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^–4. On fused silica (FS), “non-fouling” 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 ∼10^–4. Using intermolecular Förster 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 protein–protein 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 poly­(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

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 100 000 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 ∼0.1 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 (∼15 s) and short-lived (∼1.4 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) (1–50 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 >75 000 molecular trajectories for each molecular length, which were then used to calculate surface residence time and squared displacement (i.e., “step-size”) distributions. On hydrophilic OEG surfaces, the surface residence time increased systematically with ssDNA chain length, as expected due to increasing molecule–surface 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

Tuning the Flight Length of Molecules Diffusing on a Hydrophobic Surface

Transport at solid–liquid 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 “flights”. 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

Three Regimes of Polymer Surface Dynamics under Crowded Conditions

Single-molecule tracking was used to characterize the mobility of poly­(ethylene glycol) chains at a solid–liquid 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 “waiting times” 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 “site-blocking” 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 “crowding” 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 “brush” 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 solid–aqueous 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> = 4–18). 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 probe–surface 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 (16–18 carbon) alkylsilane monolayers, the probe molecule exhibited far fewer desorption-mediated “flights” than on short (4–6 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 Molecule–Surface 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 Förster 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