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A new class of peroxidase substrates has been developed which produces chemiluminescence upon enzymatic oxidation. A wide variety of N-alkylacridancarboxylic acid derivatives including esters, thioesters, and sulfonamides are efficiently oxidized by a peroxidase and a peroxide to enzymatically produce the corresponding chemiluminescent acridinium compound. In conjunction with a peroxidase enhancer, continuous light emission with high light intensities and an extended duration are produced. Alternately, an appropriately designed acridan substrate produces a stable acridinium ester intermediate which can be accumulated and the chemiluminescence elicited as a burst of light by raising the pH. The effects of leaving groups and substitution on the acridan ring on the mechanism of light production are discussed. Peroxidase-catalyzed oxidation in the presence of a peroxide permits the detection of enzyme with subattomolar sensitivity and a broad linear dynamic range.

Electrochemical oxidation of the acridan 2',6'-difluorophenyl 10-methylacridan-9-carboxylate produces the corresponding acridinium ester, which reacts with hydrogen peroxide forming a dioxetanone intermediate. Decomposition of the dioxetanone generates light at 430 nm when it relaxes to the ground state. The effect of pH and hydrogen peroxide concentration on this ECL reaction and on the stability of the acridan were investigated. At pH 8.0 and a hydrogen peroxide concentration of 10 mM, light emission from the ECL reaction was used to determine the acridan concentration with a detection limit of 54 pmol L(-1). Results suggest that acridan esters could be used as labels in ECL immunoassays and nucleotide assays.

Electrochemical oxidation of the acridine ester 2A,6A-difluorophenyl 10-methyl-9,10-dihydroacridine-9-carboxylate yields the corresponding acridinium ester which reacts with H 2 O 2 to generate intense chemiluminescence.