Construction
of AG/PyOx/CHIT bi-enzymatic sensor For the construction of the bioactive layer, the graphite electrodes were polished and washed thoroughly with distilled water and sonicated for 2 min [30]. 20 μl of CHIT or CHIT–CNT solutions (1.0%), 10 μl of AG solution (0.6 U) and 10 μl of PyOx solution (9.0 U) were placed on the graphite electrode and allowed to dry at+4 °C for 18 h. Then 20 μl of glutaraldehyde solution (1.0%; in potassium phosphate buffer, pH 7.5, 50 mM) spread evenly onto the CHIT modified graphite electrode and allowed to stand at +4 °C for 30 min [29]. Then, it was
washed with distilled water to remove excess of glutaraldehyde and unbound enzymes. Protein analysis was carried out in washing solusolution to check the availability of unbound protein amount by Bradford assay [31]. During the experimental steps daily prepared enzyme sensors were used to obtain more reproducible data.