title: Detection of microRNA in tumor cells using exonuclease III and graphene oxide-regulated signal amplification.
abstract: In this study, we developed a label-free, ultrasensitive graphene oxide (GO)-based probe for the detection of oligonucleotides by laser desorption/ionization mass spectrometry (LDI-MS). On the basis of simple π-π stacking and electrostatic interactions between rhodamine 6G (R6G) and GO, we prepared the nanocomposite R6G-modified GO (R6G-GO). Signal intensities of R6G increased in mass spectra in the presence of single-stranded oligonucleotides under pulsed laser irradiation (355 nm) of R6G-GO. In addition, the signal intensity of R6G was stronger in the presence of short oligonucleotides. Because small oligonucleotides improve the LDI efficiency of R6G on GO, we designed an enzyme-amplified signal transduction probe system for the detection of microRNA (miRNA). After specific digestion of the probe DNA (pDNA) strand from pDNA/miRNA-hybridized complexes by exonuclease III (Exo III), the resulting small oligonucleotide fragments increased the R6G signal during LDI-MS of R6G-GO. In addition, the signal intensity of the R6G ions increased with increasing concentrations of the target miRNA. Coupling this enzyme reaction and R6G-GO with LDI-MS enabled the detection of miRNA at concentrations of the femtomolar (fM) level. We also demonstrated the analysis of miRNA in tumor cells and utilized this R6G-GO probe in the detection of a single-nucleotide polymorphism (SNP) in the Arg249Ser unit of the TP53 gene. This simple, rapid, and sensitive detection system based on the coupling of functional GO with LDI-MS appears to have great potential as a tool for the bioanalyses of oligonucleotides and proteins.
authors.affiliation: Institute of Bioscience and Biotechnology and ‡Center of Excellence for the Oceans, National Taiwan Ocean University , Keelung 20224, Taiwan.
authors.forename: Rong-Cing
authors.initials: RC
authors.lastname: Huang
authors.forename: Wei-Jane
authors.initials: WJ
authors.lastname: Chiu
authors.forename: Yu-Jia
authors.initials: YJ
authors.lastname: Li
authors.forename: Chih-Ching
authors.initials: CC
authors.lastname: Huang
mesh_terms: D045744:Cell Line, Tumor; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006108:Graphite / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D035683:MicroRNAs / Q000032:analysis*; D046529:Microscopy, Electron, Transmission; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D010087:Oxides / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D010087:Oxides; D006108:Graphite; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III
keywords: graphene oxide; mass spectrometry; microRNA; oligonucleotides; single-nucleotide polymorphism; tumor cells