title: A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease.
abstract: An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5 fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer.
authors.affiliation: Department of Pharmaceutical Analysis, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350004, China.
authors.forename: Liqing
authors.initials: L
authors.lastname: Lin
authors.forename: Ailin
authors.initials: A
authors.lastname: Liu
authors.forename: Chengfei
authors.initials: C
authors.lastname: Zhao
authors.forename: Shaohuang
authors.initials: S
authors.lastname: Weng
authors.forename: Yun
authors.initials: Y
authors.lastname: Lei
authors.forename: Qicai
authors.initials: Q
authors.lastname: Liu
authors.forename: Xinhua
authors.initials: X
authors.lastname: Lin
authors.forename: Yuanzhong
authors.initials: Y
authors.lastname: Chen
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D053837:DNA Cleavage; D055664:Electrochemical Techniques; D006046:Gold / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry*; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D017354:Point Mutation; D018631:ras Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D006046:Gold; D018631:ras Proteins