|
|||||
|
|
High Sensitive Electrochemiluminescence Biosensor Based on Ru(phen)32+-loaded Double Strand DNA as Signal Tags use to Detect DNA Methyltransferase Activity |
|
| Authors: | Ming Yang Liping Chen Longhua Guo Bin Qiu Zhenyu Lin |
| Institution: | 1. The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003 China
These authors contributed equally to this work.;2. Ministry of Education Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116 China These authors contributed equally to this work.;3. College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing, 314001 China;4. Ministry of Education Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116 China |
| Abstract: | DNA methyltransferase (DNA MTase) can act as biomarker for many diseases and it is important to develop some new methods for sensitive detection of DNA MTase. In this work, a highly efficient electrochemiluminescence (ECL) sensor had been designed for detection of DNA MTase based on Ru(phen)32+ loaded double strand DNA (dsDNA- Ru(phen)32+) as signal tags. Ru(phen)32+ had been efficiently embed in the dsDNA produced through a simple hybridization chain reaction. First, a hairpin probe was designed, which can be specifically recognized by Dam MTase and modified with -SH at one end. It was modified on the surface of gold electrode by -SH as an immobilization probe (IP). This IP will be methylated in the present of Dam MTase and digested by DpnI following. Results in the release of capture probe (CP) which remains on the surface of gold electrode. The CP can hybridize with the single stand part of the dsDNA- Ru(phen)32+ and make the immobilization of ECL tags on the electrode surface, which results in a strong ECL signals detected. However, without the effect of Dam MTase, the hairpin structure of IP remains stable and cannot capture signal tags, and can only detecte weak ECL signals. The biosensor can detect the activity of Dam MTase in the concentration range of 0.01 U/mL to 20 U/mL with the ECL intensity and the logarithm of the concentration have a linear relationship, and the detection limit is calculated to be 7.6 mU/mL. The developed sensor has the ability to specifically detect Dam MTase, which can be differentiated from other types of DNA MTase. In addition, the designed method has good applicability to detect Dam MTase activity in serum samples and been applied to detect its inhibitor with high efficiency. |
| Keywords: | Electrochemiluminescence biosensor Dam MTase Inhibitor Hybridization chain reaction amplification Methylation |