基于共轭聚电解质荧光恢复测定胰蛋白酶及其抑制剂
Determination of Trypsin and Trypsin Inhibitor by Fluorescence Quenching and Recovering of the Conjugated Poly-electrolyte
摘 要
在1.0×10-5mol·L-1共轭聚电解质(CPE)溶液中加入聚精氨酸(Arg6)溶液至其质量浓度达15 mg·L-1,反应稳定后,CPE的荧光发生猝灭,猝灭程度达90%。加入不同浓度的胰蛋白酶(TPS)溶液反应,当TPS质量浓度达1 mg·L-1时,该体系的荧光强度可恢复至原来的80%。基于上述以CPE作为荧光探针和TPS对CPE-Arg6反应体系的荧光恢复效应,可实现对CPS的实时、免标记检测,其检出限(3S/N)达0.1 mg·L-1。在上述反应体系中,将CPS的质量浓度固定在1 mg·L-1,然后加入不同浓度的胰蛋白酶抑制剂(TPSI)溶液,随TPSI浓度的增加,CPE的荧光恢复速率明显减慢,根据这一现象,实现了实时检测TPSI。
胰蛋白酶
胰蛋白酶抑制剂
荧光猝灭
Conjugated poly-electrolyte
Trypsin
Trypsin in hibitor
Fluorescence quenching
Abstract
As poly-arginine (Arg6) solution was added to a solution of 1.0×10-5mol·L-1 conjugated poly-electrolyte (CPE) up to the concentration of 15 mg·L-1, the fluorescence of CPE was quenched to an extent of 90% at the equilibrium of the reaction between CPE and Arg6. Various concentrations of trypsin (TPS) solution was added, and finally attained to an extent of 80% when 1 mg·L-1 of TPS was added. Based on these facts, a real-time and non-labelling determination of TPS with its detection limit (3S/N) of 0.1 mg·L-1 was proposed. In the above reactions system, 1 mg·L-1 TPS was fixed, and various concentrations of TPS inhibitor (TPSI) was added, the fluorescence recovery was retarded, and the magnitude of retardation was related with the amount of TPSI added, hence this phenomenon was utilised as a method for determination of TPSI.
中图分类号 O657.3
所属栏目 试验与研究
基金项目 国家自然科学基金(20805014);湖南省高校科技创新团队支持计划、湖南省教育厅科学研究基金(11C0809;12B077);湖南大学国家重点实验室开放课题(201103)资助项目
收稿日期 2013/11/20
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备注肖毅(1973-),男,湖南澧县人,讲师,博士,主要研究方向为荧光生化传感技术。
引用该论文: XIAO Yi,XUE Zun-zun,HUANG Hong-mei,ZHANG You-yu,HE Xiao-xiao. Determination of Trypsin and Trypsin Inhibitor by Fluorescence Quenching and Recovering of the Conjugated Poly-electrolyte[J]. Physical Testing and Chemical Analysis part B:Chemical Analysis, 2014, 50(12): 1486～1490
肖毅,薛遵遵,黄红梅,张友玉,何晓晓. 基于共轭聚电解质荧光恢复测定胰蛋白酶及其抑制剂[J]. 理化检验－化学分册, 2014, 50(12): 1486～1490
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【3】MAO Xiao-wei, SU Hai-yan, TIAN De-mei, et al. Bipyrene-functionalized graphene as a “turn-on” fluorescence sensor for manganese(Ⅱ) ions in living cells［J］. ACS Applied Materials and Interfaces, 2013(5):592-597.
【4】JIN Yan, LI Hong-yan, BAI Jin-yan. Homogeneous selecting of a quadruplex-binding ligand-based gold nanoparticle fluorescence resonance energy transfer assay［J］. Analytical Chemistry, 2009,81(14):5709-5715.
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【6】WANG Yan-yan, ZHANG Yong, LIU Bin. Conjugated polyelectrolyte based fluorescence turn-on assay for real-time monitoring of protease activity［J］. Analytical Chemistry, 2010,82(20):8604-8610.
【7】LONESCU R E, COSNIER S, MARKS R S. Protease amperometric sensor［J］. Analytical Chemistry, 2006,78(18):6327-6331.
【8】HERNNDEZ J, VALERO M L, ANDREU D, et al. Antibody and host cell recognition of foot-and-mouth disease virus (serotype C) cleaved at the Arg-Gly-Asp (RGD) motif: a structural interpretation［J］. Journal of General Virology, 1996,77:257-264.
【9】XUE Wang-xin, ZHANG Guan-xin, ZHANG De-qing, et al. A new label-free continuous fluorometric assay for trypsin and inhibitor screening with tetraphenylethene compounds［J］. Organic Letters, 2010,12(10):2274-2277.
【10】李晔,张恒建,廖明霞,等.氨基酸分子改性的SiO2杂化材料用于胰蛋白酶固定化［J］.高等学校化学学报, 2011,32(5):1100-1105.
【11】HU Yu-qin, XIAO Yi, HUANG Hong-mei, et al. An anion-conjugated polyelectrolyte designed for the selective and sensitive detection of silver(Ⅰ)［J］. Chemistry-An Asian Journal, 2011,6:1500-1504.
【12】XIAO Yi, LAI Chun-lian, LI Sha, et al. Pyridyl-containing conjugated polyelectrolyte as a new colorimetric and fluorescent pH probe［J］. Journal of Natural Science of Hunan Normal University, 2012,35(3):49-54.
【13】XU Jian-ping, FANG Yuan, SONG Zhe-gang, et al. BSA-tetraphenylethene derivative conjugates with aggregation-induced emission properties: fluorescent probes for label-free and homogeneous detection of protease and α1-antitrypsin［J］. Analyst, 2011,136(11):2315-2321.
【14】李严,宗晖,黄小莺,等.中国野生大豆胰蛋白酶抑制剂的初步研究［J］.复旦学报:自然科学版, 1999,38(5):529-532.
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