Assessment of protein phosphatase in a re-usable rapid assay format in detecting microcystins and okadaic acid as a precursor to biosensor development.

The feasibility of developing an immobilised protein phosphatase (PP) biosensor was tested by immobilising PP onto CNBr-activated Sepharose beads placed in Millipore microfilter plate wells. Under optimised immobilised enzyme assay conditions, okadaic acid (OA) and microcystin LR (MC-LR) inhibited Upstate Biotechnology PP (PP-2A), with IC50 values of 12.5 and 11nM respectively. Similarly, immobilised recombinant PP type 1 (rec PP-1) was inhibited by MC-LR and OA, with IC50 values of 150 and >1000nM respectively. The IC50 values for free PP-2A against OA and MC-LR were 2.5 and 3.5nM, and 0.7nM and 200nM for rec PP-1 against the same substrates respectively. For free and immobilised Neptunea arthritic PP (PP-2Ana) against OA the IC50 values were 0.45 and >1000nM respectively. Of the three immobilised enzyme systems, PP-2A showed greatest sensitivity to OA and MC-LR followed by rec PP-1 and PP-2Ana. In assessments for re-usability (determined by removal of > or =70% OA or MC-LR inhibition of PP-2A by washing), <50% of the original activity remained after 20 washings. Including 1M NaCl in the wash buffer did not increase enzyme activity with wash frequency, but rather "salted in" the inhibitor. The LoD of immobilised PP-2A to MC-LR meets the WHO guideline of 1microgl(-1) for drinking water, and the sensitivity to OA (3.5microgl(-1)) would allow detection of DSP during the peak of some phytoplankton blooms.

Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis.

Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313-318 K in a flow injection analysis (FIA) system. The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions. The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD (n = 10) for 5 mM L-glutamate was 7.6%. The response to some potential interferents has been quantified. Addition of finely ground hexaammineruthenium (III) trichloride ([Ru(NH3)6]Cl3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current. The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO.

Biochemical mediator demand--a novel rapid alternative for measuring biochemical oxygen demand.

The biochemical oxygen demand (BOD) test (BOD5) is a crucial environmental index for monitoring organic pollutants in waste water but is limited by the 5-day requirement for completing the test. We have optimised a rapid microbial technique for measuring the BOD of a standard BOD5 substrate (150 mg glucose/l, 150 mg glutamic acid/l) by quantifying an equivalent biochemical mediator demand in the absence of oxygen. Elevated concentrations of Escherichia coli were incubated with an excess of redox mediator, potassium hexacyanoferrate(III), and a known substrate for 1 h at 37 degrees C without oxygen. The addition of substrate increased the respiratory activity of the microorganisms and the accumulation of reduced mediator; the mediator was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. Catabolic conversion efficiencies exceeding 75% were observed for the oxidation of the standard substrate. The inclusion of a mediator allowed a higher co-substrate concentration compared to oxygen and substantially reduced the incubation time from 5 days to 1 h. The technique replicates the traditional BOD5 method, except that a mediator is substituted for oxygen, and we aim to apply the principle to measure the BOD of real waste streams in future work.

The use of microorganisms with broad range substrate utilisation for the ferricyanide-mediated rapid determination of biochemical oxygen demand.

The feasibility of replacing oxygen with a synthetic electron acceptor in microbial catabolism was investigated as a rapid method for the determination of biochemical oxygen demand (BOD). Microorganisms known for their broad range organic substrate utilisation were investigated. It was shown that Trichosporon cutaneum, Pseudomonas putida and Bacillus licheniformis could utilize the ferricyanide ion as an alternative electron acceptor, in place of oxygen, for the catabolic oxidation of a range of simple organic compounds. The biochemical reactions were monitored by measuring the amount of microbially produced ferrocyanide using amperometry at a Pt disk microelectrode. Catabolic degradation efficiencies approaching those of the conventional 5-day assay were achieved in 1 h. BOD(5) equivalent values for a range of simple organic solutions were determined for each of the microorganisms. The effect of increased incubation time and the choice of appropriate calibration standards for rapid BOD assays were also considered.

Detection of two distinct substrate-dependent catabolic responses in yeast cells using a mediated electrochemical method.

Mediated electrochemical detection of catabolism in prokaryotic cells is well documented; however, the application of this technique to eukaryotic cells has received less attention. Two catabolic substrate-dependent mediated electrochemical signals were detected in the yeast Saccharomyces cerevisiae. The signal using a single hydrophilic mediator (ferricyanide) is small whereas the response using a double mediator system comprising a hydrophilic and a lipophilic mediator (ferricyanide and menadione) is up to three orders of magnitude larger. The behaviour of each response during cell ageing is different: the single mediator response increases whereas the double mediator response decreases. This difference indicates that the two signals originate at different points in the catabolic pathways. In S. cerevisiae the double mediator response is proposed to originate from the reduction of the lipophilic mediator by NADPH produced in the pentose phosphate pathway. The single mediator signal arises from reduction of the hydrophilic mediator by an extracellular redox species produced in response to the presence of glucose.