Department of Chemical and Biomolecular Engineering, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Biosens Bioelectron. 2010 Apr 15;25(8):1941-6. doi: 10.1016/j.bios.2010.01.010. Epub 2010 Jan 18.
We describe here a greatly simplified colorimetric detection method to identify PCR-amplified nucleic acids. Our method relies on the PCR product having thiol group at one end, which is generated by employing thiolated PCR primer. After PCR amplification reaction, unmodified gold nanoparticles (AuNPs) are added into the reaction tube followed by the addition of NaCl to induce the aggregation of AuNPs. The PCR products strongly bind to the surface of AuNPs through the interaction of the terminal thiol groups and the long chain of DNA which has abundant negative charges enhances the electrostatic and steric repulsion among AuNPs, which consequently leads to the prevention of the salt-induced aggregation. As a result, the color of AuNPs remains red in the presence of the PCR-amplified nucleic acids, while the AuNPs change its color from red to blue due to the salt-induced aggregation in the absence of the PCR products. This simple but very efficient colorimetric strategy was successfully demonstrated by diagnosing Chlamydia infection using a real human urine sample. Since the results can be clearly seen with the naked eye without any complicated step such as surface modification of AuNPs or PCR product purification, this method can be easily applied to point-of-care diagnosis.
我们在这里描述了一种大大简化的比色检测方法，用于识别 PCR 扩增的核酸。我们的方法依赖于 PCR 产物在一端具有巯基，这是通过使用巯基化的 PCR 引物产生的。在 PCR 扩增反应后，将未修饰的金纳米粒子 (AuNPs) 加入反应管中，然后加入 NaCl 诱导 AuNPs 的聚集。PCR 产物通过末端巯基基团与带大量负电荷的 DNA 长链的相互作用强烈结合到 AuNPs 表面，这增强了 AuNPs 之间的静电和空间排斥，从而防止了盐诱导的聚集。因此，在存在 PCR 扩增的核酸的情况下，AuNPs 的颜色保持红色，而在不存在 PCR 产物的情况下，由于盐诱导的聚集，AuNPs 的颜色从红色变为蓝色。使用真实的人尿样诊断衣原体感染成功地证明了这种简单但非常有效的比色策略。由于无需进行 AuNPs 的表面修饰或 PCR 产物纯化等复杂步骤，仅凭肉眼即可清楚地看到结果，因此该方法可以轻松应用于即时诊断。
