The DAO immobilisation method consisted in depositing drop
by drop on a preactivated Immunodyne ABC membrane (1 cm2),
5 lL of an enzyme solution (110 mg DAO/mL of 100 mM sodium
phosphate buffer, pH 7.2), previously concentrated by using a
100,000 Da Ultrafree-CL centrifugal filter device (4470 rpm for
40 min). The membrane with the enzyme was air-dried for
15 min, immersed in a glutaraldehyde solution (1%) for 30 s and
immediately washed with a glycine solution (100 mM in sodium
phosphate buffer, pH 7.2) to eliminate any unreacted glutaraldehyde.
The DAO immobilised membrane was applied to an oxygen
permeable Teflon membrane placed in the oxygen electrode and
clamped into the thermostated reaction cell that was kept at
30 C. Forty microlitres of an appropriate dilution of amine standard
or sausage extract was injected directly on the DAO-membrane.
After each measurement, the enzyme sensor was washed
with the sodium phosphate buffer and dried with paper tissue.
As a result of the catalytic action of DAO on amines, which conveys
oxygen consumption, the decrease of current (mA) at 50 s was
used to detect the total amine in the enzyme sensor.