Pierre Labbé

A biosensor as warning device for the detection of cyanide, chlorophenols, atrazine and carbamate pesticides

Abstract The determination of cyanide, chlorophenols, atrazine, dithiocarbamate and carbamate pesticides is described, utilizing an amperometric biosensor constructed by the electropolymerization of a pyrrole amphiphilic monomer-tyrosinase coating. Measurements were carried out with catechol, dopamine, l -DOPA or epinephrine as an enzyme substrate; the enzymatically generated quinoid products being electroreduced at -200 mV vs. SCE. The detection of these water pollutants was performed via their inhibiting action on the tyrosinase electrode. The characterization of the inhibition processes (competitive /non-competitive) and their reversibility were examined. The detection limits are 0.4, 2, 2, 4 and 0.02 μM for 3,4-dichlorophenol, chloroisopropylphenylcarbamate, 3-chloroaniline, atrazine and cyanide, respectively.

Improvement of the analytical characteristics of an enzyme electrode for free and total cholesterol via laponite clay additives

Abstract The electropolymerization of a laponite nanoparticle-amphiphilic pyrrole derivative-enzyme mixture preadsorbed on the electrode surface provides simultaneously the immobilization of the enzyme and the laponite particles in the polypyrrolic matrix. These incorporated laponite nanoparticles greatly enhance the sensitivity and stability of a cholesterol oxidase-based biosensor. Compared to a similar biosensor without laponite, the biosensor sensitivity increased from 5.1 to 13.2 mA M −1 cm −2 . Furthermore, the presence of hydrophilic laponite additive in the polymeric matrix containing cholesterol oxidase and cholesterol esterase is essential for the successful determination of total cholesterol.

Development of a PPO-poly(amphiphilic pyrrole) electrode for on site monitoring of phenol in aqueous effluents

Abstract A feasibility study in view of a future industrial development of a phenol biosensor is presented. The biosensor construction is based on the electropolymerization of a pyrrole amphiphilic monomer–tyrosinase (EC.1.14.18.1) mixture previously adsorbed on a glassy carbon electrode surface. The optimized biosensor provides a low detection limit (10 nM) and a linear response up to 10 μM of phenol. The procedure allows the construction of biosensors exhibiting very reproducible characteristics. These bioelectrodes are characterized by exceptional long-term stability since they could be stored for 1 year without significant loss of their electroenzymatic activity.

Improvement of poly(amphiphilic pyrrole) enzyme electrodes via the incorporation of synthetic laponite-clay-nanoparticles

The electropolymerization of an enzyme-amphiphilic pyrrole ammonium-laponite nanoparticles mixture preadsorbed on the electrode surface provides the simultaneous immobilization of the enzyme and the hydrophilic laponite-clay-nanoparticles in a functionalized polypyrrole film. The presence of incorporated laponite particles within the electrogenerated polymer induces a strong improvement of the analytical performances (I(max) and sensitivity) of amperometric biosensors based on polyphenol oxidase. These beneficial effects have been attributed to a marked enhancement of the apparent specific activity of the immobilized enzyme (from 0.21 to 0.85% of the specific activity of the free enzyme), the permeability of the host polymer being unchanged. This strategy of biosensor performance improvement was tested with cholesterol oxidase as an enzyme model. The presence of laponite additive in the poly(amphiphilic pyrrole) host matrix induces a similar enhancement of sensitivity and I(max) for cholesterol biosensing as well as a large improvement of the storage stability of the polypyrrole-cholesterol oxidase electrode.

Template-assembled synthetic G-quadruplex (TASQ): a useful system for investigating the interactions of ligands with constrained quadruplex topologies.

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular-like G-quadruplex motif 1 (parallel G-quadruplex conformation), an intramolecular G-quadruplex 2, and a duplex DNA 3 have been designed and developed. The method is based on the concept of template-assembled synthetic G-quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G-quadruplex conformation. Various known G-quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a pi-stacking binding mode showed a higher binding affinity for intermolecular-like G-quadruplexes 1, whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2. In addition, the present method has also provided information about the selectivity of ligands for G-quadruplex DNA over the duplex DNA. A numerical parameter, termed the G-quadruplex binding mode index (G4-BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G-quadruplex 1 against intramolecular G-quadruplex 2. The G-quadruplex binding mode index (G4-BMI) of a ligand is defined as follows: G4-BMI=K(D)(intra)/K(D)(inter), where K(D)(intra) is the dissociation constant for intramolecular G-quadruplex 2 and K(D)(inter) is the dissociation constant for intermolecular G-quadruplex 1. In summary, the present work has demonstrated that the use of parallel-constrained quadruplex topology provides more precise information about the binding modes of ligands.

Characterization of organosilasesquioxane-intercalated-laponite-clay modified electrodes and (bio)electrochemical applications

Abstract Electrode surface modification by organo-inorganic layered coatings can be achieved readily by drying a completely delaminated laponite clay sol mixed with polycationic silasesquioxane oligomers. Oligosilsesquioxanes were synthesized by hydrolytic polycondensation of trialkoxysilanes bearing an alkylamino or alkyltrimethylammonium function. Films of excellent quality with exceptional adhesion and mechanical properties can be obtained. The intercalation of organosiloxane oligomers is accompanied by the expansion of the film and by the existence of a mesoporosity as shown by XRD measurements and N 2 adsorption experiments. The ion-exchange properties of the resulting coatings as well as permeation of neutral molecules were studied in aqueous and non-aqueous electrolytes as a function of the oligomer loading. For oligomer loadings higher than the cation-exchange capacity (cec) of laponite, the coatings behave as anion-exchangers which allows the binding a wide range of redox anions. Incorporated anions remain electroactive not only in aqueous but also in non aqueous electrolytes as a consequence of a fixed pore size and permanent interlayer spacing of oligomer-expanded laponite. On the other hand intercalation of oligomers allows us to modulate the permeability of the coating as shown by permeation experiments using neutral electroactive probes in non aqueous electrolytes. In the field of electroanalysis, amperometric biosensors made by the entrapment of glucose oxidase inside the hybrid material have been investigated successfully. Such enzymatic films exhibit enhanced analytical performances as compared with those obtained using native sodic laponite. The potential applications of this new hybrid material in the field of electrocatalysis have been exemplified by the electroprecipitation of catalytic nanoparticles such as Pt(0) obtained from the incorporation of the anionic precursor PtCl 4 2− .

Determination of phenol and chlorinated phenolic compounds based on a PPO-bioelectrode and its inhibition

Abstract A polyphenol oxidase (PPO) enzyme electrode constructed by the electropolymerization of a pyrrole amphiphilic monomer-PPO mixture, previously adsorbed on a glassy carbon electrode, is applied for the direct amperometric response of phenol, 3-chlorophenol and 4-chlorophenol. Furthermore, the detection of 2-chlorophenol, several polychlorophenols and pentachlorophenol is carried out by an inhibition process of the bioelectrode functioning. The mechanism of the bioelectrode inhibition is investigated in terms of enzymatic inhibition and polymer fouling. The capacities of this system for the resolution of mixtures of phenolic pollutants are also explored on the basis of the kinetic behavior of the bioelectrode response.

Multilayer films based on host–guest interactions between biocompatible polymers

Multilayer films are formed using host-guest interaction between two derivatized chitosans, one, with beta-cyclodextrin cavities and the other with adamantyl moieties.

Amplification of amperometric biosensor responses by electrochemical substrate recycling - Part I. Theoretical treatment of the catechol-polyphenol oxidase system

Abstract The sensitivity of enzyme electrodes can be increased substantially by incorporation of a substrate recycling scheme. One possible strategy to achieve this goal is based on amperometric enzyme electrodes involving during the transduction step an electrochemical recycling of enzyme substrate. In this kind of device, the shuttle analyte is not only measured once but is reconverted to be measured again leading to an amplification of the transduction signal. In this paper we present a theoretical analysis of this kind of device using the catechol–polyphenol oxidase model system immobilized as an enzyme layer at the surface of a rotating-disk electrode. For low concentrations of substrate, which correspond to practical conditions, we derive analytical expressions for the response of such a sensor. On the basis of the model, we are able to quantify the respective influence of mass transport, partition coefficient, enzyme and electron transfer kinetics on the metrological characteristics of the bioelectrode. This is especially relevant when optimizing biosensor construction.

A novel conformationally constrained parallel g quadruplex.

Guanine-rich DNA sequences are known to form highly ordered structures called G quadruplexes. These structures play an important role in many relevant biological processes, such as telomere stabilization, oncogene activation, and the regulation of the immunoglobulin switch region. The G-quadruplex motif is based on the association of planar G quartets of four guanine residues that are held together by eight Hoogsteen-type hydrogen bonds (Figure 1A). The G-quadruplex motif requires monovalent cations, such as Na and K , for stabilization. A wide variety of topologies can be adopted depending on the number of strands involved in the structure, the strand direction, as well as variations in loop size and sequence (Figure 1). The structure of parallel-stranded as well as antiparallel-stranded quadruplexes have been extensively studied by using different methods, such as NMR spectroscopy, X-ray diffraction and circular dichroism, but the exact conformation present in vivo is still under discussion. The design of small molecules that can bind to G quadruplexes has thus received attention because these nucleic acid motifs represent valuable pharmaceutical targets. For this purpose, a large number of small molecules has been evaluated for their binding with these particular DNA structures. However, as mentioned, the G quadruplex can adopt different topologies that can confuse the study of recognition phenomena. The design of a system that is able to mimic a well-defined conformation of G quadruplex is thus of great interest to precisely study the molecular interactions that can occur with small organic molecules. In 1985, Mutter proposed the TASP concept (template-assembled synthetic proteins) for the design of folded proteins. These pioneering works described the use of a cyclodecapeptide that allows the preparation of artificial proteins with a predetermined three-dimensional structure. Despite a large number of examples that use this template, to our knowledge, it has not been applied for the design of a specific folded structure of nucleic acid. With this in mind, we investigated the use of a peptidic scaffold as a topological template that directs the intramolecular assembly of covalently attached oligonucleotides into a single characteristic folding topology of G quadruplex. We anticipated that the scaffold should permit the preorganization of the DNA strands and the stabilization of the quadruplex structure. We report herein the synthesis and characterization of the novel water-soluble peptidic scaffold–oligonucleotide conjugate 1 that mimics the parallel-stranded conformation of G quadruplex (Scheme 1). We demonstrate that the use of the scaffold allows the precise control of the conformation of the quadruplex and dramatically increases the stability of the motif—all the more so as the formation of the quadruplex motif is possible even without the addition of any monovalent cations, such as K . We also show that mimic 1 can be used for surface functionalization, and this permits the study of the molecular interaction with G-quadruplex ligands by using surface plasmon resonance (SPR). The scaffold used for the synthesis of mimic 1 is a cyclic decapeptide with two independently functionalizable faces, which are due to the orientation of the lysine side-chains. On one side, the four oligonucleotides derived from the human telomeric sequence d(TTAGGGT) were anchored by using oxime bond formation, and a biotin residue was incorporated on the other side for attachment to streptavidin-immobilized surfaces. Earlier work from our laboratory has demonstrated that the oxime coupling strategy allows the efficient preparation of peptide–oligonucleotide conjugates. Figure 1. G-quartet motif and possible folded structures of the G quadruplex. A) G quartet; B) intermolecular parallel form; C) intramolecular parallel form; D) intramolecular antiparallel form.