QUANTITATION OF ACRIDINIUM ESTERS USING ELECTROGENERATED CHEMILUMINESCENCE AND FLOW-INJECTION

Acridinium esters are used as chemiluminescence (CL) labels in immunoassay. Acridinium ester CL is traditionally triggered by addition of a solution of H2O2. This paper is concerned with the generation of the reaction-initiating species in situ (electrochemically) to eliminate problems associated with solution addition. Phenyl acridinium-9-carboxylate shows no electrochemistry over the range -1.0 to +1.0 V. In the presence of dissolved oxygen, as the applied potential is stepped negatively, electrogenerated chemiluminescence (ECL) emission intensity increases to a plateau region corresponding to the peroxide plateau of electrochemical oxygen reduction. ECL emission intensity increases as pH increases from 9 to 12, but decreases at higher pH. The rate of formation of nonchemiluminescent pseudobase between sample injection and CL observation was studied; at pH 12, that delay time should be limited to under 0.5 s. The working curve dynamic range covers 4 decades in concentration. The detection limit for acridinium ester-labeled lysine is 10 fmol.