A digital immuno-PCR assay for simultaneous determination of 5-methylcytosine and 5-hydroxymethylcytosine in human serum

Highlights: • Simultaneous detection of 5mC and 5hmC were of great importance for physiological and pathological study. • We successfully developed a novel digital immuno-PCR assay for simultaneous detection of 5mC and 5hmC in human serum. • The LOD of digital immuno-PCR signal readout for 5mC was 6.17 × 10⁻¹⁴ mol/L (61.7 fmol/L) and for 5hmC was 1.11 × 10⁻¹³ mol/L (0.111 pmol/L). This work aimed to develop an ultrasensitive and specific immunosorbent assay for simultaneous detection of double DNA methylation marks. Being considered the most important indicators in disease diagnosis, clinical treatment, and prognosis, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were chosen as the proof-of-concept targets. The described strategy consisted of Phos-tag Biotin anchoring at streptavidin-magnetic nanoparticles, specific immune recognition of anti-5mC antibody and anti-5hmC antibody and labeling of Barcode-antibody, signal amplification of immune PCR and digital PCR machine. Under optimal conditions, the digital immuno-PCR assay showed a board dynamic range from 2.7 × 10⁻¹³ mol/L to 2.7 × 10⁻⁹ mol/L and the detection limits were 61.7 fmol/L for 5mC, and of 0.111 pmol/L for 5hmC. A 16-fold and 186-fold improvement of LOD were obtained by the proposed approach for 5mC and 5hmC detection compared with real-time immune PCR. The approach also showed ideal specificity, repeatability and stability. The recovery test demonstrated that the digital immuno-PCR assay is a promising platform for the simultaneous determination of the two DNA methylation marks in human serum sample.