Overcoming charge screening via porous nanostructures for attomolar microRNA detection

Electronic biosensors offer a compact, integrable platform for real-time biomolecular detection, yet their performance is often limited by charge screening in physiological environments. Here, we report a porous ZnO/Al2O3 biosensor with grain boundary-engineered nanostructures that enables attomolar-level (0.5 aM) detection of microRNAs (miRNAs). The porous architecture forms spontaneously during thermal annealing through Zn2+ diffusion toward the Al2O3 layer along the ZnO grain boundaries and through lateral surface diffusion, generating localized porosity at structurally defined sites. These regions disrupt charge neutrality and introduce energy barriers, thereby enhancing surface potential shifts upon miRNA hybridization. The biosensor maintains high sensitivity in high-ionic-strength buffers, indicating effective suppression of Debye screening. Using the CRISPR-Cas13a system, we functionalized the surface with a crRNA targeting miR-17, thereby achieving sequence-specific detection and demonstrating compatibility with programmable molecular recognition. This grain boundary-guided nanostructuring strategy establishes a scalable route to engineering biosensors with localized sensitivity and target selectivity. Our approach provides a platform for ultrasensitive nucleic acid detection and offers strong potential for integration into miniaturized diagnostic systems.