Label-Free and Template-Free Chemiluminescent Biosensor for Sensitive Detection of 5-Hydroxymethylcytosine in Genomic DNA

5-Hydroxymethylcytosine (5hmC) is a modified base present at low levels in various mammalian cells, and it plays essential roles gene expression, DNA demethylation, genomic reprogramming. Herein, we develop label-free template-free chemiluminescent biosensor for sensitive detection of 5hmC DNAs based on 5hmC-specific glucosylation, periodate (IO4+) oxidation, biotinylation, terminal deoxynucleotidyl transferase (TdT)-assisted isothermal amplification strategy, which term hmC-GLIB-IAS. This hmC-GLIB-IAS exhibits distinct advantages bisulfite-free, improved sensitivity, genome-wide analysis constant reaction temperature without the involvement either specially labeled nucleic acid probes or specific templates signal amplification. method can sensitively detect with limit 2.07 × 10–13 M, whole genome 3.92 10–5 ng/μL. Moreover, this distinguish from 5-methylcytosine (5mC) cytosine (C) even discriminate 0.1% mixture 5hmC-DNA 5mC-DNA. Importantly, strategy enables isotope-labeled substrates antibodies, providing powerful platform to real high reproducibility accuracy.