Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, PR China.
Biosens Bioelectron. 2010 Apr 15;25(8):1934-40. doi: 10.1016/j.bios.2010.01.007. Epub 2010 Jan 18.
We report here a quantum dots-DNA (QDs-DNA) nanosensor based on fluorescence resonance energy transfer (FRET) for the detection of the target DNA and single mismatch in hepatitis B virus (HBV) gene. The proposed one-pot DNA detection method is simple, rapid and efficient due to the elimination of the washing and separation steps. In this study, the water-soluble CdSe/ZnS QDs were prepared by replacing the trioctylphosphine oxide (TOPO) on the surface of QDs with 3-mercaptopropionic acid (MPA). Subsequently, oligonucleotides were attached to the QDs surface to form functional QDs-DNA conjugates. Along with the addition of DNA targets and Cy5-modified signal DNAs into the QDs-DNA conjugates, sandwiched hybrids were formed. The resulting assembly brings the Cy5 fluorophore, the acceptor, and the QDs, the donor, into proximity, leading to fluorescence emission from the acceptor by means of FRET on illumination of the donor. In order to efficiently detect single-base mutants in HBV gene, oligonucleotide ligation assay was employed. If there existed a single-base mismatch, which could be recognized by the ligase, the detection probe was not ligated and no Cy5 emission was produced due to the lack of FRET. The feasibility of the proposed method was also demonstrated in the detection of synthetic 30-mer oliginucleotide targets derived from the HBV with a sensitivity of 4.0nM by using a multilabel counter. The method enables a simple and efficient detection that could be potentially used for high throughput and multiplex detections of target DNA and the mutants.
我们在此报告了一种基于荧光共振能量转移（FRET）的量子点-DNA（QDs-DNA）纳米传感器，用于检测乙型肝炎病毒（HBV）基因中的靶 DNA 和单碱基错配。由于消除了洗涤和分离步骤，所提出的一锅法 DNA 检测方法简单、快速且高效。在这项研究中，通过用 3-巯基丙酸（MPA）取代 QDs 表面上的三辛基氧磷（TOPO）来制备水溶性 CdSe/ZnS QDs。随后，寡核苷酸被连接到 QDs 表面上以形成功能性 QDs-DNA 缀合物。随着 DNA 靶标和 Cy5 修饰的信号 DNA 被添加到 QDs-DNA 缀合物中，形成夹心杂交体。所得组装使 Cy5 荧光团、受体和 QDs、供体接近，导致在供体的照射下通过 FRET 从受体发出荧光。为了有效地检测 HBV 基因中的单碱基突变，采用了寡核苷酸连接测定法。如果存在单碱基错配，其可以被连接酶识别，则由于缺乏 FRET，检测探针不会连接并且不会产生 Cy5 发射。该方法的可行性也通过使用多标记计数器从 HBV 衍生的合成 30 碱基 oliginucleotide 靶标检测中得到了证明，其灵敏度为 4.0nM。该方法能够进行简单高效的检测，有可能用于高通量和多重靶标 DNA 和突变体的检测。
