A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

Transport Effects on Multiple-Component Reactions in Optical Biosensors

Optical biosensors are often used to measure kinetic rate constants associated with chemical reactions. Such instruments operate in the surface–volume configuration, in which ligand molecules are convected through a fluid-filled volume over a surface to which receptors are confined. Currently, scientists are using optical biosensors to measure the kinetic rate constants associated with DNA translesion synthesis—a process critical to DNA damage repair. Biosensor experiments to study this process involve multiple interacting components on the sensor surface. This multiple-component biosensor experiment is modeled with a set of nonlinear integrodifferential equations (IDEs). It is shown that in physically relevant asymptotic limits these equations reduce to a much simpler set of ordinary differential equations (ODEs). To verify the validity of our ODE approximation, a numerical method for the IDE system is developed and studied. Results from the ODE model agree with simulations of the IDE model, rendering our ODE model useful for parameter estimation.     

Thermodynamic analysis of protein interactions with biosensor technology

A methodology using biosensor technology for combined kinetic and thermodynamic analysis of biomolecular interactions is described. Rate and affinity constants are determined with BIAcore. Thermodynamics parameters, changes in free energy, enthalpy and entropy, are evaluated from equilibrium data and by using rate constants and transition state theory. The methodology using van't Hoff theory gives complementary information to microcalorimetry, since only the direct binding is measured with BIAcore whereas microcalorimetry measures all components, including e.g. hydration effects. Furthermore, BIAcore gives possibilities to gain new information by thermodynamic analysis of the rate constants.     

Application of cell culture toxicity tests to the development of implantable biosensors

Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established. The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers. A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers. However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate, buffer, and a non-toxic sensor was readily obtained.     

A biosensor for the detection of gas toxicity using a recombinant bioluminescent bacterium

A whole-cell biosensor was developed for the detection of gas toxicity using a recombinant bioluminescent Escherichia coli harboring a lac::luxCDABE fusion. Immobilization of the cells within LB agar has been done to maintain the activity of the microorganisms and to detect the toxicity of chemicals through the direct contact with gas. Benzene, known as a representative volatile organic compound, was chosen as a sample toxic gas to evaluate the performance of this biosensor based on the bioluminescent response. This biosensor showed a dose-dependent response, and was found to be reproducible. The immobilizing matrices of this biosensor were stored at 4 degrees C and were maintained for at least a month without any noticeable change in its activity. The optimal temperature for sensing was 37 degrees C. A small size of this sensor kit has been successfully fabricated, and found to be applicable as a disposable and portable biosensor to monitor the atmospheric environment of a workplace in which high concentrations of toxic gases could be discharged.     

A New,Sensitive Marine Microalgal Recombinant Biosensor Using Luminescence Monitoring for Toxicity Testing of Antifouling Biocides

In this study, we propose the use of the marine green alga Ostreococcus tauri, the smallest free-living eukaryotic cell known to date, as a new luminescent biosensor for toxicity testing in the environment. Diuron and Irgarol 1051, two antifouling biocides commonly encountered in coastal waters, were chosen to test this new biosensor along with two degradation products of diuron. The effects of various concentrations of the antifoulants on four genetic constructs of O. tauri (based on genes involved in photosynthesis, cell cycle, and circadian clock) were compared using 96-well culture microplates and a luminometer to automatically measure luminescence over 3 days. This was compared to growth inhibition of O. tauri wild type under the same conditions. Luminescence appeared to be more sensitive than growth inhibition as an indicator of toxicity. Cyclin-dependent kinase (CDKA), a protein involved in the cell cycle, fused to luciferase (CDKA-Luc) was found to be the most sensitive of the biosensors, allowing an accurate determination of the 50% effective concentration (EC₅₀) after only 2 days (diuron, 5.65 ± 0.44 μg/liter; Irgarol 1015, 0.76 ± 0.10 μg/liter). The effects of the antifoulants on the CDKA-Luc biosensor were then compared to growth inhibition in natural marine phytoplankton. The effective concentrations of diuron and Irgarol 1051 were found to be similar, indicating that this biosensor would be suitable as a reliable ecotoxicological test. The advantage of this biosensor over cell growth inhibition testing is that the process can be easily automated and could provide a high-throughput laboratory approach to perform short-term toxicity tests. The ability to genetically transform and culture recombinant O. tauri gives it huge potential for screening many other toxic compounds.     

Silicon-based microfabricated microbial fuel cell toxicity sensor

Microbial fuel cells (MFCs) have been used for several years as biosensors for measuring environmental parameters such as biochemical oxygen demand and water toxicity. The present study is focused on the detection of toxic matter using a novel silicon-based MFC. Like other existing toxicity sensors based on MFCs, this device is capable of detecting the variation on the current produced by the cell when toxic compounds are present in the medium. The MFC approach presented in this work aims to obtain a simple, compact and planar device for its further application as a biosensor in the design and fabrication of equipment for toxicity monitoring. It consists on a proton exchange membrane placed between two microfabricated silicon plates that act as current collectors. An array of square 80 μm × 80 μm vertical channels, 300 μm deep, have been defined trough the plates over an area of 6 mm × 6 mm. The final testing assembly incorporates two perspex pieces positioned onto the plates as reservoirs with a working volume of 144 μL per compartment. The operation of the microdevice as a direct electron transfer MFC has been validated by comparing its performance against a larger scale MFC, run under the same conditions. The device has been tested as a toxicity sensor by setting it at a fixed current while monitoring changes in the output power. A drop in the power production is observed when a toxic compound is added to the anode compartment. The compact design of the device makes it suitable for its incorporation into measurement equipment either as an individual device or as an array of sensors for high throughput processing.     

Development of amperometric α-ketoglutarate biosensor based on ruthenium-rhodium modified carbon fiber enzyme microelectrode

A rapid and highly sensitive miniaturized amperometric biosensor for the detection of α-ketoglutarate (α-KG) based on a carbon fiber electrode (CFE) is presented. The biosensor is constructed by immobilizing the enzyme, glutamate dehydrogenase (GLUD) on the surface of single carbon fiber modified by co-deposition of ruthenium (Ru) and rhodium (Rh) nanoparticles. SEM and EDX shed useful insights into the morphology and composition of the modified microelectrode. The mixed Ru/Rh coating offers a greatly enhanced electrocatalytic activity towards the detection of β-nicotinamide adenine dinucleotide (NADH), with a substantial decrease in overpotential of ～ 400 mV compared to the unmodified CFE. It also imparts higher stability with minimal surface fouling, common to NADH oxidation. Further modification with the enzyme, GLUD leads to effective amperometric biosensing of α-KG through monitoring of the NADH consumption. A very rapid response to dynamic changes in the α-KG concentrations is observed with a response time of 6s. The current response is linear between 100 and 600 μM with a sensitivity of 42 μAM(-1) and a detection limit of 20 μM. This proof of concept study indicates that the GLUD-Ru/Rh-CFE biosensor holds great promise for real-time electrochemical measurements of α-KG.     

Enhancement in the sensitivity of a gas biosensor by using an advanced immobilization of a recombinant bioluminescent bacterium

A genetically engineered bioluminescent bacterium (lac::luxCDABE) was immobilized to develop a whole cell biosensor for the detection of toxic gaseous chemicals. The toxicity of chemicals can be evaluated through the bioluminescent reaction as it reduces in intensity when the cells experience toxic or lethal conditions. This whole cell biosensor was fabricated, using an immobilization technique utilizing solid agar medium, for the measurement of toxicity through direct contact of the cells with the gas. To enhance the sensitivity of the biosenor, glass beads were used and the thickness of the agar layer was reduced. The bioluminescent response was measured using a fiber optic probe connected between the biosensor kit and a luminometer. As sample gaseous toxic chemicals, BTEX (Benzene, Toluene, Ethylbenzene, and Xylene) gases were selected and their vapors were produced by a gas generation system. The concentrations of the gaseous chemicals injected into the chamber were controlled by the time of exposure and were measured using a portable gas chromatograph (Allstech., USA). Additions of glass beads facilitated gas diffusion through the solid medium, making the biosensor more sensitive. In addition, a thinner matrix layer was more advantageous for the detection of gas toxicity.     

Fabrication of a highly sensitive penicillin sensor based on charge transfer techniques

A highly sensitive penicillin biosensor based on a charge-transfer technique (CTTPS) has been fabricated and demonstrated in this paper. CTTPS comprised a charge accumulation technique for penicilloic acid and H(+) ions perception system. With the proposed CTTPS, it is possible to amplify the sensing signals without external amplifier by using the charge accumulation cycles. The fabricated CTTPS exhibits excellent performance for penicillin detection and exhibit a high-sensitivity (47.852 mV/mM), high signal-to-noise ratio (SNR), large span (1445 mV), wide linear range (0-25 mM), fast response time (<3s), and very good reproducibility. A very lower detection limit of about 0.01 mM was observed from the proposed sensor. Under optimum conditions, the proposed CTTPS outstripped the performance of the widely used ISFET penicillin sensor and exhibited almost eight times greater sensitivity as compared to ISFET (6.56 mV/mM). The sensor system is implemented for the measurement of the penicillin concentration in penicillin fermentation broth.     

S-system-based analysis of the robust properties common to many biochemical network models

Robustness is a key feature to characterize the adaptation of organisms to changes in their internal and external environments. A broad range of kinetic or dynamic models of biochemical systems have been developed. Robustness analyses are attractive for exploring some common properties of many biochemical models. To reveal such features, we transform different types of mathematical equations into a standard or intelligible formula and use the multiple parameter sensitivity (MPS) to identify some factors critically responsible for the total robustness to many perturbations. The MPS would be determined by the top quarter of the highly sensitive parameters rather than the single parameter with the maximum sensitivity. The MPS did not show any correlation to the network size. The MPS is closely related to the standard deviation of the sensitivity profile. A decrease in the standard deviation enhanced the total robustness, which shows the hallmark of distributed robustness that many factors (pathways) involve the total robustness.     

Three-dimensional interdigitated electrode array as a transducer for label-free biosensors

A new transducer for biosensor applications has been developed based on a three-dimensional interdigitated electrode array (IDEA) with electrode digits separated by an insulating barrier. Binding of molecules to a chemically modified surface of the transducer induces important changes in conductivity between the electrodes. Three-dimensional sensor shows considerable improvement compared with a standard planar IDEA design. The potential of the developed device as a sensor transducer to detect immunochemical and enzymatic reactions, as well as DNA hybridization events is demonstrated. The immunosensor allows direct detection of the antibiotic sulfapyridine and shows the IC(50) parameter value of 5.6 microgL(-1) in a buffer. Immunochemical determination occurs under competitive configurations and without the use of any label. Each modified sensor is of a single use. Nevertheless, biochemical reagents can be easily cleaned off the sensor surface for its reuse. Layer-by-layer method of used to deposit polyethyleneimine and glucose oxidase showed that the sensor is also highly effective for detecting single and multilayered molecular assemblies.     

Time-dependent outward current in guinea pig ventricular myocytes. Gating kinetics of the delayed rectifier

Several conflicting models have been used to characterize the gating behavior of the cardiac delayed rectifier. In this study, whole-cell delayed rectifier currents were measured in voltage-clamped guinea pig ventricular myocytes, and a minimal model which reproduced the observed kinetic behavior was identified. First, whole-cell potassium currents between -10 and +70 mV were recorded using external solutions designed to eliminate Na and Ca currents and two components of time-dependent outward current were found. One component was a La3(+)-sensitive current which inactivated and resembled the transient outward current described in other cell types; single-channel observations confirmed the presence of a transient outward current in these guinea pig ventricular cells (gamma = 9.9 pS, [K]o = 4.5 mM). Analysis of envelopes of tail amplitudes demonstrated that this component was absent in solutions containing 30-100 microM La3+. The remaining time-dependent current, IK, activated with a sigmoidal time course that was well-characterized by three time constants. Nonlinear least-squares fits of a four-state Markovian chain model (closed - closed - closed - open) to IK activation were therefore compared to other models previously used to characterize IK gating: n2 and n4 Hodgkin-Huxley models and a Markovian chain model with only two closed states. In each case the four-state model was significantly better (P less than 0.05). The failure of the Hodgkin-Huxley models to adequately describe the macroscopic current indicates that identical and independent gating particles should not be assumed for this K channel. The voltage-dependent terms describing the rate constants for the four-state model were then derived using a global fitting approach for IK data obtained over a wide range of potentials (-80 to +70 mV). The fit was significantly improved by including a term representing the membrane dipole forces (P less than 0.01). The resulting rate constants predicted long single-channel openings (greater than 1 s) at voltages greater than 0 mV. In cell-attached patches, single delayed rectifier channels which had a mean chord conductance of 5.4 pS at +60 mV ([K]o = 4.5 mM) were recorded for brief periods. These channels exhibited behavior predicted by the four-state model: long openings and latency distributions with delayed peaks. These results suggest that the cardiac delayed rectifier undergoes at least two major transitions between closed states before opening upon depolarization.     

Platelet oxygen consumption as a peripheral blood marker of brain energetics in a mouse model of severe neurotoxicity

Interactions of chemicals with cerebral cellular systems are often accompanied by similar changes involving components in non-neural tissues. On this basis, indirect strategies have been developed to investigate neural cell function parameters by methods using accessible cells, including platelets and/or peripheral blood lymphocytes. Therefore, here it was investigated whether peripheral blood markers may be useful for assessing the central toxic effects of methylmercury (MeHg). For this purpose, we investigated platelet mitochondrial physiology in a well-established mouse model of MeHg-induced neurotoxicity, and correlated this peripheral activity with behavioural and central biochemical parameters. In order to characterize the cortical toxicity induced by MeHg (20 and 40 mg/L in drinking water, 21 days), the behavioral parameter namely, short-term object recognition, and the central mitochondrial impairment assessed by measuring respiratory complexes I-IV enzyme activities were determined in MeHg-poisoned animals. Neurotoxicity induced by MeHg exposure provoked compromised cortical activity (memory impairment) and reduced NADH dehydrogenase, complex II and II-III activities in the cerebral cortex. These alterations correlated with impaired systemic platelet oxygen consumption of intoxicated mice, which was characterized by reduced electron transfer activity and uncoupled mitochondria. The data brought here demonstrated that impaired systemic platelet oxygen consumption is a sensitive and non-invasive marker of the brain energy deficits induced by MeHg poisoning. Finally, brain and platelets biochemical alterations significantly correlated with cognitive behavior in poisoned mice. Therefore, it could be proposed the use of platelet oxygen consumption as a peripheral blood marker of brain function in a mouse model MeHg-induced neurotoxicity.     

Kinetic characterization of TAR RNA-Tat peptide and neomycin interactions by acoustic wave biosensor

The kinetics of binding of short Tat peptides and an aminoglycoside molecule to the human immunodeficiency virus-type 1(HIV-1) TAR RNA and to a bulge mutant analogue (MTAR) is studied in a biosensor format by monitoring the time course of the response in a series resonance frequency, using an acoustic wave biosensor. Association and dissociation rate constants are evaluated by fitting the experimental data to a simple 1:1 (Langmuir) model. Kinetic rate and equilibrium dissociation constants show that MTAR-peptide complexes are subject to a higher dissociation rate and are less stable compared to the corresponding TAR-peptide complexes. In addition, longer peptides display enhanced discrimination ability than a shorter peptide according to the equilibrium dissociation constants evaluated using this technique. K(D) values for TAR-Tat vs. MTAR-Tat complexes are 2.6 vs. 3.8 microM for Tat-12, 0.87 vs. 4.3 microM for Tat-18 and 0.93 vs. 1.6 microM for Tat-20. The equilibrium dissociation constant for TAR-neomycin complex is 12.4 microM and it is comparable to the values obtained from non-biosensor type assays. These findings are in parallel with those cited in the literature and the results from this study underline the potential of the acoustic wave sensor for detailed biophysical analysis of nucleic acid-ligand binding.     

Robustness,flexibility, and the role of lateral inhibition in the neurogenic network

BACKGROUND: Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? We showed previously that a model of the segment polarity network in Drosophila is robust to parameter variation and is likely to act as a semiautonomous patterning module. Is this true of other networks as well? RESULTS: We present a model of the core neurogenic network in Drosophila. Our model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, we find that it exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms our test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the network's repertoire. We introduce two new measures for quantifying network robustness that mimic recombination and allelic divergence and use these to reveal the shape of the domain in the parameter space in which the model functions. We show that lateral inhibition yields robustness to changes in prepatterns and suggest a reconciliation of two divergent sets of experimental results. Finally, we show that, for this model, robustness confers functional flexibility. CONCLUSIONS: The neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. Our model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification.     

Co-immobilized microbial biosensor for BOD estimation based on sol-gel derived composite material

A novel type of biochemical oxygen demand (BOD) biosensor was developed for water monitor, based on co-immobilizing of Trichosporon cutaneum and Bacillus subtilis in the sol-gel derived composite material which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)). Factors that influence the performance of the resulting biosensor were examined. The biodegradable substrate spectrum could be expanded by the co-immobilized microorganisms. The biosensor prepared also exhibited good reproducibility and long-term stability. Good agreement was obtained between the results of the sensor BOD measurement and those obtained from conventional BOD(5) method for water samples.     

Description of Na, K-ATPase activation by monovalent cations using a simplified mathematical model

A simple mathematic model describing the activation Na,K-ATPase system by univalent cations is proposed. The constants for the enzyme activation values by each of the ions in the presence of a fixed concentration of the other ion have been calculated. The substitution of these values into the common equation describing the behaviour of the whole system according to the given model gives the curve of Na,K-ATPase activity change in dependence of Na/K ration at the same total concentration 150 mM. The experimental points correspond to the curve.     

Robustness Analysis of Stochastic Biochemical Systems

We propose a new framework for rigorous robustness analysis of stochastic biochemical systems that is based on probabilistic model checking techniques. We adapt the general definition of robustness introduced by Kitano to the class of stochastic systems modelled as continuous time Markov Chains in order to extensively analyse and compare robustness of biological models with uncertain parameters. The framework utilises novel computational methods that enable to effectively evaluate the robustness of models with respect to quantitative temporal properties and parameters such as reaction rate constants and initial conditions. We have applied the framework to gene regulation as an example of a central biological mechanism where intrinsic and extrinsic stochasticity plays crucial role due to low numbers of DNA and RNA molecules. Using our methods we have obtained a comprehensive and precise analysis of stochastic dynamics under parameter uncertainty. Furthermore, we apply our framework to compare several variants of two-component signalling networks from the perspective of robustness with respect to intrinsic noise caused by low populations of signalling components. We have successfully extended previous studies performed on deterministic models (ODE) and showed that stochasticity may significantly affect obtained predictions. Our case studies demonstrate that the framework can provide deeper insight into the role of key parameters in maintaining the system functionality and thus it significantly contributes to formal methods in computational systems biology.     

Application of luminescent biosensors for monitoring the degradation and toxicity of BTEX compounds in soils

Aims:  To assess the changes in acute toxicity and biodegradation of benzene, toluene, ethylbenzene and xylene (collectively referred to as BTEX) compounds in soil over time and compare the performances of biological and chemical techniques.
Methods and Results:  Biological methods ( lux -based bacterial biosensors, basal respiration and dehydrogenase activity) were related to changes in the concentration of the target compounds. There was an initial increase in toxicity determined by the constitutively expressed biosensor, followed by a continual reduction as degradation proceeded. The biosensor with the BTEX-specific promoter was most induced when BTEX concentrations were highest. The treatment with nutrient amendment had a significant increase in microbial activity, while the sterile control produced the lowest level of degradation.
Significance and Impact of the Study:  Luminescent biosensors were able to monitor changes in contaminant toxicity and bioavailability in aqueous extracts from BTEX-impacted soils as degradation proceeded. The integration of biological tests with chemical analysis enables a fuller understanding of the biodegradation processes occurring at their relative rates.
Conclusions:  The biological methods were successfully used in assessing the performance of different treatments for enhancing natural attenuation of BTEX from contaminated soils. While, chemical analysis showed biodegradation of parent BTEX compounds in biologically active soils, the biosensor assays reported on changes in bioavailability and potentially toxic intermediate fractions as they estimated the integrative effect of contaminants.