Luminol chemiluminescence-based porphyrin assays without hydrogen peroxide: a spectral study of mechanism and enhancement

Abstract

In order to use metalloporphyrins as labels in immunoassays or in nucleic probes, a detection method based on luminol chemiluminescence (CL) at alkaline pH was developed. It was found that omitting H₂O₂ improves the light yield, and that a dramatic enhancement of CL is achieved by the association of nicotinamide adenine dinucleotide (NAD) and flavins [flavin mononucleotide (FMN) or riboflavin]. To explain the mechanism of the catalytic cycle in the absence of H₂O₂, electron spin resonance (ESR) measurements were performed. Using 5,5′-dimethyl-1-pyrroline N-oxide as a spin trap agent, ESR showed the production of OH· and O₂·^– radicals. This free radical production may explain the luminol oxidation by metalloporphyrins. The role of O₂·^– was confirmed by almost complete inhibition of light emission when superoxide dismutase was added to the CL reaction. The contribution of oxygen was also confirmed by the large decrease in the CL emission when deaerated solutions were used. A reaction scheme is proposed in which a redox cycle between Fe^II and Fe^III porphyrins is coupled to the production of oxy radicals. Owing to their redox properties, FMN and NAD enhancers could act at this level through an increase of the exchange rate between Fe^II and Fe^III. Moreover, in the presence of FMN, a significant red shift and shape change of the luminol emission spectrum is observed, which arises from an energy transfer phenomenon in the final luminescent step of the reaction. On the other hand, neither a spectral shape change nor a shift was observed in the presence of NAD. These data were collected by using a high-performance laboratory-made spectroluminometer.