A selective potentiometric copper (II) ion sensor based on the functionalized ZnO nanorods.

In this work, ZnO nanorods were hydrothermally grown on the gold-coated glass substrate and characterized by field emission scanning electron microscopy (FESEM) and X-ray diffraction (XRD) techniques. The ZnO nanorods were functionalized by two different approaches and performance of the sensor electrode was monitored. Fourier transform infrared spectroscopy (FTIR) was carried out for the confirmation of interaction between the ionophore molecules and ZnO nanorods. In addition to this, the surface of the electrode was characterized by X-ray photoelectron spectroscopy (XPS) showing the chemical and electronic state of the ionophore and ZnO nanorod components. The ionophore solution was prepared in the stabilizer, poly vinyl chloride (PVC) and additives, and then functionalized on the ZnO nanorods that have shown the Nernstian response with the slope of 31 mV/decade. However, the Cu2+ ion sensor was fabricated only by immobilizing the selective copper ion ionophore membrane without the use of PVC, plasticizers, additives and stabilizers and the sensor electrode showed a linear potentiometric response with a slope of 56.4 mV/decade within a large dynamic concentration range (from 1.0 x 10(-6) to 1.0 x 10(-1) M) of copper (II) nitrate solutions. The sensor showed excellent repeatability and reproducibility with response time of less than 10 s. The negligible response to potentially interfering metal ions such as calcium (Ca2+), magnesium (Mg2+), potassium (K+), iron (Fe3+), zinc (Zn2+), and sodium (Na+) allows this sensor to be used in biological studies. It may also be used as an indicator electrode in the potentiometric titration.

Trapping and detection of single molecules in water.

An innovative nanoprobe-based device that can measure and adjust the pH, can mimic biochemistry, can create microscale vortices in water, and can be used to trap single molecules is presented. Because the analytes in question to trap and detect are small in dimensions, we start by presenting scaling issues and challenging limitations for miniaturized chemical nanosensors. Advantages of using nanoprobes e.g., isolated nanowires, as the components in chemical sensing are discussed. How the observation of the physical property can beneficially change with isomorphic scaling is highlighted. Some of the technology-related constrains are presented for specific sensors. Solutions to overcome such problems are also given. Different aspects, e.g., sample size and sensitivity, for chemical sensing at the nanoscale are highlighted.

Ondansetron and teratogenicity in rats: Evidence for a mechanism mediated via embryonic hERG blockade.

The potent hERG channel blocking drug ondansetron is used off-label for treatment of nausea and vomiting in early pregnancy. Some human epidemiological studies have associated ondansetron with fetal cardiovascular defects and orofacial clefts. This study investigated the effects of ondanestron on embryonic heart rhythm of gestational day (GD) 13 rat embryos in vitro and then integrated the results with published animal teratology, and animal and human pharmacokinetic studies to perform a risk evaluation. Ondansetron caused concentration dependent bradycardia and arrhythmia. Cardiovascular malformations in rats occurred at exposures slightly higher than those in early human pregnancy. Together the results suggest that ondansetron can have teratogenic potential in rats and humans mediated via hERG block and severe heart rhythm disturbances in the embryo. The risk may be increased in human pregnancy if additional risk factors are present such as hypokalemia.

The region ion sensitive field effect transistor, a novel bioelectronic nanosensor.

A novel type of bioelectronic region ion sensitive field effect transistor (RISFET) nanosensor was constructed and demonstrated on two different sensor chips that could measure glucose with good linearity in the range of 0-0.6mM and 0-0.3mM with a limit of detection of 0.1 and 0.04 mM, respectively. The sensor is based on the principle of focusing charged reaction products with an electrical field in a region between the sensing electrodes. For glucose measurements, negatively charged gluconate ions were gathered between the sensing electrodes. The signal current response was measured using a low-noise pico ammeter (pA). Two different sizes of the RISFET sensor chips were constructed using conventional electron beam lithography. The measurements are done in partial volumes mainly restricted by the working distance between the sensing electrodes (790 and 2500 nm, respectively) and the influence of electrical fields that are concentrating the ions. The sensitivity was 28 pA/mM (2500 nm) and 830 pA/mM (790 nm), respectively. That is an increase in field strength by five times between the sensing electrodes increased the sensitivity by 30 times. The volumes expressed in this way are in low or sub femtoliter range. Preliminary studies revealed that with suitable modification and control of parameters such as the electric control signals and the chip electrode dimensions this sensor could also be used as a nanobiosensor by applying single enzyme molecule trapping. Hypotheses are given for impedance factors of the RISFET conducting channel.

Determination of heat changes in the proximity of immobilised enzymes with an enzyme thermistor and its use for the assay of metabolites.

A device, the enzyme thermistor, is described which is capable of measuring changes in heat due to enzymic reactions. The sensor, a thermistor, is in direct contact with the site of reaction through its placement in a microcolumn filled with an immobilised enzyme preparation. The substrate solution flows past the thermistor tip, and as much as approx. one half of the total heat evolved can be registered as temperature change, deltat. Glass-bound glucose oxidase (EC 1.1.3.4), penicillinase (EC 3.5.2.6), trypsin (EC 3.4.21.4) and urease (EC 3.5.1.5) were used for the determination of glucose, penicillin G, benzoyl-L-arginine ethyl ester and urea respectively. Linear relationships between the deltat recorded and the concentration of substrate were obtained in all cases.

Induction of rhythm abnormalities in the fetal rat heart. A tentative mechanism for the embryotoxic effect of the class III antiarrhythmic agent almokalant.

OBJECTIVES: The aim was to test the hypothesis that the recently reported embryotoxic effect of class III antiarrhythmic agents may be a result of electrophysiological disturbances induced by these agents. METHODS: Comparative studies of drug effects in the adult and fetal rat were performed using three experimental models: (1) effects of almokalant upon pregnancy and fetal mortality in rats given daily doses of 0, 10, 50, 100, or 400 mumol.kg-1 orally in the diet on days 6-15 of pregnancy; (2) effects of d-sotalol (1-1000 microM), almokalant (0.1-100 microM) and dofetilide (0.01-10 microM) on the adult and fetal cardiac action potential in vitro; (3) voltage clamp recordings in single fetal and adult ventricular myocytes superfused with almokalant (0.5 microM). RESULTS: In the groups of rats treated with 100 and 400 mumol.kg-1, respectively, the body weight gain was decreased from day 12 of gestation, and there were no viable fetuses at termination of pregnancy. In atrial as well as ventricular tissue, the class III agents induced a concentration dependent prolongation of the fetal action potential duration, accompanied by a reduction in heart rate and eventually the appearance of rhythm abnormalities and/or early afterdepolarisations. The adult action potential duration remained unaffected. An almokalant sensitive current (probably the delayed rectifier, IK) could be evoked both in the fetal and in the adult ventricular cells. CONCLUSIONS: Class III antiarrhythmic agents were shown to induce fetal mortality and rhythm abnormalities in the rat heart. Although they do not prove a causal relationship between these effects, our observations may have implications for the clinical use of class III antiarrhythmic agents in women of childbearing potential.

The development and applications of thermal biosensors for bioprocess monitoring.

Enzyme thermistors are biosensors that use thermal resistors to measure the heat change caused by an enzymatic reaction. They combine the selectivity of enzymes with the sensitivity of biosensors and allow continuous analysis in a flow-injection mode. They can be used to monitor fermentation systems, biocatalysis, enzyme-catalysed synthesis and clinical and food technology. This article gives an overview of the general principles of enzyme thermistors, the sampling process and the ongoing developments in the field of bioprocess monitoring.

Class III antiarrhythmics and phenytoin: teratogenicity due to embryonic cardiac dysrhythmia and reoxygenation damage.

Class III antiarrhythmic drugs, like almokalant, dofetilide and ibutilide, cause a spectrum of malformations in experimental teratology studies. The pattern of developmental toxic effects is very similar to those reported for phenytoin, which is an established human and animal teratogen. The toxic effects are characterised by embryonic death, decreased fetal weights, and stage specific malformations, such as distal digital reductions, orofacial clefts and cardiovascular defects. Class III antiarrhythmics decrease the excitability of cardiac cells by selectively blocking the rapid component of the delayed rectified potassium channel (IKr), resulting in prolongation of the repolarisation phase of the action potential. Phenytoin, which decrease the excitability of neurones, has recently also been shown to block IKr, in addition to its known blockade of sodium channels. Animal studies indicate that IKr is expressed in the embryo and that the embryonic heart is extremely susceptible to IKr-blockers during a restricted period in early development. At concentrations not affecting the maternal heart, the embryonic heart reacts with bradycardia, arrhythmia and cardiac arrest when exposed to such drugs. Available studies strongly support the idea that birth defects after in utero exposure to both selective and non-selective IKr-blockers (like phenytoin) are initiated by concentration dependent embryonic bradycardia/arrhythmia resulting in 1) hypoxia; explaining embryonic death and growth retardation, 2) episodes of severe hypoxia, followed by generation of reactive oxygen species within the embryo during reoxygenation, causing orofacial clefts and distal digital reductions, and 3) alterations in embryonic blood flow and blood pressure, inducing cardiovascular defects.

Thermistor-based biosensing.

In this review a universal thermistor-based biosensor system is described with examples from clinical chemistry, bioprocess monitoring and environmental control. The technique is based on the measurement of the small temperature changes associated with enzymatic reactions occurring in a microreactor with immobilized enzyme. The system has good operational stability and a sensitivity that permits measurements down to 1 microM concentrations. Current developments include devices constructed by micromachining for multisensing purposes and miniaturised instrumentation intended for use in portable monitoring. With use of special supports for enzyme immobilisation even untreated whole blood samples can be applied. Another current line of investigation involves hybrid biosensors, such as combinations of electrochemistry and calorimetry into bioelectrocalorimetric devices with interesting new properties.

Drugs during pregnancy: an issue of risk classification and information to prescribers.

The Swedish system for the classification of fetal risk of drugs was the first of its kind and was implemented in 1978. Drugs for use in pregnant women are classified in 4 general categories--A to D. The US Food and Drug Administration (FDA) introduced a system in 1979 also using the letters A to D, together with an X category. However, the definitions differ considerably between the FDA system and the Swedish system, resulting in a very different allocation of drugs to the respective categories. In the Swedish system, category A includes drugs that have been extensively used and/or for which there are reliable clinical data indicating no evidence of disturbance of the reproductive process. Category B includes drugs for which data from pregnant women are insufficient for making any solid estimation of human teratogenic risk, and classification is therefore based on animal data, with allocation to 3 subgroups. For products in category C, the pharmacological action of the drug may have undesirable effects on the human fetus or newborn infant. Finally, category D contains drugs for which human data indicate an increased incidence of malformations. The categorisation statement is always followed by a short explanatory text. In contrast to the FDA system, the Swedish system has been well accepted, as judged by an interview study including 934 physicians and pharmacists. We believe that much of the American dissatisfaction may be a consequence of shortcomings in the category definitions of the FDA system. The FDA system requires an unrealistically high quality of data, e.g. the availability of controlled studies in pregnant women that fail to demonstrate a risk to the fetus are needed for a drug to be assigned to category A. Consequently, the majority of drugs on the US market are allocated to category C, interpreted as 'risk cannot be ruled out'. The distribution of drugs into the various categories is thus very different between the Swedish and FDA systems. We think that the issue of this debate reflects a fundamental problem related to public health information: how should a large, compounded, changing and difficult to evaluate databank be organised before it is made available to professionals and secondarily to lay people?

Principles and applications of thermal biosensors.

A review of thermistor-based calorimetric measurement is presented. The principles of thermometric measurements are highlighted in the introduction followed by the instrumentation, materials and methods. Various applications relating to enzyme activity measurements, clinical monitoring, process monitoring, multianalyte determination, hybrid sensing, environmental monitoring, non-aqueous measurements and other miscellaneous applications are described. A brief note on future developments and a detailed reference list is also included.

Implementation of a thermal biosensor in a process environment: on-line monitoring of penicillin V in production-scale fermentations.

The production of penicillin V was monitored in 0.5 m3 and 160 m3 bioreactors. The thermal biosensor was an enzyme thermistor modified for split-flow analysis. The heat signal generated in the enzyme column was corrected for any nonspecific heat with the use of an identical but inactive reference column. The on-line monitoring was performed in the fermentation pilot plant and in a fermentation plant of Novo Nordisk A/S. Immobilized beta-lactamase was used to monitor three consecutive 0.5 m3 penicillin fermentations. Broth samples were continuously filtered through a tangential flow filtration unit in a sterile external loop. The on-line penicillin V values were 10% higher than those obtained by off-line HPLC analysis. Alternatively a polypropylene filtration probe was inserted into a 160 m3 bioreactor and samples were withdrawn at 0.5 ml/min. The same experiments were repeated with purified and immobilized penicillin V acylase. The on-line penicillin V values obtained with this enzyme correlated very well with those from HPLC analysis. The on-line monitoring was controlled and analysed by a software program written in Labtech Notebook.

Surface plasmon resonance based pesticide assay on a renewable biosensing surface using the reversible concanavalin A monosaccharide interaction.

A competitive immunoassay based on surface plasmon resonance (SPR) for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The novelty of the assay is based on the regeneration of the chip surface by the reversible interaction between monosaccharide (D-glucose) and lectin (Concanavalin A). Concanavalin A-2,4-D conjugate was chemically synthesized, purified and used for binding to the SPR chip modified with covalently bound alpha-D-glucose. The interaction between anti-2,4-D antibody and the surface-bound concanavalin A-2,4-D conjugate was monitored by surface plasmon resonance and the response was used for the quantification of 2,4-D. The dynamic range of the calibration curve was between 3 and 100 ng/ml. The demonstrated principle of surface regeneration based on the reversible sugar-lectin interaction may be of more general applicability in immunoassays.

Helicobacter pylori vacuolating cytotoxin binding to a putative cell surface receptor, heparan sulfate, studied by surface plasmon resonance.

The Helicobacter pylori vacuolating cytotoxin or VacA toxin is a major virulence factor in H. pylori infection and type B gastritis. We predicted heparin/heparan sulfate (H/HS) binding properties of the 58-kDa subunit of VacA cytotoxin using bioinformatics tools and showed this by surface plasmon resonance (SPR)-based biosensor studies. Putative H/HS binding peptides were synthesized and binding to HS was shown by SPR in the absence or presence of trifluoroethanol. We found that a recombinant cytotoxin VacA polypeptide binds to surface-immobilized HS and propose that HS might be a receptor/co-receptor for H. pylori VacA cytotoxin.

Occurrence of endotoxin in dialysis fluid from 39 dialysis units.

Endotoxin exposure during haemodialysis may cause acute and chronic adverse reactions. In order to estimate the risk to the patient, samples of dialysis fluid from 39 of the 45 dialysis units in Sweden were analysed by the chromogenic Limulus amoebocyte lysate assay. Higher levels were obtained after the usual weekend shutdowns. The length of the tubing delivering the reverse osmosis water seemed to influence the extent of contamination. Fifty-nine percent of the units showed low mean endotoxin levels (i.e. mean concentration below the recommended limit in Sweden: < 25 ng l-1), while 18% of units had high levels (mean concentration > 100 ng l-1).

Evaluation of a miniaturized thermal biosensor for the determination of glucose in whole blood.

A miniaturized thermal biosensor has been evaluated as part of a flow-injection analysis system for the determination of glucose in whole blood. Glucose was determined by measuring the heat evolved when samples containing glucose passed through a small column with immobilized glucose oxidase and catalase. Samples of whole blood (1 microliter) can be measured directly, without any pretreatment. The correlation in the response between the thermal biosensor, the Reflolux S meter (Boehringer Mannheim), the Granutest 100 glucose test kit (Merck Diagnostica) and the Ektachem (Kodak) instrument was evaluated. The influence of the hematocrit value and of possible interferences is reported. The correlation measurements show that the thermal biosensor calibrated with aqueous glucose standards generally gives lower values on blood glucose than the reference methods calibrated for serum or blood measurements. Mean negative biases range from 0.53 to 1.16 mmol/l. Differences in sample treatment clearly complicate comparisons and the proper choice of reference method. There was no influence from substances such as ascorbic acid (0.11 mmol/l), uric acid (0.48 mmol/l), urea (4.3 mmol/l) and acetaminophen (0.17 mmol/l) on the response to 5 mmol/l glucose. The hematocrit value does not influence the glucose determination, for hematocrit values of between 13 and 53%.

Enzyme thermistor determination of glucose in serum using immobilized glucose oxidase.

An enzyme thermistor assay for serum glucose is described. The glucose present in the sample is reacted in a small column containing glucose oxidase immobilized to controlled pore glass (single thermistor device). The heat produced in the primary reaction is measured directly in the column without any need for coupling reactions. The useful linear range is 0.01-0.45 mM glucose, permitting 50-fold dilution of serum samples. Advantages are low enzyme cost, due to the immobilization, insensitivity for the color or any turbidity of the sample, and no requirement for coenzyme or any ancillary reaction. Improved sensitivity and extended linear range (0.01--0.9 mM) can be attained through a secondary reaction using catalase. The application to glucose analysis of a split-flow enzyme thermistor equipped with a reference column to eliminate unspecific heat effects is also described. The enzyme thermistor determinations were also compared with a spectrophotometric continuous flow technique using a small column with immobilized glucose oxidase and 4-aminoantipyrine and phenol as color reagents.

An enzyme thermistor-based assay for total and free cholesterol.

A method to evaluate the free (FC) and total cholesterol (TC) in human serum, bile and gallstone extract using an enzyme thermistor (ET)-based flow injection analysis (FIA) is presented. The cholesterol in high-density (HDL-C) and low density lipoprotein (LDL-C) have also been evaluated. A heparin functionalized Sepharose column was employed for the isolation of HDL and LDL fractions from serum. The estimation of cholesterol and its esters was based on their reaction with cholesterol oxidase (CO), cholesterol esterase (CE) and catalase (CAT). Three different enzyme columns, i.e. co-immobilized CO/CAT (column A), only CE (column B) and co-immobilized CO/CE/CAT (column C) were prepared by cross-linking the enzymes on glass beads using glutaraldehyde. Column A was used for estimating FC and column C was used for estimating total cholesterol (cholesterol plus esterified cholesterol). Column B was used as a pre-column which could be switched 'in' or 'out' in conjunction with column A for the estimation of TC or FC, respectively. A calibration between 1.0 and 8.0 mmol/l for FC and 0. 25 and 4.0 mmol/l for TC was obtained. For more than 2000 assays with the ET device a C.V. of less than 4% was obtained. The assay time was approximately 4 min per assay. The cholesterol estimations on the ET correlated well with similar estimations using a commercially available cholesterol diagnostic kit.

Use of an enzyme thermistor for semi-continuous blood glucose measurements.

A method to monitor glucose in whole blood is presented. The aim of the project was to develop a prototype for a bedside monitor system for semi-continuous monitoring of the blood glucose concentration, requiring only one calibration. This was made possible by using the special advantage of the thermal sensor technique in combination with the adjustment of flow. The glucose concentration was determined from the difference between the sensor response and an estimated background signal. Using standard addition technique, calibration factors for background and sensitivity were set and remained unchanged during the monitoring. The background signal was 45 +/- 8 mV (mean +/- S.D., n = 8) and the sensitivity was 28 +/- 1 mV/mmol (mean +/- S.D., n = 4). Recovery in whole blood was 90-98% (mean 94%, n = 12). With an injection interval of 3 min the precision with the sensor was < 3% over more than 100 blood samples. Response time was about 60 s. The calculated glucose values correlated, r = 0.98, with the values obtained with an YSI glucose analyser (Yellow Springs Instruments. Yellow Springs, OH, USA), over the range 2-20 mmol/l.

Zonal ultracentrifugation of plasma lipoproteins from normal and cholestatic pigs.

The plasma lipoproteins in normal and cholestatic pigs were isolated by zonal ultracentrifugation and studied with respect to apoprotein and lipid composition. In contrast with the distribution in human plasma, only one HDL-population but two LDL-populations were demonstrated. No cholestatic lipoprotein similar to the human lipoprotein X was observed. HDL and the lighter LDL component were largely unchanged in cholestasis. The immunochemical properties were changed to some extent. Thus HDL-reacting material was found in the heavier LDL-component and VLDL after cholestasis but not before.