The Design Of G-quadruplex-hemin DNAzyme-based Metal Ions Sensors And Screening Of Fluoregenic Substrates For This DNAzyme

G-quadruplex is one of the specific DNA secondary structures and can bind with hemin to form the G-quadruplex DNAzyme. It has been reported that G-quadruplex DNAzyme can catalyze the oxidation of ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) in the presence of H2O2to generate a colored product ABTS+, accompanied by an increase in absorption signal around420nm. G-quadruplex DNAzyme has been widely used in the development of different kinds of sensors. In this work, two kinds of sensors were designed for the fast and convenient detection of Pb2+and Cu2+respectively, which based on G-quadruplex-hemin DNAzyme and corresponding metal ion-triggered DNA/RNA cleaving DNAzymes. Besides, to improve the sensitivity of the sensors based on the G-quadruplex-hemin DNAzyme, the suitable fluorogenic substrates were screened.The Pb2+"turn-on" sensor was developed on the basis of the Pb2+specific RNA-cleaving DNAzyme and a G-quadruplex-hemin DNAzyme. In this strategy, the substrate strand of the RNA-cleaving DNAzyme was designed as an intramolecular stem-loop structure, and a G-rich sequence was caged in the double-stranded stem and could not form catalytically active G-quadruplex DNAzyme. The Pb2+-triggered cleavage of the substrate strand could result in the release of the G-rich sequence and subsequent formation of a catalytic G-quadruplex-hemin DNAzyme. The self-blocking mechanism of the G-quadruplex DNAzyme provided the sensing system with a low background signal. The signal amplifications of both the RNA-cleaving DNAzyme and the G-quadruplex-hemin DNAzyme provided the sensing system with a high level of sensitivity. This "turn-on" colorimetric sensor can detect Pb2+with detection limit of14nM. This sensor design strategy can be used for other metal ions with reported specific DNA/RNA-cleaving DNAzymes.The Cu2+"turn-off’ sensor was developed by reconstructing the original Cu2+specific DNA-cleaving DNAzyme. One oligonucleotides was designed to fold to an intramolecular stem-loop structure, which could effectively form the Cu2+-triggered DNA-cleaving DNAzyme when hybridized with the oligonucleotides named PPO. Once the stem-loop structure formed, the G-rich sequences at the two ends of double-stranded stem contacted closely to each other and produced the dimolecular G-quadruplex. The Cu2+-triggered cleavage of the substrate region could result in the release of one G-rich sequence and the destruction of G-quadruplex. This "turn-off" colorimetric sensor can detect Cu2+with high levels of sensitivity and selectivity, with detection limit of16.6nM.Seven candidates were tested to determine the possibilities of them as flourogenic substrates for G-quadruplex-hemin DNAzymes. Among these candidates, tyramine hydro chloride gave the maximum signal-to-background ratio for the sensing systems with and without G-quadruplex, and thus was recommended as the fluorogenic substrates for the sensors that were developed on the basis of target-triggered G-quadruplex formation or destruction.10-acetyl-3’,7’-dihydroxyphenoxazine (ADHP) gave the maximum fluorescence signal change between the sensing system without and with H2O2, thus was recommended as the fluorogenic substrate for the sensors targeting the detection of H2O2or H2O2-related analytes. In a model system of G-quadruplex-hemin DNAzyme-based Cu2+sensor, fluorescence detection using tyramine hydrochloride as fluorogenic substrate could decrease the detection limit from4nM to0.7nM compared with the colorimetric detection.