Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
Sci Rep. 2024 Oct 3;14(1):22980. doi: 10.1038/s41598-024-74227-5.
Catalytic hairpin assembly (CHA)-DNA walker allows nanostructures to spontaneously hybridize to the nucleic acids. The localized surface plasmon resonance provides the ability of color-shift for Au nanoparticles (AuNPs) to design a colorimetric biosensor by implementing CHA-DNA walker reaction on AuNPs. A target gene in Klebsiella pneumoniae as the reaction cascade trigger, was selected. H1 and H2 oligonucleotides as the components of the system were designed and verified by NUPACK. The AuNPs were conjugated to H1. The conjugation of the probes to the AuNPs was evaluated using FT-IR. The signal amplification process was conducted at 25℃. TEM imaging, zeta potential, spectroscopy, and gel-electrophoresis were used to examine the conduction of the reaction cascade and specificity. The sensitivity of the method was analyzed using serial dilution of the target. The formation of over-52 bp intermediate secondary structures (which only exist when the reaction happens) was confirmed by gel-electrophoresis. The color distinction between positive (0.08 to 0.058) and negative samples (0.098 to 0.05) was evidenced instantly and in a period of 90 min of the reaction as a drop change of 520 nm intensity absorbance. TEM imaging confirmed the further distance of AuNPs in the positive sample in comparison to that of the negative sample which reveals effective detection of the pathogen. The LOD of the technique was measured as 2.5 nM of the target sequence. The diagnostic approach is a label-free, enzyme-independent approach and can be executed in a single step. It has been designed by employing the CHA-DNA walker system along with the colorimetric properties of AuNPs for the first time, thereby paving the way for more rapid and accurate diagnostic kits.
催化发夹组装 (CHA)-DNA walker 允许纳米结构自发杂交到核酸上。局部表面等离子体共振为金纳米粒子 (AuNPs) 提供了颜色移动的能力，通过在 AuNPs 上实施 CHA-DNA walker 反应来设计比色生物传感器。选择了肺炎克雷伯氏菌中的靶基因作为反应级联触发器。设计并通过 NUPACK 验证了 H1 和 H2 寡核苷酸作为系统的组成部分。将 AuNPs 与 H1 缀合。使用 FT-IR 评估了探针与 AuNPs 的缀合。在 25℃下进行信号放大过程。使用 TEM 成像、Zeta 电位、光谱和凝胶电泳来检查反应级联的进行和特异性。通过目标的连续稀释来分析该方法的灵敏度。通过凝胶电泳证实了超过 52bp 中间二级结构的形成（仅在反应发生时存在）。阳性（0.08 至 0.058）和阴性样本（0.098 至 0.05）之间的颜色差异可以立即区分，并在反应 90 分钟内观察到 520nm 强度吸光度的 52nm 变化。TEM 成像证实了阳性样本中 AuNPs 的进一步距离，与阴性样本相比，这揭示了对病原体的有效检测。该技术的 LOD 测量值为目标序列的 2.5 nM。该诊断方法是一种无标记、无酶的方法，可以一步完成。它首次采用 CHA-DNA walker 系统和 AuNPs 的比色特性设计，为更快速、更准确的诊断试剂盒铺平了道路。
