Single quantum dot-based nanosensor with multi-layer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase

The base excision repair (BER) is an important DNA repair pathway involved in the maintenance of genome stability. As the initiator of BER, DNA glycosylase can remove a damaged base from DNA through cleaving the N-glycosidic bond between the sugar moiety and the damaged base. Accurate quantification of DNA glycosylase is essential to the early diagnosis of various human diseases. However, conventional methods for DNA glycosylase assay usually suffer from poor sensitivity and complex probe design. Herein, we develop a single quantum dot-based nanosensor with multi-layer of multiple acceptors for ultrasensitive detection of human alkyladenine DNA glycosylase (hAAG) using apurinic/apyrimidinic endonuclease 1 (APE1)-assisted cyclic cleavage-mediated signal amplification in combination with the DNA polymerase-assisted multiple cyanine 5 (Cy5)-mediated fluorescence resonance energy transfer (FRET). The presence of hAAG induces the cleavage of hairpin substrate, generating a trigger. The resultant trigger can hybridize with a probe modified with an AP site, initiating the APE1-mediated cyclic cleavage to produce a large number of primers. The primers can subsequently initiate the polymerase-mediated signal amplification with a biotin-modified capture probe as the template, generating the biotin-/multiple Cy5-labeled double-stranded DNAs (dsDNAs). The resultant dsDNAs can assemble onto the QD surface to form the QD-dsDNA-Cy5 nanostructure, leading to efficient FRET from the QD to Cy5 under the excitation of 405 nm. In contrast to the typical QD-based FRET approaches, the assembly of multi-layer of multiple Cy5 molecules onto a single QD significantly amplify the FRET signal. We further verify the FRET model with one donor and multi-layer acceptors theoretically and experimentally. This single QD-based nanosensor can sensitively detect hAAG with a detection limit of as low as 4.42 × 10-12 U/μL. Moreover, it can detect hAAG even in single cancer cell, and distinguish the cancer cells from the normal cells. Importantly, this single QD-based nanosensor can be used for kinetic study and inhibition assay, and it may become a universal platform for the detection of other DNA repair enzymes by designing appropriate DNA substrates.