An ultrasensitive FEN1-aided LCR-electrochemical biosensor enables detection of BCR/ABL1 fusion transcripts in clinical samples for early diagnosis and minimal residual monitoring of CML.

Ligase chain reaction (LCR) is a promising nucleic acid amplification technique widely applied in molecular diagnosis. However, a critical limitation of LCR is target-independent amplification, primarily caused by the blunt-end ligation activity of thermophilic DNA ligases, which results in false positives and compromises sensitivity. To overcome this challenge, we developed a thermostable flap endonuclease 1 (FEN1)-aided LCR (FALCR) employing DNA probes with non-phosphorylated sticky ends. In each amplification cycle, FEN1 selectively cleaves the 5' DNA flap of the downstream probe in the presence of a DNA template, generating 5'-phosphorylated DNA nicks that enable ligation. In the absence of a DNA template, the non-phosphorylated sticky ends of the probes prevent ligation, effectively eliminating nonspecific amplification. We further integrated FALCR with a magnetic platform-based electrochemical biosensor (FA-eLCR), achieving ultrasensitive detection of DNA targets with a limit of detection (LOD) of 0.1 aM and a dynamic range spanning 10 aM to 10 fM. Furthermore, the designing of a duplex FALCR enabled the detection of BCR/ABL1p210 comprising e13a2/e14a2/both isoforms in a single assay. Finally, the duplex FA-eLCR successfully detected the BCR/ABL1p210 transcripts in patients with chronic myeloid leukemia (CML) and monitored the change of transcripts during treatment, with 100 % concordance to reverse transcription-quantitative polymerase chain reaction, highlighting its high potential in the early diagnosis and minimal residual disease (MRD) monitoring of CML. This work presents a novel strategy to address nonspecific amplification in LCR and introduces a cost-effective, highly sensitive platform for molecular diagnosis in clinical setting.