Search

International audience; Over the years, click and bioorthogonal reactions have been the subject of considerable research efforts. These high-performance chemical reactions have been developed to meet requirements not often provided by the chemical reactions commonly used today in the biological environment, such as selectivity, rapid reaction rate and biocompatibility. Click and bioorthogonal reactions have been attracting increasing attention in the biomedical field for the engineering of nanomedicines. In this review, we study a compilation of articles from 2014 to the present, using the terms "click chemistry and nanoparticles (NPs)" to highlight the application of this type of chemistry for applications involving NPs intended for biomedical applications. This study identifies the main strategies offered by click and bioorthogonal chemistry, with respect to passive and active targeting, for NP functionalization with specific and multiple properties for imaging and cancer therapy. In the final part, a novel and promising approach for "two step" targeting of NPs, called pretargeting (PT), is also discussed; the principle of this strategy as well as all the studies listed from 2014 to the present are presented in more detail.

This study reports the development of a sensitive magnetic bead-based enzyme-linked immunoassay (MELISA) for the pan-reactive detection of the Influenza A virus. The assay combines immunomagnetic beads and biotin-nanoparticle-based detection to quantify a highly conserved viral nucleoprotein in virus lysates. At the capture step, monoclonal antibody-coated magnetic microbeads were used to bind and concentrate the nucleoprotein in samples. The colorimetric detection signal was amplified using biotinylated silica nanoparticles (NP). These nanoparticles were functionalized on the surface with short DNA spacers bearing biotin groups by an automated supported synthesis method performed on nano-on-micro assemblies with a DNA/RNA synthesizer. A biotin-nanoparticle and immunomagnetic bead-based assay was developed. We succeeded in detecting Influenza A viruses directly in the lysis buffer supplemented with 10% saliva to simulate the clinical context. The biotin-nanoparticle amplification step enabled detection limits as low as 3 × 103 PFU mL−1 and 4 × 104 PFU mL−1 to be achieved for the H1N1 and H3N2 strains respectively. In contrast, a classical ELISA test based on the same antibody sandwich showed detection limit of 1.2 × 107 PFU mL−1 for H1N1. The new enhanced MELISA proved to be specific, as no cross-reactivity was found with a porcine respiratory virus (PRRSV). Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-020-03081-x.

International audience; In this work, surface-enhanced Raman spectroscopy (SERS) method based on uncoated spherical gold nanoparticles as SERS substrate was applied for the detection and discrimination of three different bacterial strains: Gram-positive Bacillus subtilis and Lactobacillus rhamnosus GG and Gram-negative Escherichia coli. The exploitation of bacteria SERS spectra by multivariate data analysis (principal component analysis and partial least squares-discriminant analysis) proved the robustness of the developed method and its suitability to discriminate between the three different strains despite their relatively similar Raman signals. The SERS performance of the developed method was compared with that of the classical silver nanoparticles indicating a better bacteria signal enhancement, up to 15 times higher in case of E. coli, likely due to stronger interactions of gold nanoparticles with components such as purine molecules that show dominant bands in the bacterial SERS spectra. K E Y W O R D S bacteria detection, bacteria discrimination, gold nanoparticles, SERS

The interactions of mono-rhamnolipids (mono-RLs) with model membranes were investigated through a biomimetic approach using phospholipid-based liposomes immobilized on a gold substrate and also by the multiparametric surface plasmon resonance (MP-SPR) technique. Biotinylated liposomes were bound onto an SPR gold chip surface coated with a streptavidin layer. The resulting MP-SPR signal proved the efficient binding of the liposomes. The thickness of the liposome layer calculated by modeling the MP-SPR signal was about 80 nm, which matched the average diameter of the liposomes. The mono-RL binding to the film of the phospholipid liposomes was monitored by SPR and the morphological changes of the liposome layer were assessed by modeling the SPR signal. We demonstrated the capacity of the MP-SPR technique to characterize the different steps of the liposome architecture evolution, i.e., from a monolayer of phospholipid liposomes to a single phospholipid bilayer induced by the interaction with mono-RLs. Further washing treatment with Triton X-100 detergent left a monolayer of phospholipid on the surface. As a possible practical application, our method based on a biomimetic membrane coupled to an SPR measurement proved to be a robust and sensitive analytical tool for the detection of mono-RLs with a limit of detection of 2 μg mL-1.

During the design of graphene (G), their electronic, chemical and electrochemical properties among others can be tuned by incorporating heteroatoms in the honeycomb structure. In this study, we investigated boron doped graphene (BG) synthesis and the effect of boron content on the electrochemical property of graphene. To this end, BG was synthesized using pulsed laser deposition (PLD) with carbon and boron as solid sources, followed by rapid thermal annealing in the presence of nickel catalyst. Raman spectroscopy revealed that the BG films consisted of few-layer graphene and boron doping increased the defects level in the films. X-ray photoelectron spectroscopy confirmed the successful boron doping into the graphene with the control of boron content. Electrochemical characterization demonstrated that BG electrodes exhibit higher reversibility and electron transfer constant than the G electrode. All these results prove that PLD method is a viable alternative way to produce BG films with excellent electrochemical properties.

The authors thank the Centre Technologique des Microstructures of the Lyon 1 University for providing access to its TEM facilities, and the MIMA2 platform Jouy en Josas for access to electron microscopy equipment (MIMA2, INRAE, 2018. Microscopy and Imaging Facility for Microbes, Animals and Foods, https://doi.org/10.15454/1. 5572348210007727E12). JV thanks Maria-Vesna Nikolic (University of Belgrade, Serbia) for English editing.; International audience; Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/μL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.

Paper-based DNA biosensors are powerful tools in point-of-care diagnostics since they are affordable, portable, user-friendly, rapid and robust. However, their sensitivity is not always as high as required to enable DNA quantification. To improve the response of standard dot blots, we have applied a new enhancement strategy that increases the sensitivity of assays based on the use of biotinylated silica-nanoparticles (biotin-Si-NPs). After immobilization of a genomic Campylobacter DNA onto a paper membrane, and addition of a biotinylated-DNA detection probe, hybridization was evidenced using streptavidin-conjugated to horseradish peroxidase (HRP) in the presence of luminol and H2O2. Replacement of the single biotin by the biotin-Si-NPs boosted on average a 30 fold chemiluminescent read-out of the biosensor. Characterization of biotin-Si-NPs onto a paper with immobilized DNA was done using a scanning electron microscope. A limit of detection of 3 pg/μL of DNA, similar to the available qPCR kits, is achieved, but it is cheaper, easier and avoids inhibition of DNA polymerase by molecules from the food matrices. We demonstrated that the new dot blot coupled to biotin-Si-NPs successfully detected Campylobacter from naturally contaminated chicken meat, without needing a PCR step. Hence, such an enhanced dot blot paves the path to the development of a portable and multiplex paper based platform for point-of-care screening of chicken carcasses for Campylobacter.Graphical abstract

The authors would like to express their sincere gratitude to Gaelle-Anne Cremer for furnishing the streptavidin-antibody conjugates, Patrice Sarfati and Pierre Sonigo for their expert supervision of the work, François Rieunier and Stephane Rihet for their pertinent advices on the work.; International audience; A nanoparticle-based electrochemical sandwich immunoassay was developed for bacteria detection in platelet concentrates. For the assay, magnetic beads were functionalized with antibodies to allow the specific capture of bacteria from the complex matrix, and innovative methylene blue-DNA/nanoparticle assemblies provided the electrochemical response for amplified detection. This nanoparticular system was designed as a temperature-sensitive nano-tool for electrochemical detection. First, oligonucleotide-functionalized nanoparticles were obtained by direct synthesis of the DNA strands on the nanoparticle surface using an automated oligonucleotide synthesizer. Densely packed DNA coverage was thus obtained. Then, DNA duplexes were constructed on the NP surface with a complementary strand bearing a 3 methylene blue tag. This strategy ultimately produced highly functionalized nanoparticles with electrochemical markers. These assemblies enabled amplification of the electrochemical signal, resulting in a very good sensitivity. A proof-of-concept was carried out for E. coli detection in human platelet concentrates. Bacterial contamination of this complex biological matrix is the highest residual infectious risk in blood transfusion. The development of a rapid assay that could reach 10–102 CFU mL−1 sensitivity is a great challenge. The nanoparticle-based electrochemical sandwich immunoassay carried out on a boron doped diamond electrode proved to be sensitive for E. coli detection in human platelets. Two antibody pairs were used to develop either a generic assay against certain Gram negative strains or a specific assay for E. coli. The methylene blue-DNA/nanoparticles amplify sensitivity ×1000 compared with the assay run without NPs for electrochemical detection. A limit of detection of 10 CFU mL−1 in a biological matrix was achieved for E. coli using the highly specific antibody pair.

BACKGROUND Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. STUDY DESIGN AND METHODS A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. RESULTS Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. CONCLUSION In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

International audience; Polydiacetylene (PDA) inserted in films or in vesicles has received increasing attention due to its property to undergo a blue-to-red colorimetric transition along with a change from non-fluorescent to fluorescent upon application of various stimuli. In this review paper, the principle for the detection of various microorganisms (bacteria, directly detected or detected through the emitted toxins or through their DNA, and viruses) and of antibacterial and antiviral peptides based on these responsive PDA vesicles are detailed. The analytical performances obtained, when vesicles are in suspension or immobilized, are given and compared to those of the responsive vesicles mainly based on the vesicle encapsulation method. Many future challenges are then discussed

Polydiacetylene (PDA) inserted in films or in vesicles have received increasing attention due to PDA property to undergo a blue-to-red colorimetric transition along with a change from non-fluorescent to fluorescent upon application of various stimuli. In this review paper, the principle of the detection of various microorganisms (bacteria: directly detected or detected through the emitted toxins or through their DNA, and viruses) and of antibacterial and antiviral peptides based on these responsive PDA vesicles are detailed. The obtained analytical performances, when vesicles are in suspension or immobilized, are given and compared to those of the responsive vesicles mainly based on the vesicle encapsulation method. Many future challenges are then discussed.

International audience; In this work, using electrochemical impedance spectroscopy (EIS), we have, for the first time, label-free monitored protein immobilization on a gold surface through a strategy of electroaddressing, compatible with the production of microarrays for multi-detection. This functionalization is achieved via the alkyne/azide cycloaddition, better known as the "click" reaction. The electroaddressing was applied to a polythiol hexynyl derivative previously grafted onto the gold surface. This compound consists of two dithiol phosphate groups and a hexynyl function and was synthesized through a supported synthesis approach, from a dithiol reagent, phosphoramidite (DTPA), and a hexynyl phosphoramidite. Next, an azide-PEG3-biotin derivative was grafted onto the modified gold surface by electro-chronocoulometry. The "click" reaction was controlled by electrochemical impedance spectroscopy, showing the change in impedance only when the electroaddressing was performed at − 300 mV. No effect on the EIS signal was observed when a positive potential was applied, confirming the specificity of the electroactivation. Biotin-modified electrodes were used to fix streptavidin and the immobilization was monitored using EIS. Fluorescent streptavidin-functionalized silica nanoparticles were also specifically grafted onto the biotinylated gold surface in order to confirm the "click" reaction using fluorescence microscopy. The obtained streptavidin platform was used to detect the surface coverage by biotinylated human serum albumin (HSA). The lowest detectable concentration is 10 pg/mL, and surface saturation is obtained with concentrations higher than 100 ng/mL

International audience; The potential-assisted copper-catalyzed alkyne-azide cycloaddition was investigated to modify a gold electrode surface. Firstly, a tetrathiol-hexynyl derivative was used to introduce alkyne functions on the surface. This anchor proved its robustness in conditions used for the "click" reaction and in wet storage. Then, the potential-assisted "click" reaction was studied with an azido ferrocene derivative. The experimental conditions were optimized according to the electrochemical response of the sensor by cyclic voltammetry and the "click" reaction yield was established. After functionalization, the presence of the 1,2,3-triazole group was confirmed by XPS. A fluorescent azido oligonucleotide was grafted onto the gold surface allowing visualization of the reaction by fluorescence microscopy.

Development of the enzyme-containing nanocomposites provides an excellent opportunity for the development of the sensitive and effective analytical devices – biosensors. In the present work, we used the nanocomposites that contained two enzymes (glucose oxidase and hexokinase), polymers, and gold nanoparticles (GNPs). Such nanostructure was expected to increase electron transfer in the bioselective element of biosensor and to improve the enzyme stability during the immobilization process. An amperometric biosensor based on the bienzyme system has been developed. The biosensor is sensitive to adenosine-5′-triphosphate (ATP) and glucose. The enzymes were immobilized onto the surface of a platinum disk electrode that was used as amperometric transducer. Three different methods of immobilization were investigated: cross-linking of the enzymes in the presence of bovine serum albumin, entrapment in a photo-cross-linkable modified polyvinyl alcohol (PVA-SbQ) matrix, and entrapment of the enzymes in a PVA/polyethylenimine matrix. The best results during the ATP determination were obtained with PVA-SbQ, and this immobilization method was modified by addition of 18-nm GNPs to the reacting mixture. The ATP detection could be achieved in all cases except for PVA/polyethylenimine, the best sensitivity and linear range being achieved by co-immobilizing glucose oxidase/hexokinase into the photo-cross-linked PVA-SbQ/GNP polymer matrix.

International audience; Interleukin-8 (IL-8), a dimeric protein composed of two identical subunits, is overexpressed in different human cancers, among them pancreatic cancers and head and neck cancers. There is a strong demand for monitoring of this biomarker protein for early cancer detection. A new concept of label-free impedimetric immunosensors for IL-8 detection is described in this work. A nanocomposite of gold nanoparticles (AuNPs) decorating a magnetic Fe3O4 core is synthesized using cysteamine as linker, and characterized using TEM and UV-Vis spectrometry. After immobilization of these Fe3O4@Au magnetic nanocomposites on a boron doped diamond (BDD) electrode using a simple magnet, anti-human IL-8 monoclonal antibody is linked to the AuNPs, previously functionalized with thioctic acid. The functionalization of the immunosensor was characterized using two electrochemical techniques: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), performed in phosphate buffer with ferro/ferricyanide as the redox probe. The EIS technique was used for affinity assays: antibody-Antigen binding. A linear relationship between the increment in the electron transfer resistance (R-CT) and the logarithmic value of IL-8 concentration was observed between 0.1pg/mL and 1000pg/mL. The limit of detection (LOD) was estimated at 0.03pg/mL.

International audience; We demonstrate here the possibility of designing semiconducting thin films with controlled electrochemical properties. The thin films are composed of (i) an insulating binder and (ii) a semiconductor nanopowder which enables the fine tuning of the semiconducting properties of the layers. Thus, p-and n-type silicon particles (obtained from a top-down technique), or metal-oxide ZnO, SnO(2) and NiO nanoparticles (synthesized using a bottom-up protocol) are successfully integrated into spin-coated or screen-printed thin films and used as semiconducting materials. The flat band potential (Vfb) of the films is then easily tuned from DV to -1.138 V. (C) 2010 Elsevier B.V. All rights reserved.

International audience; In conductometric enzymatic biosensors, enzymatic reaction is confined close to the interdigitated electrode surface, because enzyme is cross-linked in contact with this surface in the presence or absence of nanoparticles. The effect of the use of a new type of doubly-functionalized gold nanoparticles (PF-HEG-Au NPs) on the response of conductometric biosensor based on interdigitated electrodes (IDEs), for the detection of enzymatic substrates was studied. Gold nanoparticles (AuNPs) were first synthesized following the citrate process, with an average diameter of 14 nm. AuNPs were then functionalized with 11-mercaptoundecylhexaethyleneglycol (HEG) and then with 1H,1H,2H,2H-perfluorodecanethiol (PF). The doubly-functionalized AuNPs were characterized using TEM, UV-Vis spectrophotometry and FTIR spectroscopy. Urease, mixed with these doubly functionalized AuNPs, was then cross-linked with glutaraldhedyde vapor on the IDE surface. In the presence of urea, the conductometric response was measured in a differential mode. The best sensitivities for urea detection were obtained with PF-HEG-Au NPs (520 µS /mM and 0.5µM of detection limit), as compared to 284µS/mM and 2µM of detection limit with bare Au NPs, PF-AuNPs and HEG-AuNPs, and 1.07µS/mM and 100 µM of detection limit with urease directly crosslinked on IDEs.When stored in phosphate buffer (5 mM, pH 6.7) at 4 °C, the biosensor with PF-HEG-Au NPs showed good stability for more than 12 days.

International audience; A sensitive electrochemical molecularly imprinted sensor was developed for the detection of glyphosate (Gly), by electropolymerisation of p-aminothiophenol-functionalised gold nanoparticles in the presence of Gly as template molecule. The extraction of the template leads to the formation of cavities that are able to specifically recognise and bind Gly through hydrogen bonds between Gly molecules and aniline moieties. The performance of the developed sensor for the detection of Gly was investigated by linear sweep voltammetry using a hexacyanoferrate/hexacyanoferrite solution as redox probe, the electron transfer rate increasing when concentration of Gly increases, due to a p-doping effect. The molecularly imprinted sensor exhibits a broad linear range, between 1 pg/L and 1 mu g/L and a quantification limit of 0.8 pg/L. The selectivity of the proposed sensor was investigated towards the binding of Gly metabolite, aminomethylphosphonic acid, revealing excellent selectivity towards Gly. The developed sensor was successfully applied to detect Gly in tap water samples.

International audience; A sensitive electrochemical molecularly-imprinted sensor was developed for the detection of aflatoxin B1 (AFB1), by electropolymerization of p-aminothiophenol-functionalized gold nanoparticles in the presence of AFB1 as a template molecule. The extraction of the template leads to the formation of cavities that are able to specifically recognize and bind AFB1 through - interactions between AFB1 molecules and aniline moities. The performance of the developed sensor for the detection of AFB1 was investigated by linear sweep voltammetry using a hexacyanoferrate/hexacyanoferrite solution as a redox probe, the electron transfer rate increasing when the concentration of AFB1 increases, due to a p-doping effect. The molecularly-imprinted sensor exhibits a broad linear range, between 3.2 fM and 3.2 mu M, and a quantification limit of 3 fM. Compared to the non-imprinted sensor, the imprinting factor was found to be 10. Selectivity studies were also performed towards the binding of other aflatoxins and ochratoxin A, proving good selectivity.

We thank Oceane Clabaux and Alexandre Escoffier for their contribution to the work.; International audience; We report the first synthesis of a methylene blue (MB) phosphoramidite derivative suitable for DNA solid-phase synthesis. The electrochemical and optical properties of the resulting MB modified oligonucleotides were confirmed. This new molecule is an important breakthrough in the design of new probes labelled with MB.

The authors thank INL institute (Institut des Nanotechnologies de Lyon, France) for providing the facilities for the electrospinning technique, the required access to the SEM facilities and for their remarkable assistance whenever it was needed. The Centre Technologique des Microstructures of Lyon 1 University is also acknowledged for providing the authors access to its TEM facilities. Finally, the authors thank the French government for the financial support.; International audience; Herein, we report the easy and rapid fabrication of water-stable electrospun nanofibers from blends of the photochemically cross-linkable polyvinyl alcohol styrylpyridinium polymer (PVA-SbQ), carboxylated multiwall carbon nanotubes (MWCNT-COOHs) and gluccise oxidase (GOx) for electrochemical biosensor application. Different electrospinning parameters including flow rate, applied voltage, distance between tip and collector, polymer concentration and MWCNT-COOHs content in the electrospun solution were tailored to produce PVA-SbQ/MWCNT-COOH nanofibers with minimal beading and enhanced electrical properties. Optimal PVA-SbQ and MWCNT-COOHs concentrations were 6.7 wt% and 5 wt% with respect to the total solution and polymer mass, respectively. The nanofibers were rendered water insoluble via exposure to UV irradiation for 10 mm. The electrochemical properties of electrospun PVA-SbQ/MWCNT-COOH nanofibrous mats were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Scanning electron microscopy and transmission electron microscopy were used to characterize the morphology of the nanofibers. G0x, used as model enzyme, was further incorporated into the PVA-SbQ/MWCNT-COOH mixture before electrospinning. The obtained biosensor enabled successful detection of glucose by cyclic voltammetry. The resulting novel glucose biosensor revealed very good storage and operational stabilities, a low limit of detection (2 mu M) and a wide linear range (up to at least 4 mM).

International audience; The aim of this study was to develop versatile diagnostic tools based on the use of innovative polythiolated probes for the detection of multiple viruses. This approach is compatible with optical enzyme-linked oligosorbent assay (ELOSA) or electrochemical (biosensors) detection methods. The application targeted here concerns the rapid genotyping of Hepatitis C virus (HCV). HCV genotyping is one of the predictive parameters currently used to define the antiviral treatment strategy and is based on the sequencing of the viral NS5b region. Generic and specific NS5b amplicons were produced by real-time polymease chain reaction (RT-PCR) on HCV(+) human plasma. Original NS5b probes were designed for genotypes 1a/1b, 2a/2b/2c, 3a, and 4a/4d. Robust polythiolated probes were anchored with good efficacy on maleimide-activated microplates (MAM) and gold electrodes. Their grafting on MAM greatly increased the sensitivity of the ELOSA test which was able to detect HCV amplicons with good sensitivity (10 nM) and specificity. Moreover, the direct and real-time electrochemical detection by differential pulse voltammetry enabled a detection limit of 10 fM to be reached with good reproducibility. These innovative polythiolated probes have allowed us to envisage developing flexible, highly sensitive, and easy-to-handle platforms dedicated to the rapid screening and genotyping of a wide range of viral agents.

International audience; The aim of the present work is to show the interest of a generic concept of aptamers directly obtained through a supported synthesis, using phosphoramidite synthons, for biosensor application. By that way, we were able to tailor the anchoring groups and specific spacers. The concept is validated on an impedimetric aptasensor for detection of thrombin. Two aptamer sequences, selected from literature (15 and 29 mers), were synthesized with a multi-thiol modification at one extremity. After their grafting on gold electrode, the faradic electrochemical impedance measurements allow the direct detection of thrombin in the range of 3-30 ng mL(-1) ancl 3-50 ng mL(- 1) for 15 meraptamer and 29 meraptamer respectively, with a detection limit of 3.1 ng mL(-1) (80 pmol/l) for both sequences. This study demonstrates that these aptasensors exhibit high sensitivity (higher sensitivity for 29 meraptamer), high selectivity (compared to a model protein, BSA), stability and reproducibility

International audience; Oligonucleotide solid-phase synthesis is now possible on nano-sized particles, thanks to the use of controlled pore glass-nanoparticle assemblies. We succeeded in anchoring silica nanoparticles (NPs) inside the pores of micrometric glass via a reversible covalent binding. The pore diameter must be at least six times the diameter of the nanoparticle in order to maintain efficient synthesis of oligonucleotides in the synthesizer. We demonstrated that the pores protect NP anchoring during DNA synthesis without decreasing the coupling rate of the phosphoramidite synthons. This bottom-up strategy for NP functionalization with DNA results in unprecedented DNA loading efficiency. We also confirmed that the DNA synthesized on the nanoparticle surface was accessible for hybridization with its complementary DNA strand.

International audience; We report the use of a four-ferrocene modified oligonucleotide as a probe for DNA detection with a gold electrode microsystem. This oligonucleotide is synthesized by automated solid-phase synthesis with four successive ferrocene moieties at the 5′-end and a C6-thiol modifier group at the 3′-end. The grafting of this 4Fc-DNA probe on a gold electrode microsystem results in the appearance of the ferrocene redox couple in cyclic voltammetry. The probe sequence is a stem-loop structure that folds efficiently on the electrode, thus optimizing electron transfer. Such architecture serves as sensor for DNA detection which is based on hybridization. The resulting disposable voltammetric sensor allowed direct, reagentless DNA detection in a small volume (20 μL). Electrochemical response upon hybridization with complementary short sequence (30-base length) and long sequence (50-base length) strands was observed by differential pulse voltammetry. Current variations were compared. The longer the sequence, the greater the decrease in current. The system's detection limit was estimated at 3.5 pM (0.07 fmol in 20 μL) with the 50-base length target and provided a dynamic detection range between 3.5 pM and 5 nM. Single mismatch detection showed a good level of sensitivity. The system was regenerated twice with no significant loss of Fc signal. Finally, 1 pM sensitivity was reached with a long chain analog of DNA PCR products of Influenza virus.

International audience; The goal of this work is to develop an impedimetric aptasensor sensitive, selective and stable for detection of thrombin. Thiol modified aptamer sequence being grafted on gold electrode, the faradic electrochemical impedance measurements allow the direct detection of thrombin in the range of 3.1-200 ngmL(-1) with a detection limit of 3.1 ngmL(-1) (80 fM). This study demonstrates that these aptasensors exhibit high sensitivity, high selectivity (compared to a model protein, BSA), stability and reproducibility.

International audience; This article describes an original strategy to enable solid-phase oligodeoxyribonucleotide (ODN) synthesis on nanosized silica particles. It consists of the reversible immobilization of silica nanoparticles (NPs) on micrometric silica beads. The resulting assemblies, called nano-on-micro (NOM) systems, are well adapted to ODN synthesis in an automated instrument. First, NPs are derivatized with OH functions. For NOM assembly preparation, these functions react with the silanols of the microbeads under specific experimental conditions. Furthermore, OH groups allow ODN synthesis on the nanoparticles via phosphoramidite chemistry. The stability of the NOM assemblies during ODN solid-phase synthesis is confirmed by scanning and transmission electron microscopy (SEM and TEM, respectively), together with dynamic light scattering analyses. Then, the release of ODN-functionalized nanoparticles is performed under mild conditions (1% NH4OH in water, 1 h, 60 °C). Our technique provides silica nanoparticles well functionalized with oligonucleotides, as demonstrated by hybridization experiments conducted with the cDNA target.