Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
Imaging Devices Laboratory, Research Institute of Electronics, Shizuoka University, Johoku 3-5-1, Naka-ku, Hamamatsu 432-8011, Japan.
Biosens Bioelectron. 2016 Dec 15;86:135-142. doi: 10.1016/j.bios.2016.06.027. Epub 2016 Jun 11.
Ultrasensitive, rapid and selective diagnostic probes are urgently needed to overcome the limitations of traditional probes for norovirus (NV). Here, we report the detection of NV genogroup II via nucleic acid hybridization technology using a quantum dot (QD)-conjugated molecular beacon (MB) probe. To boost the sensitivity of the MB assay system, an ultrasensitive QD fluorophore with unique optical properties was synthesized, characterized and exploited as a fluorescence signal generator. Alloyed thioglycolic (TGA)-capped CdZnSeS QDs with a high photoluminescence (PL) quantum yield (QY) value of 92% were synthesized, and a modified silanization method was employed to encapsulate the thiol-capped QDs in a silica layer. The resulting highly luminescent alloyed SiO2-coated CdZnSeS QDs had a remarkable PL QY value of 98%. Transmission electron microscopy and dynamic light scattering confirmed the monodispersity of the alloyed nanocrystals, and zeta potential analysis confirmed their colloidal stability. Powder X-ray diffraction and PL lifetime measurements confirmed the surface modification of the QDs. The alloyed TGA-capped and SiO2-coated CdZnSeS QD-conjugated MB bioprobes detected extremely low concentrations of NV RNA. Ultrasensitive detection of low concentrations of NV RNA with a limit of detection (LOD) of 8.2copies/mL in human serum and a LOD of 9.3 copies/mL in buffer was achieved using the SiO2-coated CdZnSeS QD-MB probes, an increase in sensitivity of 3-fold compared with the detection limit for NV RNA using TGA-capped CdZnSeS QD-MBs. The additional merits of our detection system are rapidity, specificity and improved sensitivity over conventional molecular test probes.
超灵敏、快速且选择性的诊断探针对于克服传统诺如病毒(NV)探针的局限性至关重要。在此,我们报告了通过核酸杂交技术使用量子点(QD)缀合的分子信标(MB)探针检测 NV 基因 II 组。为了提高 MB 分析系统的灵敏度,我们合成、表征了一种具有独特光学特性的超灵敏 QD 荧光团,并将其用作荧光信号发生器。合成了具有 92%高荧光量子产率(QY)值的合金硫醇(TGA)封端 CdZnSeS QD,并采用改良的硅烷化方法将巯基封端的 QD 包封在硅层中。所得具有高发光性的合金 SiO2 涂层 CdZnSeS QD 的 PLQY 值高达 98%。透射电子显微镜和动态光散射证实了合金纳米晶体的单分散性,而zeta 电位分析证实了其胶体稳定性。粉末 X 射线衍射和 PL 寿命测量证实了 QD 的表面修饰。合金 TGA 封端和 SiO2 涂层的 CdZnSeS QD 缀合 MB 生物探针可检测到极低浓度的 NV RNA。使用 SiO2 涂层的 CdZnSeS QD-MB 探针可检测到人血清中 NV RNA 的检测限(LOD)低至 8.2copies/mL,缓冲液中 LOD 低至 9.3copies/mL,与 TGA 封端的 CdZnSeS QD-MB 相比,灵敏度提高了 3 倍,实现了对 NV RNA 的超灵敏检测。与传统的分子检测探针相比,我们的检测系统具有快速、特异性和提高的灵敏度等额外优点。