Sensitive and accurate detection of ALP activity using a fluorescence on–off–on switch and mass barcode signal amplification

Abstract

Alkaline phosphatase (ALP) is an important biomarker for many diseases. Therefore, the sensitive and accurate detection of ALP activity is essential for fundamental biochemical processes and clinic diagnosis. Herein, we design a fluorescent on–off–on switch for sensitive and visual detection of ALP activity. Meanwhile, mass barcode-modified quantum dots (QDs) amplified the LC-MS/MS detection signal in complex biological samples. Firstly, the QDs were modified with phosphorylated Gly-Gly-Phe-Phe-Tyr (OPO₃H₂) peptide (GGFFYp) and the mass barcode. The fluorescence of QDs-SS-Yp was quenched by fluorescence resonance energy transfer (FRET) between QDs-SS-Yp and dansyl chloride (DNS). ALP can hydrolyze the phosphorylated peptide to form peptide self-assemblies on the QDs-SS-Yp surfaces. The effective separation distance between the QDs-SS-Yp donor and DNS acceptor becomes larger, restricting FRET between the QDs-SS-Yp and DNS. At this point, the obvious QDs-SS-Yp fluorescence signal can be restored. However, the absence of ALP results in no peptide self-assembly on the QDs-SS-Yp surface and no obvious QDs-SS-Yp fluorescence signal was detected. Therefore, the ALP activity can be analyzed according to the degree of fluorescence restoration by the fluorescence on–off–on switch. Finally, the small tag molecules obtained by cleaving the disulfide bond of the QDs-SS-Yp as a mass barcode were used to amplify the LC-MS/MS detection signal. The proposed approach shows a good linear relationship (from 0.01 to 2.4 U L⁻¹) and has the significant advantage of a low detection limit of 0.001 U L⁻¹.