Dual-SplitNluc: A Spectrally Resolved Bioluminescence Platform Enabling Duplex Homogeneous Immunoassays

Bioluminescence (BL)-based assays are broadly used for genetic reporter assays, imaging, as well as in vitro biomarker or serological analysis. However, nearly all existing BL assays are operated in a single-channel mode, which limits the detection capacity to one analyte each time. To extend the potential of BL assays, we developed the Dual-SplitNluc platform, a spectrally resolved BL reporter system which enables simultaneous detection of two analytes in homogeneous format based on spectral unmixing. Dual-SplitNluc represents the integration of our previously reported SplitNluc system together with SplitNanoBRET, an orange-emitting complementation-based immunoassay platform derived from SplitNluc. With chemical modification of the SplitNluc reporter by adding a fluorophore acting as a BL resonance energy transfer (BRET) acceptor, red-shifted photon emission can be generated and spectrally separated from the conventional SplitNluc signal. Through mathematical deconvolution of the integrated luminescence readouts, the original BL signals from each individual analyte can be extracted and utilized for ratiometric or quantitative analysis of both analytes. Collectively, Dual-SplitNluc extends BL assays beyond the single-analyte mode and provides a generalizable strategy for duplex and potentially higher-order multiplex homogeneous detection using spectral resolution as an orthogonal analytical dimension.