Parallel plate equipment for preparation of uniform gel sheets.

A method for the preparation of uniform gel-disks for enzyme and cell immobilisation, as well as for characterisation of gel mechanical stability, is described. The apparatus comprises a stainless steel base unit and glass parallel plates, designed to permit easy and fast production of multiple homogeneous gel sheets of variable thickness.

Acetylecholinesterase-based biosensor electrodes for organophosphate pesticide detection. II. Immobilization and stabilization of acetylecholinesterase.

The dry and wet stability of Drosophila acetylcholinesterase non-covalently immobilized onto polyethyleneimine modified screen-printed carbon electrodes was improved when compared to non-immobilized acetylcholinesterase, and acetylcholinesterase covalently immobilized onto dialdehyde and polyethyleneimine modified electrodes. Stabilizer mixtures were characterized for additional stabilization of acetylcholinesterase during storage in the dry state, with dextran-sulphate/sucrose and polygalacturonic acid/sucrose mixtures proving highly effective for long-term storage of biosensor electrodes.

Acetylcholinesterase-based biosensor electrodes for organophosphate pesticide detection. I. Modification of carbon surface for immobilization of acetylcholinesterase.

Screen-printed carbon electrodes modified with the dialdehydes, glutaraldehyde and terephthaldicarboxaldehyde, and then polyethyleneimine have been utilized for production of pesticide biosensors based on acetylcholinesterase. To improve the extent of dialdehyde modification, the electrodes were NH2-derivatized, initially by electrochemical reduction of 4-nitrobenzenediazonium to a nitroaryl radical permitting attachment to the carbon surface. Subsequent reduction of the 4-nitrobenzene yields a 4-aminobenzene modified carbon surface. Drosophila melanogaster acetylcholinesterase was immobilized either covalently onto dialdehyde modified electrodes or non-covalently onto polyethyleneimine modified electrodes. Internal diffusion limitations due to the dialdehyde and polyethyleneimine modifications increased the apparent Km of the immobilized enzyme. The thiocholine sensitivity was about 90% for dialdehyde modified electrodes and about 10% for polyethyleneimine modified electrodes as compared with non-modified carbon electrodes. The detection limit of the biosensors produced by non-covalent immobilization of acetylcholinesterase onto polyethyleneimine modified carbon electrodes was found to be about 10(-10) M for the organophosphate pesticide dichlorvos.

Development of highly selective and stable potentiometric sensors for formaldehyde determination.

Two types of biosensors selective to formaldehyde have been developed on the basis of pH-sensitive field effect transistor as a transducer. Highly or partially purified alcohol oxidase (AOX) and the permeabilised cells of methylotrophic yeast Hansenula polymorpha (as a source of AOX) have been used as sensitive elements. The response time in steady-state measurement mode is in the range of 10-60 s for the enzyme-based sensors and 60-120 s for the cell-based sensor. When measured in kinetic mode the response time of all biosensors developed was less than 5 s. The linear dynamic range of the sensor output signals corresponds to 5-200 mM formaldehyde for highly and partially purified alcohol oxidase, and 5-50 mM formaldehyde for the cells. The operational stability of the biosensors is not less than 7 h, and the relative standard deviation of intra-sensor response is approximately 2 and 5% for the enzyme- and cell-based sensors, respectively. When stored at 4 degrees C, the enzyme and cell sensor responses have been found stable for more than 60 and 30 days, respectively. Both types of biosensors demonstrate a high selectivity to formaldehyde with no potentiometric response to primary alcohols, including methanol, or glycerol and glucose. The possible reasons of such unexpected high selectivity of AOX-based FET-sensors to formaldehyde are discussed. The influence of the biomembrane composition and the effect of different buffers on the sensor response to formaldehyde are also discussed.

Improved operational stability of biosensors based on enzyme-polyelectrolyte complex adsorbed into a porous carbon electrode.

A novel porous active carbon is utilized in order to adsorb the diethylaminoethyl-dextran (DEAE-dextran)-enzyme stabilized complexes, for the construction of highly stable biosensors. The interaction of DEAE-dextran with the examined enzymes increases dramatically the operational stabilization of the sensors, without adverse effects on the enzyme activity. At the same time, the porous active carbon allows for high enzyme loading, good electrical contact and low resistance throughout the sensing element. Glucose oxidase and horseradish peroxidase are used as model enzymes in this study to construct biosensors, with very good reproducibility (less than 5% RSD). As a result, the glucose sensor exhibits very long operational stability (over a period of 5 months), while the hydrogen peroxide sensor retains its initial activity after several weeks.

Flow-injection detector incorporating a screen-printed disposable amperometric biosensor for monitoring organophosphate pesticides.

The construction of a wall-jet flow cell, which houses a screen-printed amperometric pesticide biosensor, together with a complete flow-injection system, is described. This system was initially employed in studies to stabilise the enzyme acetylcholinesterase (AChE), which was immobilised on a cobalt phthalocyanine screen-printed carbon electrode to form a biosensor. A combination of dextran sulfate and lactitol, and carbodiimide for enzyme immobilisation, resulted in biosensor lifetimes of at least 76 d (at 37 degrees C). Flow-injection and biosensor conditions were optimised, then the system was evaluated by monitoring the model organophosphate pesticides (OP) dichlorvos and paraoxon. The detection limits were 7 x 10(-11) mol dm-3 (for 1 U of AChE) and 4 x 10(-11) mol dm-3 (for 0.05 U of AChE), respectively, which are better than for other electrochemical methods. Initial evaluations on two river water samples have been carried out to test the validity of the system for OP determination in field samples.

Protein stabilisation using additives based on multiple electrostatic interactions.

A method of elevating the storage lifetime of purified proteins has been discovered which appears to confer stability to all proteins investigated and may therefore be classed as generic in action. The basic methodology involves the formation of multiple electrostatic complexes between the protein and selected soluble polyelectrolytes to give protein-polyelectrolyte (PP) complexes and then to add solutions of polyalcohols or other compounds containing multiple hydroxyl groups. Dehydration of the resulting solution by vacuum evaporation, freeze drying or forced air convection produces a dry film or powder of stabilised protein. The method has been used mainly in the preparation of active enzymes for analytical tests. It has also been found that the formation of PP complexes also enhances the stability of enzymes in solution and the technique may be applicable to the stabilisation of virus suspensions by polycations. Examples of stabilised enzymes prepared by these methods are given and the proposed mechanism of stabilisation and applicability of the method to shelf-stable vaccine products are discussed.