Capacitive Approach To Determine Phospholipase A2 Activity toward Artificial and Natural Substrates

Abstract

A capacitive approach has been employed to develop a novel method to determine phospholipase activity. The sensing electrodes have a structure like Au/S(CH(2))(17)CH(3)/substrate/electrolyte. Hydrolysis of the substrate, mediated by phospholipase A(2), leads to the formation of water-soluble products from the insoluble substrate. This results in desorption of these products into aqueous phase and corresponding increase of the electrode capacitance. The requirement of high water solubility of the reaction products can be achieved in two ways. In the first, short-chain phospholipids are used as the substrate, in which case, water-soluble products are formed and no additional reagents are required to promote desorption of these products. The sensors prepared by this strategy provide sensitive qualitative detection of phospholipases. The second way is based on the use of a water-soluble acceptor (for example, β-cyclodextrin) to solubilize the products of hydrolysis. It allows semiquantitative detection of phospholipase activity toward long-chain natural substrates. The reaction kinetics for this case was found to be monoexponential and linearly dependent on the phospholipase concentration. The detection limit of this method, as tested with phospholipase A(2) from bee venom and soy bean lecithin as the substrate, is ∼0.5 ng/mL (500 μunits/mL).