Target inhibition of rolling circle amplification generates G43 long nanowires for reverse signaling determination of alkaline phosphatase

Abstract

Background

Alkaline phosphatase (ALP) can catalyze the hydrolysis of phosphate esters under alkaline conditions, mediate the dephosphorylation of substances such as proteins and nucleic acids. ALP can participate in physiological activities such as bone mineralization, cell division, and immune regulation. Abnormal ALP activity is associated with various diseases, making it an important clinical biomarker. In recent years, highly sensitive detection technologies based on isothermal amplification have developed rapidly. The Rolling Circle Amplification (RCA) technology uses T4 DNA ligase to connect the 5′-phosphate group and 3′-hydroxyl terminus of the padlock probe into a ring, achieving efficient amplification through continuous rolling circle replication.

Results

Leveraging this characteristic, we developed a highly sensitive sensing method (RCA-LG) based on RCA-generated G43 long nanowires for ALP detection. T4 DNA ligase circularizes the RCT into a template. Subsequently, Phi29 DNA polymerase amplifies the primer along the circular template, producing G43 long nanowires that bind with Thioflavin T (ThT) to generate fluorescent signals. When ALP is present, it dephosphorylates the 5′ end of RCT, preventing circular template formation and thereby inhibiting the amplification reaction. The RCA-LG method can detect ALP concentrations as low as 1.0 × 10⁻⁶ U/mL within 2.5 h. We propose a novel one-step RCA approach that integrates ligation and amplification reactions into a single step, simplifying operations while enhancing amplification efficiency. Furthermore, we physically separated the ALP dephosphorylation and RCA processes to avoid potential contamination from repeated sample handling.

Significance and novelty

This strategy takes advantage of the characteristics of RCA without the need for redundant phosphorylation modifications. Moreover, we have proposed an improved one-step RCA method combined with physical partitioning, enabling highly sensitive and specific detection of ALP and allowing the detection of this target in complex samples. Furthermore, the RCA-LG strategy can also play an important role in the screening of enzyme inhibitors.