2.3. Comparison of Anti-S100β immobilization on IDE through amine linker

Amine surface was used to capture anti-S100β on the IDE surface and two different methods were compared for antibody immobilization. In the first method without pre-mixing APTES and antibody, initially the electrode was rinsed with distilled water before being immersed in diluted potassium hydroxide solution (1%, diluted in water). Further, the KOH-treated substrate was functionalized by amine groups by treating with 5 μl of 2% v/v APTES at 60 °C for 1 h to reach the maximum process of salinization. The amine-modified surface was rinsed with distilled water to remove the unbound APTES. At the same time, an anti-S100β antibody (5 μl of 4 μg/mL) was combined with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide(EDC) and N-Hydroxysuccinimide (NHS; 10 min at 37 °C). The obtained EDC cross-linked antibody was dropped on the amine-modified substrate and incubated at 37 °C for 1 h. Finally, the antibody-coated substrate was washed with by10 mM PBS to remove the excess antibody.

The second strategy (with pre-mixing APTES and antibody) was followed with the same steps until KOH activation. After that, anti- S100β antibody (5 μl of 8 g/mL in PBS) was combined in a 1:1 (v/v) ratio with 1% APTES. On the KOH-treated substrate, the final antibody concentration of 4 μg/mL in 0.5% APTES was applied, and the substrate was then left at room temperature for 30 min. Each experiment's current response was noted.