Papers by Rosina Georgiadis
Kinetics of DNA hybridization in solution
<b>Copyright information:</b>Taken from "Secondary structure effects on DNA hybr... more <b>Copyright information:</b>Taken from "Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison"Nucleic Acids Research 2006;34(11):3370-3377.Published online 5 Jul 2006PMCID:PMC1488884.© 2006 The Author(s) [Probe] = [Target] = 1 μM, = 20°C, and [NaCl] = 0.5 M. () Normalized absorbance change upon injection or hand mixing of single strands as a function of time. The raw values of Δ at 'infinite time' or equilibrium are 0.079, 0.063, 0.049 and 0.053 for PT, PT, PT and PT, respectively. These values generally scale with the net number of Watson–Crick base pairs formed during hybridization (for details see text). () Linearized absorbance data plotted as inverse concentration versus time. Solid lines are fits to the data and the slopes correspond to the second-order hybridization rate constant for each set of sequences. Mixing of PT, PT and PT was done by hand pipetting, while PT was carried out by syringe injection.
Surface hybridization kinetics
<b>Copyright information:</b>Taken from "Secondary structure effects on DNA hybr... more <b>Copyright information:</b>Taken from "Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison"Nucleic Acids Research 2006;34(11):3370-3377.Published online 5 Jul 2006PMCID:PMC1488884.© 2006 The Author(s) Hybridizations are monitored for many hours, but only shown on a 30 min time-scale here. Hybridization of duplexes PT and PT is very fast, while hybridization of PT is very slow. Duplex PT reaches 20% hybridization efficiency, but only after overnight target exposure. For all hybridizations, [Target] = 1 μM, T = 20°C, and [NaCl] = 0.5 M. Exposure of each probe surface to control target strands yields no significant binding (data not shown). Probe densities are 4.5 × 10, 6.8 × 10 and 5.7 × 10 molecules/cm for P, P and P probe surfaces, respectively. (Inset) Hybridizations normalized at 30 min for comparison of kinetic rates.
Dpal
Distributed Pharmaceutical Analysis Lab
In-Situ Two-Color Surface Plasmon Resonance Spectroscopy of Molecular Processes in Organic Thin Films
Distance- and Wavelength-Dependent Dielectric Function of Au Nanoparticles by Angle-Resolved Surface Plasmon Resonance Imaging
The Journal of Physical Chemistry C, 2010
ABSTRACT The optical properties of nanoparticles (NPs) near metal surfaces must be better underst... more ABSTRACT The optical properties of nanoparticles (NPs) near metal surfaces must be better understood in order to fully exploit their signal-enhancing capabilities in optical biosensors, such as in surface plasmon resonance studies. We use angle-resolved SPR imaging and a modified Maxwell−Garnett model to determine the optical properties of 10-nm-diameter gold NPs deposited on planar Au coated with various thicknesses of SiO2. We investigate how the intrinsic NP dielectric constants and extinction coefficients vary as a function of particle-to-metal-substrate distance (dP−S) for short distances that span the dimension of the NP (0−26 nm) and as a function of excitation wavelength (λexc) for values that span the NP absorbance spectrum. When the NPs are deposited almost directly onto the Au substrate, the NP dielectric function shows anomalous dispersion. At distances far from the planar Au, normal dispersion is observed. The distance dependence, viewed in terms of extinction coefficients, is consistent with a red shift in the NP absorption as dP−S decreases. Although this shift has been predicted and observed, the distance dependence of the intrinsic NP dielectric function is not known. Indeed, NP dielectric functions, which are exquisitely sensitive to experimental conditions, are generally not available. Such fundamental NP properties are needed for quantitative applications in high-sensitivity refractive-index-based biosensors and may also be useful for testing theoretical models.
Two-color approach for determination of thickness and dielectric constant of thin films using surface plasmon resonance spectroscopy
Optics Communications, 1996
ABSTRACT
Nucleic Acids Research, 2001
The hybridization of complementary strands of DNA is the underlying principle of all microarray-b... more The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.
Nucleic Acids Research, 2006
The hybridization kinetics for a series of designed 25mer probeÀtarget pairs having varying degre... more The hybridization kinetics for a series of designed 25mer probeÀtarget pairs having varying degrees of secondary structure have been measured by UV absorbance and surface plasmon resonance (SPR) spectroscopy in solution and on the surface, respectively. Kinetic rate constants derived from the resultant data decrease with increasing probe and target secondary structure similarly in both solution and surface environments. Specifically, addition of three intramolecular base pairs in the probe and target structure slow hybridization by a factor of two. For individual strands containing four or more intramolecular base pairs, hybridization cannot be described by a traditional two-state model in solutionphase nor on the surface. Surface hybridization rates are also 20-to 40-fold slower than solution-phase rates for identical sequences and conditions. These quantitative findings may have implications for the design of better biosensors, particularly those using probes with deliberate secondary structure.
Langmuir, 2000
Several groups have shown that alkanethiol-modified pyrroles can be tethered to a gold surface, b... more Several groups have shown that alkanethiol-modified pyrroles can be tethered to a gold surface, but there is often little evidence that, once oxidized, the resulting monolayer film is an organic conducting polymer. Using surface plasma resonance (SPR) spectroscopy, we demonstrate for the first time that, upon electrochemical oxidation, self-assembled alkanethiol-pyrrole films on gold show behavior characteristic of organic conducting polymers: we observe reversible changes in the optical constants of the organic film upon doping/dedoping. Since the optical constants are related to film conductivity, we show that the effective isotropic dielectric constant of the film obtained in the standard SPR data analysis can be interpreted in terms of in-plane and out-of-plane contributions to film conductivity. We find that the in-plane conductivity of oxidized 3-(ω-mercaptoundecyl)pyrrole is smaller, but of the same order of magnitude, than that found for thick films of polypyrrole. Most importantly, we observe reversible changes in the optical constants of the polymerized film, which are consistent with electrochemical switching of an organic conducting polymer whose conductivity is largest for the doped state and decreases for the dedoped state.
In Situ Kinetics of Self-Assembly by Surface Plasmon Resonance Spectroscopy
Langmuir, 1996
... and R. Georgiadis* . ... Other high index coupling fluids flowed out too quickly for our mea... more ... and R. Georgiadis* . ... Other high index coupling fluids flowed out too quickly for our measurements. ... Gold-coated sapphire windows were prepared by electron beam evaporation of 10 Å of Cr followed by 500 Å gold (99.99%) (1−2 Å/s) at a base pressure of 7 × 10 - 7 Torr. ...
Journal of the American Chemical Society, 2002
We investigate how probe density influences hybridization for unlabeled target oligonucleotides t... more We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sampleto-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.
Observation of Hybridization and Dehybridization of Thiol-Tethered DNA Using Two-Color Surface Plasmon Resonance Spectroscopy
Journal of the American Chemical Society, 1997
Biosensor arrays of immobilized single-stranded DNA (ss-DNA) have the potential for quickly seque... more Biosensor arrays of immobilized single-stranded DNA (ss-DNA) have the potential for quickly sequencing and assaying DNA samples by selective hybridization. 1, 2 Applications of this technology include the sequencing of the entire human genome (The Human ...
Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator/IKKβ Protein–Protein Interface
Journal of the American Chemical Society, 2013
We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction... more We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between nuclear factor kappa B (NF-κB) essential modulator (NEMO) and IκB kinase subunit β (IKKβ), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NEMO binding domain (NBD) region of IKKβ contains the highest concentration of hot-spot residues, the strongest of which are W739, W741, and L742 (ΔΔG = 4.3, 3.5, and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKβ L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot-spot regions centered on IKKβ residues L708/V709 and L719/I723. The computational approach successfully identified all three hot-spot regions on IKKβ. Moreover, the method was able to accurately quantify the energetic importance of all hot-spot residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small-molecule inhibitors that target the NEMO/IKKβ interaction. They additionally clarify the structural and energetic complementarity between &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;pocket-forming&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;pocket-occupying&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; hot-spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.
Analytical Chemistry, 2008
Here we present an automated angle-scanning surface plasmon resonance imaging (SPRi) instrument w... more Here we present an automated angle-scanning surface plasmon resonance imaging (SPRi) instrument which provides multiplexed, quantitative reflectance data over a wide angular range. Angle-dependent artifacts, which arise from the simple optical setup, are corrected using software. This enables monitoring of significantly different surface coatings in many solvents, which would be outside the dynamic range of typical fixed-angle instruments. Operation in the visible to near-infrared range without the need for reconfiguration extends the instrument capabilities to increase sensitivity or to investigate the optical properties of surface films. This instrument provides maximum flexibility to study a wide range of systems with full exploitation of the quantitative capabilities of SPRi achieved by fitting data to the Fresnel model.
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Papers by Rosina Georgiadis