Quantitative analysis of enzymatic assays using indoxyl-based substrates

Abstract

Hydrolysis of indoxyl-based substrates by hydrolytic enzymes is a commonly used semiquantitative detection system that generates a water-insoluble indigo dye which is difficult to quantify. This work describes the quantitative analysis and enzyme kinetics for alkaline phosphatase (AP) and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) in solution obtained by applying known solubilization methodology from the textiles industry to the enzymatic product. This proposal is based on the reduction of the tetrahalo-indigo blue dye in a basic medium with the aim of generating its aqueous-soluble parent compound termed indigo white, which gives a rich yellow color in solution and is fluorescent. A quantitative ELISA (where a soluble end product is required) is accomplished for first time using BCIP as substrate.

Dose-Dependent Effects of Triiodothyronine on the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

“…The intermediate is then oxidized by NBT to produce a dimer, which is an intense purple dye [13, 14]. Thus, this assay is an indirect method of verifying alkaline phosphatase activity.…”

A digital microfluidic electrochemical immunoassay

“…We hypothesized that the system described here would be useful for amperometric detection of immunoassay reaction products with comparable performance to those described previously, with sensitivity adequate for detecting small amounts of analyte in microfluidic samples. Building from previous work, 45,46 an amperometric detection system was developed that relies on the action of the enzyme, horseradish peroxidase (HRP), on the substrate, 3,3′,5,5′-tetramethylbenzidine (TMB), in the presence of H 2 O 2 . This reaction generates a blue intermediate which turns yellow upon termination of the reaction by acidification.…”