PADLOCK: A protein-enzyme-free dual-DNAzyme cascade for ultrasensitive and field-deployable Pb2+ detection

Ultrasensitive and field-deployable detection of Pb 2+ is highly desirable for environmental monitoring and public health protection. Nucleic acid-based sensors are promising for this purpose because they are programmable and compatible with isothermal analysis. However, current amplification strategies face a fundamental trade-off: enzyme-assisted methods provide high sensitivity yet require unstable protein catalysts and cold-chain logistics, whereas enzyme-free circuits are operationally robust but suffer from thermodynamic background leakage. To address this sensitivity–stability paradox, we propose PADLOCK (Pb 2+ -Activated Dual-DNAzyme Locked On-site Cascade Kit), a protein-enzyme-free amplification strategy operating at ambient temperature (25 °C). PADLOCK uses a two-stage catalytic cascade for high-gain signal amplification. Specifically, the upstream GR-5 DNAzyme module was optimized by tuning hairpin stem complementarity and loop geometry to suppress premature trigger release, while the downstream self-locked DNAzyme (SLD) was redesigned through base-pair engineering to improve the signal-to-noise ratio. This cascaded “cleavage-then-unlock” architecture achieves amplification gains substantially exceeding those of conventional enzyme-free circuits such as catalytic hairpin assembly (CHA) or hybridization chain reaction (HCR), while maintaining near-zero background, a combination that is difficult to achieve in enzyme-free systems. Under optimal conditions, PADLOCK demonstrates a sensitivity range of 1 pM to 1 μM with a detection limit of 342 fM and exceptional selectivity over eight competing ions. Reliable quantification in real water and soil samples yielded spike recoveries of 98.17%–111.69%. Furthermore, integration with a custom-built smartphone imaging interface further enables visual monitoring across 100 pM–1 μM, providing a robust, point-of-care testing (POCT) platform that eliminates cold-chain logistics and enables ambient-temperature operation for decentralized heavy-metal screening in resource-limited settings.