Enzyme spheres as novel tracing tags coupled with target-induced DNAzyme assembly for ultrasensitive electrochemical microRNA assay

•A highly loaded HRP sphere was synthesized for the first time and was used as bifunctional label.•Pd@HRP served as signal indicator and electrocatalyst for the fabrication of biosensor.•DNAzyme-aided target recycling amplification and rolling circle amplification further amplify the signal.•The biosensor displayed an improved sensitivity for microRNA detection.

In this work, an ultrasensitive electrochemical microRNA detection strategy was developed based on porous palladium-modified horseradish peroxidase sphere (Pd@HRP) and target-induced assembly of DNAzyme. A highly loaded HRP sphere was prepared by covalent layer-by-layer assembly with CaCO3 as sacrificial template for the first time, and was further modified with porous Pd. Notably, Pd@HRP composite showed a good redox activity of HRP and electrocatalytic activity toward H2O2. The utilization of Pd@HRP as electrochemical signal indicator and enhancer to fabricate biosensor could avoid the need for additional redox mediator and amplify the detection sensitivity. Moreover, target recycling amplification was achieved by Pb2+-induced cleavage of ternary “Y” structure, circumventing the use of labile nuclease. Subsequent DNA concatamer synthesized through rolling circle amplification (RCA) reaction with cleaved hairpin probe as primer, hybridized with plentiful Pd@HRP-DNA probes, which led to the increased loading of redox-active and electrocatalytic Pd@HRP for sensitivity improvement. So the proposed electrochemical biosensor detected miRNA-24 down to 0.2 fM (S/N = 3) with a wide linear range from 3 fM to 1 nM. With bifunctional Pd@HRP tag, DNAzyme-aided target recycle and programmable junction probe, this strategy possessed the advantages of high efficiency, high sensitivity, low cost and versatility, and thus held great promise for other low-abundance nucleic acids determination.

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