A novel signal-on strategy for M.SssI methyltransfease activity analysis and inhibitor screening based on photoelectrochemical immunosensor
Autor: | Shiyun Ai, Huanshun Yin, Zhiqing Yang, Mo Wang, Fengrui Wang |
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Rok vydání: | 2015 |
Předmět: |
Streptavidin
DNA-Cytosine Methylases Light Drug Evaluation Preclinical Biomedical Engineering Biophysics Electron donor Biosensing Techniques chemistry.chemical_compound Biotin Limit of Detection Electrochemistry Humans Enzyme Inhibitors Electrodes Palindromic sequence Immunoassay Detection limit Electrochemical Techniques General Medicine DNA Methylation Ascorbic acid Combinatorial chemistry Molecular biology chemistry 5-Methylcytosine Biosensor DNA Biotechnology |
Zdroj: | Biosensors and Bioelectronics. 66:109-114 |
ISSN: | 0956-5663 |
Popis: | 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. |
Databáze: | OpenAIRE |
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