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Improving Stability and Sensitivity of SERS-Based Biosensors
302a
Monday, February 13, 2017
1482-Pos Board B550 Modulating Protein-Nanoparticle Interaction Energetics using Site Directed Mutagenesis Yasiru R. Perera, Ailin Wang, Alex Hughes, Nicholas C. Fitzkee. Department of Chemistry, Mississippi State University, Mississippi State, MS, USA. Nanoparticle technology has been a growing field in biomedical research. This is in part due to potential applications in drug delivery, biosensing, diagnostics, and imaging. Our long-term goal is to use protein functionalized AuNPs as a general tool for molecular sensing and drug delivery. The ability to use nanomaterials as biosensors and drug delivery methods in cellular uptake is directly dependent on the amount of protein that is able to bind to the surface of any given nanoparticle. It is hypothesized that electrostatic interactions play a significant role in protein-AuNP interactions, since citrate-stabilized AuNPs carry a net negative charge. Our group has developed an NMR-based approach to rapidly quantify bound protein to AuNP. To understand the above phenomenon, GB3 was chosen as our model protein and it contains seven lysine residues. These positively-charged lysine residues are involved in protein-AuNP binding, and a potential binding site was identified using APBS calculations which contain lysine residues. This hypothesis was tested by mutating the lysine residues to alanine one at a time using site-directed mutagenesis. NMR experiments were carried out to observe how the binding capacities of each of these variants change relative to wild type GB3. Notably K4A, K13A and K50A variants has significantly reduced binding, while the binding capacity of other lysine to alanine variants was on par with wild-type GB3. To obtain better understanding, GB3 variants were competed with wild type GB3 in the same solution with AuNP to observe how the binding capacities vary with wild type GB3. As predicted, the binding capacity ratio was lower for lysine residues in the proposed binding site were changed to alanine. A reduced binding capacity ratio was not observed for other lysine variants. The results reported are significant in establishing our original hypothesis, and suggest that GB3 adopts a specific orientation on the AuNP surface. 1483-Pos Board B551 Electrostatics of Dna-Wrapped Cationically Stabilized Gold Nanospheres Savannah Miller, Celina Harris, Lucas B. Thompson, Kurt Andresen. Gettysburg College, Gettysburg, PA, USA. Gold nanoparticles have a unique set of properties that allow them to be utilized in a wide array of research and medical applications. The interface of positively stabilized gold nanoparticles with DNA is of particular interest for a variety of applications such as the study of artificial forms of DNA packing and the design of new vectors for therapeutic gene delivery. DNA wraps electrostatically around gold nanoparticles that are stabilized or layered with cationic molecules. Depending on the size of the gold nanoparticles, the length of the DNA, and the ionic content of the solution, these complexes can vary in stability, morphology, and mechanism of formation. To investigate these complexes, we incubated small cetrimonium bromide (CTAB) stabilized gold nanospheres in solutions of sheared calf thymus DNA. We removed unwrapped DNA and introduced a background concentration of sodium chloride using equilibrium dialysis by serial centrifugation. We characterized the gold nanospheres at all stages of the process using UV spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. Following centrifugation, we analyzed the purified solutions of DNA wrapped gold nanospheres using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The goal of these measurements is to elucidate the electrostatics of these complexes by analyzing the effect of DNA wrapping on the properties of the gold nanospheres within the complex, the incorporation of stabilizing sodium cations into the complex, and the ratio of DNA to nanoparticles utilized in each complex. 1484-Pos Board B552 Modulation of Fluorescence Emission Rate using Nano-Antenna Wenqi Zhao, Xiaochaoran Tian, Meng Qiu, Yuanbo Zhang, Lei Zhou, Yanwen Tan. State Key Laboratory of Surface Physics, Department of Physics, Fudan University, Shanghai, China. Enhancing fluorescence through engineering plasmonic modes in nanostructures has been actively pursued in the past decade. Such fluorescence enhancement can be achieved by two separate mechanisms: 1) the enhancement of local electromagnetic field and 2) the enhancement of spontaneous emission rate, i.e. the Purcell effect. While most experimental studies focused on local-field enhancement, the Purcell enhancement offers distinct advantages: it improves the quantum yield of the emitter and avoids the damaging effect of a strong local field. This makes the Purcell enhancement well suited for fluorescent proteins and NIR-fluorophores. Here, we engineer the plasmonic
modes of asymmetric nano-antennas, and modulate the fluorescence emission rate of fluorophores. We demonstrated a useful general strategy for fluorescence enhancement using nano-antennas. Our method has the potential to be applicable to photo-sensitive molecular studies, like bio-chip sensing, DNA sequencing, single-molecule detection /etc. 1485-Pos Board B553 Improving Stability and Sensitivity of SERS-Based Biosensors Joseph Smolsky1, Zakhar Reveguk2, Zachary Sabata1, Alexey Krasnoslobodtsev1. 1 UNO, Omaha, NE, USA, 2St. Petersburg State University, St. Petersburg, Russian Federation. Detection and monitoring of disease biomarkers increases probability of successful disease treatment. Among available readout strategies utilized in biosensors - surface enhanced Raman scattering (SERS) has several advantages over conventional techniques. In particular, multiplexing capabilities of SERS readout is a very attractive feature. Our improvements to SERS readout include protection of SERS labels and temperature assisted plasmonic coupling of DNA modified AuNP contributing to better reproducibility and higher sensitivity of biosensors. We have introduced several protection strategies to overcome SERS signal deterioration under prolonged exposure to intense laser light. Simple protection strategy involves using either polymer or graphene monolayer as a thin protective layer applied on top of the assay that renders signal to be more stable against photo-induced damage. Another protection strategy involves coadsorption of thiolated polyetheleneglycol (PEG) together with Raman reporter molecule (RRM): 4 nitrobenzenethiol (NBT) onto AuNP. These protection strategies improve stability of the assay measured as the time dependence of the Raman signal intensity at 1336 cm 1 (the most intense band for NBT) on duration of sample exposure to laser light. Protection also reduces RRM desorption from the surface of AuNP, photodamage, and catalytic photoconversion of NBT to diazobenzene. We discuss possible mechanisms of such a conversion and effectiveness of PEG protection against it. We have evaluated the size and amount of PEG molecules for optimal protection of SERS labels. Higher sensitivity of the biosensor was achieved via DNA assisted coupling between individual nanoparticles. Our design allowed for controllable plasmonic coupling which resulted in further several fold enhancement of Raman signal, thus improving sensitivity of the biosensor. Practical applications of SERSbased biosensor for in vivo and multiplex detection of biomarkers are also discussed. 1486-Pos Board B554 Controlled Biomolecule Release from a Liposomal Nanocarrier Modulated with Pulsed NIR Light Jeongeun Shin, Maria Olubunmi Ogunyankin, Joseph A. Zasadzinski. Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, USA. A generic delivery platform that could deliver any biomolecule, independent of its chemical constitution, would be a significant advance for researchers in cell biology and neuroscience. At present, small molecule delivery is done via ‘‘caged’’ compounds. However, each bioactive requires the chemical synthesis of its own ‘‘cage’’. As yet, no NIR triggered caged compounds are effective under typical experimental conditions. To address these issues, we have developed liposomes tethered to plasmon-resonant Hollow Gold Nanoshells (HGN) that can be triggered to release their contents by picosecond pulses of physiologically friendly, deeply penetrating near infrared (NIR) light. Liposomes tethered to HGN can encapsulate almost any water-soluble biologically active molecule by confining high concentrations in liposomes tethered to HGN. A major benefit of this technique is the universal mechanism of liposome contents release via nanobubble rupture following pulsed NIR light triggering: any molecule will be released by liposome rupture, so release rates, timing, laser fluence, etc. will be similar for all compounds of interest. By modifying the laser fluence, HGN properties, and liposome membrane composition, we can alter the energy threshold for triggering release, enabling delivery of multiple agents at different times and locations, which is impossible with current liposome or caged compound technologies. Chemically disparate calcium, ATP, carboxyfluorescein (CF), and cisplatin are all released at near 100 % efficiency from liposomes within msec. For a given HGN tethered to the liposome, the threshold energy is lowest at the wavelength corresponding to the maximum adsorption wavelength of the HGN; the threshold energy increases as the wavelength of the NIR light moves away from the maximum. This allows us to create liposomes that can release at different laser fluences so that we could control release rates and windows of each biomolecule in a mixture independently, by delivering two species or even changing the order of release. In this way, we can release one compound at one place and time, then a second compound at the same place at a different time simply by modulating the laser
Monday, February 13, 2017
1482-Pos Board B550 Modulating Protein-Nanoparticle Interaction Energetics using Site Directed Mutagenesis Yasiru R. Perera, Ailin Wang, Alex Hughes, Nicholas C. Fitzkee. Department of Chemistry, Mississippi State University, Mississippi State, MS, USA. Nanoparticle technology has been a growing field in biomedical research. This is in part due to potential applications in drug delivery, biosensing, diagnostics, and imaging. Our long-term goal is to use protein functionalized AuNPs as a general tool for molecular sensing and drug delivery. The ability to use nanomaterials as biosensors and drug delivery methods in cellular uptake is directly dependent on the amount of protein that is able to bind to the surface of any given nanoparticle. It is hypothesized that electrostatic interactions play a significant role in protein-AuNP interactions, since citrate-stabilized AuNPs carry a net negative charge. Our group has developed an NMR-based approach to rapidly quantify bound protein to AuNP. To understand the above phenomenon, GB3 was chosen as our model protein and it contains seven lysine residues. These positively-charged lysine residues are involved in protein-AuNP binding, and a potential binding site was identified using APBS calculations which contain lysine residues. This hypothesis was tested by mutating the lysine residues to alanine one at a time using site-directed mutagenesis. NMR experiments were carried out to observe how the binding capacities of each of these variants change relative to wild type GB3. Notably K4A, K13A and K50A variants has significantly reduced binding, while the binding capacity of other lysine to alanine variants was on par with wild-type GB3. To obtain better understanding, GB3 variants were competed with wild type GB3 in the same solution with AuNP to observe how the binding capacities vary with wild type GB3. As predicted, the binding capacity ratio was lower for lysine residues in the proposed binding site were changed to alanine. A reduced binding capacity ratio was not observed for other lysine variants. The results reported are significant in establishing our original hypothesis, and suggest that GB3 adopts a specific orientation on the AuNP surface. 1483-Pos Board B551 Electrostatics of Dna-Wrapped Cationically Stabilized Gold Nanospheres Savannah Miller, Celina Harris, Lucas B. Thompson, Kurt Andresen. Gettysburg College, Gettysburg, PA, USA. Gold nanoparticles have a unique set of properties that allow them to be utilized in a wide array of research and medical applications. The interface of positively stabilized gold nanoparticles with DNA is of particular interest for a variety of applications such as the study of artificial forms of DNA packing and the design of new vectors for therapeutic gene delivery. DNA wraps electrostatically around gold nanoparticles that are stabilized or layered with cationic molecules. Depending on the size of the gold nanoparticles, the length of the DNA, and the ionic content of the solution, these complexes can vary in stability, morphology, and mechanism of formation. To investigate these complexes, we incubated small cetrimonium bromide (CTAB) stabilized gold nanospheres in solutions of sheared calf thymus DNA. We removed unwrapped DNA and introduced a background concentration of sodium chloride using equilibrium dialysis by serial centrifugation. We characterized the gold nanospheres at all stages of the process using UV spectroscopy, dynamic light scattering (DLS), and zeta potential measurements. Following centrifugation, we analyzed the purified solutions of DNA wrapped gold nanospheres using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The goal of these measurements is to elucidate the electrostatics of these complexes by analyzing the effect of DNA wrapping on the properties of the gold nanospheres within the complex, the incorporation of stabilizing sodium cations into the complex, and the ratio of DNA to nanoparticles utilized in each complex. 1484-Pos Board B552 Modulation of Fluorescence Emission Rate using Nano-Antenna Wenqi Zhao, Xiaochaoran Tian, Meng Qiu, Yuanbo Zhang, Lei Zhou, Yanwen Tan. State Key Laboratory of Surface Physics, Department of Physics, Fudan University, Shanghai, China. Enhancing fluorescence through engineering plasmonic modes in nanostructures has been actively pursued in the past decade. Such fluorescence enhancement can be achieved by two separate mechanisms: 1) the enhancement of local electromagnetic field and 2) the enhancement of spontaneous emission rate, i.e. the Purcell effect. While most experimental studies focused on local-field enhancement, the Purcell enhancement offers distinct advantages: it improves the quantum yield of the emitter and avoids the damaging effect of a strong local field. This makes the Purcell enhancement well suited for fluorescent proteins and NIR-fluorophores. Here, we engineer the plasmonic
modes of asymmetric nano-antennas, and modulate the fluorescence emission rate of fluorophores. We demonstrated a useful general strategy for fluorescence enhancement using nano-antennas. Our method has the potential to be applicable to photo-sensitive molecular studies, like bio-chip sensing, DNA sequencing, single-molecule detection /etc. 1485-Pos Board B553 Improving Stability and Sensitivity of SERS-Based Biosensors Joseph Smolsky1, Zakhar Reveguk2, Zachary Sabata1, Alexey Krasnoslobodtsev1. 1 UNO, Omaha, NE, USA, 2St. Petersburg State University, St. Petersburg, Russian Federation. Detection and monitoring of disease biomarkers increases probability of successful disease treatment. Among available readout strategies utilized in biosensors - surface enhanced Raman scattering (SERS) has several advantages over conventional techniques. In particular, multiplexing capabilities of SERS readout is a very attractive feature. Our improvements to SERS readout include protection of SERS labels and temperature assisted plasmonic coupling of DNA modified AuNP contributing to better reproducibility and higher sensitivity of biosensors. We have introduced several protection strategies to overcome SERS signal deterioration under prolonged exposure to intense laser light. Simple protection strategy involves using either polymer or graphene monolayer as a thin protective layer applied on top of the assay that renders signal to be more stable against photo-induced damage. Another protection strategy involves coadsorption of thiolated polyetheleneglycol (PEG) together with Raman reporter molecule (RRM): 4 nitrobenzenethiol (NBT) onto AuNP. These protection strategies improve stability of the assay measured as the time dependence of the Raman signal intensity at 1336 cm 1 (the most intense band for NBT) on duration of sample exposure to laser light. Protection also reduces RRM desorption from the surface of AuNP, photodamage, and catalytic photoconversion of NBT to diazobenzene. We discuss possible mechanisms of such a conversion and effectiveness of PEG protection against it. We have evaluated the size and amount of PEG molecules for optimal protection of SERS labels. Higher sensitivity of the biosensor was achieved via DNA assisted coupling between individual nanoparticles. Our design allowed for controllable plasmonic coupling which resulted in further several fold enhancement of Raman signal, thus improving sensitivity of the biosensor. Practical applications of SERSbased biosensor for in vivo and multiplex detection of biomarkers are also discussed. 1486-Pos Board B554 Controlled Biomolecule Release from a Liposomal Nanocarrier Modulated with Pulsed NIR Light Jeongeun Shin, Maria Olubunmi Ogunyankin, Joseph A. Zasadzinski. Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, USA. A generic delivery platform that could deliver any biomolecule, independent of its chemical constitution, would be a significant advance for researchers in cell biology and neuroscience. At present, small molecule delivery is done via ‘‘caged’’ compounds. However, each bioactive requires the chemical synthesis of its own ‘‘cage’’. As yet, no NIR triggered caged compounds are effective under typical experimental conditions. To address these issues, we have developed liposomes tethered to plasmon-resonant Hollow Gold Nanoshells (HGN) that can be triggered to release their contents by picosecond pulses of physiologically friendly, deeply penetrating near infrared (NIR) light. Liposomes tethered to HGN can encapsulate almost any water-soluble biologically active molecule by confining high concentrations in liposomes tethered to HGN. A major benefit of this technique is the universal mechanism of liposome contents release via nanobubble rupture following pulsed NIR light triggering: any molecule will be released by liposome rupture, so release rates, timing, laser fluence, etc. will be similar for all compounds of interest. By modifying the laser fluence, HGN properties, and liposome membrane composition, we can alter the energy threshold for triggering release, enabling delivery of multiple agents at different times and locations, which is impossible with current liposome or caged compound technologies. Chemically disparate calcium, ATP, carboxyfluorescein (CF), and cisplatin are all released at near 100 % efficiency from liposomes within msec. For a given HGN tethered to the liposome, the threshold energy is lowest at the wavelength corresponding to the maximum adsorption wavelength of the HGN; the threshold energy increases as the wavelength of the NIR light moves away from the maximum. This allows us to create liposomes that can release at different laser fluences so that we could control release rates and windows of each biomolecule in a mixture independently, by delivering two species or even changing the order of release. In this way, we can release one compound at one place and time, then a second compound at the same place at a different time simply by modulating the laser