A highly sensitive homogeneous electrochemical assay for alkaline phosphatase activity based on single molecular beacon-initiated T7 exonuclease-mediated signal amplification

Abstract

Alkaline phosphatase (ALP), a class of enzymes that catalyzes the dephosphorylation of a variety of substrates, is one of the most commonly assayed enzymes in routine clinical practice, and an important biomarker related to many human diseases. Herein, a facile and highly sensitive homogeneous electrochemical biosensing strategy was proposed for the ALP activity detection based on single molecular beacon-initiated T7 exonuclease-assisted signal amplification. One 3′-phosphorylated and 5′-methylene blue (MB) labeled hairpin probe (HP) is ingeniously designed. In the presence of ALP, the dephosphorylation of HP, the subsequent Klenow fragment (KF) polymerase-catalyzed elongation and T7 exonuclease-catalyzed digestion of the duplex stem of HP take place, releasing MB-labeled mononucleotides and the trigger DNA (tDNA). tDNA then hybridizes with another HP and initiates the subsequent cycling cleavage process. As a result, a large amount of MB-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the ALP activity assay. A directly measured detection limit as low as 0.1 U L⁻¹ is obtained, which is comparable to that of the fluorescence method and up to three orders of magnitude lower than that of the immobilization-based electrochemical strategy previously reported. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits the advantages of simplicity and convenience, because the assay is carried out in the homogeneous solution phase and sophisticated electrode modification processes are avoided. Therefore, the homogeneous electrochemical method we proposed here is an ideal candidate for ALP activity detection in biochemical research and clinical practices.