Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 µM) underlines potential interest of this nanoplatform for biomedical applications.

Dynamic Electrochemiluminescence Imaging of Single Giant Liposome Opening at Polarized Electrodes.

In this work, the characterization of release events from liposomes has been addressed quantitatively by an electrochemiluminescence (ECL) imaging strategy. First, ECL reagents ([Ru(bpy)3]2+ and tripropylamine) were encapsulated in sealed giant asymmetrical liposomes (100 µm in diameter) made of DOPG/DOPC phospholipids. After sedimentation on an indium tin oxide electrode material, the opening of liposomes was triggered by polarization of the surface. Under these conditions, amperometry, epifluorescence imaging, and ECL imaging were combined and synchronized to monitor and image the rupture of giant liposomes during the release and subsequent ECL emission of their redox content. Amperometry allowed the quantification of the content released from single liposomes. The location and status of liposomes (closed or opened) were assessed by epifluorescence imaging. ECL provided the image of the efflux of matter after liposome opening. This original ECL imaging approach favorably compares with strictly photoluminescent or electrochemical techniques and appears to be adapted for the investigation of membrane rupture/permeation events.

Photothermally Reconfigurable Colloidal Crystals at a Fluid Interface, a Generic Approach for Optically Tunable Lattice Properties.

Optically addressable colloidal assembly at fluid interfaces is a highly desired component to generate reconfigurable 2D materials but has rarely been achieved and only with specific interface engineering. Here we describe a generic method to get optically reconfigurable colloidal crystals at the air/water interface and emphasize a new mechanism to convert light into tunable lattice properties. We use light-absorbing anionic particles adsorbed at the air/water interface in the presence of minute amounts of cationic surfactant, which self-assembled into closely packed polycrystalline structures by collectively deforming the surrounding interface. Low-intensity irradiation of these colloidal crystals results in unprecedented control of the interparticle spacing in a preserved crystalline state while, at a higher intensity, cycles of melting/recrystallization with a controllable transition kinetics can be achieved upon successive on/off stimulations. We show that this photoreversible melting originates from an initial thermocapillary stress, expanding the colloidal assembly against the local confinement, and an increase in particles diffusivity imposing the transition kinetics. With this mechanism, local irradiation leads to highly dynamic patterns, including self-healing or self-fed "living" crystals, while multiresponsive assembly is also achieved by controlling particle organization with both light and magnetic stimuli.

Enzymatically Active DNA-Protein Nanogels with Tunable Cross-Linking Density.

Hybrid DNA-protein nanogels represent potential protein vectors and enzymatic nanoreactors for modern biotechnology. Here, we describe a new, easy, and robust method for preparation of tunable DNA-protein nanogels with controllable size and density. For this purpose, polymerase chain reaction is used to prepare highly biotinylated DNA as a soft biopolymeric backbone, which can be efficiently cross-linked via streptavidin-biotin binding. This approach enables us to control both the density and size of the resulting nanogels not only by adjusting the amount of the cross-linking streptavidin but also by using different rates of DNA biotinylation. This gives DNA-streptavidin nanogels with the size ranging from 80 nm, for the most compact state, to up to 200 nm. Furthermore, using streptavidin-enzyme conjugates allows the straightforward one-pot incorporation of enzymes during the preparation of the nanogels. Monoenzymatic and multienzymatic nanogels have been obtained in this manner, and their catalytic activities have been characterized. All tested enzymes (alkaline phosphatase (AP), horseradish peroxidase (HRP), and ß-galactosidase (ßGal)), incorporated individually or in a coupled manner (glucose oxidase (GOx)-HRP cascade), were shown to remain functional. The activities of AP and ßGal were unchanged while that of HRP was slightly improved inside the nanogels. We demonstrate that, for HRP, it is not the DNA-to-enzyme ratio but the physical density of the functionalized DNA nanogels that is responsible for the improvement of its enzymatic activity.

Reversible Supra-Folding of User-Programmed Functional DNA Nanostructures on Fuzzy Cationic Substrates.

We report that user-defined DNA nanostructures, such as two-dimensional (2D) origamis and nanogrids, undergo a rapid higher-order folding transition, referred to as supra-folding, into three-dimensional (3D) compact structures (origamis) or well-defined µm-long ribbons (nanogrids), when they adsorb on a soft cationic substrate prepared by layer-by-layer deposition of polyelectrolytes. Once supra-folded, origamis can be switched back on the surface into their 2D original shape through addition of heparin, a highly charged anionic polyelectrolyte known as an efficient competitor of DNA-polyelectrolyte complexation. Orthogonal to DNA base-pairing principles, this reversible structural reconfiguration is also versatile; we show in particular that 1) it is compatible with various origami shapes, 2) it perfectly preserves fine structural details as well as site-specific functionality, and 3) it can be applied to dynamically address the spatial distribution of origami-tethered proteins.

Self-Propelled Water Drops on Bare Glass Substrates in Air: Fast, Controllable, and Easy Transport Powered by Surfactants.

Self-propelled drops are capable of motion without external intervention. As such, they constitute attractive entities for fundamental investigations in active soft matter, hydrodynamics, and surface sciences, as well as promising systems for autonomous microfluidic operations. In contrast with most of the examples relying on organic drops or specifically treated substrates, here we describe the first system of nonreactive water drops in air that can propel themselves on a commercially available ordinary glass substrate that was used as received. This is achieved by exploiting the dynamic adsorption behavior of common n-alkyltrimethylammonium bromide (CnTAB) surfactants added to the drop. We precisely analyze the drop motion for a broad series of surfactants carrying n = 6 to 18 carbon atoms in their tail and establish how the motion characteristics (speed, probability of motion) are tuned by both the hydrophobicity and the concentration of the surfactant. We show that motion occurs regardless of the n value but only in a specific concentration range with a maximum speed at around one tenth of the critical micelle concentration (CMC/10) for most of the tested surfactants. Surfactants of intermediate hydrophobicity are shown to be the best candidates to power drops that can move at a high speed (1-10 cm s-1), the optimal performance being reached with [C12TAB] = 800 µM. We propose a mechanism where the motion originates from the anisotropic wettability of the substrate created by the electrostatic adsorption of surfactants beneath the moving drop. Simply drawing lines with a marker pen allows us to create guiding paths for drop motion and to achieve operations such as complex trajectory control, programmed drop fusion, drop refilling, as well as drop moving vertically against gravity. This work revisits the role of surfactants in dynamic wetting and self-propelled motion as well as brings an original strategy to build the future of microfluidics with lower-cost, simpler, and more autonomous portable devices that could be made available to everyone and everywhere.

Photoswitchable Fluorescent Crystals Obtained by the Photoreversible Coassembly of a Nucleobase and an Azobenzene Intercalator.

Self-assembled nucleobases, such as G-quartets or quadruplexes, have numerous applications, but light-responsive structures are limited to small, noncrystalline motifs. In addition, the assembly of the widely exploited azobenzene photochromic compounds can produce fluorescent crystals of extended dimensions but at the prize of sacrificing their photoswitchability. Here, we overcome inherent limitations of self-assembly with a new concept of supramolecular coassembly leading to materials with unprecedented properties. We show that the coassembly of guanosine monophosphate (GMP) with an azobenzene-containing DNA intercalator produces supramolecular crystals arranged through a combination of π-π, electrostatic, and hydrogen-bond interactions. The resulting crystals are 100 µm long, pH-sensitive, fluorescent, and can be photoreversibly disassembled/reassembled upon UV/blue irradiation. This allows us to perform operations such as dynamic photocontrol of a single-crystal growth, light-gated permeability in membrane-like materials, and photoswitchable fluorescence. We believe this concept critically expands the breadth of multifunctional materials attainable by self-assembly.

Cellular heterogeneity mediates inherent sensitivity-specificity tradeoff in cancer targeting by synthetic circuits.

Synthetic gene circuits are emerging as a versatile means to target cancer with enhanced specificity by combinatorial integration of multiple expression markers. Such circuits must also be tuned to be highly sensitive because escape of even a few cells might be detrimental. However, the error rates of decision-making circuits in light of cellular variability in gene expression have so far remained unexplored. Here, we measure the single-cell response function of a tunable logic AND gate acting on two promoters in heterogeneous cell populations. Our analysis reveals an inherent tradeoff between specificity and sensitivity that is controlled by the AND gate amplification gain and activation threshold. We implement a tumor-mimicking cell-culture model of cancer cells emerging in a background of normal ones, and show that molecular parameters of the synthetic circuits control specificity and sensitivity in a killing assay. This suggests that, beyond the inherent tradeoff, synthetic circuits operating in a heterogeneous environment could be optimized to efficiently target malignant state with minimal loss of specificity.

Photoswitchable Dissipative Two-Dimensional Colloidal Crystals.

Control over particle interactions and organization at fluid interfaces is of great importance both for fundamental studies and practical applications. Rendering these systems stimulus-responsive is thus a desired challenge both for investigating dynamic phenomena and realizing reconfigurable materials. Here, we describe the first reversible photocontrol of two-dimensional colloidal crystallization at the air/water interface, where millimeter-sized assemblies of microparticles can be actuated through the dynamic adsorption/desorption behavior of a photosensitive surfactant added to the suspension. This allows us to dynamically switch the particle organization between a highly crystalline (under light) and a disordered (in the dark) phase with a fast response time (crystallization in ≈10 s, disassembly in ≈1 min). These results evidence a new kind of dissipative system where the crystalline state can be maintained only upon energy supply.

Intramolecularly Protein-Crosslinked DNA Gels: New Biohybrid Nanomaterials with Controllable Size and Catalytic Activity.

DNA micro- and nanogels-small-sized hydrogels made of a crosslinked DNA backbone-constitute new promising materials, but their functions have mainly been limited to those brought by DNA. Here a new way is described to prepare sub-micrometer-sized DNA gels of controllable crosslinking density that are able to embed novel functions, such as an enzymatic activity. It consists of using proteins, instead of traditional base-pairing assembly or covalent approaches, to form crosslinks inside individual DNA molecules, resulting in structures referred to as intramolecularly protein-crosslinked DNA gels (IPDGs). It is first shown that the addition of streptavidin to biotinylated T4DNA results in the successful formation of thermally stable IPDGs with a controllable crosslinking density, forming structures ranging from elongated to raspberry-shaped and pearl-necklace-like morphologies. Using reversible DNA condensation strategies, this paper shows that the gels can be reversibly actuated at a low crosslinking density, or further stabilized when they are highly crosslinked. Finally, by using streptavidin-protein conjugates, IPDGs with various enzymes are successfully functionalized. It is demonstrated that the enzymes keep their catalytic activity upon their incorporation into the gels, opening perspectives ranging from biotechnologies (e.g., enzyme manipulation) to nanomedicine (e.g., vectorization).

Light-Directed Particle Patterning by Evaporative Optical Marangoni Assembly.

Controlled particle deposition on surfaces is crucial for both exploiting collective properties of particles and their integration into devices. Most available methods depend on intrinsic properties of either the substrate or the particles to be deposited making them difficult to apply to complex, naturally occurring or industrial formulations. Here we describe a new strategy to pattern particles from an evaporating drop, regardless of inherent particle characteristics and suspension composition. We use light to generate Marangoni surface stresses resulting in flow patterns that accumulate particles at predefined positions. Using projected images, we generate a broad variety of complex patterns, including multiple spots, lines and letters. Strikingly, this method, which we call evaporative optical Marangoni assembly (eOMA), allows us to pattern particles regardless of their size or surface properties, in model suspensions as well as in complex, real-world formulations such as commercial coffee.

Photodependent Melting of Unmodified DNA Using a Photosensitive Intercalator: A New and Generic Tool for Photoreversible Assembly of DNA Nanostructures at Constant Temperature.

External control of DNA melting and hybridization, a key step in bio- and DNA nanotechnology, is commonly achieved with temperature. The use of light to direct this process is a challenging alternative, which has been only possible with a DNA modification, such as covalent grafting or mismatch introduction, so far. Here we describe the first photocontrol of DNA melting that relies on the addition of a molecule that noncovalently interacts with unmodified DNA and affects its melting properties in a photoreversible and highly robust manner, without any prerequisite in the length or sequence of the target DNA molecule. We synthesize azobenzene-containing guanidinium derivatives and show that a bivalent molecule with a conformation-dependent binding mode, AzoDiGua, strongly increases the melting temperature (Tm) of DNA under dark conditions because its trans isomer intercalates in the DNA double helix. Upon UV irradiation at 365 nm, the trans-cis isomerization induced the ejection of AzoDiGua from the intercalation binding sites, resulting in a decrease in Tm up to 18 °C. This illumination-dependent Tm shift is observed on many types of DNA, from self-complementary single-stranded or double-stranded oligonucleotides to long genomic duplex DNA molecules. Finally, we show that simply adding AzoDiGua allows us to photoreversibly control the assembly/disassembly of a DNA nanostructure at constant temperature, as demonstrated here with a self-hybridized DNA hairpin. We anticipate that this strategy will be the key ingredient in a new and generic way of placing DNA-based bio- and nanotechnologies under dynamic control by light.

Magnetic Actuation of Drops and Liquid Marbles Using a Deformable Paramagnetic Liquid Substrate.

The magnetic actuation of deposited drops has mainly relied on volume forces exerted on the liquid to be transported, which is poorly efficient with conventional diamagnetic liquids such as water and oil, unless magnetosensitive particles are added. Herein, we describe a new and additive-free way to magnetically control the motion of discrete liquid entities. Our strategy consists of using a paramagnetic liquid as a deformable substrate to direct, using a magnet, the motion of various floating liquid entities, ranging from naked drops to liquid marbles. A broad variety of liquids, including diamagnetic (water, oil) and nonmagnetic ones, can be efficiently transported using the moderate magnetic field (ca. 50 mT) produced by a small permanent magnet. Complex trajectories can be achieved in a reliable manner and multiplexing potential is demonstrated through on-demand drop fusion. Our paramagnetofluidic method advantageously works without any complex equipment or electric power, in phase with the necessary development of robust and low-cost analytical and diagnostic fluidic devices.

Protein Adsorption and Reorganization on Nanoparticles Probed by the Coffee-Ring Effect: Application to Single Point Mutation Detection.

The coffee-ring effect denotes the accumulation of particles at the edge of an evaporating sessile drop pinned on a substrate. Because it can be detected by simple visual inspection, this ubiquitous phenomenon can be envisioned as a robust and cost-effective diagnostic tool. Toward this direction, here we systematically analyze the deposit morphology of drying drops containing polystyrene particles of different surface properties with various proteins (bovine serum albumin (BSA) and different forms of hemoglobin). We show that deposit patterns reveal information on both the adsorption of proteins onto particles and their reorganization following adsorption. By combining pattern analysis with adsorption isotherm and zeta potential measurements, we show that the suppression of the coffee-ring effect and the formation of a disk-shaped pattern is primarily associated with particle neutralization by protein adsorption. However, our findings also suggest that protein reorganization following adsorption can dramatically invert this tendency. Exposure of hydrophobic (respectively charged) residues can lead to disk (respectively ring) deposit morphologies independently of the global particle charge. Surface tension measurements and microscopic observations of the evaporating drops show that the determinant factor of the deposit morphology is the accumulation of particles at the liquid/gas interface during evaporation. This general behavior opens the possibility to probe protein adsorption and reorganization on particles by the analysis of the deposit patterns, the formation of a disk being the robust signature of particles rendered hydrophobic by protein adsorption. We show that this method is sensitive enough to detect a single point mutation in a protein, as demonstrated here by the distinct patterns formed by human native hemoglobin h-HbA and its mutant form h-HbS, which is responsible for sickle cell anemia.

Light-Driven Transport of a Liquid Marble with and against Surface Flows.

Liquid marbles, that is, liquid drops coated by a hydrophobic powder, do not wet any solid or liquid substrate, making their transport and manipulation both highly desirable and challenging. Herein, we describe the light-driven transport of floating liquid marbles and emphasize a surprising motion behavior. Liquid marbles are deposited on a water solution containing photosensitive surfactants. Irradiation of the solution generates photoreversible Marangoni flows that transport the liquid marbles toward UV light and away from blue light when the thickness of the liquid substrate is large enough (Marangoni regime). Below a critical thickness, the liquid marbles move in the opposite direction to that of the surface flow at a speed increasing with decreasing liquid thickness (anti-Marangoni). We demonstrate that the anti-Marangoni motion is driven by the free surface deformation, which propels the non-wetting marble against the surface flow. We call this behavior "slide effect".

Modulation of the coffee-ring effect in particle/surfactant mixtures: the importance of particle-interface interactions.

We study the effect of surfactants on the deposits formed after the evaporation of colloidal suspension drops, at initial concentrations lower than the critical micellar concentrations, for various particle/surfactant mixtures. We show that the surfactant-mediated interactions between particles and the liquid-gas (LG) and liquid-solid (LS) interfaces, rather than the flow patterns, primarily define the morphology of the dry deposit in a robust and reproducible manner. For like-charged particle/surfactant mixtures, most of the particles form a ring-shaped deposit (according to the so-called "Coffee-Ring Effect"), but some particles can also be deposited inside the ring in a way that is modulated by electrostatic interactions between the particles and the LS interface. For oppositely charged systems, surfactant adsorption to the particle surface strongly affects particle-LG interface interactions, which in turn control the deposition pattern. For low surfactant concentrations, coffee-rings are systematically observed. For intermediate concentrations, the charge of surfactant-decorated particles becomes nearly neutral, and their hydrophobicity is enhanced, which promotes particle trapping at the LG interface. A particle skin is formed and its deposition upon drying leads to homogeneous disk-like patterns. For high surfactant concentrations, particle charge is reversed, and coffee-rings are observed again. Notably, this ring-disk-ring evolution of the deposition behavior as a function of surfactant concentration is observed in a variety of mixtures, regardless of particle absolute charge and surface chemistry as well as of surfactant charge and hydrophobicity. Its apparent universal character makes it a promising strategy for a robust control of particle deposition from evaporating drops.

ATP/azobenzene-guanidinium self-assembly into fluorescent and multi-stimuli-responsive supramolecular aggregates.

Building stimuli-responsive supramolecular systems is a way for chemists to achieve spatio-temporal control over complex systems as well as a promising strategy for applications ranging from sensing to drug-delivery. For its large spectrum of biological and biomedical implications, adenosine 5'-triphosphate (ATP) is a particularly interesting target for such a purpose but photoresponsive ATP-based systems have mainly been relying on covalent modification of ATP. Here, we show that simply mixing ATP with AzoDiGua, an azobenzene-guanidium compound with photodependent nucleotide binding affinity, results in the spontaneous self-assembly of the two non-fluorescent compounds into photoreversible, micrometer-sized and fluorescent aggregates. Obtained in water at room temperature and physiological pH, these supramolecular structures are dynamic and respond to several chemical, physical and biological stimuli. The presence of azobenzene allows a fast and photoreversible control of their assembly. ATP chelating properties to metal dications enable ion-triggered disassembly and fluorescence control with valence-selectivity. Finally, the supramolecular aggregates are disassembled by alkaline phosphatase in a few minutes at room temperature, resulting in enzymatic control of fluorescence. These results highlight the interest of using a photoswitchable nucleotide binding partner as a self-assembly brick to build highly responsive supramolecular entities involving biological targets without the need to covalently modify them.

Versatile Deposition of Complex Colloidal Assemblies from the Evaporation of Hanging Drops.

Existing strategies designed to produce ordered arrangements of colloidal particles on solid supports are of great interest for their wide range of applications, from colloidal lithography, plasmonic and biomimetic surfaces to tags for anti-counterfeiting, but they all share various degrees of complexity hampering their facile implementation. Here, a drastically simplified methodology is presented to achieve ordered particle deposition, consisting in adding micromolar amounts of cationic surfactant to a colloidal suspension drop and let it evaporate in an upside-down configuration. Confinement at the air/water interface enables particle assembly into monolayers, which are then transferred on the substrate producing highly ordered structures displaying vivid, orientation-dependent structural colors. The method is compatible with many particle types and substrates, while controlling system parameters allows tuning the deposit size and morphology, from monocrystals to polycrystalline disks and "irises", from single-component to crystal alloys with Moiré patterns, demonstrating its practicality for a variety of processes.

DNA-Encoded Immunoassay in Picoliter Drops: A Minimal Cell-Free Approach.

Immunoassays have emerged as indispensable bioanalytical tools but necessitate long preliminary steps for the selection, production, and purification of the antibody(ies) to be used. Here is explored the paradigm shift of creating a rapid and purification-free assay in picoliter drops where the antibody is expressed from coding DNA and its binding to antigens concomitantly characterized in situ. Efficient synthesis in bulk of various functional variable domains of heavy-chain only antibodies (VHH) using reconstituted cell-free expression media, including an anti-green fluorescent protein VHH, is shown first. A microfluidic device is then used to generate monodisperse drops (30 pL) containing all the assay components, including a capture scaffold, onto which the accumulation of VHH:antigen produces a specific fluorescent signal. This allows to assess, in parallel or sequentially at high throughput (500 Hz), the VHH-antigen binding and its specificity in less than 3 h, directly from a VHH-coding DNA, for multiple VHH sequences, various antigens and down to DNA concentrations as low as 12 plasmids per drop. It is anticipated that the ultraminiaturized format, robustness, and programmability of this novel cell-free immunoassay concept will constitute valuable assets in fields as diverse as antibody discovery, point-of-care diagnostics, synthetic biology, and/or bioanalytical assays.

Isothermal self-assembly of multicomponent and evolutive DNA nanostructures.

Thermal annealing is usually needed to direct the assembly of multiple complementary DNA strands into desired entities. We show that, with a magnesium-free buffer containing NaCl, complex cocktails of DNA strands and proteins can self-assemble isothermally, at room or physiological temperature, into user-defined nanostructures, such as DNA origamis, single-stranded tile assemblies and nanogrids. In situ, time-resolved observation reveals that this self-assembly is thermodynamically controlled, proceeds through multiple folding pathways and leads to highly reconfigurable nanostructures. It allows a given system to self-select its most stable shape in a large pool of competitive DNA strands. Strikingly, upon the appearance of a new energy minimum, DNA origamis isothermally shift from one initially stable shape to a radically different one, by massive exchange of their constitutive staple strands. This method expands the repertoire of shapes and functions attainable by isothermal self-assembly and creates a basis for adaptive nanomachines and nanostructure discovery by evolution.