Exponential amplification by redox cross-catalysis and unmasking of doubly protected molecular probes

The strength of autocatalytic reactions lies in their ability to provide a powerful means of molecular amplification, which can be very useful for improving the analytical performances of a multitude of analytical and bioanalytical methods. However, one of the major difficulties in designing an efficient autocatalytic amplification system is the requirement for reactants that are both highly reactive and chemically stable in order to avoid limitations imposed by undesirable background amplifications. In the present work, we devised a reaction network based on a redox cross-catalysis principle, in which two catalytic loops activate each other. The first loop, catalyzed by H₂O₂, involves the oxidative deprotection of a naphthylboronate ester probe into a redox-active naphthohydroquinone, which in turn catalyzes the production of H₂O₂ by redox cycling in the presence of a reducing enzyme/substrate couple. We present here a set of new molecular probes with improved reactivity and stability, resulting in particularly steep sigmoidal kinetic traces and enhanced discrimination between specific and nonspecific responses. This translates into the sensitive detection of H₂O₂ down to a few nM in less than 10 minutes or a redox cycling compound such as the 2-amino-3-chloro-1,4-naphthoquinone down to 50 pM in less than 30 minutes. The critical reason leading to these remarkably good performances is the extended stability stemming from the double masking of the naphthohydroquinone core by two boronate groups, a counterintuitive strategy if we consider the need for two equivalents of H₂O₂ for full deprotection. An in-depth study of the mechanism and dynamics of this complex reaction network is conducted in order to better understand, predict and optimize its functioning. From this investigation, the time response as well as detection limit are found to be highly dependent on pH, nature of the buffer, and concentration of the reducing enzyme.