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Acta Chimica Sinica >

2012 , Vol. 70 >Issue 07: 859 - 863

DOI: https://doi.org/10.6023/A1107231

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An Enzyme-Catalyzed Reaction System Using Puerarin as Substrates for Horseradish Peroxidase and Its Application in Immunoassays

  • Gong Fuchun ,
  • Liu Ping ,
  • Lin Youyi ,
  • Tang Zhijiao ,
  • Liu Jianping
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  • College of Chemistry and Biologic Engineering, Changsha University of Science and Technology, Changsha 410004

Received date: 2011-07-03

  Revised date: 2011-11-25

  Online published: 2011-12-06

Supported by

Project supported by the Natural Science Foundation of Hunan Province (No. 10JJ5006).

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Abstract

An enzyme-catalyzed reaction system puerarin-horseradish peroxidase (HRP)-hydrogen peroxide, which used puerarin as substrates for HRP, was developed. In enzymatic reaction procedure, HRP catalyze the polymerization of puerarin by H2O2, and the puerarin is partly converted to dimers, a significantly fluorescent species. The increase of the fluorescence of the HRP-enzymatic reaction solution at emission of 478 nm (excitation: 315 nm) is proportional to the concentration of HRP. The enzyme-linked fluoroimmunoassay based on the principle mentioned above and competitive immunoassay model for the determination of Brucella melitensis antibodies (BrAb) was established. The results of the property investigation of puerarin demonstrate the advantages in stability and reactivity with HRP over conventional substrates such as p-hydroxyphenylacetic acid (p-HPA), Amplex red and homovanillic acid. The experimental parameters such as BrAb-HRP, temperature were optimized. The proposed method has been successfully used for analysis of BrAb in rabbit plasma sample. The linear range of determination for BrAb is 1.3～120 ng/mL with the relative standard deviation of 3.8%. The detection limit is 1.3 ng/mL (3σ).

Key words： puerarin; HRP fluorogenic substrate; Brucella melitensis antibody; enzyme-linked fluoroimmunoassay

Cite this article

Gong Fuchun , Liu Ping , Lin Youyi , Tang Zhijiao , Liu Jianping . An Enzyme-Catalyzed Reaction System Using Puerarin as Substrates for Horseradish Peroxidase and Its Application in Immunoassays[J]. Acta Chimica Sinica, 2012 , 70(07) : 859 -863 . DOI: 10.6023/A1107231

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