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.

In situ enzymatic control of colloidal phoresis and catalysis through hydrolysis of ATP.

The ability to sense chemical gradients and respond with directional motility and chemical activity is a defining feature of complex living systems. There is a strong interest among scientists to design synthetic systems that emulate these properties. Here, we realize and control such behaviors in a synthetic system by tailoring multivalent interactions of adenosine nucleotides with catalytic microbeads. We first show that multivalent interactions of the bead with gradients of adenosine mono-, di- and trinucleotides (AM/D/TP) control both the phoretic motion and a proton-transfer catalytic reaction, and find that both effects are diminished greatly with increasing valence of phosphates. We exploit this behavior by using enzymatic hydrolysis of ATP to AMP, which downregulates multivalent interactivity in situ. This produces a sudden increase in transport of the catalytic microbeads (a phoretic jump), which is accompanied by increased catalytic activity. Finally, we show how this enzymatic activity can be systematically tuned, leading to simultaneous in situ spatial and temporal control of the location of the microbeads, as well as the products of the reaction that they catalyze. These findings open up new avenues for utilizing multivalent interaction-mediated programming of complex chemo-mechanical behaviors into active systems.

Enzyme-Mediated Temporal Control over the Conformational Disposition of a Condensed Protein in Macromolecular Crowded Media.

Temporal regulation between input and output signals is one of the hallmarks of complex biological processes. Herein, we report that the conformational disposition of a protein in macromolecularly crowded media can be controlled with time using enzymes. First, we demonstrate the pH dependence of bovine serum albumin (BSA) condensation and conformational alteration in the presence of poly(ethylene glycol) as a crowder. However, by exploiting the strength of pH-modulatory enzymatic reactions (glucose oxidase and urease), the conversion time between the condensed and free forms can be tuned. Additionally, we demonstrate that the trapping of intermediate states with respect to the overall system at a particular α-helix or ß-sheet composition and rotational mobility can be possible simply by altering the substrate concentration. Finally, we show that the intrinsic catalytic ability of BSA toward the Kemp elimination (KE) reaction is inhibited in the aggregated form but regained in the free form. In fact, the rate of KE reaction can also be actuated enzymatically in a temporal fashion, therefore demonstrating the programmability of a cascade of biochemical events in crowded media.

Self-Regulatory Micro- and Macroscale Patterning of ATP-Mediated Nanobioconjugate.

Directional interactions and the assembly of a nanobioconjugate in clusters at a specific location are important for patterning and microarrays in biomedical research. Herein, we report that self-assembly and spatial control in surface patterning of the surfactant-functionalized nanoparticles can be governed in micro- and macroscale environments by two factors, synergistic enzyme-substrate-nanoparticle affinity and the phoretic effect. First, we show that aggregation of cationic gold nanoparticles (GNP) can be modulated by multivalent anionic nanoparticle binding of an adenosine-based nucleotide and enzyme, alkaline phosphatase. We further demonstrate two different types of their autonomous aggregation pattern: (i) by introducing an enzyme gradient that modulates the synergistic nonequilibrium interactivity of the nanoparticle, nucleotide, and enzyme both in microfluidic conditions and at the macroscale; and (ii) the surface deposition pattern from evaporating droplets via the coffee ring effect. Here, temporal control over the width and site of the patterning area inside the microfluidic channel under catalytic and noncatalytic conditions has also been demonstrated. Finally, we show a change in capillary phoresis parameters responsible for the coffee ring due to introduction of ATP-loaded GNP in the blood serum, showing applicability in low-cost disease diagnostics. Overall, an enzyme-actuated surface nanobiopatterning method has been demonstrated that has potential application in controlled micro- and macroscale area patterning with a diverse cascade catalytic surface and spatiotemporal multisensory-based application.

Directional migration propensity of calf thymus DNA in a gradient of metal ions.

Herein we report the migrational behaviour and spatial distribution of calf thymus DNA in a gradient of different physiologically relevant monovalent and divalent cations under two different conditions - (i) microfluidic and (ii) evaporating droplets. Amplified phoresis toward high concentrations of Mg2+ and Ca2+ compared to other ions and non-uniform DNA coating in a gradient of ions were observed. This process, which was governed by the effective charge and diffusion coefficient of the DNA-metal ion complex, can have potential applications in nucleic acid-based spatiotemporal surface patterning, biosensors, and dynamic biocolloidal assembly and transport.

Perpetuating enzymatically induced spatiotemporal pH and catalytic heterogeneity of a hydrogel by nanoparticles.

The attainment of spatiotemporally inhomogeneous chemical and physical properties within a system is gaining attention across disciplines due to the resemblance to environmental and biological heterogeneity. Notably, the origin of natural pH gradients and how they have been incorporated in cellular systems is one of the most important questions in understanding the prebiotic origin of life. Herein, we have demonstrated a spatiotemporal pH gradient formation pattern on a hydrogel surface by employing two different enzymatic reactions, namely, the reactions of glucose oxidase (pH decreasing) and urease (pH increasing). We found here a generic pattern of spatiotemporal change in pH and proton transfer catalytic activity that was completely altered in a cationic gold nanoparticle containing hydrogel. In the absence of nanoparticles, the gradually generated macroscopic pH gradient slowly diminished with time, whereas the presence of nanoparticles helped to perpetuate the generated gradient effect. This behavior is due to the differential responsiveness of the interface of the cationic nanoparticle in temporally changing surroundings with increasing or decreasing pH or ionic contents. Moreover, the catalytic proton transfer ability of the nanoparticle showed a concerted kinetic response following the spatiotemporal pH dynamics in the gel matrix. Notably, this nanoparticle-driven spatiotemporally resolved gel matrix will find applicability in the area of the membrane-free generation and control of spatially segregated chemistry at the macroscopic scale.

Regulating Spatial Localization and Reactivity Biasness of DNAzymes by Metal Ions and Oligonucleotides.

Chemical gradient sensing behavior of catalytically active colloids and enzymes is an area of immense interest owing to their importance in understanding fundamental spatiotemporal complexity patterns in living systems and designing dynamic materials. Herein, we have shown the peroxidase activity of DNAzyme (G-quadruplex-hemin complex tagged in a micron-sized glass bead) can be modulated by metal ions and metal ion-binding oligonucleotides. Next we demonstrated both experimentally and theoretically, that the localization and product formation ability of the DNAzyme-containing particle remains biased to the more catalytically active zone where the concentration of metal ion (Hg2+ ) inhibitor is low. Interestingly, this biased localization can be broken by introduction of Hg2+ binding oligonucleotide in the system. Additionally, a macroscopically asymmetric catalytic product distributed zone has been achieved with this process, showing the possibility of regulation in autonomous spatially controlled chemical processes. This demonstration of autonomous modulation of the localization pattern and spatially specific enhanced product forming ability of DNAzymes will further enable the design of responsive nucleic acid-based motile materials and surfaces.

Interconnectivity between Surface Reactivity and Self-Assembly of Kemp Elimination Catalyzing Nanorods.

Understanding the fundamental facts behind dynamicity of catalytic processes has been a longstanding quest across disciplines. Herein, we report self-assembly of catalytically active gold nanorods that can be regulated by tuning its reactivity towards a proton transfer reaction at different pH. Unlike substrate-induced templating and co-operativity, the enhanced aggregation rate is due to alteration of catalytic surface charge only during reactivity as negatively charged transition state of reactant (5-nitrobenzisoxazole) is formed on positively charged nanorod while undergoing a concerted E2-pathway. Herein, enhanced diffusivity during catalytic processes might also act as an additional contributing factor. Furthermore, we have also shown that nanosized hydrophobic cavities of clustered nanorods can also efficiently accelerate the rate of an aromatic nucleophilic substitution reaction, which also demonstrates a catalytic phenomenon that can lead to cascading of other reactions where substrates and products of the starting reactions are not directly involved.

Spatiotemporal dynamics of self-assembled structures in enzymatically induced agonistic and antagonistic conditions.

Predicting and designing systems with dynamic self-assembly properties in a spatiotemporal fashion is an important research area across disciplines ranging from understanding the fundamental non-equilibrium features of life to the fabrication of next-generation materials with life-like properties. Herein, we demonstrate a spatiotemporal dynamics pattern in the self-assembly behavior of a surfactant from an unorganized assembly, induced by adenosine triphosphate (ATP) and enzymes responsible for the degradation or conversion of ATP. We report the different behavior of two enzymes, alkaline phosphatase (ALP) and hexokinase (HK), towards adenosine triphosphate (ATP)-driven surfactant assembly, which also results in contrasting spatiotemporal dynamic assembly behavior. Here, ALP acts antagonistically, resulting in transient self-assemblies, whereas HK shows agonistic action with the ability to sustain the assemblies. This dynamic assembly behavior was then used to program the time-dependent emergence of a self-assembled structure in a two-dimensional space by maintaining concentration gradients of the enzymes and surfactant at different locations, demonstrating a new route for obtaining 'spatial' organizational adaptability in a self-organized system of interacting components for the incorporation of programmed functionality.