Butyrylcholine chloride

In the work, the following enzymes were used for biosensor creation: urease (EC 3.5.1.5) from Canavalia ensiformis, activity 66.3 U/mg (Fluka, Switzerland) and activity 22 U/mg and 35 U/mg (Sigma, Switzerland) and butyrylcholinesterase (EC 3.1.1.8) from equine serum, activity 13 U/mg and activity 20 U/mg (Sigma, Germany). Glycerol, bovine serum albumin (BSA; fraction V), 50% aqueous solution of glutaraldehyde (GA), sodium and potassium chlorides, urea, and butyrylcholine chloride were from Sigma-Aldrich Chemie (Steinheim, Germany). Potassium-phosphate buffer (KH₂PO₄-K₂HPO₄) and NaOH were produced by Helicon (Moscow, Russia) and Sigma-Aldrich Chemie (Steinheim, Germany). Glycoalkaloids α-chaconine (purity 95%) and α-solanine (purity 95%) from potato sprouts were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and used as inhibitors for BuChE. Mercury nitrate (Hg(NO₃)₂) from Himlaborreaktiv (Kiev, Ukraine) was used as an activity inhibitor for urease. Other inorganic substances were of analytical grade (>98%).

All the solvents used in this assays were of analytical grade while the chemicals/reagents such as AChE (Electric-eel EC 3.1.1.7), BChE (horse serum EC 3.1.1.8), DTNB, Acetyl choline iodide (AChI), butyryl choline chloride (BChI) and the reference, galantamine were purchased from Sigma–Aldrich (St. Louis, MO, USA). The inhibition was obtained through spectroscopic measurements [31 (link)]. Standard procedures and conditions of the assays were applied throughout the experiments [32 ]. Various dilutions of the tested compounds (62.5, 125, 250, 500 and 1000 μg/mL) were used in this assay. AChI and BChl were used as substates in this assay.
In brief, 880 µL of sodium phosphate buffer solution (62 mM, pH 8) containing 0.2 mM DNB was mixed with 40 µL solution of compound and 40 µL AChE or BChE solutions. This reaction mixture was incubated at 25 °C for 15 min followed by initiation of the reaction through addition of acetyl choline (ACh) or butyryl choline (BCh) (40 μL) in each experiment. The formation of yellow colored product (5-thio-2-nitorbenzoate anion) from reaction of DTNB with acetylcholine/butyrylcholine were observed through naked eyes, and the absorbance was measured at 412 nm using UV/Visible spectrophotometer (BMS-USA, New York, NY, USA). All the experiments were carried out in triplicate.

In the work, the following enzymes were used for biosensor creation: urease (EC 3.5.1.5) from Canavalia ensiformis, activity 66.3 U/mg (Fluka, Buchs, Switzerland); glucose oxidase (EC 1.1.3.4) from Penicillum vitale, activity 130 U/mg (Diagnosticum, Lviv, Ukraine); acetylcholinesterase (EC 3.1.1.7) from electric eel, activity 426 U/mg (Sigma, Seelze, Germany); and butyrylcholinesterase (EC 3.1.1.8) from equine serum, activity 13 U/mg (Sigma, Seelze, Germany). Glycerol, bovine serum albumin (BSA, fraction V), urea, acetylcholine chloride, and butyrylcholine chloride were from Sigma-Aldrich Chemie (Seelze, Germany). Potassium-phosphate buffer (KH₂PO₄-K₂HPO₄), NaOH, and glucose were produced by Helicon (Russia). Other inorganic substances were of analytical grade (>98%).
All nanoparticles were synthesized in the Middle-East Technical University (Ankara, Turkey) according to the procedures described below.