Active biocompatible systems are of great current interest for their possible applications in drug or antidote delivery at specific locations. Herein, we report the synthesis and study of self-propelled microparticles powered by enzymatic reactions and their directed movement in substrate concentration gradient. Polystyrene microparticles were functionalized with the enzymes urease and catalase using a biotin-streptavidin linkage procedure. The motion of the enzyme-coated particles was studied in the presence of the respective substrates, using optical microscopy and dynamic light scattering analysis. The diffusion of the particles was found to increase in a substrate concentration dependent manner. The directed chemotactic movement of these enzyme-powered motors up the substrate gradient was studied using three-inlet microfluidic channel architecture.

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

Recent experiments have revealed that the diffusivity of exothermic and fast enzymes is enhanced when they are catalytically active, and different physical mechanisms have been explored and quantified to account for this observation. We perform measurements on the endothermic and relatively slow enzyme aldolase, which also shows substrate-induced enhanced diffusion. We propose a new physical paradigm, which reveals that the diffusion coefficient of a model enzyme hydrodynamically coupled to its environment increases significantly when undergoing changes in conformational fluctuations in a substrate-dependent manner, and is independent of the overall turnover rate of the underlying enzymatic reaction. Our results show that substrate-induced enhanced diffusion of enzyme molecules can be explained within an equilibrium picture, and that the exothermicity of the catalyzed reaction is not a necessary condition for the observation of this phenomenon.In a quest for understanding nonequilibrium processes encountered in biology and chemistry, the study of active matter, namely systems constituted of agents able to consume and convert energy extracted from their environment, has been a major focus of the contemporary physical sciences [1,2]. Recent progress led to the design, fabrication and characterization of synthetic micro-and nano-machines relying on different propulsion mechanisms, and able to reproduce functions inspired from molecular biology, such as cargo transport or chemical sensing [3,4]. Such autonomous objects could have major technological applications, provided that they are small enough and fully biocompatible. In this context, and going down in scale, enzyme molecules have received a lot of attention, as models of biological nanoscale transducers able to convert chemical energy into mechanical work. Biomolecules typically perform cyclic turnovers in which they bind to substrates and catalytically convert them to products while undergoing conformational changes [5][6][7][8]. Recently, in vitro studies of enzymes using fluorescence correlation spectroscopy (FCS) have revealed that their diffusion coefficient is enhanced in a substrate-dependent manner [9][10][11][12], and that the diffusion enhancement ∆D at substrate saturation was typically of the order of the bare diffusion coefficient of the enzyme D 0 measured in the absence of substrate molecules. This observation holds for a wide range of enzymes, which typically catalyze fast and exothermic chemical reactions, with reaction enthalpies that can reach 40k B T per molecule and catalytic rates up to ∼ 10 4 s −1 for the particular case of catalase [12].This intriguing phenomenon, that could have major implications in the spatial organisation of biological processes [13], was subsequently investigated from a theoretical point of view. It was first suggested that the enhancement of the * P.I. and X.Z. contributed equally to this work. † Present address: Indian Institute of Technology Gandhinagar, Palaj Campus, Gandhinagar, Gujarat 382 355, India. ‡ Corre...

Conspectus Enzymes are ubiquitous in living systems. Apart from traditional motor proteins, the function of enzymes was assumed to be confined to the promotion of biochemical reactions. Recent work shows that free swimming enzymes, when catalyzing reactions, generate enough mechanical force to cause their own movement, typically observed as substrate-concentration-dependent enhanced diffusion. Preliminary indication is that the impulsive force generated per turnover is comparable to the force produced by motor proteins and is within the range to activate biological adhesion molecules responsible for mechanosensation by cells, making force generation by enzymatic catalysis a novel mechanobiology-relevant event. Furthermore, when exposed to a gradient in substrate concentration, enzymes move up the gradient: an example of chemotaxis at the molecular level. The driving force for molecular chemotaxis appears to be the lowering of chemical potential due to thermodynamically favorable enzyme–substrate interactions and we suggest that chemotaxis promotes enzymatic catalysis by directing the motion of the catalyst and substrates toward each other. Enzymes that are part of a reaction cascade have been shown to assemble through sequential chemotaxis; each enzyme follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Thus, sequential chemotaxis in catalytic cascades allows time-dependent, self-assembly of specific catalyst particles. This is an example of how information can arise from chemical gradients, and it is tempting to suggest that similar mechanisms underlie the organization of living systems. On a practical level, chemotaxis can be used to separate out active catalysts from their less active or inactive counterparts in the presence of their respective substrates and should, therefore, find wide applicability. When attached to bigger particles, enzyme ensembles act as “engines”, imparting motility to the particles and moving them directionally in a substrate gradient. The impulsive force generated by enzyme catalysis can also be transmitted to the surrounding fluid and molecular and colloidal tracers, resulting in convective fluid pumping and enhanced tracer diffusion. Enzyme-powered pumps that transport fluid directionally can be fabricated by anchoring enzymes onto a solid support and supplying the substrate. Thus, enzyme pumps constitute a novel platform that combines sensing and microfluidic pumping into a single self-powered microdevice. Taken in its entirety, force generation by active enzymes has potential applications ranging from nanomachinery, nanoscale assembly, cargo transport, drug delivery, micro- and nanofluidics, and chemical/biochemical sensing. We also hypothesize that, in vivo, enzymes may be responsible for the stochastic motion of the cytoplasm, the organization of metabolons and signaling complexes, and the convective transport of fluid in cells. A detailed understanding of how enzymes convert chemical energy to directional mechanical force ...

Enzymes show two distinct transport behaviors in the presence of their substrates in solution. First, their diffusivity enhances with an increasing substrate concentration. In addition, enzymes perform directional motion toward regions with a high substrate concentration, termed as chemotaxis. While a variety of enzymes has been shown to undergo chemotaxis, there remains a lack of quantitative understanding of the phenomenon. Here, we derive a general expression for the active movement of an enzyme in a concentration gradient of its substrate. The proposed model takes into account both the substrate-binding and catalytic turnover step, as well as the enhanced diffusion of the enzyme. We have experimentally measured the chemotaxis of a fast and a slow enzyme: urease under catalytic conditions and hexokinase for both full catalysis and for simple noncatalytic substrate binding. There is good agreement between the proposed model and the experiments. The model is general, has no adjustable parameters, and only requires three experimentally defined constants to quantify chemotaxis: enzyme–substrate binding affinity (K d), Michaelis–Menten constant (K M), and level of diffusion enhancement in the associated substrate (α).

Colloidal suspensions containing microscopic swimmers have been the focus of recent studies aimed at understanding the principles of energy transfer in fluidic media at low Reynolds number conditions. Going down in scale, active enzymes have been shown to be force-generating, nonequilibrium systems, thus offering opportunity to examine energy transfer at the ultralow Reynolds number regime. By monitoring the change of diffusion of inert tracers dispersed in active enzyme solutions, we demonstrate that the nature of energy transfer in these systems is similar to that reported for larger microscopic active systems, despite the large differences in scale, modes of energy transduction, and propulsion. Additionally, even an enzyme that catalyzes an endothermic reaction behaves analogously, suggesting that heat generation is not the primary factor for the observed enhanced tracer diffusion. Our results provide new insights into the mechanism of energy transfer at the molecular level.

The determination of solubility of acetamiprid dissolved in thirteen pure solvents such as isobutanol, methanol, ethanol, n-butanol, isopropanol, acetone, ethylene glycol, 1-methyl-2-pyrrolidinone, ethyl acetate, dimethyl sulfoxide (DMSO), 1,4-dioxane, N,N-dimethylformamide, and water and binary liquid mixtures (ethanol + water) was carried out using the shake-flask method at the temperatures from 278.15 to 318.15 K under local atmospheric pressure of 101.2 kPa. The observed values of mole fraction solubility were the highest in DMSO and the lowest in water. A linear solvation energy relationship analysis was carried out to reveal how much and what type of intermolecular solvent−solvent and solute−solvent interactions were responsible for the solubility variation in these thirteen pure solvents. The results exhibited that the solubility of acetamiprid in these thirteen monosolvents depends significantly upon the Hildebrand parameter and nonspecific dipolarity/polarizability interactions of the solvents. The experimental solubility data in pure solvents were correlated through the Apelblat equation, while those in liquid mixtures through the Apelblat−Jouyban−Acree model, the van't Hoff−Jouyban−Acree model, and the Jouyban−Acree model. For the selected pure solvents, the largest values of relative average deviation and root-mean-square deviation were, respectively, 1.77% and 36.21 × 10 −4 and for the mixtures were 5.15% and 4.81 × 10 −4 , respectively. In the ethanol + water mixtures, the log x 12 values positively deviated from the average ones of log x 12 , which showed that the acetamiprid was preferentially solvated by ethanol.

A self-powered polymeric micropump based on boronate chemistry is described. The pump is triggered by the presence of glucose in ambient conditions and induces convective fluid flows, with pumping velocity proportional to the glucose concentration. The pumping is due to buoyancy convection that originates from reaction-associated heat flux, as verified from experiments and finite difference modeling. As predicted, the fluid flow increases with increasing height of the chamber. In addition, pumping velocity is enhanced on replacing glucose with mannitol because of the enhanced exothermicity associated with the reaction of the latter.