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

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 H₂O₂ from oxygen. The coupling of a concomitant H₂O₂-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 H₂O₂-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

Photoelectrochemical Biosensors Without External Irradiation: Probing Enzyme Activities and DNA Sensing Using Hemin/G-Quadruplex-Stimulated Chemiluminescence Resonance Energy Transfer (CRET) Generation of Photocurrents

A hemin/G-quadruplex nanostructure that is immobilized on CdS quantum dots (QDs) associated with an electrode leads, in the presence of luminol, H₂O₂, and triethanolamine as an electron donor, to the generation of photocurrents with no external irradiation of the QDs. The hemin/G-quadruplex-catalyzed generation of chemiluminescence leads to the chemiluminescence resonance energy transfer (CRET) to the QDs, resulting in the photoexcitation of the QDs and the generation of electron–hole pairs. The transfer of the conduction-band electrons to the electrode, and the concomitant scavenging of the valence-band holes by the triethanolamine electron donor result in the generation of photocurrents. The CRET-stimulated generation of photocurrents is applied to sense DNA by the labeling of the probe–analyte complex with a hemin/G-quadruple, and is also implemented to follow the activity of glucose oxidase and to sense glucose, by the labeling of the enzyme with the hemin/G-quadruplex catalyst

Nucleoapzymes: Hemin/G-Quadruplex DNAzyme–Aptamer Binding Site Conjugates with Superior Enzyme-like Catalytic Functions

A novel concept to improve the catalytic functions of nucleic acids (DNAzymes) is introduced. The method involves the conjugation of a DNA recognition sequence (aptamer) to the catalytic DNAzyme, yielding a hybrid structure termed “nucleoapzyme”. Concentrating the substrate within the “nucleoapzyme” leads to enhanced catalytic activity, displaying saturation kinetics. Different conjugation modes of the aptamer/DNAzyme units and the availability of different aptamer sequences for a substrate provide diverse means to design improved catalysts. This is exemplified with (i) The H₂O₂-mediated oxidation of dopamine to aminochrome using a series of hemin/G-quadruplex-dopamine aptamer nucleoapzymes. All nucleoapzymes reveal enhanced catalytic activities as compared to the separated DNAzyme/aptamer units, and the most active nucleoapzyme reveals a 20-fold enhanced activity. Molecular dynamics simulations provide rational assessment of the activity of the various nucleoapzymes. The hemin/G-quadruplex–aptamer nucleoapzyme also stimulates the chiroselective oxidation of l- vs d-DOPA by H₂O₂. (ii) The H₂O₂-mediated oxidation of <i>N</i>-hydroxy-l-arginine to l-citrulline by a series of hemin/G-quadruplex–arginine aptamer conjugated nucleoapzymes