Acetylcholinesterase enzyme

Acetylcholinesterase enzyme (EC:232-559-3) from Electrophorus electricus (electric eel) Type VI-S (lyophilized powder, 200–1000 units/mg protein), Acetylthiocholine iodide (AChI), Carbaryl PESTANAL^® were obtained from Sigma-Aldrich (Darmstadt, Germany). Electrochemical experiments were performed in a conventional three-electrode system in the Autolab 128N electrochemical system. A bare glassy carbon (GC) electrode was modified with the renewable carbon and AChE enzyme (GC/RC/AChE), or functionalized renewable carbon and AChE enzyme (GC/RCF/AChE) as the working electrode, the Ag/AgCl/ KCl (3.0 mol L⁻¹) as the reference electrode, and a platinum plate as the auxiliary electrode. The resulting data were analyzed via NOVA 2.1 software.

Donepezil standard was prepared at the concentration of 5 mM to serve as a positive control. Larvae samples were prepared as mentioned before. Acetylcholinesterase enzyme was purchased from Sigma-Aldrich from the Electrophorus electrics. Cat number: 3389. Aceylthiocholine iodide substrate and the indicator 3,3′-Dithiodipropionic acid di (N-hydroxysuccinimide ester) (DTNB) were purchased from Sigma-Aldrich. Briefly, 10µL of the indicator solution (0.4 mM in buffer (1): 100 mM tris buffer pH-7.5) was transferred to a 96-well plate followed by 20µL of enzyme solution (acetylcholine esterase 0.02U/mL final concentration in buffer (2): 50 mM tris buffer PH = 7.5 containing 0.1% bovine serum albumin). Following that, 140 mL of buffer was added, then 20 mL of the sample/standard solution (1). The mixture could stand for 15 min at room temperature. The substrate (0.4 mM acetylcholine iodide buffer (1)) was then added to each well in an instant, totaling 10 L. The plate was incubated in a dark chamber for 20 min at room temperature. At the end of the incubation period, the color was measured at 412 nm. Data are represented as means ± SD. The results were recorded using a microplate reader, FluoStar Omega^{21 (link),22 (link)}.