In spite of the DNA walkers executing the signal accumulation task in the process of moving along the predetermined paths, the enhancement of walking dynamics and walking path controllability are still challenging due to the unprogrammed arrangements of DNA orbits. Taking these dilemmas into account, a bipedal DNA walker was designed skillfully by the virtue of wireframe orbits assembled by DNA cubes in order, which improved the efficiency and the continuity of walking. It could be attributed to the fact that both the contact chance and the dynamic interaction between walking strands and designated orbits were beneficial to minimize the possibility of derailment and improve the accumulation of signal. In addition, the hollow titanium dioxide nanospheres coated with rubrene (Rub@TiO2 NSs) were prepared by the etching of inner silicon dioxide nanoparticles (SiO2 NPs) to regulate the distribution pattern of rubrene (Rub) molecules and expose more electrochemically active sites for high-efficient electrochemiluminescence (ECL). Benefiting by the pore confinement-enhanced ECL, the electron and mass transfer was significantly accelerated because of the hollow structure of Rub@TiO2 NSs. Subsequently, endogenous dissolved oxygen as the coreactant and palladium nanoparticles (Pd NPs) as the coreaction accelerator were employed to constitute a ternary ECL system with explosive signal response. Combining with this ECL platform, the bipedal walker activated by the target can autonomously and directionally move on the DNA wireframe orbits to release the quenching probes continuously. In this way, the biosensor displayed a low detection limit (2.30 × 10–8 U·mL–1) and a wide linear range (1.0 × 10–7 to 1.0 × 10–1 U·mL–1) for the sensitive detection of Dam methyltransferase (Dam MTase) activity. Therefore, a novel strategy for the accurate quantification of epigenetic targets was developed by virtue of improving the walking dynamics of DNA walker and amplifying the ECL of Rub molecules.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are two of the most abundant epigenetic marks in mammalian genomes, and it has been proven that these dual epigenetic marks give a more accurate prediction of recurrence and survival in cancer than the individual mark. However, due to the similar structure and low expression of 5mC and 5hmC, it is challenging to distinguish and quantify the two methylation modifications. Herein, we employed the ten-eleven translocation family dioxygenases (TET) to convert 5mC to 5hmC via a specific labeling process, which realized the identification of the two marks based on a nanoconfined electrochemiluminescence (ECL) platform combined with the amplification strategy of a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system. Benefiting from the TET-mediated conversion strategy, a highly consistent labeling pathway was developed for identifying dual epigenetic marks on random sequence, which reduced the system error effectively. The ECL platform was established via preparing a carbonized polymer dot embedded SiO2 nanonetwork (CPDs@SiO2), which exhibited higher ECL efficiencies and more stable ECL performance compared to those of the scattered emitters due to the nanoconfinement-enhanced ECL effect. The proposed bioanalysis strategy could be employed for the identification and quantification of 5mC and 5hmC in the range from 100 aM to 100 pM, respectively, which provides a promising tool for early diagnosis of diseases associated with abnormal methylation.