Characterization of Acridancarboxylic Acid Derivatives as Chemiluminescent Peroxidase Substrates

Abstract

A new class of peroxidase substrates has been developed which produces chemiluminescence upon enzymatic oxidation. A wide variety of N-alkylacridancarboxylic acid derivatives including esters, thioesters, and sulfonamides are efficiently oxidized by a peroxidase and a peroxide to enzymatically produce the corresponding chemiluminescent acridinium compound. In conjunction with a peroxidase enhancer, continuous light emission with high light intensities and an extended duration are produced. Alternately, an appropriately designed acridan substrate produces a stable acridinium ester intermediate which can be accumulated and the chemiluminescence elicited as a burst of light by raising the pH. The effects of leaving groups and substitution on the acridan ring on the mechanism of light production are discussed. Peroxidase-catalyzed oxidation in the presence of a peroxide permits the detection of enzyme with subattomolar sensitivity and a broad linear dynamic range.

Comparison Between Acridan Ester, Luminol, and Ruthenium Chelate Electrochemiluminescence

“…Tris(2,2 0 -bipyridyl)dichlororuthenium(II) hexahydrate ðRuðbpyÞ 2þ 3 Þ and tripropylamine (TPA) were from Aldrich; stock solutions were prepared in 0.15 M phosphate buffer, pH 7.0, which contained 0.05 % Tween-20. 2 0 ,3 0 ,6 0 -Trifluorophenyl 10-methylacridan-9-carboxylate (TMC) was synthesized as described previously [31]; stock solutions were prepared in 1:1 ethanol: 1,4-dioxane.…”

“…Inevitably this double dilution protocol decreases the sensitivity of the assay, but attomoles of these compounds can be detected, which makes them suitable as labels for the majority of immunoassays [1]. 2 0 ,3 0 ,6 0 -Trifluorophenyl 10-methylacridan-9-carboxylate (TMC) is one of a class of compounds known as acridans that can be prepared by reducing the corresponding acridinium ester with a reducing agent such as powdered zinc in acetic acid [31]. Unlike acridinium esters they are relatively stable in the presence of hydrogen peroxide and are not converted to an inactive pseudobase under the conditions necessary for enzyme and immunochemical reactions.…”

“…Unlike acridinium esters they are relatively stable in the presence of hydrogen peroxide and are not converted to an inactive pseudobase under the conditions necessary for enzyme and immunochemical reactions. They can, however, be reconverted to the acridinium ester by oxidizing agents such as horseradish peroxidase (HRP) [31], and therefore they can be used as substrates for this enzyme when it is used as a label in DNA assays [32,33]. In the presence of an enhancer such as p-iodophenol it is possible to detect less than an amol of HRP with an acridan as the substrate [31].…”

“…They can, however, be reconverted to the acridinium ester by oxidizing agents such as horseradish peroxidase (HRP) [31], and therefore they can be used as substrates for this enzyme when it is used as a label in DNA assays [32,33]. In the presence of an enhancer such as p-iodophenol it is possible to detect less than an amol of HRP with an acridan as the substrate [31]. Recently it was shown that acridans can also be converted to the corresponding acridinium ester by electrochemical oxidation [34].…”

Nucleic Acids Analysis with Nano-Ag-Tb(III) by a Resonance Light Scattering Technique

“…Up to now, various analytical methods have been developed for the determination of nucleic acids, such as fluorometry, 1,2 chemiluminescence, 3 and electrochemistry. 4 Recently, fluorescent probes including organic dyes, such as yellow orange dyes, 5,6 metal ions, such as Tb(III), 7 and a metal complex 8 are frequently employed to investigate nucleic acids.…”