辣根过氧化物酶和荧光碳点的复合物研究  

Research on the complex of horseradish peroxidase with fluorescent carbon dots

作　　者：刘利 丁海贞 房伟[2,3] 毕红 LIU Li;DING Hai-zhen;FANG Wei;BI Hong(School of Chemistry and Chemical Engineering,Anhui University,Hefei 230601;School of Life Sciences,Anhui University,Hefei 230601;Anhui Key Laboratory of Modern Biomanufacturing,Hefei 230601,China)

机构地区：[1]安徽大学化学化工学院,合肥230601 [2]安徽大学生命科学学院,合肥230601 [3]现代生物制造安徽省重点实验室,合肥230601

出　　处：《生物学杂志》2020年第4期75-78,共4页Journal of Biology

基　　金：国家自然科学基金项目(51772001)。

摘　　要：辣根过氧化物酶(HRP)是一种来源于辣根的含有铁离子的糖蛋白,可用于生化分析且在过氧化氢存在时能催化苯酚等物质氧化,HRP可与许多物质复合进而改善性能并拓宽其应用。采用一步水热法合成了平均尺寸约2 nm的氟掺杂的荧光碳点(F-CDs)。所得F-CDs在紫外灯下发黄绿色荧光且具有明显的激发光依赖性和浓度依赖性,以硫酸喹啉盐为参比计算出其相对量子产率高达45.6%。研究表明,F-CDs在Hela细胞内具有较好显影性能,F-CDs可与HRP形成稳定的复合物(F-CDs/HRP),该复合物不仅保留酶氧化2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)的能力,还保留着F-CDs优异的荧光性能。此外,加入过氧化氢的浓度影响F-CDs/HRP催化氧化ABTS的能力。在低温如4℃时,F-CDs/HRP复合物仍然可以迅速催化氧化ABTS,能通过溶液显色程度来定性分析F-CDs/HRP中HRP的催化活性。因此,F-CDs/HRP复合物有应用于免疫荧光分析的潜力。Horseradish peroxidase(HRP)is a glycoprotein containing iron ions derived from horseradish that can be used for biochemical analysis,and catalyze the phenol in the presence of hydrogen peroxide.HRP can be combined with many substances to improve its performance and application.This work used a one-step hydrothermal method to easily synthesize fluorine-doped fluorescent carbon dots(F-CDs)with an average size of 2 nm.The as-synthesized F-CDs had obvious excitation light dependence,showing yellow-green fluorescence under ultraviolet light,and concentration dependence.The relative quantum yield was calculated to be as high as 45.6%with the quinoline sulfate as a reference.The results indicated that F-CDs have better fluorescence imaging performance in Hela cells,and F-CDs can form stable complexes(F-CDs/HRP)with HRP.The F-CDs/HRP complex not only preserved the ability of the enzyme to oxidize 2,2′-diazide-bis-3-ethylbenzothiazoline-6-sulfonic acid(ABTS),but also retained the excellent fluorescence performance of F-CDs.Moreover,the added hydrogen peroxide concentration will affect the ability of F-CDs/HRP to catalyze the oxidation of ABTS.At the temperature as low as 4℃,HRP still kept the activity to catalyze the oxidation of ABTS.Therefore,the catalytic activity of HRP in the obtained F-CDs/HRP could be analyzed qualitatively by the degree of chromogenic reaction.Therefore,the F-CDs/HRP complex has potential for application in immunofluorescence analysis.

关 键 词：辣根过氧化物酶 碳点 荧光 免疫荧光分析 

分 类 号：Q55[生物学—生物化学]