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 (â¼100 µM) underlines potential interest of this nanoplatform for biomedical applications.
Biotin , Polyethylene Glycols , Animals , Mice , Nanogels , Streptavidin , Proteins , DNA/metabolism , Lipids , Drug Carriers
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.
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.
Nanostructures , Nanotechnology , Nucleic Acid Conformation , DNA/chemistry , Nanostructures/chemistry , Temperature
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.
Antibodies , Antigens , Immunoassay , Immunoglobulin Heavy Chains/genetics , DNA
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.
Liposomes , Luminescent Measurements , Electrochemical Techniques/methods , Electrodes , Luminescent Measurements/methods , Photometry
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.
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.
DNA , Glucose Oxidase , Horseradish Peroxidase , Nanogels , Streptavidin
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)â it is compatible with various origami shapes, 2)â it perfectly preserves fine structural details as well as site-specific functionality, and 3)â it can be applied to dynamically address the spatial distribution of origami-tethered proteins.
BACKGROUND: Opioid-free anesthesia (OFA) is a new method of anesthesia based on a paradigm shift. Under general anesthesia, the physiology and/or the pathophysiological variations clinically observed are more a reflection of a systemic reaction to the stress (surgical and anesthesia stresses) than a true reflection of pain. OBJECTIVE: To report the results of a large monocenter, retrospective, non-interventional observational study of all consecutive patients who received a total intravenous (IV)-OFA protocol for the surgical management of major open abdominal and urological surgery. PATIENTS AND METHODS: We retrospectively extracted the anesthesia files of 311 consecutive patients (regional anesthesia excluded). No opioids were administered to any of these patients during the surgery. IV morphine administered in the recovery room was the primary endpoint of the study. The secondary endpoints included the amount of opioid required during the first two postoperative days, as well as the maximum pain intensity. RESULTS: Only very small doses of IV morphine were administered. The mean total morphine titration was 2 mg (1.9 ± 2.9 mg), corresponding to control of the maximal level of pain to 2.1 ± 2.6 as evaluated with a numerical scale in the postoperative care unit. Similarly, we observed a very low level of morphine consumption during the first two postoperative days. CONCLUSIONS: These results highlight the safety and the feasibility of our total IV-OFA protocol, thus confirming this new paradigm. Under general anesthesia, the cardiovascular and inflammatory response to the stress could be reliably managed through a multimodal approach without a need for opioids. In the postoperative period, very low doses of opioids were required.
The so-called coffee-ring effect (CRE) is extraordinarily common, problematic in industry and attractively puzzling for researchers, with the accepted rule that it requires two key-ingredients: solvent evaporation and contact line pinning. Here, we demonstrate that the CRE also occurs when the solvent of a pinned sessile drop transfers into another liquid, without involving any evaporation. We show that it shares all characteristic features of the evaporative CRE: solvent transfer-driven transport of solutes to the contact line, ring-shaped deposit, closely-packed particle organization at the contact line, and size-dependent particle sorting. We thus suggest expanding the definition of the coffee-ring effect to any pinned drop having its solvent transferring to an outer medium where the drop compounds cannot be dissolved.
The "flipping method" is a new straightforward way to both adsorb and organize microparticles at a liquid interface, with ultralow amounts of a surfactant and no other external forces than gravity. Here we demonstrate that it allows the adsorption of a variety of inorganic nanoparticles at an air/water interface, in an organized way, which is directly controlled by the surfactant concentration, ranging from amorphous to highly crystalline two-dimensional assemblies. With micromolar amounts of a conventional cationic surfactant (dodecyltrimethylammonium bromide, DTAB), nanoparticles of different compositions (silica, silver, and gold), sizes (down to 100 nm) and shapes (spheres and cubes) adsorb from the bulk and directly organize at the air/water interface, resulting in marked optical properties such as reflectivity or intense structural coloration.
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.
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.
G-Quadruplexes , Light , Photochemical Processes , Azo Compounds , Fluorescence , Heterocyclic Compounds , Hydrogen-Ion Concentration , Intercalating Agents
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 ≈10â s, disassembly in ≈1â min). These results evidence a new kind of dissipative system where the crystalline state can be maintained only upon energy supply.
We precisely measure the effect of moderate magnetic field intensity on the surface tension of liquids, by placing pendant drops inside uniform fields where bulk forces due to gradients are eliminated. The surface tension of water is unaffected while that of paramagnetic salt solutions slightly decreases with increasing field strength.
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. 50â 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.
We have recently devised the evaporative optical Marangoni assembly (eOMA), a novel and versatile interfacial flow-based method for directing the deposition of colloidal nanoparticles (NPs) on solid substrates from evaporating sessile drops along desired patterns using shaped UV light. Here, we focus on a fixed UV spot irradiation resulting in a cylinder-like deposit of assembled particles and show how the geometrical features of the single deposit can be tailored in three dimensions by simply adjusting the optical conditions or the sample composition, in a quantitative and reproducible manner. Sessile drops containing cationic NPs and a photosensitive surfactant at various concentrations are allowed to evaporate under a single UV beam with a diameter much smaller than that of the drop. After complete evaporation, the geometrical characteristics of the NP deposits are precisely assessed using optical profilometry. We show that both the volume and the radial size of the light-directed NP deposit can be adjusted by varying the diameter or the intensity of the UV beam or alternatively by changing the concentration of the photosensitive surfactant. Notably, in all these cases, the deposits display an almost constant median height corresponding to a few layers of particles. Moreover, both the radial and the axial extent of the patterns are tuned by changing the NP concentration. These results are explained by the correlation among the strength of Marangoni flow, the particle trapping efficiency, and the volume of the deposit, and by the role of evaporation-driven flow in strongly controlling the deposit height. Finally, we extend the versatility of eOMA by demonstrating that NPs down to 30 nm in diameter can be effectively patterned on glass or polymeric substrates.
Genes, Synthetic/physiology , Genetic Therapy/methods , Molecular Targeted Therapy/methods , Neoplasms/genetics , Neoplasms/therapy , Animals , Gene Expression Regulation, Neoplastic , Genetic Therapy/trends , Humans , Molecular Targeted Therapy/trends , Precision Medicine/methods , Precision Medicine/trends , Treatment Outcome
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).
DNA/chemistry , Gels/chemistry , Hydrogels/chemistry , Nanostructures/chemistry , Catalysis , Streptavidin/chemistry
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.
Hemoglobins/chemistry , Hemoglobins/genetics , Nanoparticles/chemistry , Point Mutation , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/genetics , Adsorption , Adult , Animals , Cattle , Humans , Models, Molecular , Polystyrenes/chemistry , Protein Conformation
