Key Laboratory of Medical Cell Biology-Ministry of Education, Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, People's Republic of China.
Nanotechnology. 2013 Sep 6;24(35):355504. doi: 10.1088/0957-4484/24/35/355504. Epub 2013 Aug 7.
Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg²⁺ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification.
量子点在医学和生命科学的成像、标记和传感方面经历了快速发展。为了适用于聚合酶链反应（PCR）检测，我们已经测试了量子点的热循环耐久性和兼容性，这在以前的报告中尚未涉及。在这项研究中，我们合成了具有高分子量两亲性共聚物表面修饰的 CdSe/ZnS 量子点，并观察到 PCR 反应中的 Mg²⁺离子会在热循环后诱导量子点沉淀，并显著降低其荧光信号。为了克服这个问题，我们使用 mPEG2000 对 QD 表面进行共轭进一步保护，并发现这种修饰使 QD 在存在其他对 PCR 反应至关重要的成分的情况下能够耐受 40 个热循环。我们还发现 QD 对 rTaq 和 Ex Taq 聚合系统有不同的影响。高浓度的 QD 可明显降低 PCR 效率，但在 Ex PCR 系统中，随着 Ex Taq 聚合酶浓度的增加，这种抑制作用得到显著缓解。实时 PCR 扩增结果表明，QD 可以提供足够可测量的荧光信号，而不会过度抑制 DNA 扩增。基于这种改进的热循环耐久性和与 PCR 系统的兼容性，QD 有可能成为 PCR 和实时 PCR 扩增中稳定的荧光传感器。
