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Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

The residence time of a drug on its target has been suggested as a more pertinent metric of therapeutic efficacy than the traditionally used affinity constant. Here, we introduce junctured-DNA tweezers as a generic platform that enables real-time observation, at the single-molecule level, of biomolecular interactions. This tool corresponds to a double-strand DNA scaffold that can be nanomanipulated and on which proteins of interest can be engrafted thanks to widely used genetic tagging strategies. Thus, junctured-DNA tweezers allow a straightforward and robust access to single-molecule force spectroscopy in drug discovery, and more generally in biophysics. Proof-of-principle experiments are provided for the rapamycin-mediated association between FKBP12 and FRB, a system relevant in both medicine and chemical biology. Individual interactions were monitored under a range of applied forces and temperatures, yielding after analysis the characteristic features of the energy profile along the dissociation landscape.

Living systems offer attractive strategies to generate nanoscale structures because of their innate functional properties such as the dynamic assembly of ordered nanometer fibers, the generation of mechanical forces, or the directional transport mediated by molecular motors. The design of hybrid systems, capable of interfacing artificial building blocks with biomolecules, may be a key step toward the rational design of nanoscale devices and materials. Here, we have designed a bottom-up approach to organize cytoskeletal elements in space using the self-assembly properties of magnetic nanoparticles conjugated to signaling proteins involved in microtubule nucleation. We show that magnetic nanoparticles conjugated to signaling proteins involved in microtubule nucleation can control the positioning of microtubule assembly. Under a magnetic field, a self-organized pattern of biofunctionalized nanoparticles is formed and leads to the nucleation of a periodical network of microtubules in Xenopus laevisegg extract. Our method shows how bioactive nanoparticles can generate a biochemically active pattern upon magnetic actuation, which triggers the spatial organization of nonequilibrium biological structures.

We have recently developed a microsystem to electroporate a few cells at the surface of early post-implantation mouse embryos. We could achieve the efficient, reproducible, and safe transfection of various genetic markers, which allowed single cell fate studies during morphogenesis. However, our single-use polymeric device necessitated to be fabricated in a clean room the day before each experiment. Thus, we here introduce an all-glass chip that any biologist can easily recycle in its laboratory. Most importantly, during the technological evolution process we could validate a comprehensive design strategy based on finite element model simulations. Indeed, both the embryo and the microsystem were represented as very simple electric objects and stationary computations enabled to properly predict the voltage pulse amplitude that would yield optimal device performances.

We describe a method of controlled evaporation on a textured substrate for self-assembling and shaping gold-nanorod-based materials. Tridimensional wall features are formed over areas as large as several square millimeters. Furthermore, analyses by small-angle X-ray scattering and scanning electron microscopy techniques demonstrate that colloids are locally ordered as a smectic B phase. Such crystallization is in fact possible because we could finely adjust the nanoparticle charge, knowledge that additionally enables tuning the lattice parameters. In the future, the type of ordered self-assemblies of gold nanorods we have prepared could be used for amplifying optical signals.

When placed in a Hele-Shaw cell and submitted to a uniform magnetic field, a suspension of superparamagnetic particles self-organizes into a 2D array of columns with hexagonal order on short distances and glasslike order on long distances. Such arrays were usefully used for fast electrophoretic separation of long DNA molecules. Although self-assembled matrices have the advantages of simplicity and tunability, it is impossible to have the precisely controlled order and pore size obtainable in a microlithographic array. We address the latter challenge here. A magnetic nickel template is prepared on one cell surface by lift-off. Under a uniform external field, the nickel dots create local magnetic field gradients that attracts the beads, imposing a predefined order. We demonstrate the ability to separate long DNA molecules with these matrices and achieve the first steps of studying the resolution of the DNA separation over a wide range of posts density.

Cantilevers and optical tweezers are widely used for micromanipulating cells or biomolecules for measuring their mechanical properties. However, they do not allow easy rotary motion and can sometimes damage the handled material. We present here a system of magnetic tweezers that overcomes those drawbacks while retaining most of the previous dynamometers properties. Electromagnets are coupled to a microscope-based particle tracking system through a digital feedback loop. Magnetic beads are first trapped in a potential well of stiffness ∼10−7N/m. Thus, they can be manipulated in three dimensions at a speed of ∼10μm/s and rotated along the optical axis at a frequency of 10Hz. In addition, our apparatus can work as a dynamometer relying on either usual calibration against the viscous drag or complete calibration using Brownian fluctuations. By stretching a DNA molecule between a magnetic particle and a glass surface, we applied and measured vertical forces ranging from 50fN to 20pN. Similarly, nearly horizontal forces up to 5pN were obtained. From those experiments, we conclude that magnetic tweezers represent a low-cost and biocompatible setup that could become a suitable alternative to the other available micromanipulators.

We have recently developed a microsystem to electroporate a few cells at the surface of early post-implantation mouse embryos. We could achieve the efficient, reproducible, and safe transfection of various genetic markers, which allowed single cell fate studies during morphogenesis. However, our single-use polymeric device necessitated to be fabricated in a clean room the day before each experiment. Thus, we here introduce an all-glass chip that any biologist can easily recycle in its laboratory. Most importantly, during the technological evolution process we could validate a comprehensive design strategy based on finite element model simulations. Indeed, both the embryo and the microsystem were represented as very simple electric objects and stationary computations enabled to properly predict the voltage pulse amplitude that would yield optimal device performances.