Hemin/G-Quadruplex-Catalyzed Aerobic Oxidation of
Thiols to Disulfides: Application
of the Process for the Development of Sensors and Aptasensors and
for Probing Acetylcholine Esterase Activity
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Abstract
This
study describes the novel hemin/G-quadruplex DNAzyme-catalyzed
aerobic oxidation of thiols to disulfides and the respective mechanism.
The mechanism of the reaction involves the DNAzyme-catalyzed oxidation
of thiols to disulfides and the thiol-mediated autocatalytic generation
of H<sub>2</sub>O<sub>2</sub> from oxygen. The coupling of a concomitant
H<sub>2</sub>O<sub>2</sub>-mediated hemin/G-quadruplex-catalyzed oxidation
of Amplex Red to the fluorescent resorufin as a transduction module
provides a fluorescent signal for probing the catalyzed oxidation
of the thiol to disulfides and for probing sensing processes that
yield the hemin/G-quadruplex as a functional label. Accordingly, a
versatile sensing method for analyzing thiols (l-cysteine,
glutathione) using the H<sub>2</sub>O<sub>2</sub>-mediated DNAzyme-catalyzed
oxidation of Amplex Red to the resorufin was developed. Also, the l-cysteine and Amplex Red system was implemented as an auxiliary
fluorescent transduction module for probing recognition events that
form the catalytic hemin/G-quadruplex structures. This is exemplified
with the development of thrombin aptasensor. The thrombin/thrombin
binding aptamer recognition complex binds hemin, and the resulting
catalytic complex activates the auxiliary transduction module, involving
the aerobic oxidation of l-cysteine and the concomitant formation
of the fluorescent resorufin. Finally, the hemin/G-quadruplex DNAzyme/Amplex
Red system was used to follow the activity of acetylcholine esterase,
AChE, and to probe its inhibition. The AChE-catalyzed hydrolysis of
acetylthiocholine to the thiol-functionalized thiocholine enabled
the probing of the enzymatic activity of AChE through the hemin/G-quadruplex-catalyzed
aerobic oxidation of thiocholine to the respective disulfide and the
concomitant generation of the fluorescent resorufin product