Béatrice D. Leca-Bouvier

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. 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.

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. 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 for Protein Detection: A Review

Abstract There is considerable demand for the rapid low‐cost determination of proteins, particularly in the food and beverage industry. The most widely used tests are based on colorimetric procedures in which proteins react to produce colored complexes. These methods (Lowry et al. 1951; Bradford 1976; Gornall et al. 1949; Smith et al. 1985) are dependent upon a number of factors other than absolute protein quantity, including amino acid composition, protein purity, and association state. The successful application of these methods relies on using representative calibration standards. Time‐consuming and complex methodologies such as the Kjeldahl technique and quantitative amino acid analysis procedures have been also reported. Apart from these methods, biosensors are interesting tools offering certain operational advantages over standard photometric methods, notably with respect to rapidity, ease‐of‐use, cost, simplicity, portability, and ease of mass manufacture. By appropriate recognition element selection, it is possible to detect either a particular target protein or a broad range of proteins. This review presents an overview of the different biological recognition elements and the various transduction systems that have been reported in the literature to design biosensors for protein detection. Examples are given to illustrate the different possibilities. It must be noticed that this review reports detection of proteins in solution and not proteins immobilized on membranes. Briefly, immobilized proteins can be detected by antibodies (Western blotting) or oligonucleotides such as aptamers (“Eastern blotting”) bearing reporter molecules, fluorescently labeled or radiolabeled.

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 H2SO4 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 × 10−8 to 1.3 × 10−4 M H2O2 concentrations. Based on this performant sensor for hydrogen peroxide detection, an enzymatic biosensor has been developed by associating the polyluminol film with an H2O2-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 × 10−8 to 1.3 × 10−4 M.

Kinetics study of Bungarus fasciatus venom acetylcholinesterase immobilised on a Langmuir-Blodgett proteo-glycolipidic bilayer.

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.

Enzyme for Biosensing Applications

Enzymes are very efficient biocatalysts, which have the ability to specifically recognize their substrates and to catalyze their transformation. These unique properties make the enzymes powerful tools to develop analytical devices. Enzyme-based biosensors associate intimately a biocatalyst-containing sensing layer with a transducer. The transformations catalyzed by an enzyme come with the variations of some physicochemical parameters. The role of the transducer is to convert those physicochemical signals into a measurable electrical signal. In biosensors, enzymes are generally immobilized on or close to the transducer. Depending on the chemical and physical characteristics of the enzyme support, different immobilization techniques can be implemented. The transduction mode will be adapted to the physicochemical parameter that is monitored. The variation of the concentration of a substrate or a product in the course of an enzymatic reaction can be detected with the help of a physical or chemical sensor, which then acts as a transducer. Electrochemical biosensors have been then developed involving oxidoreduction reactions. Optical biosensors are based either on fluorescence, absorbance, and bioluminescence or chemiluminescence measurements. Enzymatic reactions are usually associated with a high enthalpy change, which results in a temperature variation that can be recorded using a thermistor. Gravimetric biosensors are based on a mass variation induced by an enzymatic reaction.

PCB-based integration of electrochemiluminescence detection for microfluidic systems.

This communication presents an instrumental development based on the printed circuit board (PCB) technology to integrate electrochemiluminescence (ECL) analysis in microfluidic systems. 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.

CMOS optical detector system for capillary fluorescence measurements

We present a fluorescence detection system for capillary analysis. It is designed using a CMOS BDJ (Buried Double p-n Junction) detector which can be operated either as a photodiode or as a wavelength-sensitive device. Noise-reduction techniques such as signal pre-amplification and synchronous detection are implemented to boost the sensitivity of measurements. The system indicates fluorescence intensity for concentration determination, and average wavelength of fluorescence spectrum for molecular discrimination. The system has been tested by measuring two widely used fluorophores (FITC and Rhodamine B) in different concentrations. A 407-nm blue laser diode and a 532-nm green YAG compact laser have been respectively employed for their excitation. The illuminated volume inside the capillary is about 5 nl. The best results have been obtained with FITC, enabling as low as 10-10 M to be detectable.

Combining microfluidics and electrochemical detection

This paper describes two configurations that integrate electrochemical detection into microfluidic devices. The first configuration is a low-cost approach based on the use of PCB technology. This device was applied to electrochemiluminescence detection. The second configuration was used to carry out amperometric quantification of electroactive species using a serial dilution microfluidic system.

Implementation of Electrochemiluminescence Microanalysis in PCB Technology

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.