Improving chemiluminescence in heterogeneous HRP-catalyzed enhanced chemiluminescence reaction by replacement of luminol with 5-(4-hydroxy-1,3-dioxoisoindolin-2-yl)-2,3-dihydrophthalazine-1,4-dione (HDDD).

Previously, 5-(4-hydroxy-1,3-dioxoisoindolin-2-yl)-2,3-dihydrophthalazine-1,4-dione (HDDD) was shown to be a more efficient emitter than luminol (Lum) in the homogeneous enhanced chemiluminescence reaction (ECR). In this study, conditions of heterogeneous HDDD-assisted ECR (ECRHDDD) catalyzed by horseradish peroxidase (HRP) immobilized on a polystyrene microtiter plate were optimized using a full-factorial design. The favorable composition of reactants for heterogeneous ECRHDDD differed from that for homogeneous one. A comparison of HDDD and Lum as chemiluminescent emitters showed that in heterogeneous ECR, the light output produced by HDDD was significantly higher than that produced by Lum. Long-term chemiluminescent signal was formed in heterogeneous ECRHDDD. The advantages of HDDD over Lum were demonstrated in a chemiluminescent enzyme-linked oligonucleotide sorbent assay (ELONA) for the detection of miRNA-141 and in an ELISA for the detection of prostate-specific antigen.