- Email: [email protected]
Therapeutic Enhancement with Nuclear Targeted Gold Nanoparticles
502a
Tuesday, March 1, 2016
changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to transcription factor (TF) binding. Initial experiments using the nucleosomecaliper construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation, shows that the angular distribution broadens as a result of increasing TF concentrations. Our measurements revealed a dissociation constant in the range of 1-10nM, which agrees with bulk measurements. These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm. 2474-Pos Board B618 Therapeutic Enhancement with Nuclear Targeted Gold Nanoparticles Celina J. Yang, Devika B. Chithrani. Biomedical Physics, Ryerson University, Toronto, ON, Canada. Nanoparticles have been used as a platform to improve therapeutic results in medical research. Gold nanoparticles (GNPs) have been extensively studied among other nanoparticles in cancer research due to its property being radiosensitizing by producing more secondary electrons in response to irradiation. It is predicted that closer the GNPs can get to the DNA, the extra secondary electrons will cause more breaks in the DNA. However, the regular uptake pathway of GNPs into the cell is known to be through receptor mediated endocytosis, where GNPs follow the endo-lyso pathway. An effective design of GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. An effective design of a GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. Each of the peptides is used for enhancing intracellular retention, inducing nuclear delivery and stabilization. When in vitro cells were irradiated with 2 Gy of radiation, the cells incubated with peptidemodified GNPs had an improved therapeutic result, not only compared to the control sample (no incubation of GNP) but to cells incubated with unmodified GNPs. This research will provide insight to a more successful NP-based platform for combining treatment modalities that could eventually lead to a more effective approach in the treatment of cancer. 2475-Pos Board B619 Microcarrier-Guided Nanopore Dielectrophoresis for Selective Nucleic Acid Detection Kai Tian1, Karl Decker2, Aleksei Aksimentiev2, Liqun Gu1. 1 Biological Engineering, University of Missouri, Columbia, MO, USA, 2 Physics, University of Illinois at Urbana-Champaign, Champaign, IL, USA. Dielectrophoresis (DEP) is the motion of a polarizable particle in a nonuniform electric field due to an unbalanced electrostatic force on the particle’s induced dipole. The DEP mechanism has been extensively utilized for manipulation of biological particles, from cancer cells and viruses to biomolecules such as DNAs and proteins, for their concentration, separation, sorting, and transport. However, current DEP approaches to molecular manipulation are not selective, as DEP is not sensitive enough to discriminate among the induced dipoles of different molecules. Here we explore a novel single-molecule DEP mechanism, carrier-guided nanopore dielectrophoresis, for selective nucleic acid sequence detection. Rather than rely on a target’s native polarizability, we designed a polycationic carrier to impart a tunable synthetic dipole to the target nucleic acid molecule; carrier sensitivity and selectivity are both programmable. Such synthetic dipoles can be captured in an engineered nanopore, which acts as an ideal foulfree point source to generate an extremely high field gradient (DE~107 V$m-1 per nanometer or ~1016 V$m-2) for molecular dipole manipulation. Non-target nucleic acids do not bind the carriers and hence are repelled from the pore by electrophoresis. To elucidate the mechanism of the dipole capture by the nanopore, we took an all-atom molecular dynamics (MD) simulation approach to observe the movements of and forces on the dipole. Simulation results predicted significantly increased force attracting the probe into the engineered nanopore as opposed to the wild type, consistent with the increased capture rates observed in experiment. Most strikingly of all, we find that a carrier with only a few positive charges can drive any length of DNA or RNA with equal capture efficiency. Development of nanopore dielectrophoretic detection thus offers ready medical applications of nanopore technology.
2476-Pos Board B620 Dipole Effects on Ion Transport Demonstrated in Aprotic Solvents Timothy S. Plett1, Wenqing Shi2, Yuhan Zeng2, William Mann1, Ivan Vlassiouk3, Lane Baker2, Zuzanna S. Siwy1. 1 Physics, University of California, Irvine, Irvine, CA, USA, 2Chemistry, Indiana University, Bloomington, IN, USA, 3Oak Ridge National Laboratory, Oak Ridge, TN, USA. Dipole interactions play a significant role in biological systems, influencing key functions such as protein folding and selective transport of ions through channels, among many others. Possible importance of the presence of dipoles in systems of synthetic nanopores has not yet been explored. Here, we report experiments and modeling of ionic current through nanopores in polymer films as well as glass nanopipettes. In order to probe importance of dipoles for ionic transport, experiments were performed with aprotic solvents characterized with dipole moments between 5D and 1.7D. LiClO4 was used as the electrolyte due to its high solubility in a wide range of solvents. The conical nanopores used in the experiments rectify the curent with the direction and degree being very sensitive to the surface properties of the pore walls. Current-voltage curves in aprotic solvents with high dipole moments provided evidence that the solvent and possible cation adsorption caused formation of an effectively positive surface potential, which was further confirmed by scanning ion conductance microscopy. Continuum modeling of ion current using models developed for biological systems confirmed that presence of dipoles on pore walls can indeed modify local ionic concentrations and the recorded current. 2477-Pos Board B621 A Novel Multi-Layer Microfluidic Pipette Aspiration Device for Studying Mechanosensitive Vesicles Lap Man Lee1, Danielle Chase2, Allen Liu1. 1 Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA, 2 Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA. Mechanosensitive channel of large conductance (MscL) is a prokaryotic channel that opens due to increased membrane tension. It has been reconstituted in vesicles to study how various biophysical factors influence their mechanical gating properties. In recent years, several theoretical and computational models based on molecular dynamics and continuum mechanics have been developed to understand the underlying mechanisms for the activation of MscLs in vesicles. Experimental approaches for MscL gating studies have focused on cell-by-cell techniques, such as pressure patch clamp, on reconstituted MscL vesicles. Due to the limitation of throughput, such studies are time intensive with low sample numbers. Recently, a microfluidic pipette aspiration array device, based on PDMS multi-layer soft-lithography technique, has been developed in our group to trap and apply mechanical perturbation to single cells in a parallel manner1. This device was used to study the stiffness of human breast cancer cell lines and mechanical gating threshold of reconstituted MscL on infected mammalian cell lines. In this work, we have developed a new device that incorporates a pressure valve and a smaller micropipette dimension to increase trapping efficiency of vesicles. Using this device, we demonstrate stable trapping of single vesicles and expand our efforts to study mechanical gating threshold of reconstituted MscL in vesicles formed by electroformation. Development of novel microtechnology tools that can trap single vesicles and exert tension will have numerous applications to the study of other mechanosensitive channels. [1] Lee & Liu, Lab Chip, 2015, 15, 264-273. 2478-Pos Board B622 An Ion-Specific Effect on Polymer-Protein Interaction Enhances Resolution of Nanopore-Based Detection Aleksandra Dylewska-Chaumeil1, Gerhard Baaken2, Jan C. Behrends1. 1 Physiology, University of Freiburg, Freiburg, Germany, 2Ionera Technologies GmbH, Freiburg, Germany. Poly(ethyleneglycol) (PEG) oligomers partition into acqueous transmembrane channels formed by alpha-Hemolysin (aHL) and Aerolysin (AeL) proteins and transiently bind the pore to induce reductions in conductance, i.e. resistive pulses, the amplitude of which is strongly related to polymer chain length. Histograms of such data for PEG yield mass spectrograms with single monomer resolution between approximately 20 and 60 repeat units (r.u.). For chain lengths <20 the duration of resistive pulses becomes too short (e.g.<50 ms) to be fully resolved using state-of-the art electrophysiological recording technology. Recently, computational reconstruction of unresolved events has revealed the presence in a sample of PEG species down to
Tuesday, March 1, 2016
changes in nucleosomes. Our immediate goal is to measure the structural changes in single nucleosomes and nucleosome arrays in response to transcription factor (TF) binding. Initial experiments using the nucleosomecaliper construct with GAL4-VP16, a hybrid transcription factor capable of highly-efficient transcription activation, shows that the angular distribution broadens as a result of increasing TF concentrations. Our measurements revealed a dissociation constant in the range of 1-10nM, which agrees with bulk measurements. These results demonstrate the potential of a DNA origami device probing chromatin structure and function in vitro, in particular, the ability to measure structural changes in the range of 10-100nm. 2474-Pos Board B618 Therapeutic Enhancement with Nuclear Targeted Gold Nanoparticles Celina J. Yang, Devika B. Chithrani. Biomedical Physics, Ryerson University, Toronto, ON, Canada. Nanoparticles have been used as a platform to improve therapeutic results in medical research. Gold nanoparticles (GNPs) have been extensively studied among other nanoparticles in cancer research due to its property being radiosensitizing by producing more secondary electrons in response to irradiation. It is predicted that closer the GNPs can get to the DNA, the extra secondary electrons will cause more breaks in the DNA. However, the regular uptake pathway of GNPs into the cell is known to be through receptor mediated endocytosis, where GNPs follow the endo-lyso pathway. An effective design of GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. An effective design of a GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. Each of the peptides is used for enhancing intracellular retention, inducing nuclear delivery and stabilization. When in vitro cells were irradiated with 2 Gy of radiation, the cells incubated with peptidemodified GNPs had an improved therapeutic result, not only compared to the control sample (no incubation of GNP) but to cells incubated with unmodified GNPs. This research will provide insight to a more successful NP-based platform for combining treatment modalities that could eventually lead to a more effective approach in the treatment of cancer. 2475-Pos Board B619 Microcarrier-Guided Nanopore Dielectrophoresis for Selective Nucleic Acid Detection Kai Tian1, Karl Decker2, Aleksei Aksimentiev2, Liqun Gu1. 1 Biological Engineering, University of Missouri, Columbia, MO, USA, 2 Physics, University of Illinois at Urbana-Champaign, Champaign, IL, USA. Dielectrophoresis (DEP) is the motion of a polarizable particle in a nonuniform electric field due to an unbalanced electrostatic force on the particle’s induced dipole. The DEP mechanism has been extensively utilized for manipulation of biological particles, from cancer cells and viruses to biomolecules such as DNAs and proteins, for their concentration, separation, sorting, and transport. However, current DEP approaches to molecular manipulation are not selective, as DEP is not sensitive enough to discriminate among the induced dipoles of different molecules. Here we explore a novel single-molecule DEP mechanism, carrier-guided nanopore dielectrophoresis, for selective nucleic acid sequence detection. Rather than rely on a target’s native polarizability, we designed a polycationic carrier to impart a tunable synthetic dipole to the target nucleic acid molecule; carrier sensitivity and selectivity are both programmable. Such synthetic dipoles can be captured in an engineered nanopore, which acts as an ideal foulfree point source to generate an extremely high field gradient (DE~107 V$m-1 per nanometer or ~1016 V$m-2) for molecular dipole manipulation. Non-target nucleic acids do not bind the carriers and hence are repelled from the pore by electrophoresis. To elucidate the mechanism of the dipole capture by the nanopore, we took an all-atom molecular dynamics (MD) simulation approach to observe the movements of and forces on the dipole. Simulation results predicted significantly increased force attracting the probe into the engineered nanopore as opposed to the wild type, consistent with the increased capture rates observed in experiment. Most strikingly of all, we find that a carrier with only a few positive charges can drive any length of DNA or RNA with equal capture efficiency. Development of nanopore dielectrophoretic detection thus offers ready medical applications of nanopore technology.
2476-Pos Board B620 Dipole Effects on Ion Transport Demonstrated in Aprotic Solvents Timothy S. Plett1, Wenqing Shi2, Yuhan Zeng2, William Mann1, Ivan Vlassiouk3, Lane Baker2, Zuzanna S. Siwy1. 1 Physics, University of California, Irvine, Irvine, CA, USA, 2Chemistry, Indiana University, Bloomington, IN, USA, 3Oak Ridge National Laboratory, Oak Ridge, TN, USA. Dipole interactions play a significant role in biological systems, influencing key functions such as protein folding and selective transport of ions through channels, among many others. Possible importance of the presence of dipoles in systems of synthetic nanopores has not yet been explored. Here, we report experiments and modeling of ionic current through nanopores in polymer films as well as glass nanopipettes. In order to probe importance of dipoles for ionic transport, experiments were performed with aprotic solvents characterized with dipole moments between 5D and 1.7D. LiClO4 was used as the electrolyte due to its high solubility in a wide range of solvents. The conical nanopores used in the experiments rectify the curent with the direction and degree being very sensitive to the surface properties of the pore walls. Current-voltage curves in aprotic solvents with high dipole moments provided evidence that the solvent and possible cation adsorption caused formation of an effectively positive surface potential, which was further confirmed by scanning ion conductance microscopy. Continuum modeling of ion current using models developed for biological systems confirmed that presence of dipoles on pore walls can indeed modify local ionic concentrations and the recorded current. 2477-Pos Board B621 A Novel Multi-Layer Microfluidic Pipette Aspiration Device for Studying Mechanosensitive Vesicles Lap Man Lee1, Danielle Chase2, Allen Liu1. 1 Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA, 2 Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA. Mechanosensitive channel of large conductance (MscL) is a prokaryotic channel that opens due to increased membrane tension. It has been reconstituted in vesicles to study how various biophysical factors influence their mechanical gating properties. In recent years, several theoretical and computational models based on molecular dynamics and continuum mechanics have been developed to understand the underlying mechanisms for the activation of MscLs in vesicles. Experimental approaches for MscL gating studies have focused on cell-by-cell techniques, such as pressure patch clamp, on reconstituted MscL vesicles. Due to the limitation of throughput, such studies are time intensive with low sample numbers. Recently, a microfluidic pipette aspiration array device, based on PDMS multi-layer soft-lithography technique, has been developed in our group to trap and apply mechanical perturbation to single cells in a parallel manner1. This device was used to study the stiffness of human breast cancer cell lines and mechanical gating threshold of reconstituted MscL on infected mammalian cell lines. In this work, we have developed a new device that incorporates a pressure valve and a smaller micropipette dimension to increase trapping efficiency of vesicles. Using this device, we demonstrate stable trapping of single vesicles and expand our efforts to study mechanical gating threshold of reconstituted MscL in vesicles formed by electroformation. Development of novel microtechnology tools that can trap single vesicles and exert tension will have numerous applications to the study of other mechanosensitive channels. [1] Lee & Liu, Lab Chip, 2015, 15, 264-273. 2478-Pos Board B622 An Ion-Specific Effect on Polymer-Protein Interaction Enhances Resolution of Nanopore-Based Detection Aleksandra Dylewska-Chaumeil1, Gerhard Baaken2, Jan C. Behrends1. 1 Physiology, University of Freiburg, Freiburg, Germany, 2Ionera Technologies GmbH, Freiburg, Germany. Poly(ethyleneglycol) (PEG) oligomers partition into acqueous transmembrane channels formed by alpha-Hemolysin (aHL) and Aerolysin (AeL) proteins and transiently bind the pore to induce reductions in conductance, i.e. resistive pulses, the amplitude of which is strongly related to polymer chain length. Histograms of such data for PEG yield mass spectrograms with single monomer resolution between approximately 20 and 60 repeat units (r.u.). For chain lengths <20 the duration of resistive pulses becomes too short (e.g.<50 ms) to be fully resolved using state-of-the art electrophysiological recording technology. Recently, computational reconstruction of unresolved events has revealed the presence in a sample of PEG species down to