Target-triggered DNAzyme walker with 3D walking unit for copper species sensing in serum: A multivalent binding strategy for improving the detection performance

Abstract Monitoring copper (Cu) species in bio-samples is critical for the investigation and auxiliary diagnosis of Cu-metabolism disorders. Herein, a target-triggered DNAzyme walker, consisting of Carboxyfluorescein (FAM)-labeled substrates of the Cu-DNAzyme (Cu-Sub) coated gold nanoparticles (AuNPs) (AuNPs@FAM-Cu-Sub track) and the enzyme chain of the Cu-DNAzyme (Cu-Enzy) functionalized magnetic beads (MBs@Cu-Enzy 3D walking unit), was designed for the sensitive detection of copper species. Upon addition of copper ion (Cu2+), DNAzyme was activated and triggered the walking process, thereby releasing FAM-labeled fragments and recovering fluorescence. When compared with free-running Cu-Enzy or co-conjugated Cu-Enzy on AuNPs walking units, the MBs@Cu-Enzy walking unit exhibited a higher cleavage efficiency, shorter detection time (40 min), and a lower limit of detection (LOD = 3 nM). This improved performance was mainly attributed to multivalent interactions between the 3D track and the 3D walking unit which dramatically increased the affinity and cleavage efficiency. Importantly, good accuracy was observed for copper species analysis in clinical serum samples (n = 130), relative to other instrumentation, inductively coupled plasma atomic emission spectrometry (ICP-AES) (P > 0.01). The serum detection data indicated that total, exchangeable and relative exchangeable Cu variables could serve as potential indicators for the auxiliary diagnosis of Cu-related disorders. Thus, our target-triggered DNAzyme walker provides improved technical support for the in-depth study of clinical Cu metabolic-related diseases.