Electrochemiluminescent/Electrochemical ratiometric biosensor for extremely specific and ultrasensitive detection of circulating tumor DNA

Herein, an electrochemiluminescent (ECL)/electrochemical (EC) ratiometric biosensor was developed for the ultrasensitive determination of circulating tumor DNA (ctDNA) by means of an exonuclease III (Exo III)-assisted target recycling strategy. The ratiometric biosensing was realized by flexibly integrating the electrochemical activity and ECL quenching property of ferrocene (Fc). In addition, novel aggregation-induced electrochemiluminescence (AIECL) molecule of 4,4′-(benzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N, N-diphenylaniline) (TPA-BT) was designed, which possessed a symmetric donor-acceptor-donor (D-A-D) structure with a narrow band gap, which was beneficial to further enhance the AIECL emission. Further, the TPA-BT dots were synthesized to construct the ratiometric biosensor. Amazingly, a testing range of 100 aM-100 pM and a detection limit of 11.2 aM were finally obtained from the as-optimized biosensor for the analysis of ctDNA (KRAS gene). The strategy can be easily expanded for multiplexed detection of other ctDNAs by altering the hairpin DNA sequence with the assistance of Exo III-assisted target recycling, which has tremendous versatility and broad application prospects.