Dual-Mode CRISPR/Cas12a-Mediated Alkaline Phosphatase Detection (CAD) Biosensor

Abstract

Alkaline phosphatase (ALP), a crucial biomarker for hepatobiliary disorders, bone diseases, and cancer progression, demands ultrasensitive detection methods to meet clinical diagnostic requirements. Current methodologies predominantly depend on single-readout mechanisms, which fail to address the growing requirements for sensitivity, operational simplicity, and adaptability to resource-limited settings. Herein，we present a CRISPR/Cas12a-mediated ALP Detection (CAD) isothermal amplification system that overcomes these challenges through a novel dual-signal (fluorescence and lateral flow immunoassay (LFIA)) readout mechanism. The system features a rationally engineered hairpin DNA probe (HPP) that initiates Klenow(exo-)-driven polymerase elongation upon ALP recognition, subsequently activating Cas12a's trans-cleavage activity for exponential signal amplification. With fluorescence readout, this cascade amplification strategy achieves unprecedented sensitivity with a detection limit of 0.1 U/L and a wide linear range (0.1-10 U/L), outperforming conventional colorimetric methods by one order of magnitude while maintaining exceptional specificity against biological interferents. Furthermore, the LFIA adaptation of readout bridges the gap between laboratory-based detection and point-of-care applications. This user-friendly adaptation enables instrument-free visual detection with a clear cut-off value at about 7 U/L, offering the potential to effectively differentiate pathological samples from normal physiological levels with an appropriate dilution factor of clinical samples. Our dual-mode biosensing strategy not only enables high-precision quantitative analysis in clinical settings but also facilitates rapid qualitative detection under resource-limited conditions, offering significant potential for early disease diagnosis and long-term therapeutic monitoring.