Chemical-chemical redox cycling for improving the sensitivity of the fluorescent assay: A proof-of-concept towards DNA methylation detection

Ultrasensitive analytical methods are still urgent for the discovery of trace level biomarkers and the early clinical diagnosis of disease. In this work, an ultrasensitive universal sensing platform was constructed by integrating fluorescent assay with chemical-chemical redox cycling signal amplification strategy. Using Ru@SiO2 nanoparticles wrapped by MnO2 nanosheets (Ru@SiO2@MnO2) as fluorescent probe, the chemical-chemical redox cycling system was conducted upon ascorbic acid (AA) and tris(2-carboxyethyl)phosphine (TCEP) as reductants and MnO2 nanosheets as oxidant. The MnO2 nanosheets not only could quench the fluorescence of Ru@SiO2 nanoparticles to reduce the background, but also could serve as oxidants to react with AA, generating dehydroascorbic acid (DHA). The DHA was reduced by TCEP in turn to form AA that participated in the next cycling of chemical-chemical redox reaction. Thus, the constantly released AA from the chemical-chemical redox cycling system could massively etch MnO2 nanosheets on Ru@SiO2 surface, making the fluorescence of Ru@SiO2 nanoparticles greatly recovered. It was shown that the sensitivity of the fluorescent assay was improved almost 52 times by utilizing the chemical-chemical redox cycling signal amplification strategy. This strategy was further employed to detect DNA methylation with the aid of AA-encapsulated liposomes that were modified with 5 mC antibodies to bind with the methylated DNA captured in 96-well plate. A detection of limit down to 16.2 fM was achieved for the detection of methylated DNA. It's believed that the incorporation of chemical-chemical redox cycling signal amplification strategy into fluorescent sensing paves a new way for ultrasensitive detection of biomarkers.