Localization of multiple DNAzymes as a branchedzyme-powered nanodevice for the immunoassay of tumor biomarkers

Traditional immunoassay methods often face challenges due to the labeling procedure of protein enzymes, the use of multiple antibodies, and severe conditions. To address these limitations, we propose the concept of incorporating branchedzyme-powered nanodevices into immunoassays. In this strategy, multiple DNAzymes are localized onto gold nanoparticles (AuNPs) along with substrates. The localization format facilitates intramolecular reactions between DNAzymes and substrates, leading to accelerated kinetics of the nanodevice. Upon the formation of an immunocomplex with an antibody on a 96-well plate, the branchedzyme-powered nanodevice catalytically releases multiple fluorescent signals under ambient temperature, eliminating the need for secondary antibodies. The branched DNAzymes exhibit catalytic properties similar to those of protein enzymes, thus simplifying the assay procedure and achieving isothermal detection. Furthermore, the detection process can be controlled by the addition or deletion of cofactors. Additionally, the affinity ligand can be easily modified to construct nanodevices specific to different targets without requiring extensive redesign. This strategy has demonstrated successful quantification of tumor biomarkers such as alpha-fetoprotein (AFP) and prostatespecific antigen (PSA) at subpicomolar concentrations, showcasing its suitability for clinical applications. Consequently, the branchedzyme-powered nanodevice represents a valuable addition to the immunoassay toolbox, opening new possibilities for clinical diagnostics.