An Ultrasensitive Light-up Cu²⁺ Biosensor Using a New DNAzyme Cleaving a Phosphorothioate-Modified Substrate

Abstract

Cu 2+ is a very important metal ion in biology, environmental science, and industry. Developing biosensors for Cu 2+ is a key topic in analytical chemistry. DNAzyme-based sensors are highly attractive for their excellent sensitivity, stability, and programmability. In the past decade, a few Cu 2+ biosensors were reported using DNAzymes with DNA cleavage or DNA ligation activity.However, they require unstable ascorbate or imidazole activation. So far, no RNA-cleaving DNAzymes specific for Cu 2+ are known. In this work, a phosphorothioate (PS) RNA containing library was used for in vitro selection, and a few new Cu 2+ -specific RNA-cleaving DNAzymes were isolated. Among them, a DNAzyme named PSCu10 was studied further. It has only eight nucleotides in the enzyme loop with a cleavage rate of 0.1 min -1 in the presence of 1 µM Cu 2+ at pH 6.0 (its optimal pH). Between the two diastereomers of the PS RNA chiral center, the Rp isomer is 37 times more active than the Sp one. Among the other divalent metal ions, only Hg 2+ can cleave the substrate due to its extremely high thiophilicity. A catalytic beacon sensor was designed with a detection limit of 1.6 nM Cu 2+ and extremely high selectivity. PSCu10 is specific for Cu 2+ and it has no cleavage in the presence of ascorbate, which reduces Cu 2+ to Cu + .3

Theranostic DNAzymes

“…Many interesting progresses were made by using transition metal ions 7, 11, 35. Many DNAzymes were reported to use Zn 2+ 26, 36, UO 2 2+ 14, Cu 2+ 37, Cd 2+ 29, 38, Hg 2+ 39, and Ag + 40. We also isolated a series of lanthanide-dependent DNAzymes 15, 41-43.…”

DNAzymes: Selected for Applications

“…In addition to obtaining RNA‐cleaving DNAzymes from random‐sequence pools, the Liu group has also made considerable post‐selection efforts to produce phosphorothioate RNA‐cleaving DNAzymes. For example, his group has developed two phosphorothioate RNA‐cleaving DNAzymes, one for Cd 2+ and the other for Cu 2+ , both of which are highly specific …”

DNA‐Templated Copper Nanoprobes: Overview, Feature, Application, and Current Development in Detection Technologies

“…Copper ions (Cu 2+ ), are essential micronutrient for plants and animals at low concentration levels, as they act as an essential cofactor for many protein enzymes in living organisms . However, excess Cu 2+ has been reported to be associated with many serious diseases, and Cu is one of the significant heavy‐metal environmental pollutants . A visual and portable strategy for Cu 2+ detection was based on using a strip‐like hydrogel (Figure (C)) .…”