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At least 343 records · Page 19Linked to original sources
PubMed2001

Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports: routes for enzyme sensors, immunosensors and DNA sensors.

The biocatalyzed precipitation of an insoluble product produced on electrode supports is used as an amplification path for biosensing. Enzyme-based electrodes, immunosensors and DNA sensors are developed using this biocatalytic precipitation route. Faradaic impedance spectroscopy and chronopotentiometry are used as transduction methods to follow the precipitation processes. While Faradaic impedance spectroscopy leads to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provides the total resistance at the interfaces of the modified electrodes. A horseradish peroxidase, HRP, monolayer-functionalized electrode is used to sense H(2)O(2) by the biocatalyzed oxidation of 4-chloro-1-naphthol (1), to the insoluble product benzo-4-chlorohexadienone (2). An antigen monolayer electrode is used to sense the dinitrophenyl antibody, DNP-Ab, applying an anti-antibody-HRP conjugate as a biocatalyst for the oxidative precipitation of 1 by H(2)O(2) to yield the insoluble product 2. An oligonucleotide (3) functionalized monolayer electrode is used to sense the DNA-analyte (4), that is one of the Tay-Sachs genetic disorder mutants. Association of a biotin-labeled oligonucleotide to the sensing interface, followed by the association of the avidin-HRP conjugate and the biocatalyzed precipitation of 2 leads to the amplified sensing of 4. The amount of the precipitate accumulated on the conductive support is controlled by the concentration of the respective analytes and the time intervals employed for the biocatalytic precipitation of 2. The electron-transfer resistances of the electrodes covered by the insoluble product (2) are derived from Faradaic impedance measurements, whereas the total electrode resistances are extracted from chronopotentiometric experiments. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports are found. Chronopotentiometry is suggested as a rapid transduction means (a few seconds). The precautions needed to apply chronopotentiometry in biosensors are discussed.

Animals↗
PubMed2002

A novel ISFET-type biosensor based on P450 monooxygenases.

We made a biosensor based on ion-sensitive field effect transistor (ISFET) using P450 monooxygenase. ISFETs are electrical devices and have been used as pH sensors. We used genetically engineered P450 monooxygenase for our research because of its high enzymatic activity. The fusion enzyme between rat CYP1A1P450 monooxygenase and yeast NADPH-cytochrome P450 oxidoreductase was expressed in yeast Saccharomyces cerevisiae strain AH22. Yeast microsomal membranes were immobilized in an agarose layer on the ISFET. o-Deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin was catalyzed by the enzyme in the presence of nicotinamide adenine dinucleotide phosphate reduced form (NADPH). Formation of 7-hydroxycoumarin from 7-ethoxycoumarin was also measured by fluorescence. The difference of the voltage between the ISFET device and control device without enzymes showed a voltage increase along with the enzymatic reaction of P450 monooxygenases, and this voltage increase in the device was inhibited by addition of MnCl(2), an inhibitor of P450 monooxygenase. There was a positive correlation between the voltage increase in the ISFET device and the fluorescence intensity. This is the first electrochemical biosensing using P450 monooxygenases immobilized on the ISFET, and is applicable to the sensing of chlorophenol compounds.

Biosensing Techniques↗
PubMed2002

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria.

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 (lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 (fabA::luxCDABE), TV1061 (grpE::luxCDABE), DPD2794 (recA::luxCDABE), and DPD2511 (katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.

Biosensing Techniques↗
PubMed2002

Bio-smart hydrogels: co-joined molecular recognition and signal transduction in biosensor fabrication and drug delivery.

Two classes of polymers that are currently receiving widespread attention in biosensor development are hydrogels and conducting electroactive polymers. The present study reports on the integration of these two materials to produce electroactive hydrogel composites that physically entrap enzymes within their matrices for biosensor construction and chemically stimulated controlled release. Enhanced biosensing capabilities of these membranes have been demonstrated in the fabrication of glucose, cholesterol and galactose amperometric biosensors. All biosensors displayed extended linear response ranges (10(-5)-10(-2) M), rapid response times (<60 s), retained storage stabilities of up to 1 year, and excellent screening of the physiological interferents ascorbic acid, uric acid, and acetaminophen. When the cross-linked hydrogel components of these composite membranes were prepared with the amine containing dimethylaminoethyl methacrylate monomer the result was polymeric devices that swelled in response to pH changes (neutral to acidic). Entrapment of glucose oxidase within these materials made them glucose-responsive through the formation of gluconic acid. When insulin was co-loaded with glucose oxidase into these "bio-smart" devices, there was a twofold increase in insulin release rate when the devices were immersed in glucose solutions. This demonstrates the potential of such systems to function as a chemically-synthesized artificial pancreas.

Biocompatible Materials↗
PubMed2002

Oxygen-independent poly(dimethylsiloxane)-based carbon-paste glucose biosensors.

Several silicone oils have been assessed and compared as an internal source of oxygen in connection to their use as binders for carbon-paste glucose biosensors. All four poly(dimethylsiloxane) (PDMS) oils tested a dramatic increase in the oxygen capacity of carbon-paste enzyme electrodes to allow convenient biosensing under severe oxygen-deficit conditions. The resulting oxygen independence is better than that exerted by perfluorocarbon binders or that displayed by mediator-based bioelectrodes. The resistance to oxygen effects is indicated from the identical response (observed in the presence and absence of oxygen) up to 2 x 10(-2) M glucose and the slight (12%) sensitivity loss at 4 x 10(-2) M. The influence of the viscosity of the PDMS binder upon the internal oxygen supply is examined. The PDMS carbon-paste enzyme electrode displays a stable glucose response over prolonged (15 h) operation in an oxygen-free solution. On-line continuous testing indicates favorable dynamic properties with no carry-over effects over the physiological and pathophysiological range (3-12 mM glucose).

Biosensing Techniques↗
PubMed2003

Application of atomic force microscopy and grating coupler for the characterization of biosensor surfaces.

Atomic force microscopy (AFM) and an optical grating coupler system were used to improve the understanding of the biosensing layer on a Ta(2)O(5)-light-guiding surface. Exemplary, we investigated the immobilization of the protein avidin, the subsequent binding of biotinylated oligonucleotides and hybridization of a complementary 12-mer. The AFM measurements revealed the height of approximately 1.6 nm for a single avidin molecule, while the thickness of the avidin layer on the biosensor surface seemed to be 2.8-3.0 nm. This result lead to the conclusion that the protein was not forming a simple monolayer. However, the thickness of the avidin layer could not be determined directly, but only after shifting of protein by the tip of the AFM leading to grooves of 1 micro m(2) and approximately 3 nm depth. As the height of oxide particles forming the waveguide surface was also in the range of 1.5 nm, the depth of these grooves could also be a result of the deposition of proteins on top of the oxide particles. This was consistent with the increased roughness of the surface after protein binding. Thus, investigations with the grating coupler were used to determine quantitatively the amount of immobilized avidin. On a biotinylated surface the amount of immobilized avidin lead to the assumption of a complete monolayer, whereas simple adsorption proved to be less efficient. A binding ratio of 1:1.3 for avidin and a biotinylated oligonucleotide was achieved. Up to 83% of the bound single strand were accessible for a subsequent hybridization reaction with a 12-mer. These results supported the model of avidin being deposited mainly on top of the oxide particles leading to the picture of a 'rough' complete protein monolayer, which was postulated from the AFM investigations.

Avidin↗
PubMed2003

Independently-addressable micron-sized biosensor elements.

With the continuing development of micro-total analysis systems and sensitive biosensing technologies, it is often desirable to immobilize biomolecules onto a surface in a small well-defined area. A novel method was developed to electrochemically attach DNA probes to micron-sized regions of a gold surface using biotin-LC-hydrazide (BH). Previously, we have found that the radical produced during the oxidation of BH will attach to a wide variety of electroactive surfaces. An array of micron-sized gold band electrodes (75 microm wide) was fabricated onto glass microscope slides and BH was deposited onto each electrode through the application of an oxidizing potential. Subsequent attachment of avidin to the biotinylated surface created the 'molecular sandwich' architecture necessary for further immobilization of biotinylated biomolecules to the surface. In this work, we utilized biotinylated DNA probes of varying sequence to illustrate the specificity of the attachment scheme. The immobilization of avidin, DNA probe, and hybridization of DNA target is visualized with fluorescence tags and the spatially selective attachment and hybridization of unique DNA sequences is demonstrated.

Avidin↗
PubMed2004

Pulse mode shear horizontal-surface acoustic wave (SH-SAW) system for liquid based sensing applications.

In this work, we describe a novel pulse mode shear horizontal-surface acoustic wave (SH-SAW) polymer coated biosensor that monitors rapid changes in both amplitude and phase. The SH-SAW sensors were fabricated on 36 degrees rotated Y-cut X propagating lithium tantalate (36 YX.LT). The sensitivity of the device to both mass loading and visco-elastic effects may be increased by using a thin guiding layer of cross-linked polymer. Two acoustic modes are excited by the electrodes in this crystalline direction. Metallisation of the propagation path of the 36 YX.LT devices allows the two modes to be discriminated. Successive polymer coatings resulted in the observation of resonant conditions in both modes as the layer thickness was increased. Using the 36 YX.LT devices, we have investigated the application of a novel pulse mode system by sensing a sequence of deposition and removal of a biological layer consisting of vesicles of the phospholipid POPC. A continuous wave system was used to verify the accuracy of the pulse mode system by sensing a series of poly(ethylene glycol) (PEG) solutions. The data clearly demonstrates the ability of the 36 YX.LT pulse mode system to provide rapid measurements of both amplitude and phase for biosensing applications.

Acoustics↗
PubMed1997

Free cholesterol fiber-optic biosensor for serum samples with simplex optimization.

An optical fiber biosensor for free cholesterol monitoring in serum samples is described. Silicone-entrapped tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) complex, the luminescence of which is sensitive to oxygen changes, is used as an optical transducer of the oxidation of cholesterol by cholesterol oxidase. The biocatalyst is entrapped in a graphite powder layer deposited onto the dyed silicone film. Optimization of some interdependent chemical variables which affect the performance of the biosensor has been achieved by application of a super-modified simplex method. The dynamic range of the biosensing membranes is found to be 0.15-3.0 mM of free cholesterol. Studies of the reproducibility, stability and interferences of the device, as well as the application of the sensor to measurements in serum samples, are reported. Simplex optimization has proven to be a very useful tool in the search for the optimal conditions for performing analyses with the optical fiber biosensor.

Biosensing Techniques↗
PubMed1999

Specific bio-recognition reactions observed with an integrated Mach-Zehnder interferometer.

The combination of an integrated Mach-Zehnder-interferometer (iMZI) at the bottom of a fluidic microchannel system with supramolecular interfacial binding layers optimized for biosensing purposes is described. The model system used is based on the highly specific interaction of streptavidin to its 'ligand' biotin: a single monolayer of a correspondingly derivatized silane-compound is formed by a self-assembly procedure on top of the channel rib guiding the light through the channels. Injection of a streptavidin solution which leads to the formation of a protein monolayer of d = 2.8 nm in effective thickness results in a phase shift of the sample light relative to the reference channel of delta phi = 6 pi, in good agreement with the theoretical sensitivity of delta phi/delta df = 2.9 pi/nm for a protein layer (n = 1.45) calculated for the device.

Biosensing Techniques↗
PubMed1999

A contactless surface acoustic wave biosensor.

Surface acoustic wave sensors operating in liquid generally cause problems resulting from wire bonding. The authors present an approach for a biosensor where the need for bonding wires is eliminated by utilizing inductive coupling of the sensor device to the RF circuitry. Protection of the electrodes from the liquid is achieved by coating the device surface with a SiO2 layer, resulting in a simplified handling of the devices. The first measurements with a sensor operating at 420 MHz are presented, demonstrating the potential of this operating principle for biosensing.

Acoustics↗
PubMed1999

The use of regenerable, affinity ligand-based surfaces for immunosensor applications.

The regeneration of antibody-binding surfaces is of major importance for re-usable sensor formats such as required for direct 'real-time' biosensing technologies and is often difficult to achieve. Antibodies commonly bind the antigen with high avidity and may themselves be sensitive to regeneration conditions. The interaction of polyclonal anti-chlorpyriphos antibody with an immobilised chlorpyriphos-ovalbumin (chlor-oval) conjugate and the interaction of soluble recombinant CD4 with covalently immobilised anti-CD4 IgG are presented in order to highlight these difficulties. Affinity-capture is suggested as an alternative format as it facilitates surface regeneration, directed immobilisation and the attainment of interaction progress curves that conform to the ideal pseudo-first-order kinetic interaction model. Protein A, protein G and polyclonal anti-mouse Fe-coated surfaces were used to observe the interaction of captured anti-GST monoclonal antibody with glutathione-s-transferase (GST). It was shown that a protein A affinity-capture surface produced ideal interaction progress curves while both protein G and polyclonal anti-mouse Fe resulted in systemic deviations.

Affinity Labels↗
PubMed2000

A binderless, bulk-modified, renewable surface amperometric sensor for NADH and ethanol.

Graphite particles are exfoliated and subsequently functionalized with toluidine blue. The resulting covalently modified graphite particles are restacked without any binder to form a surface-renewable, bulk-modified electrode. Electrocatalytic oxidation of NADH and its application in the amperometric biosensing of ethanol using alcohol dehydrogenase enzyme have been demonstrated with this material.

Alcohol Dehydrogenase↗
PubMed1993

Biomagnetic neurosensors.

In this report we demonstrate the first analytical application of biomagnetic field detection at nerve fibers for biosensing purposes. A ferrite core toroid surrounding the nerve, coupled to a low-noise, low-input-impedance amplifier, is used to inductively detect the compound action current (CAC) in crayfish giant axons upon stimulation of nerve firing. Detection of the local anesthetic lidocaine, which blocks neuronal conduction by binding in the ion channel of the voltage-gated sodium channel receptor, is achieved by monitoring the disappearance of the CAC. The application of this novel detection principle to the screening of neurotoxic and neuromodulatory drugs and natural product extracts is proposed.

Action Potentials↗
PubMed1989

Conductometric transducers for enzyme-based biosensors.

The use of alternating current conductometric transducers in biosensing devices has been investigated for urea and D-amino acid sensors using the enzyme systems urease and D-amino acid oxidase/catalase. Transducers with copper and platinum electrodes were constructed and characterized, and two enzyme immobilization methods were tested. Detection limits of 1 x 10(-6)M and linear ranges of 2 orders of magnitude were routinely achieved for these model sensors with enzymes covalently immobilized on collagen films.

Amino Acids↗
PubMed2001

Adaptation of a surface plasmon resonance biosensor with microfluidics for use with small sample volumes and long contact times.

The efficient delivery of sample to surface-immobilized sites is a key element in biosensing. For a surface plasmon resonance (SPR) biosensor, this has been addressed by constant flow through a microfluidic system with a sample injection loop (Sjölander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345). The present study describes an alternative mode of sample delivery without constant unidirectional flow. It was implemented on a commercial Biacore X SPR biosensor equipped with a microfluidic cartridge, but with the fluidic handling performed by an externally computer-controlled syringe pump. We demonstrate that sample volumes as low as 2 microL can be reproducibly positioned to cover the sensor surfaces, manipulated in a serial fashion, efficiently mixed by applying an oscillatory flow pattern, and fully recovered. Compared to the traditional continuous unidirectional flow configuration, we found very similar kinetic responses at high analyte concentrations and slightly slower responses at low concentrations, most likely due to depletion of analyte from the small sample volumes due to surface binding. With the antibody-antigen systems tested, binding parameters were obtained that are generally within 10% of those from conventional experiments. In the new configuration, biosensor experiments can be conducted without the usual constraints in the surface contact time that are correlated with sample volume and mass transport rate. This can translate to improved detection limits for slow reactions and can facilitate kinetic and thermodynamic binding studies.

Biosensing Techniques↗
PubMed2002

Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films.

A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (approximately 99%) and large enzyme activity (60 units cm(-2)). The latter translated into a high sensitivity (42 mA M(-1) cm(-2)) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t90% < or = 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 microM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability, e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.

Biosensing Techniques↗
PubMed2003

Analysis of protein adsorption and binding at biosensor polymer interfaces using X-ray photon spectroscopy and scanning electrochemical microscopy.

We describe a method, based on X-ray photoelectron spectroscopy (XPS) measurements, to assess the extent of protein adsorption or binding on a variety of different muTAS and biosensor interfaces. Underpinning this method is the labeling of protein molecules with either iodine- or bromine-containing motifs by using protocols previously developed for radiotracer studies. Using this method, we have examined the adsorption and binding properties of a variety of modified electrodeposited polymer interfaces as well as other materials used in muTAS device fabrication. Using polymer interfaces modified with poly(propylene glycol) (PPG) chains, our results indicate that a chain of at least approximately 30 monomer units is required to inhibit nonspecific adsorption from concentrated protein solutions. The XPS methodology was also used to probe specific binding of avidins and enzyme conjugates thereof to biotinylated and mixed biotin/PPG-modified polymer interfaces. In one example, using competitive binding, it was established that the mode of binding of a peroxidase-streptavidin conjugate to a biotinylated modified polymer interface was primarily via the streptavidin moiety (as opposed to nonspecific binding via the enzyme conjugate). XPS evaluation of nonspecific and specific peroxidase-streptavidin immobilization on various functionalized polymers has guided the design and fabrication of functionalized interdigitated electrodes in a biosensing muTAS device. Subsequent characterization of this device using scanning electrochemical microscopy (SECM) corroborated the adsorption and binding previously inferred from XPS measurements on macroscale electrodes.

Adsorption↗