18 research outputs found
Transient Three-Dimensional Orientation of Molecular Ions in an Ordered Polyelectrolyte Membrane
Single-molecule fluorescence spectroscopy is employed to reveal 3D details of the mechanisms underpinning ion transport in a polyelectrolyte thin film possessing polymer-brush nanoscale order. The ability to resolve fluorescence emission over three discrete polarization angles reveals that these ordered materials impart 3D orientation to charged, diffusing molecules. The experiments, supported by simulations, report global orientation parameters for molecular transport, track dipole angle progressions over time, and identify a unique transport mechanism: translational diffusion with restricted rotation. In general, realization of this experimental method for translational diffusion in systems exhibiting basic orientation should lend itself to evaluation of transport in a variety of important, ordered, functional materials
In Situ Measurement of Bovine Serum Albumin Interaction with Gold Nanospheres
We present in situ observations of adsorption of bovine
serum albumin
(BSA) on citrate-stabilized gold nanospheres. We implemented scattering
correlation spectroscopy as a tool to quantify changes in the nanoparticle
Brownian motion resulting from BSA adsorption onto the nanoparticle
surface. Protein binding was observed as an increase in the nanoparticle
hydrodynamic radius. Our results
indicate the formation of a protein monolayer at similar albumin concentrations
as those found in human blood. Additionally, by monitoring the frequency
and intensity of individual scattering events caused by single gold
nanoparticles passing the observation volume, we found that BSA did
not induce colloidal aggregation, a relevant result from the toxicological
viewpoint. Moreover, to elucidate the thermodynamics of the gold nanoparticleâBSA
association, we measured an adsorption isotherm which was best described
by an anticooperative binding model. The number of binding sites based
on this model was consistent with a BSA monolayer in its native state.
In contrast, experiments using polyÂ(ethylene glycol)-capped gold nanoparticles
revealed no evidence for adsorption of BSA
Adsorption of a Protein Monolayer via Hydrophobic Interactions Prevents Nanoparticle Aggregation under Harsh Environmental Conditions
We
find that citrate-stabilized gold nanoparticles aggregate and
precipitate in saline solutions below the NaCl concentration of many
bodily fluids and blood plasma. Our experiments indicate that this
is due to complexation of the citrate anions with Na<sup>+</sup> cations
in solution. A dramatically enhanced colloidal stability is achieved
when bovine serum albumin is adsorbed to the gold nanoparticle surface,
completely preventing nanoparticle aggregation under harsh environmental
conditions where the NaCl concentration is well beyond the isotonic
point. Furthermore, we explore the mechanism of the formation of this
albumin âcoronaâ and find that monolayer protein adsorption
is most likely ruled by hydrophobic interactions. As for many nanotechnology-based
biomedical and environmental applications, particle aggregation and
sedimentation are undesirable and could substantially increase the
risk of toxicological side effects; the formation of the BSA corona
presented here provides a low-cost biocompatible strategy for nanoparticle
stabilization and transport in highly ionic environments
A Two-Step Method for smFRET Data Analysis
We demonstrate a two-step data analysis
method to increase the
accuracy of single-molecule FoÌrster Resonance Energy Transfer
(smFRET) experiments. Most current smFRET studies are at a time resolution
on the millisecond level. When the system also contains molecular
dynamics on the millisecond level, simulations show that large errors
are present (e.g., > 40%) because false state assignment becomes
significant
during data analysis. We introduce and confirm an additional step
after normal smFRET data analysis that is able to reduce the error
(e.g., < 10%). The idea is to use Monte Carlo simulation to search
ideal smFRET trajectories and compare them to the experimental data.
Using a mathematical model, we are able to find the matches between
these two sets, and back guess the hidden rate constants in the experimental
results
A Two-Step Method for smFRET Data Analysis
We demonstrate a two-step data analysis
method to increase the
accuracy of single-molecule FoÌrster Resonance Energy Transfer
(smFRET) experiments. Most current smFRET studies are at a time resolution
on the millisecond level. When the system also contains molecular
dynamics on the millisecond level, simulations show that large errors
are present (e.g., > 40%) because false state assignment becomes
significant
during data analysis. We introduce and confirm an additional step
after normal smFRET data analysis that is able to reduce the error
(e.g., < 10%). The idea is to use Monte Carlo simulation to search
ideal smFRET trajectories and compare them to the experimental data.
Using a mathematical model, we are able to find the matches between
these two sets, and back guess the hidden rate constants in the experimental
results
Single-Molecule FRET Studies of HIV TARâDNA Hairpin Unfolding Dynamics
We directly measure the dynamics
of the HIV trans-activation response
(TAR)âDNA hairpin with multiple loops using single-molecule
FoÌrster resonance energy transfer (smFRET) methods. Multiple
FRET states are identified that correspond to intermediate melting
states of the hairpin. The stability of each intermediate state is
calculated from the smFRET data. The results indicate that hairpin
unfolding obeys a âfraying and peelingâ mechanism, and
evidence for the collapse of the ends of the hairpin during folding
is observed. These results suggest a possible biological function
for hairpin loops serving as additional fraying centers to increase
unfolding rates in otherwise stable systems. The experimental and
analytical approaches developed in this article provide useful tools
for studying the mechanism of multistate DNA hairpin dynamics and
of other general systems with multiple parallel pathways of chemical
reactions
Variable Lysozyme Transport Dynamics on Oxidatively Functionalized Polystyrene Films
Tuning protein adsorption
dynamics at polymeric interfaces is of
great interest to many biomedical and material applications. Functionalization
of polymer surfaces is a common method to introduce application-specific
surface chemistries to a polymer interface. In this work, single-molecule
fluorescence microscopy is utilized to determine the adsorption dynamics
of lysozyme, a well-studied antibacterial protein, at the interface
of polystyrene oxidized via UV exposure and oxygen plasma and functionalized
by ligand grafting to produce varying degrees of surface hydrophilicity,
surface roughness, and induced oxygen content. Single-molecule tracking
indicates lysozyme loading capacities, and surface mobility at the
polymer interface is hindered as a result of all functionalization
techniques. Adsorption dynamics of lysozyme depend on the extent and
the specificity of the oxygen functionalities introduced to the polystyrene
surface. Hindered adsorption and mobility are dominated by hydrophobic
effects attributed to water hydration layer formation at the functionalized
polystyrene surfaces
High-Throughput Screening of Optical Properties of Glass-Supported Plasmonic Nanoparticles Fabricated by Polymer Pen Lithography
Optical applications of plasmonic nanoparticles depend
critically
on particle properties such as relative proximity, composition, crystallinity,
and shape. The most common nanoparticle fabrication techniques, colloidal
synthesis and electron beam lithography, allow the tailoring of some
of these parameters, yet do not provide control over all of them.
Scanning probe block copolymer lithography (SPBCL), a technique that
grows nanoparticles on substrates from precisely deposited precursor
droplets, merges the advantages of colloidal synthesis and electron
beam lithography, and offers high throughput, precise particle positioning,
and composition control. A few challenges with the SBCL method remain:
fabrication of optically relevant particle sizes on optically transparent
supports, and detailed correlation of their optical and morphological
properties. Here, we adapt SPBCL to fabricate large arrays of gold
nanoparticles on glass supports. The resulting nanoparticles have
varying shapes, and at âŒ100 nm in diameter, they support strong
plasmon resonances. In order to fully exploit the high-throughput
fabrication method, we designed an automated dark-field microscope
and correlated the optical behavior to the mechanical properties as
determined through electron and pumpâprobe microscopy. We find
that the SPBCL-synthesized nanoparticles are highly crystalline, supporting
both plasmon oscillations and mechanical vibrations with lifetimes
comparable to colloidal nanospheres. Our work highlights SPBCL as
a promising and versatile synthesis approach for plasmonic nanoparticles,
leading the way toward extensive screening capabilities for optical
properties and hence improved potential applications
Super-Temporal-Resolved Microscopy Reveals Multistep Desorption Kinetics of 뱉Lactalbumin from Nylon
Insight
into the mechanisms driving proteinâpolymer interactions
is constantly improving due to advances in experimental and computational
methods. In this study, we used super-temporal-resolved microscopy
(STReM) to study the interfacial kinetics of a globular protein, α-lactalbumin
(α-LA), adsorbing at the waterânylon 6,6 interface. The
improved temporal resolution of STReM revealed that residence time
distributions involve an additional step in the desorption process.
Increasing the ionic strength in the bulk solution accelerated the
desorption rate of α-LA, attributed to adsorption-induced conformational
changes. Ensemble circular dichroism measurements were used to support
a consecutive reaction mechanism. Without the improved temporal resolution
of STReM, the desorption intermediate was not resolvable, highlighting
both STReMâs potential to uncover new kinetic mechanisms and
the continuing need to push for better time and space resolution
Super Temporal-Resolved Microscopy (STReM)
Super-resolution
microscopy typically achieves high spatial resolution,
but the temporal resolution remains low. We report super temporal-resolved
microscopy (STReM) to improve the temporal resolution of 2D super-resolution
microscopy by a factor of 20 compared to that of the traditional camera-limited
frame rate. This is achieved by rotating a phase mask in the Fourier
plane during data acquisition and then recovering the temporal information
by fitting the point spread function (PSF) orientations. The feasibility
of this technique is verified with both simulated and experimental
2D adsorption/desorption and 2D emitter transport. When STReM is applied
to measure protein adsorption at a glass surface, previously unseen
dynamics are revealed