Lee, Gil U.
Lee, Gil U.
Lee, Gil U.
Now showing 1 - 10 of 18
- PublicationAffinity Separation: M13 Bacteriophage-Activated Superparamagnetic Beads for Affinity Separation(Wiley, 2012-08-06)
; ;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 283
- PublicationNeuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics(Frontiers, 2017-01-05)
; ;Netrin-1 modulates axonal growth direction and speed. Its best characterized receptor, Deleted in Colorectal Cancer (DCC), is localized to growth cones, but also observed in the cell bodies. We hypothesized that cell bodies sense Netrin-1 and contribute to axon growth rate modulation, mediated by the second messenger system. We cultured mouse cortical neurons in microfluidic devices to isolate distal axon and cell body microenvironments. Compared to isolated axonal treatment, global Netrin-1 treatment decreased the axon elongation rate and affected the dynamics of total and membranous DCC, calcium, and cyclic nucleotides. Signals induced by locally applied Netrin-1 propagated in both anterograde and retrograde directions, demonstrated by the long-range increase in DCC and by the increased frequency of calcium transients in cell bodies, evoked by axonal Netrin-1. Blocking the calcium efflux from endoplasmic reticulum suppressed the membranous DCC response. Our findings support the notion that neurons sense Netrin-1 along their entire lengths in making axonal growth decisions. 285Scopus© Citations 11
- PublicationAdvances in magnetic tweezers for single molecule and cell biophysics(Royal Society of Chemistry, 2014-11)
;Magnetic tweezers (MTW) enable highly accurate forces to be transduced to molecules to study mechanotransduction at the molecular or cellular level. We review recent MTW studies in single molecule and cell biophysics that demonstrate the flexibility of this technique. We also discuss technical advances in the method on several fronts, i.e., from novel approaches for the measurement of torque to multiplexed biophysical assays. Finally, we describe multi-component nanorods with enhanced optical and magnetic properties and discuss their potential as future MTW probes. 516Scopus© Citations 66
- PublicationCharacterization of carboxylate nanoparticle adhesion with the fungal pathogen Candida albicans(Royal Society of Chemistry, 2017-10-11)
; ; ; ; ; ;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. 375Scopus© Citations 14
- PublicationBio-Nano-Magnetic Materials for Localized Mechanochemical Stimulation of Cell Growth and Death(Wiley, 2016-07-20)
; ;Magnetic nanoparticles are promising new tools for therapeutic applications, such as magnetic nanoparticle hyperthermia therapy and targeted drug delivery. Recent in vitro studies have demonstrated that a force application with magnetic tweezers can also affect cell fate, suggesting a therapeutic potential for magnetically modulated mechanical stimulation. The magnetic properties of nanoparticles that induce physical responses and the subtle responses that result from mechanically induced membrane damage and/or intracellular signaling are evaluated. Magnetic particles with various physical, geometric, and magnetic properties and specific functionalization can now be used to apply mechanical force to specific regions of cells, which permit the modulation of cellular behavior through the use of spatially and time controlled magnetic fields. On one hand, mechanochemical stimulation has been used to direct the outgrowth on neuronal growth cones, indicating a therapeutic potential for neural repair. On the other hand, it has been used to kill cancer cells that preferentially express specific receptors. Advances made in the synthesis and characterization of magnetic nanomaterials and a better understanding of cellular mechanotransduction mechanisms may support the translation of mechanochemical stimulation into the clinic as an emerging therapeutic approach. 625Scopus© Citations 40
- PublicationMagnetic Tweezers-Based Force Clamp Reveals Mechanically Distinct apCAM Domain Interactions(Biophysical Society, 2012-09-19)
; ; ; ;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. 403Scopus© Citations 15
- PublicationResistive Pulse Sensing of Analyte-Induced Multicomponent Rod Aggregation Using Tunable Pores(Wiley, 2012-07-06)
; ;Resistive pulse sensing is used to monitor individual and aggregated rod-shaped nanoparticles as they move through tunable pores in elastomeric membranes. By comparing particles of similar dimensions, it is demonstrated that the resistive pulse signal of a rod is fundamentally different from that of a sphere. Rods can be distinguished using two measurements: the blockade event magnitude (Δip), which reveals the particle's size, and the full width at half maximum (FWHM) duration, which relates to the particle's speed and length. While the observed Δip values agree well with simulations, the measured FWHM times are much larger than expected. This increase in dwell time, caused by rods moving through the pore in various orientations, is not observed for spherical particles. These differences are exploited in a new agglutination assay using rod-shaped particles. By controlling the surface chemistry and location of the capture ligand, rods are made to form either long “end-on-end” or wide 'side-on' aggregates upon the addition of an analyte. This observation will facilitate multiplexed detection in agglutination assays, as particles with a particular aspect ratio can be distinguished by two measurements. This is first demonstrated with a biotinylated target and avidin capture probe, followed by the detection of platelet-derived growth factor (PDGF-BB) using an aptamer capture probe, with limits of detection down to femtomolar levels. 388Scopus© Citations 73
- PublicationFlow-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. 286Scopus© Citations 15
- PublicationMicromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and single cells(Royal Society of Chemistry, 2015-07-10)
; ; ; ; ;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
- PublicationNeuron 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. 587Scopus© Citations 11