Highly sensitive closed-tube detection of alkaline phosphatase based on phosphate-mediated CRISPR/Cas12a regulation

Recently, we reported that a 5'-terminal phosphate at the junction of split activators inhibits the trans-cleavage activity of Cas12a. In this work, we discovered that phosphate-mediated Cas12a inhibition has two key features: strong position dependence and notable dual-phosphate enhancement. AlphaFold 3 (AF3) structural predictions indicate that the inhibition is mediated by interactions between the phosphate and specific local residues on Cas12a. On the basis of these findings, we developed a highly sensitive alkaline phosphatase (ALP) biosensor by harnessing the principle that ALP controls Cas12a activation through dephosphorylating activators. Without the need for upstream signal amplification, the biosensor achieves a high sensitivity of 6.07 × 10^-6 U/L for ALP across a broad dynamic range of 7.5 × 10^-6 to 2.5 × 10^-2 U/L. Furthermore, the assay is readily extendable to the evaluation of ALP inhibitors. Notably, a closed-tube assay system with lyospheres (CASL) was developed. Our biosensor equipped with the CASL enables closed-tube ALP detection in human serum, effectively preventing environmental contamination and interference. Owing to its simple closed-tube workflow and excellent performance, this biosensor holds great potential for point-of-care clinical diagnostics. Beyond its application, the phosphate-mediated CRISPR/Cas12a regulation strategy elucidated herein offers deeper mechanistic insight and a versatile blueprint for future biosensor design.