A bisulfite-free sequence-independent biosensor for genomic 5-hydroxymethylcytosine detection via triple-cascade...

5-Hydroxymethylcytosine (5hmC) is a crucial epigenetic modification involved in various biological processes and diseases, but its accurate detection remains challenging due to difficulty in distinguishing it from 5-methylcytosine (5mC). Herein, we develop a bisulfite-free, sequence-independent biosensor for genomic 5hmC detection via triple-cascade signal amplification. This strategy integrates 5hmC-specific glycosylation-mediated magnetic separation with terminal deoxynucleotidyl transferase (TdT)-assisted extension, rolling circle amplification (RCA), and DNAzyme catalysis. Upon recognition, 5hmC is azide-labeled by T4 phage β-glucosyltransferase (T4 β-GT) and covalently captured by dibenzocyclooctyne (DBCO)-modified magnetic beads via click chemistry. The captured DNA undergoes TdT-catalyzed poly(A) extension, triggering apurinic/apyrimidinic endonuclease 1 (APE1)-mediated cleavage of AP probes to release primers. These primers initiate RCA, generating abundant DNAzymes that cleave fluorogenic probes for fluorescence readout. This assay is bisulfite-free and sequence-independent without the need for either specific antibodies or DNA degradation, facilitating genome-wide 5hmC analysis. The biosensor achieves a detection limit of 4.41 fM, discriminates 5hmC from 5mC and C, distinguishes 5hmC levels among cell lines with single-cell resolution, and quantifies 5hmC in human tissues with results consistent with gold-standard ELISA. This work provides a powerful platform for low-abundance epigenetic analysis in clinical diagnosis and biomedical research.