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PubMed2003

A metal dispersed sol-gel biocomposite amperometric glucose biosensor.

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a copper dispersed sol-gel derived ceramic-graphite composite. The copper in the biocomposite offers excellent electrocatalytic activity towards the reduction (at -0.2 V) as well as oxidation (at +0.45 V) of hydrogen peroxide liberated in the enzymatic reaction enabling sensitive and selective determination of glucose. A linear response to glucose in the concentration range between 2.7 x 10(-5) to 4.0 x 10(-3) M with a correlation coefficient of 0.9987 and 4.0 x 10(-5) to 5.6 x 10(-3) M with a correlation coefficient of 0.9989 were observed with the electrocatalytic reduction and oxidation, respectively. Ascorbic acid and uric acid did not interfere with the glucose measurement during catalytic reduction at -0.2 V, a Nafion membrane was used to eliminate these interferences during the electrocatalytic oxidation at +0.45 V. The combination of copper catalysis and the promising feature of sol-gel biocomposite favor the sensitive and selective determination of glucose with improved analytical capabilities.

Biosensing Techniques↗

PubMed2003

New highly sensitive and selective catalytic DNA biosensors for metal ions.

While remarkable progress has been made in developing sensors for metal ions such as Ca(II) and Zn(II), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specific and strong metal binding. Since our knowledge about the construction of metal-binding sites in general is limited, searching for sensors in a combinatorial way is of significant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain catalytic DNA that can bind a metal ion of choice strongly and specifically. The metal ion selectivity of the catalytic DNA was further improved using a 'negative selection' strategy where catalytic DNA that are selective for competing metal ions are discarded in the in vitro selection processes. By labeling the resulting catalytic DNA with a fluorophore/quencher pair, we have made a new class of metal ion fluorescent sensors that are the first examples of catalytic DNA biosensors for metal ions. The sensors combine the high selectivity of catalytic DNA with the high sensitivity of fluorescent detection, and can be applied to the quantitative detection of metal ions over a wide concentration range and with high selectivity. The use of DNA sensors in detection and quantification of lead ions in environmental samples such as water from Lake Michigan has been demonstrated. DNA is stable, cost-effective, environmentally benign, and easily adaptable to optical fiber and microarray technology for device manufacture. Thus, the DNA sensors explained here hold great promise for on-site and real-time monitoring of metal ions in the fields of environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial process monitoring.

Biosensing Techniques↗

PubMed2003

An oxygen-rich fill-and-flow channel biosensor.

An oxygen-rich fill-and-flow channel biosensor has been developed for the measurement of glucose in wine. Glucose oxidase (GOD), immobilised in carbon paste (CP), was located in a well adjacent to a downstream detector electrode. When the analyte solution flows, hydrogen peroxide produced in the enzyme reaction is swept down to the detector electrode. Mineral oil and Kel-F oil (poly(chlorotrifluorethylene)) were used to prepare an enzyme layer of GOD within a CP. The hydrophobicity of the CP confined the reaction between the enzyme and its substrate to the surface of the enzyme layer. The oxidation current of hydrogen peroxide was sensitive to the enzyme loading but insensitive to mass transport variations such as flow rate. This response was, therefore, limited by the kinetics of the reaction between the enzyme and the substrate. For Kel-F oil, which can support a high concentration of dissolved oxygen, good reproducibility and greater dynamic range was obtained and the response did not decrease after degassing for 40 min with argon. Analysis of wine samples showed good agreement with the values obtained by spectrophotometric enzyme assay.

Biosensing Techniques↗

PubMed2003

The effect of an HMG-CoA reductase inhibitor on arteriosclerotic nanoplaque formation and size in a biosensor model.

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution, thereby representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques. Low-density lipoprotein (LDL) was found to deposit strongly at the proteoheparan sulfate-coated surface, particularly in the presence of Ca(2+), apparently through complex formation 'proteoglycan-LDL-calcium'. This ternary complex build-up may be interpreted as arteriosclerotic nanoplaque formation on the molecular level responsible for the arteriosclerotic primary lesion. HDL bound to heparan sulfate proteoglycan protected against LDL deposition and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL was able to decelerate the ternary complex deposition and to disrupt newly formed nanoplaques. Therefore, HDL attached to its proteoglycan receptor sites is thought to raise a multidomain barrier, selection and control motif for transmembrane and paracellular lipoprotein uptake into the arterial wall. The molecular arteriosclerosis model was tested on its reliability in a biosensor application in order to unveil possible acute pleiotropic effects of the lipid lowering drug fluvastatin. The very low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)/LDL and VLDL/IDL/LDL/HDL plasma fractions from a high-risk patient with dyslipoproteinemia and type 2 diabetes mellitus showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca(2+) concentration, with a strong increase at higher Ca(2+) concentrations. Nanoplaque formation and size of the HDL-containing lipid fraction remained well below that of the LDL-containing lipid fraction. Fluvastatin, whether applied acutely to the patient (one single 80 mg slow release matrix tablet) or in a 2-months medication regimen, markedly slowed down this process of ternary aggregational nanoplaque build-up and substantially inhibited nanoplaque size development at all Ca(2+) concentrations used. The acute action resulted without any significant change in lipid concentrations of the patient. Furthermore, after nanoplaque generation, fluvastatin, similar to HDL, was able to reduce nanoplaque formation and size. These immediate effects of fluvastatin have to be taken into consideration while interpreting the clinical outcome of long-term studies.

Adsorption↗

PubMed2003

Oxidase enzyme immobilisation through electropolymerised films to assemble biosensors for batch and flow injection analysis.

Glucose oxidase, lactate oxidase, L-aminoacid oxidase and alcohol oxidase were immobilised on new films based on 2,6-dihydroxynaphthalene (2,6-DHN) copolymerised with 2-(4-aminophenyl)-ethylamine (AP-EA) onto the Pt electrodes. The electropolymerisation was performed by cyclic voltammetry. Different scan rates and scan potential ranges were investigated and selected according to the monomers used. These sensors were tested for hydrogen peroxide, ascorbic acid and acetaminophen by cyclic voltammetry and amperometry. The amperometric studies were carried out in batch as well as in a flow injection analysis (FIA) system. Analytical parameters such as reproducibility, interference rejection, response time, buffer, storage and operational time of the sensors have been studied. These films were also characterised by X-ray photoelectron spectroscopy (XPS). Different strategies for enzyme immobilisation were performed and discussed: enzyme entrapment in the film during the electropolymerisation and covalent attachment of the enzyme to the film via a carbodiimide (1-ethtl-3-(3-dimethylaminopropyl)carbodiimide, EDC) or glutaraldehyde. Different parameters were considered in order to optimise the immobilisation procedures. Results provide a guide to design high sensitive, stable and interference-free biosensors. In addition, studies were performed using these probes in an original FIA based on solenoidal valves. Sensor stability, life time and dynamic range were also optimised in these conditions.

Biosensing Techniques↗

PubMed2003

Development of a biosensor specific for cysteine sulfoxides.

S-Alk(en)yl cysteine sulfoxides have been observed in several plants, mainly belonging to the onion family (Alliaceae), which are of high commercial interest (e.g. garlic, Allium sativum). The quality of most garlic containing herbal remedies produced from garlic powder is determined by their content of the cysteine sulfoxide alliin. Therefore, a comprehensive method for the documentation of alliin amounts present in the fresh plant material through to the final remedy is desirable. The newly developed biosensoric method described in this paper was designed in order to fulfil these demands. In contrast to conventional HPLC-methods, neither a pre-column derivatization nor a chromatographic separation are required allowing a high throughput of samples. This technique is based on immobilized alliinase (EC 4.4.1.4), which was combined with an ammonia-gas electrode. The enzyme was either placed in a small cartridge or was immobilized in direct contact of the electrode surface giving detection limits of 3.7 x 10(-7) and 5.9 x 10(-6) M. Founded on these experiments, a pH-sensitive electrolyte/insulator/semiconductor (EIS) layer structure made of Al/p-Si/SiO(2)/Si(3)N(4) was also combined with immobilized alliinase. Measurements could be performed in a range between 1 x 10(-5) and 1 x 10(-3) M alliin. All sensors were operated in the flow-through modus. A high specificity for alliin could be demonstrated for the electrode and a number of garlic samples were analyzed. Results gained with the new method showed a good correlation with those obtained with conventional HPLC-methods. In addition, onion and a variety of wild Allium species were analyzed in order to determine the amount of isoalliin or total cysteine sulfoxides present, respectively.

Allium↗

PubMed2003

A disposable biosensor employing a glucose-sensitive biochemomechanical gel.

A novel approach to construct a disposable biosensor is proposed. By immobilizing glucose oxidase in a pH-sensitive copolymerized poly(N-isopropylacrylamide)/acrylic acid gel, a glucose-sensitive gel can be obtained. When the gel makes contact with a sample solution containing glucose, the pH of the gel decreases as a result of the enzymatic reaction, inducing the collapse of the gel. The response time to the shrinkage depended upon the activity of the enzyme and the concentration of glucose, although the final ratio of shrinkage settled to approximately the same value irrespective of the concentration of glucose. The transient stage can be used for sensing. The volume change of the gel was transduced into the movement of a colored solution in a flow channel with the help of a silicone rubber diaphragm. The mechanism was used to construct a disposable glucose sensor. A clear dependence of the column length of the colored solution on the concentration of glucose was observed. By measuring the change at a predetermined time, the glucose concentration was obtained. This sensor can be considered the ultimate style in disposable sensors, as detection does not require any electrical measurement or spectroscopic procedures.

Acrylamides↗

PubMed2003

Graphite-Teflon composite bienzyme amperometric biosensors for monitoring of alcohols.

Composite graphite-Teflon electrodes, in which the enzymes alcohol oxidase (AOD) and horseradish peroxidase (HRP), as well as the mediator ferrocene, are incorporated into the electrode matrix, are reported for the reliable monitoring of alcohols in food and beverages. The bienzyme electrodes are constructed by simple physical inclusion of the enzymes and the mediator in the bulk of graphite-70% Teflon rigid cylindrical pellets. The composite biosensors are robust and reusable because of the renewability of the electrode surface by polishing. Reproducible amperometric responses at 0.00 V were obtained with different electrodes constructed from the same pellet and from different pellets. No significant loss of the enzymes activities was found after at least 3 months of storage at 0 degrees C. The detection limits obtained by amperometry in stirred solutions can be advantageously compared with those achieved with commercial sensors for alcohols. The bienzyme electrodes are suitable to be used under flow-injection conditions, as well as for amperometric detection in HPLC. The bioelectrodes were employed for the determination of ethanol in beers, wines and liquors, using both batch- and flow-injection modes, and for the determination of methanol in wines and liquors by HPLC with amperometric detection. Only a dilution of the beverages was needed as sample treatment in all cases.

Alcoholic Beverages↗

PubMed2003

Sensitivity of the acoustic waveguide biosensor to protein binding as a function of the waveguide properties.

The aim of this work is to study the effect of operating frequency, piezoelectric substrate and waveguide layer thickness on the sensitivity of the acoustic waveguide sensor during the specific binding of an antibody by a protein. Shear horizontal (SH) wave devices consisting of (a) a LiTaO3 substrate operating at 104 MHz, (b) a quartz substrate operating at 108 MHz and (c) a quartz substrate operating at 155 MHz were coated with a photoresist polymer layer in order to produce acoustic waveguide devices supporting a Love wave. The effect of the thickness of the polymer layer on the Love wave was assessed by measuring the amplitude and phase of the wave before and after coating. The sensitivity of the above three biosensors was compared during the detection of the specific binding of different concentrations of Immunoglobulin G in the range of 0.7-667 nM to a protein A modified surface. Results indicate that the thickness of the polymer guiding layer is critical for obtaining the maximum sensitivity for a given geometry but a trade-off has to be made between the theoretically determined optimum thickness for waveguiding and the device insertion loss. It was also found that increasing the frequency of operation results in a further increase in the device sensitivity to protein detection.

Acoustics↗

PubMed2003

Encapsulation of glucose oxidase microparticles within a nanoscale layer-by-layer film: immobilization and biosensor applications.

We report on an immobilization strategy utilizing layer-by-layer encapsulated microparticles of enzymes within a nanoscale polyelectrolyte film. Encapsulation of glucose oxidase (GOD) microparticles was achieved by the sequential adsorption of oppositely charged polyelectrolytes onto the GOD biocrystal surface. The polyelectrolyte system polyallylamine/polystyrene sulfonate was used under high salt conditions to preserve the solid state of the highly water soluble GOD biocrystals during the encapsulation process. The resulting polymer multilayer capsule of about 15 nm wall thickness is permeable for small molecules (glucose), but non-permeable for macromolecules thus preventing the enzyme from leakage and at the same time shielding it from the outer environment e.g., from protease or microbial activity. Decrease of the buffer salt concentration leads to the dissolution of the enzyme under formation of mu-bioreactors. The spherical mu-bioreactors are bearing an extremely high loading of biocompound per volume. Encapsulated GOD was subsequently used to construct a biosensor by nanoengineered immobilisation of mu-bioreactor capsules onto an electrode surface. The presented approach demonstrates a general method to encapsulate highly soluble solid biomaterials and an immobilization strategy with the potential to create highly active thin and stable films of biomaterial.

Biosensing Techniques↗

PubMed2003

Quartz crystal biosensor for real-time monitoring of molecular recognition between protein and small molecular medicinal agents.

A quartz crystal microbalance (QCM) biosensor integrated into a flow injection analysis (FIA) system was used for the real-time investigation of molecular recognition between a protein and small molecular medicinal agents. Two sulfa-drugs, sulfamethazine (SMZ) and sulfamethoxazole (SMO), were, respectively, immobilized on the gold electrodes of the piezoelectric crystals using appropriate procedures based on self-assembly of the dithiothreitol (DTT). The binding interactions of the two immobilized drug ligands, with various proteins in solution, were followed as changes in the resonant frequency of the modified crystals. Results obtained from this rapid screen analysis clearly indicated that the two drug ligands appeared quite different in this molecular recognition procedure although their structures were similar. SMZ-immobilized sensor showed specific interaction only with IgG, while SMO-immobilized sensor showed negligible specific binding with IgG, but binding with trypsin and chymotrypsin. Further studies on the specific interaction between immobilized SMZ and three different species of IgG--human IgG, goat IgG and mouse IgG were carried out and the marked species-dependent difference was observed. The resultant sensorgrams were rapidly analyzed by using an in-house kinetic analysis software based on genetic algorithm (GA) to derive both the kinetic rate constants (kass and kdiss) and equilibrium association constants (KA) for IgG-SMZ interactions. For the interactions, KA were 5.48 x 10(5), 2.75 x 10(5) and 1.86 x 10(5) M(-1) for human IgG, goat IgG and mouse IgG, respectively. The kinetic data provided further insight into the structural/functional relationships of different IgG on a molecular level.

Adsorption↗

PubMed2003

Chemisorptions of bacterial receptors for hydrophobic amino acids and sugars on gold for biosensor applications: a surface plasmon resonance study of genetically engineered proteins.

This paper demonstrates potential applications of two periplasmic receptor proteins from E. coli as sensing elements for biosensors using the surface plasmon resonance (SPR) technique. These molecules, namely the aspartate to cysteine mutant of the leucine-specific receptor (LS-D1C) and the glutamine to cysteine mutant of the D-glucose/D-galactose receptor (GGR-Q26C) proteins, are chemisorbed on a thin (approximately 40 nm) Au film in neutral K2HPO4 buffers. Using angle and time resolved SPR measurements; we show that adsorption behaviors of both proteins are dominated by diffusion-free second order Langmuir kinetics. We also show that the protein-modified Au films exhibit measurable SPR shifts upon binding to their respective target ligands. According to these SPR data, the kinetics of ligand binding for both LS-D1C and GGR-Q26C are governed by irreversible first order diffusion limited Langmuir model. The utility of the SPR technique for studying reactions of biological molecules is further illustrated in this work.

Adsorption↗

PubMed2003

The influence of antimicrobial treatments on the cytocompatibility of polyurethane biosensor membranes.

The cytocompatibility of polyurethane membranes was tested following ultraviolet or gamma irradiation as well as treatment with hydrogen peroxide or glutaraldehyde containing solutions. Despite the fact that all of the methods had been recommended for antimicrobial treatment of glucose biosensors, the treatments investigated significantly influenced cytocompatibility characteristics. Cytotoxicity of membrane eluates was not observed following irradiation treatments. This was also the case when the membranes were repeatedly washed following chemical treatment. Cell growth upon the membranes was stimulated to a different extent after gamma and UV irradiation as well as following hydrogen peroxide treatments. Residues of an urea-based hydrogen peroxide inclusion compound caused a restriction in cell growth upon the membranes as was similarly observed with 2 and 4% glutaraldehyde solutions acting over 2 and 4 h, respectively. It is concluded that cytocompatibility in vitro reflecting the host response against a biomaterial in vivo does not only depend upon the material itself but also upon antimicrobial treatments which could have consequences for its bioperformance characteristics.

Animals↗

PubMed2003

Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate.

A multifunctional bio-sensing chip was designed based on the electrochemiluminescent (ECL) detection of enzymatically produced hydrogen peroxide. Six different oxidases specific for choline, glucose, glutamate, lactate, lysine and urate were non-covalently immobilised on imidodiacetic acid chelating beads (glucose oxidase only) or on diethylaminoethyl (DEAE) anion exchanger beads, and spotted on the surface of a glassy carbon foil (25 mm(2) square), entrapped in PVA-SbQ photopolymer. The chip measurement was achieved by applying during 3 min a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference, while capturing a numeric image of the multifunctional bio-sensing chip with a CCD camera. The use of luminol supporting beads (DEAE-Sepharose) included in the sensing layer was shown to enable the achievement of spatially well defined signals, and to solve the hydrogen peroxide parasite signal which appeared between contiguous spots using luminol free in solution. The detection limits of the different biosensor were found to be 1 microM for glutamate, lysine and uric acid, 20 microM for glucose and 2 microM for choline and lactate. The detection ranges were 1-25 microM (uric acid), 1 microM-0.5 mM (glutamate and lysine), 20 microM-2 mM (glucose) and 2 microM-0.2 mM (choline and lactate). The ECL chip was used for the detection of glucose, lactate and uric acid in human serum matrix. Good correlations between measured and expected values were found without the need of internal calibration of the sample, demonstrating the potentiality of the ECL multifunctional bio-sensing chip.

Biosensing Techniques↗

PubMed1997

Effectiveness of protein A for antibody immobilization for a fiber optic biosensor.

The fiber optic biosensor performs fluoroimmunoassays at the surface of multimode optical fibers. The effectiveness of protein A, an immunoglobulin binding protein, for antibody immobilization on the surface of these fiber probes has been investigated. No difference was observed in the binding of fluorescently-labeled goat-IgG by rabbit anti-goat IgG regardless of whether the capture antibody was bound to the probe surface via protein A or covalently attached. However, in a sandwich immunoassay for the F1 antigen of Yersinia pestis, probes with rabbit anti-plague IgG bound to the surface via protein A generated twice the signal as probes with the antibody covalently attached. Assay regeneration was also examined with protein A probes since antibody-antigen complexes have been successfully eluted from protein A under low pH conditions. Protein A probes coated with rabbit anti-goat IgG obtained nearly identical signal levels at 500 and 5000 ng/ml of Cy5.5 goat IgG five consecutive times following regeneration with glycine-HCl, 2% acetic acid, pH 2.5.

Antigen-Antibody Reactions↗

PubMed1997

Studies on dynamic behavior of the biosensor based on immobilized glucoamylase-glucose oxidase membrane.

Frequency response of the glucose/maltose biosensors based on immobilized glucoamylase and glucose oxidase membranes of different thickness were investigated with the sinusoidal signal generator of substrate concentration. The results were modeled by the serial combination of the dead time and the first order response blocks, and time constants and dead times were estimated. The estimated value of time constant ranged from 4.5 to 13.2 s and that of the dead time from 3.2 to 7.7 s with the tendency that both time constant and dead time increase as membrane thickness increases and number of enzymatic reaction steps increases.

Biosensing Techniques↗

PubMed1997

Fiber optic-based biosensor for ricin.

We report on an evanescent wave fiber-optic biosensor for detecting a potently toxic protein, ricin, in the picograms per milliliter range. A sandwich immunoassay scheme was used to detect ricin. First, an anti-ricin IgG was immobilized onto the surface of an optical fiber in two different ways. In the first method, the antibody was directly coated to the silanized fiber using a crosslinker. Second, avidin-coated fibers were incubated with biotinylated anti-ricin IgG to immobilize the antibody using an avidin-biotin bridge. The assay using the avidin-biotin linked antibody demonstrated higher sensitivity and wider linear dynamic range than the assay using antibody directly conjugated to the surface. The linear dynamic range of detection for ricin in buffer using the avidin-biotin chemistry is 100 pg/ml-250 ng/ml. The limits of detection for ricin in buffer solution and river water are 100 pg/ml and 1 ng/ml, respectively. At higher concentrations of ricin (> 50 ng/ml), we observe a strong interaction of ricin with the avidin coated on the surface of the fibers. We have demonstrated that this interaction is primarily due to the lectin activity of ricin and is significantly reduced using fibers coated with neutravidin or by adding galactose to the ricin samples.

Adsorption↗

PubMed1997

Studies towards a disposable screen-printed amperometric biosensor for progesterone.

A screen-printed carbon electrode (SPCE) has been investigated as the base transducer for a disposable amperometric progesterone biosensor. The biorecognition element was a monoclonal sheep anti-progesterone antibody (mAb). This was immobilized onto the transducer by interaction with a layer of rabbit IgG which had been previously coated onto the SPCE; optimum conditions for these loadings were deduced experimentally. The device was employed in a competitive assay using alkaline phosphatase-labelled progester-one. Three possible substrates for the enzyme were considered, namely, phenyl phosphate, phenolphthalein phosphate and 4-aminophenol phosphate. Cyclic voltammetry and amperometry were carried out on the corresponding aromatic phenols and phenol itself was found to give the best electrochemical characteristics; consequently, phenyl phosphate was employed as the substrate. Chronoamperometry was used to measure the phenol produced by the reaction of bound enzyme-labelled progesterone and substrate. The chronoamperometric response was dependent on unlabelled progesterone over at least three orders of magnitude with a detection limit of about 1 x 10(-9) mol/dm3. This suggests that the device may have applications for the analysis of biological fluids.

Aminophenols↗