Hybridization chain reaction-DNAzyme amplified switch microplate assay for ultrasensitive detection of magnesium ions

It is well-recognized that metal ion contaminants present in food and the environment pose a serious threat to human health and contribute to huge economic losses. Therefore, the development of simple, rapid, sensitive, and on-site methods for the detection of metal ions has become an urgent need. Herein, we combined the isothermal hybridization chain reaction (HCR) and a DNAzyme to develop a dual-signal amplification sensing assay for ultrasensitive Mg²⁺ detection on microplates. In this assay, the linker DNA strand (LDNA) that triggered the formation of the HCR structure was immobilized on a microplate via the biotin-streptavidin conjugation. Upon addition of the H5 sequence substrate strand to form a DNAzyme structure, an amplification switch microplate with 2n signaling amplification sites was established. The HCR-DNAzyme switch was activated by capturing Mg²⁺, and the methylene blue (MB)-labeled H5 was released. It generated an electrochemical signal after being captured by the reporter electrode attached to its complementary sequence (CDNA), accomplishing an efficient detection of Mg²⁺. Moreover, owing to the 2n signal amplification of the HCR-DNAzyme system with the simple separation and purification processing of the microplate, the Mg²⁺ detection limit of this strategy was as low as 0.6 fM. Furthermore, this method could be employed for other targets by simply changing the recognition structure of the DNAzyme, revealing the potential practical applications of this strategy in a wide range of fields.