Fluorescent Labeling of Human Serum Albumin by Thiol-Cyanimide Addition and Its Application in the Fluorescence Quenching Method for Nanoparticle–Protein Interactions

• Published in: Analytical chemistry (Washington) Vol. 94; no. 7; pp. 3111 - 3119
• Main Authors: Gao, Lian-Xun, Chen, Wen-Qi, Liu, Yi, Jiang, Feng-Lei
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.02.2022
• Subjects: Fluorescent Dyes; Gold - chemistry; Humans; Metal Nanoparticles - chemistry; Reproducibility of Results; Serum Albumin, Human - chemistry; Spectrometry, Fluorescence - methods; Sulfhydryl Compounds
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.1c04231

A boron-dipyrromethene (BODIPY)-based fluorescent probe, BDP-CN, was synthesized in this work. It had a fluorescence emission maximum at 512 nm and a high quantum yield (48%). As evidenced by agarose gel electrophoresis and liquid chromatography–mass spectrometry, it could realize the fluorescent labeling of human serum albumin (HSA) through a thiol-cyanimide addition. Interestingly, f-HSA, defined as HSA labeled by BDP-CN, had an even higher quantum yield (77%). In addition, BDP-CN would not affect the secondary structure of HSA. Based on the successful formation of f-HSA, it was further applied to study the interactions with nanoparticles. The fluorescence quenching of f-HSA by dihydrolipoic acid-coated gold nanoclusters (DHLA-AuNCs) obeyed a dynamic mechanism, consistent with the intrinsic fluorescence quenching of HSA by DHLA-AuNCs. The association constant K a between f-HSA and DHLA-AuNCs at 298 K was 1.5 × 105 M–1, which was the same order of magnitude as that between HSA and DHLA-AuNCs. Moreover, the interactions of f-HSA with glutathione-coated gold nanoclusters confirmed that the labeled fluorescence could replace the intrinsic fluorescence to monitor the interactions between proteins and nanoparticles. By this method, strong fluorescence ensures better stability and reproducibility, excitation at a longer wavelength reduces the damage to the proteins, and covalent conjugation with cysteine residues eliminates the inner filter effects to a great extent. Therefore, the strategy for the fluorescent labeling of HSA can be expanded to investigate a broad class of nanoparticle–protein interactions and inspire even more fluorescent labeling methods with organic dyes.