Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China.
Biosens Bioelectron. 2010 Oct 15;26(2):491-6. doi: 10.1016/j.bios.2010.07.067. Epub 2010 Jul 24.
Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect β-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5α. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5α was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization.
量子点（QD）是无机荧光纳米晶体，与有机染料相比具有可调发射光谱和光漂白抗性等优异性能，这使得它们适用于分子信标应用。在这项工作中，制备了基于量子点的分子信标（QD 基 MBs），以特异性检测位于 pUC18 中的β-内酰胺酶基因，该基因负责细菌大肠杆菌（E. coli）DH5α中的抗生素耐药性。QD 基 MBs 通过将巯基乙酸-量子点（MAA-QD）与黑洞猝灭剂 2（BHQ2）标记的硫醇 DNA 管金属-硫醇键偶联而构建。通过凝胶电泳观察到产物表征中的两种分子信标，双链信标和发夹信标。首次在微小细菌 DH5α 中实现了基于 QD 的一步 FISH。QD 基 MBs 在与互补靶 DNA 杂交时保持其生物活性，这显示出消除非特异性生物探针吸附引起的背景噪声并实现质粒 pUC18 中基因更清晰聚焦的优异优势，以及在一步原位杂交中穿透细菌细胞和信号特异性的能力。
