Water-soluble π-conjugated polymers are increasingly envisioned in biosensors, in which their unique optical and electronic properties permit a highly sensitive detection of biomolecular targets. In particular, cationic π-conjugated polymers are attractive for DNA sensing technologies, through the use of the fluorescence signals either in physiological solutions or in thin films. However, in the context of enzymatic activity assays, fluorescence-based methods require covalently labeling DNA with a dye or an antibody and are limited to short time scale due to dye photobleaching. In this frame, we report here a novel possible approach to probe the cleavage of DNA by a restriction enzyme, in continuous and without covalently labeled DNA substrate. This is achieved by exploiting unique chiroptical signals arising from the chiral induction of DNA to a poly[3-(6′-(trimethylphosphonium)hexyl)thiophene-2,5-diyl] upon interaction. The cleavage of DNA by HpaI, an endonuclease enzyme, is monitored through circular dichroism (CD) signals in the spectral range where the polymer absorbs light, i.e., far away from the spectral ranges of both DNA and the enzyme. We compare the results to a conventional noncontinuous assay by polyacrylamide gel electrophoresis, and we demonstrate that induced CD signals are effective in probing the enzymatic activity. By means of molecular dynamics simulations and calculations of CD spectra, we bring molecular insights into the structure of DNA/polymer supramolecular complexes before and after the cleavage of DNA. We show that the cleavage of DNA modifies the dynamics and the organization of the polymer backbone induced by the DNA helix. Altogether, our results provide detailed spectroscopic and structural insights into the enzymatic cleavage of DNA in interaction with a π-conjugated polymer, which could be helpful for developing chiroptical detection tools to monitor the catalytic activity in real time.
We report on the construction of multiarm colloidal molecules by tip-linking filamentous bacteriophages, functionalized either by biological engineering or chemical conjugation. The affinity for streptavidin of a genetically modified vector phage displaying Strep-tags fused to one end of the viral particle is measured by determining the dissociation constant, K. In order to improve both the colloidal stability and the efficiency of the self-assembly process, a biotinylation protocol having a chemical yield higher than 90% is presented to regioselectively functionalize the cystein residues located at one end of the bacteriophages. For both viral systems, a theoretical comparison is performed by developing a quantitative model of the self-assembly and interaction of the modified viruses with streptavidin compounds, which accurately accounts for our experimental results. Multiarm colloidal structures of different valencies are then produced by conjugation of these tip-functionalized viruses with streptavidin activated nanoparticles. We succeed to form stable virus-based colloidal molecules, whose number of arms, called valency, is solely controlled by tuning the molar excess. Thanks to a fluorescent labeling of the viral arms, the dynamics of such systems is also presented in real time by fluorescence microscopy.
International audienceA simple method combining magnetic field and microparticles has been designed for assembling, locating and grafting permanent microfilaments onto a silanised glass surface. Filaments orthogonal to a glass support are implemented by assembling magnetic carboxylatex particles under magnetic field. They exhibit an aspect ratio up to 14. Carboxyl moieties on beads and silanised glass are activated using the carbodiimide method. A further reaction with long chain diamines allows the formation of permanent filaments covalently anchored onto the glass support. Grafted microfilaments can withstand intense washing with detergent solutions under ultrasound. 500 mm diameter filament spots are formed using ferromagnetic tips connected to an electromagnet. After their formation, filaments can be actuated by an external magnetic field: their orientation is tunable in real time within a 2 pi solid angle. Filaments can be biofunctionalised in order to perform biomolecular recognition on the support. We show that nucleic acid probes immobilised onto these filaments hybridize with labelled oligonucleotide targets. Due to an increase of the specific surface, the fluorescence signal of targets is 3-fold higher with filaments than with a planar glass support biofunctionalised with the same oligonucleotides. Our methodology thus provides an easy way to create filaments usable for both actuation and biodetection onto a planar support. We expect our strategy to be compatible with the formation of nanofilaments using nanoparticles
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