Sandra Michel-Souzy

Measuring blood glutamine (GLN) levels is valuable for managing urea cycle disorders. Currently, patients visit the hospital to have their GLN levels checked using analytical techniques that, despite being highly accurate and reliable, lead to long waiting times and delay clinical decisions. A point‐of‐care device would enable patients to monitor GLN levels at home, allowing for greater control over the condition. Here, we made use of a highly sensitive GLN biosensor system based on dodecameric Cytolysin A variant AS (ClyA‐AS) nanopore coupled with the GLN‐specific protein, the substrate‐binding domain 2 (SBD2). Measurements are performed on a single ClyA‐AS nanopore embedded into an artificial lipid bilayer, where the application of a small voltage generates ionic currents that report on the binding and unbinding of GLN (added in the trans side of ClyA‐AS) to SBD2 (added in cis ). We demonstrate that the ClyA‐AS and SBD2 biosensor pair can quantify GLN levels in patients’ blood plasma. To facilitate the use of this sensor in clinical and point‐of‐care settings, we also developed a dedicated algorithm that accelerates signal processing and data analysis. Together, these advances represent a significant step toward a point‐of‐care device for GLN monitoring, both at the hospital and home‐care levels.

Atomic-scale perturbations on plasmonic surfaces can give rise to enhanced optical fields on the picoscale due to adatom formation or metallic crystal rearrangements, called picocavity and flare, respectively. To date, these atomic-scale effects have been observed exclusively in closed nanogaps upon interaction with small organic molecules. Here, we generate these atomic-scale effects on an open plasmonic metasurface, which is a monolayer of Au nanoparticles on a Au film. We observe picocavity and flare spectral signatures for proteins spanning 2 orders of magnitude in mass, from lysozyme (14 kDa) to encapsulin (1932 kDa). The atomic-scale interactions reveal 1 order of magnitude picocavity-enhanced Raman intensity, a single-exponential spectral wandering distribution, and a biexponential picocavity lifetime. Our results demonstrate the plasmonic metasurface as a platform to access picoscale-enhanced optical fields that can be harnessed to study light–matter interactions and applications for sensing of large molecules and particles.

Le SST2 permet la sécrétion de protéines nécessitant un repliement périplasmique. Cette sécrétion s’effectue en deux étapes avec l’export de la protéine à travers la membrane interne puis son transport à travers la membrane externe grâce à une machinerie trans-enveloppe appelée sécréton. Les interactions mises en évidence entre les différents composants du sécréton ont permis d’établir un modèle statique de cette machinerie. Cependant les mécanismes d’assemblage et de fonctionnement du SST2 restent peu documentés. Les interactions rapportées entre le substrat et certains composants de la machinerie suggèrent une reconnaissance séquentielle du substrat au cours du processus de sécrétion.Nous avons observé 2 nouvelles interactions entre le substrat et les composants XcpY et XcpZ. Ces nouvelles données, associées aux données bibliographiques, suggèrent que la fixation du substrat sur ces deux composants induirait des changements conformationnels au niveau de la plateforme de membrane interne pouvant être nécessaires à l’énergisation du système. J’ai contribué à 2 autres projets. Le premier, toujours en développement, a consisté en l’étude du cheminement du substrat in vivo chez P. aeruginosa via la technique du photocrosslinking. Cette étude devrait permettre la mise en évidence des régions impliquées dans la reconnaissance de substrat ainsi qu’établir une potentielle chronologie dans le processus de sécrétion. Le deuxième projet a porté sur l’étude de l’assemblage et adressage de la sécrétine XcpQ. Grâce à l’utilisation d’anticorps de camélidés nous avons mis en évidence l’existence d’une pilotine et identifié le site d’interaction du partenaire XcpP sur la sécrétine.

Polymer microstructures rely on tacticity, yet exploration in polyamines has focused predominantly on atactic polymers. We introduce a method to synthesize a diverse library of ortho and para-cyanobenzenesulfonyl-activated-methyl aziridines using R, S, and racemic alaninol. Living anionic ring-opening polymerization of racemic sulfonyl aziridines yields soluble polymers, while enantiomerically-pure sulfonyl aziridines follow a dispersion polymerization with complete monomer conversion giving access to stereoblock copolymers. Removal of activation groups is achieved using dodecanethiol and tert-butylimino-tri(pyrrolidino)phosphorane to obtain isotactic or atactic linear polypropylene imines (LPPIs). High-purity L-PPIs are obtained in salt and neutral forms with high yields. Stereoblock copolymers of poly-R-block-S-polysulfonamides and respective polypropylene imine stereoblocks are synthesized, revealing helical structures in water influenced by the monomer type and sequence in CD spectroscopy. Molecular dynamics simulations confirm the helical nature of isotactic LPPIs in water. Bulk characterization demonstrates the first crystalline isotactic polyamines via spherulite growth in polarized light, atomic force microscopy and XRD analyses. In cell-transfection studies, the synthesized isotactic LPPIs exhibit lower toxicity and transfection efficiency than commercial hyperbranched polyethylene imine, with longer chains showing increased transfection efficiency. These isotactic polymers open avenues for complex macromolecular architectures with optically active polyamines akin to poly(amino acid)s but lacking hydrolytically cleavable amide links.

Gene therapy is widely recognized as a promising method in combating diseases caused by gene abnormalities or deletions. The effects of these deletions and mutations are ameliorated through gene therapy by means of transfection vectors. These delivery vehicles are tasked with protecting the gene and transporting it to the cell nucleus when necessary. Nano-sized hydrogel particles, also known as nanogels, are crosslinked polymeric nanoparticles that are promising materials for such biomedical applications. Whereas most cationic carriers for gene delivery are nitrogen-based, we are interested in utilizing a sulfonium moiety to this end. Diversifying the available gene vectors not only satisfies scientific curiosity, it could also offer improved gene delivery efficiencies. Here we describe the synthesis of glycidyl methacrylate (GMA) nanogels as a platform for subsequent functionalization. Ring-opening reactions with diethyl sulfide were carried out to install permanent cationic sulfonium groups on the nanogels, yielding readily water-soluble nanogels with a zeta potential of ζ = +40 ± 0.5 mV at neutral pH and a mean diameter of D = 29 ± 10 nm as determined by transmission electron microscopy (TEM). The degree of functionalization with sulfonium groups was found to be tunable. These nanogels were subjected to post-synthesis modifications resulting in biocompatible sulfonium nanogels containing a thioglycerol moiety. Polyplexes were formed by successful incubation with plasmid DNA encoding for green fluorescent protein (pCMV-GFP), at various ratios. In a next step, nucleic acid delivery by sulfonium nanogels was probed for various cell lines for the first time, showing poor delivery properties.

Encapsulins are a class of protein nanocages that are found in bacteria, which are easy to produce and engineer in E. coli expression systems. The encapsulin from Thermotoga maritima (Tm) is well studied, its structure is available, and without modification it is barely taken up by cells, making it promising candidates for targeted drug delivery. In recent years, encapsulins are engineered and studied for potential use as drug delivery carriers, imaging agents, and as nanoreactors. Consequently, it is important to be able to modify the surface of these encapsulins, for example, by inserting a peptide sequence for targeting or other functions. Ideally, this is combined with high production yields and straightforward purification methods. In this chapter, we describe a method to genetically modify the surface of Tm and Brevibacterium linens (Bl) encapsulins, as model systems, to purify them and characterize the obtain nanocages. Key words Nanocages Encapsulin Targeting Drug encapsulation Genetic modification

A dopamine based macromolecular glue is used to facilitate the construction of hybrid nanomaterials that are coated with virus capsid proteins, with the aim to improve stability, biocompatibility, and function.

Charge transport across proteins can be surprisingly efficient over long distances—so-called long-range tunneling—but it is still unclear as to why and under which conditions (e.g., presence of co-factors, type of cargo) the long-range tunneling regime can be accessed. This paper describes molecular tunneling junctions based on an encapsulin (Enc), which is a large protein cage with a diameter of 24 nm that can be loaded with various types of (small) proteins, also referred to as “cargo”. We demonstrate with dynamic light scattering, transmission electron microscopy, and atomic force microscopy that Enc, with and without cargo, can be made stable in solution and immobilized on metal electrodes without aggregation. We investigated the electronic properties of Enc in EGaIn-based tunnel junctions (EGaIn = eutectic alloy of Ga and In that is widely used to contact (bio)molecular monolayers) by measuring the current density for a large range of applied bias of ±2.5 V. The encapsulated cargo has an important effect on the electrical properties of the junctions. The measured current densities are higher for junctions with Enc loaded with redox-active cargo (ferritin-like protein) than those junctions without cargo or redox-inactive cargo (green fluorescent protein). These findings open the door to charge transport studies across complex biomolecular hierarchical structures.

Using the biomarker hypermethylated DNA (hmDNA) for cancer detection requires a pretreatment to isolate or concentrate hmDNA from nonmethylated DNA. Affinity chromatography using a methyl binding domain-2 (MBD2) protein can be used, but the relatively low enrichment selectivity of MBD2 limits its clinical applicability. Here, we developed a superselective, multivalent, MBD2-coated platform to improve the selectivity of hmDNA enrichment. The multivalent platform employs control over the MBD2 surface receptor density, which is shown to strongly affect the binding of DNA with varying degrees of methylation, improving both the selectivity and the affinity of DNAs with higher numbers of methylation sites. Histidine-10-tagged MBD2 was immobilized on gold surfaces with receptor density control by tuning the amount of nickel nitrilotriacetic acid (NiNTA)-functionalized thiols in a thiol-based self-assembled monolayer. The required MBD2 surface receptor densities for DNA surface binding decreases for DNA with higher degrees of methylation. Both higher degrees of superselectivity and surface coverages were observed upon DNA binding at increasing methylation levels. Adopting the findings of this study into hmDNA enrichment of clinical samples has the potential to become more selective and sensitive than current MBD2-based methods and, therefore, to improve cancer diagnostics.