Highly sensitive and specific fluorescence biosensor for global 5–hydroxymethylcytosine detection based on M.HhaI methyltransferase catalysis and the improved isothermal exponential amplification reaction

5-Hydroxymethylcytosine (5hmC) is closely correlated with cancer occurrence and development. Monitoring 5hmC level is of great significance for understanding the epigenetic status of cancer development. Here, a sensitive and specific fluorescence strategy for 5hmC assay was developed based on M.HhaI methyltransferase catalysis and the efficient amplification of the improved isothermal exponential amplification reaction (im-EXPAR). M.HhaI catalyzed the sulfhydryl group in cysteamine to replace the hydroxyl group in 5hmC, producing amino-derivatized 5hmC-dsDNA (NH2-5hmC-dsDNA). The 5′-carboxyl-modified anchor probe was incorporated with NH2-5hmC-dsDNA by amide reaction, forming compound DNA. Then, the anchor DNA in compound DNA hybridized with the circular template and served as primers to trigger im-EXPAR. The linear range of the assay was 100 aM-1 nM, spanning 7 orders of magnitude. The limit of detection was 37.4 aM, exhibiting high sensitivity. Compared with the linear templates, the utilization of circular templates elevated the sensitivity by two orders of magnitude. Moreover, the sensor showed good stability and excellent selectivity in distinguishing down to 0.001% of 5hmC from 5mC. Additionally, the method also demonstrated good analytical performance in human serum samples, and was successfully applied to genomic 5hmC analysis in various mice tissues with satisfactory results, showing its potential in clinical analysis.