A two-dimensional fluorescence and chemiluminescence orthogonal probe for discriminating and quantifying similar proteins

Given that proteins with minor variations in amino acid sequences cause distinct functional outcomes, identifying and quantifying similar proteins is crucial, but remains a long-standing challenge. Herein, we present a two-dimensional orthogonal fluorescence and chemiluminescence design strategy for the probe DCM-SA, which is sequentially activated by albumin-mediated hydrolysis, exhibiting light-up fluorescence and photo-induced cycloaddition generating chemiluminescence, enabling orthogonal signal amplification for discrimination of subtle differences between similar proteins. By orthogonalizing these dual-mode signals, a two-dimensional work curve of fluorescence and chemiluminescence is established to distinguish and quantify similar proteins HSA and BSA. Importantly, the dual-mode signals of DCM-SA exhibit contrary incremental trends towards HSA and BSA. Molecular docking and femtosecond transient absorbance spectroscopy reveal that the lower <i>K</i> _D value of DCM-SA with HSA and the longer excited-state lifetime of DCM-SA with BSA underlie the distinct dual-mode responses. Using two-dimensional orthogonal signals, for the first time, we precisely measure the HSA/BSA ratio in mixed serum. This method facilitates rapid blood source identification and trace HSA quantitation in human urine. Our two-dimensional orthogonal amplification approach offers a powerful tool for distinguishing and quantifying subtle differences among highly similar proteins, demonstrating great potential for both basic life science research and clinical applications.