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- Pour toute publication de résultats ayant reçu l’aide de l’IPGG (présence dans les locaux de l’IPGG, passage sur la plateforme technologique de l’IPGG, collaboration inter équipes IPGG, lié à une bourse doctorale ou postdoctorale IPGG, ou encore utilisation des espaces communs), il vous faut indiquer  cette phrase « Ce travail a été réalisé avec le soutien du laboratoire d’excellence Institut Pierre-Gilles de Gennes (programme Investissements d’avenir ANR-10-IDEX-0001-02 PSL et ANR-10-LABX-31). » / « This work has received the support of "Institut Pierre-Gilles de Gennes" (laboratoire d’excellence, “Investissements d’avenir” program ANR-10-IDEX-0001-02 PSL and ANR-10-LABX-31.). ».

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Protein-protected metal nanoclusters as diagnostic and therapeutic platforms for biomedical applications
ImanZare, Daniel M.Chevrier, Anna Cifuentes-Rius, NasrinMoradi, Yunlei Xianyu, SubhadipGhosh, LauraTrapiella-Alfonso, Ye Tian, Alireza Shourangiz-Haghighi, Saptarshi Mukherjee, Kelong Fan, Michael R.Hamblin
Materials Today - - doi.org/10.1016/j.mattod.2020.10.027 - 2021
The use of protein templates for the controlled synthesis of inorganic nanostructures has gained considerable attention in multidisciplinary fields, including electronics, optics, energy, sensing, and biomedicine, owing to their biocompatibility and structural programmability. The possible synergistic combination of protein scaffolds (and other biomolecules/biopolymers) with metal nanoclusters (MNCs) has created a new class of highly photoluminescent nanoprobes and nanodevices. For the first time, we will discuss the different types of protein templates used for MNC preparation with an emphasis on their optoelectronic properties for application. In particular, applications of protein-coated MNCs for chemosensing or biosensing of cancer biomarkers, neurotransmitters, pathogenic microorganisms, biomolecules, pharmaceutical compounds, and immunoassays are discussed in detail herein. Fluorescence-based and multimodal molecular imaging, both in vitro and in vivo based on functional proteins are also covered. Furthermore, we discuss the burgeoning growth of protein-coated MNCs (e.g., gold (Au) and silver (Ag) NCs) to develop synergistic nanotherapeutics with potential biomedical applications in chemotherapy, radiotherapy, photodynamic therapy (PDT), photothermal therapy (PTT), and antibacterial activity, as well as MNC-containing nanocomposites for enhanced bioimaging and controlled drug release. Overall, the proposed review highlights the recent progress, technical challenges and new horizons in this field, and summarizes our understanding of how MNC properties interact with the biological function of protein scaffolds to develop synergistic nanotherapeutics towards clinical translation.
Superparamagnetic iron oxide nanoparticles functionalized with a binary alkoxysilane array and poly(4-vinylpyridine) for magnetic targeting and pH-responsive release of doxorubicin
Fernando Espinola-Portilla,ab Oracio Serrano-Torres, Gilberto F. Hurtado-López, Uriel Sierra, Anne Varenne, Fanny d’Orlyé, Laura Trapiella-Alfonso, Silvia Gutiérrez-Granados and Gonzalo Ramírez-García
New Journal of Chemistry - 45 3600-3609 - doi.org/10.1039/D0NJ05227B - 2021
Chemotherapeutic drugs cause harmful side effects in cancer patients due to their low specificity, calling for the development of more effective strategies for their dosage and administration. In this work, a smart drug nanocarrier was synthesized through the covalent functionalization of superparamagnetic iron oxide nanoparticles with a triblock copolymer, which includes a dual alkoxysilane array, ((3-aminopropyl)triethoxysilane and (trimethoxysilyl)propyl methacrylate), and the pH-responsive poly(4-vinylpyridine). The synthetic conditions were optimized through structural and physicochemical characterization after every functionalization step. Afterward, the systematic loading, capture, and release of the anticancer drug doxorubicin (Dox) were demonstrated at relevant pH values using a specially designed square wave voltammetry technique. This strategy revealed that the P4VP polymeric chains underwent reversible hydrophobic to hydrophilic transitions in acidic media, triggering a molecular distention driven by the induced intermolecular electro-repulsive forces. Thereafter, the Dox solution can easily penetrate the polymeric layer at pH values below 5.62 (the pKa of poly(4-vinylpyridine)), allowing a loading of 61.9 ± 5.4 mg g−1 in the nanocomplex. After deprotonation in a pH 7.4 buffer solution, the polymer chains underwent intermolecular interactions again, capturing the drug molecules. Subsequently, 93.5 ± 3.5% of the payload was released upon suspension of the nanocomplex in pH 4.0 media, which is significantly more acidic than healthy tissues. Since the magnetic properties of the MNPs were practically unaffected by the surface modification, this nanocomplex offers a versatile strategy for the pH-selective and magnetically-guided release of drugs.

Non-amplified impedimetric genosensor for quantification of miRNA-21 based on the use of reduced graphene oxide modified with chitosan
Michael López Mujica Yuanyuan Zhang Fabiana Gutierrez Féthi Bédioui Gustavo Rivasa
Microchemical Journal - 160 105596 - https://doi.org/10.1016/j.microc.2020.105596 - 2021
We report here an impedimetric genosensor for the quantification of microRNA-21 using [Fe(CN)6]3−/4− as redox probe to transduce the hybridization event. The biosensing platform was built at a thiolated-gold electrode by covalent bond of reduced graphene oxide (RGO) modified with chitosan (CHIT) and further covalent attachment of the aminated DNA probe. GO was used to provide the carboxylic groups for the covalent attachment of CHIT and, once reduced, to improve the electroactivity of the resulting platform, while CHIT served as a bridge between the thiol and the aminated probe DNA. The proposed bioanalytical platform allows the label-free, non-amplified, simple and fast biosensing of microRNA-21, with a linear range between 1.0 × 10−12 M and 1.0 × 10−8 M, a sensitivity of (134 ± 4) ΩM−1 (r2 = 0.996), a detection limit of 300 fM, and a reproducibility of 5.9% for 1.0 × 10−12 M miRNA-21 and 2.2% for 1.0 × 10−9 M miRNA-21. The genosensor was successfully used for the quantification of microRNA-21 in enriched human blood serum, urine and saliva samples.


Corrosion analysis of AISI 430 stainless steel in the presence of Escherichia coli and Staphylococcus aureus
C.Guerra A.Ringuedé M.I.Azocar M.Walter C.Galarce F.Bedioui M.Cassir M.Sancy
Corrosion Science - 181 109204 - doi.org/10.1016/j.corsci.2020.109204 - 2021
AISI 430 stainless steel is an attractive material to be used in the healthcare industry, particularly as a sensor due to its low cost, corrosion resistance, as well as being Ni-free. AISI 430 was evaluated in an artificial sweat solution with the presence of Escherichia coli, and Staphylococcus aureus. Surface microbial analyses did not reveal colonization of bacteria on metallic surfaces, even when bacteria adhesion was investigated in a Müeller-Hinton solution. However, by electrochemical techniques, the AISI 430 surfaces demonstrated clear signs of corrosion mainly in a sterile medium after two weeks of exposure.



Stereo Darkfield Interferometry : a versatile localization method for subnanometer force spectroscopy of single molecules and 3D-tracking of single cells
Martin Rieu, Thibault Vieille, Gaël Radou, Raphaël Jeanneret, Nadia Ruiz, Bertrand Ducos, Jean-François Allemand, Vincent Croquette
Biological Physics - - DOI:10.1126/sciadv.abe3902 - 2020
Super-resolutive 3D tracking, such as PSF engineering or evanescent field imaging has long been used to track microparticles and to enhance the throughput of single molecules force spectroscopic measurements. However, current methods present two drawbacks. First, they lack precision compared with optical tweezers or AFM. Second, the dependence of their signal upon the position is complex creating the need for a time-consuming calibration step.
Here, we introduce a new optical technique that circumvents both issues and allows for a simple, versatile and efficient 3D tracking of diluted particles while offering a sub-nanometer frame-to-frame precision in all three spatial directions. The principle is to combine stereoscopy and interferometry, such that the z (axial) position is measured through the distance between two interferometric fringe patterns. The linearity of this stereoscopy technique alleviates the need for lookup tables while the structured interferometric pattern enhances precision. On the other hand, the extended spatial footprint of this PSF maximizes the number of photons detected per frame without the need of fancy cameras, nor the need for complex hardware. Hence, thanks to its simplicity and versatility, we believe that SDI (Stereo Darkfield Interferometry) technology has the potential to significantly enhance the spreading of 3D tracking.
We demonstrate the efficiency of this technique on various single-molecule measurements thanks to magnetic tweezers. In particular we demonstrate the precise quantification of two-state dynamics involving axial steps as short as 1 nm. We then show that SDI can be directly embedded in a commercial objective providing a means to track multiple single cells in 3D .
Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST
Chekli, Yankel; Peron-Cane, Caroline; Dell'Arciprete, Dario; Allemand, Jean-François; Li, Chenge; Ghigo, Jean-Marc; Gautier, Arnaud; Lebreton, Alice; Desprat, Nicolas; Beloin, Christophe
Scientific Reports - 10(1) 15791 - 10.1038/s41598-020-72498-2 - 2020
Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and used to specifically tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins.
Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells
Peron-Cane, Caroline; Fernandez, José-Carlos; Leblanc, Julien; Wingertsmann, Laure; Gautier, Arnaud; Desprat, Nicolas; Lebreton, Alice
journal article - 16(10) e1009001 - 10.1371/journal.ppat.1009001 - 2020
Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.s.
Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking
Martin Rieu; Valle-Orero, Jessica; Ducos, Bertrand; Allemand, Jean-François; Croquette, Vincent
bioRxiv - - 10.1101/2020.09.30.321232 - 2020
Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.Competing Interest StatementThe authors have declared no competing interest.
Performance evaluation of a MIP for the MISPE-LC determination of p-[18F]MPPF and a potential metabolite in human plasma
F.Lecomte J.Aerts Plenevaux .Defraiteur. Chapuis-Hugonc. Rozetd. Chiape. Luxen. Pichon, Ph.Huberta C.Huberta
ELSEVIER - 180 113015 - https://doi.org/10.1016/j.jpba.2019.113015 - 2020
Within the family of serotonin (5-HT) receptors, the 5-HT1A subtype is particularly interesting as it may be involved in various physiological processes or psychological disorders. The p-[18F]MPPF, a highly selective 5-HT1A antagonist, is used for in vivo studies in human or animal by means of positron emission tomography (PET) [1].
In order to selectively extract p-[18F]MPPF and its main metabolites from plasma, molecularly imprinted polymer (MIP) was prepared against these compounds by using the p-MPPF as template. For the control of the selectivity, non-imprinted polymer (NIP) was also synthesized without template. The MIP sorbent, packed in disposable extraction cartridges (DECs), was then evaluated as molecularly imprinted solid-phase extraction (MISPE) prior to the LC determination. The conditions of extraction were evaluated in order to obtain the highest selective retention of the p-[18F]MPPF and its metabolites on this MIP. The MIP selectivity was exploited in the loading and washing steps by adjusting the pH of plasma samples at a suitable value and by selecting mixtures for the washing step to limit the contribution of non-specific interactions. Other important parameters involved in the conditioning and elution steps were also studied. Finally, a pre-validation was carried out with optimal extraction conditions to demonstrate the performance of this MISPE-LC method as a generic method in the context of evaluation of new MISPE for p-[18F]MPPF and its potential for metabolites extraction from human plasma.
Identification and semi-relative quantification of intact glycoforms by nano-LC–(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone
Amira Al Matari, Audrey Combès, Julien Camperi, Thierry Fournier, Valérie Pichon & Nathalie Delaunay
ELSEVIER - 412 5729–5741 - , 10.1007/s00216-020-02794-3 - 2020
Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level. A recombinant hCG-based drug, Ovitrelle®, was analyzed. This method combined with an appropriate data treatment allowed the detection of not only the major isoforms but also the minority ones with a high mass accuracy. More than 30 hCGα glycoforms were detected without overlapping of the isotopic patterns. The figures of merit of the method were assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.1 and 6.08% (n = 3), with an average of 0.4%. The RSDs of the peak area measured on the extracted ion chromatogram of each glycoform are below 38% (n = 3), with an average of 16%, thus allowing semi-relative quantification. The ability to accurately profile glycosylated variants of hCGα was next demonstrated by comparing qualitatively and semi-quantitatively 3 batches of Ovitrelle®. The method was also used to analyze 3 batches of a recombinant FSH-based drug, Puregon®, and 30 FSHα glycoforms were detected and semi-quantified. This demonstrates the high potential of this method for fast quality control or comparison of the glycosylation of glycoprotein-based pharmaceutical preparations.

606 publications.