Engineering a fluorescent probe with symmetric D-π-A-π-D configuration for fluorescence discrimination of HSA from...

Human serum albumin (HSA) levels serve as critical biomarkers for disease diagnosis, highlighting the need for both continuous monitoring and novel detection methods. Numerous studies have reported that D-π-A structural fluorescent probes can be used for visualization and real-time monitoring of HSA in complex biological environments by detecting characteristic changes in emission color and intensity. However, designing fluorescent probes that can discriminate between HSA and bovine serum albumin (BSA) remains challenging due to their structural similarities. Moreover, visual on-site HSA detection using a smartphone and portable sensing device offers substantial benefits, particularly for point-of-care testing (POCT) in clinical settings. Therefore, there is an urgent need to enhance detection specificity, and develop POCT systems. In this study, a symmetrical D-π-A-π-D structured fluorescent probe (designated APA) with low background fluorescence was developed for the selective detection and quantification of HSA. APA exhibited a 100-fold fluorescence enhancement in the presence of HSA but only a 5-fold enhancement with BSA, resulting in a selectivity index of 20 for highly discriminative detection of HSA versus BSA. The fluorescence intensity of APA demonstrated a linear relationship with HSA concentration up to 8 μM, with a detection limit of 14.3 nM. The interaction studies confirmed that APA specifically binds to Site III of HSA in a 1:1 M ratio. Given the excellent performance of APA, it was further applied to quantify HSA in serum, assess sample purity and develop a point-of-care testing (POCT) system. This work establishes a foundation for designing next-generation fluorescent probes with enhanced specificity for HSA.