title: A novel signal-on strategy for M.SssI methyltransfease activity analysis and inhibitor screening based on photoelectrochemical immunosensor.
abstract: 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 0.33unit/mL for M.SssI MTase. Furthermore, the inhibition research suggested that RG108 could inhibit the M.SssI MTase activity with the IC50 value of 152.54nM.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Yang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Fengrui
authors.initials: F
authors.lastname: Wang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Mo
authors.initials: M
authors.lastname: Wang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: yinhs@sdau.edu.cn.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: ashy@sdau.edu.cn.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis; D015374:Biosensing Techniques / Q000379:methods; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008027:Light; D057230:Limit of Detection
chemical_list: D004791:Enzyme Inhibitors; D044503:5-Methylcytosine; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases
keywords: Anti-5-methylcytosine antibody; M.SssI MTase; Photoelectrochemical immunosensor; RG108