NW1 6DT, U.K
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information on biosensor regeneration
Deposition of the diaminobenzene membrane
Electropolymerisation of the diaminobenzene requires
the working electrode to be cleaned and polished with very fine sandpaper,
which means that the enzyme layer also has to be removed. Next, the following
steps must be strictly followed:
A 3 mM solution of 1-2,diaminobenzene in potassium
phosphate buffer is deoxygenated by bubbling nitrogen through for about
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.
The enzyme layer is deposited as follows:
Gelatin is dissolved at a concentration of 50 mg/ml
in phosphate buffer at a temperature of 40-50oC (solution 1).
Bovine serum albumin is then dissolved in solution
1 at a concentration of 10 mg/ml at a temperature of 30-40oC
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
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.
The whole procedure takes about 4 hours.
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.