Just-in-Time\nGeneration of Nanolabels via In Situ\nBiomineralization of ZIF‑8 Enabling Ultrasensitive MicroRNA\nDetection on Unmodified Electrodes

Nanolabels can enhance the detection performance of electrochemical\nbiosensing methods, yet their practical application is hindered by\ncomplex preparation, batch-to-batch variability, and poor long-term\nstorage stability. Herein, we present a novel electrochemical method\nfor miRNA detection based on the just-in-time generation of zeolitic\nimidazolate framework-8 (ZIF-8) nanolabels initiated by nucleic acids.\nIn this design, the target miRNA-21 is captured with magnetic beads\nand polyadenylated by Escherichia coli Poly(A) polymerase (<i>E</i>PP), producing miRNA-21 molecules\nwith poly(A) tails (miR-21-poly(A)). These molecules are then adsorbed\nonto a bare gold electrode (AuE) surface via adenine–gold affinity\ninteractions, serving as nucleation sites for the rapid <i>in\nsitu</i> formation of ZIF-8 nanoparticles. The ZIF-8 nanoparticles\nfunction as signal labels, impeding electron transfer at the electrode\ninterfaces and thereby generating a notable electrochemical signal.\nThe developed method demonstrated exceptional sensitivity, with a\ndetection limit (LOD) as low as 2.3 aM and a linear detection range\nfrom 10 aM to 1000 fM. The practical application of the developed\nmethod was validated by using it to evaluate miRNA-21 expression levels\nin various biological samples, including cell lines, tumor tissues,\nand clinical blood samples from non-small cell lung cancer (NSCLC)\npatients. This approach simplifies the detection process by eliminating\nthe need for presynthesized nanomaterials and premodified electrodes.\nIts simplicity and high sensitivity make this method a promising tool\nfor point-of-care testing and a wide range of biomedical research\napplications.