Single Quantum Dot-Based Nanosensor For Rapid And Sensitive Detection Of Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase（TdT）is a unique template-free polymerase and is responsible for catalyzing random incorporation of nucleotides into the 3’-hydroxyl（OH）termini of single-stranded DNA（ssDNA）to generate random genetic information.The aberrant expression of TdT is associated with human leukemic disease,and thus the sensitive detection of TdT activity is of great importance to biomedical research and clinical diagnosis.Quantum dots（QDs）are novel semiconductor nanocrystals composed of elements from the periodic groups of II-VI and III-V,which have been frequently used as fluorescent labels with wide applications in the fields of chemistry,biology and medical research.Here,we develop a simple and rapid method for sensitive detection of TdT activity on the basis of polymerization-directed exonuclease-assisted construction of single QD-based fluorescence resonance energy transfer（FRET）nanosensor.This assay involves three steps:（1）TdT-catalyzede longation of DNA primers,（2）exonuclease III（Exo III）-mediated cyclic release of Cy5 molecules to form the Cy5-double stranded（dsDNA）-QD nanostructure,and（3）the measurement of QD-based FRET.The presence of TdT enables the addition of 2’-deoxythymidine 5’-triphosphates（dTTPs）to3’-hydroxyl（OH）terminus of DNA primer,generating a poly-T DNA sequence which may hybridize with the QD-modified capture probe to form a Cy5-dsDNA-QD nanostructure with efficient FRET between the QD and Cy5.The resultant dsDNA duplexes may function as the substrates of Exo III,and their subsequent digestion by Exo III may initiate the cycles of hybridization-digestion-release,leading to the release of T-rich DNA sequence and Cy5molecules from the Cy5-dsDNA-QD nanostructure and consequently the reduction of FRET efficiency.The TdT activity may be simply quantified through the measurement of QD-based FRET by total internal reflection fluorescence（TIRF）-based single-molecule imaging.This method is very simple without the involvement of any separation steps,rapid with the entire experimental time of 50 min,and sensitive with a detection limit of 1×10^-6 U/μL.Moreover,this method can be used for the screening of potential TdT inhibitors and accurate quantitation of TdT activity in only 5 cancer cells.