Circulating tumor DNA (ctDNA) bearing tumor-specific mutation and methylation 
are promising biomarkers for noninvasive cancer assessment. However, existing 
methods for ctDNA detection are restricted to genetic mutations. Recently, 
nanoplasmonics has emerged as a platform for one-step dual detection with high 
sensitivity and specificity. Here we present a strategy for ultrasensitive 
detection of tumor-specific mutations (E542K and E545K) and methylation of ctDNA 
of PIK3CA gene based on localized surface plasmon resonance (LSPR) and the 
coupling plasmon mode of gold nanoparticles (AuNPs). Peptide nucleic acids (PNA) 
is used as a probe to capture and enrich the 69-bp PIK3CA ctDNA. The exposure of 
PNA-probed AuNPs to 200 fM ctDNA generates LSPR-peak shift of 4.3 nm, 
corresponding to the primary response. Immunogold colloids are exploited as 
methylation detectors and plasmon coupling based enhancement for secondary 
response. LSPR-peak shifted from 4.3 nm to 11.4 nm upon the immunogold colloids 
binding to two methylcytosines (mCpG), which is an approximately 107% increase, 
compared to that of the primary response. This enhancement leads to four times 
(~50 fM) improvement of sensitivity and because of two mCpG sites, ctDNA was 
detected. These results demonstrate that the sensor can simultaneously detect 
the hot-spot mutation and epigenetic changes on the ctDNA. Promisingly, other 
specific-tumor mutants and epigenetic changes can be detected at low 
concentration with this platform.