title: DNAzyme-based probe for circulating microRNA detection in peripheral blood.
abstract: BACKGROUND
  The recent discovery of microRNAs (miRNAs) and their extracellular presence suggest a potential role of these regulatory molecules in defining the metastatic potential of cancer cells and mediating the cancer-host communication. This study aims to improve the sensitivity of miRNA detection via DNAzyme-based method and enhance the selectivity by using the DNAzyme-based probe to reduce nonspecific amplification.
  
  
  METHODS
  The miRNA probes were chemically synthesized with a phosphate at the 5' end and purified by polyacrylamide gel electrophoresis. Exosomal RNA from peripheral blood was isolated. Carboxylated magnetic microsphere beads (MBs) were functionalized with streptavidin (SA) according to a previously reported method with some modification. T capture probe-coated SA-MBs (DNA-MBs) were also prepared. The fluorescent spectra were measured using a spectrofluorophotometer.
  
  
  RESULTS
  We designed an incomplete DNAzyme probe with two stems and one bubble structure as a recognition element for the specific detection of miRNA with high sensitivity. The background effects were decreased with increase of the added of DNA-MBs and capturing times. Therefore, 20 minutes was selected as the optimal concentration in the current study. The fluorescence intensity increases as the hybridization time changed and reached a constant level at 40 minutes, and 1 μM is the optimum signal probe concentration for self-assembled DNA concatemers formation. In the presence of miRNA, the fluorescence of the solution increased with increasing miRNA concentration. There is no obvious fluorescence in the presence of 10 mM of other nontarget DNA.
  
  
  CONCLUSIONS
  A simple, rapid method with high performance has been constructed based on identified circulating miRNA signatures using miRNA-induced DNAzyme. This assay is simple, inexpensive, and sensitive, enabling quantitative detection of as low as 10 fM miRNA.
authors.affiliation: Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Guoli
authors.initials: G
authors.lastname: Shao
authors.affiliation: Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Shufeng
authors.initials: S
authors.lastname: Ji
authors.affiliation: Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Aiguo
authors.initials: A
authors.lastname: Wu
authors.affiliation: Department of Critical Care Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Cuiping
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Mengchuan
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Pusheng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Qingli
authors.initials: Q
authors.lastname: Jiao
authors.affiliation: Department of Critical Care Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong Province, People's Republic of China.
authors.forename: Yuzhan
authors.initials: Y
authors.lastname: Kang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D021881:DNA, Catalytic / Q000097:blood / Q000138:chemical synthesis / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D035683:MicroRNAs / Q000097:blood*; D013050:Spectrometry, Fluorescence
chemical_list: D021881:DNA, Catalytic; D005456:Fluorescent Dyes; D035683:MicroRNAs
keywords: DNAzyme; breast cancer; detection; miRNAs