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DNA Nanostructures for Single Molecule Protein Sensing with Nanopores
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Wednesday, March 2, 2016
aggregation process. The experimental results indicate that it is the presence of 20% 1,2-dioleoyl-sn- glycero-3- [phospho-L-serine] (DOPS) dramatically enhances the aggregation rate of a-Syn, while the aggregation pathway is similar to a-Syn only samples. 3229-Pos Board B606 Controllable Plasmonic Enhancement and Hot Spots of Metallic Nanoclusters Assembled by Green Fluorescent Proteins Taerin Chung, Tugba Koker, Fabien Pinaud. University of Southern California, Los angeles, CA, USA. Biocompatible metallic nanoparticles in optical regime have been incessantly explored for the state of art technologies of non-invasive sensing, high resolution imaging and surface-enhanced Raman spectroscopy. We propose threedimensional numerical modeling to unveil the physical phenomena of gold nanoclusters assembled by green fluorescent protein (GFP) with FDTD technique. GFPs over metallic nanoparticles are physically modeled as a nanometer cylindrical shape with estimated refractive index depending on the molecular concentration of buffer solution. Electromagnetic field distributions in the vicinity of metallic nanoparticles accompanying with GFPs exhibit localized hot spot intensity and locations as a function of gap length between nanoparticles, incident polarization state of light, and array types. Calculated spectra is verified with experimental data in terms of buffer concentration with and without gold nanoparticles. In addition, plasmonic tunability within visible range is observed in randomized hetero gold nanoclusters assembled by specific binding between split-GFP with 40 nm gold particle and M3 peptide with 20 nm gold particle, giving rise to major electric capacitive coupling. SERS enhancement factor relies on the coupling location of hetero gold nanoparticles. Interparticle electric couplings between 40 nm gold particles and 20 nm gold particles in randomized hetero gold nanoclusters produce localized hot spots in response to the interparticle distance, polarization orientation, and nanoparticle size. In case of gold nanoheptamer, which is also assembled by site-specific GFP binding, symmetric localized hot spots are observed corresponding to the couplings between center gold nanoparticle and surrounding gold nanoparticles. By the variation of surrounding gold nanoparticle size, the number and position of localized hot spots, which can be called Raman active site, is controllable. According to numerical analysis of GFP-driven gold nanocluster, tailored gold nanoclusters are offered. 3230-Pos Board B607 DNA Nanostructures for Single Molecule Protein Sensing with Nanopores Nicholas A.W. Bell, Jinglin Kong, Ulrich F. Keyser. Physics, Cambridge University, Cambridge, United Kingdom. Solid-state nanopore sensing of proteins presents challenges due to the fast timescale of translocation and the difficulty in discriminating signals from different proteins. In this work we describe a new method for nanopore protein sensing which allows us to address these issues by introducing chemical selectivity into solid-state nanopore measurements. We designed linear DNA nanostructures by hybridising nearly two hundred oligonucleotides to the m13mp18 virus genome. This engineered DNA nanostructure allows positioning of protein binding sites at nanometre accurate intervals along its contour via DNA conjugation chemistry. The ionic current signal of each translocating DNA nanostructure shows an extra characteristic modulation after incubation with a target protein due its binding at the desired position. We also show how multiple protein species can be simultaneously detected at nanomolar concentration levels using this technique. 3231-Pos Board B608 Single-Site Resolution Detection of Methylation in DNA with Graphene Nanopores Aditya Sarathy1,2, Hu Qiu2, Klaus Schulten2,3, Jean-Pierre Leburton1,2. 1 Department of Electrical and Computer Engineering, University of Illinois, Urbana-Champaign, Champaign, IL, USA, 2Beckman Institute of Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL, USA, 3Department of Physics, University of Illinois, Urbana-Champaign, Urbana, IL, USA. Epigenetic modification of DNA where methyl groups are added at the 5carbon position of cytosine is known as DNA methylation. It is associated with carcinogenesis, thus capable of serving as markers for detection of cancer. Nanopore analytics provides an easier and quicker route to detect methylated sites by avoiding complicated bisulfite treatment and polymerase chain reaction amplification. Graphene has a thickness of a single atom, thereby holding the potential to detect the methylation at single-site resolu-
tion. In this work, we use a self-consistent Poisson-Boltzmann formalism to simulate the translocation of methyl-CpG (MBD) proteins bound to a DNA molecule in a graphene nanopore, and detect the methylated sites along the DNA strand by computing both ionic current using molecular dynamics simulations and transverse sheet current via tight binding Hamiltonian based none-equilibrium Green’s function approach.1 Evident dips are recorded in the ionic current trace through the nanopore for each methylation site, as expected, suggesting a single-site resolution. The graphene membrane with added quantum point contacts, by means of transverse sheet currents, can also detect the methylated site through local jumps in the variance of the measured transverse current. The proposed measurement strategy allows for real time, fast and high resolution DNA methylation detection.2 1. Girdhar, A., Sathe, C., Schulten, K., & Leburton, J. P. (2013). Graphene quantum point contact transistor for DNA sensing. Proceedings of the National Academy of Sciences, 110(42), 16748-16753. 2. Sarathy, A., Qiu, H., Schulten, K., & Leburton, J. P. Single-site detection of methylation in DNA with graphene nanopores. To be published. 3232-Pos Board B609 Conductance Modulation in Silicon-on-Insulator Solid-State Nanopores Coated with Electroactive Polymers Xiaofeng Wang, Michael Goryll. Electrical Engineering, Arizona State University, Tempe, AZ, USA. Solid-state nanopores are an ideal platform for bioanalytical studies. However, only very few solid-state nanopores allow the ionic conductance through the nanopore to be modulated, which is a feature of most transmembrane protein channels. Using silicon-on-insulator (SOI) substrates, a substrate bias can be applied to the top silicon layer in which cylindrical nanopores have been formed, leading to a modulation of the ionic conductance through the nanopore. The effect of the electrostatic modulation on the nanopore conductance, however, is small unless the electrolyte concentration is low (<100 mM) or the pore diameter is below 10 nm. To increase the effect of the conductance modulation, polyelectrolyte brushes tethered to the inside of the nanopore can be used, which actively change the diameter of the lumen of the nanopore. Since the polymer brushes are charged, a change in surface charge due to an electrostatic bias is expected to influence the length of the brushes extending into the nanopore, thereby changing its diameter. In our experiments, poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) polyelectrolyte brushes were deposited on the inner walls of thermally oxidized SOI cylindrical nanopores using surface-initiated atom transfer radical polymerization (SI-ATRP). After polymerization, the ionic conductance of the nanopore was characterized using DC conductance and Electrochemical Impedance Spectroscopy (EIS). The EIS data allowed a separation of the influence of the polyelectrolyte brushes on the nanopore conductance from the influence on the capacitance of the complete device, enabling the development of an equivalent electrical circuit model. Ion transport studies under both pH and gate bias stimuli revealed a significant conductance increase in strong acidic solutions (100 mM HCl) and a conductance modulation upon application of a positive gate bias in 100 mM HCl solutions, which was not observable without the polyelectrolyte brush layer. 3233-Pos Board B610 Single Oligonucleotide Discrimination with Aerolysin Nanopore Chan Cao, Yi-Lun Ying, Yi-Tao Long. East China University of Science and Technology, shanghai, China. Nanopore technique is an emerging platform for inexpensive and ultrafast analysis for single oligonucleotides 1, 2. Aerolysin has been used as a biological nanopore for studying peptides, proteins and oligosaccharides in the past decade 3. Here, we report for the first time that wild-type aerolysin performs a high current and temporary resolution for oligonucleotides discrimination 4. Further applications of aerolysin nanopores were achieved to distinguish individual oligonucleotides from mixtures and to monitor the enzyme related step-wise cleavage process. Our findings would promotes the application of aerolysin to accurate analyses of nucleic acids. 1. Kasianowicz, John J, Brandin, Eric, Branton, Daniel, Deamer, David W., Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13770. 2. Yi-LunYing, Jun-Ji Zhang, Rui Gao, Yi-TaoLong. Angew. Chem. Int. Ed., 2013, 52, 13154. 3. Stefureac, Radu, Long, Yi-Tao Long, Kraatz, Heinz-Bernhard, Howard Peter, Lee, Jeremy S., Biochemistry, 2006, 45, 9172. 4. Yi-Tao Long, Chan Cao, Yongxu Hu. CN 201510047662.
Wednesday, March 2, 2016
aggregation process. The experimental results indicate that it is the presence of 20% 1,2-dioleoyl-sn- glycero-3- [phospho-L-serine] (DOPS) dramatically enhances the aggregation rate of a-Syn, while the aggregation pathway is similar to a-Syn only samples. 3229-Pos Board B606 Controllable Plasmonic Enhancement and Hot Spots of Metallic Nanoclusters Assembled by Green Fluorescent Proteins Taerin Chung, Tugba Koker, Fabien Pinaud. University of Southern California, Los angeles, CA, USA. Biocompatible metallic nanoparticles in optical regime have been incessantly explored for the state of art technologies of non-invasive sensing, high resolution imaging and surface-enhanced Raman spectroscopy. We propose threedimensional numerical modeling to unveil the physical phenomena of gold nanoclusters assembled by green fluorescent protein (GFP) with FDTD technique. GFPs over metallic nanoparticles are physically modeled as a nanometer cylindrical shape with estimated refractive index depending on the molecular concentration of buffer solution. Electromagnetic field distributions in the vicinity of metallic nanoparticles accompanying with GFPs exhibit localized hot spot intensity and locations as a function of gap length between nanoparticles, incident polarization state of light, and array types. Calculated spectra is verified with experimental data in terms of buffer concentration with and without gold nanoparticles. In addition, plasmonic tunability within visible range is observed in randomized hetero gold nanoclusters assembled by specific binding between split-GFP with 40 nm gold particle and M3 peptide with 20 nm gold particle, giving rise to major electric capacitive coupling. SERS enhancement factor relies on the coupling location of hetero gold nanoparticles. Interparticle electric couplings between 40 nm gold particles and 20 nm gold particles in randomized hetero gold nanoclusters produce localized hot spots in response to the interparticle distance, polarization orientation, and nanoparticle size. In case of gold nanoheptamer, which is also assembled by site-specific GFP binding, symmetric localized hot spots are observed corresponding to the couplings between center gold nanoparticle and surrounding gold nanoparticles. By the variation of surrounding gold nanoparticle size, the number and position of localized hot spots, which can be called Raman active site, is controllable. According to numerical analysis of GFP-driven gold nanocluster, tailored gold nanoclusters are offered. 3230-Pos Board B607 DNA Nanostructures for Single Molecule Protein Sensing with Nanopores Nicholas A.W. Bell, Jinglin Kong, Ulrich F. Keyser. Physics, Cambridge University, Cambridge, United Kingdom. Solid-state nanopore sensing of proteins presents challenges due to the fast timescale of translocation and the difficulty in discriminating signals from different proteins. In this work we describe a new method for nanopore protein sensing which allows us to address these issues by introducing chemical selectivity into solid-state nanopore measurements. We designed linear DNA nanostructures by hybridising nearly two hundred oligonucleotides to the m13mp18 virus genome. This engineered DNA nanostructure allows positioning of protein binding sites at nanometre accurate intervals along its contour via DNA conjugation chemistry. The ionic current signal of each translocating DNA nanostructure shows an extra characteristic modulation after incubation with a target protein due its binding at the desired position. We also show how multiple protein species can be simultaneously detected at nanomolar concentration levels using this technique. 3231-Pos Board B608 Single-Site Resolution Detection of Methylation in DNA with Graphene Nanopores Aditya Sarathy1,2, Hu Qiu2, Klaus Schulten2,3, Jean-Pierre Leburton1,2. 1 Department of Electrical and Computer Engineering, University of Illinois, Urbana-Champaign, Champaign, IL, USA, 2Beckman Institute of Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL, USA, 3Department of Physics, University of Illinois, Urbana-Champaign, Urbana, IL, USA. Epigenetic modification of DNA where methyl groups are added at the 5carbon position of cytosine is known as DNA methylation. It is associated with carcinogenesis, thus capable of serving as markers for detection of cancer. Nanopore analytics provides an easier and quicker route to detect methylated sites by avoiding complicated bisulfite treatment and polymerase chain reaction amplification. Graphene has a thickness of a single atom, thereby holding the potential to detect the methylation at single-site resolu-
tion. In this work, we use a self-consistent Poisson-Boltzmann formalism to simulate the translocation of methyl-CpG (MBD) proteins bound to a DNA molecule in a graphene nanopore, and detect the methylated sites along the DNA strand by computing both ionic current using molecular dynamics simulations and transverse sheet current via tight binding Hamiltonian based none-equilibrium Green’s function approach.1 Evident dips are recorded in the ionic current trace through the nanopore for each methylation site, as expected, suggesting a single-site resolution. The graphene membrane with added quantum point contacts, by means of transverse sheet currents, can also detect the methylated site through local jumps in the variance of the measured transverse current. The proposed measurement strategy allows for real time, fast and high resolution DNA methylation detection.2 1. Girdhar, A., Sathe, C., Schulten, K., & Leburton, J. P. (2013). Graphene quantum point contact transistor for DNA sensing. Proceedings of the National Academy of Sciences, 110(42), 16748-16753. 2. Sarathy, A., Qiu, H., Schulten, K., & Leburton, J. P. Single-site detection of methylation in DNA with graphene nanopores. To be published. 3232-Pos Board B609 Conductance Modulation in Silicon-on-Insulator Solid-State Nanopores Coated with Electroactive Polymers Xiaofeng Wang, Michael Goryll. Electrical Engineering, Arizona State University, Tempe, AZ, USA. Solid-state nanopores are an ideal platform for bioanalytical studies. However, only very few solid-state nanopores allow the ionic conductance through the nanopore to be modulated, which is a feature of most transmembrane protein channels. Using silicon-on-insulator (SOI) substrates, a substrate bias can be applied to the top silicon layer in which cylindrical nanopores have been formed, leading to a modulation of the ionic conductance through the nanopore. The effect of the electrostatic modulation on the nanopore conductance, however, is small unless the electrolyte concentration is low (<100 mM) or the pore diameter is below 10 nm. To increase the effect of the conductance modulation, polyelectrolyte brushes tethered to the inside of the nanopore can be used, which actively change the diameter of the lumen of the nanopore. Since the polymer brushes are charged, a change in surface charge due to an electrostatic bias is expected to influence the length of the brushes extending into the nanopore, thereby changing its diameter. In our experiments, poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) polyelectrolyte brushes were deposited on the inner walls of thermally oxidized SOI cylindrical nanopores using surface-initiated atom transfer radical polymerization (SI-ATRP). After polymerization, the ionic conductance of the nanopore was characterized using DC conductance and Electrochemical Impedance Spectroscopy (EIS). The EIS data allowed a separation of the influence of the polyelectrolyte brushes on the nanopore conductance from the influence on the capacitance of the complete device, enabling the development of an equivalent electrical circuit model. Ion transport studies under both pH and gate bias stimuli revealed a significant conductance increase in strong acidic solutions (100 mM HCl) and a conductance modulation upon application of a positive gate bias in 100 mM HCl solutions, which was not observable without the polyelectrolyte brush layer. 3233-Pos Board B610 Single Oligonucleotide Discrimination with Aerolysin Nanopore Chan Cao, Yi-Lun Ying, Yi-Tao Long. East China University of Science and Technology, shanghai, China. Nanopore technique is an emerging platform for inexpensive and ultrafast analysis for single oligonucleotides 1, 2. Aerolysin has been used as a biological nanopore for studying peptides, proteins and oligosaccharides in the past decade 3. Here, we report for the first time that wild-type aerolysin performs a high current and temporary resolution for oligonucleotides discrimination 4. Further applications of aerolysin nanopores were achieved to distinguish individual oligonucleotides from mixtures and to monitor the enzyme related step-wise cleavage process. Our findings would promotes the application of aerolysin to accurate analyses of nucleic acids. 1. Kasianowicz, John J, Brandin, Eric, Branton, Daniel, Deamer, David W., Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13770. 2. Yi-LunYing, Jun-Ji Zhang, Rui Gao, Yi-TaoLong. Angew. Chem. Int. Ed., 2013, 52, 13154. 3. Stefureac, Radu, Long, Yi-Tao Long, Kraatz, Heinz-Bernhard, Howard Peter, Lee, Jeremy S., Biochemistry, 2006, 45, 9172. 4. Yi-Tao Long, Chan Cao, Yongxu Hu. CN 201510047662.