3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

Abstract

In this work 3-indoxyl phosphate (3-IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3-IP in presence of hydrogen peroxide (H 2 O 2 ) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of À 0.15 V (vs. Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V max , K M ) for both substrates (3-IP and H 2 O 2 ) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86 Â 10 À12 and 5.68 Â 10 À12 M, respectively. Thus, 3-IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.

Electrochemical characterization of different screen-printed gold electrodes

“…Another substrate of the AP, that can also been used in combination with HRP systems, is 3-indoxyl phosphate [29]. The product, indigo blue, is not soluble in aqueous solutions and is commonly solubilised in sulphuric acid.…”

Effect of cadmium binding form on rabbit aminolevulinate dehydratase in high molecular weight fraction of rainbow trout liver

“…Based on these findings, MTs prevent several metals from inhibiting some vital enzymes [26], so the presence of Cd bound to MT does not give any information about the toxic effects [27]. On the other hand, the enzyme activities are affected by conditions such as heat, pH, salt levels, and solvents [28,29]. In addition to these factors, protein structure is also easily changed by physical factors, comparatively small molecules such as cofactors and metal ions, other regulatory proteins, and covalent modification, and these changes can affect enzymatic activity [30].…”

Gold Electrodes for Detection of Enzyme Assays with 3‐Indoxylphosphate as Substrate

“…Although 3-IP results in a more adequate substrate for AP since the K m is lower [39,20], is also appropriate for HRP, constituting this detection a valid possibility for the most common ELISAs developed for clinical determinations.…”

“…Enzyme-catalyzed reaction conditions optimized in a previous paper [20]: reaction time of 30 min, reaction buffer containing 0.1% of BSA and 10 À4 M of H 2 O 2 were employed. The hydrolysis was performed at 37 8C using a concentration of 3-IP of 3.7 mM.…”

“…Moreover, it has been demonstrated that it is appropriate not only for AP but also for HRP [20].The successful detection of HRP using 3-IP substrate appears promising for future enzyme immunoassays. Therefore, this substrate can be used for two most common labels employed in enzymeimmunoassays.…”