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

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 H(2)O(2) (HP), using a fluorescent europium-tetracycline (Eu(3)TC) complex as indicator. On addition of HP, this complex forms a highly fluorescent adduct (Eu(3)TC-HP), which is decomposed in the presence of POx to form the weakly fluorescent europium-tetracycline (Eu(3)TC). Hence, the activity of the enzyme can be directly determined by means of the luminescent Eu(3)TC 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 x 10(-5) to 5.9 x 10(-3) U mL(-1), with a limit of detection of 1.0 x 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.

Luminescent Chemical and Physical Sensors Based on Lanthanide Complexes

“…This is the first reported Eu 3+ probe that can be excited at wavelengths >400 nm. It can be used to detect hydrogen peroxide in river water [39] or to monitor the activity of enzymes that convert or produce hydrogen peroxide such as glucose oxidase, catalases, or peroxidases [40][41][42]. Due to its quenching effect, the determination of copper ions also can be achieved by the EuTc hydrogen peroxide complex [43].…”

A New Fluorescent PET Probe for Hydrogen Peroxide and its Use in Enzymatic Assays for L‐Lactate and D‐Glucose

“…[5,6] Optical methods are an attractive alternative and widely used in the fields of imaging and high-throughput screening. [7,8] Photometric methods [9] are less sensitive than fluorometry and cannot be applied to strongly colored and scattering media such as cells or tissue. Some fluorophores can detect HP directly without the use of peroxidase.…”

Gold Nanolabels for New Enhanced Chemiluminescence Immunoassay of Alpha‐Fetoprotein Based on Magnetic Beads

“…[3][4][5] The improvement of EIA in terms of reducing assay time and simplifying operations is one of the major trends in development of immunoassay technology. [6] In recent years, many improvements have been made by using magnetic beads (MBs) and gold nanoparticles (AuNPs) in order to enhance sensitivity and reduce detection time. [7] Magnetic beads (MBs), as a special biomolecular immobilizing carrier, have been widely applied in immunoassays; enzyme, DNA and protein immobilization and purification; and magnetically controlled transport of anticancer drugs.…”