Comparison of nanowire pellicles for plasma membrane enrichment: coating nanowires on cell

Abstract

A study is reported on the effect of nanowire density on the ease of pellicle formation and the enrichment of plasma membrane proteins for analysis by mass spectrometry. An optimized synthesis is reported for iron silicate nanowires with a narrow size range of 900 ±400 nm in length and 200 nm diameter. The nanowires were coated with Al 2 O 3 and used to form pellicles around suspended multiple myeloma cells, which acted as a model for cells recovered from tissue samples. Lighter alumina-coated silica nanowires were also synthesized , which allowed a comparison of the construction of the two pellicles and of the effect of nanowire density on plasma membrane enrichment. Evidence is offered that the dense nanowire pellicle does not crush or distort these mammalian cells. Finally, the pellicles were incorporated into a massspectrometry-based proteomic workflow to analyze transmembrane proteins in the plasma membrane. In contrast to a prior comparison of the effect of density with nanoparticles pellicles , nanowire density was not found to significantly affect the enrichment of the plasma membrane. However, nanowires with a favorable aspect for pellicle formation are more easily and reliably produced with iron silicate than with silica. Additionally, the method for pellicle formation was optimized through the use of iron silicate nanowires (ISNW), which is crucial to the improvement of PM protein enrichment and analysis.

Analytical Protocols for Separation and Electron Microscopy of Nanoparticles Interacting with Bacterial Cells

“…Samples with low concentrations of both cells and NPs (Figure e and f) are typically the easiest to image at high resolution; , however, isolated individual areas of such samples (e.g., Figure S2b, SI) may not be representative of the whole and the low concentration of NPs may have limited their opportunity to interact with particular cells in each FOV (Figure f). Conversely, having a high concentration of NPs (or cells) in mixed samples increases the possibility of observing artifacts from both preparation and imaging procedures (Figure a–d). , While it obfuscates evidence of specific NP–cell interactions, coating of cells with a dense layer of NPs is successfully used, for example, in membrane-protein enrichment protocols, which are commonly illustrated with SEM images resembling our Figure a–c. − A high concentration of NPs during incubation, therefore, provides a simple general method for ensuring that all the cells had the opportunity to interact with NPs before having been prepared for imaging.…”

“…The importance of ensuring that all the free NPs have been removed after co-incubation depends on the context of an experiment. For example, when verifying high loading of each cell with NPs, − including NPs in a form of dense layers (Figure a and b) or aggregates (Figure d), even a relatively high fraction (perhaps, as high as 30%) of free NPs among all NPs sedimented with cells would not affect the interpretation of results dramatically. The salient common characteristic of such experiments is the relative unimportance of distinguishing between NP–NP and NP–cell interactions.…”

“…For separating free NPs from a co-incubation mixture, we propose centrifugation because it relies on the colloidal stability of bacteria and NPs; the latter can be controlled via physicochemical properties of NPs and the solvent . Furthermore, centrifugation is routinely used for separation and concentration of bacteria , as well as NPs , or NP-loaded fragments of cell membranes. − For larger (∼5–15 μm) cells, dense coating with NPs can be sufficient to sediment the NP-loaded cells from an NP-cell mixture based on their higher density. ,, For mixtures of NPs and submicrometer-sized bacteria with relatively low NP loading, however, density-based sedimentation may not be very effective, so more general colloidal stability differences have to be used for differential sedimentation, especially when a nearly complete removal of free NPs, as discussed above, is the goal. Here, we demonstrate a basic example of a systematic methodology for performing a deterministic separation of free NPs from a co-incubation mixture, whereby the initial separate measurements of NPs and cells are used (1) to narrow down the mixed NP-bacteria samples parameter space (which, a priori, is vast, owing to the combinatorics of possible mixtures) and (2) to predict the parameters for separation of mixed NP-bacteria samples.…”