Ultra-sensitive detection of alkaline phosphatase activity using a SAHATA-Cas12a-ECL biosensor

• Published in: Microchemical journal 2024-12, Vol.207, p.112282, Article 112282
• Main Authors: Zhuo, Chenyi, Yang, Wujia, Sun, Guangrong, Zhang, Jiayi, Song, Zichun, Wei, Jihua, Gong, Yuanxun, Tang, Qianli, Zhang, Kai, Liao, Xianjiu
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase (ALP); CRISPR-Cas12a; Electrochemiluminescence (ECL); Split Activators for Highly Accessible Target Analysis (SAHATA)
• ISSN: 0026-265X
• DOI: 10.1016/j.microc.2024.112282

[Display omitted] •SAHATA-Cas12a-ECL biosensor achieves ultra-sensitive ALP detection with a 1.7 U/L limit.•Triple signal amplification enhances detection sensitivity and specificity.•High precision and reliability demonstrated in complex biological samples.•Unique CRISPR-Cas12a activation mechanism ensures accurate signal detection.•Simplified assay design reduces costs, suitable for clinical diagnostics and research. This study introduces a novel detection method combining the Split Activators for Highly Accessible Target Analysis (SAHATA) strategy with electrochemiluminescence (ECL) for sensitive and specific detection of alkaline phosphatase (ALP). ALP is a crucial enzyme involved in various physiological processes, and its activity levels serve as important biomarkers for several diseases. Traditional methods for measuring ALP activity have limitations, prompting the development of more reliable, accurate, and sensitive approaches. The developed method leverages the unique properties of CRISPR-Cas12a, which exhibits non-specific degradation of single-stranded DNA or RNA upon recognizing target nucleic acids. This property enables a triple signal amplification strategy, where T7 transcription generates RNA, CRISPR-Cas12a performs non-specific cleavage of single-stranded DNA, and these steps collectively enhance the ECL signal, ensuring high sensitivity and specificity. Key features of the SAHATA-Cas12a system include high sensitivity, with the biosensor showing a clear increase in ECL signal with higher ALP concentrations, achieving an exceptional detection limit of 1.7 U·L−1. The biosensor demonstrates negligible ECL signal enhancement in the presence of various interferents, confirming its robustness in selective ALP detection. The method’s performance was validated in complex biological samples, achieving high precision and recovery rates, which highlights its potential for clinical diagnostics and biological research.