Loïc J. Blum

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.

Luminescence Fiber-Optic Biosensor

Abstract A novel type of biosensors involving immobilized bioluminescence enzymes and a fiber-optic probe has been developed. The enzymes were immobilized on preactivated Nylon membranes placed in close contact with the tip of a bundle of optical glass fibers. The fiber-optic sensor was immersed in a stirred and thermostated cell protected from ambient light by a PVC jacket. The light emitted by the luminescence reactions was conducted through the fiber bundle to the photomultiplier tube of a luminometer. With immobilized firefly luciferase from Photinus pyralis, light emission could be linearly related to ATP concentration in the range 2.8 × 10−10 − 1.4 × 10−6 M. When co-immobilizing bacterial luciferase and oxidoreductase from Vibrio fischeri, NADH measurements could be performed from 3 × 10−10 M to 3 × 10−6 M. The luminol chemiluminescence reaction catalyzed by immobilized horseradish peroxidase has also been used for hydrogen peroxide determination. The standard curve was linear from 2 × 10−8 M to 2 ×...

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.

An electrochemiluminescence-based fibre optic biosensor for choline flow injection analysis

A fibre optic biosensor based on luminol electrochemiluminescence (ECL) integrated in a flow injection analysis (FIA) system was developed for the detection of choline. The electrochemiluminescence of luminol was generated by a glassy carbon electrode polarised at +425 mV vs. a platinum pseudo-reference electrode. Choline oxidase (Chx) was immobilised either covalently on polyamide (ABC type) or on UltraBind preactivated membranes, or by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) alone or after absorption on a weak anion exchanger, DEAE (diethylaminoethyl) Sepharose. The optimisation of the reaction conditions and physicochemical parameters influencing the FIA biosensor response demonstrated that the choline biosensor exhibited the best performances in a 30 mM veronal buffer containing 30 mM KCl and 1.5 mM MgCl2, at pH 9. The use of a 0.5 ml min-1 flow rate enabled the measurement of choline by the membrane-based ECL biosensors in 8 or 5 min, with ABC or UltraBind membranes, respectively, whereas the measurement required only 3 min with the DEAE-PVA system. For comparison, the detection of choline was performed with Chx immobilised using the four different supports. The best performances were obtained with the DEAE-PVA-Chx sensing layer, which allowed a detection limit of 10 pmol, whereas with the ABC, the UltraBind and the PVA systems, the detection limits were 300 pmol, 75 pmol and 220 pmol, respectively. The DEAE-based system also exhibited a good operational stability since 160 repeated measurements of 3 nmol of choline could be performed with an RSD of 4.5% whereas the stability under the best conditions was 45 assays with the other supports.

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.

Luminol electrochemiluminescence with screen-printedelectrodes for low-cost disposable oxidase-based optical sensors

This paper reports the use of screen-printed electrodes polarised at a fixed potential to trigger luminol electrochemiluminescence in a batch system. Choline oxidase is used as a model H2O2-generating oxidase. The enzyme is immobilised by ionic interactions in DEAE–Sepharose beads before entrapment in a poly(vinyl alcohol) bearing styrylpyridinium groups (PVA–SbQ) photocrosslinked polymer. The light signal is transmitted to a photomultiplier tube via a fibre optic facing the electrode. Under optimised conditions, at 450 mV vs. Ag/AgCl, in 30 mM veronal–HCl buffer, pH 9, containing 50 μM luminol, the linear range extends from 2 × 10−8 to 1 × 10−4 M choline.

“Escherichia coli‐milk” biofilm removal from stainless steel surfaces: Synergism between ultrasonic waves and enzymes

Three different methods to standardize biofilm removal for in situ sanitary control of closed surfaces in the food industry have been developed and compared, i.e. sonication, enzymatic treatment and a combined treatment which involved the application of ultrasound to enzyme preparations. The biofilm studied was an Escherichia coli model biofilm, made with milk on stainless steel sheets. Plate counting and epifluorescence microscopy were used to assess the efficiency of each treatment. The results are expressed in percentages, 100% denoting total removal, obtained with a flat ultrasonic transducer (T1) developed and presented in a previous study. The application of ultrasound by a patented curved transducer, T2 (10 s, 40 kHz), specifically devised for closed surfaces, was not sufficient to completely remove the biofilm (30 ± 7%). This biofilm was dislodged by two proteolytic enzyme preparations tested by immersion, viz. a 15‐min application of protease (84±1%) and a 30‐min trypsin application (95±8%). Using a combined treatment, the results showed a synergism between ultrasonic waves and proteolytic or glycolytic enzyme preparations, with removal of a significant amount of biofilm, i.e. 61–96% depending on the conditions tested, i.e. two to three times greater compared to sonication alone (30%). This application was in agreement with an industrial control, i.e. a good reproducible recovery of the biofilm in 10 s compared with 30 or 15 min with the enzyme alone.

The development of an ultrasonic apparatus for the non-invasive and repeatable removal of fouling in food processing equipment

N. OULAHAL‐LAGSIR, A. MARTIAL‐GROS, E. BOISTIER, L.J. BLUM and M. BONNEAU.2000.A new ultrasonic apparatus operating at a frequency of 40 kHz was developed to dislodge biofilms from food processing equipment in order to assess the effectiveness of cleaning protocols. Sonication conditions to remove biofilms and quantification by ATP‐bioluminescence are described. An industrial meat process was developed at the laboratory level to form a biofilm with industrial characteristics. Our results show that the biofilm removal by sonication during 10 s is reproducible and four times greater compared to the swabbing method (83% removal of fouling material against 20%). Unlike the swabbing method, this ultrasonic apparatus permitted the immediately demonstration of the inefficiency (within 1 min) of an industrial meat cleaning protocol. This apparatus is portable, easy to use and can be operated by unskilled users.

Paper electrodes for bioelectrochemistry: Biosensors and biofuel cells.

Paper-based analytical devices (PAD) emerge in the scientific community since 2007 as low-cost, wearable and disposable devices for point-of-care diagnostic due to the widespread availability, long-time knowledge and easy manufacturing of cellulose. Rapidly, electrodes were introduced in PAD for electrochemical measurements. Together with biological components, a new generation of electrochemical biosensors was born. This review aims to take an inventory of existing electrochemical paper-based biosensors and biofuel cells and to identify, at the light of newly acquired data, suitable methodologies and crucial parameters in this field. Paper selection, electrode material, hydrophobization of cellulose, dedicated electrochemical devices and electrode configuration in biosensors and biofuel cells will be discussed.

Langmuir-Blodgett Technique for Synthesis of Biomimetic Lipid Membranes

Abbreviations: A: Area per molecule LB: Langmuir-Blodgett AFM: Atomic Force Microscopy LC: Liquid Condensed DLPE: Dilauroylphosphatidylethanolamine LE: Liquid Expanded DMPC: Dimyristoylphosphatidylcholine LS: Langmuir-Schaefer DMPE: Dimyristoylphosphatidylethanolamine MGDG: Monogalactosyldiglyceride DOPC: Dioleoylphosphatidylcholine DGDG: Digalactosyldiglyceride DOPE: Dioleoylphosphatidylethanolamine MLV: Multilamellar Vesicle DPG: diposphatidylglycerol OTS: Octadecyltrichlorosilane DPPA: Dipalmitoylphosphatidic acid PC: Phosphatidylcholine DPPC: Dipalmitoylphosphatidylcholine PE: Phosphatidylethanolamine DPPE: Dipalmitoylphosphatidylethanolamine PG: Phosphatidylglycerol DPPS: Dipalmitoylphosphatidylserine PI: Phosphatidylinositol DSPC: Distearoylphosphatidylcholine PS: Phosphatidylserine DSPE: Distearoylphosphatidylethanolamine RD: Monolayer deposition Rate ESP: Equilibrium Spreading Pressure SAM: Self-Assembled Monolayer FSB: Free Supported Bilayer SFA: Surface Force Apparatus GM1: Monosialoganglioside-GM1 : Surface pressure IgG: Immunoglobulin G : Surface tension incl.: included SPM: Palmitoyl-Sphingomyeline