Collective chemomechanical oscillations in active hydrogels.

We report on the collective response of an assembly of chemomechanical Belousov-Zhabotinsky (BZ) hydrogel beads. We first demonstrate that a single isolated spherical BZ hydrogel bead with a radius below a critical value does not oscillate, whereas an assembly of the same BZ hydrogel beads presents chemical oscillation. A BZ chemical model with an additional flux of chemicals out of the BZ hydrogel captures the experimentally observed transition from oxidized nonoscillating to oscillating BZ hydrogels and shows this transition is due to a flux of inhibitors out of the BZ hydrogel. The model also captures the role of neighboring BZ hydrogel beads in decreasing the critical size for an assembly of BZ hydrogel beads to oscillate. We finally leverage the quorum sensing behavior of the collective to trigger their chemomechanical oscillation and discuss how this collective effect can be used to enhance the oscillatory strain of these active BZ hydrogels. These findings could help guide the eventual fabrication of a swarm of autonomous, communicating, and motile hydrogels.

Crosslinking and depletion determine spatial instabilities in cytoskeletal active matter.

Active gels made of cytoskeletal proteins are valuable materials with attractive non-equilibrium properties such as spatial self-organization and self-propulsion. At least four typical routes to spatial patterning have been reported to date in different types of cytoskeletal active gels: bending and buckling instabilities in extensile systems, and global and local contraction instabilities in contractile gels. Here we report the observation of these four instabilities in a single type of active gel and we show that they are controlled by two parameters: the concentrations of ATP and depletion agent. We demonstrate that as the ATP concentration decreases, the concentration of passive motors increases until the gel undergoes a gelation transition. At this point, buckling is selected against bending, while global contraction is favored over local ones. Our observations are coherent with a hydrodynamic model of a viscoelastic active gel where the filaments are crosslinked with a characteristic time that diverges as the ATP concentration decreases. Our work thus provides a unified view of spatial instabilities in cytoskeletal active matter.

Long-Lasting and Responsive DNA/Enzyme-Based Programs in Serum-Supplemented Extracellular Media.

DNA molecular programs are emerging as promising pharmaceutical approaches due to their versatility for biomolecular sensing and actuation. However, the implementation of DNA programs has been mainly limited to serum-deprived in vitro assays due to the fast deterioration of the DNA reaction networks by the nucleases present in the serum. Here, we show that DNA/enzyme programs are functional in serum for 24 h but are later disrupted by nucleases that give rise to parasitic amplification. To overcome this, we implement three-letter code networks that suppress autocatalytic parasites while still conserving the functionality of DNA/enzyme programs for at least 3 days in the presence of 10% serum. In addition, we define a new buffer that further increases the biocompatibility and conserves responsiveness to changes in molecular composition across time. Finally, we demonstrate how serum-supplemented extracellular DNA molecular programs remain responsive to molecular inputs in the presence of living cells, having responses 6-fold faster than the cellular division rate, and are sustainable for at least three cellular divisions. This demonstrates the possibility of implementing in situ biomolecular characterization tools for serum-demanding in vitro models. We foresee that the coupling of chemical reactivity to our DNA programs by aptamers or oligonucleotide conjugations will allow the implementation of extracellular synthetic biology tools, which will offer new biomolecular pharmaceutical approaches and the emergence of complex and autonomous in vitro models.

Programmed mechano-chemical coupling in reaction-diffusion active matter.

Embryo morphogenesis involves a complex combination of self-organization mechanisms that generate a great diversity of patterns. However, classical in vitro patterning experiments explore only one self-organization mechanism at a time, thus missing coupling effects. Here, we conjugate two major out-of-equilibrium patterning mechanisms­reaction-diffusion and active matter­by integrating dissipative DNA/enzyme reaction networks within an active gel composed of cytoskeletal motors and filaments. We show that the strength of the flow generated by the active gel controls the mechano-chemical coupling between the two subsystems. This property was used to engineer a synthetic material where contractions trigger chemical reaction networks both in time and space, thus mimicking key aspects of the polarization mechanism observed in C. elegans oocytes. We anticipate that reaction-diffusion active matter will promote the investigation of mechano-chemical transduction and the design of new materials with life-like properties.

DNA-Controlled Spatiotemporal Patterning of a Cytoskeletal Active Gel.

Living cells move and change their shape because signaling chemical reactions modify the state of their cytoskeleton, an active gel that converts chemical energy into mechanical forces. To create life-like materials, it is thus key to engineer chemical pathways that drive active gels. Here we describe the preparation of DNA-responsive surfaces that control the activity of a cytoskeletal active gel composed of microtubules: A DNA signal triggers the release of molecular motors from the surface into the gel bulk, generating forces that structure the gel. Depending on the DNA sequence and concentration, the gel forms a periodic band pattern or contracts globally. Finally, we show that the structuration of the active gel can be spatially controlled in the presence of a gradient of DNA concentration. We anticipate that such DNA-controlled active matter will contribute to the development of life-like materials with self-shaping properties.

Spatiotemporal Patterning of Living Cells with Extracellular DNA Programs.

Reactive extracellular media focus on engineering reaction networks outside the cell to control intracellular chemical composition across time and space. However, current implementations lack the feedback loops and out-of-equilibrium molecular dynamics for encoding spatiotemporal control. Here, we demonstrate that enzyme-DNA molecular programs combining these qualities are functional in an extracellular medium where human cells can grow. With this approach, we construct an internalization program that delivers fluorescent DNA inside living cells and remains functional for at least 48 h. Its nonequilibrium dynamics allows us to control both the time and position of cell internalization. In particular, a spatially inhomogeneous version of this program generates a tunable reaction-diffusion two-band pattern of cell internalization. This demonstrates that a synthetic extracellular program can provide temporal and positional information to living cells, emulating archetypal mechanisms observed during embryo development. We foresee that nonequilibrium reactive extracellular media could be advantageously applied to in vitro biomolecular tracking, tissue engineering, or smart bandages.

DNA-based long-lived reaction-diffusion patterning in a host hydrogel.

The development of living organisms is a source of inspiration for the creation of synthetic life-like materials. Embryo development is divided into three stages that are inextricably linked: patterning, differentiation and growth. During patterning, sustained out-of-equilibrium molecular programs interpret underlying molecular cues to create well-defined concentration profiles. Implementing this patterning stage in an autonomous synthetic material is a challenge that at least requires a programmable and long-lasting out-of-equilibrium chemistry compatible with a host material. Here, we show that DNA/enzyme reactions can create reaction-diffusion patterns that are extraordinarily long-lasting both in solution and inside an autonomous hydrogel. The life-time and stability of these patterns - here, traveling fronts and two-band patterns - are significantly increased by blocking parasitic side reactions and by dramatically reducing the diffusion coefficient of specific DNA strands. Immersed in oil, hydrogels pattern autonomously with limited evaporation, but can also exchange chemical information with other gels when brought into contact. Providing a certain degree of autonomy and being capable of interacting with each other, we believe these out-of-equilibrium hydrogels open the way for the rational design of primitive metabolic materials.

Tunable corrugated patterns in an active nematic sheet.

Active matter locally converts chemical energy into mechanical work and, for this reason, it provides new mechanisms of pattern formation. In particular, active nematic fluids made of protein motors and filaments are far-from-equilibrium systems that may exhibit spontaneous motion, leading to actively driven spatiotemporally chaotic states in 2 and 3 dimensions and coherent flows in 3 dimensions (3D). Although these dynamic flows reveal a characteristic length scale resulting from the interplay between active forcing and passive restoring forces, the observation of static and large-scale spatial patterns in active nematic fluids has remained elusive. In this work, we demonstrate that a 3D solution of kinesin motors and microtubule filaments spontaneously forms a 2D free-standing nematic active sheet that actively buckles out of plane into a centimeter-sized periodic corrugated sheet that is stable for several days at low activity. Importantly, the nematic orientational field does not display topological defects in the corrugated state and the wavelength and stability of the corrugations are controlled by the motor concentration, in agreement with a hydrodynamic theory. At higher activities these patterns are transient and chaotic flows are observed at longer times. Our results underline the importance of both passive and active forces in shaping active matter and demonstrate that a spontaneously flowing active fluid can be sculpted into a static material through an active mechanism.

rEXPAR: An Isothermal Amplification Scheme That Is Robust to Autocatalytic Parasites.

In the absence of DNA, a solution containing the four deoxynucleotidetriphosphates (dNTPs), a DNA polymerase, and a nicking enzyme generates a self-replicating mixture of DNA species called parasite. Parasites are problematic in template-based isothermal amplification schemes such as EXPAR as well as in related molecular programming approaches, such as the PEN DNA toolbox. Here we show that using a nicking enzyme with only three letters (C, G, T) in the top strand of its recognition site, such as Nb.BssSI, allows us to change the sequence design of EXPAR templates in a way that prevents the formation of parasites when dATP is removed from the solution. This method allows us to make the EXPAR reaction robust to parasite contamination, a common feature in the laboratory, while keeping it compatible with PEN programs, which we demonstrate by engineering a parasite-proof bistable reaction network.

Synthesis and materialization of a reaction-diffusion French flag pattern.

During embryo development, patterns of protein concentration appear in response to morphogen gradients. These patterns provide spatial and chemical information that directs the fate of the underlying cells. Here, we emulate this process within non-living matter and demonstrate the autonomous structuration of a synthetic material. First, we use DNA-based reaction networks to synthesize a French flag, an archetypal pattern composed of three chemically distinct zones with sharp borders whose synthetic analogue has remained elusive. A bistable network within a shallow concentration gradient creates an immobile, sharp and long-lasting concentration front through a reaction-diffusion mechanism. The combination of two bistable circuits generates a French flag pattern whose 'phenotype' can be reprogrammed by network mutation. Second, these concentration patterns control the macroscopic organization of DNA-decorated particles, inducing a French flag pattern of colloidal aggregation. This experimental framework could be used to test reaction-diffusion models and fabricate soft materials following an autonomous developmental programme.

Synthesis of programmable reaction-diffusion fronts using DNA catalyzers.

We introduce a DNA-based reaction-diffusion (RD) system in which reaction and diffusion terms can be precisely and independently controlled. The effective diffusion coefficient of an individual reaction component, as we demonstrate on a traveling wave, can be reduced up to 2.7-fold using a self-assembled hydrodynamic drag. The intrinsic programmability of this RD system allows us to engineer, for the first time, orthogonal autocatalysts that counterpropagate with minimal interaction. Our results are in excellent quantitative agreement with predictions of the Fisher-Kolmogorov-Petrovskii-Piscunov model. These advances open the way for the rational engineering of pattern formation in pure chemical RD systems.

A microdevice for parallelized pulmonary permeability studies.

We describe a compartmentalized microdevice specifically designed to perform permeability studies across a model of lung barrier. Epithelial cell barriers were reproduced by culturing Calu-3 cells at the air-liquid interface (AIC) in 1 mm² microwells made from a perforated glass slide with an embedded porous membrane. We created a single basolateral reservoir for all microwells which eliminated the need to renew the growth medium during the culture growth phase. To perform drug permeability studies on confluent cell layers, the cell culture slide was aligned and joined to a collection platform consisting in 35 µL collection reservoirs connected at the top and bottom with microchannels. The integrity and functionality of the cell barriers were demonstrated by measurement of trans-epithelial electrical resistance (TEER), confocal imaging and permeability assays of ¹4C-sucrose. Micro-cell barriers were able to form confluent layers in 1 week, demonstrating a similar bioelectrical evolution as the Transwell systems used as controls. Tight junctions were observed throughout the cell-cell interfaces, and the low permeability coefficients of ¹4C-sucrose confirmed their functional presence, creating a primary barrier to the diffusion of solutes. This microdevice could facilitate the monitoring of biomolecule transport and the screening of formulations promoting their passage across the pulmonary barrier, in order to select candidates for pulmonary administration to patients.

Pressure-assisted selective preconcentration in a straight nanochannel.

We investigate the preconcentration profiles of a fluorescein and bovine serum albumin derivatized with this fluorescent tag in a microfluidic chip bearing a nanoslit. A new preconcentration method in which a hydrodynamic pressure is added to both electroosmotic and electrophoretic contributions is proposed to monitor the location of the preconcentration frontline. A simple predictive model of this pressure-assisted electropreconcentration is proposed for the evolution of the flow profile along this micro/nano/microfluidic structure. We show with a small analyte such as fluorescein that the additional hydrostatic pressure mode enables to stabilize the concentration polarization (CP) effect, resulting in better control of the cathodic focusing (CF) peak. For BSA (bovine serum albumin), we exhibit that the variation of the hydrodynamic pressure can have an even more drastic effect on the preconcentration. We show that, depending on this hydrodynamic pressure, the preconcentration can be chosen, either in the cathodic side or in the anodic one. For the first time, we prove here that both anodic focusing (AF) and cathodic focusing (CF) regimes can be reached in the same structures. These results also open new routes for the detection and the quantification of low abundance biomarkers.

A nanoliter-scale open chemical reactor.

An open chemical reactor is a container that exchanges matter with the exterior. Well-mixed open chemical reactors, called continuous stirred tank reactors (CSTR), have been instrumental for investigating the dynamics of out-of-equilibrium chemical processes, such as oscillations, bistability, and chaos. Here, we introduce a microfluidic CSTR, called µCSTR, that reduces reagent consumption by six orders of magnitude. It consists of an annular reactor with four inlets and one outlet fabricated in PDMS using multi-layer soft lithography. A monolithic peristaltic pump feeds fresh reagents into the reactor through the inlets. After each injection the content of the reactor is continuously mixed with a second peristaltic pump. The efficiency of the µCSTR is experimentally characterized using a bromate, sulfite, ferrocyanide pH oscillator. Simulations accounting for the digital injection process are in agreement with experimental results. The low consumption of the µCSTR will be advantageous for investigating out-of-equilibrium dynamics of chemical processes involving biomolecules. These studies have been scarce so far because a miniaturized version of a CSTR was not available.

Amplification and temporal filtering during gradient sensing by nerve growth cones probed with a microfluidic assay.

Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during γ-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA(A) chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA(A) receptors.

Concentration landscape generators for shear free dynamic chemical stimulation.

In this paper we first introduce a novel fabrication process, which allows for easy integration of thin track-etched nanoporous membranes, within 2D or 3D microchannel networks. In these networks, soluble chemical compounds can diffuse out of the channels through well-defined and spatially organized microfabricated porous openings. Interestingly, multiple micron-scale porous areas can be integrated in the same device and each of these areas can be connected to a different microfluidic channel and reservoir. We then present and characterize several membrane-based microdevices and their use for the generation of stable diffusible concentration gradients and complex dynamic chemical landscapes under shear free conditions. We also demonstrate how a simple flow-focusing geometry can be used to generate "on-demand" concentration profiles. In turn, these devices should provide an ideal experimental framework for high throughput cell-based assays: long term high-resolution video microscopy experiments can be performed, under multiple spatially and temporally controlled chemical conditions, with simple protocols and in a cell-friendly environment.

Thermoplastic elastomers for microfluidics: towards a high-throughput fabrication method of multilayered microfluidic devices.

Multilayer soft lithography of polydimethylsiloxane (PDMS) is a well-known method for the fabrication of complex fluidic functions. With advantages and drawbacks, this technique allows fabrication of valves, pumps and micro-mixers. However, the process is inadequate for industrial applications. Here, we report a rapid prototyping technique for the fabrication of multilayer microfluidic devices, using a different and promising class of polymers. Using styrenic thermoplastic elastomers (TPE), we demonstrate a rapid technique for the fabrication and assembly of pneumatically driven valves in a multilayer microfluidic device made completely from thermoplastics. This material solution is transparent, biocompatible and as flexible as PDMS, and has high throughput thermoforming processing characteristics. We established a proof of principle for valving and mixing with three different grades of TPE using an SU-8 master mold. Specific viscoelastic properties of each grade allow us to report enhanced bonding capabilities from room temperature bonding to free pressure thermally assisted bonding. In terms of microfabrication, beyond classically embossing means, we demonstrate a high-throughput thermoforming method, where TPE molding experiments have been carried out without applied pressure and vacuum assistance within an overall cycle time of 180 s. The quality of the obtained thermoplastic systems show robust behavior and an opening/closing frequency of 5 Hz.

Microfluidic stickers for cell- and tissue-based assays in microchannels.

Difficulties in culturing cells inside microchannels is a major obstacle for the wide use of microfluidic technology in cell biology. Here, we present a simple and versatile method to interface closed microchannels with cellular and multicellular systems. Our approach, based on microfluidic stickers which can adhere to wet glass coverslips, eliminates the need to adapt cell culture conditions to microchannels and greatly facilitates the methods required to position cells into microcircuits. We demonstrate the simplicity and efficiency of the method with HeLa cells, primary cultured neurons and Drosophila tissues.

Microfluidic patterning of miniaturized DNA arrays on plastic substrates.

This paper describes the patterning of DNA arrays on plastic surfaces using an elastomeric, two-dimensional microcapillary system (muCS). Fluidic structures were realized through hot-embossing lithography using Versaflex CL30. Like elastomers based on poly(dimethylsiloxane), this thermoplastic block copolymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspotting techniques. We focus on muCSs that support arrays comprising up to 2 x 48 spots, each being 45 mum in diameter. Substrates were fabricated from two hard thermoplastic materials, poly(methylmethacrylate) and a polycyclic olefin (e.g., Zeonor 1060R), which were both activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride and N-hydroxysuccinimide to mediate covalent attachment of DNA molecules. The approach was exemplified by using 0.25-32 muM solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate-buffered saline, allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to >48 h at 22, 30, and 40 degrees C to probe the conditions for obtaining uniform spots of high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation of the solvent. While selective activation of the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of the liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity. Moreover, we show that resultant arrays are compatible with standard hybridization protocols, which allowed for reliable discrimination of individual strands when exposed to a specific ssDNA target molecule.