Nanoplasmonic biosensor: detection and amplification of dual bio-signatures of circulating tumor DNA.

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.