Ultrasensitive TALEs-mediated electrochemical sensor for DNA methylation detection based on RCA reaction and Exo III...

Pancreatic cancer has become one of the most challenging malignant tumors. This type of cancer shows a high mortality paralleling morbidity and a rather low 5-year survival rate. Early diagnosis of the disease is the key to prevent and treat the pancreatic cancer. Cellular free DNA (cfDNA) methylation detection is a promising non-invasive method in early cancer screening, diagnosis, and prognostic monitoring. However, there are still shortcomings in developing fast, sensitive, low-cost, and quantitative methods for detecting target methylated DNA. Herein, new transcription activator-like effectors (TALEs)-mediated electrochemical sensor by rolling circle amplification (RCA)-assisted "silver-link" crossing electrode was developed for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The TALEs-Ni magnetic beads could specifically recognize R-5mC and bind to target methylated DNA to trigger the RCA and catalytic hairpin assembly (CHA), thereby duplexes (H1-H2) were generated. By introducing H3 probe to cause CHA amplification and Exo III-assisted cyclic amplification strategy, large amounts of H2 probes were generated to trigger a new round of RCA reaction, long crossing DNA strands were formed between the gaps of electrodes. The "silver-link" crossing electrode was formed by metallization of "gene-link" and target was detected by recording this change in conductivity. The limit of detection (LOD) was 0.5 fM. The proposed electrochemical sensor showed good stability, high sensitivity and low detected background signal in the R-5mC detection. In addition, the R-5mC detection of cfDNA in plasma samples of cancer patients was satisfactory. This strategy is of great significance in the early screening, treatment and prognosis monitoring of pancreatic cancer.