Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose.

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.

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...

Self-propelled, synthetic active matters that transduce chemical energy into mechanical motion are examples of biomimetic nonequilibrium systems. They are of great current interest, with potential applications in nanomachinery, nanoscale assembly, fluidics, and chemical/biochemical sensing. Many of the physical challenges associated with generating motility on the micro- and nanoscale have recently been overcome, leading to the first generation of autonomous motors and pumps on scales ranging from microns to nanometers. This perspective focuses on catalytically powered motile systems, outlining major advances to date in motor/pump design, propulsion mechanisms and directional control, and intermotor communications leading to collective behavior. We conclude by discussing the possible future directions, from the fundamental questions that remain to be addressed to the design principles required for useful applications.

DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid pumps using enzymes as engines.

We demonstrate a procedure for the separation of enzymes based on their chemotactic response toward an imposed substrate concentration gradient. The separation is observed within a two-inlet, five-outlet microfluidic network, designed to allow mixtures of active (ones that catalyze substrate turnover) and inactive (ones that do not catalyze substrate turnover) enzymes, labeled with different fluorophores, to flow through one of the inlets. Substrate solution prepared in phosphate buffer was introduced through the other inlet of the device at the same flow rate. The steady-state concentration profiles of the enzymes were obtained at specific positions within the outlets of the microchannel using fluorescence microscopy. In the presence of a substrate concentration gradient, active enzyme molecules migrated preferentially toward the substrate channel. The excess migration of the active enzyme molecules was quantified in terms of an enrichment coefficient. Experiments were carried out with different pairs of enzymes. Coupling the physics of laminar flow of liquid and molecular diffusion, multiphysics simulations were carried out to estimate the extent of the chemotactic separation. Our results show that, with appropriate microfluidic arrangement, molecular chemotaxis leads to spontaneous separation of active enzyme molecules from their inactive counterparts of similar charge and size.

A Pd nanoparticle‐containing polymer microsphere moves with increasing speed across a pH gradient, following differential catalytic decomposition of aqueous hydrogen peroxide. The directional motion is akin to the pH taxis of living microorganisms. The artificial pH taxis exhibits random walk, translation, vertical, hopping, and pulsed motion, when the size of the motor and the imposed pH gradient are modulated.