The new method detects messenger RNA, produced by active genes, without the need for amplification, relying instead on optical sensors attached to tiny silicon cantilevers that are only 500 nanometers long, 100 nanometers wide, and 450 nanometers thick.
Writing in the journal Nature Nanotechnology, a research team headed by Christoph Gerber, Ph.D., describes its use of gold-coated nanocantilevers to detect activity of the gene known as 1-8U produced by human melanoma cells. The researchers attached to the gold coating a short stretch of single-stranded DNA that binds specifically to messenger RNA produced by the 1-8U gene. As an internal control that provides a baseline reference measurement, the investigators attached a nonsense DNA probe “ one that does not bind to known messenger RNA sequences “ to an adjacent cantilever. The researchers mounted an array of eight nanocantilevers within a microcapillary tube connected to an injection port.
Initial characterization of this device showed it is capable of detecting picomolar levels of messenger RNA, comparable to conventional gene chip technologies. The researchers then showed that the device was capable of distinguishing between messenger RNA coding for a human protein and messenger RNA coding for a similar but not identical rat protein “ the two messenger RNAs differed by a mere four bases. The investigators also showed they could detect the human messenger RNA in a sample containing a total extract of all human RNAs. Finally, the researchers showed they could determine when the I-8U gene became active in human melanoma cells that were responding to interferon therapy.
This work is detailed in a paper titled, Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA. Investigators from IBM Research, in Zurich, the Roche Centre for Medical Genomics, in Basel, and the London Centre for Nanotechnology, in the United Kingdom, also participated in this study. An abstract of this paper is available at the journal ™s website. View abstract.
nanoncer
Researchers are aware that cells on flat surfaces have skewed metabolisms, gene expression and growing patterns. But the only choices have been glass labware and a product called Matrigel, a gelatinous protein mixture secreted by mouse tumor cells. While Matrigel does resemble a complex extracellular environment, it also contains growth factors and unknown proteins that limit its desirability for experiments requiring precise conditions.
"Synthetic biopolymer microfiber scaffolds have been studied for more than 30 years to mimic a living 3D microenvironment, but concerns exist about their degradation products and chemicals," the authors wrote in the paper.
Other synthetic polymer biomaterials are simply too big. Getting cells to grow on them is like forcing spiders to build webs on skyscraper girders. Zhang's nanofiber scaffold, around 1,000 times smaller than the existing systems, is much closer in size to the extracellular matrices that living cells manufacture themselves.
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