Highly sensitive and high-throughput detection of human alkyladenine DNA glycosylase via hybridization chain reaction-assisted quantum dot fluorescent assay.

Human alkyladenine DNA glycosylase (hAAG) is an important enzyme in the base excision repair (BER) pathway, and its abnormal expression is correlated with various human diseases. While several methods have been developed for hAAG detection, constructing low-background, highly sensitive, and high-throughput techniques remains a significant challenge. Herein, we introduce a highly sensitive and high-throughput platform for hAAG activity detection, utilizing quantum dots (QDs) as the signal sensitizer, the hybridization chain reaction (HCR) for signal amplification, and microplate wells for high-throughput analysis. The custom-designed hairpin DNA substrate with a glycosylase recognition site undergoes a conformational change upon the addition of hAAG and apurinic/apyrimidinic endonuclease 1 (APE1), resulting in the generation of primer chains. These released primer chains then initiate HCR-mediated signal amplification, creating numerous binding sites for DNA-functionalized QD (DNA-QD) probes. This method displays minimal background signal owing to the stable structure of the hairpin substrate and demonstrates excellent sensitivity with a detection limit of 0.012 U/mL. Notably, this strategy enables versatile evaluation of the hAAG inhibitor as well as the detection of endogenous hAAG from cancer cells, highlighting its potential for early clinical diagnosis. Additionally, this strategy could be adapted to quantify various DNA repair enzymes by rationally modifying the DNA substrates.