The concept that catalytic enzymes can act as molecular machines transducing chemical activity into motion has conceptual and experimental support, but much of the claimed support comes from experimental conditions where the substrate concentration is higher than biologically relevant and accordingly exceeds kM, the Michaelis-Menten constant. Moreover, many of the enzymes studied experimentally to date are oligomeric. Urease, a hexamer of subunits, has been considered to be the gold standard demonstrating enhanced diffusion. Here we show that urease and certain other oligomeric enzymes of high catalytic activity above kM dissociate into their smaller subunit fragments that diffuse more rapidly, thus providing a simple physical mechanism of enhanced diffusion in this regime of concentrations. Mindful that this conclusion may be controversial, our findings are supported by four independent analytical techniques, static light scattering, dynamic light scattering (DLS), size-exclusion chromatography (SEC), and fluorescence correlation spectroscopy (FCS). Data for urease are presented in the main text and the conclusion is validated for hexokinase and acetylcholinesterase with data presented in supplementary information. For substrate concentration regimes below kM at which these enzymes do not dissociate, our findings from both FCS and DLS validate that enzymatic catalysis does lead to the enhanced diffusion phenomenon.

It has long been documented that the reduced viscosity of polyelectrolyte has an anomalous dependence on its concentration, i.e., the Fuoss law. To explore the molecular mechanism, the counterion distribution of sodium polystyrenesulfonate (NaPSS) as a function of concentration is investigated at the single-molecule level. By examination of the fluorescence resonance energy transfer (FRET) between a fluorescence donor on NaPSS chain and an acceptor in the counterions using single-molecule fluorescence spectroscopy, an increase of average counterion−chain distance is discovered upon dilution, indicating the expansion of counterion cloud. By photon counting histogram, an increase of effective charge of the NaPSS chain during dilution is exposed. The variation of these parameters agrees well with that of the reduced viscosity, helping to shed light on the molecular mechanism of the Fuoss law: the expansion of the counterion cloud increases hydrodynamic friction, and the increase of effective charges of NaPSS due to desorption of counterions brings about the stronger interchain coupling.

The single most intrinsic property of nonrigid polymer chains is their ability to adopt enormous numbers of chain conformations, resulting in huge conformational entropy. When such macromolecules move in media with restrictive spatial constraints, their trajectories are subjected to reductions in their conformational entropy. The corresponding free energy landscapes are interrupted by entropic barriers separating consecutive spatial domains which function as entropic traps where macromolecules can adopt their conformations more favorably. Movement of macromolecules by negotiating a sequence of entropic barriers is a common paradigm for polymer dynamics in restrictive media. However, if a single chain is simultaneously trapped by many entropic traps, it has recently been suggested that the macromolecule does not undergo diffusion and is localized into a topologically frustrated dynamical state, in apparent violation of Einstein’s theorem. Using fluorescently labeled λ-DNA as the guest macromolecule embedded inside a similarly charged hydrogel with more than 95% water content, we present direct evidence for this new state of polymer dynamics at intermediate confinements. Furthermore, using a combination of theory and experiments, we measure the entropic barrier for a single macromolecule as several tens of thermal energy, which is responsible for the extraordinarily long extreme metastability. The combined theory–experiment protocol presented here is a determination of single-molecule entropic barriers in polymer dynamics. Furthermore, this method offers a convenient general procedure to quantify the underlying free energy landscapes behind the ubiquitous phenomenon of movement of single charged macromolecules in crowded environments.

The physical mechanism of multiple modes in dynamics of polyelectrolyte aqueous solutions has been drawing extensive research attention for decades. This unsolved mystery makes it highly desirable to use new techniques to conduct investigations. In this study, dual-color fluorescence cross-correlation spectroscopy is applied to study the dynamics of individual molecules of a model polyelectrolyte, sodium poly(styrene sulfonate), in aqueous solutions. Anticorrelation in the cross-correlation function is discovered as a result of motion coupling due to interchain electrostatic repulsion. After correction, the self-part of the autocorrelation function is obtained, and the calculated mean square displacement data demonstrate a two-stage diffusion processa fast one at short time lag and a slow one at long time lag. The two processes are attributed to a faster diffusion inside the cage formed by neighboring chains and a slower diffusion beyond the cage. Effects of the salt level and polyelectrolyte concentration are investigated with the comparison with the results of light scattering, showing the connection of the bimodal dynamics and the local ordering in the polyelectrolyte solution.

A negative correlation between the water content inside polymer brushes and protein adsorption.

Using fluorescence microscopy and single-particle tracking, we have directly observed the dynamics of λ-DNA trapped inside poly­(acrylamide-co-acrylate) hydrogels under an externally applied electric field. Congruent with the recent discovery of the nondiffusive topologically frustrated dynamical state (TFDS) that emerges at intermediate confinements between the traditional entropic barrier and reptation regimes, we observe the immobility of λ-DNA in the absence of an electric field. The electrophoretic mobility of the molecule is triggered upon application of an electric field with strength above a threshold value E c. The existence of the threshold value to elicit mobility is attributed to a large entropic barrier, arising from many entropic traps acting simultaneously on a single molecule. Using the measured E c which depends on the extent of confinement, we have determined the net entropic barrier of up to 130 k B T, which is responsible for the long-lived metastable TFDS. The net entropic barrier from multiple entropic traps is nonmonotonic with the extent of confinement and tends to vanish at the boundaries of the TFDS with the single-entropic barrier regime at lower confinements and the reptation regime at higher confinements. We present an estimate of the mesh size of the hydrogel that switches off the nondiffusive TFDS and releases chin diffusion in the heavily entangled state.

One of the major challenges in the technology of sequencing DNA using single-molecule electrophoresis through a nanopore is to control the translocation of the macromolecule across the pore in order to allow sufficient time for accurate sequence reading at limited recording bandwidths. If the translocation speed is too fast, the signatures of the bases passing through the sensing region of the nanopore overlap in time, presenting difficulties in accurately identifying the bases in a sequential manner. Even though several strategies, such as enzyme ratcheting, have been implemented to reduce the translocation speed, the challenge to achieve a substantial reduction in the translocation speed continues to be of paramount significance. Toward achieving this goal, we have fabricated a nonenzymatic hybrid device that can reduce the translocation speed of long DNAs by more than 2 orders of magnitude, in comparison with the current status of the art. This device is made of a tetra-PEG hydrogel that is chemically anchored to the donor side of a solid-state nanopore. The idea behind this device is based on the recent discovery of the topologically frustrated dynamical state of confined polymers, whereby the front hydrogel matter of the hybrid device provides multiple entropic traps for a single DNA molecule holding it back against the electrophoretic driving force that pulls the DNA through the solid-state nanopore portion of the device. As a demonstration of slowing DNA translocation by a factor of about 500, we find the average translocation time realized in the present hybrid device for 3 kbp DNA as 23.4 ms, whereas the corresponding time for the bare solid-state nanopore under otherwise identical conditions is 0.047 ms. Our measurements on 1 kbp DNA and λ-DNA show that such a slowing down of DNA translocation with our hybrid device is general. An additional feature of our hybrid device is its incorporation of all features of the conventional gel electrophoresis to separate different DNA sizes in a clump of DNAs and to streamline them in an orderly and slow manner into the nanopore. Our results suggest the high potential of our hydrogel-nanopore hybrid device in further advancing the single-molecule electrophoresis technology to accurately sequence very large biological polymers.

For the purpose of understanding the molecular origins of the rheological properties of polyelectrolyte solutions, an in situ investigation into the distribution of counterions of a polyelectrolyte under shear is conducted. An apparatus named rheo-microspectrometer is constructed by integrating an advanced rheometer with multiple highly sensitive single-molecule fluorescence microscopic and spectroscopic techniques, including fluorescence correlation spectroscopy, photon counting histogram, single-molecule fluorescence imaging, and fluorescence emission spectroscopy. It has the capacity of measuring the directional and diffusive motions of single fluorescence-labeled polymer molecules as well as their fluorescence emission spectra when the samples are under shear. The shear-induced changes in counterion distribution of a model polyelectrolyte, sodium polystyrene sulfonate (NaPSS), is investigated at the single molecular level. Fluorescence resonance energy transfer between the fluorescence donor attached at the PSS– chain end and the cationic acceptor serving as the counterion probe in solution shows the increase of average counterion–chain distance. The emission spectra of the pH-responsive fluorophore labeled at the PSS– chain end show the dilution of hydronium ions near the chain by shear. The results indicate that the shear applied to the NaPSS solution releases the counterions from the PSS– chain. The mechanism of counterions’ redistribution is attributed to the shear-induced transient overlap of electric fields of multiple PSS– chains. The results indicate that the effective charge of a polyelectrolyte molecule increases with shear, in contrast to the conventional assumption of the invariable charges.