RESUMO
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.
Assuntos
Técnicas Biossensoriais/instrumentação , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Medições Luminescentes/instrumentação , Luminol/química , Polímeros/química , Oxirredutases do Álcool/química , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Peróxido de Hidrogênio/química , Medições Luminescentes/métodos , Polímeros/síntese químicaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Animais , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Colorimetria/métodos , Técnicas Eletroquímicas/métodos , Fluorescência , Humanos , Imageamento por Ressonância Magnética/métodos , Dados de Sequência Molecular , RNA Catalítico/metabolismoRESUMO
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.
Assuntos
Oxirredutases do Álcool/metabolismo , Técnicas Biossensoriais/métodos , Eletroquímica/instrumentação , Enzimas Imobilizadas/metabolismo , Medições Luminescentes/métodos , Luminol/química , Polímeros/química , Oxirredutases do Álcool/química , Colina/análise , Eletrodos , Enzimas Imobilizadas/química , Peróxido de Hidrogênio/análise , Concentração de Íons de Hidrogênio , Luminol/síntese química , Polímeros/síntese química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
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. Polymerization conditions have been optimized in order to obtain the best ECL responses to H(2)O(2). By performing electrodeposition in a potentiostatic mode, at 425 mV vs. Ag|AgCl, in 0.1 mol L(-1) phosphate/0.1 mol L(-1) KCl pH 6 and 1 mmol L(-1) luminol, with a total charge of 0.5 mC, the linear range for H(2)O(2) detection extends from 7.9 x 10(-8) mol L(-1) to 1.3 x 10(-3) mol L(-1). Such performant disposable reagentless easy-to-use miniaturized systems based on SPEs should be applicable to the electrochemiluminescent detection of many oxidase-substrate compounds.
Assuntos
Técnicas Biossensoriais , Eletroquímica/métodos , Luminescência , Luminol/química , Eletrodos , Desenho de Equipamento , Peróxido de Hidrogênio/química , Modelos Químicos , Oxigênio/química , Polímeros/química , Cloreto de Potássio/química , Potenciometria/métodos , Prata/química , Compostos de Prata/químicaRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Enzimas Imobilizadas/química , Polímeros/química , Adsorção , Técnicas Biossensoriais/classificação , Estrutura Molecular , Nanoestruturas/química , Transição de FaseRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Dispositivos Ópticos , Ressonância de Plasmônio de Superfície/métodos , Colorimetria/métodos , Nanopartículas/química , Trombina/química , Trombina/isolamento & purificaçãoRESUMO
We present an instrumental development to implement electrochemiluminescence (ECL) microanalysis using printed circuit board (PCB) technology. PCB gold macro-(10 mm2) and micro- (0.09 mm2) electrodes and two ECL microfluidic devices are designed, fabricated and tested via luminol ECL detection. Potential modulation is performed between 0.7 and 0 V vs. Ag/AgCl for luminol oxidation, thus giving rise to on/off ECL responses in the presence of hydrogen peroxide. Synchronous detection is adopted to allow weak ECL signal recovery at a very low signal-to-noise ratio (SNR). The detection limit obtained with the two ECL microfluidic devices is 50 nM and 100 nM H2O2 for macroelectrodes and microelectrodes, respectively.
Assuntos
Medições Luminescentes/métodos , Técnicas Analíticas Microfluídicas , Eletroquímica , Eletrônica , Peróxido de Hidrogênio/química , Substâncias Luminescentes/química , Medições Luminescentes/instrumentação , Luminol/química , Microeletrodos , Prata/química , Compostos de Prata/químicaRESUMO
This study deals with the kinetics properties of an enzyme immobilised in a defined orientation in a biomimetic environment. For this purpose, acetylcholinesterase (AChE) was captured at the surface of a nanostructured proteo-glycolipidic Langmuir-Blodgett film through specific recognition by a noninhibitor monoclonal antibody (IgG) inserted in a neoglycolipid bilayer. Modelling of this molecular assembly provided a plausible interpretation of the functional orientation of the enzyme. The AChE activity being stable for several weeks, the enzyme kinetics were investigated, and fitted perfectly with heterogeneous biocatalytic behaviour representative of cellular enzymatic catalysis. The AChE-IgG-glycolipid nanostructure was directly interfaced with an efficient optical device. Such an association, leading to an intimate contact between the nanostructure and the biochemical signal transducer, gives direct access to the intrinsic AChE behaviour. This study thus demonstrates the potential for direct investigation of the kinetic behaviour of an immobilised enzyme on a lipid bilayer through an efficient transduction system.