39 research outputs found
Mapping the Functional Tortuosity and Spatiotemporal Heterogeneity of Porous Polymer Membranes with Super-Resolution Nanoparticle Tracking
As particles flow
through porous media, they follow complex pathways
and experience heterogeneous environments that are challenging to
characterize. Tortuosity is often used as a parameter to characterize
the complexity of pathways in porous materials and is useful in understanding
hindered mass transport in industrial filtration and mass separation
processes. However, conventional calculations of tortuosity provide
only average values under static conditions; they are insensitive
to the intrinsic heterogeneity of porous media and do not account
for potential effects of operating conditions. Here, we employ a high-throughput
nanoparticle tracking method which enables the observation of actual
particle trajectories in polymer membranes under relevant operating
conditions. Our results indicate that tortuosity is not simply a structural
material property but is instead a functional property that depends
on flow rate and particle size. We also resolved the spatiotemporal
heterogeneity of flowing particles in these porous media. The distributions
of tortuosity and of local residence/retention times were surprisingly
broad, exhibiting heavy tails representing a population of highly
tortuous trajectories and local regions with anomalously long residence
times. Interestingly, local tortuosity and residence times were directly
correlated, suggesting the presence of highly confining regions that
cause more meandering trajectories and longer retention times. The
comprehensive information about tortuosity and spatiotemporal heterogeneity
provided by these methods will advance the understanding of complex
mass transport and assist rational design and synthesis of porous
materials
Influence of Protein Surface Coverage on Anomalously Strong Adsorption Sites
Serum albumin is commonly used as
a blocking agent to reduce nonspecific protein adsorption in bioassays
and biodevices; however, the details of this process remain poorly
understood. Using single molecule techniques, we investigated the
dynamics of human serum albumin (HSA) on four model surfaces as a
function of protein concentration. By constructing super-resolution
maps, identifying anomalously strong adsorption sites, and quantifying
surface heterogeneity, we found that the concentration required for
site blocking varied dramatically with surface chemistry. When expressed
in terms of protein surface coverage, however, a more consistent picture
emerged, where a significant fraction of strong sites were passivated
at a fractional coverage of 10<sup>â4</sup>. On fused silica
(FS), ânon-foulingâ oligo (ethylene glycol) functionalized
FS, and hydrophobically modified FS, a modest additional site blocking
effect continued at higher coverage. However, on amine-functionalized
surfaces, the surface heterogeneity exhibited a minimum at a coverage
of âŒ10<sup>â4</sup>. Using intermolecular FoÌrster
resonance energy transfer (FRET), we determined that new anomalous
strong sites were created at higher coverage on amine surfaces and
that adsorption to these sites was associated with proteinâprotein
interactions, i.e., surface-induced aggregation
Unbiased Clustering of Molecular Dynamics for Spatially Resolved Analysis of Chemically Heterogeneous Surfaces
A technique is described for resolving
and interpreting molecular
interactions with a chemically heterogeneous surface. Using total
internal reflection fluorescence microscopy, dynamic single molecule
trajectories were accumulated simultaneously for fluorescently labeled
fatty acid (interacting primarily via hydrophobic interactions) and
dextran (interacting via hydrogen-bonding interactions) probe molecules
at the interface between an aqueous solvent and a photopatterned solid
surface with distinct regions of amine-terminated and polyÂ(ethylene
glycol) self-assembled monolayers. Using dynamic properties of the
probe molecules (adsorption rate, surface diffusion coefficient, residence
time), an unsupervised Gaussian mixture model algorithm was used to
identify areas of the surface that were chemically related to each
other, and the dynamic behaviors of the probe molecules were studied
statistically on these distinct regions. The dynamic data were compared
to data from homogeneous surfaces of known chemistry to provide a
chemical identification of each location on the surface. Spatial maps
were also constructed, allowing for spatial visualization of surface
chemistry on a hydrophilic substrate. This work enables the direct
study of interactions between single-molecule probes and distinct
surface chemistries, even in the presence of spatial heterogeneity,
without human bias, assumptions about surface structure, or model-dependent
analysis
Tracking Nanoparticle Diffusion in Porous Filtration Media
Porous materials are used extensively
in industrial filtration
and mass separation processes, but it is often difficult to predict
their mass transport behavior because porous materials are an inherently
heterogeneous medium and multiple microscopic mechanisms can lead
to macroscopic changes in transport. To provide a microscopic view
of hindered porous transport, we present the results of single-particle
tracking experiments in which we followed the diffusive motion of
individual nanoparticles in commercial filtration media. We compared
two materials, glass fiber and nitrocellulose, with similar nominal
characteristics, but we found that the diffusion behavior of the embedded
particles differed significantly. While diffusion in the glass fiber
material was nearly unhindered, the dynamics were heterogeneous and
significantly slowed in the nitrocellulose. We rationalized the observations
based on differences in geometric hindrance, particle binding, and
hydrodynamic interactions. Our results highlight the ability of single-particle
tracking to differentiate between distinct dynamic mechanisms, and
they suggest that nominal material characteristics may be a poor predictor
of transport properties
Tuning the Flight Length of Molecules Diffusing on a Hydrophobic Surface
Transport at solidâliquid
interfaces is critical to self-assembly,
biosensing, and heterogeneous catalysis, but surface diffusion remains
difficult to characterize and rationally manipulate, due to the inherent
heterogeneity of adsorption on solid surfaces. Using single-molecule
tracking, we characterized the diffusion of a fluorescent long-chain
surfactant on a hydrophobic surface, which involved periods of confinement
alternating with bulk-mediated âflightsâ. The concentration
of methanol in solution was varied to tune the strength of the hydrophobic
surface-molecule interaction. The frequency of confinement had a nonmonotonic
dependence on methanol concentration that reflected the relative influence
of anomalously strong adsorption sites. By carefully accounting for
the effect of this surface heterogeneity, we demonstrated that flight
lengths increased monotonically as the hydrophobic attraction decreased,
in agreement with theoretical predictions for bulk-mediated surface
diffusion. The theory provided an accurate description of surface
diffusion, despite the system being heterogeneous, and can be leveraged
to optimize molecular search and assembly processes
Mechanisms of Surface-Mediated DNA Hybridization
Single-molecule total internal reflection fluorescence microscopy was employed in conjunction with resonance energy transfer (RET) to observe the dynamic behavior of donor-labeled ssDNA at the interface between aqueous solution and a solid surface decorated with complementary acceptor-labeled ssDNA. At least 100â000 molecular trajectories were determined for both complementary strands and negative control ssDNA. RET was used to identify trajectory segments corresponding to the hybridized state. The vast majority of molecules from solution adsorbed nonspecifically to the surface, where a brief two-dimensional search was performed with a 7% chance of hybridization. Successful hybridization events occurred with a characteristic search time of âŒ0.1 s, and unsuccessful searches resulted in desorption from the surface, ultimately repeating the adsorption and search process. Hybridization was reversible, and two distinct modes of melting (<i>i</i>.<i>e</i>., dehybridization) were observed, corresponding to long-lived (âŒ15 s) and short-lived (âŒ1.4 s) hybridized time intervals. A strand that melted back onto the surface could rehybridize after a brief search or desorb from the interface. These mechanistic observations provide guidance for technologies that involve DNA interactions in the near-surface region, suggesting a need to design surfaces that both enhance the complex multidimensional search process and stabilize the hybridized state
Effects of Molecular Size and Surface Hydrophobicity on Oligonucleotide Interfacial Dynamics
Single-molecule total internal reflection fluorescence
microscopy
was used to observe the dynamic behavior of polycytosine single-stranded
DNA (ssDNA) (1â50 nucleotides long) at the interface between
aqueous solution and hydrophilic (oligoethylene glycol-modified fused
silica, OEG) and hydrophobic (octadecyltriethoxysilane-modified fused
silica, OTES) solid surfaces. High throughput molecular tracking was
used to determine >75â000 molecular trajectories for each
molecular
length, which were then used to calculate surface residence time and
squared displacement (i.e., âstep-sizeâ) distributions.
On hydrophilic OEG surfaces, the surface residence time increased
systematically with ssDNA chain length, as expected due to increasing
moleculeâsurface interactions. Interestingly, the residence
time decreased with increasing ssDNA length on the hydrophobic OTES
surface, particularly for longer chains. Similarly, the interfacial
mobility of polynucleotides slowed with increasing chain length on
OEG, but became faster on OTES. On OTES surfaces, the rates associated
with desorption and surface diffusion exhibited the distinctive anomalous
temperature dependence that is characteristic of hydrophobic interactions
for short-chain species but not for longer chains. These combined
observations suggest that long oligonucleotides adopt conformations
minimizing hydrophobic interactions, e.g., by internal sequestration
of hydrophobic nucleobases
Three Regimes of Polymer Surface Dynamics under Crowded Conditions
Single-molecule tracking
was used to characterize the mobility
of polyÂ(ethylene glycol) chains at a solidâliquid interface
over a wide range of surface coverage. Trajectories exhibited intermittent
motion consistent with a generalized continuous time random walk (CTRW)
model, where strongly confined âwaiting timesâ alternated
with rapid flights. The presence of three characteristic regimes emerged
as a function of surface coverage, based on an analysis of effective
short-time diffusion coefficients, mean-squared displacement, and
CTRW distributions. The dilute âsite-blockingâ regime
exhibited increasing short-time diffusion, less confined behavior,
and shorter waiting times with higher surface coverage, as anomalously
strong adsorption sites were increasingly passivated. At intermediate
values of surface coverage, the âcrowdingâ regime was
distinguished by the exact opposite trends (slower, more confined
mobility), presumably due to increasing intermolecular interactions.
The trends reversed yet again in the âbrushâ regime,
where adsorbing molecules interacted weakly with a layer of extended
overlapping chains
Single Molecule Dynamics on Hydrophobic Self-Assembled Monolayers
The interactions between adsorbate molecules and hydrophobic
surfaces
are of significant interest due to their importance in a variety of
biological and separation processes. However, it is challenging to
extrapolate macroscopic ensemble-averaged force measurements to molecular-level
phenomena. Using total internal reflection fluorescence microscopy
to image individual molecules at hydrophobic solidâaqueous
interfaces, we directly observed dynamic behavior associated with
the interactions between fluorescently labeled dodecanoic acid (our
probe molecules) and self-assembled monolayers (SAM) comprising <i>n</i>-alkyltriethoxysilanes with systematically increasing chain
length (from <i>n</i> = 4â18). In all cases, we observed
at least two characteristic surface residence times and two diffusive
modes, suggesting the presence of multiple distinct adsorbed populations.
In general, the mean surface residence time increased and the mobility
decreased with increasing SAM chain length, consistent with stronger
probeâsurface interactions. However, these trends were not
primarily due to changes in characteristic residence times or diffusion
coefficients associated with the individual populations but rather
to a dramatic increase in the fraction associated with the long-lived
slow-moving population(s) on long-chain SAMs. In particular, on longer
(16â18 carbon) alkylsilane monolayers, the probe molecule exhibited
far fewer desorption-mediated âflightsâ than on short
(4â6 carbon) monolayers. Additionally, probes on the longer
chain surfaces were much more likely to exhibit extended surface residence
times as opposed to short transient surface visits
Capturing Conformation-Dependent MoleculeâSurface Interactions When Surface Chemistry Is Heterogeneous
Molecular building blocks, such as carbon nanotubes and DNA origami, can be fully integrated into electronic and optical devices if they can be assembled on solid surfaces using biomolecular interactions. However, the conformation and functionality of biomolecules depend strongly on the local chemical environment, which is highly heterogeneous near a surface. To help realize the potential of biomolecular self-assembly, we introduce here a technique to spatially map molecular conformations and adsorption, based on single-molecule fluorescence microscopy. On a deliberately patterned surface, with regions of varying hydrophobicity, we characterized the conformations of adsorbed helicogenic alanine-lysine copeptides using FoÌrster resonance energy transfer. The peptides adopted helical conformations on hydrophilic regions of the surface more often than on hydrophobic regions, consistent with previous ensemble-averaged observations of α-helix surface stability. Interestingly, this dependence on surface chemistry was not due to surface-induced unfolding, as the apparent folding and unfolding dynamics were usually much slower than desorption. The most significant effect of surface chemistry was on the adsorption rate of molecules as a function of their initial conformational state. In particular, regions with higher adsorption rates attracted more molecules in compact, disordered coil states, and this difference in adsorption rates dominated the average conformation of the ensemble. The correlation between adsorption rate and average conformation was also observed on nominally uniform surfaces. Spatial variations in the functional state of adsorbed molecules would strongly affect the success rates of surface-based molecular assembly and can be fully understood using the approach developed in this work