Carole Farre

Molecularly imprinted polymer-based electrochemical sensor for the sensitive detection of glyphosate herbicide

ABSTRACT 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 µg/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.

Automated oligonucleotide solid-phase synthesis on nanosized silica particles using nano-on-micro assembled particle supports.

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% NH(4)OH in water, 1 h, 60 degrees C). Our technique provides silica nanoparticles well functionalized with oligonucleotides, as demonstrated by hybridization experiments conducted with the cDNA target.

Aflatoxin B1 Detection Using a Highly-Sensitive Molecularly-Imprinted Electrochemical Sensor Based on an Electropolymerized Metal Organic Framework

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 π-π 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 µM, 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.

Development of Innovative and Versatile Polythiol Probes for Use on ELOSA or Electrochemical Biosensors: Application in Hepatitis C Virus Genotyping

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.

Oligonucleotide solid-phase synthesis on fluorescent nanoparticles grafted on controlled pore glass{

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.

Responsive Polydiacetylene Vesicles for Biosensing Microorganisms

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.

Effect of Perfluorinated-Hexaethylene Glycol Functionalization of Gold Nanoparticles on the Enhancement of the Response of an Enzymatic Conductometric Biosensor for Urea Detection

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.

Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates

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.

Gold Nanoparticle/Polymer/Enzyme Nanocomposite for the Development of Adenosine Triphosphate Biosensor

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.