DNA glycosylases are engaged in the base excision repair process and play a vital role in maintaining genomic integrity. It remains a challenge for multiplexed detection of
DNA glycosylases in
cancer cells. Herein, we demonstrate the construction of a dephosphorylation-mediated chemiluminescent biosensor for multiplexed detection of human
alkyladenine DNA glycosylase (hAAG) and
uracil DNA glycosylase (UDG) in
cancer cells. In this biosensor, the generation of chemiluminescence signals relies on the dephosphorylation of 3-(2'-spiroadamantyl)-4-methoxy-4-(3''-phosphoryloxyphenyl)-1,2-dioxetane (
AMPPD) catalyzed by
alkaline phosphatase (ALP). We design a bifunctional
double-stranded DNA (dsDNA) substrate, a
biotin-labelled
poly-(T) probe, and two capture probes for the hAAG and UDG assay. This assay involves four steps including (1) the cleavage of the bifunctional dsDNA substrate induced by
DNA glycosylases, (2) the recognition of the 3'-OH terminus of the primer by TdT and the subsequent TdT-mediated polymerization reaction, (3) the construction of the AuNPs-dsDNA-ALP nanostructures, and (4) the
streptavidin-
alkaline phosphatase (SA-ALP)-initiated dephosphorylation of
AMPPD for the generation of an enhanced chemiluminescence signal. By taking advantage of the unique features of TdT-mediated polymerization and the intrinsic superiority of the ALP-
AMPPD-based chemiluminescence system, this biosensor exhibits good specificity and high sensitivity with a detection limit of 1.53 × 10-6 U mL-1 for hAAG and 1.77 × 10-6 U mL-1 for UDG, and it can even quantify multiple
DNA glycosylases at the single-cell level. Moreover, this biosensor can be applied for the measurement of kinetic parameters and the screening of
DNA glycosylase inhibitors, holding great potential in DNA damage-related biomedical research and disease diagnostics.