The crosstalk fluorescence spectroscopy analysis principle and an accurate fluorescence quantitative method for multi-composition fluorescence substances

Fluorescence quantitative analysis methods are extensively used in biomedicine inspection, petrochemical industry, environmental monitoring, and many other fields in the past decades. When the analyte is composed of multiple compositions, the accuracy of the conventional method declines significantly due to the fluorescence spectral crosstalk. In this research, the interactions between the light and the multiple compositions are comprehensively analyzed. The concepts of the quenching due to mutual absorption and the fluorescence overlapping are considered, and the mechanism of multi-composition fluorescence emission under single-wavelength excitation light is analyzed theoretically. The mixture experiment and the dilution experiment are designed to illustrate that the quenching due to mutual absorption has a significant nonlinear impact on fluorescence quantitative analysis and the mechanism of fluorescence spectral crosstalk gives a good explanation for these experiments. Through the in-depth theoretical analysis, the computer simulation, and the experiments, a novel principle named the Crosstalk Fluorescence Spectroscopy Analysis (CFSA) is proposed and verified, which has much higher quantitative analysis accuracy (R2>0.99 and RMSE≤0.2) than the conventional methods when analyzing the multi-composition samples. Unlike many correction approaches to fluorescence spectroscopy, the novel CFSA can serve as a complete analysis method rather than a correction method. These concepts and the principle are expected to be applied in many practical analysis fields.