DNAzyme-Au nanoprobe coupled with graphene-oxide-loaded hybridization chain reaction signal amplification for fluorometric determination of alkaline phosphatase

A sensing platform is presented for the determination of alkaline phosphatase (ALP) activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide-loaded hybridization chain reaction (HCR/GO) system to achieve good detection sensitivity and specificity. This assay takes advantage of the strong affinity of pyrophosphate (PPi) to Cu2+ ions and the fact that ALP can hydrolyze pyrophosphate (PPi) to release free Cu2+ ions. In the presence of ALP, the released Cu2+ can promote the Cu2+-dependent DNAzyme to cleave the substrate that generates a shorter DNA fragment, which is responsible for further triggering the HCR/GO system to form a long fluorescence dsDNA and thereby giving an amplified fluorescence signal. Linear calibration range was obtained from 0.2 to 10 U L-1, and the limit of detection (LOD) is about 0.14 U L-1. The feasibility of the proposed method was validated by spiking ALP standards in bovine serum. The recovery ranged from 97.2 to 104.6%, and a coefficient of variation (CV) of less than 8% (n=3) was obtained. This assay strategy was also applied to evaluate the ALP inhibitor efficiency, which indicates that the assay has potential for drug screening.Graphical abstract

Automated summary

A sensing platform was developed for determining alkaline phosphatase activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide-loaded hybridization chain reaction system. The assay utilizes the strong affinity between pyrophosphate and Cu2+ ions, as well as the hydrolysis of pyrophosphate by ALP to release Cu2+ ions, to achieve good detection sensitivity and specificity. The feasibility of the method was validated by spiking ALP standards in bovine serum, showing potential for drug screening applications.