Chemomechanical Communication between Liposomes Based on Enzyme Cascades.

Communication between cells is crucial to the survival of both uni- and multicellular organisms. The primary mode of communication involves chemical cues. There is great current interest in mimicking this behavior in synthetic cells to understand the physical basis of intercellular communication and design collective functional behavior. Using liposomal cell mimics, we demonstrate how a chemical input can elicit a mechanical response (enhanced motility). We employed a single substrate to trigger enzyme cascade-induced control of the diffusion of up to three different liposome populations. Furthermore, substrate competition allows temporal control over enhanced diffusion. The use of enzyme cascades to propagate chemical signals provides a robust and efficient mechanism for diverse populations of protocells to coordinate their motion in response to signals from each other.

Self-Propelling Macroscale Sheets Powered by Enzyme Pumps.

Nanoscale enzymes anchored to surfaces act as chemical pumps by converting chemical energy released from enzymatic reactions into spontaneous fluid flow that propels entrained nano- and microparticles. Enzymatic pumps are biocompatible, highly selective, and display unique substrate specificity. Utilizing these pumps to trigger self-propelled motion on the macroscale has, however, constituted a significant challenge and thus prevented their adaptation in macroscopic fluidic devices and soft robotics. Using experiments and simulations, we herein show that enzymatic pumps can drive centimeter-scale polymer sheets along directed linear paths and rotational trajectories. In these studies, the sheets are confined to the air/water interface. With the addition of appropriate substrate, the asymmetric enzymatic coating on the sheets induces chemically driven, buoyancy flows that controllably propel the sheet's motion on the air/water interface. The directionality and speed of the motion can be tailored by changing the pattern of the enzymatic coating, type of enzyme, and nature and concentration of the substrate. This work highlights the utility of biocompatible enzymes for generating motion in macroscale fluidic devices and robotics and indicates their potential utility for in vivo applications.

Dynamic Oscillation and Motion of Oil-in-Water Emulsion Droplets.

The interplay of interfacial tensions on droplets results in a range of self-powered motions that mimic those of living systems and serve as a tunable model to understand their complex non-equilibrium behavior. Spontaneous shape deformations and oscillations are crucial features observed in nature but difficult to incorporate in synthetic artificial systems. Here, we report sessile oil-in-water emulsions that exhibit rapid oscillating behavior. The oscillations depend on the nature and concentration of the surfactant, the chemical composition of the oil, and the wettability of the solid substrate. The rapid changes in the contact angle per oscillation are observed using side-view optical microscopy. We propose that the changes in the interfacial tension of the oil droplets is due to the partitioning of the surfactant into the oil phase and the movement of self-emulsified oil out of the parent droplets giving rise to the rhythmic variation in droplet contact-line. The ability to control and understand droplet oscillation can help model similar oscillations in out-of-equilibrium systems in nature and reproduce biomimetic behavior in artificial systems for various applications, such as microfluidic lab-on-a-chip and adaptive materials.

Kinetic Asymmetry versus Dissipation in the Evolution of Chemical Systems as Exemplified by Single Enzyme Chemotaxis.

Single enzyme chemotaxis is a phenomenon by which a nonequilibrium spatial distribution of an enzyme is created and maintained by concentration gradients of the substrate and product of the catalyzed reaction. These gradients can arise either naturally through metabolism or experimentally, e.g., by flow of materials through microfluidic channels or by use of diffusion chambers with semipermeable membranes. Numerous hypotheses regarding the mechanism of this phenomenon have been proposed. Here, we discuss a mechanism based solely on diffusion and chemical reaction and show that kinetic asymmetry, a difference in the transition state energies for dissociation/association of substrate and product, and diffusion asymmetry, a difference in the diffusivities of the bound and free forms of the enzyme, are the determinates of the direction of chemotaxis and can result in either positive or negative chemotaxis, both of which have been demonstrated experimentally. Exploration of these fundamental symmetries that govern nonequilibrium behavior helps to distinguish between possible mechanisms for the evolution of a chemical system from initial to the steady state and whether the principle that determines the direction a system shifts when exposed to an external energy source is based on thermodynamics or on kinetics with the latter being supported by the results of the present paper. Our results show that, while dissipation ineluctably accompanies nonequilibrium phenomena, including chemotaxis, systems do not evolve to maximize or minimize dissipation but rather to attain greater kinetic stability and accumulate in regions where their effective diffusion coefficient is as small as possible. The chemotactic response to the chemical gradients formed by other enzymes participating in a catalytic cascade provides a mechanism for forming loose associations known as metabolons. Significantly, the direction of the effective force due to these gradients depends on the kinetic asymmetry of the enzyme and so can be nonreciprocal, where one enzyme is attracted to another enzyme, but the other enzyme is repelled by the one, in seeming contradiction to Newtons third law. This nonreciprocity is an important ingredient in the behavior of active matter.

Ultrasound Manipulation and Extrusion of Active Nanorods.

Synthetic self-propelled nano and microparticles have a growing appeal for targeted drug delivery, collective functionality, and manipulation at the nanoscale. However, it is challenging to control their positions and orientations under confinement, e.g., in microchannels, nozzles, and microcapillaries. This study reports on the synergistic effect of acoustic and flow-induced focusing in microfluidic nozzles. In a microchannel with a nozzle, the balance between the acoustophoretic forces and the fluid drag due to streaming flows generated by the acoustic field controls the microparticle's dynamics. This study manipulates the positions and orientations of dispersed particles and dense clusters inside the channel at a fixed frequency by tuning the acoustic intensity. The main findings are: first, this study successfully manipulates the positions and orientations of individual particles and dense clusters inside the channel at a fixed frequency by tuning the acoustic intensity. Second, when an external flow is applied, the acoustic field separates and selectively extrudes shape-anisotropic passive particles and self-propelled active nanorods. Finally, the observed phenomena are explained by multiphysics finite-element modeling. The results shed light on the control and extrusion of active particles in confined geometries and enable applications for acoustic cargo (e.g., drug) delivery, particle injection, and additive manufacturing via printed self-propelled active particles.

Interactions with amyloid beta peptide and acetylcholinesterase increase alkaline phosphatase activity.

Multiple studies have shown that the activity of alkaline phosphatase (AP) increases during Alzheimer's disease (AD). In this paper, using UV-Visible spectroscopy, we show that this increase in activity is due to its interaction with key components of AD such as amyloid ß peptide and acetylcholinesterase. Activity increase also occurs due to high concentrations of acetylcholine and choline. These conditions are present in AD or could occur due to drugs used for treating AD.

Self-Generated Convective Flows Enhance the Rates of Chemical Reactions.

In chemical solutions, the products of catalytic reactions can occupy different volumes compared to the reactants and thus give rise to local density variations in the fluid. These density variations generate solutal buoyancy forces, which are exerted on the fluid and thus "pump" the fluid to flow. Herein, we examine if the reaction-induced pumping accelerates the chemical reaction by transporting the reactants to the catalyst at a rate faster than passive diffusion. Using both simulations and experiments, we show a significant increase in reaction rate when reaction-generated convective flow is present. In effect, through a feedback loop, catalysts speed up reactions not only by lowering the energy barrier but also by increasing the collision frequency between the reactants and the catalyst.

Droplet Navigation by Photothermal Pumping in an Optofluidic System.

Droplets with guided motion have potential applications as microreactors and delivery vehicles. Directing long-range migration powered solely by light is particularly advantageous since light can be applied remotely, patterned with a photomask, and readily translated to irradiate specified locations. Herein, we describe a universal platform that allows fast directional navigation and collective merging of droplets controlled by either ultraviolet or visible light. The guided motion of water and oil droplets follows density-driven convective flows arising from photothermal conversion at a light-absorbing amphiphobic substrate. Because of the relatively high photothermal efficiency, a low-intensity light beam can be employed. Further, we demonstrate that the moving droplets can function as carriers and on-demand reaction chambers.

Chemically Propelled Nano and Micromotors in the Body: Quo Vadis?

The active delivery of drugs to disease sites in response to specific biomarkers is a holy grail in theranostics. If successful, it would greatly diminish the therapeutic dosage and reduce collateral cytotoxicity. In this context, the development of nano and micromotors that are able to harvest local energy to move directionally is an important breakthrough. However, serious hurdles remain before such active systems can be employed in vivo in therapeutic applications. Such motors and their energy sources must be safe and biocompatible, they should be able to move through complex body fluids, and have the ability to reach specific cellular targets. Given the complexity in the design and deployment of nano and micromotors, it is also critically important to show that they are significantly superior to inactive "smart" nanoparticles in theranostics. Furthermore, receiving regulatory approval requires the ability to scale-up the production of nano and micromotors with uniformity in structure, function, and activity. In this essay, the limitations of the current nano and micromotors and the issues that need to be resolved before such motors are likely to find theranostic applications are discussed.

Relative Diffusivities of Bound and Unbound Protein Can Control Chemotactic Directionality.

Enzyme-based systems have been shown to undergo chemotactic motion in response to their substrate gradient. This phenomenon has been exploited to direct the motion of enzymes and enzyme-attached particles to specific locations in space. Here, we propose a new kinetic model to analyze the directional movement of an ensemble of protein molecules in response to a gradient of the ligand. We also formulate a separate model to probe the motion of enzyme molecules in response to a gradient of the substrate under catalytic conditions. The only input for the new enzymatic model is the Michaelis-Menten constant which is the relevant measurable constant for enzymatic reactions. We show how our model differs from previously proposed models in a significant manner. For both binding and catalytic reactions, a net movement up the ligand/substrate gradient is predicted when the diffusivity of the ligand/substrate-bound protein is lower than that of the unbound protein (positive chemotaxis). Conversely, movement down the ligand/substrate gradient is expected when the diffusivity of the ligand/substrate-bound protein is higher than that of the unbound protein (negative chemotaxis). However, there is no net movement of protein/enzyme when the diffusivities of the bound and free species are equal. The work underscores the critical importance of measuring the diffusivity of the bound protein and comparing it with that of the free protein.

Enzyme aggregation and fragmentation induced by catalysis relevant species.

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.

A simple theory for molecular chemotaxis driven by specific binding interactions.

Recent experiments have suggested that enzymes and other small molecules chemotax toward their substrates. However, the physical forces driving this chemotaxis are currently debated. In this work, we consider a simple thermodynamic theory for molecular chemotaxis that is based on the McMillan-Mayer theory of dilute solutions and Schellman's theory for macromolecular binding. Even in the absence of direct interactions, the chemical binding equilibrium introduces a coupling term into the relevant free energy, which then reduces the chemical potential of both enzymes and their substrates. Assuming a local thermodynamic equilibrium, this binding contribution to the chemical potential generates an effective thermodynamic force that promotes chemotaxis by driving each solute toward its binding partner. Our numerical simulations demonstrate that, although small, this thermodynamic force is qualitatively consistent with several experimental studies. Thus, our study may provide additional insight into the role of the thermodynamic binding free energy for molecular chemotaxis.

Light-Induced Dynamic Control of Particle Motion in Fluid-Filled Microchannels.

The design of remotely programmable microfluidic systems with controlled fluid flow and particle transport is a significant challenge. Herein, we describe a system that harnesses the intrinsic thermal response of a fluid to spontaneously pump solutions and regulate the transport of immersed microparticles. Irradiating a silver-coated channel with ultraviolet (UV) light generates local convective vortexes, which, in addition to the externally imposed flow, can be used to guide particles along specific trajectories or to arrest their motion. The method provides the distinct advantage that the flow and the associated convective patterns can be dynamically altered by relocating the source of UV light. Moreover, the flow can be initiated and terminated "on-demand" by turning the light on or off.

Silver-Based Self-Powered pH-Sensitive Pump and Sensor.

Nonmechanical nano/microscale pumps that provide precise control over flow rate without the aid of an external power source and that are capable of turning on in response to specific analytes in solution are needed for the next generation of smart micro- and nanoscale devices. Herein, a self-powered chemically driven silver micropump is reported that is based on the two-step catalytic decomposition of hydrogen peroxide, H2O2. The pumping direction and speed can be controlled by modulating the solution pH, and modeling and theory allow for the kinetics of the reaction steps to be connected to the fluid velocity. In addition, by changing the pH dynamically using glucose oxidase (GOx)-catalyzed oxidation of glucose to gluconic acid, the direction of fluid pumping can be altered in situ, allowing for the design of a glucose sensor. This work underscores the versatility of catalytic pumps and their ability to function as sensors.

Motility of Enzyme-Powered Vesicles.

Autonomous nanovehicles powered by energy derived from chemical catalysis have potential applications as active delivery agents. For in vivo applications, it is necessary that the engine and its fuel, as well as the chassis itself, be biocompatible. Enzyme molecules have been shown to display enhanced motility through substrate turnover and are attractive candidates as engines; phospholipid vesicles are biocompatible and can serve as cargo containers. Herein, we describe the autonomous movement of vesicles with membrane-bound enzymes in the presence of the substrate. We find that the motility of the vesicles increases with increasing enzymatic turnover rate. The enhanced diffusion of these enzyme-powered systems was further substantiated in real time by tracking the motion of the vesicles using optical microscopy. The membrane-bound protocells that move by transducing chemical energy into mechanical motion serve as models for motile living cells and are key to the elucidation of the fundamental mechanisms governing active membrane dynamics and cellular movement.

Powering Motion with Enzymes.

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 can lead us to the basic principles of fabrication, development, and monitoring of biological and biomimetic molecular machines.

Self-Organization of Fluids in a Multienzymatic Pump System.

The nascent field of microscale flow chemistry focuses on harnessing flowing fluids to optimize chemical reactions in microchambers and establish new routes for chemical synthesis. With enzymes and other catalysts anchored to the surface of microchambers, the catalytic reactions can act as pumps and propel the fluids through the containers. Hence, the flows not only affect the catalytic reactions, but these reactions also affect the flows. Understanding this dynamic interplay is vital to enhancing the accuracy and utility of flow technology. Through experiments and simulation, we design a system of three different enzymes, immobilized in separate gels, on the surface of a microchamber; with the appropriate reactants in the solution, each enzyme-filled gel acts as a pump. The system also exploits a reaction cascade that controls the temporal interactions between two pumps. With three pumps in a triangular arrangement, the spatio-temporal interactions among the chemical reactions become highly coordinated and produce well-defined fluid streams, which transport chemicals and form a fluidic "circuit". The circuit layout and flow direction of each constituent stream can be controlled through the number and placement of the gels and the types of catalysts localized in the gels. These studies provide a new route for forming self-organizing and bifurcating fluids that can yield fundamental insight into nonequilibrium, dynamical systems. Because the flows and fluidic circuits are generated by internal chemical reactions, the fluids can autonomously transport cargo to specific locations in the device. Hence, the findings also provide guidelines to facilitate further automation of microfluidic devices.

Convective flow reversal in self-powered enzyme micropumps.

Surface-bound enzymes can act as pumps that drive large-scale fluid flows in the presence of their substrates or promoters. Thus, enzymatic catalysis can be harnessed for "on demand" pumping in nano- and microfluidic devices powered by an intrinsic energy source. The mechanisms controlling the pumping have not, however, been completely elucidated. Herein, we combine theory and experiments to demonstrate a previously unreported spatiotemporal variation in pumping behavior in urease-based pumps and uncover the mechanisms behind these dynamics. We developed a theoretical model for the transduction of chemical energy into mechanical fluid flow in these systems, capturing buoyancy effects due to the solution containing nonuniform concentrations of substrate and product. We find that the qualitative features of the flow depend on the ratios of diffusivities δ=D(P)/D(S) and expansion coefficients ß=ß(P)/ß(S) of the reaction substrate (S) and product (P). If δ>1 and δ>ß (or if δ<1 and δ<ß ), an unexpected phenomenon arises: the flow direction reverses with time and distance from the pump. Our experimental results are in qualitative agreement with the model and show that both the speed and direction of fluid pumping (i) depend on the enzyme activity and coverage, (ii) vary with the distance from the pump, and (iii) evolve with time. These findings permit the rational design of enzymatic pumps that accurately control the direction and speed of fluid flow without external power sources, enabling effective, self-powered fluidic devices.

Organization of Particle Islands through Light-Powered Fluid Pumping.

The field of active matter holds promise for applications in particle assembly, cargo and drug delivery, and sensing. In pursuit of these capabilities, researchers have produced a suite of nanomotors, fluid pumps, and particle assembly strategies. Although promising, there are many challenges, especially for mechanisms that rely on chemical propulsion. One way to circumvent these issues is by the use of external energy sources. Herein, we propose a method of using freely suspended nanoparticles to generate fluid pumping towards desired point sources. The pumping rates are dependent on particle concentration and light intensity, making it highly controllable. Using these directed flows, we further demonstrate the ability to reversibly construct and move colloidal crystals.

A Theory of Enzyme Chemotaxis: From Experiments to Modeling.

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 ( Kd), Michaelis-Menten constant ( KM), and level of diffusion enhancement in the associated substrate (α).