Alkaline phosphatase (ALP) is an essential hydrolase widely present in humans, and it extensively acts as a biomarker for multiple human diseases. Conventional ALP assays suffer from complicated synthesis, tedious operation, low sensitivity, and large sample consumption. Herein, we construct an end-repairing-engineered quadratic in vitro transcription machine for single-molecule monitoring of ALP in diverse cancers with 3'-phosphoryl (PO4) nucleic acid as a macromolecular substrate. In presence of ALP, it catalyzes the removal of 3'-PO4 group to yield a 3'-hydroxyl end in hairpin probe 1 (HP1). Under the catalysis of Taq ligase, 3'-hydroxylated HP1 and hairpin probe 2 (HP2) are ligated together to form an intact transcription template. With the addition of T7 RNA polymerase, in vitro transcription amplification is activated to synthesize numerous reporter probes. Resulting reporter probes can bind with signal probes to initiate duplex-specific nuclease (DSN)-aided cyclic degradation of signal probes. Eventually, multiple cycles of degradation-liberation-hybridization induce the generation of large amounts of FAM fluorophores that are counted via single-molecule imaging. Due to high specificity of ALP-directed 3'-end dephosphorylation, high efficiency of quadratic in vitro transcription cascades, and ultrahigh signal-to-noise ratio (SNR) of single-molecule counting, this machine can detect ALP with a limit of detection (LOD) of 7.93 × 10-8 U/μL in vitro and 1 cell in vivo. Furthermore, it can be applied for the evaluation of enzyme kinetics, screening of potential antidrugs, and quantification of ALP level in various cancer cells and human serums, holding potential in 3'-phosphatases-associated biological study and clinical diagnosis.
Keywords: 3′-end repair; Alkaline phosphatase; Cancer diagnosis; In vitro transcription; Single-molecule detection.
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