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 biomarkers 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 overlapped 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 feasibility 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.