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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.89, #2-3)
Fiberoptic biosensors based on chemiluminescent reactions by Christophe A. Marquette; Agnès Degiuli; Loïc J. Blum (pp. 107-115).
The chemiluminescence of luminol in the presence of H2O2 has been exploited to develop fiberoptic biosensors associated with flow injection analysis systems. A chlorophenol sensor was developed based on the ability of certain halophenols to enhance the peroxidase-catalyzed luminol chemiluminescence. Horseradish peroxidase immobilized on a collagen membrane was used. Ten chlorophenols have been tested with this chemiluminescent-based sensor. The lower detection limit was obtained with 4-chloro-3-methylphenol and was equal to 0.01 µM. Electrochemiluminescent-based fiberoptic biosensors for glucose and lactate were also developed using glucose oxidase or lactate oxidase immobilized on polyamide membranes. In the presence of oxidase-generated H2O2, the light emission was triggered electrochemically by means of a glassy carbon electrode polarized at +425 mV vs a platinum pseudo-reference electrode. The detection limits for glucose and lactate were 150 and 60 pmol, respectively, and the dynamic ranges were linear from 150 pmol to 600 nmol and from 60 pmol to 60 nmol, respectively.
Keywords: Biosensors; chemiluminescence; chlorophenols; electrochemiluminescence; fiberoptic sensor; flow injection analysis; glucose; lactate; luminol; peroxidase
Development of a chemiluminescent optical fiber immunosensor to detect Streptococcus pneumoniae antipolysaccharide antibodies by Robert S. Marks; Alon Margalit; Alexei Bychenko; Efim Bassis; Nurith Porat; Ron Dagan (pp. 117-126).
A chemiluminescent-based optical fiber immunosensor was developed for the detection of antipneumococcal antibodies. This was accomplished by developing a different chemical procedure utilizing 3-aminopropyl trimethoxysilane and cyanuric chloride to conjugate pneumococcal cell wall polysaccharides to the optical fiber tips, and by improving the sensitivity of the photodetection system. The lowest titer of antipneumococcal antibodies detected by the optical fiber was at a 1:819,200 dilution. The lowest corresponding value by standard enzyme-linked immunosorbent assay was at a 1:98,415 dilution. It was concluded that the optical immunosensor system is an accurate and sensitive method to detect antipneumococcal antibodies and may be an adequate tool to monitor antibodies in specimens such as saliva and urine.
Keywords: Optical fiber; chemiluminescence; immunosensor; Streptococcus pneumoniae
Biosensors based on immobilization of biomolecules by electrogenerated polymer films by Serge Cosnier (pp. 127-138).
The concept and potentialities of electrochemical procedures of biomolecule immobilization are described. The entrapment of biomolecules within electropolymerized films consists of the application of an appropriate potential to an electrode soaked in an aqueous solution containing monomer and biomolecules. This method of biosensor construction is compared with a two-step procedure based on the adsorption of an aqueous amphiphilic pyrrole monomer-biomolecule mixture on an electrode followed by the electropolymerization of the adsorbed monomers. Another approach is based on the electrogeneration of polymer films functionalized by specific groups allowing subsequently the attachment of biomolecules. The immobilization of biomolecules on these films by covalent binding or noncovalent interactions is described.
Keywords: Avidin; biosensor; biotin; enzyme; functionalized polymer; polypyrrole
Immunodetection by quartz crystal microbalance by K. Bizet; C. Gabrielli; H. Perrot (pp. 139-149).
Biodetection is one of the most important challenges for the twenty-first century: many fields are concerned, mainly environmental and medical. The quartz crystal microbalance (QCM) may offer great possibilities for this purpose: a direct response signal, which characterizes the binding event between a sensitive layer, immobilized onto the surface transducer, and the analyte to be detected, can be obtained. However, for the detection of small biomolecules such as antigens, it is quite difficult to obtain an observable signal that corresponds directly to the binding event. In general, this is owing to the lack of mass sensitivity of the commonly used QCM, with 5-to 10-MHz quartz crystals. For improving this mass sensitivity, a 27-MHz quartz resonator was developed and incorporated in a flow-through microcell. Two biospecies, IgG rabbit and peroxidase enzyme, were studied with this ultrasensitive QCM in terms of specificity, detection limit, and calibration curve.
Keywords: Quartz crystal microbalance; peroxidase; rabbit IgG; immunodetection; 27-MHz resonators
Microbial sensors of ultraviolet radiation based on recA’::lux fusions by Rachel Rosen; Yaakov Davidov; Robert A. LaRossa; Shimshon Belkin (pp. 151-160).
Escherichia coli strains containing plasmid-borne fusions of the recA promoter-operator region to the Vibrio fischeri lux genes were previously shown to increase their luminescence in the presence of DNA damage hazards, and thus to be useful for genotoxicant detection. The present study expands previous work by demonstrating and investigating the luminescent response of these strains to ultraviolet radiation. Several genetic variants of the basic recA’::lux design were examined, including a tolC modification of membrane efflux capacity, a chromosomal integration of the recA’::lux fusion, a different lux reporter (Photorhabdus luminescens instead of V. fischeri, allowing the assay to be run at 37°C), and a different host bacterium (Salmonella typhimurium instead of E. coli). Generally, two modifications provided the fastest responses: the use of the S. typhimurium host or the P. luminescens lux reporter. Highest sensitivity, however, was demonstrated in an E. coli strain in which a single copy of the V. fischeri lux fusion was integrated into the bacterial chromosome.
Keywords: Bioluminescence; Escherichia coli ; DNA damage; genotoxicity; microbial biosensors; Photorhabdus luminescens ; Salmonella typhimurium ; SOS response; UV irradiation; Vibrio fischeri
Sensitive immunodetection through impedance measurements onto gold functionalized electrodes by Sami Ameur; Claude Martelet; Nicole Jaffrezic-Renault; Jean-Marc Chovelon (pp. 161-170).
This article deals with a direct electrochemical method of detecting antigens using new methods of functionalization of gold electrodes. Based on the reacting ability of gold with sulfhydryl groups, three protocols for the fixation of antibodies have been explored. They are based on either the self-assembling properties of functional thiols bearing long alkyl chains or the possibility of a direct coupling of antibody moieties. Coverage rates as high as 97% can be reached. The analysis of the electrochemical impedance behavior of such layers can lead to a sensitive method for the direct detection of the antibody/antigen interaction. The addition of a redox couple in the tested solution, acting as an amplifier, allowed detection limits for the antigens as low as a few picograms/milliliter to be reached.
Keywords: Biosensors; electrochemical impedance; modified gold electrodes; functional thiol
Biosensor for determination of glucose and sucrose in fruit juices by flow injection analysis by Yann Guémas; Mohamed Boujtita; Nabil El Murr (pp. 171-181).
Glucose and sucrose were measured with an amperometric method by using the flow injection analysis technique. A carbon paste electrode with a renewable surface containing glucose oxidase, horseradish peroxidase, and ferrocene was used in combination with the soluble enzymes invertase and mutarotase. The effect of invertase, mutarotase, and ascorbic acid on the electrode response was examined. Glucose and sucrose concentrations were determined with <3% errors. The proposed method for glucose and sucrose measurements was validated in real samples of fruitjuices. The results were also compared with those obtained with the ultraviolet method.
Keywords: Biosensors; food analysis; glucose; sucrose; carbon paste electrode
Electropolymerization as a versatile route for immobilizing biological species onto surfaces by Gérard Bidan; Martial Billon; Katia Galasso; Thierry Livache; Gérard Mathis; André Roget; Luz Maria Torres-Rodriguez; Eric Vieil (pp. 183-193).
Biosensors based on electronic conducting polymers appear particularly well suited to the requirements of modern biological analysis—multiparametric assays, high information density, and miniaturization. We describe a new methodology for the preparation of addressed DNA matrices. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5′ end a pyrrole moiety. The resulting polymer film deposited on the addressed electrode consists of pyrrole chains bearing covalently linked oligonucleotides (ODN). An oligonucleotide array was constructed on a silicon device bearing a matrix of 48 addressable 50 × 50 µm gold microelectrodes. This technology was successfully applied to the genotyping of hepatitis C virus in blood samples. Fluorescence detection results show good sensitivity and a high degree of spatial resolution. In addition, gravimetric studies carried out by the quartz crystal microbalance technique provide quantitative data on the amount of surface-immobilized species. In the case of ODN, it allows discrimination between hybridization and nonspecific adsorption. The need for versatile processes for the immobilization of biological species on surfaces led us to extend our methodology. A biotinylated surface was obtained by coelectropolymerization of pyrrole and biotin-pyrrole monomers. The efficiency for recognition (and consequently immobilization) of R-phycoerythrin-avidin was demonstrated by fluorescence detection. Copolymerization of decreasing ratios of pyrrole-biotin over pyrrole allowed us to obtain a decreasing scale of fluorescence.
Keywords: Electrochemical polymerization; functionalized polypyrrole; DNA chip; biosensors; genotyping; biotin
Comparison between electrochemical and optoelectrochemical impedance measurements for detection of DNA hybridization by E. Souteyrand; C. Chen; J. P. Cloarec; X. Nesme; P. Simonet; I. Navarro; J. R. Martin (pp. 195-207).
The principles of the electrochemical and optoelectrochemical impedance measurements on bare electrolyte/dielectric/semiconductor structures are described. The analysis of the experimental curves allows access to several indications concerning the electrical behavior of such structures. The application of these techniques to follow the electrical behavior of structures modified with two biological systems was investigated. The antibody/antigen recognition did not change the surface charge and, therefore, did not affect the impedance curves with respect to the applied potential. By contrast, the hybridization of two complementary DNA strands on the surface of the structure induced a variation of flat band potential of the semiconductor leading to a shift of impedance curves along the potential axis. This means that it is possible to detect directly the DNA hybridization without the use of labeled probes. The use of light allows the surface to be probed locally. In the future, the application of this technique for direct detection of hybridization on DNA chips should be possible.
Keywords: DNA; hybridization; biosensor; electrochemical impedances; optoelectrochemical impedances
Sol-gel-derived prussian blue-silicate amperometric glucose biosensor by Subramanian Bharathi; Ovadia Lev (pp. 209-216).
A new type of inorganic biosensor is introduced. The sensor comprises glucose oxidase enzymes encapsulated in a sol-gel-derived Prussian blue-silicate hybrid network. Glucose is detected by the biocatalytic reduction of oxygen followed by catalytic reduction of hydrogen peroxide by the Prusian blue catalyst. The sol-gel silicate entails a rigid encapsulating matrix, the Prussian blue provides chemical catalysis and charge mediation from the reduction site to the supporting electrode, and the enzyme is responsible for the biocatalysis. The feasibility of a dual optical/electrochemical mode of analysis is also demonstrated.
Keywords: Sol-gel; biosensor; Prussian blue; modified electrodes
Amperometric biosensors based on microflow injection system by Inna Eshkenazi; Vered Sacks; Tova Neufeld; Judith Rishpon (pp. 217-230).
Novel electrochemical cells based on a microflow system combined with amperometric enzyme electrodes were developed and served for quantitative determination of various compounds, such as organophosphates and lactose. The resulting biosensors are selective and efficient owing to immobilization of the sensing elements on the electrodes. The sensors are easy to operate, and the procedures are rapid, accurate, reproducible, and inexpensive, requiring neither special skills and training nor complicated instrumentation. The use of a microflow cell ensures the continuous flux of a new substrate, thus preventing the accumulation or adsorption of products to the electrode. Miniaturization of the sensor has two main advantages: (1) it is easy to carry and therefore can be used outdoors as well, and (2) it allows working with low volumes of compounds and reagents, which is highly important when dealing with hazardous compounds.
Keywords: Microflow; amperometric biosensor; injection; electrode
Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals by V. Papper; N. Medvedeva; I. Fishov; G. I. Likhtenshtein (pp. 231-248).
We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the “triple” cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer’s triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10−12 mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10−5 M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10−3 µL or less.
Keywords: Stilbene; Erythrosin B; nitroxide radical; liposome membrane; triplet-triplet energy transfer; sensitized cis-trans photoisomerization; quenching of triplet state; cascade reaction