega, until they reached an immobilization frequency, omega(nu) where the beads stopped moving. Laminar flow along the edge of the array could be tuned to sweep the beads for which omega omega(i) undisturbed. Flow-enhanced nonlinear magnetophoresis (F-NLM) promises to enable multiple superparamagnetc bead types to be used in the fractionation of cells and implementation of diagnostic assays.
      285Scopus© Citations 15