Rolling circle transcription/G-quadruplex/QnMorpholine probe for highly selective and sensitive detection of alkaline phosphatase activity

Abstract

In this study, we combined a rolling circle transcription (RCT) system producing 22AG G-quadruplex RNA with a QnMorpholine (QNM) fluorescent probe for the selective and sensitive detection of alkaline phosphatase (ALP). ALP is involved in various biological functions, with monophosphate cleavage being one of its characteristic properties. Here, we developed a padlock RCT probing system in which a large amount of RCT 22AG RNA G-quadruplex was produced in the absence of ALP, providing a high fluorescence signal. In contrast, no RNA G-quadruplex was produced in the presence of ALP, with minimal fluorescence. This huge deviation in signal intensity allowed us to identify the presence or absence of ALP in a test sample. Under practical conditions, our system allowed the differentiation for ALP even when it was present at an extremely low concentration (0.0085 U/L), along with very high specificity. The simplicity and efficiency of this approach for ALP detection suggest its potential for use as a reliable diagnostic tool.