Integrating CRISPR-Cas12a with Aptamer as a Logic Gate Biosensing Platform for the Detection of CD33 and CD123.

Molecular logic gates, as biomolecule-based computational systems, are highly suitable for multitarget detection due to their programmability and modularity. However, existing systems are primarily limited to nucleic acid detection and have not been widely applied to disease-related sensing, particularly for disease antigens. CD33 and CD123 are critical biomarkers for acute myeloid leukemia (AML), yet conventional detection methods rely on expensive equipment and complex procedures, limiting their accessibility and practicality. This study designs a DNA logic gate system integrating nucleic acid aptamers, catalytic hairpin assembly (CHA), and CRISPR-Cas12a, pioneering its use for AML antigen detection. The system comprises three modules: input recognition, signal amplification, and signal transduction. Nucleic acid aptamers specifically identify CD33 and CD123, while CHA enables efficient signal amplification and CRISPR-Cas12a generates a fluorescent output via trans-cleavage activity. The system operates stably at room temperature and implements multiple logic gate models, including YES, OR, AND, NOR, and INHIBIT, enabling the simultaneous detection of CD33 and CD123. Experimental results are visually distinguishable under blue light, and the system requires only standard fluorescence detection instruments. In serum samples, it exhibits excellent selectivity and stability, with a detection limit of 0.5 ng/mL. This study pioneers the application of logic gate technology for disease antigen detection, addressing a critical gap in AML biomarker sensing. Our study indicates that this logic detection platform, characterized by its simplicity in operation, high sensitivity, and versatility in logic functions, holds promise as a potent sensing system for the intelligent multiplex target detection of disease antigens, environmental pollutants, and heavy metals.