Acta Chimica Sinica ›› 2007, Vol. 65 ›› Issue (15): 1499-1503. Previous Articles     Next Articles

Original Articles

季胺-辣根过氧化物酶-过氧化氢显色新体系及其在酶活性检测中的应用

李建国, 刘颖, 鞠熀先*   

  1. (生命分析化学教育部重点实验室 南京大学化学系 南京 210093)
  • 投稿日期:2006-05-22 修回日期:2006-07-03 发布日期:2007-08-14
  • 通讯作者: 鞠熀先

A Novel Tetrabase-horseradish Peroxi-dase-H2O2 Spectrophotometric System and Its Application to Detection of Enzyme Activity

LI Jian-Guo; LIU Ying; JU Huang-Xian*   

  1. (Key Laboratory of Analytical Chemistry for Life Science (Ministry of Education of China), Department of Chemistry, Nanjing University, Nanjing 210093)
  • Received:2006-05-22 Revised:2006-07-03 Published:2007-08-14
  • Contact: JU Huang-Xian

A new spectrophotometric system of tetrabase [bis(4-dimethylaminophenyl)methane]-H2O2- horseradish peroxidase (HRP) was developed for the detection of HRP activity, and the reaction mechanism was discussed. The detection was based on the HRP catalyzing coloring reaction of tetrabase in pH 4.5 PBS containing KI, which behaved maximum absorption at 462 nm. The absorbance was proportional to the activity of HRP. This new tetrabase reagent is soluble and more stable than 3,3',5,5'-tetramethyl benzidine (TMB), a usually used coloring re-agent for ELISA in clinic, and could overcome the shortcomings of TMB. Under optimal conditions, the linear range for HRP detection was 2×10-9~2.5×10-7 g/mL with the detec-tion limit of 3×10-10 g/mL. This system could be used for the determination of HRP labeled horse anti-human α-fetoprotein with satisfactory results. The new system is simple, rapid and sensi-tive, and has good application prospect in clinic.

Key words: spectrophotometric enzyme-linked immunosorbent assay, horseradish peroxidase, hydrogen peroxide, tetrabase, bis(4-dimethylaminophenyl)methane