Laboratory of Microanalysis, Institute of Bioc hemistry and Biophysics, University of Tehran, P.O. Box 131451384, Tehran, Iran; Nanobiomedicine Center of Excellence,g Nanoscience and Nanotechnology Research Center, University of Tehran, P.O. Box 131451384, Tehran, Iran.
Laboratory of Microanalysis, Institute of Bioc hemistry and Biophysics, University of Tehran, P.O. Box 131451384, Tehran, Iran; Nanobiomedicine Center of Excellence,g Nanoscience and Nanotechnology Research Center, University of Tehran, P.O. Box 131451384, Tehran, Iran.
Biosens Bioelectron. 2015 Oct 15;72:121-6. doi: 10.1016/j.bios.2015.04.078. Epub 2015 Apr 25.
A new colorimetric method for monitoring of rolling circle amplification was developed. At first H5N1 target hybrids with padlock probe (PLP) and then PLP is circularized upon the action of T4 ligase enzyme. Subsequently, the circular probe is served as a template for hyperbranched rolling circle amplification (HRCA) by utilizing Bst DNA polymerase enzyme. By improving the reaction, pyrophosphate is produced via DNA polymerization and chelates the Mg(2+) in the buffer solution. This causes change in solution color in the presence of hydroxy naphthol blue (HNB) as a metal indicator. By using pH shock instead of heat shock and isothermal RCA reaction not only the procedure becomes easier, but also application of HNB for colorimetric detection of RCA reaction further simplifies the assay. The responses of the biosensor toward H5N1 were linear in the concentration range from 0.16 to 1.20 pM with a detection limit of 28 fM.
开发了一种用于监测滚环扩增的比色法。首先，与发夹探针（PLP）杂交的 H5N1 靶标，然后在 T4 连接酶的作用下 PLP 环化。随后，环状探针被用作超支化滚环扩增（HRCA）的模板，利用 Bst DNA 聚合酶酶。通过改进反应，焦磷酸通过 DNA 聚合产生，并螯合缓冲溶液中的 Mg（2+）。这会导致在羟基萘酚蓝（HNB）作为金属指示剂存在的情况下溶液颜色发生变化。通过使用 pH 冲击代替热冲击和等温 RCA 反应，不仅使该过程变得更加容易，而且 HNB 用于 RCA 反应的比色检测进一步简化了测定。该生物传感器对 H5N1 的响应在 0.16 到 1.20 pM 的浓度范围内呈线性，检测限为 28 fM。
