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At least 973 records · Page 54Linked to original sources

PubMed2006

Analyzing ligand and small molecule binding activity of solubilized GPCRs using biosensor technology.

We used Biacore technology to measure directly the binding of natural ligands and small molecules to the chemokine receptors CXCR4 and CCR5. Both G protein-coupled receptors were solubilized from whole cell pellets and captured on antibody surfaces for analysis. Our solubilization conditions maintained high-affinity binding of chemokines SDF-1alpha and RANTES to CXCR4 and CCR5, respectively. Surface density- and buffer-dependent binding responses, along with binding data for a selective ligand (RCP-168), further validated the biosensor assay. In addition, we showed that it is possible to collect high-quality binding responses for the archetypal small molecule inhibitors JM-2987 and TAK-779. Finally, using our biosensor-based method, we characterized the kinetics of 19 novel small molecule inhibitors of CCR5 and showed that their affinities correlated with values determined for the membrane-associated receptor. Together, the chemokine and small molecule binding data provide evidence that the solubilized receptors maintain native binding properties. These solubilized receptor preparations could be useful reagents for biophysical studies as well as for structural analysis.

Chemokine CCL5↗

PubMed2006

In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-catenin interactions.

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.

Biosensing Techniques↗

PubMed2006

Exploring "one-shot" kinetics and small molecule analysis using the ProteOn XPR36 array biosensor.

A ProteOn XPR36 parallel array biosensor was used to characterize the binding kinetics of a set of small molecule/enzyme interactions. Using one injection with the ProteOn's crisscrossing flow path system, we collected response data for six different concentrations of each analyte over six different target protein surfaces. This "one-shot" approach to kinetic analysis significantly improves throughput while generating high-quality data even for low-molecular-mass analytes. We found that the affinities determined for nine sulfonamide-based inhibitors of the enzyme carbonic anhydrase II were highly correlated with the values determined using isothermal titration calorimetry. We also measured the temperature dependence (from 15 to 35 degrees C) of the kinetics for four of the inhibitor/enzyme interactions. Our results illustrate the potential of this new parallel-processing biosensor to increase the speed of kinetic analysis in drug discovery and expand the applications of real-time protein interaction arrays.

Biosensing Techniques↗

PubMed2006

Immobilization of bovine serum albumin as a sensitive biosensor for the detection of trace lead ion in solution by piezoelectric quartz crystal impedance.

A biosensor based on bovine serum albumin (BSA) for the detection of lead (Pb(2+)) ion was developed and characterized. BSA was immobilized onto a colloidal Au-modified piezoelectric quartz crystal (PQC) as a biosensor for the detection of Pb(2+) ion by piezoelectric quartz crystal impedance (PQCI). Calibration curves for the quantification of Pb(2+) ion showed excellent linearity throughout the concentration range from 1.0 x 10(-7) to 3.0 x 10(-9)mol/L. The interaction between the Pb(2+) ions and the sensor chip is influenced significantly by the pH of the reaction buffer, and the optimal pH for the experiment was 5.4. Under the optimal conditions, the detection limit of 1.0 x 10(-9)mol/L for Pb(2+) was obtained. Kinetic parameters of the Pb(2+)-BSA interactions were also determined by using this chip. The sensor chip could be regenerated for use by dipping in the ethylenediaminetetraacetic acid (EDTA) solution for approximately 2h, and the chip was used to detect Pb(2+) ion for eight times without obvious signal attenuation.

Biosensing Techniques↗

PubMed2004

A stable three-enzyme creatinine biosensor. 1. Impact of structure, function and environment on PEGylated and immobilized sarcosine oxidase.

The determination of creatinine levels in biological fluids is an increasingly important clinical requirement. Amperometric biosensors have been developed based on a three-enzyme system which converts creatinine to amperometrically measurable hydrogen peroxide. The development of the amperometric creatinine biosensor has been slow due the complexity of the three-enzyme system. This paper, the first of three, discusses the chemical modification of sarcosine oxidase and the immobilization and stabilization of this enzyme using polyurethane prepolymers. Sarcosine oxidase was completely inactivated after modification using poly(ethylene glycol) activated with isocyanate. The addition of a competitive inhibitor during enzyme modification was effective in protecting the enzyme from inactivation. Computational analysis of the structure of sarcosine oxidase suggests that there is a lysine in the active site that may be hyper-reactive. The enzyme was irreversibly immobilized using polyurethane prepolymers and retained significant activity. The enzyme's half-life at 37 degrees C increased from seven days to more than 50 days after immobilization.

Biosensing Techniques↗

PubMed2004

A stable three-enzyme creatinine biosensor. 3. Immobilization of creatinine amidohydrolase and sensor development.

We describe the development of an extended use amperometric three-enzyme creatinine biosensor and the successful chemical modification and immobilization of the enzyme creatinine amidohydrolase using polyurethane prepolymers. Creatinine amidohydrolase is significantly stabilized by immobilization in polyurethane polymers. The half-life increases from six to more than 80 days in buffer at 37 degrees C. The effect of silver ions leached from amperometric reference electrodes on enzyme and sensor performance is discussed. The use of cellulose acetate cover membranes to prevent silver from reaching the enzyme is investigated. Sensors prepared with cover membranes have half-lives almost an order of magnitude greater than those prepared with no cover membrane over the silver electrode. The complete biosensor has been constructed on a clinical blood analyzer platform and is stable for many days.

Amidohydrolases↗

PubMed2005

Immobilization of native membrane-bound rhodopsin on biosensor surfaces.

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.

Animals↗

PubMed2004

Phenols monitoring and Hill coefficient evaluation using tyrosinase-based amperometric biosensors.

Sensitive amperometric biosensors for phenols compounds, based on tyrosinase (polyphenoloxidase, PPO) immobilized on a Pt electrode in an electropolymerized poly-amphiphilic pyrrole matrix or cross-linked with glutaraldehyde, were constructed and compared. Steady-state amperometric measurements, performed at -50 mV vs. SCE in aqueous phosphate buffer containing LiClO(4) 0.1 M (pH 7) as well as in a chloroform solution containing 0.1 M C(6)H(5)CH(2)N(CH(3))(3)Cl, were used in order to compare the electroanalytical and kinetic parameters of the investigated amperometric biosensors in aqueous and nonaqueous media. It was established that the polypyrrole matrix has a higher efficiency for enzyme retention resulting in higher bioelectrode sensitivity, both in aqueous buffer (690 microA M(-1)) and in chloroform (149 microA M(-1)).

Benzoic Acid↗

PubMed2004

Electrochemical study of quercetin-DNA interactions: part II. In situ sensing with DNA biosensors.

Quercetin interaction with dsDNA was investigated electrochemically using two types of DNA biosensor in order to evaluate the occurrence of DNA damage caused by oxidized quercetin. The results showed that quercetin binds to dsDNA where it can undergo oxidation. The radicals formed during quercetin oxidation cause breaks of the hydrogen bonds in the dsDNA finally giving rise to 8-oxoguanine since the DNA guanosine and adenosine nucleotides in contact with the electrode surface can easily be oxidized. A mechanism for oxidized quercetin-induced damage to dsDNA immobilized onto a glassy carbon electrode surface is proposed and the formation of 8-oxoguanine is explained. The importance of DNA-electrochemical biosensors in the determination of the interaction mechanism between DNA and quercetin is clearly demonstrated.

Biosensing Techniques↗

PubMed2005

Nucleic acid based biosensors: the desires of the user.

The need for nucleic acid based diagnostic tests has increased enormously in the last few years. On the one hand, this has been stimulated by the discovery of new hereditary genetic disease loci following the completion of the Human Genome Project, but also by the presence of new rapidly spreading viral threats, such as that of the SARS epidemic, or even micro-organisms released for the purpose of biological warfare. As in many instances rapid diagnoses of specific target genetic loci is required, new strategies have to be developed, which will allow this to be achieved directly at the point-of-care setting. One of these avenues being explored is that of biosensors. In this review, we provide an overview of the current state of the art concerning the high-throughput analysis of nucleic acids, and address future requirements, which will hopefully be met by new biosensor-based developments.

Biosensing Techniques↗

PubMed2005

New trends in nucleic acid based biosensors: University of Florence (Italy), 25-28 October 2003.

The Workshop on "New Trends in Nucleic Acids Based Biosensors" was held at the University of Florence (Italy), from 25th to 28th October 2003. The workshop was funded by the European Science Foundation (ESF) within the programme "Functional Genomics" (Programme Area: DNA arrays and chips). This report describes the main arguments discussed during the workshop considering the state of art and the future trends in the field of nucleic acid-based biosensors, with particular attention on the aptamer field.

Biosensing Techniques↗

PubMed2006

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3(2+) with DNA-binding intercalators.

This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) with a DNA-binding intercalator as a reductant of Ru(bpy)(3)(3+). Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4',6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)(3)(2+) at +1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)(3)(2+) ECL.

Biosensing Techniques↗

PubMed2006

The application of methacrylate-based polymers to enzyme biosensors.

Enzyme electrodes based on methacrylates have received significant attention in the development of biosensors. This article reviews the use and application of methacrylate and its derivatives as an immobilization system for the preparation of enzyme electrodes. Resent examples, extracted from the literature, illustrate the superior performance of such materials in the fabrication of biosensors and bioreactors.

Biocompatible Materials↗

PubMed2006

Layer-by-layer self-assembled multilayer films of carbon nanotubes and platinum nanoparticles with polyelectrolyte for the fabrication of biosensors.

Platinum nanoparticle-doped chitosan (CHIT) solution can be easily prepared by treating the CHIT solution with aqueous H2PtCl6 solution followed by chemical reduction of Pt(IV) with NaBH4. Multiwalled carbon nanotubes (MWCNT) are then dispersed in the nanoparticle-doped solution. The resulting Pt-CNT-CHIT material brings new capabilities for electrochemical devices by using the synergistic action of Pt nanaoparticles and CNT. Positively charged Pt-CNT-CHIT solution and negatively charged poly(sodium-p-styrenesulfonate) salt (PSS) have been employed to fabricate stable ultrathin multilayer films on gold electrode and quartz glass slides in a layer-by-layer fashion. Cyclic voltammetric and UV-vis adsorption spectroscopy confirms the consecutive growth of the multilayer films. The modified gold electrode allows low-potential detection of hydrogen peroxide with high sensitivity and fast response time. With the immobilization of cholesterol oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to cholesterol has been constructed. In pH 6.98 phosphate buffer, almost interference free determination of cholesterol has been realized at 0.1 V vs. SCE with a linear range from 0.01 to 3mM and response time<30s. With the immobilization of another cholesterol esterase enzyme layer, the biosensor was used to determine total cholesterol in serum samples with satisfactory results.

Biosensing Techniques↗

PubMed2004

Low-level detection of a bacillus anthracis simulant using Love-wave biosensors on 36 degrees YX LiTaO3.

We present an acoustic Love-wave biosensor for detection of the Bacillus anthracis simulant, Bacillus thuringiensis at or below inhalational infectious levels. The present work is an experimental study of 36 degrees YX cut LiTaO3 based Love-wave devices for detection of pathogenic spores in aqueous conditions. Given that the detection limit (D1) of Love-wave-based sensors is a strong function of the overlying waveguide, two waveguide materials have been investigated, which are polyimide and polystyrene. To determine the mass sensitivity of Love-wave sensor, bovine serum albumin (BSA) protein was injected into the Love-wave test cell while recording the magnitude and phase shift across each sensor. Polyimide had the lowest mass detection limit with an estimated value of 1.0-2.0 ng/cm2, as compared to polystyrene where D1 = 2.0 ng/cm2. Suitable chemistries were used to orient antibodies on the Love-wave sensor using protein G. The thickness of each biofilm was measured using ellipsometry from which the surface concentrations were calculated. The monoclonal antibody BD8 with a high degree of selectivity for anthrax spores was used to capture the non-pathogenic simulant B. thuringiensis B8 spores. Bacillus subtilis spores were used as a negative control to determine whether significant non-specific binding would occur. Spore aliquots were prepared using an optical counting method, which permitted removal of background particles for consistent sample preparation. This work demonstrates that Love-wave biosensors are promising for low-level detection for whole-cell biological pathogens.

Bacillus anthracis↗

PubMed2004

The use of yeast and moulds as sensing elements in biosensors.

Whole cell biosensors are able to provide information that sensors based on single and multiple types of molecules are unable to do. For example, broad-spectrum catabolite analysis, cell toxicity and genotoxicity are best detected in the context of a functioning cell. Most whole cell sensors have used bacterial cells as the sensing element. Fungal cells, however, can provide all of the advantages bacterial cells offer but in addition they can provide information that is more relevant to other eukaryote organisms. These cells are easy to cultivate, manipulate for sensor configurations and are amenable to a wide range of transducer methodologies. An overview of the use of yeast and filamentous fungi as the sensing element of some biosensors is presented here.

Biosensing Techniques↗

PubMed2004

Visualization of micropatterned complex biosensor sensing chemistries by means of scanning electrochemical microscopy.

Redox hydrogel-based micropatterned complex biosensor architectures, used as sensing chemistries in amperometric ethanol or glucose biosensors, were deposited on gold, graphite or glass. Well-localized immobilization of active hydrogels with variable compositions was achieved by dispensing 100 pl droplets of cocktails containing alcohol or glucose dehydrogenase, redox polymer (PVI(13)dmeOs) and crosslinker (PEGDGE) while moving the target surface relative to the position of the nozzle of a piezo-actuated microdispenser. The resulting structures were microscopic patterns of enzyme-containing lines of a redox hydrogel with a line width of about 100 microm. Scanning electrochemical microscopy (SECM) in the amperometric feedback mode was used to visualize the immobilized enzyme microstructures and their localized biochemical activity was observed with high lateral resolution by detecting the enzymatically consumed substrate using K(4)[Fe(CN)(6)] as a free-diffusing electron-transfer mediator.

Biosensing Techniques↗

PubMed2004

Stabilization of immobilized glucose oxidase against thermal inactivation by silanization for biosensor applications.

An important requirement of immobilized enzyme based biosensors is the thermal stability of the enzyme. Studies were carried out to increase thermal stability of glucose oxidase (GOD) for biosensor applications. Immobilization of the enzyme was carried out using glass beads as support and the effect of silane concentration (in the range 1-10%) during the silanization step on the thermal stability of GOD has been investigated. Upon incubation at 70 degrees C for 3h, the activity retention with 1% silane was only 23%, which increased with silane concentration to reach a maximum up to 250% of the initial activity with 4% silane. Above this concentration the activity decreased. The increased stability of the enzyme in the presence of high silane concentrations may be attributed to the increase in the surface hydrophobicity of the support. The decrease in the enzyme stability for silane concentrations above 4% was apparently due to the uneven deposition of the silane layer on the glass bead support. Further work on thermal stability above 70 degrees C was carried out by using 4% silane and it was found that the enzyme was stable up to 75 degrees C with an increased activity of 180% after 3-h incubation. Although silanization has been used for the modification of the supports for immobilization of enzymes, the use of higher concentrations to stabilize immobilized enzymes is being reported for the first time.

Biosensing Techniques↗