Gil U. Lee

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Organization: University College Dublin , Ireland

Department: University College Dublin

Title: (PhD)

TOPICS

Material Chemistry

Chemicals
References

Characterization of carboxylate nanoparticle adhesion with the fungal pathogen Candida albicans

Co-reporter:Amy Lyden;Lisa Lombardi;Wilfried Sire;Peng Li;Jeremy C. Simpson;Geraldine Butler

Nanoscale (2009-Present) 2017 vol. 9(Issue 41) pp:15911-15922

Publication Date(Web):2017/10/26

DOI:10.1039/C7NR04724J

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.

Bio-Nano-Magnetic Materials for Localized Mechanochemical Stimulation of Cell Growth and Death

Co-reporter:Devrim Kilinc;Cindi L. Dennis

Advanced Materials 2016 Volume 28( Issue 27) pp:5672-5680

Publication Date(Web):

DOI:10.1002/adma.201504845

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.

A microfluidic dual gradient generator for conducting cell-based drug combination assays

Co-reporter:Devrim Kilinc, Jefrem Schwab, Stefano Rampini, Oshoke W. Ikpekha, Ashwin Thampi, Agata Blasiak, Peng Li, Robert Schwamborn, Walter Kolch, David Matallanas and Gil U. Lee

Integrative Biology 2016 vol. 8(Issue 1) pp:39-49

Publication Date(Web):10 Nov 2015

DOI:10.1039/C5IB00209E

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.

Mechanochemical Stimulation of MCF7 Cells with Rod-Shaped FeAu Janus Particles Induces Cell Death Through Paradoxical Hyperactivation of ERK

Co-reporter:Devrim Kilinc;Anna Lesniak;Suad A. Rashdan;Dhruv Ghi;Agata Blasiak;Paul C. Fannin;Alex von Kriegsheim;Walter Kolch

Advanced Healthcare Materials 2015 Volume 4( Issue 3) pp:395-404

Publication Date(Web):

DOI:10.1002/adhm.201400391

Multifunctional nanoparticles that actively target-specific tissues are studied for cancer diagnosis and treatment. Magnetically and optically active particles are of particular interest because they enable multiple imaging modalities and physically modulated therapies, such as magnetic hyperthermia. Fe–Au nanorods are synthesized that have a long iron segment, coated with polyethylene glycol, and a short gold tip functionalized with heregulin (HRG), a known ligand of ErbB family of receptors. HRG–nanorods preferentially target MCF7 cells relative to MDA-MB-231 cells, as demonstrated in a novel microfluidics device. Targeting rates of these classical breast cancer cells correlate with their differential expression of ErbB2/3 receptors. HRG–nanorod binding stimulates the extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in MCF7 cells. The increase in ERK phosphorylation is linked to “active zones,” dynamic regions in the cell periphery, which exhibit higher rates of particle binding than the rest of the cell. Periodically stretching cells using magnetic tweezers further activates ERK, which leads to cell death in cells co-treated with B-Raf inhibitors, through ERK hyperactivation. Although to a lesser extent, cell death is also achieved through magnetic hyperthermia. These results demonstrate nanoscale targeting and localized mechanochemical treatment of specific cancer cell lines based on their receptor expression using multifunctional nanoparticles.

Neuron Subpopulations with Different Elongation Rates and DCC Dynamics Exhibit Distinct Responses to Isolated Netrin-1 Treatment

Co-reporter:Agata Blasiak, Gil U. Lee, and Devrim Kilinc

ACS Chemical Neuroscience 2015 Volume 6(Issue 9) pp:1578

Publication Date(Web):July 20, 2015

DOI:10.1021/acschemneuro.5b00142

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.Keywords: filopodia; growth cones; Microfluidics; receptor dynamics; UNC-5

Rapid, highly sensitive detection of herpes simplex virus-1 using multiple antigenic peptide-coated superparamagnetic beads

Co-reporter:Ying-Fen Ran, Conor Fields, Julien Muzard, Viktoryia Liauchuk, Michael Carr, William Hall and Gil U. Lee

Analyst 2014 vol. 139(Issue 23) pp:6126-6134

Publication Date(Web):03 Jun 2014

DOI:10.1039/C4AN00774C

A sensitive, rapid, and label free magnetic bead aggregation (MBA) assay has been developed that employs superparamagnetic (SPM) beads to capture, purify, and detect model proteins and the herpes simplex virus (HSV). The MBA assay is based on monitoring the aggregation state of a population of SPM beads using light scattering of individual aggregates. A biotin–streptavidin MBA assay had a femtomolar (fM) level sensitivity for analysis times less than 10 minutes, but the response of the assay becomes nonlinear at high analyte concentrations. A MBA assay for the detection of HSV-1 based on a novel peptide probe resulted in the selective detection of the virus at concentrations as low as 200 viral particles (vp) per mL in less than 30 min. We define the parameters that determine the sensitivity and response of the MBA assay, and the mechanism of enhanced sensitivity of the assay for HSV. The speed, relatively low cost, and ease of application of the MBA assay promise to make it useful for the identification of viral load in resource-limited and point-of-care settings where molecular diagnostics cannot be easily implemented.

Advances in magnetic tweezers for single molecule and cell biophysics

Co-reporter:Devrim Kilinc and Gil U. Lee

Integrative Biology 2014 vol. 6(Issue 1) pp:27-34

Publication Date(Web):14 Nov 2013

DOI:10.1039/C3IB40185E

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.

Flow enhanced non-linear magnetophoretic separation of beads based on magnetic susceptibility

Co-reporter:Peng Li, Devrim Kilinc, Ying-Fen Ran and Gil U. Lee

Lab on a Chip 2013 vol. 13(Issue 22) pp:4400-4408

Publication Date(Web):20 Aug 2013

DOI:10.1039/C3LC50816A

Magnetic separation provides a rapid and efficient means of isolating biomaterials from complex mixtures based on their adsorption on superparamagnetic (SPM) beads. Flow enhanced non-linear magnetophoresis (FNLM) is a high-resolution mode of separation in which hydrodynamic and magnetic fields are controlled with micron resolution to isolate SPM beads with specific physical properties. In this article we demonstrate that a change in the critical frequency of FNLM can be used to identify beads with magnetic susceptibilities between 0.01 and 1.0 with a sensitivity of 0.01 Hz−1. We derived an analytical expression for the critical frequency that explicitly incorporates the magnetic and non-magnetic composition of a complex to be separated. This expression was then applied to two cases involving the detection and separation of biological targets. This study defines the operating principles of FNLM and highlights the potential for using this technique for multiplexing diagnostic assays and isolating rare cell types.

Synthesis of Superparamagnetic Particles with Tunable Morphologies: The Role of Nanoparticle–Nanoparticle Interactions

Co-reporter:James J. O’Mahony, Mark Platt, Devrim Kilinc, and Gil Lee

Langmuir 2013 Volume 29(Issue 8) pp:2546-2553

Publication Date(Web):February 1, 2013

DOI:10.1021/la3047565

Superparamagnetic microparticles are extensively used in the purification of biomolecules due to the speed and ease of magnetic separation. It is desirable that the microparticles used in biological affinity separations have both high surface area and high magnetic mobility to facilitate a high binding capacity of target biomolecules and their rapid removal from solution, respectively. Scaling laws for conventional spherical superparamagnetic microparticles are such that increasing the microparticle specific surface area results in a significant decrease in the magnetic mobility. More favorable combinations of these key parameters can be found if alternative microparticle morphologies are developed for use in affinity separations. Emulsion-templated self-assembly of iron oxide nanoparticles into microparticles using oil-in-water emulsions was carried out using a modified Couette shear mixer with separate inlet ports for the oil and aqueous phases, enabling high throughput microparticle synthesis. By controlling the dissolved nanoparticle concentration and nanoparticle surface activity at the droplet interfaces, the resulting microparticles were tuned to spherical, dimpled, or crumpled morphologies. The specific binding capacity and magnetic mobility of each type of microparticle were measured by a peroxidase-based colorimetric assay and by their magnetic field-induced motion in a viscous fluid, respectively. Superparamagnetic microparticles with dimpled and crumpled morphologies were found to have higher specific binding capacities compared to spherical microparticles, while maintaining high magnetic field velocities due to their high iron oxide content. Superparamagnetic microparticles with these novel morphologies would make excellent tools for affinity-based bioseparations where binding capacity and magnetic mobility are key factors.

Isolation of Bowman-Birk-Inhibitor from soybean extracts using novel peptide probes and high gradient magnetic separation

Co-reporter:Conor Fields, Paul Mallee, Julien Muzard, Gil U. Lee

Food Chemistry 2012 Volume 134(Issue 4) pp:1831-1838

Publication Date(Web):15 October 2012

DOI:10.1016/j.foodchem.2012.03.085

Soybean 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.Highlights► Identification of soluble peptide structures to bind Soybean derived Bowman-Birk Inhibitor. ► Examine the functional binding proprieties of the synthetic probes in vitro. ► Isolation of BBI from soy crude protein extracts using superparamagnetic particles.

Probing the Soybean Bowman–Birk Inhibitor Using Recombinant Antibody Fragments

Co-reporter:Julien Muzard, Conor Fields, James John O’Mahony, and Gil U. Lee

Journal of Agricultural and Food Chemistry 2012 Volume 60(Issue 24) pp:6164-6172

Publication Date(Web):May 29, 2012

DOI:10.1021/jf3004724

The nutritional and health benefits of soy protein have been extensively studied over recent decades. The Bowman–Birk inhibitor (BBI), derived from soybeans, is a double-headed inhibitor of chymotrypsin and trypsin with anticarcinogenic and anti-inflammatory properties, which have been demonstrated in vitro and in vivo. However, the lack of analytical and purification methodologies complicates its potential for further functional and clinical investigations. This paper reports the construction of anti-BBI antibody fragments based on the principle of protein design. Recombinant antibody (scFv and diabody) molecules targeting soybean BBI were produced and characterized in vitro (KD ∼ 1.10–9 M), and the antibody-binding site (epitope) was identified as part of the trypsin-specific reactive loop. Finally, an extremely fast purification strategy for BBI from soybean extracts, based on superparamagnetic particles coated with antibody fragments, was developed. To the best of the authors' knowledge, this is the first report on the design and characterization of recombinant anti-BBI antibodies and their potential application in soybean processing.

Highly Ordered Fe–Au Heterostructured Nanorod Arrays and Their Exceptional Near-Infrared Plasmonic Signature

Co-reporter:Yong Zhang, Brian Ashall, Gillian Doyle, Dominic Zerulla, and Gil U. Lee

Langmuir 2012 Volume 28(Issue 49) pp:17101-17107

Publication Date(Web):October 26, 2012

DOI:10.1021/la302290v

The potential of highly ordered array nanostructures in sensing applications is well recognized, particularly with the ability to define the structural composition and arrangement of the individual nanorods accurately. The use of heterogeneous nanostructures generates an additional degree of freedom, which can be used to tailor the optical response of such arrays. In this article, we report on the fabrication and characterization of well-defined Fe–Au bisegmented nanorod arrays in a repeating hexagonal arrangement. Through an asymmetric etching method, free-standing Fe–Au nanorod arrays on a gold-coated substrate were produced with an inter-rod spacing of 26 nm. This separation distance renders the array capable of sustaining resonant electromagnetic wave coupling between individual rods. Owing to this coupling, the subwavelength arrangement, and the structural heterogeneity, the nanorod arrays exhibit unique plasmonic responses in the near-infrared (NIR) range. Enhanced sensitivity in this spectral region has not been identified for gold-only nanorods of equivalent dimensions. The NIR response offers confirmation of the potential of these highly ordered, high-density arrays for biomedical relevant applications, such as subcutaneous spectroscopy and biosensing.

Flow-Enhanced Nonlinear Magnetophoresis for High-Resolution Bioseparation

Co-reporter:Peng Li, Aamer Mahmood, and Gil U. Lee

Langmuir 2011 Volume 27(Issue 10) pp:6496-6503

Publication Date(Web):April 20, 2011

DOI:10.1021/la105126n

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, ω, until they reached an immobilization frequency, ωi, where the beads stopped moving. Laminar flow along the edge of the array could be tuned to sweep the beads for which ω < ωi downstream at a velocity that increased with size while leaving beads for which ω > ωi 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.

Magnetic Manipulation and Optical Imaging of an Active Plasmonic Single-Particle Fe–Au Nanorod

Co-reporter:Yong Zhang, Manuel DaSilva, Brian Ashall, Gillian Doyle, Dominic Zerulla, Timothy D. Sands, and Gil U. Lee

Langmuir 2011 Volume 27(Issue 24) pp:15292-15298

Publication Date(Web):November 2, 2011

DOI:10.1021/la203863p

Superparamagnetic microbeads play an important role in a number of scientific and biotechnology applications including single-molecule force measurements, affinity separation, and in vivo and in vitro diagnostics. Magneto-optically active nanorods composed of single-crystalline Au and polycrystalline Fe segments were synthesized with diameters of 60 or 295 nm using templated electrodeposition. The Fe section was magnetically soft and had a saturation magnetization of approximately 200 emu/g, resulting in a 10-fold increase in magnetization relative to that iron oxide nanoparticles. The strong plasmonic response of the Au segment of the rod in both the longitudinal and transverse directions made it possible to detect the orientation of a single rod in a polarized light microscope with nanometer resolution. These nanorods provide significantly improved physical properties over iron oxide superparamagnetic beads, making it possible to simultaneously manipulate and monitor the orientation of biomolecules with well-defined forces at the nanometer scale.

Single-Molecule Force Spectroscopy of the Aplysia Cell Adhesion Molecule Reveals Two Homophilic Bonds

Co-reporter:E. Martines, J. Zhong, J. Muzard, A.C. Lee, B.B. Akhremitchev, D.M. Suter, G.U. Lee

Biophysical Journal (22 August 2012) Volume 103(Issue 4) pp:

Publication Date(Web):22 August 2012

DOI:10.1016/j.bpj.2012.07.004

Aplysia californica neurons comprise a powerful model system for quantitative analysis of cellular and biophysical properties that are essential for neuronal development and function. The Aplysia cell adhesion molecule (apCAM), a member of the immunoglobulin superfamily of cell adhesion molecules, is present in the growth cone plasma membrane and involved in neurite growth, synapse formation, and synaptic plasticity. apCAM has been considered to be the Aplysia homolog of the vertebrate neural cell adhesion molecule (NCAM); however, whether apCAM exhibits similar binding properties and neuronal functions has not been fully established because of the lack of detailed binding data for the extracellular portion of apCAM. In this work, we used the atomic force microscope to perform single-molecule force spectroscopy of the extracellular region of apCAM and show for the first time (to our knowledge) that apCAM, like NCAM, is indeed a homophilic cell adhesion molecule. Furthermore, like NCAM, apCAM exhibits two distinct bonds in the trans configuration, although the kinetic and structural parameters of the apCAM bonds are quite different from those of NCAM. In summary, these single-molecule analyses further indicate that apCAM and NCAM are species homologs likely performing similar functions.

Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy

Co-reporter:Ying Xiong, Aih Cheun Lee, Daniel M. Suter, Gil U. Lee

Biophysical Journal (17 June 2009) Volume 96(Issue 12) pp:

Publication Date(Web):17 June 2009

DOI:10.1016/j.bpj.2009.03.032

Neuronal growth cones are motile structures located at the end of axons that translate extracellular guidance information into directional movements. Despite the important role of growth cones in neuronal development and regeneration, relatively little is known about the topography and mechanical properties of distinct subcellular growth cone regions under live conditions. In this study, we used the AFM to study the P domain, T zone, and C domain of live Aplysia growth cones. The average height of these regions was calculated from contact mode AFM images to be 183 ± 33, 690 ± 274, and 1322 ± 164 nm, respectively. These findings are consistent with data derived from dynamic mode images of live and contact mode images of fixed growth cones. Nano-indentation measurements indicate that the elastic moduli of the C domain and T zone ruffling region ranged between 3–7 and 7–23 kPa, respectively. The range of the measured elastic modulus of the P domain was 10–40 kPa. High resolution images of the P domain suggest its relatively high elastic modulus results from a dense meshwork of actin filaments in lamellipodia and from actin bundles in the filopodia. The increased mechanical stiffness of the P and T domains is likely important to support and transduce tension that develops during growth cone steering.