title: Label-free colorimetric detection of cancer related gene based on two-step amplification of molecular machine.
abstract: Highly sensitive detection of K-ras gene is of great significance in biomedical research and clinical diagnosis. Here, we developed a colorimetric biosensing system for the detection of proto-oncogene K-ras based on enhanced amplification effect of DNA molecular machine, where dual isothermal circular strand-displacement amplification (D-SDA) occurs on two arms in one-to-one correspondence. Specifically, we designed a primer-locked hairpin probe (HP) and a primer-contained linear polymerization template (PPT). In the presence of target gene, HP can hybridize with PPT, forming a DNA molecular machine with dual functional arms (called DFA-machine). Each of the two probes in this machine is able to be extended by polymerase on its counterpart species. Moreover, with the help of nicking endonuclease, the dual isothermal polymerization is converted into dual circular strand-displacement amplification, generating a large amount of anti-hemin aptamer-contained products. After binding to hemins, the aptamer/hemin duplex, horseradish peroxidase (HRP)-mimicking DNAzyme, was formed and catalyzed the oxidation of colorless ABTS by H2O2, producing a visible green color. The proposed colorimetric assay exhibits a wide linear range from 0.01 to 150nM with a low detection limit of 10pM. More interestingly, the mutations existing in target gene are easily observed by the naked eye. It should be noted that this colorimetric system was proved by the analysis of K-ras gene of SW620 cell lines. The simple and powerful DFA-machine is expected to provide promising potential in the sensitive detection of biomarkers for cancer diagnosis, prognosis and therapy.
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Huo
authors.initials: H
authors.lastname: Xu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Chen-Qiao
authors.initials: CQ
authors.lastname: Li
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Xiao-Yun
authors.initials: XY
authors.lastname: Liao
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
authors.affiliation: Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350002, China. Electronic address: wuzaisheng@163.com.
authors.forename: Zai-Sheng
authors.initials: ZS
authors.lastname: Wu
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics* / Q000302:isolation & purification; D015374:Biosensing Techniques; D003124:Colorimetry; D004247:DNA / Q000737:chemistry; D054856:G-Quadruplexes; D006427:Hemin / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D000090063:Proto-Oncogene Mas; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D006427:Hemin; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Colorimetric detection; DNA molecular machine with dual functional arms (DFA-machine); DNAzyme; Dual isothermal circular strand-displacement amplification (D-SDA); K-ras gene