Audrey Sassolas

Highly sensitive ochratoxin A impedimetric aptasensor based on the immobilization of azido-aptamer onto electrografted binary film via click chemistry.

The aptamer immobilization 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 1.25 ng/L and 500 ng/L, with a detection limit of 0.25 ng/L.

Detection of the marine toxin okadaic acid: Assessing seafood safety

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.

Development of a novel label-free amperometric immunosensor for the detection of okadaic acid

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.

Development of a colorimetric inhibition assay for microcystin-LR detection: Comparison of the sensitivity of different protein phosphatases

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.

Automated flow-through amperometric immunosensor for highly sensitive and on-line detection of okadaic acid in mussel sample

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 antibody 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 μg/L with a linear range of 0.19-25 μg/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.

Development of an efficient protein phosphatase-based colorimetric test for okadaic acid detection

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.

Immobilization of enzymes on magnetic beads through affinity interactions.

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.

Enzyme Immobilization by Entrapment Within a Gel Network

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.

Immobilization of enzymes on ethynyl-modified electrodes via click chemistry.

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.