新型酸性磷酸酶“Turn-on”型荧光底物  

A Novel “Turn on” Fluorescent Substrate for Acid Phosphatase

作　　者：李晓佩[1,2] 张勇杰 王东东[2] 卿光焱 LI Xiao-pei;ZHANG Yong-jie;WANG Dong-dong;QING Guang-yan(Dalian Polytechnic University,Dalian 116034,China;Dalian Institute of Chemical Physics,Chinese Academy of Sciences,Dalian 116023,China)

机构地区：[1]大连工业大学,辽宁大连116034 [2]中国科学院大连化学物理研究所,辽宁大连116023

出　　处：《光谱学与光谱分析》2024年第6期1607-1612,共6页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(21922411,22174138)资助。

摘　　要：酸性磷酸酶(ACP)是前列腺癌、戈谢病、肾病等疾病的重要生物标记物,因此开发灵敏、高选择性的ACP检测方法具有重要的意义。目前,已发展出多种ACP检测方法,包括免疫法、光谱法、色谱法、电化学法等;其中,荧光检测方法因灵敏度高、选择性好、快速、准确等优点备受关注。通过席夫碱反应,利用2-氨基苯硼酸(2-APBA)二聚体和磷酸吡哆醛(PLP)合成了一种新型的ACP“Turn-on”型荧光底物APBA-PLP(ABPL)。反应后,PLP的—CHO基团和2-APBA二聚体的—NH 2基团的特征峰消失,同时ABPL的C N基团的特征峰出现;此外,^(1)H和^(1)H—^(1)H COSY核磁波谱的信号峰与ABPL的结构相对应。以上表征结果证实了ABPL的成功合成。进一步,将ABPL应用于ACP检测。我们发现向ABPL水溶液中加入ACP后,体系的荧光强度增强,机理为:2-APBA二聚体为聚集诱导发光增强(AIEE)分子,其AIEE特性源于2-APBA二聚体的高度有序堆积;当2-APBA二聚体分子上引入PLP后(即合成ABPL),分子的高度有序堆积结构被破坏;加入ACP后,PLP水解为吡哆醛并从2-APBA二聚体分子上脱落,2-APBA二聚体分子又可重新进行高度有序堆积,表现为体系的荧光增强。在0~5 U·L^(-1)活性范围内,376.5 nm处的相对荧光强度与ACP活性之间呈现出良好的线性关系(R 2=0.99)。此外,当ACP、果胶酶、木瓜蛋白酶和脂肪酶的活性分别为4、50000、80000和10000 U·L^(-1)时,四种酶对应的相对荧光强度分别为0.2、-0.006、0.03和0.05。该结果表明ABPL对ACP具有高选择性。ABPL分子易合成,对ACP具有线性和高选择性响应,为设计合成可用于ACP检测的新型高效荧光底物提供了新策略。Acid phosphatase(ACP)is an important biomarker for several diseases,such as prostate cancer,Gaucher disease and kidney disease.Therefore,developing sensitive and highly selective assays for ACP is of great significance.To date,several assays for ACP have been reported,including immunoassay,spectrophotometry,chromatography and electrochemistry.Among these methods,fluorescence-based assays have gained prominence due to their high sensitivity,good selectivity,efficiency,and precision.In this study,a novel“turn-on”fluorescent substrate for ACP,named APBA-PLP(ABPL),was synthesized through the Schiff base reaction between 2-aminophenyl boronic acid(2-APBA)dimer and pyridoxal phosphate(PLP).After the reaction,the characteristic FTIR peaks associated with—CHO groups of PLP and—NH 2 groups of 2-APBA dimer disappear,and a new FTIR band,originating from C N vibrations of ABPL,appears.In addition,the ^(1)H and ^(1)H-^(1)H COSY NMR spectra display all the ABPL signals.The FTIR and NMR results indicate the successful synthesis of ABPL.Furthermore,ABPL is applied to the detection of ACP.We observe an increase in fluorescence intensity of the ABPL solution upon adding ACP.This phenomenon is attributed to the aggregation-induced enhanced emission property of the 2-APBA dimer,which arises from its highly ordered stacking.The highly ordered stacking structure is disrupted upon the introduction of PLP onto the 2-APBA dimer(i.e.,the synthesis of ABPL).Subsequent addition of ACP leads to the hydrolysis of PLP into pyridoxal,causing the detachment of PLP from the 2-APBA dimer molecule.The 2-APBA dimer molecules can then reassemble into a highly ordered structure,increasing the fluorescence intensity.The relative fluorescence intensity at 376.5 nm exhibits an excellent linear relationship(R 2=0.99)with ACP activity in the 0~5 U·L^(-1) range.Moreover,when ACP,pectinase,papain,and lipase activity were 4,50000,80000,and 10000 U·L^(-1),respectively,the corresponding relative fluorescence intensities were 0.2,-0.006,0.03,and 0.05.T

关 键 词：酸性磷酸酶 2-氨基苯硼酸二聚体 磷酸吡哆醛 荧光 

分 类 号：O652[理学—分析化学]