Department of Chemical and Biological Engineering, College of Engineering, Korea University, Seoul 136-701, Republic of Korea.
Department of Chemical and Biological Engineering, College of Engineering, Korea University, Seoul 136-701, Republic of Korea.
Biosens Bioelectron. 2015 May 15;67:443-9. doi: 10.1016/j.bios.2014.09.003. Epub 2014 Sep 6.
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.
循环肿瘤 DNA（ctDNA）携带肿瘤特异性突变和甲基化，是一种很有前途的非侵入性癌症评估生物标志物。然而，现有的 ctDNA 检测方法仅限于遗传突变。最近，纳米等离子体学作为一种具有高灵敏度和特异性的一步双检测平台已经出现。在这里，我们提出了一种基于局域表面等离子体共振（LSPR）和金纳米粒子（AuNPs）的耦合等离子体模式，用于超灵敏检测肿瘤特异性突变（E542K 和 E545K）和 PIK3CA 基因的 ctDNA 甲基化的策略。肽核酸（PNA）被用作探针来捕获和富集 69bp 的 PIK3CA ctDNA。当 PNA 探针 AuNPs 暴露于 200fM 的 ctDNA 时，会产生 4.3nm 的 LSPR 峰位移，对应于主要响应。免疫金胶体被用作甲基化探测器，并基于等离子体耦合进行二次响应的增强。当免疫金胶体结合到两个甲基胞嘧啶（mCpG）时，LSPR 峰从 4.3nm 移动到 11.4nm，相对于主要响应增加了约 107%。这种增强导致灵敏度提高了四倍（~50fM），因为有两个 mCpG 位点，所以可以检测到 ctDNA。这些结果表明，该传感器可以同时检测 ctDNA 上的热点突变和表观遗传变化。有希望的是，该平台可以以低浓度检测其他特定肿瘤突变体和表观遗传变化。
