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 oxi-dative 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 de-tection 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 H2O2 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 highly dependent on pH, nature of buffer, and concentration of the reducing enzyme.


Supplementary material

Experimental Section and complementary Figures
Experimental Section and complementary Figures (from S1 to S23) on the (i) influence of pH on the autocatalysis with P2, (ii) UV-vis spectrophometry and HPLC characterization of probes, (iii) kinetic analysis of the H2O2-mediated depro-tection of probes, (iv) numerical simulations of the autocata-lytic reactions, (v) justification of the rate constants used for the 1,4-NQ autoxidation, (vi) redox cycling experiments with the 1,4-NQ, (vii) influence of the buffer composition and pH on the autocatalysis with P3, (viii) screening of naphthoqui-nones for their redox cyling properties, and (ix) indirect detection of glucose oxidase.