With hepatitis B virus double mutation (HBVDM) and KRAS G12V point mutation as model double mutations, it is shown that PEPS was able to detect double-stranded HBVDM and KRAS with 70% detection efficiency or better at concentration as low as 10<sup>-19</sup> M against single-stranded mutation detection at the same concentrations, which was validated by the following in situ fluorescent reporter microspheres (FRMs) detection as well as microscopic visualization of the FRMs bound to the captured mutant on the PEPS surface.