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Comparative analysis of bladder wall compliance based on cystometry and biosensor measurements during the micturition cycle of the rat.

The stiffness characteristics of the empty and filling bladder and the modulating influence of oxybutynin were investigated using a new biosensor system. Studies were done comparing the stiffness measured using the pressure/volume relationship with direct biosensor monitoring on male and female rats during isovolumetric contractions elicited during the cystometrogram (CMG). Bladder stiffness at zero volume, measured in vitro using the biosensor, was evaluated and compared with the stiffness of the prostate, seminal vesicles, testicles, and uterus. In 5 small anesthetized male rats, in vivo isovolumetric studies were performed and bladder stiffness was measured during the storage and contraction phase of the CMG. In 6 mature female rats, change in bladder stiffness during isovolumetric contractions was investigated following intraarterial (i.a.) administration of 0.1 and 1.0 mg/kg of oxybutynin. After the in vivo CMG was completed, an in vitro CMG was done measuring bladder stiffness. The results show that bladder stiffness, measured during the storage phase of the CMG, increased in accordance with the stretched length of bladder wall. During the in vivo CMG, bladder stiffness increased consequent to a spontaneous contraction from 10.0 +/- 1.9 g/cm to 29.9 +/- 3.0 g/cm (P < 0.005). Oxybutynin produced a significant decrease in bladder stiffness during the storage phase of the CMG, as measured using the biosensor, which was concomitant with an increase in bladder compliance derived from pressure/volume data. The incremental change in stiffness, delta K, during isovolumetric contraction decreased due to i.a. oxybutynin in accordance with a decrease of maximum detrusor pressure. These results indicate that delta K is related to the active change of viscoelastic properties of bladder smooth muscle. These findings imply that direct measurement of the stiffness of the bladder wall possesses the potential to be an objective assessment of bladder biomechanical properties and of their functional response to obstruction and pharmacological intervention.

Animals↗

Electrogenerated chemiluminescence of luminol for oxidase-based fibre-optic biosensors.

The luminol electrochemiluminescence has been exploited for the development of several fibre-optic biosensors allowing the detection of hydrogen peroxide and of substrates of H(2)O(2)-producing oxidases. Electro-optical flow injection analysis of glucose, lactate, cholesterol and choline are thus described. To perform the experiments, a glassy carbon electrode was polarized at a fixed potential. Luminol was then electrochemically oxidized and could react in the presence of hydrogen peroxide to produce light. Several parameters had to be optimized to obtain reliable optical biosensors. An optimum applied potential of +425 mV between the glassy carbon electrode and the platinum pseudo-reference electrode was determined, allowing the best signal: noise ratio to be obtained. It was also necessary to optimize the experimental conditions for the immobilization of the different oxidases involved (preactivated membranes, chemically activated collagen membranes, photopolymerized matrix). For each biosensor developed, the optimum reaction conditions have been studied: buffer composition, pH, temperature, flow rate and luminol concentration. Under optimal conditions, the detection limits (S/N = 3) were 30 pmol, 60 pmol, 0.6 nmol and 10 pmol for lactate, glucose, cholesterol and choline, respectively. The miniaturization of electrochemiluminescence-based biosensors has been realized using screen-printed electrodes instead of a glassy carbon macroelectrode, with choline oxidase as a model H(2)O(2)-generating oxidase.

Alcohol Oxidoreductases↗

On-line biosensors for simultaneous determination of glucose, choline, and glutamate integrated with a microseparation system.

An effective microseparation system integrated with ring-disc electrodes and two microfluidic devices was fabricated for in vivo determination using a microdialysis pump. The major interference of ascorbic acid (AA) was excluded by direct oxidation with ascorbate oxidase. Glucose, glutamate, and choline were successfully determined simultaneously through the biosensors modified with a bilayer of osmium-poly(4-vinylpyridine)gel-horseradish peroxidase (Os-gel-HRP)/glucose oxidase (GOD), glutamate oxidase (GlutaOD) or choline oxidase (ChOD). To stabilize the biosensors, 0.2% polyethylenimine (PEI) was mixed with the oxidases. The cathodic currents of glucose, glutamate, and choline biosensors started to increase after the standard solutions were injected into the microseparation system. The on-line biosensors show a wide calibration range (10(-7)-10(-5) mol/L) with a detection limit of 10(-8) mol/L at the working potential of -50 mV. The variations of glucose, glutamate, and choline were determined simultaneously in a free moving rat when we perfused the medial frontal cortex with 100 micro mol/L N-methyl-D-aspartate (NMDA) solution, which is the agonist of the NMDA receptor.

Alcohol Oxidoreductases↗

Kinetic characterization of the interaction of biotinylated human interleukin 5 with an Fc chimera of its receptor alpha subunit and development of an ELISA screening assay using real-time interaction biosensor analysis.

The interaction of biotinylated human interleukin 5 ([BT]hIL5) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring an ELISA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5R alpha-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor alpha subunit to the constant domain (Fc) of immunoglobulin G), were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisfactory and led to use of the Fc chimera attached by oriented immobilization via protein A. Hence, shIL5R alpha-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BT]hIL5 to protein A/shIL5R alpha-Fc receptor complex. The binding was high affinity (Kdapp = 6 nM), reversible and saturable. The affinity of [BT]hIL5 was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BT]hIL5 with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing a microtiter plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BT]hIL5 to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Current and emerging commercial optical biosensors.

The field of commercial optical biosensors is rapidly evolving, with new systems and detection methods being developed each year. This review outlines the currently available biosensor hardware and highlights unique features of each platform. Affinity-based biosensor technology, with its high sensitivity, wide versatility and high throughput, is playing a significant role in basic research, pharmaceutical development, and the food and environmental sciences. Likewise, the increasing popularity of biosensors is prompting manufacturers to develop new instrumentation for dedicated applications. We provide a preview of some of the emerging commercial systems that are dedicated to drug discovery, proteomics, clinical diagnostics and routine biomolecular interaction analysis.

Biosensing Techniques↗

High-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions on GST-fusion protein arrays with a spectral surface plasmon resonance biosensor.

We modified gold arrays with a glutathione (GSH) surface, and investigated high-throughput protein interactions with a spectral surface plasmon resonance (SPR) biosensor. We fabricated the GSH exterior on gold surfaces by successive modification with aminoethanethiol, 4-maleimidobutyric acid N-hydroxysuccinimide ester and GSH. We immobilized GST-Rac1, GST-RhoA, the GST-Rho-binding domain of rhotekin and the GST-p21-binding domain of PAK1 onto the GSH surface, and observed specific antigen-antibody interactions on the GST-fusion protein arrays. We determined the expression of GST-fusion proteins in Escherichia coli on the GSH surface with the SPR biosensor. We then analyzed the interactions of tissue transglutaminase (tTGase), a Ca2+-dependent enzyme, with RhoA and Rac1 on the GST-fusion protein arrays with the SPR biosensor. We found that tTGase interacted with RhoA and Rac1 in a Ca2+-dependent manner, indicating that the interactions were dependent on tTGase activity. In addition, transamidation of Rac1 by tTGase was dependent on Ca2+ concentration. We obtained similar results with GST pull-down assays. Thus, protein arrays prepared on the GSH surface provide a useful system for the high-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions with the spectral SPR biosensors.

Apoptosis Regulatory Proteins↗

A cell biosensor specific for formaldehyde based on pH-sensitive transistors coupled to methylotrophic yeast cells with genetically adjusted metabolism.

A cell biosensor specific for formaldehyde was developed using double-mutant cells of the methylotrophic yeast Hansenula polymorpha A3-11. The activities of some of the enzymes in the metabolic pathway of the wild-strain cells were deliberately suppressed by introducing respective genetic blocks to optimize the selectivity and acidification rate. Mutant yeast cells produced in this way were immobilized in Ca-alginate gel on the gate of a pH-sensitive field effect transistor. The local acidification of the extracellular medium due to specific conversion of formaldehyde was recorded. The steady-state response time of the biosensor was 2-3 min, i.e., about 10 times shorter than the response time for the alcohol-specific cell biosensors described earlier. The linear dynamic range of the sensor's response corresponds to formaldehyde concentrations of 2 to 200 mM. The operational stability of the sensor was not less than 4 h. The biosensor demonstrated high specificity to formaldehyde with no response to several organic acids, methanol, and other alcohols, except for low sensitivity to ethanol. The influence of sample buffer capacity and pH on the sensor response, as well as thermostability, was investigated.

Biosensing Techniques↗

Calibration of biosensor response using simultaneous evanescent wave excitation of cyanine-labeled capture antibodies and antigens.

Fiber optic biosensors have proven their ability to detect antigens rapidly in a variety of environmental and clinical samples. These biosensors are based on the technique of covalently linking antibodies to the core of an optical fiber and detecting antigen binding via measurement of fluorescence induced in the evanescent wave. One problem associated with these biosensors is the fiber-to-fiber variability in measured signal. We have addressed this problem by labeling a portion of the immobilized capture antibody with the fluorescent cyanine dye Cy5.5 (emission lambda max = 696 nm). The antigen was then labeled with fluorescent Cy5 (emission lambda max = 668 nm). Both fluorophores were excited by 635-nm light, and their emission was collected using both a fiber optic spectrometer and a biosensor optimized to collect fluorescence at two wave-lengths. The fluorescence from the Cy5.5-labeled capture antibody served as a calibration signal for each fiber and corrected for differences in optics, fiber defects, and varying amounts of capture antibody present on the fiber. Our data show that normalizing the signal measured from Cy5-labeled antigen binding to the Cy5.5 signal provides a standardization process for greatly reducing signal variance among individual fibers.

Animals↗

Use of a resonant mirror biosensor to characterize the interaction of carboxypeptidase A with an elicited monoclonal antibody.

The binding of apocarboxypeptidase A to an immobilized form of its elicited monoclonal antibody has been used to explore the potential of a biosensor instrument (IAsys) based on resonant mirror technology for the quantitative characterization of antibody-antigen interactions. Advantage has been taken of the stirred cuvette design of the IAsys instrument to develop a stepwise titration procedure for thermodynamic characterization of the interaction, an association equilibrium constant of 3.3 (+/-0.9) x 10(7) M-1 having been obtained under the conditions studied (0.1 M Tris/HCl-0.5 M NaCl, pH 7.5, 21 degrees C). In a test of the feasibility of subjecting the time course of biosensor response to conventional pseudo-first-order kinetic analysis, nonconformity of results with such description was encountered at high and low concentrations of apocarboxypeptidase A. Whereas the deviations from Langmuirian kinetic behavior at high antigen concentrations undoubtedly stem from the same sources as those already encountered in studies with the BIA-core biosensor instrument, the deviations at the other concentration extreme occur in a range in which the assumed constancy of free antigen concentration in the liquid phase is becoming a poor approximation. An alternative approach in such circumstances has been tested in which prior thermodynamic characterization is a prerequisite for rate constant evaluation by means of a second-order kinetic analysis. Finally, the effect of soluble anticarboxypeptidase A on the pseudo-first-order kinetics of the biosensor response has been used to illustrate a simple kinetic procedure for evaluating the affinity constant for the antibody-antigen interaction in solution, a value of 1.9 (+/-0.2) x 10(8) M-1 being obtained by such means.

Antibodies, Monoclonal↗

Ferrocene-conjugated m-phenylenediamine conducting polymer-incorporated peroxidase biosensors.

The development and characteristics of a reagentless amperometric biosensor employing horseradish peroxidase incorporated in an electrochemically deposited ferrocene-modified phenylenediamine film on a glassy carbon electrode is reported. The horseradish peroxidase/poly(m-aminoanilinomethylferrocene)- modified glassy carbon electrode reagentless biosensor measured hydrogen peroxide and other organic peroxides in both aqueous and organic medium by reduction at a low applied potential of -0.05 V (vs Ag/AgCl) without interference from molecular oxygen. When modified with glucose oxidase, the new bienzyme electrode measured glucose sensitively and selectively, demonstrating the suitability of the above peroxide biosensor for other oxidoreductase enzyme-based biosensors.

Biosensing Techniques↗

Mediated, amperometric biosensor for glucose-6-phosphate monitoring based on entrapped glucose-6-phosphate dehydrogenase, Mg2+ ions, tetracyanoquinodimethane, and nicotinamide adenine dinucleotide phosphate in carbon paste.

In this study, an amperometric carbon paste biosensor is developed for glucose-6-phosphate (G6P) monitoring which is based on entrapped Mg2+ ions, G6P dehydrogenase, NADP+ polyethylenimine (PEI) and the electroactive mediator, tetracyanoquinodimethane (TCNQ). The calibration line had a slope of 1.55 x 10(-5) A. M-1 with a correlation coefficient of 0.9965. The limit of detection (defined as three times the standard deviation of the response of the electrode to blank phosphate buffer injections (noise)) of the G6P biosensor was 5.0 x 10(-5) M. The application of this biosensor for monitoring G6P in human blood using the standard addition method is also demonstrated. A two-parameter empirical equation which adequately describes the deactivation of the biosensor steady-state response with time is also proposed.

Biosensing Techniques↗

A mathematical analysis using fractals for binding interactions of nuclear estrogen receptors occurring on biosensor surfaces.

A mathematical approach using fractal concepts is presented for modeling the binding and dissociation interactions between analytes and nuclear estrogen receptors (ER) occurring on surface plasmon resonance biosensor chip surfaces. A kinetic knowledge of the binding interactions mediated by ER would help in better understanding the carcinogenicity of these steroidogenic compounds and assist in modulating these reactions. The fractal approach is applied to analyte-ER interaction data obtained from literature. Numerical values obtained for the binding and dissociation rate coefficients are linked to the degree of roughness or heterogeneity (fractal dimension, D(f)) present on the biosensor surface. For example, a single-fractal analysis is used to describe the binding and dissociation phases for the binding of estradiol and ERalpha in solution to clone 31 protein immobilized on a biosensor chip (C-S. Suen et al., 1998, J. Biol. Chem. 273(42), 27645-27653). The binding and the dissociation rate coefficients are 27.57 and 8.813, respectively, and the corresponding fractal dimensions are 1.986 and 2.268, respectively. In some examples dual-fractal models were employed to obtain a better fit of either the association or the dissociation phases or for both. Predictive relationships are developed for (a) the binding and the dissociation rate coefficients as a function of their respective fractal dimensions and (b) the ratio K(A) (= k/k(d)) as a function of the ratio of the fractal dimensions (D(f)/D(fd)). The analysis should provide further physical insights into the ER-mediated interactions occurring on biosensor and other surfaces.

Alitretinoin↗

Fiber optic biosensor using Chlorella vulgaris for determination of toxic compounds.

A new biosensor is constructed for the detection of some herbicides based on kinetic measurements of chlorophyll-a fluorescence in Chlorella vulgaris cells. The microalgae are immobilized on removable membranes placed in front of the tip of an optical fiber bundle inside a homemade microcell. C. vulgaris was easily cultivated in laboratory and very sensitive to herbicides that effect the photosynthesis process. The response of the algal biosensor is studied in terms of detection limits, reversibility, and long-term activity. The effects of temperature and pH are also reported. The biosensor can be used to measure the concentration of a toxic chemical in the form of a single drop or dissolved in a continuous flow. The detection of 0.1 microg small middle dotL(-1) of a single herbicide as is required by European Community legislation for drinking water is possible with this algal biosensor especially for atrazine, simazine, and diuron.

Biosensing Techniques↗

Analyte-Receptor Binding Kinetics for Biosensor Applications: A Single-Fractal and a Dual-Fractal Analysis of the Influence of the Fractal Dimension on the Binding Rate Coefficient.

The diffusion-limited binding kinetics of antigen (analyte) in solution to antibody (receptor) immobilized on a biosensor surface is analyzed within a fractal framework. Most of the data presented are adequately described by a single-fractal analysis. This was indicated by the regression analysis provided by Sigmaplot ("Scientific Graphing Procedure, User's Manual," Jandel Scientific, San Rafael, CA, 1993). A couple of examples of a dual-fractal analysis are also presented. It is of interest to note that the binding rate coefficient and the fractal dimension both exhibit changes in the same direction for the analyte-receptor systems analyzed. Binding rate coefficient expressions as a function of the fractal dimension developed for the analyte-receptor binding systems indicate the high sensitivity of the binding rate coefficient on the fractal dimension when both a single- and a dual-fractal analysis are used. For example, for a single-fractal analysis and for the binding of cell surface proteins from Helicobacter pylori strain in solution to sialyl-(alpha-2,3)-lactose-conjugated (20 mol%) polyacrylamide immobilized on a resonant mirror biosensor (S. Hirmo et al., Anal. Biochem. 257, 63, 1998), the order of dependence of the binding rate coefficient, k, on the fractal dimension, Df, was 14.15. The fractional order of dependence of the binding rate coefficient(s) on the fractal dimension(s) further reinforces the fractal nature of the system. The binding rate coefficient(s) expressions developed as a function of the fractal dimension(s) are of particular value since they provide a means to better control biosensor performance by linking it to the heterogeneity on the surface and further emphasize in a quantitative sense the importance of the nature of the surface in biosensor performance. Copyright 1998 Academic Press.

Journal Article↗

Reusable, real-time, immuno-optical protein C biosensor.

A Protein C (PC) biosensor can be used to diagnose PC deficiency, to monitor the PC level in the blood of PC deficient patients, and to measure the PC concentration in other PC-containing samples, such as PC producing animal cell culture broth or transgenic animal milk. A fully functional biosensor requires extremely high sensitivity and specificity, and real-time measurement. To satisfy these requirements, it is proposed to develop an immuno-optical fiber biosensor that utilizes PC-specific biomolecules (PC probes) tagged with fluorophores. The method involves immobilizing monoclonal antibody against PC (anti-PC) on the surface of an optical fiber. When PC in a sample is adsorbed to the anti-PC on the fiber, it can be reached with the fluorophore tagged PC-probe. The intensity of light transported through the optical fiber, therefore, can be correlated with the concentration of PC in the sample. The sensor will be designed so it can be reused, following a simple elution step, thus reducing diagnostic expense. The preliminary study shows encouraging future for the real-time optical PC biosensor.

Animals↗

Preparation of micro-biosensor and its application in monitoring in vivo change of dopamine.

The self-made high sensitivity and selectivity micro-biosensor was applied to monitor the change of dopamine in cerebral nucleus in rats in vivo. The micro-biosensor was prepared and used to detect dopamine level in vitro and monitor the dynamic change of dopamine in different cerebral nucleus in vivo. The results showed the lowest concentration of dopamine that could be detected by the biosensor was 32.5 nmol/L. Its positive peak was significantly different from that of AA, 5-HTP and E. The biosensor could keep working for monitoring the dopamine concentration in the cerebral tissue for more than 10 h. It was concluded that the microsensor has high sensitivity and selectivity to dopamine and can be used to dynamically monitor the change of dopamine in vivo.

Animals↗

[Evaluation of lactate measurement in blood and plasma with biosensor technology: a comparison of methods].

UNLABELLED: The introduction of biosensor technology for near bedside measurement of plasma lactate concentrations has been a promising step for critical care profiling. However, methodological drawbacks and relevant inaccuracy have been reported. With the advent of a new biosensor (Chiron Diagnostics) and a revised NOVA Biomedical device, accuracy was expected to be improved. The goal of the present investigation was to evaluate the accuracy of both methods. METHODS: Two devices (System 860, Chiron Diagnostics; StatProfile 9, NOVA Biomedical) were simultaneously analysed using 9 biosensors in both fresh frozen plasma and citrated whole blood. The results were compared with an established photometric method (Lactat PAP, Analyticon). Measurements were performed as duplicates (n = 1120) before and after the addition of 1 molar sodium lactate solution (2-24 mmol/L). For the estimation of between-day precision commercially available aqueous and serum-based quality controls were analysed daily over a period of 60 days. RESULTS: Reproducibility in blood was 2.6 +/- 2.8% (Chiron), 4.1 +/- 4.0% (NOVA) and 1.5 +/- 2.1% (Analyticon), in plasma respectively 2.1 +/- 2.4%, 2.1 +/- 2.9% and 1.0 +/- 1.1%. Mean inaccuracy in plasma presented to be -0.2 +/- 16.4% (plasma) and +7.2 +/- 13.1% (blood) for Chiron, +9.4 +/- 18.4% and +18.7 +/- 16.7% for NOVA, and -37.8 +/- 18.2% and -27.5 +/- 17.6% for Analyticon. Calculated between-day-precision (variation coefficients mean values) was 11.5 +/- 4.9% (Chiron) and 14.0 +/- 5.9% (NOVA). CONCLUSION: Although accuracy of lactate concentrations obtained with biosensor technology has improved (mean 0-18%), the variability of the results still poses a problem (mean 13-18%). Therefore, from the methodological point of view, interpretation of a single lactate value requires caution when applying to the critically ill, particularly with view to threshold values, and should be considered vis-à-vis other options.

Biosensing Techniques↗