orm with a diameter of about 12 nm, and the dense lead phosphate cores appear black and are surrounded by an amorphous protein shell. It seems to confirm that lead ions were encapsulated into the core of the apoferritin. We calculated the loading amount of metal ions per apoferritin by comparing the concentrations of apoferritin and lead ions in the nanoparticle solution. The concentrations of apoferritin and metal ions were determined by the BCA method and ICP, respectively. The numbers of lead ions are 1300 per apoferritin with a standard deviation of ±90 (n=6).

Evaluation of immunoaffinities
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Nonspecific adsorption is one of the most important factors in development of nanoparticle-based DNA biosensor. Electrochemical quartz crystal microbalance (EQCM) and electrochemical methods were used to monitor the interaction among PNA, ctDNA and anti-5-mC antibodies. As shown in Fig. [2](#F2){ref-type="fig"}A, the frequency changes (Δ*f*) included the changing trends and well reproduced EQCM response. Before adding samples, the EQCM was initially equilibrated with Tris-HCl for 30 min, until stable baseline for Δ*f* was achieved. After attaching ctDNA, as expected, it resulted great decrease in Δ*f* response, indicating successful capture of ctDNA onto the PNA-AuNPs modified SPE surface (curve a). Further decrease of Δ*f* resulted from the introduction of LPA-anti-5-mC which was recognized by ctDNA based on immnointeraction. We also observed that two recognitions were completed both within 30 min. Besides, we used ncDNA as control for comparison (curve b). It showed that the resulting response of Δ*f* is very weak, which further proved strong interaction between PNA-AuNPs and ctDNA.

Furthermore, Square-wave voltammetry (SWV) measurements were used to confirm the above immunoaffinities. Here, 20 fM ctDNA, 1000 fM ncDNA, PBS and 1% BSA were served as controls to be simultaneously examined as the challenging proteins. As shown in Fig. [2](#F2){ref-type="fig"}B, there are no detectable