Detection And Imaging Of Intracellular Zinc And Copper Ions With Fluorescent Probe Based On Deoxyribozyme

Recently, trace metal ions in the cells are becoming one of the important concerns due to their assignable function in the biological system and deleterious effects on the public health. Among these metals, Zn2+ is an essential micronutrient element for human life and plays active roles in the gene transcription and neural signal transmission. It also constitutes an important component of variety of enzymes and DNA-binding proteins. As another highly chemical reactive metal ion, Cu2+ has an important effect on the mitochondrial respiratory chain. Low-level Cu2+ is essential for biological activities such as enzyme regulation, metabolism and immune function. However, it has been demonstrated that high-level Cu2+ exposure can lead to neural disturbance, liver or kidney damage, as well as serious neurodegenerative diseases and prion diseases.Cell signaling pathways are multistage interconnection networks. During the signaling pathways, Zn2+ and Cu2+, which have widespread regulation of the biological function, are interactive and complementary to each other. Thus, it is important to simultaneously detect the cellular Zn2+ and Cu2+ on site and in real time, which would be beneficial in further understanding the Zn2+ and Cu2+ relative cellular events and biological process.Among kinds of detection methods for Zn2+ and Cu2+, molecular fluorescence imaging is undoubtedly a powerful tool for monitoring the localization and concentration of cellular Zn2+ and Cu2+ ions. Recently, great progress in the development of various imaging probes based on organic fluorophores and nanomaterials has been made. But, all of these probes could just detect cellular Zn2+ and Cu2+ individually.Based on this, this paper developed new methods for simultaneous imaging of Zn2+ and Cu2+ in living cells combined with DNAzymes and nanomaterials, such as grapheme oxide and gold nanoparticles. The main contents are as follow: 1. We present a novel two-color fluorescence nanoprobe based on the DNAzymes for. The probe consists of a 13 nm gold nanoparticle, DNAzymes that are specific for Zn2+ and Cu2+, and the substrate strands labeled with fluorophores at the 5' end and quenchers at the 3' end. The fluorescence of the fluoreophores is quenched both by the gold nanoparticle and the quencher. After the nanoprobes are transferred into the cells, the substrate strands would be cleaved in the presence of the Zn2+ and Cu2+ target, resulting in disassociation of the shorter DNA fragments containing fluorophores, which produce fluorescence signals correlated with the location and concentration of the Zn2+ and Cu2+. The nanoprobe exhibits high specificity, nuclease stability and good biocompatibility. Moreover, the nanoprobe can simultaneously monitor the cellular Zn2+ and Cu2+ with an on-site and real-time manner, providing the information of localization and concentration of targets which is significant to further research the Zn2+ and Cu2+ relative cellular events and biological process. The proposed method has shown great potential in the detection of multiple metal ions in living cells, which may help us to better understand the function of metal ions in the fields of biochemistry, molecular biology and cellular toxicology. 2. We present an amplification strategy utilizes the enzyme-free hybridization chain reaction(HCR) with graphene oxide(GO) as a carrier to image cellular. The resulting signal amplification provides excellent recognition and signal enhancement of Zn2+ and Cu2+ in living cells. As the fluorescence quencher and probe carrier, GO enables activation of the signal switch and effective intracellular delivery of amplification reagents. After the probes are transferred into the cells, the substrate strands of DNAzymes would be cleaved in the presence of the Zn2+ and Cu2+ target, resulting in disassociation of the shorter DNA fragments which could trigger hybridization chain reaction, produce fluorescence signals correlated with the location and concentration of the Zn2+ and Cu2+. This new imaging method realizes simple, sensitive and non-destructive signal amplification of Zn2+ and Cu2+ in living cells and has an ability to simultaneously image two types of Zn2+ and Cu2+ in the same cell. This method supplies accurate information regarding cellular Zn2+ and Cu2+-related biological events and provides a new tool for highly sensitive and simultaneous imaging of multiple metal ions, which may help us to better understand the function of metal ions in the fields of biochemistry, molecular biology and cellular toxicology.