Generation of colorimetric, dual signals from two different enzymes by using catalytic pH shift

Because enzymes usually require distinct pH conditions for maximum activity, an approach of spontaneous pH shift of solution was devised to derive multiple reactions in a sequence. The two enzymes selected, horseradish peroxidase (HRP) and beta-galactosidase (GAL), had dissimilar optimal pH levels, i.e., 5.1 and 7.0, respectively. In a solution, HRP initially reacted at a lower pH range and the GAL reaction was consecutively carried out at a higher range in the presence of a third enzyme, urease, which caused an increase in pH. Under optimal conditions, the multiple system provided comparable performances with those of single reactions.