Detection of leucoindigo in alkaline phosphatase and peroxidase based assays using 3-indoxyl phosphate as substrate Original Research Article

3-Indoxyl phosphate (3-IP) is the first common electrochemical substrate of the two most important label enzymes in immunoassays: alkaline phosphatase (AP) and horseradish peroxidase (HRP). The product of the enzymatic reactions is indigo blue, an aromatic heterocycle compound insoluble in aqueous solutions. Through the reduction of water insoluble indigo in an alkaline media and in presence of a dithionite salt (Na2S2O4), a water-soluble compound, called leucoindigo, is obtained. This solubilisation methodology, widely used in the textile dyeing industry, is applied in this work for the measurement of AP and HRP activity. Leucoindigo is electroactive and also develops a rich yellow color in solution with a maximum absorbance at 415 nm, so 3-IP is shown also as the first common chromogenic substrate for HRP and AP. Through an electrochemical (alternating current adsorptive stripping voltammetry) and a spectrophotometric detection of leucoindigo, picomolar levels of AP and HRP in solution can be measured. Using this approach, two ELISA assays to quantify pneumolysin (PLY) and osteocalcin (BGP) were carried out using AP and HRP, respectively as enzymatic label. Therefore, the suitability of the detection of leucoindigo in ELISAs that employ 3-IP as substrate is demonstrated. This approach can unify solutions and procedures to develop AP and HRP based affinity devices.