Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
Analyst. 2013 Sep 7;138(17):4877-84. doi: 10.1039/c3an00774j. Epub 2013 Jul 8.
We have developed a method for the detection of genetic markers associated with high pathogenicity in influenza. The assay consists of an array of 5'-thiolated ssDNA oligonucleotides immobilized on the surface of a Ag nanorod substrate that serve as capture probes for the detection of synthetic RNA sequences coding for a genetic mutation in the influenza PB1-F2 protein. Hybridization of the DNA probes to their complementary RNA sequences was detected using surface-enhanced Raman spectroscopy (SERS). Multivariate statistical analysis was used to differentiate the spectra of the complementary DNA probe-RNA target hybrids from those of the non-complementary DNA probes containing a single base pair polymorphism. Hierarchical cluster analysis (HCA) was able to distinguish with 100% accuracy the spectra of the complementary DNA probe-RNA target from the spectra of the immobilized DNA probes alone, or the DNA probes incubated with non-complementary RNA sequences. Linearity of response and limits of sensitivity of the SERS-based assays were determined using a partial least squares (PLS) regression model; detection limits computed by PLS was determined to be 10 nM. The binding affinity of the DNA probes to their complementary RNA sequences was confirmed using enzyme-linked immunosorbent assay (ELISA); however, the detection limits observed using ELISA were approximately 10× higher (100 nM) than those determined by PLS analysis of the SERS spectra.
我们开发了一种用于检测流感高致病性相关遗传标记的方法。该测定法由固定在 Ag 纳米棒基底表面的 5′-巯基化 ssDNA 寡核苷酸阵列组成，作为检测编码流感 PB1-F2 蛋白遗传突变的合成 RNA 序列的捕获探针。使用表面增强拉曼光谱（SERS）检测 DNA 探针与其互补 RNA 序列的杂交。采用多元统计分析方法区分互补 DNA 探针-RNA 靶标杂交体的光谱与包含单个碱基对多态性的非互补 DNA 探针的光谱。层次聚类分析（HCA）能够以 100%的准确度区分互补 DNA 探针-RNA 靶标与固定化 DNA 探针本身或与非互补 RNA 序列孵育的 DNA 探针的光谱。使用偏最小二乘（PLS）回归模型确定基于 SERS 的测定的线性响应和灵敏度限制；通过 PLS 计算的检测限被确定为约 10 nM。使用酶联免疫吸附测定（ELISA）证实了 DNA 探针与其互补 RNA 序列的结合亲和力；然而，ELISA 观察到的检测限比 SERS 光谱的 PLS 分析确定的检测限高约 10 倍（约 100 nM）。
