Single-Molecule Imaging

Total internal reflection fluorescence microscopy (TIRFM) allows us to image millions of individual molecules dynamically at solid/liquid and liquid/liquid interfaces.  As with all single-molecule experimental methods, these experiments go beyond understanding the average behavior of adsorbates and enable us to probe variability in surface chemistry, molecular conformations, and adsorbate dynamics.  Making such measurements, we often find that the behavior is much richer and more interesting than previously suspected.

Dynamics of Proteins on Surfaces

Protein adsorption at the solid-aqueous interface is a widely studied and very complex phenomenon, with applications including biocompatible materials, protein separations, biosensing, and biopharmaceuticals. We are interested in exploring the mechanism by which proteins adsorb to, diffuse on, and desorb from surfaces and the molecular conformations or aggregation states associated with these dynamics.  For example, we observed multiple fibrinogen residence time populations and multiple distinct diffusion modes at an interface.  By correlating these dynamics, on a molecule by molecule basis, these different dynamic populations were found to correspond to different oligomerization states (e.g. protein monomer, dimer or trimer). More recently we have employed intermolecular Förster resonance energy transfer (FRET) to observe the dynamics of protein-protein associations at the interface and found BSA-BSA associations to be heterogeneous, reversible, and highly dynamic.  We have also employed intramolecular FRET to monitor the dynamics of protein conformation changes.  A better understanding of the mechanisms involved in protein adsorption and protein layer formation ultimately can inform the design of all surfaces that come in contact with proteins.

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Mapping Using Accumulated Probe Trajectories (MAPT)

In MAPT, a time series of images is obtained using single-molecule TIFRM, and imaging processing is used to determine the locations of all the individual molecules. By tracking these locations through a time series, physical properties of probe/surface interactions (such as adsorption rates, local diffusion, desorption probability and surface coverage) are determined.  By distributing these events to appropriate locations on the surface, one can determine characteristic values as a function of position. The use of single-molecule localization makes the MAPT analysis intrinsically super-resolution and we have demonstrated resolution of ~100 nm in our un-optimized initial results.  In contrast with all previous super-resolution methods, MAPT can be used on any surface and provides absolute quantitative values of physical interaction properties. Homogenous surfaces can then be characterized and used as “calibration” points to connect small scale heterogeneities to specific surface chemistries.  Using such maps, we found the first experimental evidence for desorption-mediated diffusion at the solid-liquid interface. 

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MAPT images of a photopatterned surface using multiple modes of contrast. (a) adsorption rate (1013 µm-2s-1M-1) (b) average diffusion coefficient (µm2/s) (c) desorption probability per 400 ms and (d) surface coverage/occupancy (1013 µm-2s-1M-1).

Surface Hybridization of DNA

Various technologies rely on measurements of surface hybridization to sequence DNA, detect DNA polymorphisms, and find DNA mutations. Surface hybridization involves a target single stranded deoxyribonucleic acid (ssDNA) molecule engaging in specific interactions (hybridization) with immobilized probe ssDNA molecules. In principle hybridization relies on the thermodynamic equilibrium of hybridized and unhybridized DNA. The complex mechanisms behind this equilibrium involve molecular, electrostatic, and conformational interaction phenomena. Our single-molecule observations suggest that long oligonucleotides adopt conformations minimizing hydrophobic interactions, e.g., by internal sequestration of hydrophobic nucleobases. We are currently using FRET (2-color experiments) to explore the effects of hydrophobicity on surface hybridization.

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Nucleic Acid Binding at Liquid Crystal Interfaces

The unique physical and optical properties of liquid crystals (LCs) facilitate the transduction of molecular binding events into a measurable signal. We are interested in studying how conformational changes that occur upon nucleic acid binding (i.e. DNA hybridization and aptamer-ligand binding) affect the interfacial organization at solid and aqueous LC interfaces. For example, DNA hybridization involves a reduction in hydrophobic exposure which can induce LC reorientations at surfactant-laden aqueous/LC interfaces. Another area of interest involves studying ways to modulate fusogenic activity at aqueous/LC interfaces using specific binding events. These studies have the potential to lead to advanced biosensing strategies while also advancing the understanding of the molecular mechanisms that control bilayer/monolayer fusion.

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DNA hybridization between oligonucleotides anchored in dispersed liposomes and at an aqueous/LC interface promotes liposome fusion and induces LC re-orientation from a planar (left) to homeotropic (right) state.

Molecular Modification of Heterogeneous Catalysts

Performing selective conversions of reagents with multiple functional groups is a challenging objective since each group can potentially adsorb and react on a catalytic surface. One approach involves the modification of supported metal catalysts with organic ligands such as alkanethiols. Alkanethiols can be deposited on metal surfaces to form organized self-assembled monolayers (SAMs) that can greatly alter surface chemistry and potentially be used to significantly enhance selectivity of traditional catalytic systems. We have recently shown that such a strategy can be applied to technical supported catalysts such as Pd/Al2O3. This attachment strategy was found to dramatically enhance the selectivity to the desired products during furfural hydrogenation from < 5% on uncoated catalysts to > 90% on SAM coated catalysts by selectively blocking sites associated with the undesirable reaction pathway. By understanding how these ligands interact both with the surface as well as the reactants, we aim to tune their molecular structure to promote specific interactions for enhancing product selectivity.

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