Master curve of boosted diffusion for 10 catalytic enzymes.

Molecular agitation more rapid than thermal Brownian motion is reported for cellular environments, motor proteins, synthetic molecular motors, enzymes, and common chemical reactions, yet that chemical activity coupled to molecular motion contrasts with generations of accumulated knowledge about diffusion at equilibrium. To test the limits of this idea, a critical testbed is the mobility of catalytically active enzymes. Sentiment is divided about the reality of enhanced enzyme diffusion, with evidence for and against. Here a master curve shows that the enzyme diffusion coefficient increases in proportion to the energy release rate-the product of Michaelis-Menten reaction rate and Gibbs free energy change (ΔG)-with a highly satisfactory correlation coefficient of 0.97. For 10 catalytic enzymes (urease, acetylcholinesterase, seven enzymes from the glucose cascade cycle, and one other), our measurements span from a roughly 40% enhanced diffusion coefficient at a high turnover rate and negative ΔG to no enhancement at a slow turnover rate and positive ΔG Moreover, two independent measures of mobility show consistency, provided that one avoids undesirable fluorescence photophysics. The master curve presented here quantifies the limits of both ideas, that enzymes display enhanced diffusion and that they do not within instrumental resolution, and has possible implications for understanding enzyme mobility in cellular environments. The striking linear dependence of ΔG for the exergonic enzymes (ΔG <0), together with the vanishing effect for endergonic enzyme (ΔG >0), are consistent with a physical picture in which the mechanism boosting the diffusion is an active one, utilizing the available work from the chemical reaction.

Catalytic enzymes are active matter.

Using a microscopic theory to analyze experiments, we demonstrate that enzymes are active matter. Superresolution fluorescence measurements-performed across four orders of magnitude of substrate concentration, with emphasis on the biologically relevant regime around or below the Michaelis-Menten constant-show that catalysis boosts the motion of enzymes to be superdiffusive for a few microseconds, enhancing their effective diffusivity over longer timescales. Occurring at the catalytic turnover rate, these fast ballistic leaps maintain direction over a duration limited by rotational diffusion, driving enzymes to execute wormlike trajectories by piconewton forces performing work of a few kBT against viscosity. The boosts are more frequent at high substrate concentrations, biasing the trajectories toward substrate-poor regions, thus exhibiting antichemotaxis, demonstrated here experimentally over a wide range of aqueous concentrations. Alternative noncatalytic, passive mechanisms that predict chemotaxis, cross-diffusion, and phoresis, are critically analyzed. We examine the physical interpretation of our findings, speculate on the underlying mechanism, and discuss the avenues they open with biological and technological implications. These findings violate the classical paradigm that chemical reaction and motility are distinct processes, and suggest reaction-motion coupling as a general principle of catalysis.

Enzyme leaps fuel antichemotaxis.

There is mounting evidence that enzyme diffusivity is enhanced when the enzyme is catalytically active. Here, using superresolution microscopy [stimulated emission-depletion fluorescence correlation spectroscopy (STED-FCS)], we show that active enzymes migrate spontaneously in the direction of lower substrate concentration ("antichemotaxis") by a process analogous to the run-and-tumble foraging strategy of swimming microorganisms and our theory quantifies the mechanism. The two enzymes studied, urease and acetylcholinesterase, display two families of transit times through subdiffraction-sized focus spots, a diffusive mode and a ballistic mode, and the latter transit time is close to the inverse rate of catalytic turnover. This biochemical information-processing algorithm may be useful to design synthetic self-propelled swimmers and nanoparticles relevant to active materials. Executed by molecules lacking the decision-making circuitry of microorganisms, antichemotaxis by this run-and-tumble process offers the biological function to homogenize product concentration, which could be significant in situations when the reactant concentration varies from spot to spot.

Enhanced Diffusion and Oligomeric Enzyme Dissociation.

The concept that catalytic enzymes can act as molecular machines transducing chemical activity into motion has conceptual and experimental support, but experimental support has involved oligomeric enzymes, often studied under conditions where the substrate concentration is higher than biologically relevant and accordingly exceeds kM, the Michaelis constant. 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 dissociate above kM into their subunits that diffuse more rapidly, thus providing a simple physical mechanism that contributes to 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 emphasized and the conclusion is validated for hexokinase, acetylcholinesterase, and aldolase. For hexokinase and aldolase no enhanced diffusion is observed except under conditions when these oligomeric enzymes dissociate. At substrate concentration regimes below kM at which acetylcholinesterase and urease do not dissociate, our finding showing up to 10% enhancement of the diffusion coefficient is consistent with various theoretical scenarios in the literature.

Robustness of FCS (Fluorescence Correlation Spectroscopy) with Quenchers Present.

Inspired by recent publications doubtful of the FCS technique, we scrutinize how irreversible ("static") and reversible ("dynamic") quenching can influence the interpretation of such data. Textbook presentations often emphasize only how to analyze data in extremes, the absence of quenching or the presence of substantial quenching. Here, we consider intermediate cases where the assessment of photophysics (static quenching, blinking-like triplet-state relaxation) influence on autocorrelation curves can be delicate if dye-labeled objects diffuse on comparably rapid time scales. We used the amino acid, tryptophan, as the quencher. As our example of small-molecule dye that diffuses rapidly, we mix the quencher with the fluorescence dye, Alexa 488. The translational diffusion coefficient, inferred from fit to the standard one-component Fickian diffusion model, speeds up without the loss of quality of fit, but quenching is reflected in the fact that the data become exceptionally noisy. This reflects the bidisperse population of quenched and unquenched dyes when the time scales overlap between the processes of translational diffusion, quenching, and blinking. As our example of the large-molecule dye-labeled object that diffuses relatively slowly, we mixed the quencher with dye-labeled BSA, bovine serum albumin. Diffusion, static quenching, and blinking time scales are now separated. In spite of quenching contribution to the autocorrelation function when the delay time is relatively short, the inferred translational diffusion coefficient now depends weakly on the presence of a quencher. We conclude that when the diffusing molecule is substantially slower to diffuse than the time scale of photophysical processes of the fluorescent dye to which it is attached, the influence of quenching is self-evident and the FCS autocorrelation curves give an appropriate diffusion coefficient if correct fitting functions are chosen in the analysis.

Deep line-temporal focusing with high axial resolution and a large field-of-view using intracavity control and incoherent pulse shaping.

Line-temporal focusing has been recognized as an elegant strategy that provides two-photon microscopy with an effective means for fast imaging through parallelization, together with an improved resilience to scattering for deep imaging. However, the axial resolution remains sub-optimal, except when using high NA objectives and a small field-of-view. With the introduction of an intracavity control of the spectral width of the femtosecond laser to adaptively fill the back aperture of the objective lens, line-temporal focusing two-photon microscopy is demonstrated to reach near-diffraction-limited axial resolution with a large back-aperture objective lens, and improved immunity to sample scattering. In addition, a new incoherent flattop beam shaping method is proposed which provides a uniform contrast with little degradation of the axial resolution along the focus line, even deep in the sample. This is demonstrated in large volumetric imaging of mouse lung samples.

DNA molecules deviate from shortest trajectory when driven through hydrogel.

Dynamic fluorescence-based single-molecule imaging of λ-DNA molecules driven through agarose hydrogels by DC electric fields reveals that passage through the hydrogel (98.5% water content) induces mobility orthogonal to the external field. Tortuous paths followed by the DNA molecules, which are heavily entangled in the hydrogel mesh as their contour length is nearly 100 times the hydrogel mesh size of 200 nm, cause them to appear to diffuse orthogonal to the driving force. The higher the driving field, from 2 to 16 V/cm, the higher the off-axis dispersion is, over the same time interval. We measure the off-axis displacement distribution over 3 orders of magnitude of probability density and find a master curve after normalizing for time (t) elapsed, but the power of time for normalizing increases with the external field, from t0.25 to t0.6 with increasing field. Comparing trajectories over the same distance traveled in the electric field direction, we observe whereas for the highest field strengths DNA molecules come closest to taking the shortest trajectory between two points in space, deviations from the shortest trajectory grow larger and larger (up to 40% larger) as one approaches the case of small yet finite external field strength.

Nanoparticle puzzles and research opportunities that go beyond state of the art.

We present an overview of current progress and research challenges in the field of nanoparticle assembly, touching on the following topics: (1) historical perspective; (2) consideration of what is a nanoparticle; (3) contrast between nanoparticle self-assembly and top-down construction; (4) opportunities for nanoparticles with more intelligent sub-structures; (5) opportunities for nanoparticle systems cued to interact subtly in space and time. In this personal and subjective account, certain holy grails for nanoparticle science and technology are identified.

Colloidal-sized metal-organic frameworks: synthesis and applications.

Colloidal metal-organic frameworks (CMOFs), nanoporous colloidal-sized crystals that are uniform in both size and polyhedral shape, are crystals composed of metal ions and organic bridging ligands, which can be used as building blocks for self-assembly in organic and aqueous liquids. They stand in contrast to conventional metal-organic frameworks (MOFs), which scientists normally study in the form of bulk crystalline powders. However, powder MOFs generally have random crystal size and shape and therefore do not possess either a definite mutual arrangement with adjacent particles or uniformity. CMOFs do have this quality, which can be important in vital uptake and release kinetics. In this Account, we present the diverse methods of synthesis, pore chemistry control, surface modification, and assembly techniques of CMOFs. In addition, we survey recent achievements and future applications in this emerging field. There is potential for a paradigm shift, away from using just bulk crystalline powders, towards using particles whose size and shape are regulated. The concept of colloidal MOFs takes into account that nanoporous MOFs, conventionally prepared in the form of bulk crystalline powders with random crystal size, shape, and orientation, may also form colloidal-sized objects with uniform size and morphology. Furthermore, the traditional MOF functions that depend on porosity present additional control over those MOF functions that depend on pore interactions. They also can enable controlled spatial arrangements between neighboring particles. To begin, we discuss progress regarding synthesis of MOF nano- and microcrystals whose crystal size and shape are well regulated. Next, we review the methods to modify the surfaces with dye molecules and polymers. Dyes are useful when seeking to observe nonluminescent CMOFs in situ by optical microscopy, while polymers are useful to tune their interparticle interactions. Third, we discuss criteria to assess the stability of CMOFs for various applications. In another section of this Account, we give examples of supracrystal assembly in liquid, on substrates, at interfaces, and under external electric fields. We end this Account with discussion of possible future developments, both conceptual and technological.

Communication: Time-resolved fluorescence of highly single crystalline molecular wires of azobenzene.

We report the enhanced fluorescence with the remarkably long lifetime (1.17 ns) in the first excited state (S(1)) of highly crystalline molecular wires of azobenzene at the excitation wavelength of 467 nm for the first time. This observation suggests that trans-cis photoisomerization through the rotation or inversion mechanism may not be a favorable pathway after excitation to the S(1) state in highly single crystalline molecular wires of azobenzene due to the hindered motion within densely packed crystal structure. We also measured the fluorescence lifetime image of a single crystalline molecular wire of azobenzene, indicating that the lifetime was remarkably uniform and that there was only a very minor variation within the crystal.

Light-induced isomerization dynamics of a cyanine dye in the modulus-controlled regime.

The trans-cis isomerization of an excited molecule converts light energy into mechanical motion, which interacts cooperatively with its surroundings. To understand such a photodynamic process in solids, we investigated the internal twisting motion of 1,1'-diethyl-2,2'-cyanine iodide (DCI) in a series of poly(alkyl methacrylate) (PAMA) polymers by measuring the Young's moduli of the polymers with atomic force microscopy nanoindentation and the fluorescence lifetimes of the dye with time-correlated single photon counting. We found that the isomerization rate constant obtained from the average lifetime correlated well with the mechanical property of the matrix. Our results show that the light-induced molecular motion lies in the modulus-controlled regime in which the polymer matrix not only provides a rigid environment for the dynamics of the molecules but also participates actively in the motion. The concept of elastic modulus may be applicable to molecular rotor dynamics in any synthetic polymer and, in principle, can be extended to biopolymers such as proteins or DNA.

Photomechanical effects in a dye-doped polymer nanocomposite.

Nile Red (NR) has been widely used as a microenvironmental probe since its luminescence characteristics depend strongly on medium polarity, viscosity, and hydrophobicity. The driving source for the internal motion of NR in rigid media is an absorbed photon that induces the molecule to rotate internally, causing the matrix deformed. Reversible (elastic) deformation and irreversible (plastic) deformation will influence the twisting dynamics in a different manner. In this work, we have investigated its excited state motion in a polymer nanocomposite, wherein polyvinyl alcohol (PVA) and nanodiamonds (NDs) were used as a matrix and a filler, respectively. PVA is a hydrophilic polymer having good chemical resistance, processability, and gel formation ability. Nanodiamond is a good candidate as a nanofiller for polymer composites. The elastic modulus of the polymer nanocomposite was measured by atomic force microscopy (AFM) nanoindentation and the emission lifetime of NR embedded in the polymer nanocomposite by time-resolved emission spectroscopy. Our results show that the fluorescence lifetime of NR is correlated well to the elastic modulus of polymer nanocomposite.

Mechanical properties of polycarbonate and poly(methyl methacrylate) films reinforced with surface-functionalized nanodiamonds.

Polymer nanocomposites (PNCs) possess highly versatile characteristics, depending on the nanofiller properties such as its chemical composition, particle size, dimension, polydispersity, concentration, or surface functional groups. In comparison with micron-sized materials, the nanofiller having a large surface area facilitates stronger interaction with the matrix. In this work, various surface-functionalized nanodiamonds (sf-NDs) having hydroxyl, carboxyl, amino, and amide group were prepared, and dispersed into polycarbonate (PC) and poly(methyl methacrylate) (PMMA) polymers. The polymer nanocomposites (PNCs) which contain the ND content of 5 wt% were subjected to the measurements of mechanical properties such as hardness and Young's modulus by atomic force microscopy (AFM) nanoindentation. It was observed that the hardness and Young's modulus of the polymer nanocomposites depend on strongly the nature of functional groups. The amine or amide functionalization gives the high mechanical properties for both polymers. The interfacial interaction between sf-NDs and polymer matrices is an important factor determining the mechanical properties of the PNCs.

Mixed-Charge Nanocarriers Allow for Selective Targeting of Mitochondria by Otherwise Nonselective Dyes.

Targeted delivery of molecular cargos to specific organelles is of paramount importance for developing precise and effective therapeutics and imaging probes. This work describes a disulfide-based delivery method in which mixed-charged nanoparticles traveling through the endolysosomal tract deliver noncovalently bound dye molecules selectively into mitochondria. This system comprises three elements: (1) The nanoparticles deliver their payloads by a kiss-and-go mechanism - that is, they drop off their dye cargos proximate to mitochondria but do not localize therein; (2) the dye molecules are by themselves nonspecific to any cellular structures but become so with the help of mixed-charge nanocarriers; and (3) the dye is engineered in such a way as to remain in mitochondria for a long time, up to days, allowing for observing dynamic remodeling of mitochondrial networks and long-term tracking of mitochondria even in dividing cells. The selectivity of delivery and long-lasting staining derive from the ability to engineer charge-imbalanced, mixed [+/-] on-particle monolayers and from the structural features of the cargo. Regarding the former, the balance of [+] and [-] ligands can be adjusted to limit cytotoxicity and control the number of dye molecules adsorbed onto the particles' surfaces. Regarding the latter, comparative studies with multiple dye derivatives we synthesized rationalize the importance of polar groups, long alkyl chains, and disulfide moieties in the assembly of fluorescent nanoconstructs and long-lasting staining of mitochondria. Overall, this strategy could be useful for delivering hydrophilic and/or anionic small-molecule drugs difficult to target to mitochondria by classical approaches.

Internal motion of an electronically excited molecule in viscoelastic media.

The twisting motion of trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (4-DASPI) in the excited state was investigated in solutions and various polymers in order to understand dependence of molecular rotor dynamics on viscoelasticity. It was observed that the internal motion of electronically excited 4-DASPI correlates strongly with dynamic viscosity and elastic modulus. Our results also showed that condensed phase dynamics of 4-DASPI are governed by the explicit mode coupling between the rotamerizing coordinate and mechanical properties of viscoelastic media.

Soft-chemical exfoliation route to layered cobalt oxide monolayers and its application for film deposition and nanoparticle synthesis.

A colloidal suspension of exfoliated, layered cobalt oxide nanosheets has been synthesized through the intercalation of quaternary tetramethylammonium ions into protonated lithium cobalt oxide. According to atomic force microscopy, exfoliated nanosheets of layered cobalt oxide show a plateau-like height profile with nanometer-level height, underscoring the formation of unilamellar 2D nanosheets. The exfoliation of layered cobalt oxide was cross-confirmed by X-ray diffraction, UV/Vis spectroscopy, and transmission electron microscopy. The maintenance of the hexagonal in-plane structure of the cobalt oxide lattice after the exfoliation process was evidenced by selected-area electron diffraction and Co K-edge X-ray absorption near-edge structure analysis. The zeta-potential measurements clearly demonstrated the negative surface charge of cobalt oxide nanosheets. Adopting the nanosheets of layered cobalt oxide as a precursor, we were able to prepare the monodisperse CoO nanocrystals with a particle size of approximately 10 nm as well as the heterolayered film composed of cobalt oxide monolayer and polycation.

Molecular rotor dynamics influenced by the elastic modulus of polyethylene nanocomposites.

We observed that the excited-state twisting motion of 3,3(')-diethyloxacarbocyanine in polymer nanocomposites (PNCs) depends strongly on the elastic modulus of medium. PNCs consist of low density polyethylene dispersed with surface-functionalized nanodiamonds with various alkyl groups. The mechanical properties of the PNCs were measured by a nanoindentation method, and the photoisomerization processes of the cyanine dye doped in the composites were investigated by time-resolved fluorescence spectroscopy. It was found that the molecular rotor dynamics in rigid media should be quantitatively describable by the elastic modulus of polymer.

Comparing Geometry and Chemistry When Confined Molecules Diffuse in Monodisperse Metal-Organic Framework Pores.

The monodisperse pore structure of MOFs (metal-organic frameworks) is advantageous for investigating how porosity influences diffusion. Here we report translational and rotational diffusion using fluorescence correlation spectroscopy and time-correlated single-photon counting, using the three-dimensional pores of the zeolitic-like metal-organic framework family. We compare the influence of size and electric charge as well as dependence on pore size that we controlled through postsynthetic cation-exchange modifications. Charge-charge interactions with the MOF appeared to produce transient adsorption, manifested as a relatively fast and a slower diffusion process, but diffusants without net electric charge displayed a single diffusion process. Obtained from this family of guest molecules selected to be fluorescent, these findings suggest potentially useful general design rules to predict how pore size, guest size, and host-guest interaction control guest mobility within nanopores. With striking fidelity, diffusion coefficient scales with the ratio of cross-sectional areas of diffusant and host pores when charge is taken into account.