CRISPR/Cas13a-Programmed Cu NCs and Z-Scheme T-COF/Ag₂S for Photoelectrochemical Biosensing of circRNA

Circular RNAs (circRNAs), as a class of noncoding RNA molecules with a circular structure exhibit high stability and spatiotemporal-specific expression, making them ideal cancer biomarkers for liquid biopsy. Herein, a new photoelectrochemical (PEC) biosensor for a highly sensitive circRNA assay in the whole blood of lung cancer patients was designed based on CRISPR/Cas13a-programmed Cu nanoclusters (Cu NCs) and a Z-scheme covalent organic framework/silver sulfide (T-COF/Ag₂S) composite. This Z-scheme T-COF/Ag₂S composite accelerates electron transfer and produces an excellent initial photocurrent. When CRISPR/Cas13a precisely targets circRNA, it nonspecifically cleaves the triple-helix molecular structure to release DNA fragments (C'/C"). After the C'/C" opens the DNA hairpin probe (HP) modified on the electrode, hybridization chain reactions are performed to produce abundant AT-rich double-stranded DNA with the addition of H1 and H2 probes. Upon the incubation of Cu²⁺, Cu NCs are in situ formed via the A-Cu²⁺-T bonds and can effectively quench the photocurrent of the Z-scheme T-COF/Ag₂S due to the energy transfer process. This developed PEC biosensor for the circRNA assay shows a low limit of detection of 0.5 fM, and the reusability of DNA-modified magnetic beads (MB-DNA) reduces the detection cost. Moreover, the PEC biosensor can accurately quantify the circRNA level and distinguish the circRNA expression in whole blood from healthy controls and lung cancer patients, offering strong potential in clinical diagnosis.