细胞色素C是一种多功能酶,参与细胞的多个生理过程,具有调节细胞代谢和细胞凋亡的作用,发展简便、灵敏的细胞色素C分析方法具有重要意义。该文探究了一种基于纳米生物传感技术检测细胞色素C的新方法。首先以牛血清白蛋白(bovine serum albumin,BSA)作为还原剂和稳定剂,在碱性条件下合成具有一定荧光的金纳米簇,BSA同时具有增强金纳米簇荧光的性质。当体系中存在过氧化氢时,细胞色素C的过氧化氢酶活性可催化氧化BSA分子中的还原性官能团(如巯基、双硫键),导致BSA从金纳米簇表面脱落,进而引发荧光淬灭。在最优的实验条件下,随着细胞色素C浓度的增大,体系的荧光强度逐渐降低,细胞色素C检测范围为0.2~10 μmol/L,最低检测限为30 nmol/L。加入其他常见干扰物,无明显荧光淬灭效果,说明该方法对细胞色素C具有良好的选择性。此外,利用棒曲霉素刺激诱导HepG2细胞凋亡,不同时间产生细胞色素C的量不同,金纳米簇通过胞吞作用进入细胞后,可通过荧光成像实时监测凋亡细胞中细胞色素C的水平。该方法便捷、灵敏、选择性好,有望推广至相关生理活动中细胞色素C的实时可视化定量分析。
Cytochrome C (Cyt C) is a multifunctional enzyme that participates in multiple physiological processes of cells and has the function of regulating cell metabolism and apoptosis.The development of simple and sensitive Cyt C analysis methods is of great significance.This paper explores a novel Cyt C detection strategy using nano-biosensing technology.Au Nanoclusters (AuNCs) with intrinsic fluorescence were first synthesized under alkaline conditions using bovine serum albumin (BSA) as both reductant and stabilizer.BSA also has the property of enhancing the fluorescence of AuNCs.When hydrogen peroxide is present in the system, the catalase activity of Cyt C can oxidize the reducing functional groups (such as thiol groups and disulfide bonds) in BSA, causing BSA to detach from the surface of AuNCs and subsequently leading to fluorescence quenching.Under optimal conditions, fluorescence intensity decreased as the Cyt C concentration increased (0.2-10 μmol/L), achieving a detection limit of 30 nmol/L (S/N=3).No significant fluorescence quenching occurred in the presence of other common interferents, which confirmed the high selectivity detection toward Cyt C.In addition, Patulin-stimulated apoptosis in HepG2 cells released Cyt C at varies levels over time.After entering cells via endocytosis, AuNCs enabled real-time fluorescence imaging of Cyt C in apoptotic cells.This method is convenient, sensitive, and selective, showing potential for real-time and visual quantitative analysis of Cyt C in related physiological processes.
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