CAPACITIVE APPROACH TO DETERMINE PHOSPHOLIPASE A(2) ACTIVITY TOWARD ARTIFICIAL AND NATURAL SUBSTRATES

A capacitive approach has been employed to develop a novel method to d etermine phospholipase activity. The sensing electrodes have a structu re like Au/S(CH2)(17)CH3/substrate/electrolyte. Hydrolysis of the subs trate, mediated by phospholipase A(2), leads to the formation of water soluble products from the insoluble substrate. This results in desorpt ion of these products into aqueous phase and corresponding increase of the electrode capacitance. The requirement of high water solubility o f the reaction products can be achieved in two ways. In the first, sho rt-chain phospholipids are used as the substrate, in which case, water -soluble products are formed and no additional reagents are required t o promote desorption of these products. The sensors prepared by this s trategy provide sensitive qualitative detection of phospholipases. The second way is based on the use of a water-soluble acceptor (for examp le, beta-cyclodextrin) to solubilize the products of hydrolysis, It al lows semiquantitative detection of phospholipase activity toward long- chain natural substrates, The reaction kinetics for this case was foun d to be monoexponential and linearly dependent on the phospholipase co ncentration. The detection limit of this method, as tested with phosph olipase A(2) from bee venom and soy bean lecithin as the substrate, is similar to 0.5 ng/mL (500 mu units/mL).