摘要： Fluorescent probes have been valuable tools for bioanalytical multiplex assays. However, as a common phenomenon in multiplex fluorescence assays, spectral overlap usually leads to difficulty in spectral analysis for multiple analytes. Although multiway calibrations have provided mathematic approaches for complex spectral analysis, it remains a grand challenge for these methods in practical applications because of the problems such as prior rank estimation. Herein, we report a novel second-order calibration algorithm of alternating residual trilinearization (ART) for the decomposition of complex spectra generated from multiplex fluorescence assays. By alternating iterative convergence to the spectral profiles of each component in convergence process, ART enables automatic rank estimation for second-order calibration, thus able to avoid the risk of chemical meaningless fitting of component spectra. Combined with fluorescence excitation-emission matrix (EEM) spectroscopy, the performance of ART has been demonstrated by a simulated example and an analytical experiment performed using molecular beacons (MBs) for the simultaneous assay of three breast cancer related DNA targets. The results revealed that the proposed algorithm is capable of automatic estimating the number of underlying components during its convergence process to produce acceptable performance in spectral profile resolution and concentration estimation. Compared with other existing iterative trilinear decomposition strategies such as parallel factor analysis (PARAFAC) requiring a prior rank estimation, the proposed ART therefore provides a robust second-order calibration strategy for complex spectral analysis in multiplex fluorescence assays.