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Lee, Gil U.
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Lee, Gil U.
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Lee, Gil U.
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Now showing 1 - 10 of 18
- PublicationAdvances in Affinity Ligand-Functionalized Nanomaterials for Biomagnetic SeparationThe 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.
1009Scopus© Citations 37 - PublicationIsolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separationSoybean proteins offer exceptional promise in the area of cancer prevention and treatment. Specifically, Bowman-Birk Inhibitor (BBI) has the ability to suppress carcinogenesis in vivo, which has been attributed to BBI’s inhibition of serine protease (trypsin and chymotrypsin) activity. The lack of molecular probes for the isolation of this protein has made it difficult to work with, limiting its progress as a significant candidate in the treatment of cancer. This study has successfully identified a set of novel synthetic peptides targeting the BBI, and has demonstrated the ability to bind BBI in vitro. One of those probes has been covalently immobilised on superparamagnetic microbeads to allow the isolation of BBI from soy whey mixtures in a single step. Our ultimate goal is the use of the described synthetic probe to facilitate the isolation of this potentially therapeutic protein for low cost, scalable analysis and production of BBI.
729Scopus© Citations 24 - PublicationBio-Nano-Magnetic Materials for Localized Mechanochemical Stimulation of Cell Growth and DeathMagnetic 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.
903Scopus© Citations 52 - 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.550Scopus© Citations 18 - PublicationFlow-Enhanced Nonlinear Magnetophoresis for High-Resolution BioseparationA 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.
416Scopus© 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.504Scopus© Citations 10 - PublicationResistive pulse sensing of magnetic beads and supraparticle structures using tunable poresTunable 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.
491Scopus© Citations 32 - PublicationA microfluidic dual gradient generator for conducting cell-based drug combination assays(Royal Society of Chemistry, 2016-01-01)
; ; ; ; ; ; ; ; ; ; 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.598Scopus© Citations 24 - PublicationIn vitro study of the interaction of heregulin-functionalized magnetic-optical nanorods with MCF7 and MDA-MB- 231 cells(Royal Society of Chemistry, 2014-09)
; ; ; ; ; ; ; Multifunctional nanoparticles that actively target specific cells are promising tools for cancer diagnosis and therapy. In this article we review the synthesis and surface chemistry of Fe–Au nanorods and their characterization using microscopy. The diameter of the rods used in this study was selected to be 150–200 nm so that they did not enter the cells. The 80 nm-long Au tips of the nanorods were functionalized with heregulin (HRG), and the micron-long Fe portion was coated with a poly(ethylene glycol) monolayer to minimize non-specific interactions. Nanorods functionalized with HRG were found to preferentially bind to MCF7 cells that express high levels of the receptor tyrosine-protein kinase ErbB2/3. Magnetic tweezers measurements were used to characterize the kinetic properties of the bond between the HRG on the rods and ErbB2/3 on the surface of the cells. The strong magnetization of Fe–Au nanorods makes them excellent candidates for in-vitro and in-vivo imaging, and magnetic therapeutic applications targeting cancer cells in circulation.708Scopus© Citations 2 - PublicationResistive Pulse Sensing of Analyte-Induced Multicomponent Rod Aggregation Using Tunable PoresResistive 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.
576Scopus© Citations 79