Interference-free and simultaneous quantification of three breast cancer-related DNAs in multiple biological fluids using excitation-emission matrix fluorescence spectroscopy combined with second-order calibration method

Breast cancer is the leading cause of cancer deaths in women worldwide. Quantitation of breast cancer-related biomarkers facilitates cancer research, diagnosis, and prognosis. However, few studies have determined multiple biomarkers associated with breast cancer simultaneously. We presented a chemometrics-enhanced excitation -emission matrix fluorescence (EEMF) spectroscopy strategy for the simultaneous quantification of three bio-markers associated with breast cancer. Three molecular beacons (MBs) were designed to detect the target analytes in complex biological systems. Even in the existence of unexpected interferences and seriously over-lapped fluorescent peaks, the alternating normalization-weighted error (ANWE) method can accurately resolve the spectral profiles and provide satisfactory qualitative and quantitative results. The proposed strategy showed good linearity (r >= 0.9971) and satisfactory average spiked recoveries (92.3%-107.8%). Furthermore, figures of merit (FOM), precision, and the elliptic joint confidence region (EJCR) test were applied to evaluate the feasi-bility and reliability of the method. These results demonstrate that the proposed strategy has the advantages of being simple, rapid, cost-effective, and anti-interference. Therefore, this study provides a new pathway for the simultaneous determination of multiple cancer-related biomarkers in complex matrices, which is expected to bring a valuable reference for cancer research and disease diagnosis.