Chemiluminescent duplex analysis using phenoxy-1,2-dioxetane luminophores with color modulation

Multiplex technology is an important emerging field, in diagnostic sciences, that enables the simultaneous detection of several analytes in a single sample. The light-emission spectrum of a chemiluminescent phenoxy-dioxetane luminophore can be accurately predicted by determining the fluorescence-emission spectrum of its corresponding benzoate species, which is generated during the chemiexcitation process. Based on this observation, we designed a library of chemiluminescent dioxetane luminophores with multicolor emission wavelengths. Two dioxetane luminophores that have different emission spectra, but similar quantum yield properties, were selected from the synthesized library for a duplex analysis. The selected dioxetane luminophores were equipped with two different enzymatic substrates to generate turn-ON chemiluminescent probes. This pair of probes exhibited a promising ability to act as a chemiluminescent duplex system for the simultaneous detection of two different enzymatic activities in a physiological solution. In addition, the pair of probes were also able to simultaneously detect the activities of the two enzymes in a bacterial assay, using a blue filter slit for one enzyme and a red filter slit for the other enzyme. As far as we know, this is the first successful demonstration of a chemiluminescent duplex system composed of two-color phenoxy-1,2-dioxetane luminophores. We believe that the library of dioxetanes presented here will be beneficial for developing chemiluminescence luminophores for multiplex analysis of enzymes and bioanalytes.