Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710119, China.
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, 710119, China.
Anal Chim Acta. 2021 Jun 22;1165:338550. doi: 10.1016/j.aca.2021.338550. Epub 2021 Apr 22.
Chemiluminescence (CL) assay is a promising point-of-care testing (POCT) technology due to the fast response, high sensitivity, and easy miniaturization. The application and performance of CL POCT method were highly dependent on the CL reaction. Herein, based on the CL reaction between luminol and in-situ generated KFe(CN), a low-cost, enzyme-free, and label-free CL POCT method was explored via a portable and handheld luminometer to detect telomerase activity. Telomerase elongated telomere substrate (TS) primer to form (TTAGGG) repeats which hybridize with multiple short DNAs. The intercalation of SYBR Green I (SGI) into double-stranded DNA (dsDNA) generated singlet oxygen under the irradiation of LED light source. Singlet oxygen was then employed for in-situ oxidation of KFe(CN) to KFe(CN), which could react with luminol to generate a strong CL intensity. Thus, telomerase activity could be specifically, sensitively, and label-free detected. The detection limit was down to 98 HeLa cells. The detection process was very simple, and the cost was about $0.01 for each measurement. Furthermore, telomerase activity was detectable in human serum samples, with spike recoveries from 96% to 105%. According to our knowledge, it is the first effort to develop a low-cost, label-free and enzyme-free CL method with good repeatability for detecting biomarker based on the analyte-triggered and in-situ generated KFe(CN)/luminol CL reaction.
化学发光（CL）分析是一种很有前途的即时检测（POCT）技术，因为它具有快速响应、高灵敏度和易于小型化等优点。CL POCT 方法的应用和性能高度依赖于 CL 反应。在此，基于鲁米诺和原位生成的 KFe(CN)之间的 CL 反应，我们通过一种便携式手持发光计探索了一种低成本、无酶和无标记的 CL POCT 方法来检测端粒酶活性。端粒酶将端粒延伸底物（TS）引物延伸，形成（TTAGGG）重复序列，与多个短 DNA 杂交。SYBR Green I（SGI）插入双链 DNA（dsDNA）中，在 LED 光源照射下产生单线态氧。然后，单线态氧用于原位氧化 KFe(CN)为 KFe(CN)，它可以与鲁米诺反应生成强 CL 强度。因此，可以特异性、灵敏性和无标记地检测端粒酶活性。检测限低至 98 个 HeLa 细胞。检测过程非常简单，每次测量的成本约为 0.01 美元。此外，在人血清样本中也可以检测到端粒酶活性，回收率在 96%到 105%之间。据我们所知，这是首次开发出一种低成本、无标记和无酶的 CL 方法，具有良好的重复性，可基于分析物触发和原位生成的 KFe(CN)/鲁米诺 CL 反应来检测生物标志物。
