Audrey Sassolas

Université de Perpignan, Perpinyà, Languedoc-Roussillon, France

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Publications (23)113.9 Total impact

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    ABSTRACT: This paper reports on electrochemiluminescent sensors and biosensors based on polyluminol/hydrogel composite sensing layers using chemical or biological membranes as hydrogel matrices. In this work, luminol is electropolymerized under near-neutral conditions onto screen-printed electrode (SPE)-supported hydrogel films. The working electrode coated with a hydrogel film is soaked in a solution containing monomeric luminol units, allowing the monomeric luminol units to diffuse inside the porous matrix to the electrode surface where they are electropolymerized by cyclic voltammetry (CV). Sensors and enzymatic biosensors for H2O2 and choline detection, respectively, have been developed, using choline oxidase (ChOD) as a model enzyme. In this case, hydrogel is used both as the enzymatic immobilization matrix and as a template for the electrosynthesis of polyluminol. The enzyme was immobilized by entrapment in the gel matrix during its formation before electropolymerization of the monomer. Several parameters have been optimized in terms of polymerization conditions, enzyme loading, and average pore size. Using calcium alginate or tetramethoxysilane (TMOS)-based silica as porous matrix, H2O2 and choline detection are reported down to micromolar concentrations with three orders of magnitude wide dynamic ranges starting from 4 × 10(-7) M. Polyluminol/hydrogel composites appear as suitable electrochemiluminescence (ECL)-active sensing layers for the design of new reagentless and disposable easy-to-use optical sensors and biosensors, using conventional TMOS-based silica gel or the more original and easier to handle calcium alginate, reported here for the first time in such a configuration, as the biocompatible hydrogel matrix.
    Analytical and Bioanalytical Chemistry 06/2014; · 3.66 Impact Factor
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    ABSTRACT: Okadaic acid (OA) is a marine toxin ingested by shellfish. The consumption of contaminated seafood causes diarrheic shellfish poisoning, responsible for gastrointestinal troubles in humans. The development of fast, reliable and sensitive methods for OA analysis is an evident necessity in order to guarantee the seafood safety and to protect human health. In this work, competitive indirect immunoassays were developed for the toxin detection. Indirect and direct labeling were used and compared. With indirect labeling, the unlabeled anti-OA antibody binds to the toxin and a labeled secondary antibody is used for the signal generation. Alternatively, using the direct labeling, the label is attached via a covalent bond to the anti-OA antibody. In this work, peroxidase was conjugated to the anti-OA antibody through a periodate activation. The peroxidaseeantibody conjugate is used for both toxin recognition and signal generation. In the last case, ELISA test is simplified and the colorimetric competitive indirect immunoassay based on direct labeling is more sensitive to OA than the test based on indirect labeling. In optimized conditions, the IC50 value and detection limit were respectively 0.066 mg L�1 and 0.0061 mg L�1. Then, the colorimetric immunoassay based on direct labeling was validated with certified reference mussel samples, demonstrating the efficiency of the approach.
    Food Control 03/2013; 30(1):144-149. · 2.74 Impact Factor
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    ABSTRACT: Diarrheic Shellfish Poisoning (DSP) is a gastrointestinal illness caused by consumption of shellfish contaminated with DSP toxins such as okadaic acid (OA) and dinophysistoxins (DTX). The occurrences of OA in bivalves induce not only public health problems but also economic damages to shellfish farming. Consequently, the development of fast, reliable and sensitive detection methods is an evident necessity. The mouse bioassay has been the reference and most commonly used analysis method. However, this technique suffers from low accuracy, specificity and ethical problems due to the animal experimentation. Thus, the development of alternative and efficient detection systems is required. Several biological, chemical, and immunological methods have been developed to evaluate the presence of DSP toxins in seafood. This review gives an overview of different analytical methods and new trends for the detection of OA. Over the past decade, considerable attention has been given to the development of biosensors for the efficient detection of marine toxin. Recent advances in the field of aptamers and nanomaterial offer exciting new opportunities to develop improved and more reliable devices allowing the detection of OA.
    Talanta 02/2013; 105C:306-316. · 3.50 Impact Factor
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    ABSTRACT: The aptamer immbolization onto organized mixed layers of diazonium salts via click chemistry was explored. The immobilized aptamer was employed in the fabrication of a highly sensitive and reusable electrochemical impedimetric aptasensor for the detection of ochratoxin A (OTA). The screen-printed carbon electrodes (SPCEs) were first modified by electrografting of a protected 4-((trimethylsilyl)ethynyl) benzene (TMSi-Eth-Ar) layer followed by a second one of p-nitrobenzene (p-NO(2)-Ar) by means of electrochemical reduction of their corresponding diazonium salts, (TMSi-Eth-Ar-N(2)(+)) and (p-NO(2)-ArN(2)(+)). After deprotection, a layer with active ethynyl groups was obtained. In the presence of copper (I) catalyst, the ethynyl groups reacted efficiently with aptamer bearing an azide function, thus forming a covalent 1,2,3-triazole linkage. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of ferri/ferrocyanide redox probe [Fe(CN)(6)](4-/3-) were used to characterize each step in the aptasensor development. The increase in electron-transfer resistance (R(et)) values due to the specific aptamer-OTA interaction was proportional to the concentration of OTA in a range between 1.25ng/L and 500ng/L, with a detection limit of 0.25ng/L.
    Talanta 01/2013; 103:14-9. · 3.50 Impact Factor
  • Audrey Sassolas, Akhtar Hayat, Jean-Louis Marty
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    ABSTRACT: The development of enzyme immobilization techniques that will not affect catalytic activity and conformation is an important research task. Affinity tags that are present or added at a specific position far from the active site in the structure of the native enzyme could be used to create strong affinity bonds between the protein structure and a surface functionalized with the complementary affinity ligand. These immobilization techniques are based on affinity interactions between biotin and (strept)avidin molecules, lectins and sugars, or metal chelate and histidine tag.Recent developments involve immobilization of tagged enzymes onto magnetic nanoparticles. These supports can improve the performance of immobilized biomolecules in analytical assay because magnetic beads provide a relative large numbers of binding sites for biochemical reactions resulting in faster assay kinetics.This chapter describes immobilization procedures of tagged enzymes onto various magnetic beads.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1051:139-48. · 1.29 Impact Factor
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    ABSTRACT: This paper describes a novel, simple, and versatile protocol for covalent immobilization of enzyme on electrode. The immobilization method is based on the combination of diazonium salt electrografting and click chemistry. The ethynyl-terminated monolayers are obtained by diazonium salt electrografting, then, in the presence of copper (I) catalyst, the ethynyl modified surfaces reacts efficiently and rapidly with enzyme bearing an azide function (azido-enzyme), thus forming a covalent 1,2,3-triazole linkage by means of click chemistry. The ethynyl-terminated film preserves the activity of the immobilized enzyme. The click chemistry along with binary film of diazonium salts offers a variety of good characteristics including high sensitivity, good repeatability and reusability, rapid response and long term stability of the system. Thus, because of the chemoselective reactivity and quantitative yield of the click reaction, an ethynyl-terminated monolayer can be treated as a general platform for obtaining reliable coverage of a wide range of azido-terminated species of interest for various sensing applications.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1051:209-16. · 1.29 Impact Factor
  • Audrey Sassolas, Akhtar Hayat, Jean-Louis Marty
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    ABSTRACT: This chapter provides a detailed description of the three immobilization methods based on the biomolecules entrapment into polymer matrices. The poly (vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), a soluble pre-polymer bearing photo-cross-linkable groups, has widely been used to entrap enzymes, and several bioassays based on this immobilization matrix have been reported. Similarly, immobilization of enzymes via sol-gel has been described in this chapter. Sol-gel process is based on the ability to form solid metal or semi-metal oxides via the aqueous process of hydrolytically labile precursors. Enzymes can also be entrapped in an agarose gel. Contrary to synthetic polymers such as polyacrylamide, this matrix is biocompatible, non-toxic, provides natural microenvironment to the enzyme and also gives sufficient accessibility to electrons to shuttle between the enzyme and the electrode. The entrapment strategies are easy-to-perform, and permit to deposit enzyme, mediators, and additives in the same sensing layer. Moreover, the activity of the enzyme is preserved during the immobilization process, as biological element is not modified. Biosensors based on physically entrapped enzymes are often characterized by increased operational and storage stability.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1051:229-39. · 1.29 Impact Factor
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    ABSTRACT: a b s t r a c t An electrochemical immunosensor for okadaic acid (OA) detection has been developed, and used in an indirect competitive immunoassay format under automated flow conditions. The biosensor was fabricated by injecting OA modified magnetic beads onto screen printed carbon electrode (SPCE) in the flow system. The OA present in the sample competed with the immobilized OA to bind with anti-okadaic acid monoclonal antibody (anti-OA-MAb). The secondary alkaline phosphatase labeled anti-body was used to perform electrochemical detection. The current response obtained from the labeled alkaline phosphatase to 1–naphthyl phosphate decreased proportionally to the concentration of free OA in the sample. The calculated limit of detection (LOD) was 0.15 mg/L with a linear range of 0.19–25 mg/L. The good recoveries percentages validated the immunosensor application for real mussel samples. The developed system automatically controlled the incubation, washing and current measurement steps, showing its potential use for OA determination in field analysis. & 2012 Elsevier B.V. All rights reserved.
    Talanta 04/2012; · 3.50 Impact Factor
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    ABSTRACT: a b s t r a c t An electrochemical immunosensor for okadaic acid (OA) detection has been developed, and used in an indirect competitive immunoassay format under automated flow conditions. The biosensor was fabricated by injecting OA modified magnetic beads onto screen printed carbon electrode (SPCE) in the flow system. The OA present in the sample competed with the immobilized OA to bind with anti-okadaic acid monoclonal antibody (anti-OA-MAb). The secondary alkaline phosphatase labeled anti-body was used to perform electrochemical detection. The current response obtained from the labeled alkaline phosphatase to 1–naphthyl phosphate decreased proportionally to the concentration of free OA in the sample. The calculated limit of detection (LOD) was 0.15 mg/L with a linear range of 0.19–25 mg/L. The good recoveries percentages validated the immunosensor application for real mussel samples. The developed system automatically controlled the incubation, washing and current measurement steps, showing its potential use for OA determination in field analysis. & 2012 Elsevier B.V. All rights reserved.
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    ABSTRACT: Okadaic acid (OA), a lipophilic phycotoxin is mainly produced by toxigenic dinoflagellates. The need to develop high performing methods for OA analysis able to improve the traditional ones is evident. In this work, a novel experimental methodology for label-free detection of OA was developed. Protein G magnetic beads (protein-G-MBs) modified gold electrode was used to immobilize anti-OA monoclonal antibody (anti-OA-MAb). Preliminary, colorimetric tests were performed in order to validate protein-G-MBs and anti-OA-MAb reaction. Electrochemical detection was carried out by differential pulse voltammetry in ferri/ferrocyanide solution. The limit of detection value obtained (0.5 μg L(-1)) validated the developed electrochemical immunosensor as a promising tool for routine use. The matrix effect and the recovery rate were also assessed with real samples, showing a good percentage of recovery.
    Analytica chimica acta 04/2012; 724:92-7. · 4.31 Impact Factor
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    ABSTRACT: The inhibition characteristics of three different protein phosphatases by three microcystin (MC) variants--LR, YR, and RR--were studied. The corresponding K (I) for each enzyme-MC couple was calculated. The toxicity of MC varies in the following order: MC-LR > MC-YR > MC-RR. The sensitivity of the enzymes increased in the following order: mutant PP2A < mutant PP1 < natural PP2A. The best limit of detection obtained was 21.2 pM MC-LR using the most sensible enzyme. Methanol, ethanol, and acetonitrile up to 2 % (v/v) may be used in inhibition measurements. An artificial neural network (ANN) was used to discriminate two MC variants--LR and YR--using the differences in inhibition percentages measured with mutant PP1 and natural PP2A. The ANN is able to analyze mixtures with concentrations ranging from 8 to 98 pM MC-LR and 31 to 373 pM MC-YR.
    Analytical and Bioanalytical Chemistry 04/2012; · 3.66 Impact Factor
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    ABSTRACT: Okadaic acid (OA), responsible for gastrointestinal problems, inhibits protein phosphatase 2A (PP2A). Therefore, the inhibition exerted by the toxin on PP2A could be used to detect the presence of OA in aqueous solution and in shellfish sample. In this work, two commercial PP2As (from ZEU Immunotec and Millipore) and one produced by molecular engineering (from GTP Technology) were tested. Enzymes were used in solution and also immobilized within a polymeric gel. In solution, best performances were obtained using PP2A purchased from ZEU Immunotec (Spain). OA was detected in aqueous solution in concentration as low as 0.0124 μg L(-1) using the enzyme from ZEU Immunotec whereas the detection limits were 0.47 μg L(-1) and 0.123 μg L(-1) with PP2As from Millipore and GTP Technology, respectively. Considering that the immobilization step contributes to stabilize the PP2A activity, enzymes were entrapped within a photopolymer and an agarose gel. These different polymeric matrices were optimized, tested and compared. Agarose gel seems to be a good alternative to the photopolymer largely used in our group. For instance, the IC(50) value obtained with the test based on PP2A from ZEU Immunotec immobilized within an agarose gel was 1.98 μg L(-1). This value was 1.8-fold lower than those obtained with the photopolymer test using the same enzyme. The proposed test is sensitive, fast and does not require expensive equipment. To evaluate the efficiency of the assay, PP inhibition tests based on PP2A from ZEU Immunotec in solution or immobilized within a gel were used for OA detection in contaminated shellfish.
    Analytica chimica acta 09/2011; 702(2):262-8. · 4.31 Impact Factor
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    ABSTRACT: Immobilization of enzymes on the transducer surface is a necessary and critical step in the design of biosensors. An overview of the different immobilization techniques reported in the literature is given, dealing with classical adsorption, covalent bonds, entrapment, cross-linking or affinity as well as combination of them and focusing on new original methods as well as the recent introduction of promising nanomaterials such as conducting polymer nanowires, carbon nanotubes or nanoparticles. As indicated in this review, various immobilization methods have been used to develop optical, electrochemical or gravimetric enzymatic biosensors. The choice of the immobilization method is shown to represent an important parameter that affects biosensor performances, mainly in terms of sensitivity, selectivity and stability, by influencing enzyme orientation, loading, mobility, stability, structure and biological activity.
    Biotechnology advances 09/2011; 30(3):489-511. · 8.25 Impact Factor
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    ABSTRACT: In this work, an electrochemical method based on the diazonium-coupling reaction mechanism for the immobilization of okadaic acid (OA) on screen printed carbon electrode was developed. At first, 4-carboxyphenyl film was grafted by electrochemical reduction of 4-carboxyphenyl diazonium salt, followed by terminal carboxylic group activation by N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyle)-N'-ethyle-carbodiimide hydrochloride (EDC). Hexamethyldiamine was then covalently bound by one of its terminal amine group to the activated carboxylic group. The carboxyl group of okadaic acid was activated by EDC/NHS and then conjugated to the second terminal amine group on other side of the hexamethyldiamine through amide bond formation. After immobilization of OA, an indirect competitive immunoassay format was employed to detect OA. The immunosensor obtained using this novel approach allowed detection limit of 1.44 ng/L of OA, and was also validated with certified reference mussel samples.
    Talanta 07/2011; 85(1):513-8. · 3.50 Impact Factor
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    ABSTRACT: Advances in the development and the applications of optical biosensing systems based on immobilized aptamers are presented. These nucleic acid sequences have been used as new molecular recognition elements to develop heterogeneous assays, biosensors and microarrays. Among different detection modes that have been employed, optical ones which are described here are among the most used. Since their first report in 1996, numerous optical detection systems using aptamers and mainly based on fluorescence have been developed. Two main approaches have been used: label-based (using fluorophore, luminophore, enzyme, nanoparticles) or aptamer label-free detection systems (e.g. surface plasmon resonance, optical resonance). Most methods are based on a labeling approach. Some targets can be optically detected using not only colorimetry, chemiluminescence or the most developed fluorescence mode but also more recent non conventional optical methods such as surface plasmon-coupled directional emission (SPCDE). The first SPCDE-based aptasensor for thrombin detection has recently been reported in 2009. Aptasensors based on surface-enhanced Raman scattering spectroscopy (SERS) which presents advantages compared to fluorescence have also been described. Different label-free techniques have recently been shown to be suitable for developing performant aptasensors or aptamer-based microarrays, such as surface plasmon resonance (SPR), diffraction grating, evanescent-field-coupled (EFC) waveguide-mode, optical resonance or Brewster angle straddle interferometry (BASI). Important advances have been realized on optical aptamer-based detection systems that appear as highly efficient devices with enormous potential.
    Biosensors & Bioelectronics 05/2011; · 6.45 Impact Factor
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    ABSTRACT: Aptamers are DNA or RNA oligonucleotides that can bind with high affinity and specificity to a wide range of targets such as proteins, metal ions or pathogenic microorganisms. Soluble aptamers and aptazymes have been used as sensing elements for developing homogeneous assays in a solution phase, the whole sensing process being carried out in a homogeneous solution. Contrary to most conventional heterogeneous assays that are time-consuming and labor-intensive, aptamer-based homogeneous assays are simple, easy-to-perform, rapid and do not require immobilization nor washing steps. To our knowledge, this review is the first entirely dedicated to aptamer-based homogeneous assays. Optical detection appears as the most developed technique. Colorimetry represents the simplest sensing mode that occupies a very important position among aptamer-based assays, involving gold nanoparticle aggregation (with unmodified or aptamer-modified gold NPs), the formation of HRP-mimicking DNAzyme with hemin, dye displacement or interactions with a cationic polymer. Fluorescence that is highly sensitive offers the most developed detection mode. Aptamers can be labeled or not, to give rise to turn-on or usually less sensitive turn-off fluorescent assays. Newly reported and thus less developed non-conventional magnetic resonance imaging (MRI) and electrochemistry also recently appeared in the literature, thrombin still remains the main detected target. Homogeneous assays based on aptazyme, an aptamer sequence connected to a known ribozyme motif, are also described in this review, involving optical detection, by colorimetry or fluorescence.
    The Analyst 01/2011; 136(2):257-74. · 4.23 Impact Factor
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    ABSTRACT: A colorimetric protein phosphatase (PP) inhibition test for the detection of microcystin-LR (MC-LR) has been developed. Three PP2As, one recombinant and two natural versions, as well as one PP1 produced by molecular engineering, were tested. First, assays were performed using the enzymes in solution to compare their sensitivity to MC-LR. The PP2A purchased from ZEU Immunotec and PP1 appeared more sensitive to the toxin than the other enzymes. With PP2A from ZEU Immunotec, the colorimetric test showed a detection limit of 0.0039 μg L(-1) and an IC(50) value of 0.21 μg L(-1). With PP1, the assay gave a detection limit of 0.05 μg L(-1) and an IC(50) value of 0.56 μg L(-1). Therefore, this assay allowed the detection of lower microcystin-LR (MC-LR) concentrations than the maximum level (1 μg L(-1)) recommended by the World Health Organisation (WHO). The main drawback of this PP-based approach in solution is poor enzyme stabilisation. To overcome this problem, enzymes were entrapped within either a photopolymer or an agarose gel. PP2A from ZEU Immunotec and PP1 were immobilised at the bottom of microwells. The agarose-based tests performed better than the photopolymer-based assay for all of the enzymes. Therefore, the agarose gel is a good candidate to replace the photopolymer, which is generally used in PP-immobilising membranes. The assays based on enzyme-entrapping agarose gels showed detection limits equal to 0.17 μg L(-1) and 0.29 μg L(-1) with immobilised PP2A from ZEU and PP1, respectively. In view of these performances, these tests can potentially be used for monitoring water quality.
    Talanta 01/2011; 85(5):2498-503. · 3.50 Impact Factor
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    ABSTRACT: Performant reagentless electrochemiluminescent (ECL) (bio)sensors have been developed using polymeric luminol as the luminophore. The polyluminol film is obtained by cyclic voltammetry (CV) on a screen-printed electrode either in a commonly used H(2)SO(4) medium or under more original near-neutral buffered conditions. ECL responses obtained after performing polymerization either at acidic pH or at pH 6 have been compared. It appears that polyluminol formed in near-neutral medium gives the best responses for hydrogen peroxide detection. Polymerization at pH 6 by cyclic voltammetry gives a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M H(2)O(2) concentrations. Based on this performant sensor for hydrogen peroxide detection, an enzymatic biosensor has been developed by associating the polyluminol film with an H(2)O(2)-producing oxidase. Here, choline oxidase (ChOD) has been chosen as a model enzyme. To develop the biosensor, luminol has been polymerized at pH 6 by CV, and then an enzyme-entrapping matrix has been formed on the above modified working electrode. Different biological (chitosan, agarose, and alginate) and chemical (silica gels, photopolymers, or reticulated matrices) gels have been tested. Best performances have been obtained by associating a ChOD-immobilizing photopolymer with the polyluminol film. In this case, choline can be detected with a linear range extending from 8 x 10(-8) to 1.3 x 10(-4) M.
    Analytical and Bioanalytical Chemistry 05/2009; 394(4):971-80. · 3.66 Impact Factor
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    ABSTRACT: This review presents electrochemical aptasensors reported in the literature. Their development has been growing as evidenced by the increasing number of scientific publications on this subject since 2004. Aptasensors are presented according to their transduction mode, including the most described amperometric and impedimetric devices as well as FETs and the first very recently reported potentiometric aptasensor. ‘Signal-on’ (positive readout signal) and less sensitive ‘signal-off’ (negative readout signal) aptasensors are reviewed based on target binding-induced conformational change of aptamers or on target binding-induced strand displacement or even on both processes. Aptamer-labeled and aptamer label-free devices are presented separately.
    Electroanalysis 04/2009; 21(11):1237 - 1250. · 2.82 Impact Factor
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    ABSTRACT: A performant reagentless electrochemiluminescent (ECL) system for H2O2 detection based on electropolymerized luminol is first presented. In this work, polyluminol is formed under near-neutral conditions onto pre-treated screen-printed electrodes (SPEs). Pre-treatment conditions of the working electrode surface have been optimized so as to obtain the best ECL responses to H2O2 that were increased by a factor as high as 400. Galvanostatic polymerization has been tested as a new process to form polyluminol films. Good performances were obtained in terms of responses to H2O2, with quite the same linear range as the ones obtained under potentiostatic and potentiodynamic modes.The association of the polyluminol film with an H2O2-producing oxidase has also been investigated, using choline oxidase as a model enzyme. Silica glasses obtained by the sol–gel process have been employed for biomolecule immobilization. Polymeric luminol has been coupled with choline oxidase-immobilizing gel under bilayer or monolayer configurations. In the first case, enzyme has been immobilized in a silica gel formed on a polyluminol film. In the second case, the enzymatic gel formed on SPE was soaked in a solution containing monomeric luminol units, allowing them to diffuse and penetrate throughout the porous gel. Then, electrodeposition was performed to polymerize the luminophore within the silica matrix. In both cases, choline could be detected down to micromolar concentrations.
    Sensors and Actuators B-chemical - SENSOR ACTUATOR B-CHEM. 01/2009; 139(1):214-221.

Publication Stats

391 Citations
113.90 Total Impact Points

Institutions

  • 2011–2013
    • Université de Perpignan
      • IMAgES Institute of Modelling and Analysis in Geo- Environment and Health
      Perpinyà, Languedoc-Roussillon, France
  • 2008
    • Claude Bernard University Lyon 1
      • Institut de chimie et biochimie moléculaires et supramoléculaires (ICBMS)
      Villeurbanne, Rhône-Alpes, France
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
    • University of Lyon
      Lyons, Rhône-Alpes, France