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PubMed2006

Simultaneous determination of kinetic parameters for the binding of cholera toxin to immobilized sialic acid and monoclonal antibody using an array biosensor.

Interactions between protein toxins and carbohydrate receptors are often semi-selective processes and the kinetic parameters that define the binding of a receptor to different toxins may vary with each interaction. In this study, we have determined the affinity constants for binding of cholera toxin (CT) to immobilized sialic acid and to anti-CT antibody (as a simultaneous reference) by measuring real-time binding processes using an array biosensor. N-Acetylneuraminic acid (Neu5Ac), a member of the sialic acid family, was covalently immobilized onto maleimide-activated planar waveguides via a thiol-terminated linker attached to the anomeric carbon of the sugar. Control antibodies were immobilized using two different approaches: covalent attachment onto maleimide-activated slides via the thiol on cysteine residues and non-covalent attachment using a biotin-NeutrAvidin linkage. Cy5-labeled CT was flowed over the immobilized receptors and the fluorescent intensity of the bound CT-receptor complex was recorded as a function of time. The association constants for CT binding to covalently attached Neu5Ac, to covalently attached anti-CT monoclonal antibody, and to antibody tethered by biotin-NeutrAvidin interactions were determined to be 1.3 x 10(8), 2.1 x 10(8) and 5.7 x 10(8)M(-1), respectively.

Antibodies, Monoclonal↗

PubMed2006

Integration of a surface acoustic wave biosensor in a microfluidic polymer chip.

SAW devices based on horizontally polarized surface shear waves (HPSSW) enable label-free, sensitive and cost-effective detection of biomolecules in real time. It is known that small sampling volumes with low inner surface areas and minimal mechanical stress arising from sealing elements of miniaturized sampling chambers are important in this field. Here, we present a new approach to integrate SAW devices with sampling chamber. The sensor device is encapsulated within a polymer chip containing fluid channel and contact points for fluidic and electric connections. The chip volume is only 0.9 microl. The polymeric encapsulation was performed tailor-made by Rapid Micro Product Development 3Dimensional Chip-Size-Packaging (RMPD 3D-CSP), a 3D photopolymerisation process. The polymer housing serves as tight and durable package for HPSSW biosensors and allows the use of the complete chips as disposables. Preliminary experiments with these microfluidic chips are shown to characterise the performance for their future applications as generic bioanalytical micro devices.

Acoustics↗

PubMed2006

Poly(vinylpyrrolidone)-doped nitric oxide-releasing xerogels as glucose biosensor membranes.

Nitric oxide (NO)-releasing xerogel membranes were prepared as coatings for an electrochemical glucose biosensor to allow for enhanced biocompatibility while maintaining adequate response times and sensitivity. Formation of the NO-donor species was found to drastically decrease the permeability of the aminosilane-based xerogels to both hydrogen peroxide and glucose. The addition of poly(vinylpyrrolidone) (PVP) polymer enhanced the membrane permeability even after exposure to high pressures of NO (necessary for NO-donor synthesis). The analytical response and NO release of PVP-doped NO-releasing xerogels as glucose sensor membranes were further investigated and found to be enhanced via polymer doping. Doping of the polymer into the xerogel did not compromise the stability of the xerogel as evaluated by silicon leaching studies. Despite the addition of PVP, the NO-releasing xerogels maintained reduced bacterial adhesion characteristics analogous to previous reports for NO-releasing xerogels.

Biosensing Techniques↗

PubMed2006

Olfactory cell-based biosensor: a first step towards a neurochip of bioelectronic nose.

Human olfactory system can distinguish thousands of odors. In order to realize the biomimetic design of electronic nose on the principle of mammalian olfactory system, this article reports an olfactory cell-based biosensor as a real bionic technique for odorants detection. Effective cultures of olfactory receptor neurons and olfactory bulb cells have been achieved on the semiconductor chip. Using light-addressable potentiometric sensor (LAPS) as sensing chip to monitor extracellular potential of the neurons, the response under stimulations of the odorants or neurotransmitters, such as acetic acid and glutamic acid, was tested. The results demonstrate that this kind of hybrid system of LAPS and olfactory neurons, which is sensitive to odorous changes, has great potential and is promising to be used as a novel neurochip of bioelectronic nose for detecting odors.

Animals↗

PubMed2006

Morphological and binding properties of interleukin-6 on thin ZnO films grown on (100) silicon substrates for biosensor applications.

To develop effective protein immobilization technology with minimal amounts of protein for high sensitivity surface acoustic wave biosensors, we determined the binding properties, and morphological characteristics of human interleukin-6 (IL-6), a pro-inflammatory cytokine, on the surface of ZnO, and SiO(2) films grown onto (100) Si substrates, for the first time. Interleukin-6 was immobilized in the range of 0.276-10 pg/ml on the surface of ZnO and SiO(2), and visualized at each stage, while protein-protein interactions were measured with the antigen/antibody immunoassay of solid-phase ELISA, which we modified for these types of substrates. A relative mass value was determined in each case. ELISA detected upward of 1 and 6 ng/ml of protein applied on ZnO and SiO(2), respectively. It is concluded that the more reactive ZnO surface is a new and more effective template for protein immobilization.

Binding Sites↗

PubMed2006

On-line detection of atmospheric formaldehyde by a conductometric biosensor.

Atmospheric formaldehyde (CH(2)O) was detected under continuous flow conditions by an on-line system comprising of a wet scrubber for a continuous transfer of the pollutant to an aqueous solution, a micro-reactor containing immobilized formaldehyde dehydrogenase (FDH) and a conductometric transducer. By this system atmospheric formaldehyde concentrations in the range 0.05-2 ppm were detected with a sensitivity of 20 microS/ppm. In this concentration range the immobilized enzyme oxidized all the sampled formaldehyde molecules to formic acid, avoiding cumbersome calibration procedures. The operational stability of the biosensor was at least 3 months, working continuously 10 h/day at room temperature.

Air Pollutants↗

PubMed2006

Amperometric tyrosinase biosensor based on polyacrylamide microgels.

An amperometric enzyme sensor using tyrosinase (PPO) entrapped in polyacrylamide microgels has been developed for determination of phenolic compounds. Polyacrylamide microgels were obtained by the concentrated emulsion polymerization method. The crosslinking of the polymer matrix optimum to retain the enzyme and to allow the diffusion of the compounds involved in the enzyme reaction has been studied (4.0%) as well as the influence on the response of analytical parameters such as pH, temperature, enzyme load and working potential. The useful lifetime of the biosensor was 27 days and it was useful to determine monophenolics compounds (e.g. cresol, chlorophenol) and diphenolics compounds (e.g. catechol and dopamine) by amperometric measurements at -100mV (versus SCE) in a batch system. The results showed that the substrate structures have a great influence on the sensor response.

Acrylic Resins↗

PubMed2006

Hemoglobin niobate composite based biosensor for efficient determination of hydrogen peroxide in a broad pH range.

Inorganic layered niobates (HCa2Nb3O10) were used as immobilization matrices of hemoglobin (Hb) because of their tunable interlayer spaces, large surface areas and good biocompatibilities. A pair of well-defined, quasi-reversible cycle voltammertric peaks were obtained at the Hb-HCa2Nb3O10 modified pyrolytic graphite electrode, suggesting that the layered niobates facilitate the electron transfer between the proteins and the electrode. Hb-HCa2Nb3O10 modified electrode exhibited electrocatalytic response for monitoring H2O2 with a large linear detection range from 25 microM to 3.0 mM and a relatively high sensitivity of 172 microA mM-1 cm-2. Based on the stabilizing effect of the layered niobates, Hb-HCa2Nb3O10 modified electrode can detect H2O2 in strongly acidic and basic solutions with pH of 1-12, which greatly expands the application fields of biosensors.

Animals↗

PubMed2006

Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor.

A strategy for patterned cell adhesion based on chemical surface modification is presented. To confine cell adhesion to specific locations, an engineered surface for high-contrast protein adsorption and, hence, cell attachment has been developed. Surface functionalization is based on selective molecular-assembly patterning (SMAP). An amine-terminated self-assembled monolayer is used to define areas of cell adhesion. A protein-repellent grafted copolymer, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), is used to render the surrounding silicon dioxide resistant to protein adsorption. X-ray photoelectron spectroscopy, scanning ellipsometry and fluorescence microscopy techniques were used to monitor the individual steps of the patterning process. Successful guided growth using these layers is demonstrated with primary neonatal rat cardiomyocytes, up to 4 days in vitro, and with the HL-1 cardiomyocyte cell line, up to 7 days in vitro. The advantage of the presented method is that high-resolution engineered surfaces can be realized using a simple, cost-effective, dip-and-rinse process. The technique has been developed for application on a CMOS cell-based biosensor, which comprises an array of microelectrodes to extracellularly record electrical activity from cardiomyocytes.

Animals↗

PubMed2006

Computational selection of nucleic acid biosensors via a slip structure model.

Aptamers have been shown to undergo ligand-dependent conformational changes, and can be joined to ribozymes to create allosteric ribozymes (aptazymes). An anti-flavin (FMN) aptamer joined to the hammerhead ribozyme yielded an aptazyme that underwent small, FMN-dependent displacements in the helix that joined the aptamer and ribozyme. This 'slip structure' model in which alternative sets of base-pairs are formed in the absence and presence of ligand proved amenable to energetic and computational modeling. Initial successes in modeling the activities of known aptazymes led to the in silico selection of new ligand-dependent aptazymes from virtual pools that contained millions of members. Those aptazymes that were predicted to best fit the slip structure model were synthesized and assayed, and the best-designed aptazyme was activated 60-fold by FMN. The slip structure model proved to be generalizable, and could be applied with equal facility to computationally generate aptazymes that proved to be experimentally activated by other ligands (theophylline) or that contained other catalytic cores (hairpin ribozyme). Moreover, the slip structure model could be applied to the prediction of a ligand-dependent aptamer beacon biosensor in which the addition of the protein vascular endothelial growth factor (VegF) led to a 10-fold increase in fluorescent signal.

Aptamers, Nucleotide↗

PubMed2006

Laccase immobilization in redox active layered double hydroxides: a reagentless amperometric biosensor.

This paper describes a new system for amperometric determination of dissolved oxygen and its application for the detection of anionic toxic substances, which are known as enzyme inhibitors. This biosensor is based on the co-immobilization of laccase from Trametes versicolor and a redox active layered double hydroxide [Zn-Cr-ABTS] on a glassy carbon electrode. The electrochemical transduction step corresponds to the electrocatalytic reduction of O2 at 0.2V by laccase as catalyst and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as mediator. Such device provides a fast and a sensitive response for dissolved oxygen determination between 6 x 10(-8) and 4 x 10(-6)M and very low detection limits for azide (5.5 nM), fluoride (6.9 nM) and cyanide (6.2 nM).

Biosensing Techniques↗

PubMed2006

Toward continuous glucose monitoring with planar modified biosensors and microdialysis. Study of temperature, oxygen dependence and in vivo experiment.

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.

Animals↗

PubMed2006

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

Biosensing Techniques↗

PubMed2006

Uncoated quartz resonator as a universal biosensor.

We studied dynamic processes in drying drops of model protein-salt solutions, using an uncoated quartz resonator as a biosensor. To measure these processes we developed a method based on recording the dynamics of the Acoustic-Mechanical Impedance (AMI) of a drop as it dried on the surface of a quartz resonator oscillating at a resonant frequency of 60 kHz. The aim of this work was to highlight the role of some components of serum in self-organization processes. Human serum albumin (HSA), fibronectin (Fn), immunoglobulin G (IgG), immunoglobulin M (IgM), bovine serum albumin (BSA), sodium chloride (NaCl), Potassium Chloride (KCl), and nonionic surfactant O(CH(2)CH(2))(n)CH(2)CH(2)OH were used as components of the tested solutions. It was shown that dynamics of the AMI in drying drops were closely related to liquid composition. This approach allowed us to distinguish with good accuracy solutions in which one or more components (proteins or salts) were replaced by other components with the same mass concentration. We assumed that these differences were due to different surface properties and native functions of proteins, and different positions of salts in the Hofmeister line. Our preliminary work demonstrated that the dynamics of phase transitions in drying drops of serum could be used as an informative parameter for medical diagnostics. In this study, we highlight some positions in this cause-effect chain.

Biosensing Techniques↗

PubMed2006

The development of a real-time biosensor for the detection of trace levels of trinitrotoluene (TNT) in aquatic environments.

This paper describes the development of a highly sensitive TNT immunosensor consisting of a highly specific monoclonal antibody coupled with a prototype fluorescence-based detector system (KinExA Inline Biosensor, Sapidyne Instrument Inc). The antibody developed possesses a high affinity for TNT (association constant, aK 8.2) with minimal cross reactivity with other compounds such as tetryl, 2,4-dinitrotoluene and 2-amino-4,6-dinitrotoluene. This system provides sample assessment within 160 s from source acquisition and possesses sensitivity for TNT of 0.05 microg/L in ground water. The sensor can be regenerated in 8 min, allows a minimum of 40 repeated readings, and has a standard error of 0.1-0.4% between repeat readings. The fluidics and software allow samples to be obtained from up to eight different sources allowing the user to examine the stratification of the pollutant in the water column. We believe that this immunosensor can be used to rapidly assess trace levels of TNT in environmental water samples.

Biosensing Techniques↗

PubMed2006

Poly-(3-hexylthiophene) self-assembled monolayer based cholesterol biosensor using surface plasmon resonance technique.

Cholesterol oxidase (ChOx) has been covalently immobilized onto 1-fluoro-2-nitro-4-azidobenzene (FNAB) modified poly-(3-hexylthiophene) (P3HT) self-assembled monolayer (SAM) onto gold coated glass plates. These ChOx/FNAB/P3HT/Au bio-electrodes have been characterized using contact angle (CA) measurements, UV-vis spectroscopy, electrochemical impedance technique, cyclic voltammetric technique and atomic force microscopic (AFM) technique, respectively. The ChOx/FNAB/P3HT/Au bio-electrodes were utilized for the estimation of cholesterol concentration in standard solutions using surface plasmon resonance (SPR) technique. It is shown that this SPR biosensor has linearity from 50 to 500 mg/dl of cholesterol in solution with detection limit of 50 mg/dl, sensitivity of 1.0 4 m degrees /(mg dl), reusability of around 15 times and a shelf-life of about 10 weeks when stored at 4 degrees C.

Adsorption↗

PubMed2006

Interferometric biosensor based on planar optical waveguide sensor chips for label-free detection of surface bound bioreactions.

A label free optical biosensor based on a free-space Young interferometer configuration is presented. Commercial planar Ta(2) O(5) waveguides are used as sensing elements and allow the investigation of surface bound bioreactions like immunoreactions or biological affinity systems. Design criteria are discussed and a detailed characterization of the sensor performance is presented. The developed interferometer yields an effective refractive index resolution of 9 x 10(-9), corresponding to a surface coverage of approximately 13 fg/mm(2). The performance of the system is characterized by two different affinity systems: the antibody-antigen complex protein G-immunoglobulin G is used as a model system for monitoring reaction kinetics. Further measurements on a silanized surface show the formation of a streptavidin monolayer on a biotinylated surface.

Biosensing Techniques↗

PubMed2006

Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase.

This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP(+). The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 microM and 1000 microM with a low detection limit of 1.2 microM. Also, it has a fast measuring time which can achieve 95% of the maximum current response in 20s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min).

4-Hydroxybenzoate-3-Monooxygenase↗