The use of this second membrane allowed both pyruvate and NADH www.selleckchem.com/products/Tipifarnib(R115777).html to be immobilized but their free diffusion in the membrane would also be Inhibitors,Modulators,Libraries ensured to maintain a rapid reaction rate. The biosensor was thus designed to provide a reagentless system specific for NH4+ ion. Figure 1 represents the mechanism of the enzymic reaction involved in the developed NH4+ ion biosensor based on a stacked membrane system.Figure 1.The mechanism of enzymatic reaction involved in the NH4+ biosensor based on stacked membranes.2.?Experimental2.1. ReagentsChemicals were obtained from commercial sources and were used without further purification. ��-Nicotinamide adenine dinucleotide, reduced form (NADH, 98%) was purchased from Sigma. Stock solution of L-alanine dehydrogenase enzyme (AlaDH, E.C. 1.4.1.
1, from Bacillus subtillis, Sigma) was prepared by mixing an appropriate amount of AlaDH enzyme Inhibitors,Modulators,Libraries solution with 10 mM phosphate buffer pH 7 in an Eppendorf tube and stored at 4 ��C [23]. Sodium pyruvate (C3H3NaO3, 99%, Sigma) stock solution was prepared by dissolving an appropriate amount of pyruvate salt in deionised water. pH 7, 10 mM phosphate buffer was prepared by adding 10 mM dipotassium hydrogen phosphate (K2HPO4, 98%, Fluka) to 10 mM potassium dihydrogen phosphate (KH2PO4, 99.5%, Fluka) and adjusting to the required pH value [23]. A standard stock ammonia solution was prepared by dissolving the required amount of concentrated ammonia solution (NH4OH, 25%, Merck) in deionised water. The ammonia solution used was standardised by the Nessler method using ammonium chloride (NH4Cl, 99.5%) salt.
A homogeneous stock solution of 2-hydroxyethyl methacrylate monomer (HEMA, C(CH3)COOCH2CH2OH, 97%, Aldrich) was prepared by mixing appropriate amounts of HEMA monomer and initiator (2,2-dimethoxy-2-phenylacetophenone, DMPP, C16H16O3, Inhibitors,Modulators,Libraries 98%, Fluka) in a vial wrapped with aluminium foil. The mixture was then stirred gently for a few minutes and stored at 4 ��C. Poly(2-hydroxyethyl methacrylate) (pHEMA) was obtained commercially from Aldrich.2.2. InstrumentationElectrochemical behaviours Inhibitors,Modulators,Libraries of the reagentless biosensor were characterised using a chronoamperometry technique with an Autolab PG12 (AUT 71681) Potentiostat/Galvanostat. A conventional three-electrode electrochemical cell was used Carfilzomib with a glassy carbon electrode as an auxiliary electrode.
A Ag/AgCl electrode saturated with KCl was thoroughly used as a reference and a modified carbon paste electrode was used as a working electrode. The SPEs used were designed by Universiti Kebangsaan Malaysia and manufactured by Scrint Print Co. All potentials were measured and reported versus a Ag/AgCl electrode (saturated by KCl). During the constant potential experiments, a magnetic stir bar was used and the background current was allowed to decay to a constant value before NH4+ ion was added to the buffer solution.