【作者】 王芳芳； 郜捷； 赵建伟； 张文月； 白洁； 贾红霞； 王愈聪；

【Author】 Fangfang Wang;Jie Gao;Jianwei Zhao;Wenyue Zhang;Jie Bai;Hongxia Jia;Yucong Wang;College of Chemistry and Environment Science, Hebei University;Medical Comprehensive Experimental Center, Hebei University;

【机构】 河北大学化学与环境科学学院； 河北大学医学部综合实验中心；

【摘要】 首先我们设计了一条标记有荧光素的羧肽酶特异性的多肽底物。氧化石墨烯能够吸附标记有荧光素的底物多肽,并猝灭荧光素的荧光。当目标蛋白酶-羧肽酶Y（CPY）存在时,标记有荧光素的底物多肽被CPY水解,荧光素被释放,产生荧光。CPY的水解作用导致了荧光的增强。更有趣的是,水解导致的荧光增强甚至超过了标记在多肽底物上的荧光素本身的荧光。我们推测这意外的荧光增强同样是由CPY对底物多肽的水解作用造成的。水解作用降低了氨基酸残基对荧光素荧光量子产率的影响,所以荧光进一步增强。同时,氧化石墨烯优越的荧光猝灭能力有效的提高了方法的信噪比。将基于氧化石墨烯的荧光打开机制与基于CPY水解的信号扩增机制相结合,我们提出的方法在CPY检测上获得了高的灵敏度和好的选择性。该方法检出限可以达到1.0×10^-5 UμL^-1 CPY。考查的其它蛋白、蛋白酶,以及复杂基质都不干扰CPY的检测。更多还原

【Abstract】 GO can adsorb the FITC-labeled substrate peptide and quench the fluorescence of FITC. In the presence of target protein, carboxypeptidase Y（CPY）, the FITC-labeled substrate peptide is hydrolyzed by CPY, leading to the turn-on of fluorescence. The fluorescence intensity increases significantly after the hydrolysis. More interestingly, it is even much higher than the fluorescence intensity of the added FITC-labeled substrate peptides. It is deduced that the extraordinary growing of fluorescence intensity is attributed to the hydrolysis also. The strong quenching efficiency of GO significantly improved the the signal-to-noise ratio（SNR） of the proposed method for protease analysis. By combining the GO-based fluorescence turn-on with the fluorescence signal amplification induced by hydrolysis, the proposed method obtained higher sensitivity and specificity for CPY activity detection. The detection limit for CPY activity assay is estimated to be 1.0×10^-5 U μL^-1. The other proteins, proteases and a complicated matrix cannot disturb the assay of CPY activity.更多还原