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.

Polyluminol/hydrogel composites as new electrochemiluminescent-active sensing layers.

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).

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.

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.

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.

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.

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)(+)).

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).

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.

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.

Immobilization strategies to develop enzymatic biosensors.

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.

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.

An electrochemical immunosensor based on covalent immobilization of okadaic acid onto screen printed carbon electrode via diazotization-coupling reaction.

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.

Optical detection systems using immobilized aptamers.

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.

New electrochemiluminescent biosensors combining polyluminol and an enzymatic matrix.

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.

Electrogeneration of polyluminol and chemiluminescence for new disposable reagentless optical sensors.

A performant reagentless electrochemiluminescent (ECL) detection system for H(2)O(2) is presented, based on an electropolymerized polyluminol film prepared under near-neutral conditions. Such an original polyluminol electrodeposition is reported for the first time and on a screen-printed electrode (SPE) surface. Electropolymerized luminol acts as an active luminophore of the electrochemiluminescent reaction, as the monomer does.