基于多肽微阵列芯片的荧光和共振光散射法筛选凝血酶抑制剂  

A Peptide Microarray-Based Fluorescent and Resonance Light Scattering Assay for Screening Thrombin Inhibitor

作　　者：苏敏[1,2] 李桃[1] 刘殿骏[1] 王振新[1] 

机构地区：[1]中国科学院长春应用化学研究所电分析化学国家重点实验室,长春130022 [2]中国科学院大学,北京100049

出　　处：《分析化学》2015年第2期199-206,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.21075118;21205113)资助~~

摘　　要：研制了一种基于多肽微阵列芯片的荧光和共振光散射双通路检测方法,对血液样品中的凝血酶抑制剂进行检测。以生物素化的多肽微阵列芯片为反应平台,加入凝血酶水解多肽上的特异位点使其C末端的生物素解离,通过亲和素和生物素之间的特异性结合,用荧光探针和30 nm金纳米粒子探针标记此反应过程。凝血酶抑制剂阻止凝血酶对底物多肽的水解反应,通过荧光和共振光散射信号强度的变化检测抑制剂的抑制能力。酶溶液和加标人血清中测定的半抑制浓度(IC50)值:阿加曲班<抗凝血酶Ⅲ<4-(2-氨乙基)苯磺酰氟盐酸盐,IC50差的绝对值随抑制剂特异性的减弱而增大。当抑制剂浓度为7.5μmol/L时,血浆中5种化合物的抑制能力:阿加曲班>抗凝血酶Ⅲ>胰蛋白酶抑制剂>N-(反式-环氧丁二酰基)-L-亮氨酸-4-胍基丁基酰胺>4-(2-氨乙基)苯磺酰氟盐酸盐。在血浆中考察了阿加曲班和抗凝血酶Ⅲ的可逆性。对比荧光法和共振光散射法在血液样品中的检测结果,用30A peptide microarray-based fluorescence and resonance light scattering （ RLS ） two readout assay was developed for screening thrombin inhibitors in blood samples. In this assay, the biotinylated peptide microarray was used as the platform. The peptide C-terminal fragments carried biotin sites departed from the slide when the biotinylated peptides were digested by thrombin hydrolysis reaction. The hydrolysis progress was labeled by fluorescence and 30 nm peptide-stabilized gold nanoparticles through the biotin-avidin reaction. In the presence of thrombin inhibitors, the hydrolysis reactions were blocked, and the inhibition capability of inhibitors could be detected by the fluorescent and RLS signal changes. The order of the half maximal inhibitory concentration （ IC50 ） of thrombin inhibitors in pure thrombin solution and spiked human serum were argatroban〈human antithrombin-Ⅲ （ HAT-Ⅲ）〈AEBSF. The absolute differences of IC50 between pure thrombin solution and spiked human serum increased with the decreasing inhibition specificity. The order of the inhibition activities of five compounds （7. 5 μmol/L） in human plasma were argatroban〉HAT-Ⅲ〉trypsin inhibitor〉E-64〉AEBSF. The reversible or irreversible characters of argatroban and HAT-Ⅲ had been estimated in human plasma. Compared with the experimental data of fluorescent and RLS assay in blood sample, the RLS assay labeled by 30 nm gold nanoparticles are more suitable for the inhibitor detection in complicated blood sample.

关 键 词：多肽微阵列 金纳米粒子探针 共振光散射 半抑制浓度 凝血酶抑制剂 

分 类 号：R927.2[医药卫生—药学] O657.3[理学—分析化学]