Dual-mode CRISPR/Cas12a-mediated alkaline phosphatase detection (CAD) biosensor.

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensing

Background:

• Alkaline phosphatase (ALP) is a vital biomarker for diagnosing hepatobiliary disorders, bone diseases, and monitoring cancer progression.
• Existing detection methods for ALP often lack the required sensitivity, simplicity, and adaptability for diverse clinical and resource-limited settings.
• There is a critical need for advanced diagnostic tools that provide ultrasensitive and versatile ALP detection.

Purpose of the Study:

• To develop and validate a novel CRISPR/Cas12a-mediated ALP detection (CAD) system with a dual-signal readout (fluorescence and lateral flow immunoassay).
• To enhance sensitivity, specificity, and operational simplicity for ALP detection compared to existing methodologies.
• To enable both high-precision quantitative analysis in clinical settings and rapid qualitative detection in resource-limited environments.

Main Methods:

• A CRISPR/Cas12a-mediated isothermal amplification system was designed, utilizing a hairpin DNA probe (HPP) for ALP recognition.
• Upon ALP binding, Klenow (exo-) polymerase initiates elongation, activating Cas12a's *trans*-cleavage activity for signal amplification.
• The system was adapted for both fluorescence-based quantitative detection and lateral flow immunoassay (LFIA) for visual, instrument-free qualitative detection.

Main Results:

• The fluorescence readout achieved a highly sensitive detection limit of 0.1 U L^-1 with a wide linear range (0.1-10 U L^-1), surpassing conventional methods by an order of magnitude.
• The system demonstrated exceptional specificity against potential biological interferents.
• The LFIA adaptation provided a user-friendly, instrument-free visual detection with a cut-off value around 7 U L^-1, suitable for point-of-care applications.

Conclusions:

• The dual-mode CAD biosensing strategy offers a significant advancement in ALP detection, combining high precision with operational simplicity.
• This approach effectively addresses the limitations of current single-readout methods, providing a versatile tool for diverse diagnostic needs.
• The CAD system holds substantial potential for early disease diagnosis, patient monitoring, and point-of-care testing, particularly in resource-limited settings.