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

In this work, an ultrasensitive electrochemical microRNA detection strategy was developed based on porous palladium-modified horseradish peroxidase sphere ( [email protected] ) and target-induced assembly of DNAzyme. A highly loaded HRP sphere was prepared by covalent layer-by-layer assembly with CaCO 3 as sacrificial template for the first time, and was further modified with porous Pd. Notably, [email protected] composite showed a good redox activity of HRP and electrocatalytic activity toward H 2 O 2 . The utilization of [email protected] 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 Pb 2+ -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 [email protected] probes, which led to the increased loading of redox-active and electrocatalytic [email protected] 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 [email protected] 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.