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Towards a “Green” Antimicrobial Therapy: Study of Graphene Nanosheets Interaction with Human Pathogens
530a
Wednesday, March 2, 2016
process with multiple discrete steps. Future investigations will focus on whether the principles derived here can be used to design functional proteinbased nanoconjugates. 2609-Plat Biointeractions of Ultrasmall Gold Nanoparticles: Influence of Nanoparticle Size and Surface Chemistry Luiza L. Knittel1, Sergio A. Hassan2, Maria A. Aronova3, Peter Schuck3, Richard D. Leapman3, Alioscka A. Sousa1. 1 Biochemistry, UNIFESP, Sao Paulo, Brazil, 2CIT, National Institutes of Health, Bethesda, MD, USA, 3NIBIB, National Institutes of Health, Bethesda, MD, USA. Recent studies have shown that gold nanoparticles (AuNPs) in the ultrasmall size regime (< 3 nm in diameter) can present distinct advantages for applications in nanomedicine, such as an efficient renal clearance in vivo. Herein we perform a systematic investigation of the effects of size and surface chemistry on the in vitro biointeractions of ultrasmall AuNPs. Nanoparticle surface chemistry was modulated using small peptides as passivating ligands; size and uniformity were carefully characterized with dark-field scanning transmission electron microscopy (STEM) and analytical ultracentrifugation; and nanoparticle aggregation and serum protein adsorption were evaluated both experimentally by analytical ultracentrifugation as well as computationally by molecular dynamics simulations. First, our results showed that the colloidal stability of ultrasmall AuNPs in biological media can depend on seemingly small variations in core diameter. Our investigations also established how the surface chemistry could be tuned to produce ultrasmall AuNPs highly resistant to aggregation and adsorption from serum proteins. 2610-Plat Towards a ‘‘Green’’ Antimicrobial Therapy: Study of Graphene Nanosheets Interaction with Human Pathogens Valentina Palmieri1,2, Massimiliano Papi1,2, Francesca Bugli1, Mariacarmela Lauriola1, Claudio Conti2, Gabriele Ciasca1, Giuseppe Maulucci1, Maurizio Sanguinetti1, Marco De Spirito1. 1 Universita` Cattolica del Sacro Cuore, Roma, Italy, 2Institute for Complex Systems (ISC-CNR), Roma, Italy. Graphene and its derivatives are versatile materials promising candidates for important biomedical applications. Graphene is composed of sp2-hybridized carbon atoms hexagonally arranged in a two-dimensional structure, resulting in a large surface area on both sides of the planar axis. Among the materials of the graphene family, graphene oxide (GO), because of the formation of hydrogen bonds between polar functional groups on its surface and water molecules, is stable in solution and has more advantages for potential biomedical applications. However studies concerning antimicrobial effects of GO evidenced contradictory results and toxic and nontoxic effects were simultaneously observed. Goal of this work is the precise analysis of size, buffer and concentration dependence of GO antimicrobial effects on important human pathogens, such as E. Coli, C. Albicans, E. Faecalis and S. Aureus. Different sizes of GO nanosheets have been produced by sonication and characterized by Dynamic Light Scattering and Atomic Force Microscopy. Subsequently GO sheets having size comprised between 1 mm and 50 nm have been incubated with pathogens in ddH20, saline solution or in LB Broth, i.e. in different ionic strength conditions, to follow the growth kinetics in presence of GO. We analysed in detail how the GO aggregation influences experimental results and optimized an experimental protocol and analysis method. Moreover, since high GO concentration caused the formation of complexes between pathogens and GO nanosheets, we characterized the surface covering of the bacteria and the leakage of genetic material, membrane damaging and biofilm formation. These results allow the optimization GO-based nanomaterial design for a new ‘‘green’’ antimicrobial therapy. 2611-Plat Tracking Single-Particle Rotation during Macrophage Uptake Lucero Sanchez, Yan Yu. Chemistry, Indiana University, Bloomington, IN, USA. Phagocytosis is a cell function used by immune cells, such as macrophages, to engulf and degrade foreign pathogens and particles. It is also the cellular process that leads to the failure of many drug- or gene-delivery particles; the drug carriers are cleared by immune cells before reaching their intended destination. Therefore, understanding phagocytosis of particles has significant implications in both fundamental understanding and biomedical engineering. In this presentation, I will show our progress in developing novel
quantitative methods to probe dynamics and mechanism of phagocytosis. By using particles that display two fluorescent patches on opposing poles as rotational probes, we investigated the rotational dynamics of single microparticles during their internalization by macrophage cells. We show that particles exhibit a mixture of fast and slow rotation and transiently undergo directional rotation as they are internalized by macrophages. Results showing the effect of the particle size and the surface presentation of ligands will also be presented. 2612-Plat Peptide Translocation through Single Lysenin Channels Nisha Shrestha1, Sheenah Bryant1, Xinzhu Pu2, Paul Carnig3, Juliette Tinker4, Charles Hanna3, Daniel Fologea5. 1 Biomolecular Sciences Ph.D. Program, Boise State University, Boise, ID, USA, 2Biomolecular Research Center, Boise State University, Boise, ID, USA, 3Department of Physics, Boise State University, Boise, ID, USA, 4 Biomolecular Sciences Ph.D. Program/Department of Biology, Boise State University, Boise, ID, USA, 5Biomolecular Sciences Ph.D. Program/ Department of Physics, Boise State University, Boise, ID, USA. Nano-sized pore forming proteins (PFPs) inserted into lipid membranes are excellent tools for single molecule detection and characterization. However, difficult insertion, reduced transport capabilities, and undesired interactions with analytes are among the major reasons for which only several PFPs are currently studied for nanobiotechnology applications. In order to overcome these inherent limitations, scientists are actively looking for alternative PFPs that will allow expanding their use for biosensing, sequencing, and diagnostic. To address this problem, our work focused on investigating lysenin channels as stochastic sensors for peptide molecules. Lysenin, a 297 amino acid pore forming toxin extracted from the earthworm E. foetida self-inserts large conductance pores (~3 nm diameter) in artificial and natural lipid membranes containing sphingomyelin. The channel’s larger opening might accommodate the electrophoretical-driven passage of larger analytes thus their translocation may be electronically detected by using the long-revered Coulter approach. In the present study, we investigated translocation of angiotensin II, a short positively charged peptide, through single lysenin channels inserted into planar lipid bilayer membranes. Our data show that the average current blockage and dwell time recorded during translocation depends on the magnitude of the applied transmembrane voltage, consistent with previous studies employing either synthetic or biological nanopores. In addition, mass spectroscopy analysis of angiotensin II translocated for extended time through large populations of inserted lysenin channels indicated that successful translocation is conditioned by both the presence of channels and proper electric field orientation. These results warrant further studies on using lysenin channels for single molecule detection and characterization with a special emphasis on peptide molecules. 2613-Plat Nanopore Zero-Mode Waveguides for DNA Sequencing and Beyond Joseph Larkin1, Robert Y. Henley1, Jonas Korlach2, Meni Wanunu1. 1 Physics, Northeastern University, Boston, MA, USA, 2Pacific Biosciences, Menlo Park, CA, USA. Single-molecule techniques are becoming more practical and useful in academic research and commercial healthcare devices. A prime example of this is single-molecule DNA sequencing, which is now achievable using either nanopores or single-molecule real time (SMRT) methods. In these platforms, read lengths exceed those of bulk sequencing methods by orders of magnitude, and sensitivity to epigenetic modifications is allowed in cases where DNA amplification is not necessary. However, since sequencing and epigenetic analysis require native DNA fragments, a critical part of accessing this information is loading of the DNA into a readout device with high sensitivity. While typical DNA library input requirements are in the 100’s of ng, ideally one would want to probe single cell amounts, or pg-level DNA. In this work, we have utilized micro and nano fabrication to construct zero-mode waveguides that contain nanopores in their base, and show that these devices can be used for extremely sensitive DNA capture and sequencing. The device, a nanopore-zero-mode waveguide (NZMW), allows sub nanogram levels of DNA to be efficiently drawn for analysis in seconds, and does not exhibit a bias towards lowmolecular weight DNA as in diffusion-based methods. We will discuss the implications of such a device on DNA sequencing, RNA sequencing, as well as other single-molecule biophysical studies where only precious samples are available.
Wednesday, March 2, 2016
process with multiple discrete steps. Future investigations will focus on whether the principles derived here can be used to design functional proteinbased nanoconjugates. 2609-Plat Biointeractions of Ultrasmall Gold Nanoparticles: Influence of Nanoparticle Size and Surface Chemistry Luiza L. Knittel1, Sergio A. Hassan2, Maria A. Aronova3, Peter Schuck3, Richard D. Leapman3, Alioscka A. Sousa1. 1 Biochemistry, UNIFESP, Sao Paulo, Brazil, 2CIT, National Institutes of Health, Bethesda, MD, USA, 3NIBIB, National Institutes of Health, Bethesda, MD, USA. Recent studies have shown that gold nanoparticles (AuNPs) in the ultrasmall size regime (< 3 nm in diameter) can present distinct advantages for applications in nanomedicine, such as an efficient renal clearance in vivo. Herein we perform a systematic investigation of the effects of size and surface chemistry on the in vitro biointeractions of ultrasmall AuNPs. Nanoparticle surface chemistry was modulated using small peptides as passivating ligands; size and uniformity were carefully characterized with dark-field scanning transmission electron microscopy (STEM) and analytical ultracentrifugation; and nanoparticle aggregation and serum protein adsorption were evaluated both experimentally by analytical ultracentrifugation as well as computationally by molecular dynamics simulations. First, our results showed that the colloidal stability of ultrasmall AuNPs in biological media can depend on seemingly small variations in core diameter. Our investigations also established how the surface chemistry could be tuned to produce ultrasmall AuNPs highly resistant to aggregation and adsorption from serum proteins. 2610-Plat Towards a ‘‘Green’’ Antimicrobial Therapy: Study of Graphene Nanosheets Interaction with Human Pathogens Valentina Palmieri1,2, Massimiliano Papi1,2, Francesca Bugli1, Mariacarmela Lauriola1, Claudio Conti2, Gabriele Ciasca1, Giuseppe Maulucci1, Maurizio Sanguinetti1, Marco De Spirito1. 1 Universita` Cattolica del Sacro Cuore, Roma, Italy, 2Institute for Complex Systems (ISC-CNR), Roma, Italy. Graphene and its derivatives are versatile materials promising candidates for important biomedical applications. Graphene is composed of sp2-hybridized carbon atoms hexagonally arranged in a two-dimensional structure, resulting in a large surface area on both sides of the planar axis. Among the materials of the graphene family, graphene oxide (GO), because of the formation of hydrogen bonds between polar functional groups on its surface and water molecules, is stable in solution and has more advantages for potential biomedical applications. However studies concerning antimicrobial effects of GO evidenced contradictory results and toxic and nontoxic effects were simultaneously observed. Goal of this work is the precise analysis of size, buffer and concentration dependence of GO antimicrobial effects on important human pathogens, such as E. Coli, C. Albicans, E. Faecalis and S. Aureus. Different sizes of GO nanosheets have been produced by sonication and characterized by Dynamic Light Scattering and Atomic Force Microscopy. Subsequently GO sheets having size comprised between 1 mm and 50 nm have been incubated with pathogens in ddH20, saline solution or in LB Broth, i.e. in different ionic strength conditions, to follow the growth kinetics in presence of GO. We analysed in detail how the GO aggregation influences experimental results and optimized an experimental protocol and analysis method. Moreover, since high GO concentration caused the formation of complexes between pathogens and GO nanosheets, we characterized the surface covering of the bacteria and the leakage of genetic material, membrane damaging and biofilm formation. These results allow the optimization GO-based nanomaterial design for a new ‘‘green’’ antimicrobial therapy. 2611-Plat Tracking Single-Particle Rotation during Macrophage Uptake Lucero Sanchez, Yan Yu. Chemistry, Indiana University, Bloomington, IN, USA. Phagocytosis is a cell function used by immune cells, such as macrophages, to engulf and degrade foreign pathogens and particles. It is also the cellular process that leads to the failure of many drug- or gene-delivery particles; the drug carriers are cleared by immune cells before reaching their intended destination. Therefore, understanding phagocytosis of particles has significant implications in both fundamental understanding and biomedical engineering. In this presentation, I will show our progress in developing novel
quantitative methods to probe dynamics and mechanism of phagocytosis. By using particles that display two fluorescent patches on opposing poles as rotational probes, we investigated the rotational dynamics of single microparticles during their internalization by macrophage cells. We show that particles exhibit a mixture of fast and slow rotation and transiently undergo directional rotation as they are internalized by macrophages. Results showing the effect of the particle size and the surface presentation of ligands will also be presented. 2612-Plat Peptide Translocation through Single Lysenin Channels Nisha Shrestha1, Sheenah Bryant1, Xinzhu Pu2, Paul Carnig3, Juliette Tinker4, Charles Hanna3, Daniel Fologea5. 1 Biomolecular Sciences Ph.D. Program, Boise State University, Boise, ID, USA, 2Biomolecular Research Center, Boise State University, Boise, ID, USA, 3Department of Physics, Boise State University, Boise, ID, USA, 4 Biomolecular Sciences Ph.D. Program/Department of Biology, Boise State University, Boise, ID, USA, 5Biomolecular Sciences Ph.D. Program/ Department of Physics, Boise State University, Boise, ID, USA. Nano-sized pore forming proteins (PFPs) inserted into lipid membranes are excellent tools for single molecule detection and characterization. However, difficult insertion, reduced transport capabilities, and undesired interactions with analytes are among the major reasons for which only several PFPs are currently studied for nanobiotechnology applications. In order to overcome these inherent limitations, scientists are actively looking for alternative PFPs that will allow expanding their use for biosensing, sequencing, and diagnostic. To address this problem, our work focused on investigating lysenin channels as stochastic sensors for peptide molecules. Lysenin, a 297 amino acid pore forming toxin extracted from the earthworm E. foetida self-inserts large conductance pores (~3 nm diameter) in artificial and natural lipid membranes containing sphingomyelin. The channel’s larger opening might accommodate the electrophoretical-driven passage of larger analytes thus their translocation may be electronically detected by using the long-revered Coulter approach. In the present study, we investigated translocation of angiotensin II, a short positively charged peptide, through single lysenin channels inserted into planar lipid bilayer membranes. Our data show that the average current blockage and dwell time recorded during translocation depends on the magnitude of the applied transmembrane voltage, consistent with previous studies employing either synthetic or biological nanopores. In addition, mass spectroscopy analysis of angiotensin II translocated for extended time through large populations of inserted lysenin channels indicated that successful translocation is conditioned by both the presence of channels and proper electric field orientation. These results warrant further studies on using lysenin channels for single molecule detection and characterization with a special emphasis on peptide molecules. 2613-Plat Nanopore Zero-Mode Waveguides for DNA Sequencing and Beyond Joseph Larkin1, Robert Y. Henley1, Jonas Korlach2, Meni Wanunu1. 1 Physics, Northeastern University, Boston, MA, USA, 2Pacific Biosciences, Menlo Park, CA, USA. Single-molecule techniques are becoming more practical and useful in academic research and commercial healthcare devices. A prime example of this is single-molecule DNA sequencing, which is now achievable using either nanopores or single-molecule real time (SMRT) methods. In these platforms, read lengths exceed those of bulk sequencing methods by orders of magnitude, and sensitivity to epigenetic modifications is allowed in cases where DNA amplification is not necessary. However, since sequencing and epigenetic analysis require native DNA fragments, a critical part of accessing this information is loading of the DNA into a readout device with high sensitivity. While typical DNA library input requirements are in the 100’s of ng, ideally one would want to probe single cell amounts, or pg-level DNA. In this work, we have utilized micro and nano fabrication to construct zero-mode waveguides that contain nanopores in their base, and show that these devices can be used for extremely sensitive DNA capture and sequencing. The device, a nanopore-zero-mode waveguide (NZMW), allows sub nanogram levels of DNA to be efficiently drawn for analysis in seconds, and does not exhibit a bias towards lowmolecular weight DNA as in diffusion-based methods. We will discuss the implications of such a device on DNA sequencing, RNA sequencing, as well as other single-molecule biophysical studies where only precious samples are available.