A novel signal-on strategy for M.SssI methyltransfease activity analysis and inhibitor screening based on photoelectrochemical immunosensor

作者：Yang, Zhiqing; Wang, Fengrui; Wang, Mo; Yin, Huanshun^*; Ai, Shiyun

来源：Biosensors and Bioelectronics, 2015, 66: 109-114.

DOI：10.1016/j.bios.2014.11.015

• Photoelectrochemical immunosensor
• M.SssI MTase
• Anti-5-methylcytosine antibody
• RG108

In this work, a novel signal-on photoelectrochemical (PEC) immunosensor was fabricated for M.SssI methyltransfease (MTase) activity analysis and inhibitor screening based on an in situ electron donor producing strategy, where the anti-5-methylcytosine antibody was selected as DNA CpG methylation recognition unit, gold nanoparticle labeled streptavidin (SA-AuNPs) as signal amplification unit and alkaline phosphatase conjugated biotin (ALP-Biotin) as enzymatic unit. In the presence of M.SssI MTase, hairpin DNA1 containing the palindromic sequences of 5'-CCGG-3' in its stem was methylated. After hybridization with biotin-conjugated DNA2, the stem-loop structure of the hairpin DNA1 was unfolded and the duplex strand DNA (dsDNA) was formed. Then, the dsDNA was captured on the surface of anti-5-methylcytosine antibody modified electrode through the specific immuno-reaction. Afterwards, SA-AuNPs and ALP-Biotin was further captured on the electrode surface through the specific reaction between biotin and streptavidin. Under the catalysis effect of ALP towards ascorbic acid 2-phosphate trisodium salt (AAP), ascorbic acid (AA) was in situ produced as electron donor and a strong PEC response was obtained. The fabricated biosensor showed high detection sensitivity with low detection limit of 0.33 unit/mL for M.SssI MTase. Furthermore, the inhibition research suggested that RG108 could inhibit the M.SssI MTase activity with the IC50 value of 152.54 nM.

• 出版日期2015-4-15
• 单位山东农业大学