title: DNA-AuNPs based signal amplification for highly sensitive detection of DNA methylation, methyltransferase activity and inhibitor screening.
abstract: A sensitive and selective electrochemical method was developed for the detection of DNA methylation, determination of DNA methyltransferase (MTase) activity and screening of MTase inhibitor. Methylene blue (MB) was employed as electrochemical indicator and DNA-modified gold nanoparticles (AuNPs) were used as signal amplification unit because the DNA strands in this composite have strong adsorption ability for MB. First, the thiolated single-stranded DNA S1 was self-assembled on gold electrode, hybridization between the lower portion of DNA S1 and its complementary DNA S2 formed an identical double-stranded tetranucleotide target sequence for both DNA adenine methylation (Dam) MTase and methylation-resistant endonuclease Mbo I, then the upper portion of DNA S1 was hybridized with its complementary DNA S3 modified on AuNPs to bring the DNA S3-AuNPs amplification units onto the electrode. The DNA S1/S2/S3-AuNPs bioconjugate has lots of DNA strands, and they can adsorb abundant MB. Mbo I endounuclease could not cleave the identical target sequence after it was methylated by Dam MTase. On the contrary, the sequence without methylation could be cleaved, which would decrease the amount of adsorbed MB. The presence of redox-active MB was detected electrochemically by differential pulse voltammetry (DPV). Thus, the activity of Dam MTase and methylation status were sensitively converted to the DNA S3-AuNPs amplified DPV signals. The DPV signal demonstrated a linear relationship with logarithm of Dam concentration ranging from 0.075 to 30U/mL, achieving a detection limit of 0.02U/mL (S/N=3). Also, screening of Dam MTase inhibitor 5-fluorouracil was successfully investigated using this fabricated sensor.
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, No. 1 Yi Xue Yuan Road, Chongqing 400016, PR China.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Jing
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, No. 1 Yi Xue Yuan Road, Chongqing 400016, PR China.
authors.forename: Xianqing
authors.initials: X
authors.lastname: Cao
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, No. 1 Yi Xue Yuan Road, Chongqing 400016, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
authors.forename: Tian
authors.initials: T
authors.lastname: Lan
authors.affiliation: Nevada Nanotechnology Center, College of Engineering, University of Nevada, Las Vegas, NV 89154-4026, USA.
authors.forename: Yiyan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics of Education, Department of Laboratory Medicine, Chongqing Medical University, No. 1 Yi Xue Yuan Road, Chongqing 400016, PR China. Electronic address: guomingxie@cqmu.edu.cn.
authors.forename: Guoming
authors.initials: G
authors.lastname: Xie
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors*; D019175:DNA Methylation / Q000187:drug effects; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005472:Fluorouracil / Q000032:analysis / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D008751:Methylene Blue / Q000737:chemistry; D010316:Particle Size
chemical_list: D006046:Gold; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D008751:Methylene Blue; D005472:Fluorouracil
keywords: DNA methylation; DNA-AuNPs amplification unit; Electrochemical assay; Inhibitor screening; Methylene blue; Methyltransferase activity