Multispatially Localized DNA Walker Coupling Covalent\nOrganic Framework for Dual-Mode Detection of Nucleocapsid Protein\nUsing the Walking-Recycling-Conversion Strategy

DNA\nwalkers have emerged as a powerful tool in bioanalysis; however,\nmany existing approaches are still restricted by low reaction kinetics\nand inaccurate single-mode detection. Herein, a fluorescence (FL)\nand electrochemical (EC) dual-mode biosensor was proposed based on\na multispatially localized DNA walker (m-DNA walker) coupling covalent\norganic framework (COF) using the walking-recycling-conversion strategy.\nSpecifically, the functionalized COF not only served as a three-dimensional\nnanocarrier but also acted as an effective quencher of the walking\ntracks. In the presence of the target, the activated m-DNA walker\nmoved fast along the numerous quenching tracks, leading to the cleavage\nof Cy3-H1 and the recovery of the FL signal. To further improve the\ndetection sensitivity, the Cy3-H1 fragments’ recycling process\nwas implemented with the generation of a large amount of S1 and S2,\nwhich caused the assembly of DNA-Fe<sup>3+</sup>-polydopamine network\namplifiers on the electrode. The rapid electrochemical conversion\nwas introduced to convert DNA-Fe<sup>3+</sup>-polydopamine into electroactive\nPrussian Blue, providing a significant EC signal output. Using nucleocapsid\nprotein (N-protein) as the model target, the designed biosensing platform\nproduced a FL/EC dual-mode readout with the detection limits of 65.0\nfg/mL for FL mode and 2.3 fg/mL for EC mode, which could eliminate\nthe interference from different reactive pathways and improve the\ndetection accuracy, holding potential application in early disease\ndiagnosis and treatment.