Liliane Coche-Guérente

Development of amperometric biosensors based on the immobilization of enzymes in polymer films electrogenerated from a series of amphiphilic pyrrole derivatives

Abstract A series of amphiphilic pyrrolyl-alkylammonium ions differing in the size of their ammonium heads has been used for the immobilization at the electrode surface of horseradish peroxidase, galactose oxidase, polyphenol oxidase, glucose oxidase and xanthine oxidase in polypyrrolic films electrogenerated from adsorbed amphiphilic pyrrole-enzyme mixtures. The enzyme retention properties of the different polymers have been determined indicating that the less hydrophobic monomer has the best immobilization efficiency. The electrochemical assays performed for galactose, glucose and hypoxanthine detection show clearly that the bioelectrode sensitivity is related to the permeability of the host polymer. The selectivity of the glucose oxidase electrodes towards interfering agents like ascorbate, urate and acetaminophen has also been examined, these agents interfering to some extent when present at physiological concentrations.

Incorporation of anionic metalloporphyrins into poly(pyrrole-alkylammonium) films—Part 2. Characterization of the reactivity of the iron(III) porphyrininc-based polymer

Abstract Polypyrrole films containing alkylammonium groups have been deposited on graphite and ITO transparent electrodes by a simple and monomer saving oxidative electropolymerisation of an adsorbed amphiphilic N-substituted pyrrole. The irreversible incorporation of tetra anionic manganese, zinc and iron porphyrins is easily achieved in the pre-formed films and these modified electrodes have been characterized by cyclic voltammetry and absorptiometric measurements. Finally, the reactivity of the polymer containing iron(III) porphyrin towards nitric oxide in acidic aqueous solution has been proved by the formation of the iron-nitrosyl complex within the polypyrrole film.

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− .

Amplification of amperometric biosensor responses by electrochemical substrate recycling. Part II. Experimental study of the catechol-polyphenol oxidase system immobilized in a laponite clay matrix

Abstract Amperometric catechol biosensors can be constructed by drying onto the surface of a glassy carbon rotating-disk electrode an aqueous sol of synthetic laponite clay containing controlled amounts of polyphenol oxidase (PPO) and polycationic oligosilasesquioxane additive. The procedure allows the electrode surface to be coated with composite enzyme–laponite clay films exhibiting improved adhesion, enhanced mechanical strength and high enzymatic activity. Electrodes prepared in this manner can be used to detect catechol in the range 0.5 nM to 10 μM. The low detection limit of 0.5 nM results from an efficient signal amplification as a consequence of the electrochemical recycling of catechol substrate. An intrinsic amplification factor of 3.35 has been measured. The observed responses from such an electrode as a function of applied potential, enzyme activity and electrode rotation rate are in excellent agreement with theory. From a comparison of the experimental results with theory, we are able to characterize diffusion and enzyme kinetics in the enzymatic layer. The results are consistent with a microporous structure of the enzymatic layer in which microchannels are distributed. Diffusion of catechol substrate and orthoquinone product occurs within the microchannels filled by electrolyte and can be described using a pinhole model. The study shows that only a fraction of PPO, the one which is entrapped in open micropores with interconnected microchannels, is accessible to catechol substrate while keeping its full enzymatic activity. The other fraction should correspond to inaccessible proteins enclosed within the walls of microchannels.

Characterization of a modified gold platform for the development of a label-free anti-thrombin aptasensor.

This work reports the characterization of a modified gold surface as a platform for the development of a label free aptasensor for thrombin detection. The biorecognition platform was obtained by the self-assembly of 4-mercaptobenzoic acid onto a gold surface, covalent attachment of streptavidin and further immobilization of the biotinylated anti-thrombin aptamer. The biosensing platform was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring. The biorecognition event aptamer-thrombin was detected from changes in the SPR angle produced as a consequence of the molecular interaction between the aptasensor and the target protein. The biosensing platform demonstrated to be highly selective for human thrombin even in the presence of large excess of bovine thrombin, bovine serum albumin, cytochrome C, lysozyme and myoglobin. The relationship between the changes in the SPR angle and thrombin concentration was linear up to 0.19 μmol L(-1) (R(2)=0.992) while the detection limit was of 12.0 nmol L(-1) (240 fmol in the sample). This new sensing approach represents an interesting and promising alternative for the SPR-based quantification of thrombin.

Unlimited growth of host–guest multilayer films based on functionalized neutral polymers

Multilayer polymer films based on host–guest interactions were formed on gold surfaces covered with self-assembled monolayers terminated by β-cyclodextrin groups using poly(N-hydroxypropylmethacrylamide) derivatives bearing ferrocene or β-cyclodextrin moieties.

Poly (Amphiphilic Pyrr0Le)-PPO Electrodes for Organic-Phase Enzymatic Assay

Abstract The utility of amphiphilic pyrrole monomers to elaborate stable organic-phase polymers containing enzyme is described. Four amphiphilic pyrrole monomers differing in the size of their ammonium groups were applied to the immobilization of a preadsorbed enzyme-monomer coating. The peiformances of the different biosensors towards the detection of catechol, 4-tert-butylcatechol and phenol have been examined in either anhydrous or wet chloroform solutions.

Organosilasesquioxane-laponite clay sols: a versatile approach for electrode surface modification

Abstract Electrode surface modification by organo-inorganic layered coatings can be readily achieved by drying a completely delaminated laponite clay sol mixed with neutral octa(3-aminopropylsilasesquioxane) (OAPS) cubane-like octameric pillar precursors. Films of excellent quality with exceptional adhesion and mechanical properties could be obtained. Under mild acidic conditions, protonation of the octamer amino groups occurs. The ion-exchange properties of the resulting coatings were studied in aqueous electrolyte as a function of the octamer loading using the cationic Ru(NH 3 ) 3+ 6 and the anionic Mo(CN) 4− 8 electroactive probes. For octamer loadings higher than the cation exchange capacity (cec) of laponite, the octamer-laponite coating behaves as an anion exchanger which allows the efficient incorporation of a wide range of redox anions, e.g. RuO 2− 4 , PtCl 2− 4 , PdCl 2− 4 , Fe(CN) 3− 6 , SiW 12 O 4− 40 polyoxometallate, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) disodium salt (ABTS) or anthraquinone sulfonate (AQS). The apparent diffusion coefficient of Mo(CN) 4− 8 varies from 4 × 10 −10 to 8 × 10 −10 depending on the amount of intercalated octamer. The original use of such a modified electrode to operate in organic medium has been examined with RuO 2− 4 , ABTS and AQS as electroactive immobilized species.

Influence of the interaction strength between supramolecular complexes on the topography of neutral polymer multilayer films.

Step-by-step polymer film buildup processes lead to polymer coatings, e.g., polyelectrolyte multilayers, of various structures ranging from continuous smooth films to droplet like discontinuous coatings. Yet, the origin of these different behaviors depending upon the system is not yet known. This study is a first attempt to rationalize the evolution of the coating structure as a function of the strength of the interactions between the polymers constituting the film. We investigated the influence of the strength of noncovalent host-guest interactions between cyclodextrin (CD) and pyrene (Py), ferrocene (Fc) or adamantane (Ad) on the structure of neutral poly(N-hydroxypropylmethacrylamide) (PHPMA) multilayers films formed in a step-by-step manner. In solution, the strength of the inclusion complex (measured by log K where K is the complex association constant) is increasing in the order Py/β-CD < Fc/β-CD < Ad/β-CD and can be further varied in the presence of different sodium salts at different ionic strengths. Depending upon this strength, the buildup process is limited to the formation of isolated aggregates for PHPMA-CD/PHPMA-Py, leading to smooth continuous films for PHPMA-CD/PHPMA-Fc and to droplet-like films, not entirely covering the substrate, for PHPMA-CD/PHPMA-Ad. To study the influence of the strength of the host-guest interactions on the film topography, PHPMA-CD/PHPMA-Fc films were built in the presence of different sodium salts at different ionic strengths. For low host-guest interactions, only isolated aggregates are formed on the substrate. As the strength of the host-guest interactions increases (increase of log K), the formed films go through a droplet-like structure, before becoming continuous but rough for stronger interactions. When the interaction strength is further increased, the roughness of the films decreases, leading to a smooth continuous film before becoming rough again at still higher interaction strength. Smooth continuous multilayers seem thus to be obtained for an optimal range of the interaction strength.

Layer-by-layer deposition of chitosan derivatives and DNA on gold surfaces for the development of biorecognition layers

Abstract We describe a new supramolecular architecture obtained by deposition of double‐stranded deoxyribonucleic acid (dsDNA) and chitosan derivatives on a thiolated gold surface. Surface plasmon resonance was used to monitor, in real time, the construction of the supramolecular architecture. Three chitosan derivatives were used, a quaternized N‐substituted by three methyl groups in a 40.0 mol%, and two others N‐substituted with an octyl chain in a 5.0 and 25.0 mol%. The multilayer formation depends on the degree of substitution of chitosan as well as on the nature of the substituents. The adsorption of dsDNA is slower than that of chitosan. The best immobilization of dsDNA is obtained by using a quaternized chitosan and dsDNA prepared in 0.050 M acetate buffer pH 5.00. The system is stable in buffer solution independently of the nature of the chitosan derivative. The effect of the nature of the electrolyte and ionic strength is also discussed.