Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Anal Biochem. 2010 Jun 15;401(2):182-7. doi: 10.1016/j.ab.2010.02.041. Epub 2010 Mar 4.
The preparation of DNA-tagged liposomes containing an encapsulated prosthetic group tracer, pyrroloquinoline quinone (PQQ), and their application to the development of a sandwich-type hybridization assay for the visual detection of single-stranded DNA are described. Capture DNA is conjugated to the surface of microtiter plate wells through a biotin-streptavidin interaction. Target DNA is incubated with the plate in high salt concentrations. The reporter DNA-tagged liposomes encapsulating PQQ, the prosthetic group of the apo-enzyme glucose dehydrogenase (GDH), are used as the label to probe for bound target DNA. After washing away unbound liposomes and subsequent lysis of the bound fraction by surfactant, PQQ is released and available to activate the apo-enzyme. In the presence of glucose and a redox dye, 2,6-dichlorophenol indophenol (DCPIP), the dye is reduced to yield an optical color change from blue to colorless. This transition is observed visually or spectrophotometrically. The degree of optical change is proportional to the amount of PQQ present, which directly relates to the number of liposomes and, thus, the total amount of target DNA. An arbitrary target DNA sequence is used as a model system, and a limit of detection of 62 fmol is achieved.
描述了一种制备含有包裹 prosthetic 组示踪剂吡咯并喹啉醌（PQQ）的 DNA 标记脂质体的方法，并将其应用于开发用于可视化检测单链 DNA 的夹心型杂交测定法。通过生物素-链霉亲和素相互作用将捕获 DNA 连接到微孔板孔的表面。将靶 DNA 与平板在高盐浓度下孵育。使用包封 PQQ 的报告 DNA 标记脂质体作为探针，探测结合的靶 DNA，PQQ 是脱辅基葡萄糖脱氢酶（GDH）的辅酶。在洗掉未结合的脂质体并通过表面活性剂裂解结合的部分后，释放出 PQQ，可激活脱辅基酶。在葡萄糖和氧化还原染料 2,6-二氯苯酚靛酚（DCPIP）存在下，染料被还原为从蓝色变为无色的光学颜色变化。可以通过肉眼观察或分光光度法观察到这种转变。光变化的程度与存在的 PQQ 量成正比，这直接与脂质体的数量以及因此与靶 DNA 的总量相关。使用任意靶 DNA 序列作为模型系统，检测限达到 62 fmol。
