Contribution of Ribonucleic Acid (RNA) to the Fourier Transform Infrared (FTIR) Spectrum of Eukaryotic Cells.

We report on an optimized protocol for the digestion of cellular RNA, which minimally affects the cell membrane integrity, maintaining substantially unaltered the vibrational contributions of the other cellular macromolecules. The design of this protocol allowed us to collect the first Fourier transform infrared (FTIR) spectra of intact hydrated B16 mouse melanoma cells deprived of RNA and to highlight the in-cell diagnostic spectral features of it. Complementing the cellular results with the FTIR analysis of extracted RNA, ds-DNA, ss-cDNA and isolated nuclei, we verified that the spectral component centered at ∼1220 cm-1 is a good qualitative and semiquantitative marker of cellular DNA, since it is minimally affected by cellular RNA removal. Conversely, the band centered at ∼1240 cm-1, conventionally attributed to RNA, is only a qualitative marker of it, since its intensity is majorly influenced by other macromolecules containing diverse phosphate groups, such as phospholipids and phosphorylated proteins. On the other hand, we proved that the spectral contribution centered at ∼1120 cm-1 is the most reliable indicator of variations in cellular RNA levels, that better correlates with cellular metabolic activity. The achievement of these results have been made possible also by the implementation of new methods for baseline correction and automated peak fitting, presented in this paper.

SU-8 bonding protocol for the fabrication of microfluidic devices dedicated to FTIR microspectroscopy of live cells.

Here we present a new bonding protocol for SU-8 negative tone photoresist that exploits the chemical modifications induced in the resin by exposure to 254 nm (UVC) light. Fourier Transform Infrared microspectroscopy (µ-FTIR) was used to carry out a thorough study on the chemical processes and modifications occurring within the epoxy resin by exposure to 365 nm and 254 nm light. In particular, we established that UVC light promotes the opening of the epoxy rings bypassing the post-exposure bake. The possibility to promote a further activation of the resin, already patterned with standard UV lithography, was exploited to produce closed microfluidic devices. Specifically, we were able to fabricate fluidic chips, characterized by broadband transparency from mid-IR to UV and long term stability in continuous flow conditions. CaF2 was used as substrate, coated by sputtering with a nanometric silicon film, in order to make surface properties of this material more suitable for standard fabrication processes with respect to the original substrate. The fabricated microfluidic chips were used to study by µ-FTIR the biochemical response of live breast cancer MCF-7 cells to osmotic stress and their subsequent lysis induced by the injection of deionized water in the device. µ-FTIR analyses detected fast changes in protein, lipid and nucleic acid content as well as cytosol acidification.