Sensitive and non-separation 5-hydroxymethylcytosine double-stranded DNA sensing in different mice tissues: Electrogenerated chemiluminescence biosensing method incorporating DNA nanomachine amplification and nanocomposite magnetic separation

Herein, a sensitive and non-separation electrogenerated chemiluminescence (ECL) biosensing method that combines poly (diallyl dimethyl ammonium chloride)/ferrosoferric oxide/molybdenum disulfide (PDDA/Fe3O4/MoS2) nanocomposite with DNA nanomachine was developed to determine 5-hydroxymethylcytosine modified double-strand DNA (5-hmC-dsDNA). A positively charged and magnetic PDDA/Fe3O4/MoS2 nanocomposite was proposed, which can be used to fix negatively charged 5-hmC-dsDNA to overcome the steric resistance problem and is available for magnetic fields separation. In addition, the DNA nanomachine with three elements was fabricated: a DNA walking probe for signal amplification, an ECL reagent labeled S1 DNA (Ru-S1), 5-hmC antibody (Ab) for specifically recognizing 5-hmC modification. In the presence of 5-hmC-dsDNA, sandwich-type conjugates were generated to incubate PDDA/Fe3O4/MoS2 and DNA nanomachine. After magnetic separation and the DNA walking driven by enzyme cleavage, a lot of ECL reagents were released and increased the ECL signal. ECL measurement was performed in a separation-free format. The method showed a broader linear range (0.0090–0.5761 %) and a low limit of detection (0.0024 %). Furthermore, the determination of 5-hmC-dsDNA in different mice tissues using the ECL biosensing method was also carried out, demonstrating that the established strategy may pave a novel avenue for the early clinical diagnosis of disease.