Multifunctional Photoelectrochemical Biosensor Based on ZnIn2S4/ZnS QDs@Au–Ag-Reversed Photocurrent of Cu-Metal–Organic Framework Coupled with CRISPR/Cas-12a-Shearing for Assay of Dual Targets

False positives and negatives in bioanalytical assays remain a persistent problem. Herein, a multifunctional photoelectrochemical (PEC) biosensor based on ZnIn2S4 (ZIS)/ZnS quantum dots (QDs)@Au–Ag-reversed photocurrent of Cu-metal–organic framework (MOF) coupled with CRISPR/Cas-12a-shearing was innovatively developed for assay of dual targets. First, Cu-MOF as a good PEC material shows cathodic photocurrent. Then, numerous ZIS/ZnS QDs were assembled to the Au–Ag nanoparticles (NPs) to prepare a stable and highly amplified signal probe, which can just match the energy level of Cu-MOFs and realized the polarity-reversed photocurrent of Cu-MOF for the first time. As the empty-core nanostructure of Au–Ag NPs has a high specific surface area and low material density, the bimetallic nanocrystal can much increase the reaction rate and improve the redox efficiency. When target CEA-produced cDNA opened the hairpin DNA (HP1 DNA) on the electrode, the ZIS/ZnS QDs@Au–Ag signal probe was conjugated to the electrode via DNA hybridization, achieving a significantly reversed PEC current for CEA detection. Moreover, the specific binding of kanamycin/aptamer generated the acDNA (activator), which can activate the trans-cleavage activity of the CRISPR-CAS12a system on ssDNA, so the signal probe was sheared and caused the obvious decrease of PEC signal for kanamycin detection. The newly developed ZIS/ZnS QDs@Au–Ag NPs displayed excellent PEC properties and reversed photocurrent to MOF and were combined with the unique CRISPR-Cas12a system to achieve sensitive detection of dual targets, which can open a new polarity-reversed PEC sensing platform for rapid and accurate analysis of multiple targets and can effectively avoid false positives results in clinical testing.