In-situ generation of potassium ferricyanide for label-free and enzyme-free chemiluminescence detection of telomerase activity

Abstract

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 K3Fe(CN)6, 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)n 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 K4Fe(CN)6 to K3Fe(CN)6, 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 K3Fe(CN)6/luminol CL reaction.