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PubMed2004

Development of biosensors with aptamers as bio-recognition element: the case of HIV-1 Tat protein.

The in vitro selection of combinatorial libraries of RNA/DNA, has allowed the identification of specific nucleic acids (aptamers) which bind to a wide range of target molecules with high affinity and specificity. In this work, an RNA aptamer, specific for the protein trans-activator of transcription (Tat) of HIV-1, has been used as bio-recognition element to develop a biosensor (aptasensor). The biosensor was optimised using piezoelectric quartz-crystals as transducers and the aptamer was immobilised on the gold electrode of the crystal. The immobilisation procedure was based on the interaction between the biotinylated aptamer and streptavidin previously deposited on the electrode. The main analytical characteristics of the biosensor, such as sensitivity, selectivity and reproducibility, have been studied in details. An optimised regeneration procedure allowed the multiple use of the aptamer-coated crystal. The aptasensor has been compared with the corresponding immunosensor, based on the specific monoclonal anti-Tat antibody. The antibody was immobilised on a layer of carboxylated dextran previously deposited on the gold electrode. The results demonstrated that the use of a biosensor with a specific aptamer as bio-recognition element could be an interesting approach in the detection of proteins, which has been here examined considering a model system.

Biosensing Techniques↗

PubMed2005

Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor.

The steady rise of observations of harmful or toxic algal blooms throughout the world in the past decades constitute a menace for coastal ecosystems and human interests. As a consequence, a number of programs have been launched to monitor the occurrence of harmful and toxic algae. However, the identification is currently done by microscopic examination, which requires a broad taxonomic knowledge, expensive equipment and is very time consuming. In order to facilitate the identification of toxic algae, an inexpensive and easy-to-handle DNA-biosensor has been adapted for the electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii. The detection of the toxic algae is based on a sandwich hybridisation, which is carried out on a disposable sensor chip. A set of two probes for the species-specific identification of A. ostenfeldii was developed. The specificity of the probes could be shown in dot-blot hybridisations and with the DNA-biosensor. The sensitivity of the DNA-biosensor was optimised with respect to hybridisation temperature and NaCl-concentration and a significant increase of the sensitivity of the DNA-biosensor could be obtained by a fragmentation of the rRNA prior to the hybridisation and by adding a helper oligonucleotide, which binds in close proximity to the probes to the hybridisation.

Animals↗

PubMed2004

Design of an amperometric biosensor using polypyrrole-microgel composites containing glucose oxidase.

A new material consisting of a water-dispersed complex of polypyrrole-polystyrensulfonate (PPy) embedded in polyacrylamide (PA) has been prepared and tested as enzyme immobilizing system for its use in amperometric biosensors. Glucose oxidase (GOx) and the water-dispersed polypyrrole complex were entrapped within polyacrylamide microgels by polymerization of acrylamide in the dispersed phase of concentrated emulsions containing GOx and PPy. Polymerization of the dispersed phase provides microparticles whose size lies between 3.5 and 7 microm. The aim of incorporating polypyrrole into the polyacrylamide microparticles was to facilitate the direct transfer of the electrons released in the enzymatic reaction from the catalytic site to the platinum electrode surface. The conductivity of the microparticles was measured by a four-point probe method and confirmed by the successful anaerobic detection of glucose by the biosensor. Thus, the polyacrylamide-polypyrrole (PAPPy) microparticles combine the conductivity of polypyrrole and the pore size control of polyacrylamide. The effects of the polyacrylamide-polypyrrole ratio and cross-linking on the biosensor response have been investigated, as well as the influence of analytical parameters such as pH and enzymatic loading. The PAPPy biosensor is free of interferences arising from ascorbic and uric acids, which allows its use for quantitative analysis in human blood serum.

Acrylic Resins↗

PubMed2005

Cell-based biosensors based on light-addressable potentiometric sensors for single cell monitoring.

Cell-based biosensors incorporate cells as sensing elements that convert changes in immediate environment to signals for processing. This paper reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible cell-base biosensor that will enable us to monitor extracellular action potential of single living cell under stimulant. In order to modify chip surface and immobilize cells, we coat a layer of poly-L-ornithine and laminin on surface of LAPS chip on which rat cortical cells are grown well. When 10 microg/ml acetylcholine solution is administrated, the light pointer is focused on a single neuronal cell and the extracellular action potential of the targeted cell is recorded with cell-based biosensor based on LAPS. The results demonstrate that this kind of biosensor has potential to monitor electrophysiology of living cell non-invasive for a long term, and to evaluate drugs primarily.

Acetylcholine↗

PubMed2005

Determination of phenolic compounds by a polyphenol oxidase amperometric biosensor and artificial neural network analysis.

The determination of phenolic compounds is significant given its toxicity, even at very low concentration levels. Amperometric determination of phenols is a simple technique available. Direct oxidation of phenols can be used, but another possibility is the use of polyphenol oxidase (tyrosinase) enzyme biosensors that oxidises the phenolic compounds into their corresponding quinones. Reduction of the resulting quinones accomplishes the amplification of the amperometric signal, as long as the result of the reduction process is the corresponding cathecol, this being able to be oxidised again by the polyphenol oxidase immobilized on the surface of the biosensor. In this communication, simultaneous determination of different phenols was carried out combining biosensor measurements with chemometric tools, in what is known as electronic tongue. The departure information used was the overlapped reduction voltammogram generated with the amperometric biosensor based on polyphenol oxidase. Artificial Neural Networks (ANN) were used for extraction and quantification of each compound. Phenol, cathecol and m-cresol formed the three-analyte study case resolved in this work. Good prediction ability was attained, and so, the separate quantification of these three phenols was accomplished.

Algorithms↗

PubMed2005

Quantitative analysis of the response of an electrochemical biosensor for progesterone in milk.

An electrochemical biosensor for progesterone in cow's milk was developed and used in a competitive immunoassay by Hart et al. (1977, Studies towards a disposable screenprinted amperometric biosensor for progesterone, Biosens. Bioelectron. 12, 1113-1121). The sensor was fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs) which were coated with rabbit anti-sheep IgG (rIgG). This sensor was operated following the steps of competitive binding between sample and conjugate (alkaline-phosphatase-labelled progesterone) for the immobilised mAb sites and measurements of an amperometric signal in the presence of p-nitrophenylphosphate using either colorimetric assays or cyclic voltammetry. The hook effect of the progesterone biosensor was found in the concentration range of milk progesterone between 0 and 5 ng/ml when the sensor was fabricated using a loading of 25 ng rIgG per electrode of a diameter of 3 mm and a 1/50 dilution of mAb. A computer model has been developed in this study to simulate the operation of this progesterone biosensor with consideration of the fabrication processes. This paper presents the results of validating the computer model and the model has predicted the hook effect as observed in tests. The model thus reveals that the hook effect is determined by the total number of binding sites available and the rates of labelled and unlabelled progesterone diffusing towards the sensor surface and the binding rates.

Animals↗

PubMed2004

Fluorescence detection of enzymatic activity within a liposome based nano-biosensor.

The encapsulation of enzymes in microenvironments and especially in liposomes, has proven to greatly improve enzyme stabilization against unfolding, denaturation and dilution effects. Combining this stabilization effect, with the fact that liposomes are optically translucent, we have designed nano-sized spherical biosensors. In this work liposome-based biosensors are prepared by encapsulating the enzyme acetylcholinesterase (AChE) in L-a phosphatidylcholine liposomes resulting in spherical optical biosensors with an average diameter of 300+/-4 nm. Porins are embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The enzyme activity within the liposome is monitored using pyranine, a fluorescent pH indicator. The response of the liposome biosensor to the substrate acetylthiocholine chloride is relatively fast and reproducible, while the system is stable as has been shown by immobilization within sol-gel.

Acetylcholinesterase↗

PubMed2004

Semi disposable reactor biosensors for detecting carbamate pesticides in water.

Two flow-injection biosensor systems using semi disposable enzyme reactor have been developed to determine carbamate pesticides in water samples. Acetylcholinesterase was immobilized on silica gel by covalent binding. pH and conductivity electrodes were used to detect the ionic change of the sample solution due to hydrolysis of acetylcholine. Carbamate pesticides inhibited acetylcholinesterase and the decrease in the enzyme activity was used to determine these pesticides. Parameters influencing the performance of the systems were optimized to be used in the inhibition procedure. Carbofuran and carbaryl were used to test these systems. Detection limits for the potentiometric and conductimetric systems were both at 10% inhibition corresponding to 0.02 and 0.3 ppm of carbofuran and carbaryl, respectively. Both systems also provided the same linear ranges, 0.02-8.0 ppm for carbofuran, and 0.3-10 ppm for carbaryl. The analysis of pesticides was done a few times before the reactor was disposed. Percentages of inhibition obtained from different reactors were reproducible, therefore, no recalibration was necessary when changing the reactor. The biosensors were used to analyze carbaryl in water samples from six wells in a vegetable growing area. Both systems could detect the presence of carbaryl in the samples and provided good recoveries of the added carbaryl, i.e., 80-106% for the potentiometric system and 75-105% for the conductimetric system. The presence of carbaryl in water samples analyzed by the biosensors was confirmed by gas chromatography-mass spectrometric system. These biosensors do not require any sample preconcentration and are suitable for detecting pesticides in real water samples.

Acetylcholinesterase↗

PubMed2005

Sensor and biosensor preparation, optimisation and applications of Prussian Blue modified electrodes.

Being one of the most commonly used electrochemical mediators for analytical applications, Prussian Blue has found a wide use in the biosensor field during the last years. Its particular characteristic of catalysing hydrogen peroxide reduction has been applied in the construction of a large number of oxidase enzyme-based biosensors for clinical, environmental and food analysis. By modifying an electrode surface with Prussian Blue, it is in fact possible to easily detect hydrogen peroxide at an applied potential around 0.0 V versus Ag/AgCl, thus making possible coupling with oxidase enzymes while also avoiding or reducing electrochemical interferences. Papers dealing with glucose, lactate, cholesterol and galactose biosensors that are based on the use of Prussian Blue have recently appeared in the most important analytical chemistry journals. Another recent trend is the use of a choline probe based on choline oxidase for pesticide determination to exploit the inhibition of acetylcholinesterase by these compounds. In addition, the use of Prussian Blue in the development of biosensors for food analysis has captured the interest of many research groups and led to improved methods for the detection of glutamate, galactose, alcohol, fructosyl amine, formate, lysine and oxalate. This review will focus on the biosensing aspects of Prussian Blue-based sensors giving a general overview of the advantages provided by such mediator as well as its drawbacks. A comprehensive bibliographic reference list is presented together with the most up to date research findings in this field and possible future applications. The commercial potential of sensors based on this mediator will also be discussed.

Biosensing Techniques↗

PubMed2005

A carbon fiber microelectrode-based third-generation biosensor for superoxide anion.

Implantable and miniature carbon fiber microelectrode (CFME)-based third-generation biosensor for superoxide anion (O(2)(-)) was fabricated for the first time. The CFME-based biosensor was constructed by electro-deposition of Au nanoparticles on the CFMEs and then modification of the Au nanoparticles by cysteine followed by immobilization of superoxide dismutase (SOD) on the electrodes. The direct electrochemistry of the SOD immobilized on the CFME-based electrodes was efficiently realized by electron transfer promoter - cysteine molecules confined on the Au nanoparticles deposited on the CFMEs. The CFME-based biosensors were demonstrated to possess striking analytical properties for O(2)(-) determination, such as optional operation potentials, high selectivity and sensitivity as well as good stability. Along with the implantable capacity inherent in the CFMEs, these striking analytical properties of the CFME-based biosensors substantially make them potential for in vivo determination of O(2)(-).

Adsorption↗

PubMed2005

A novel, disposable, screen-printed amperometric biosensor for glucose in serum fabricated using a water-based carbon ink.

Screen-printed amperometric glucose biosensors have been fabricated using a water-based carbon ink. The enzyme glucose oxidase (GOD) and the electro-catalyst cobalt phthalocyanine were mixed with the carbon ink prior to the screen-printing process; therefore, biosensors are prepared in a one-step fabrication procedure. Optimisation of the biosensor performance was achieved by studying the effects of pH, buffer strength, and applied potential on the analytical response. Calibration studies were performed under optimum conditions, using amperometry in stirred solution, with an operating potential of +500 mV versus SCE. The sensitivity was found to be 1170 nA mM(-1), with a linear range of 0.025-2 mM; the former represents the detection limit. The disposable amperometric biosensor was evaluated by carrying out replicate determinations on a sample of bovine serum. This was achieved by the method of multiple standard additions and included a correction for background currents arising from oxidizable serum components. The mean serum concentration was calculated to be 8.63 mM and compared well with the supplier's value of 8.3 mM; the coefficient of variation was calculated to be 3.3% (n=6).

Biosensing Techniques↗

PubMed2006

Platinum nanoparticles-doped sol-gel/carbon nanotubes composite electrochemical sensors and biosensors.

Platinum nanoparticle-doped sol-gel solution is prepared and used as a binder for multi-walled carbon nanotubes (CNT) for the fabrication of electrochemical sensors. Amine group containing sol-gel solution is selected to utilize the affinity of -NH(2) groups toward metal nanoparticles for stabilization the nanoparticles in solution. The resulting CNT-silicate material brings new capabilities for electrochemical devices by using the synergistic action of the electrocatalytic activity of Pt nanoparticles and CNT. The combined electrocatalytic activity permits low-potential detection of hydrogen peroxide with remarkably improved sensitivity. With the incorporation of glucose oxidase within the Pt-CNT-silicate matrix, a Pt-CNT paste-based biosensor has been constructed that responds more sensitively to glucose than CNT-based biosensor. The influences of the composite of the sol-gel solution, the quantity of the solution and the Pt nanoparticles loading are examined. In pH 6.98 phosphate buffer, almost interference free determination of glucose is realized at 0.1 V versus SCE with a linear range from 1 to 25 mM, a response time <15s, and the sensitivity is 0.98 microA mM(-1)cm(-2). The sensitivity of the Pt-CNT paste-based biosensor is almost four times larger than that of the CNT-based biosensor (0.27 microA mM(-1)cm(-2) at 0.1 V). The improved electrocatalytic activity and surface renewability made the Pt-CNT-silicate system a potential platform to immobilize different enzymes for other bioelectrochemical applications.

Adsorption↗

PubMed2005

A fluorescent glucose biosensor based on immobilized glucose oxidase on bamboo inner shell membrane.

A fluorescent glucose biosensor was constructed by immobilizing glucose oxidase on a bamboo inner shell membrane with glutaraldehyde as a cross-linker. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution with a concomitant increase in the fluorescence intensity of an oxygen transducer, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(Pi) ditetrakis(4-chlorophenyl)borate. The enzyme immobilization, effect of pH, temperature and ionic strength have been studied in detail. The biosensor exhibited repeatable response to a 2.0 mM glucose solution with a relative standard deviation of 3.0% (n = 10). It showed good storage stability and maintained 95% of its initial response after it had been kept at 4 degrees C for 8 months. The biosensor has a linear response range of 0.0-0.6 mM glucose with a detection limit of 58 microM (S/N = 3). Common potential interferants in samples do not pose any significant interference on the response of the glucose biosensor. It was successfully applied to the determination of glucose content in some commercial wines and medical glucose injections.

Biosensing Techniques↗

PubMed2005

Glucose biosensor based on Au nanoparticles-conductive polyaniline nanocomposite.

In this paper, we report a novel glucose biosensor based on composite of Au nanoparticles (NPs)-conductive polyaniline (PANI) nanofibers. Immobilized with glucose oxidase (GOx) and Nafion on the surface of nanocomposite, a sensitive and selective biosensor for glucose was successfully developed by electrochemical oxidation of H2O2. The glucose biosensor shows a linear calibration curve over the range from 1.0x10(-6) to 8.0x10(-4) mol/L, with a slope and detection limit (S/N=3) of 2.3 mA/M and 5.0x10(-7) M, respectively. In addition, the glucose biosensor system indicates excellent reproducibility (less than 5% R.S.D.) and good operational stability (over 2 weeks).

Aniline Compounds↗

PubMed2005

A high sensitivity amperometric biosensor using laccase as biorecognition element.

An amperometric flow biosensor, using laccase from Rigidoporus lignosus as bioelement was developed. The laccase was kinetically characterized towards various phenolics both in solution and immobilized to a hydrophilic matrix by carbodiimide chemistry. A bioreactor connected to an amperometric flow cell by a FIA system was filled with the immobilized enzyme and the operational conditions of this biosensor were optimized as regards pH. Under the adopted experimental conditions, the immobilized enzyme oxidizes all the substrate molecules avoiding the need of cumbersome calibration procedures. The biosensor sensitivity, which was found to be 100 nA/microM for some of the tested substrates, resulted to be constant for more than 100 working days. This biosensor permits the detection of phenolics in aqueous solutions at concentrations in the nanomolar range and was successfully used to detect phenolics in wastewaters from olive oil mill without sample preparation.

Biosensing Techniques↗

PubMed2005

Strategies to develop malic acid biosensors based on malate quinone oxidoreductase (MQO).

An amperometric biosensor based on malate quinone oxidoreductase (MQO) was developed for monitoring of the malolactic fermentation of wines. Screen-printed electrodes coupled with appropriate mediators were used as transducers for this novel biosensor. MQO was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer (PVA-SbQ) on the surface of the working electrode. Several electrochemical mediators were studied in order to lower the applied potential and minimise the matrix effects. Among them, 2,6-dichlorophenol indophenol (DPIP) and phenazine methosulfate (PMS) were chosen for further development. The working conditions (mediator concentration, applied potential and pH) were optimised for both DPIP and PMS. Detection limits for both types of biosensors were of 5 microM malic acid. Sensitivities obtained for the linear part of the calibration curve were 0.85 and 1.7 mA/M for the biosensors based on DPIP and PMS, respectively. Interferences due to non-specific oxidations were shown to be negligible when using PMS as mediator.

Biosensing Techniques↗

PubMed2005

Epinephrine quantification in pharmaceutical formulations utilizing plant tissue biosensors.

A plant tissue biosensor associated with flow injection analysis is proposed to determine epinephrine in pharmaceutical samples. The polyphenol oxidase enzymes present in the fibers of a palm tree fruits (Livistona chinensis), catalyses the oxidation of epinephrine to epinephrinequinone as a primary product. This product is then electrochemically reduced (at -0.10 V versus Ag/AgClsat) on the biosensor surface and the resulting current is used for the quantification of epinephrine. The biosensor provides a linear response for epinephrine in the concentration range from 5.0 x 10(-5) to 3.5 x 10(-4) mol l(-1). The limit of detection estimated for this interval was 1.5 x 10(-5) mol l(-1) and the correlation coefficient of 0.998, working under a flow rate of 2.0 ml min(-1) and using a sample loop of 100 microl. The repeatability (R.S.D. for 10 consecutive determinations of a 3.0 x 10(-4) mol l(-1) epinephrine solution) was 3.1%. The results obtained by the method here proposed were compared with the official UV spectrophotometric procedure and also using a plant tissue reactor. The responses obtained with the proposed strategies were in good agreement with both ways of analyses, whereas the values obtained by the official spectrophotometric method was strongly affected by benzoic acid, present in the formulation of pharmaceutical product utilized for inhalation. Such favorable results obtained with the carbon paste biosensor or utilizing the bioreactor, joined with the simplicity of its preparation turns these procedures very attractive for epinephrine quantification in pharmaceutical products.

Arecaceae↗

PubMed2006

Electrochemical microfluidic biosensor for nucleic acid detection with integrated minipotentiostat.

An electrochemical microfluidic biosensor with an integrated minipotentiostat for the quantification of RNA was developed based on nucleic acid hybridization and liposome signal amplification. Specificity of the biosensor was ensured by short DNA probes that hybridize with the target RNA or DNA sequence. The reporter probe was coupled to liposomes entrapping the electrochemically active redox couple potassium ferri/ferrohexacyanide. The capture probes were coupled to superparamagnetic beads that were isolated on a magnet in the biosensor. Upon capture, the liposomes were lysed to release the electrochemical markers that were detected on an interdigitated ultramicroelectrode array in the biosensor just downstream of the magnet. The current was measured, stored and displayed by miniaturized instrumentation (miniEC). The accuracy of the miniEC was evaluated by comparing its performance to a standard bench-top electrochemical workstation in static and dynamic DC amperometric experiments. In both sets of experiments, the inexpensive miniEC performance was comparable in signal strength to that of the electrochemical workstation. In fact, the miniEC achieved a detection limit of 0.01 microM combined ferri/ferrohexacyanide concentration which was 10 x lower than that of the standard lab-bench system. The response time of the miniEC system was the same for low concentrations taking about 10 s to steady state. It was, however, slower at higher concentrations, taking 5 s versus only 1 s for the bench-top system. Finally, the functionality of the miniEC was successfully demonstrated with the detection of Dengue virus RNA.

Biosensing Techniques↗