title: Dual amplified and ultrasensitive electrochemical detection of mutant DNA Biomarkers based on nuclease-assisted target recycling and rolling circle amplifications.
abstract: Based on nicking endonuclease (NEase)-assisted target recycling and rolling circle amplification (RCA) for in situ generation of numerous G-quadruplex/hemin complexes, we developed a new, dual amplified and ultrasensitive electrochemical biosensor for mutant human p53 gene. The target mutant DNA hybridizes with the loop portion of a dithiol-modified hairpin probe (HP) self-assembled on a gold sensing electrode and forms nicking site for the NEase, which cleaves the HP and releases the target DNA. The released target DNA again hybridizes with the intact HP and initiates the DNA recycling process with the assistance of the NEase, leading to the cleavage of a large number of the HPs and the generation of numerous primers for RCA. With rationally designed, G-quadruplex complementary sequence-encoded RCA circular template, subsequent RCA results in the formation of long DNA sequences with massive tandem-repeat G-quadruplex sequences, which further associate with hemin and generate significantly amplified current response for highly sensitive DNA detection down to 0.25 fM. The developed method also exhibits high specificity for the target DNA against single-base mismatched sequence. With the ultrahigh sensitivity feature induced by the dual signal amplification, the proposed method can thus offer new opportunities for the detection of trace amounts of DNA.
authors.affiliation: Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
authors.forename: Cuiyun
authors.initials: C
authors.lastname: Yang
authors.affiliation: Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. Electronic address: yunatswu@swu.edu.cn.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xiang
authors.affiliation: Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
authors.forename: Ruo
authors.initials: R
authors.lastname: Yuan
authors.affiliation: Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Chai
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation; D003217:Conductometry / Q000295:instrumentation*; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018390:Gene Targeting / Q000295:instrumentation*; D016158:Genes, p53 / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics*; D006427:Hemin / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D005819:Genetic Markers; D006427:Hemin; D004247:DNA
keywords: Electrochemical; G-quadruplex; Rolling circle amplification; Target recycling