A Novel Method for Time-Resolved Fluorimetric Determination and Imaging of the Activity of Peroxidase, and Its Application to an Enzyme-Linked Immunosorbent Assay

Lin, Zhihong ; Wu, Meng ; Wolfbeis, Otto S. ; Schäferling, Michael

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Date of publication of this fulltext: 04 Apr 2011 10:29

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Details

Item type:	Article
Journal or Publication Title:	Chemistry: a European journal
Publisher:	Wiley-VCH Verl.
Volume:	12
Number of Issue or Book Chapter:	10
Page Range:	pp. 2730-2738
Date:	20 March 2006
Institutions:	Chemistry and Pharmacy > Institut für Analytische Chemie, Chemo- und Biosensorik > Chemo- und Biosensorik (Prof. Antje J. Bäumner, formerly Prof. Wolfbeis)
Identification Number:	Value Type 10.1002/chem.200500884 DOI
Keywords:	fluorescent probes; imaging agents; immunoassays; peroxidase activity; time-resolved spectrometry
Dewey Decimal Classification:	500 Science > 540 Chemistry & allied sciences
Status:	Published
Refereed:	Unknown
Created at the University of Regensburg:	Unknown
Item ID:	20295

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Abstract

A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H2O2 (HP), using a fluorescent europium–tetracycline (Eu3TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu3TC–HP), which is decomposed in the presence of POx to form the weakly ...

Abstract

A new type of fluorescence assay for the determination of peroxidase (POx) activity is presented. The assay is based on the indication of the enzymatic consumption of H2O2 (HP), using a fluorescent europium–tetracycline (Eu3TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu3TC–HP), which is decomposed in the presence of POx to form the weakly fluorescent europium–tetracycline (Eu3TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent Eu3TC complex as indicator. The POx assay demonstrated herein was elaborated starting from a spectral characterization of the complex systems involved. Due to the long lifetime of lanthanide luminescence, both steady-state and time-resolved luminescence assays can easily be performed. The time-resolved assay can quantify POx in the range from 4.0×10−5 to 5.9×10−3 U mL−1, with a limit of detection of 1.0×10−5 U mL−1. The effects of POx inhibitors such as cyanide, hydroxylamine, and azide have also been studied. In addition, a time-resolved fluorescent detection method for a POx-based enzyme-linked immunosorbent assay (ELISA) has been developed, which is demonstrated in a sandwich model assay with bovine IgG serving as analyte. Furthermore, a time-resolved fluorescent imaging method is demonstrated that makes use of a straightforward imaging set-up adjusted to the optical properties of the europium reagent.

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Metadata last modified: 03 Jun 2018 05:21

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