Enzyme-responsive aptasensor based on the functionalized fluorescent protein chromophore derivative for the detection of alkaline phosphatase activity

Abstract

Fluorescent proteins (FPs) based sensing strategies have been widely developed in bioanalytical applications. However, it still remains a challenge to realize the efficient and precise analysis. Recently, FPs chromophore and its derivatives with favorable optical properties are well-designed to simulate and replace the function of FPs by specifically binding to the selected fluorescent light-up aptamers (FLAPs), it is worthy note that there is still a lack of systematic approaches to remold these FPs mimics with enzyme-responsive functions. Taking alkaline phosphatase (ALP) as a research model, we here propose an ALP-responsive fluorescent DNA aptasensor (p-DAA) by utilizing a truncated Lettuce (tLet-10) FLAP and a phosphorylated DFHBI-derivative (ALP-DFSS) for achieving ALP activity analysis. Taking the HBI-chromophore as the remolding molecular template, ALP-DFSS has been successfully synthesized and characterized to response to ALP activity. With phosphate group substitution in ALP-DFSS, the decayed fluorescence of p-DAA can be observed by forming the complex of tLet-10/ALP-DFSS. While the turn-on fluorescence of p-DAA will be obtained by the ALP-triggered dephosphorylation process and further exhibit the label-free, fast-response, specific, and sensitive detection performances for both evaluating ALP activity and the inhibition efficiency of ALP inhibitor. Moreover, p-DAA allow the spiked ALP analysis in serum samples and is also applied for realizing the visual differentiation between normal cell line and cancer cell line by responding the endogenous ALP, which holds the great potential in guiding the chromophores functionalization and exploring aptasensing related biological research.