Li Juan, Zhao Xiuyan, Zhang Yutao, Lu Yao, Xue Haoyun, Li Dan, Liu Qiang, Yan Chenxu, Chi Weijie, Xiao Xingqing, Zhu Wei-Hong, Guo Zhiqian
Key Laboratory for Advanced Materials, Institute of Fine Chemicals, Feringa Nobel Prize Scientist Joint Research Center, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, Center of Photosensitive Chemicals Engineering, East China University of Science and Technology Shanghai 200237 China
Department of Chemistry, School of Chemistry and Chemical Engineering, Hainan University Haikou City Hainan Province 570228 China
Chem Sci. 2025 Jan 7;16(7):3228-3237. doi: 10.1039/d4sc07714h. eCollection 2025 Feb 12.
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 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.
鉴于氨基酸序列存在微小差异的蛋白质会导致截然不同的功能结果，识别和定量相似蛋白质至关重要，但这仍是一个长期存在的挑战。在此，我们提出了一种用于探针DCM-SA的二维正交荧光和化学发光设计策略，该探针由白蛋白介导的水解顺序激活，呈现点亮荧光并通过光诱导环加成产生化学发光，能够进行正交信号放大以区分相似蛋白质之间的细微差异。通过将这些双模式信号正交化，建立了荧光和化学发光的二维工作曲线以区分和定量相似蛋白质HSA和BSA。重要的是，DCM-SA的双模式信号对HSA和BSA呈现相反的增量趋势。分子对接和飞秒瞬态吸收光谱表明，DCM-SA与HSA的较低 值以及DCM-SA与BSA的较长激发态寿命是产生不同双模式响应的基础。利用二维正交信号，我们首次精确测量了混合血清中的HSA/BSA比值。该方法有助于快速进行血源鉴定和对人尿中的痕量HSA进行定量。我们的二维正交放大方法为区分和定量高度相似蛋白质之间的细微差异提供了一个强大的工具，在基础生命科学研究和临床应用中均显示出巨大潜力。
