34 results on '"Eunkeu Oh"'
Search Results
2. Self-assembled nanoparticle-enzyme aggregates enhance functional protein production in pure transcription-translation systems
- Author
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Meghna Thakur, Joyce C. Breger, Kimihiro Susumu, Eunkeu Oh, Joseph R. Spangler, Igor L. Medintz, Scott A. Walper, and Gregory A. Ellis
- Subjects
Medicine ,Science - Abstract
Cell-free protein synthesis systems (CFPS) utilize cellular transcription and translation (TX-TL) machinery to synthesize proteins in vitro. These systems are useful for multiple applications including production of difficult proteins, as high-throughput tools for genetic circuit screening, and as systems for biosensor development. Though rapidly evolving, CFPS suffer from some disadvantages such as limited reaction rates due to longer diffusion times, significant cost per assay when using commercially sourced materials, and reduced reagent stability over prolonged periods. To address some of these challenges, we conducted a series of proof-of-concept experiments to demonstrate enhancement of CFPS productivity via nanoparticle assembly driven nanoaggregation of its constituent proteins. We combined a commercially available CFPS that utilizes purified polyhistidine-tagged (His-tag) TX-TL machinery with CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) known to readily coordinate His-tagged proteins in an oriented fashion. We show that nanoparticle scaffolding of the CFPS cross-links the QDs into nanoaggregate structures while enhancing the production of functional recombinant super-folder green fluorescent protein and phosphotriesterase, an organophosphate hydrolase; the latter by up to 12-fold. This enhancement, which occurs by an undetermined mechanism, has the potential to improve CFPS in general and specifically CFPS-based biosensors (faster response time) while also enabling rapid detoxification/bioremediation through point-of-concern synthesis of similar catalytic enzymes. We further show that such nanoaggregates improve production in diluted CFPS reactions, which can help to save money and extend the amount of these costly reagents. The results are discussed in the context of what may contribute mechanistically to the enhancement and how this can be applied to other CFPS application scenarios.
- Published
- 2022
3. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.
- Author
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Ying Fu, Juliana da Fonseca Rezende E Mello, Bryan D Fleming, Alex Renn, Catherine Z Chen, Xin Hu, Miao Xu, Kirill Gorshkov, Quinlin Hanson, Wei Zheng, Emily M Lee, Lalith Perera, Robert Petrovich, Manisha Pradhan, Richard T Eastman, Zina Itkin, Thomas B Stanley, Allen Hsu, Venkata Dandey, Kedar Sharma, William Gillette, Troy Taylor, Nitya Ramakrishnan, Shelley Perkins, Dominic Esposito, Eunkeu Oh, Kimihiro Susumu, Mason Wolak, Marc Ferrer, Matthew D Hall, Mario J Borgnia, and Anton Simeonov
- Subjects
Medicine ,Science - Abstract
Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.
- Published
- 2022
- Full Text
- View/download PDF
4. Liquid Crystal Nanoparticle Conjugates for Scavenging Reactive Oxygen Species in Live Cells
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Okhil K. Nag, Jawad Naciri, Kwahun Lee, Eunkeu Oh, Bethany Almeida, and James B. Delehanty
- Subjects
liquid crystal nanoparticles ,ROS scavenger ,oxidative stress ,TEMPO ,lipid peroxidation ,reactive oxygen species ,Medicine ,Pharmacy and materia medica ,RS1-441 - Abstract
The elevated intracellular production of or extracellular exposure to reactive oxygen species (ROS) causes oxidative stress to cells, resulting in deleterious irreversible biomolecular reactions (e.g., lipid peroxidation) and disease progression. The use of low-molecular weight antioxidants, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), as ROS scavengers fails to achieve the desired efficacy because of their poor or uncontrolled cellular uptake and off-target effects, such as dysfunction of essential redox homeostasis. In this study, we fabricated a liquid crystal nanoparticle (LCNP) conjugate system with the fluorescent dye perylene (PY) loaded in the interior and poly (ethylene glycol) (PEG) decorated on the surface along with multiple molecules of TEMPO (PY-LCNP-PEG/TEMPO). PY-LCNP-PEG/TEMPO exhibit enhanced cellular uptake, and efficient ROS-scavenging activity in live cells. On average, the 120 nm diameter PY-LCNPs were conjugated with >1800 molecules of TEMPO moieties on their surface. PY-LCNP-PEG/TEMPO showed significantly greater reduction in ROS activity and lipid peroxidation compared to free TEMPO when the cells were challenged with ROS generating agents, such as hydrogen peroxide (H2O2). We suggest that this is due to the increased local concentration of TEMPO molecules on the surface of the PY-LCNP-PEG/TEMPO NPs, which efficiently bind to the plasma membrane and enter cells. Overall, these results demonstrate the enhanced capability of TEMPO-conjugated LCNPs to protect live cells from oxidative stress by effectively scavenging ROS and reducing lipid peroxidation.
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- 2022
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5. Fabrication of Photoluminescent Quantum Dot Thiol–yne Nanocomposites via Thermal Curing or Photopolymerization
- Author
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Michael H. Stewart, Kimihiro Susumu, Eunkeu Oh, Christopher G. Brown, Collin C. McClain, Edward P. Gorzkowski, and Darryl A. Boyd
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Chemistry ,QD1-999 - Published
- 2018
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- View/download PDF
6. Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions
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Jinny L. Liu, Scott A. Walper, Kendrick B. Turner, Audrey Brozozog Lee, Igor L. Medintz, Kimihiro Susumu, Eunkeu Oh, Dan Zabetakis, Ellen R. Goldman, and George P. Anderson
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Single domain antibodies ,Rhizavidin ,Quantum dots ,Surface plasmon resonance ,Biotechnology ,TP248.13-248.65 - Abstract
Straightforward and effective methods are required for the bioconjugation of proteins to surfaces and particles. Previously we demonstrated that the fusion of a single domain antibody with the biotin binding molecule rhizavidin provided a facile method to coat biotin-modified surfaces with a highly active and oriented antibody. Here, we constructed similar single domain antibody—rhizavidin fusions as well as unfused rhizavidin with a His-tag. The unfused rhizavidin produced efficiently and its utility for assay development was demonstrated in surface plasmon resonance experiments. The single domain antibody-rhizavidin fusions were utilized to coat quantum dots that had been prepared with surface biotins. Preparation of antibody coated quantum dots by this means was found to be both easy and effective. The prepared single domain antibody-quantum dot reagent was characterized by surface plasmon resonance and applied to toxin detection in a fluoroimmunoassay sensing format.
- Published
- 2016
- Full Text
- View/download PDF
7. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly
- Author
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Lauren D. Field, Scott A. Walper, Kimihiro Susumu, Eunkeu Oh, Igor L. Medintz, and James B. Delehanty
- Subjects
fluorescence ,sensor ,FRET ,quantum dot ,protein ,membrane ,assembly ,noncovalent ,ligand ,Chemical technology ,TP1-1185 - Abstract
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing.
- Published
- 2015
- Full Text
- View/download PDF
8. Inhibiting SARS‐CoV‐2 infection with lysosomal alkalizers
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Donald C. Lo, Catherine Chen, Kirill Gorshkov, Mason A. Wolak, Zina Itkin, Carleen Klumpp-Thomas, Eunkeu Oh, Min Shen, Robert Bostwick, Khalida Shamim, Kimihiro Susumu, Anton Simeonov, Samuel G. Michael, Bruce Nguyen Tran, Wei Zhu, Wenwei Huang, Yu-Shan Cheng, Mark J. Henderson, Wei Zheng, Lynn Rasmussen, Paul Shinn, Matthew Hall, Xin Hu, Juan Carlos de la Torre, Manisha Pradhan, and Miao Xu
- Subjects
Pharmacology ,business.industry ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Genetics ,Medicine ,business ,Molecular Biology ,Biochemistry ,Virology ,Pharmacology ‐ COVID‐19 ,Biotechnology - Abstract
Understanding the SARS‐CoV‐2 virus’ routes of infection, virus–host–protein interactions, and mechanisms of virus‐induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID‐19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID‐19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose‐limiting toxicities in patients. To identify an alternative lysosome‐based drug repurposing opportunity we evaluated additional lysosomotropic compounds . We found that six of these compounds blocked the cytopathic effect of SARS‐CoV‐2 in Vero E6 cells with half‐maximal effective concentration (EC50) values ranging from 2.0 to 13 μM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5‐ to >10‐fold. We demonstrate how the compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) that ROC‐325 reduced viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV‐NL63 cytopathic effect in LLC‐MK2 cells. Moreover, an analysis of SARS‐CoV‐2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS‐CoV‐2. Our findings support targeting the lysosome to combat SARS‐CoV‐2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID‐19.
- Published
- 2021
9. Isolation and characterization of Lactobacillus-derived membrane vesicles
- Author
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Scott A. Walper, Eunkeu Oh, Scott N. Dean, Dagmar H. Leary, and Claretta J. Sullivan
- Subjects
0301 basic medicine ,Limosilactobacillus reuteri ,lcsh:Medicine ,Proteomics ,Article ,Biological pathway ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Lactobacillus ,medicine ,lcsh:Science ,Multidisciplinary ,biology ,Bacteria ,Chemistry ,Vesicle ,Probiotics ,lcsh:R ,Membrane Proteins ,food and beverages ,biology.organism_classification ,Subcellular localization ,Lactobacillus acidophilus ,Lacticaseibacillus casei ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,lcsh:Q ,030217 neurology & neurosurgery ,Lactobacillus plantarum - Abstract
Bacterial membrane vesicles have been implicated in a broad range of functions in microbial communities from pathogenesis to gene transfer. Though first thought to be a phenomenon associated with Gram-negative bacteria, vesicle production in Staphylococcus aureus, Lactobacillus plantarum, and other Gram-positives has recently been described. Given that many Lactobacillus species are Generally Regarded as Safe and often employed as probiotics, the engineering of Lactobacillus membrane vesicles presents a new avenue for the development of therapeutics and vaccines. Here we characterize and compare the membrane vesicles (MVs) from three different Lactobacillus species (L. acidophilus ATCC 53544, L. casei ATCC 393, and L. reuteri ATCC 23272), with the aim of developing future strategies for vesicle engineering. We characterize the vesicles from each Lactobacillus species comparing the physiochemical properties and protein composition of each. More than 80 protein components from Lactobacillus-derived MVs were identified, including some that were enriched in the vesicles themselves suggesting vesicles as a vehicle for antimicrobial delivery. Additionally, for each species vesicular proteins were categorized based on biological pathway and examined for subcellular localization signals in an effort to identify possible sorting mechanisms for MV proteins.
- Published
- 2019
10. The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules
- Author
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Mark J. Henderson, Manisha Pradhan, Sam Michael, Miao Xu, Paul Shinn, Zina Itkin, Kirill Gorshkov, Mason A. Wolak, Min Shen, Carleen Klumpp-Thomas, Juan Carlos de la Torre, Robert Bostwick, Lynn Rasmussen, Catherine Z. Chen, Wei Zheng, Wenwei Huang, Yu-Shan Cheng, Matthew D. Hall, Khalida Shamim, Wei Zhu, Donald C. Lo, Kimihiro Susumu, Anton Simeonov, Bruce Nguyen Tran, Xin Hu, and Eunkeu Oh
- Subjects
0301 basic medicine ,autophagy ,Chemistry ,SARS-CoV-2 ,030106 microbiology ,Autophagy ,coronavirus ,Pharmacology ,medicine.disease_cause ,small molecule inhibitors ,Virus ,Article ,cytopathic effect ,03 medical and health sciences ,Drug repositioning ,030104 developmental biology ,medicine.anatomical_structure ,Infectious Diseases ,Chloroquine ,Lysosome ,medicine ,Vero cell ,Coronavirus ,medicine.drug ,Cytopathic effect - Abstract
Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 μM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.
- Published
- 2020
11. Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis
- Author
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Mason A. Wolak, Wei Zhu, Manisha Pradhan, Miao Xu, Eunkeu Oh, Xin Hu, Kimihiro Susumu, Jiji Chen, Joyce C. Breger, and Kirill Gorshkov
- Subjects
Cell ,Pneumonia, Viral ,General Physics and Astronomy ,Metal Nanoparticles ,02 engineering and technology ,Plasma protein binding ,Peptidyl-Dipeptidase A ,010402 general chemistry ,Endocytosis ,01 natural sciences ,Article ,law.invention ,angiotensin converting enzyme 2 ,Betacoronavirus ,law ,Quantum Dots ,medicine ,Humans ,endocytosis ,General Materials Science ,Receptor ,Pandemics ,Quenching (fluorescence) ,receptor binding domain ,Chemistry ,SARS-CoV-2 ,General Engineering ,Virion ,COVID-19 ,quantum dot ,Transfection ,spike ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,medicine.anatomical_structure ,Colloidal gold ,Spike Glycoprotein, Coronavirus ,Biophysics ,Recombinant DNA ,Angiotensin-Converting Enzyme 2 ,Gold ,fluorescence ,0210 nano-technology ,Coronavirus Infections ,hormones, hormone substitutes, and hormone antagonists ,Protein Binding - Abstract
The first step of SARS-CoV-2 infection is binding of the spike protein's receptor binding domain to the host cell's ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.
- Published
- 2020
12. A Multiparametric Evaluation of Quantum Dot Size and Surface-Grafted Peptide Density on Cellular Uptake and Cytotoxicity
- Author
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Bella B. Manshian, Christy Maksoudian, Kimihiro Susumu, Stefaan J. Soenen, Igor L. Medintz, and Eunkeu Oh
- Subjects
Chemistry, Multidisciplinary ,Pharmaceutical Science ,Nanoparticle ,02 engineering and technology ,Cell-Penetrating Peptides ,01 natural sciences ,Mice ,BIODISTRIBUTION ,Materials Testing ,Cytotoxicity ,INTRACELLULAR DELIVERY ,chemistry.chemical_classification ,DAMAGE ,Chemistry ,LOCALIZATION ,021001 nanoscience & nanotechnology ,Mitochondria ,Protein Transport ,Colloidal gold ,Physical Sciences ,Nanomedicine ,tat Gene Products, Human Immunodeficiency Virus ,0210 nano-technology ,Life Sciences & Biomedicine ,Biotechnology ,Biodistribution ,Biochemistry & Molecular Biology ,Biocompatibility ,Cell Survival ,Surface Properties ,Biomedical Engineering ,Chemistry, Organic ,Bioengineering ,Biochemical Research Methods ,NANOMEDICINE ,Cell Line ,CADMIUM ,Quantum Dots ,Autophagy ,Animals ,Particle Size ,Pharmacology ,Reactive oxygen species ,Science & Technology ,010405 organic chemistry ,Organic Chemistry ,IN-VITRO ,0104 chemical sciences ,Quantum dot ,GOLD NANOPARTICLES ,CELLS ,Biophysics ,PENETRATING PEPTIDES ,Reactive Oxygen Species - Abstract
Despite the progress in nanotechnology for biomedical applications, great efforts are still being employed in optimizing nanoparticle (NP) design parameters to improve functionality and minimize bionanotoxicity. In this study, we developed CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) that are compact ligand-coated and surface-functionalized with an HIV-1-derived TAT cell-penetrating peptide (CPP) analog to improve both biocompatibility and cellular uptake. Multiparametric studies were performed in different mammalian and murine cell lines to compare the effects of varying QD size and number of surface CPPs on cellular uptake, viability, generation of reactive oxygen species, mitochondrial health, cell area, and autophagy. Our results showed that the number of cell-associated NPs and their respective toxicity are higher for the larger QDs. Meanwhile, increasing the number of surface CPPs also enhanced cellular uptake and induced cytotoxicity through the generation of mitoROS and autophagy. Thus, here we report the optimal size and surface CPP combinations for improved QD cellular uptake. ispartof: BIOCONJUGATE CHEMISTRY vol:31 issue:4 pages:1077-1087 ispartof: location:United States status: published
- Published
- 2020
13. Water-soluble, thermostable, photomodulated color-switching quantum dots
- Author
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Thomas M. Jovin, Igor L. Medintz, Florencia Gillanders, Kimihiro Susumu, Sebastián A. Díaz, and Eunkeu Oh
- Subjects
Light ,Ultraviolet Rays ,Analytical chemistry ,02 engineering and technology ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Catalysis ,Spectral line ,Isomerism ,Quantum Dots ,Fluorescence Resonance Energy Transfer ,Irradiation ,Absorption (electromagnetic radiation) ,Fluorescent Dyes ,Chemistry ,Organic Chemistry ,Water ,General Chemistry ,021001 nanoscience & nanotechnology ,Fluorescence ,Acceptor ,0104 chemical sciences ,Förster resonance energy transfer ,Indenes ,Solubility ,Quantum dot ,0210 nano-technology ,Visible spectrum - Abstract
Photoswitchable probes are of great utility in fluorescence microscopy, permitting numerous determinations, including molecular localization for super-resolution, based on their modifiable emission intensity and spectra. We have coated a blue (425 nm) emitting quantum dot (QD) with a diheteroarylethene photochrome (PCf), the closed form isomer of which has absorption and emission maxima at 440 and 520-530 nm, respectively, and thus functions as a fluorescent acceptor for the QD donor in Förster resonance energy transfer (FRET). The transition from the non-absorbing, non-fluorescent open state to the fluorescent closed state is achieved by irradiation in the near-UV and reversed by visible light. The PCf is coupled to an amphiphilic polymer that stably coats the QD, thereby creating a water soluble color switching QD (csQD) emitting in the blue after visible light irradiation and in the green after UV irradiation. Thus, csQDs photomodulate between two observable states, i.e. without the "off" state of previous constructs. The resulting change in the emission ratios of the QD and PCf is up to 180% and the csQD can undergo multiple photocycles with minimal fatigue.
- Published
- 2017
14. Energy Transfer Sensitization of Luminescent Gold Nanoclusters: More than Just the Classical Förster Mechanism
- Author
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Marc Currie, Ramasis Goswami, Igor L. Medintz, Alan L. Huston, Alexander L. Efros, Eunkeu Oh, Fredrik K. Fatemi, Kimihiro Susumu, Andrew Shabaev, and Konrad Bussmann
- Subjects
Multidisciplinary ,Quenching (fluorescence) ,Materials science ,Dephasing ,02 engineering and technology ,010402 general chemistry ,021001 nanoscience & nanotechnology ,01 natural sciences ,Acceptor ,Article ,0104 chemical sciences ,Nanoclusters ,Intersystem crossing ,Förster resonance energy transfer ,Nanocrystal ,Chemical physics ,0210 nano-technology ,Luminescence - Abstract
Luminescent gold nanocrystals (AuNCs) are a recently-developed material with potential optic, electronic and biological applications. They also demonstrate energy transfer (ET) acceptor/sensitization properties which have been ascribed to Förster resonance energy transfer (FRET) and, to a lesser extent, nanosurface energy transfer (NSET). Here, we investigate AuNC acceptor interactions with three structurally/functionally-distinct donor classes including organic dyes, metal chelates and semiconductor quantum dots (QDs). Donor quenching was observed for every donor-acceptor pair although AuNC sensitization was only observed from metal-chelates and QDs. FRET theory dramatically underestimated the observed energy transfer while NSET-based damping models provided better fits but could not reproduce the experimental data. We consider additional factors including AuNC magnetic dipoles, density of excited-states, dephasing time, and enhanced intersystem crossing that can also influence ET. Cumulatively, data suggests that AuNC sensitization is not by classical FRET or NSET and we provide a simplified distance-independent ET model to fit such experimental data.
- Published
- 2016
- Full Text
- View/download PDF
15. Influence of luminescence quantum yield, surface coating, and functionalization of quantum dots on the sensitivity of time-resolved FRET bioassays
- Author
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Nicolas Lequeux, Emerson Giovanelli, Thomas Pons, Simon M. Fairclough, Travis L. Jennings, Vaibhav Jain, Phung Thi Lanh, Niko Hildebrandt, K. David Wegner, Jason M. Smith, and Eunkeu Oh
- Subjects
Materials science ,Luminescence ,chemistry.chemical_element ,Terbium ,Nanotechnology ,02 engineering and technology ,Luminescence quantum yield ,engineering.material ,010402 general chemistry ,01 natural sciences ,Sensitivity and Specificity ,Coating ,Quantum Dots ,Fluorescence Resonance Energy Transfer ,General Materials Science ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Surface coating ,Förster resonance energy transfer ,chemistry ,Quantum dot ,engineering ,Surface modification ,Nanoparticles ,Biological Assay ,0210 nano-technology ,Europium - Abstract
In clinical diagnostics, homogeneous time-resolved (TR) FRET immunoassays are used for fast and highly sensitive detection of biomarkers in serum samples. The most common immunoassay format is based on europium chelate or cryptate donors and allophycocyanin acceptors. Replacing europium donors with terbium complexes and the acceptors with QDs offers large photophysical advantages for multiplexed diagnostics, because the Tb-complex can be used as FRET donor for QD acceptors of different colors. Water-soluble and biocompatible QDs are commercially available or can be synthesized in the laboratory using many available recipes from the literature. Apart from the semiconductor material composition, an important aspect of choosing the right QD for TR-FRET assays is the thickness of the QD coating, which will influence the photophysical properties and long-term stability as well as the donor-acceptor distance and FRET efficiency. Here we present a detailed time-resolved spectroscopic study of three different QDs with an emission maximum around 605 nm for their application as FRET acceptors (using a common Tb donor) in TR-bioassays: (i) Invitrogen/Life Technologies Qdot605, (ii) eBioscience eFluorNC605 and iii) ter-polymer stabilized CdSe/CdS/ZnS QDs synthesized in our laboratories. All FRET systems are very stable and possess large Förster distances (7.4-9.1 nm), high FRET efficiencies (0.63-0.80) and low detection limits (0.06-2.0 pM) within the FRET-bioassays. Shapes, sizes and the biotin/QD ratio of the biocompatible QDs could be determined directly in the solution phase bioassays at subnanomolar concentrations. Both commercial amphiphilic polymer/lipid encapsulated QDs and self-made ligand-exchanged QDs provide extremely low detection limits for highly sensitive TR-FRET bioassays.
- Published
- 2016
16. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly
- Author
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Igor L. Medintz, Scott A. Walper, Lauren D. Field, Kimihiro Susumu, James B. Delehanty, and Eunkeu Oh
- Subjects
assembly ,Recombinant Fusion Proteins ,Nanotechnology ,lcsh:Chemical technology ,Ligands ,ligand ,Biochemistry ,Theranostic Nanomedicine ,Article ,Analytical Chemistry ,sensor ,Chlorocebus aethiops ,Quantum Dots ,Fluorescence Resonance Energy Transfer ,Animals ,lcsh:TP1-1185 ,Histidine ,Electrical and Electronic Engineering ,Instrumentation ,membrane ,Chemistry ,Ligand ,quantum dot ,fluorescence ,FRET ,protein ,noncovalent ,Fluorescence ,Acceptor ,Atomic and Molecular Physics, and Optics ,Luminescent Proteins ,Membrane ,Förster resonance energy transfer ,Quantum dot ,Molecular Probes ,COS Cells ,Biophysics ,mCherry ,Intracellular - Abstract
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing.
- Published
- 2015
- Full Text
- View/download PDF
17. Inhibition assay of biomolecules based on fluorescence resonance energy transfer (FRET) between quantum dots and gold nanoparticlest4
- Author
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Eunkeu Oh, Mi-Young Hong, Dohoon Lee, Sung-Hun Nam, Hyun C. Yoon, and Hak-Sung Kim
- Subjects
Photoluminescence -- Research ,Quantum theory -- Research ,Gold compounds -- Spectra ,Energy transformation -- Research ,Chemistry - Abstract
An inhibition assay method based on the modulation in fluorescence resonance energy transfer (FRET) efficiency between quantum dots (QDs) and gold nanoparticles (AuNPs) in the presence of the molecules which inhibit the interactions between QD- and AuNP conjugated biomolecules is reported. The externally added avidin specifically bound to the surface of biotin-AuNPs resulted in reduction of the photoluminescence (PL) quenching of SA-QDs.
- Published
- 2005
18. Probing the kinetics of quantum dot-based proteolytic sensors
- Author
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Kimihiro Susumu, Eunkeu Oh, Igor L. Medintz, Sebastián A. Díaz, Romina V. Hofele, and Anthony P. Malonoski
- Subjects
medicine.diagnostic_test ,Chemistry ,Proteolysis ,Elastase ,Proteolytic enzymes ,Substrate (chemistry) ,Biosensing Techniques ,Biochemistry ,Michaelis–Menten kinetics ,Analytical Chemistry ,Kinetics ,Förster resonance energy transfer ,Spectrometry, Fluorescence ,Yield (chemistry) ,Quantum Dots ,medicine ,Biophysics ,Enzyme kinetics - Abstract
As an enzyme superfamily, proteases are rivaled only by kinases in terms of their abundance within the human genome. Two ratiometric quantum dot (QD) Forster resonance energy transfer-based sensors designed to monitor the activity of the proteolytic enzymes collagenase and elastase are investigated here. Given the unique material constraints of these sensing constructs, assays are realized utilizing excess enzyme and fixed substrate in progress curve format to yield enzyme specificity or k cat/K m ratios. The range of k cat/Km values derived is 0.5–1.1 mM−1 s−1 for the collagenase sensor and 3.7–4.2 mM−1 s−1 for the elastase sensor. Of greater interest is the observation that the elastase sensor can be well represented by the Michaelis-Menten model while the collagenase sensor cannot. The latter demonstrates increased specificity at higher peptide substrate/QD loading values and an apparent QD-caused reversible inhibition as the reaction progresses. Understanding the detailed kinetic mechanisms that underpin these types of sensors will be important especially for their further quantitative utilization.
- Published
- 2015
19. Exploring attachment chemistry with FRET in hybrid quantum dot dye-labeled DNA dendrimer composites.
- Author
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Samanta, Anirban, Buckhout-White, Susan, Eunkeu Oh, Kimihiro Susumu, and Medintz, Igor L.
- Published
- 2018
- Full Text
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20. Intracellularly Actuated Quantum Dot-Peptide-Doxorubicin Nanobioconjugates for Controlled Drug Delivery via the Endocytic Pathway.
- Author
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Sangtani, Ajmeeta, Petryayeva, Eleonora, Miao Wu, Susumu, Kimihiro, Eunkeu Oh, Huston, Alan L., Lasarte-Aragones, Guillermo, Medintz, Igor L., Algar, W. Russ, and Delehanty, James B.
- Published
- 2018
- Full Text
- View/download PDF
21. Purple-, Blue-, and Green-Emitting Multishell Alloyed Quantum Dots: Synthesis, Characterization, and Application for Ratiometric Extracellular pH Sensing.
- Author
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Kimihiro Susumu, Field, Lauren D., Eunkeu Oh, Hunt, Michael, Delehanty, James B., Palomo, Valle, Dawson, Philip E., Huston, Alan L., and Medintz, Igor L.
- Published
- 2017
- Full Text
- View/download PDF
22. Concurrent Modulation of Quantum Dot Photoluminescence Using a Combination of Charge Transfer and Förster Resonance Energy Transfer: Competitive Quenching and Multiplexed Biosensing Modality.
- Author
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Algar, W. Russ, Khachatrian, Ani, Melinger, Joseph S., Huston, Alan L., Stewart, Michael H., Kimihiro Susumu, Blanco-Canosa, Juan B., Eunkeu Oh, Dawson, Philip E., and Medintz, Igor L.
- Published
- 2017
- Full Text
- View/download PDF
23. Synthesis and Characterization of PEGylated Luminescent Gold Nanoclusters Doped with Silver and Other Metals.
- Author
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Eunkeu Oh, Delehanty, James B., Field, Lauren D., Mäkinen, Antti J., Goswami, Ramasis, Huston, Alan L., and Medintz, Igor L.
- Subjects
- *
GOLD nanoparticle synthesis , *POLYETHYLENE glycol , *PHOTOLUMINESCENCE , *QUANTUM states , *SILVER nanoparticles , *DOPED semiconductors - Abstract
Doping of fluorescent noble metal nanoclusters is being pursued to manipulate the structure of such materials along with improving physicochemical characteristics such as long-term stability and photoluminescence quantum yield. Here, we synthesize metal-doped and alloyed ultrasmall gold nanoclusters (AuNCs) directly in water using a facile one-step coreduction reaction with bidentate dithiolane PEGylated ligands that terminate in different functional groups including a methoxy, carboxy, amine, and azide. Two primary types of cluster materials were the focus of synthesis and characterization: first, a series of doped/alloyed Ag-doped AuNCs, where the ratio of Au:Ag was varied across a wide range including 99:1, 98:2, 90:10, 80:20, 50:50, 20:80, 10:90, and 2:98 along with pure AuNC and AgNC controls; second, doped Au:D NCs, where D included Pt, Cu, Zn, and Cd. Physical characterization of the modified AuNCs included TEM analysis of size, XPS/EDX analysis of dopant content, and a detailed analysis of photophysical properties including absorption and photoluminescence profiles, quantum yields over time, photoluminescence lifetimes, and examination of energy levels for selected materials. The addition of just a few Ag dopant atoms per AuNC yielded significant enhancement in quantum yield along with improving long-term photostability especially in comparison to materials with a very high Ag content. Preliminary cell imaging applications of the Ag-doped AuNCs were also investigated. Facilitated cellular uptake by mammalian cells via endocytosis following modification with cell penetrating peptides was confirmed by colabeling with specific cellular markers. Long-term intracellular photostability and lack of aggregation were confirmed with microinjection studies, and cytoviability assays showed the doped clusters to be minimally toxic. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
24. Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor-Acceptor Assembly.
- Author
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Field, Lauren D., Walper, Scott A., Kimihiro Susumu, Eunkeu Oh, Medintz, Igor L., and Delehanty, James B.
- Subjects
QUANTUM dots ,FLUORESCENT proteins ,FLUORESCENCE resonance energy transfer ,LIGANDS (Chemistry) ,ELECTRON donor-acceptor complexes ,ZWITTERIONS - Abstract
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor-acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
25. Electric Field Modulation of Semiconductor QuantumDot Photoluminescence: Insights Into the Design of Robust Voltage-SensitiveCellular Imaging Probes.
- Author
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Clare E. Rowland, Kimihiro Susumu, MichaelH. Stewart, Eunkeu Oh, Antti J. Mäkinen, Thomas J. O’Shaughnessy, Gary Kushto, Mason A. Wolak, Jeffrey S. Erickson, Alexander L. Efros, Alan L. Huston, and James B. Delehanty
- Published
- 2015
- Full Text
- View/download PDF
26. Chemoenzymatic Sensitization of DNA Photonic WiresMediated through Quantum Dot Energy Transfer Relays.
- Author
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ChrisL. Dwyer, Sebastián A. Díaz, Scott A. Walper, Anirban Samanta, Kimihiro Susumu, Eunkeu Oh, Susan Buckhout-White, and Igor L. Medintz
- Published
- 2015
- Full Text
- View/download PDF
27. One-Phase Synthesis of Water-Soluble Gold Nanoparticles with Control over Size and Surface Functionalities.
- Author
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Eunkeu Oh, Kimihiro Susumu, Ramasis Goswami, and Hedi Mattoussi
- Subjects
- *
SURFACES (Technology) , *WATER , *COLLOIDAL gold , *CHLORIDES , *POLYETHYLENE glycol , *PARTICLE size distribution , *NANOCRYSTALS , *LIGANDS (Chemistry) - Abstract
We report a simple and efficient synthetic method to prepare gold nanoparticles (AuNPs) in aqueous phase using HAuCl4and poly(ethylene glycol) (PEG) ligands appended with bidentate anchoring groups. Our approach provides narrow size distribution nanocrystals over the size range between 1.5 and 18 nm; this range is much wider than those achieved using other small molecules and polymer ligands. The NP size was simply controlled by varying the molar ratio of Au-to-PEG ligand precursors. Further passivation of the as-prepared AuNPs permitted in situfunctionalization of the NP surface with the desired functional groups. The prepared AuNPs exhibit remarkable stability in the presence of high salt concentrations, over a wide range of pHs (2â13), and a strong resistance to competition from dithiothreitol (DTT). These results are a clear manifestation of the advantages offered by our synthetic approach to prepare biocompatible AuNPs, where modular, multifunctional ligands presenting strong anchoring groups and hydrophilic PEG chains are used. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
28. Energy Transfer-Based Multiplexed Assay of Proteases by Using Gold Nanoparticle and Quantum Dot Conjugates on a Surface.
- Author
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Young-Pilu Kim, Young-Hee Oh, Eunkeu Oh, Sungho Ko, Min-Kyu Han, and Hak-Sung Kim
- Published
- 2008
- Full Text
- View/download PDF
29. Gold Nanoparticle-Enhanced Secondary Ion Mass Spectrometry Imaging of Peptides on Self-Assembled Monolayers.
- Author
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Young-Pil Kim, Eunkeu Oh, Mi-Young Hong, Dohoon Lee, Min-Kyu Han, Hyun Kyong Shon, Dae Won Moon, Hak-Sung Kim, and Tae Geol Lee
- Subjects
- *
MONOMOLECULAR films , *NANOPARTICLES , *SECONDARY ion mass spectrometry , *QUARTZ crystal microbalances , *SURFACE plasmon resonance , *MOLECULAR self-assembly , *ADSORPTION (Chemistry) , *MICROFLUIDICS , *PEPTIDES - Abstract
We demonstrate the use of gold nanoparticles (AuNPs) to enhance the secondary ion emission of peptides in time-of-flight secondary ion mass spectrometry (TOF-SIMS). The signal intensity of peptides adsorbed onto AuNPs was significantly increased when compared to that of self-assembled monolayers (SAMs). This gold nanoparticle-enhanced SIMS, termed NE-SIMS, enabled the sensitive detection of subtle modifications of peptides, such as phosphorylation. From a quantitative analysis of the amounts of adsorbed peptides and AuNPs on SAMs using quartz crystal microbalance and surface plasmon resonance spectroscopy, the ratio of peptide molecule to AuNP on amine-SAMs was revealed to be 18–19:1. When considering the ratio of peptide to matrix (1:10³–106) employed in a matrix-enhanced SIMS, the use of AuNPs gave rise to a significantly increased secondary ion emission of peptides. Peptides were adsorbed onto patterned AuNPs on SAMs using a microfluidic system, and well-contrasted molecular ion images were obtained. NE-SIMS is expected to be applied to a chip-based analysis of modification of biomolecules in a label-free manner. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
30. Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures.
- Author
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Joyce C Breger, Susan Buckhout-White, Scott A Walper, Eunkeu Oh, Kimihiro Susumu, Mario G Ancona, and Igor L Medintz
- Published
- 2017
- Full Text
- View/download PDF
31. Preparation of a Magnetically Switchable Bio-electrocatalytic System Employing Cross-linked Enzyme Aggregates in Magnetic Mesocellular Carbon Foam.
- Author
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Jinwoo Lee, Dohoon Lee, Eunkeu Oh, Jaeyun Kim, Young-Pil Kim, Sunmi Jin, Hak-Sung Kim, Yosun Hwang, Ja Hun Kwak, Je-Geun Park, Chae-Ho Shin, Jungbae Kim, and Taeghwan Hyeon
- Published
- 2005
- Full Text
- View/download PDF
32. Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application.
- Author
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Zheng Wang, Tanya Tschirhart, Schultzhaus, Zachary, Kelly, Erin E., Amy Chen, Eunkeu Oh, Nag, Okhil, Glaser, Evan R., Eunkyoung Kim, Lloyd, Pamela F., Charles, Paul T., Weiyao Li, Leary, Dagmar, Compton, Jaimee, Phillips, Daniel A., Dhinojwala, Ali, Payne, Gregory F., and Vora, Gary J.
- Subjects
- *
MELANINS , *MARINE bacteria , *BIOSYNTHESIS , *VIBRIO , *VIBRIO harveyi , *MARINE engineering , *REVERSE engineering - Abstract
Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the largescale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as "melanin ghost" structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens. Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
33. Magnetophoretic Immunoassay of Allergen-Specific IgE in an Enhanced Magnetic Field Gradient.
- Author
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Young Ki Hahn, Zongwen Jin, Kang, Joo H., Eunkeu Oh, Min-Kyu Han, Hak-Sung Kim, Jung-Tak Jang, Jae-Hyun Lee, Jinwoo Cheon, Seung Hyun Kim, Hae-Sim Park, and Je-Kyun Park
- Subjects
- *
IMMUNOASSAY , *ALLERGENS , *ANTIGENS , *BLOOD plasma , *BLOOD proteins , *IMMUNE serums , *BIOLOGY , *CHEMISTRY , *BIOCHEMISTRY , *SERUM - Abstract
We demonstrate a novel magnetophoretic immunoassay of allergen-specific immunoglobulin E (IgE) based on the magnetophoretic deflection velocity of a microbead that is proportional to the associated magnetic nanoparticles under enhanced magnetic field gradient in a microchannel. In this detection scheme, two types of house dust mites, Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus), were used as the model allergens. Polystyrene microbeads were conjugated with each of the mite extracts followed by incubation with serum samples. The resulting mixture was then reacted with magnetic nanoparticle-conjugated anti-human IgE for detection of allergen-specific IgE by using sandwich immuno-reactions. A ferromagnetic microstructure combined with a permanent magnet was employed to increase the magnetic field gradient (∼104 T/m) in a microfluidic device. The magnetophoretic velocities of microbeads were measured in a microchannel under applied magnetic field, and the averaged velocity was well correlated with the concentration of allergen-specific IgE in serum. From the analysis of pooled sera obtained from 44 patients, the detection limits of the allergen-specific human IgEs for D. farinae and D. pteronyssinus were determined to be 565 (0.045 IU/mL) and 268 fM (0.021 IU/mL), respectively. These values are 1 order of magnitude lower than those by a conventional CAP system. For evaluation of reproducibility and accuracy, unknown sera were subjected to a blind test by using the developed assay system, and they were compared with the CAP system. As a result, coefficient of variance was less than 10%, and the developed method enabled a fast assay with a tiny amount of serum (∼10 μL). [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
34. Quantitative Analysis of Surface-Immobilized Protein by TOF-SIMS: Effects of Protein Orientation and Trehalose Additive.
- Author
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Young-Pu Kim, Mi-Young Hong, Jinmo Kim, Eunkeu Oh, Hyun Kyong Shon, Dae Won Moon, Hak-Sung Kim, and Tae Geol Lee
- Subjects
- *
IMMOBILIZED proteins , *SPECTRUM analysis , *MEDICAL equipment , *DENATURATION of proteins , *IONS , *MASS spectrometry , *BACTERIAL proteins , *STREPTAVIDIN , *BIOSENSORS - Abstract
We demonstrate the effects of protein orientation and trehalose on a quantitative analysis of surface-immobilized proteins by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As our model protein, streptavidin (SA) was quantitatively immobilized on a solid surface at different configurations by random or oriented immobilization and subsequently treated with trehalose. The resulting surface was analyzed by using TOF-SIMS and surface plasmon resonance (SPR) spectroscopy, where the secondary ion spectra from SA were compared with the surface density of the protein. In the case of oriented immobilization, the ion peak intensities measured by TOF-SIMS were correlated well with the SPR data, regardless of the presence of trehalose. Alternatively, trehalose significantly increased correlation between TOF-SIMS and SPR data for the randomly immobilized SA. It is likely that a trehalose-treated surface is less vulnerable to denaturation, thus leading to a reliable quantification of surface immobilized proteins by TOF-SIMS. Our results show that TOF-SIMS can be used for understanding biophysical states such as orientation and denaturation of surface immobilized proteins as well as for quantifying proteins within the field of biosensors and biochips. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
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