• A 3 mM solution of 1-2,diaminobenzene in potassium phosphate buffer is deoxygenated by bubbling nitrogen through for about 30 minutes.
• The biosensor is then assembled using a special silicone rubber gasket and, after flushing through the deoxygenated solution, it is continously perfused with this solution at about 5 µl/min. The electropolymerisation is accomplished by applying a voltage 'ramp' rising from 0 to 0.8 V (versus a Ag/AgCl reference electrode) over a period of 7 minutes. When the voltage reaches its maximum it is quickly returned to 0 and the cycle repeated 14 times. Further cycling does not appear to give any improved performance. The peak current drawn during the 1st cycle is about 100 µA.

Enzyme immobilisation
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The enzyme layer is deposited as follows:
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• Gelatin is dissolved at a concentration of 50 mg/ml in phosphate buffer at a temperature of 40-50^oC (solution 1).
• Bovine serum albumin is then dissolved in solution 1 at a concentration of 10 mg/ml at a temperature of 30-40^oC (solution 2).
• 5 µl of solution 2 is then added to a 5 µl solution of enzyme in buffer containing 1 unit of enzyme, and this solution diluted by half by addition of 10 µl of distilled water (solution 3). Dilution with distilled water is necessary to reduce the chance of the solution solidifying as it is being applied to the surface at room temperature.
• Approximately 0.5 µl of solution 3 is applied over each of the two gold surfaces using a 10 µl pipette tip. This is then allowed to dry for about 30 minutes after which a 2.5% solution of glutaraldehyde in phosphate buffer is applied for 2 minutes, and then washed thouroughly with phosphate buffer.

NOTE: The above treatment is critical. All the above steps must be strictly followed. In addition, the use of the biosensor requires great care in order to avoid air pockets. These are favoured by the very fine 50 µm thick teflon gasket, and the very fine inlet and outlet tubes, that we use to provide very fast response.

Applied Neuroscience