Counting the number of enzymes immobilized onto a nanoparticle-coated electrode.

To immobilize enzymes at the surface of a nanoparticle-based electrochemical sensor is a common method to construct biosensors for non-electroactive analytes. Studying the interactions between the enzymes and nanoparticle support is of great importance in optimizing the conditions for biosensor design. This can be achieved by using a combination of analytical methods to carefully characterize the enzyme nanoparticle coating at the sensor surface while studying the optimal conditions for enzyme immobilization. From this analytical approach, it was found that controlling the enzyme coverage to a monolayer was a key factor to significantly improve the temporal resolution of biosensors. However, these characterization methods involve both tedious methodologies and working with toxic cyanide solutions. Here we introduce a new analytical method that allows direct quantification of the number of immobilized enzymes (glucose oxidase) at the surface of a gold nanoparticle coated glassy carbon electrode. This was achieved by exploiting an electrochemical stripping method for the direct quantification of the density and size of gold nanoparticles coating the electrode surface and combining this information with quantification of fluorophore-labeled enzymes bound to the sensor surface after stripping off their nanoparticle support. This method is both significantly much faster compared to previously reported methods and with the advantage that this method presented is non-toxic. Graphical abstract A new analytical method for direct quantification of the number of enzymes immobilized at the surface of gold nanoparticles covering a glassy carbon electrode using anodic stripping and fluorimetry.

Prevention of urinary tract infections in nursing homes: lack of evidence-based prescription?

BACKGROUND: Urinary tract infections (UTIs, including upper and lower symptomatic) are the most common infections in nursing homes and prevention may reduce patient suffering, antibiotic use and resistance. The spectre of agents used in preventing UTIs in nursing homes is scarcely documented and the aim of this study was to explore which agents are prescribed for this purpose. METHODS: We conducted a one-day, point-prevalence study in 44 Norwegian nursing homes during April-May 2006. Nursing home residents prescribed any agent for UTI prophylaxis were included. Information recorded was type of agent and dose, patient age and gender, together with nursing home characteristics. Appropriateness of prophylactic prescribing was evaluated with references to evidence in the literature and current national guidelines. RESULTS: The study included 1473 residents. 18% (n = 269) of the residents had at least one agent recorded as prophylaxis of UTI, varying between 0-50% among the nursing homes. Methenamine was used by 48% of residents prescribed prophylaxis, vitamin C by 32%, and cranberry products by 10%. Estrogens were used by 30% but only one third was for vaginal administration. Trimethoprim and nitrofurantoin were used as prophylaxis by 5% and 4%, respectively. CONCLUSIONS: The agents frequently prescribed to prevent UTIs in Norwegian nursing homes lack documented efficacy including methenamine and vitamin C. Recommended agents like trimethoprim, nitrofurantoin and vaginal estrogens are infrequently used. We conclude that prescribing of prophylactic agents for UTIs in nursing homes is not evidence-based.

How are antibacterials used in nursing homes? Results from a point-prevalence prescription study in 44 Norwegian nursing homes.

PURPOSE: To describe the use of antibacterials among nursing home residents in Norway according to diagnosis, therapy choice, doses and expected duration of treatment. METHODS: A one-day point-prevalence study was carried out in 44 Norwegian nursing homes, spring 2006. Use of systemic antibacterials was recorded by indication, antibiotic name, dose and expected length of treatment. RESULTS: Of the 1473 nursing home residents, 224 (15%) were prescribed antibiotics. 149 (10%) were given antibiotics as prophylaxis, while 85 residents (6%) were treated with an antibiotic for an infection. 10 residents received both prophylaxis and treatment simultaneously. Antibiotics for treatment were in 66% of the cases for urinary tract infections (UTI) and in 20% for respiratory tract infections (RTI) with pivmecillinam and phenoxymethylpenicillin most frequently used, respectively. CONCLUSIONS: Antibiotics are often used in nursing homes, both as treatment and prophylaxis. The most common infections treated with antibiotics were UTIs, followed by RTIs. Choice of antibacterial, dosage and duration of treatment were in accordance with recommendations in guidelines.

Ultrafast Glutamate Biosensor Recordings in Brain Slices Reveal Complex Single Exocytosis Transients.

Neuronal communication relies on vesicular neurotransmitter release from signaling neurons and detection of these molecules by neighboring neurons. Glutamate, the main excitatory neurotransmitter in the mammalian brain, is involved in nearly all brain functions. However, glutamate has suffered from detection schemes that lack temporal and spatial resolution allowed by electrochemistry. Here we show an amperometric, novel, ultrafast enzyme-based nanoparticle modified sensor, measuring random bursts of hundreds to thousands of rapid spontaneous glutamate exocytotic release events at approximately 30 Hz frequency in the nucleus accumbens of rodent brain slices. Characterizing these single submillisecond exocytosis events revealed a great diversity in spike shape characteristics and size of quantal release, suggesting variability in fusion pore dynamics controlling the glutamate release by cells in this brain region. Hence, this novel biosensor allows recording of rapid single glutamate exocytosis events in the brain tissue and offers insight on regulatory aspects of exocytotic glutamate release, which is critical to understanding of brain glutamate function and dysfunction.

Amperometric detection of single vesicle acetylcholine release events from an artificial cell.

Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells.

Joint medicine-information and pharmacovigilance services could improve detection and communication about drug-safety problems.

BACKGROUND: RELIS is a Norwegian network of four regional medicine-information and pharmacovigilance centers where pharmacists and clinical pharmacologists provide feedback to health care professionals in spontaneous drug-related questions and adverse drug-reaction (ADR) reports published in a question-answer pair (QAP) database (the RELIS database) and the Norwegian ADR database, respectively. OBJECTIVE: To describe the potential of RELIS's dual service to improve detection and communication of drug-safety problems. MATERIALS AND METHODS: We searched the RELIS database for QAPs about ADRs with use of the Norwegian ADR database as a reference. We also searched the Norwegian ADR database for reports that used the RELIS database as a reference. Both searches were limited to the years 2003-2012. We then selected the example of pregabalin and drug abuse after the marketing of Lyrica in Norway in September 2004 to illustrate RELIS's potential to detect new drug-safety information through a limited number of QAPs and ADR reports. RESULTS: A total of 5,427 (26%) of 21,071 QAPs in the RELIS database concerned ADRs. QAPs from this database were used as references in 791 (4%) of a total of 22,090 reports in the Norwegian ADR database. The Norwegian ADR database was used as a reference in 363 (7%) of 5,427 QAPs that concerned ADRs. Between September 2004 and September 2008, RELIS received eleven questions and 13 ADR reports about suspicion of Lyrica (pregabalin) and different aspects of abuse. CONCLUSION: RELIS processes data through two databases that facilitate communication about ADRs. Our service also has the potential to detect new drug-safety problems with a limited number of questions and ADR reports.

Hepatitis caused by Lotus-f3?

We present a case of hepatitis and jaundice are associated with ingestion of Lotus-f3 submitted to our regional pharmacovigilance centre. A 56-year-old woman with psoriatic arthritis developed increased liver enzymes and jaundice 3 weeks after having started to take the product. The woman had been treated with etanercept for more than a year. She was hospitalized with hepatitis, and viral causes were ruled out. Liver biopsy suggested autoimmune or toxic hepatitis. Both etanercept and Lotus-f3 were withdrawn, and 6 weeks later the liver enzymes were normalized without any treatment. Etanercept was subsequently successfully reintroduced, and based on the rapid resolution of the hepatitis, a toxic effect of Lotus-f3 was suggested. This was the first report in the national adverse drug reaction database for this product, but three similar cases have now been reported. Lotus-f3 contains an extract of green tea, which has been associated with hepatotoxicity. The Norwegian adverse drug reaction database contains nine reports of hepatitis or jaundice associated with natural products. Four different natural products containing extracts of green tea have been suspected in eight out of these nine reports.