Abstract
Here we present new applications for silicon nitride (SiN) membranes to evaluate biological processes. We determined that 50-nanometer thin films of SiN produced from silicon wafers were sufficiently durable to bind active rotavirus assemblies. A direct comparison of SiN microchips with conventional carbon support films indicated that SiN performs equivalent to the traditional substrate to prepare samples for Electron Microscopy (EM) imaging. Likewise, SiN films coated with Ni-NTA affinity layers concentrated rotavirus particles similarly to affinity-coated carbon films. However, affinity-coated SiN membranes outperformed glow-discharged conventional carbon films 5-fold as indicated by the number of viral particles quantified in EM images. In addition, we were able to recapitulate viral uncoating and transcription mechanisms directed onto the microchip surfaces. EM images of these processes revealed the production of RNA transcripts emerging from active rotavirus complexes. These results were confirmed by the functional incorporation of radiolabeled nucleotides into the nascent RNA transcripts. Collectively, we demonstrate new uses for SiN membranes to perform molecular surveillance on life processes in real-time.
Highlights
Experiments performed at the level of individual cells and molecules have the capacity to shed new light on elusive biological processes
We tested the use of silicon nitride (SiN) microchips for biological applications using rotavirus transcription complexes (i.e., double-layered particles (DLPs)) as a model system (Figure 1A)
We performed a direct comparison of SiN surfaces with graphitized carbon films, which are the most common substrate used in electron microscopy
Summary
Experiments performed at the level of individual cells and molecules have the capacity to shed new light on elusive biological processes. Fluidic chambers composed of silicon nitride (SiN)-based microchips with nanometer-thin windows provide new tools to view the progression of chemical processes using electron microscopy [5,6]. These devices are currently being used to evaluate properties of hard materials [7,8], they are not as widely utilized to assess biological polymers. Amorphous carbon is the conventional support material used to prepare biological specimens for transmission electron microscopy (EM) imaging. Alternatives support films have been recently introduced to mitigate this effect They include conductive materials composed of titanium-silicon metal glass (Ti88Si12) [10]
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