A nucleic acid biosensor for gene expression analysis in nanograms of mRNA.
An ultrasensitive nucleic acid biosensor for direct detection of genes in mRNA extracted from animal tissues is described. It is based on amperometric detection of a target gene by forming an mRNA/redox polymer bilayer on a gold electrode. The mRNA was directly labeled with cisplatin-biotin conjugates through coordinative bonds with purine bases in the mRNA molecules. A subsequent binding of glucose oxidase-avidin conjugates to the labeled mRNA and the introduction of a poly(vinylimidazole-co-acrylamide) partially imidazole-complexed with [Os(bpy)(2)(im)] (bpy = 2,2'-bipyridine, im = imidazole) redox polymer overcoating to the electrode allowed for electrochemical detection of the oxidation current of glucose in solution. Depending on individual genes, detection limits of subfemtograms were achieved. As compared to a sandwich-type assay, the sensitivity was improved by as much as 25-fold through the incorporation of multiple enzyme labels to the mRNA molecules. Less than 2-fold gene expression difference was unambiguously differentiated in as little as 5.0 ng of mRNA. With the greatly improved sensitivity, at least 1000-fold more sensitive than fluorescence-based techniques, the amount of mRNA needed in the assay was cut down from microgram to nanogram levels.
Xie
Hong
H
Institute of Bioengineering and Nanotechnology, 51 Science Park Road, Singapore 117586, Republic of Singapore.
Yu
Yuan Hong
YH
Xie
Fang
F
Lao
Yuan Zhi
YZ
Gao
Zhiqiang
Z
D000818:Animals; D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001710:Biotin / Q000378:metabolism; D002945:Cisplatin / Q000378:metabolism; D004247:DNA / Q000032:analysis; D015870:Gene Expression; D005986:Glyceraldehyde 3-Phosphate / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D005986:Glyceraldehyde 3-Phosphate; D001710:Biotin; D004247:DNA; D002945:Cisplatin