The interaction of fluorescent nanodiamond probes with cellular media

Abstract

Fluorescent nanodiamonds (FNDs) are promising tools to image cells, bioanalytes and physical quantities such as temperature, pressure, and electric or magnetic fields with nanometer resolution. To exploit their potential for intracellular applications, the FNDs have to be brought into contact with cell culture media. The interactions between the medium and the diamonds crucially influence sensitivity as well as the ability to enter cells. The authors demonstrate that certain proteins and salts spontaneously adhere to the FNDs and may cause aggregation. This is a first investigation on the fundamental questions on how (a) FNDs interact with the medium, and (b) which proteins and salts are being attracted. A differentiation between strongly binding and weakly binding proteins is made. Not all proteins participate in the formation of FND aggregates. Surprisingly, some main components in the medium seem to play no role in aggregation. Simple strategies to prevent aggregation are discussed. These include adding the proteins, which are naturally present in the cell culture to the diamonds first and then inserting them in the full medium. Graphical abstractSchematic of the interaction of nanodiamonds with cell culture medium. Certain proteins and salts adhere to the diamond surface and lead to aggregation or to formation of a protein corona.

Keywords: Aggregation; Cellular uptake; Corona formation; Fluorescent nanodiamonds; Imaging; Microscopy; Nanoscale sensing; Proteins; Surface analysis.

Graphical abstract

Schematic of the interaction of nanodiamonds with cell culture medium. Certain proteins and salts adhere to the diamond surface and lead to aggregation or to formation of a protein corona.

Fig. 1

Schematic representation of nanodiamond aggregation. a depicts the bare Nanodiamond particle. In figure b the nanodiamond particle with proteins adhering to the surface is shown. Figure c represents the nanodiamond aggregation by interaction between multiple diamond particles through protein connections

Fig. 2

Hydrodynamic diameters measured using cumulant analysis for ND particles (Microdiamant MSY 0–0,05, hydrodynamic diameter 25 nm) in different media. Error bars correspond to the standard deviation. The schematics on top show the composition of the particles we found. Samples (1) and (2) are reference measurements, the diamonds were suspended in water. If not stated differently the concentration of FBS was 10% (prepared in distilled water to eliminate the salt effect). The samples which are labeled with “wash” are measured after resuspending the particles. For the exact measurement protocol see the supporting information

Fig. 3

Electron microscopy images of FND and FND aggregates. Fluorescent nanodiamonds spotted on a silicon surface and imaged with an SEM (Leo 1530 Gemini, Carl Zeiss AG) (3a). FND aggregate imaged using Philips CM12 (Philips, Eindhoven, The Netherlands) on holey carbon grids (3b). The arrow indicates the protein corona

Fig. 4

Most abundant proteins and their theoretical isoelectrical point present in aggregates from FND in DMEM +10% FBS (sample 3) calculated from a typical mass spectrum (for details see supplementary information)

Fig. 5

Determining the salt contribution in aggregates: left: XPS spectrum of FND in DMEM +10% FBS (sample 3). Also here, sodium chloride remains the main compound in the aggregates (apart from the carbon mainly present as diamond, protein and amino acids). Right: Comparison of the element composition of DMEM medium, and the measured elemental ratios in aggregate samples

Fig. 6

Cellular uptake of nanodiamonds into HeLa Cells. (red: nanodiamonds, green: Phalloidin-FITC (stains the actin cytoskeleton), blue: DAPI (stains the nucleus)). Arrows indicate diamond particles. In the lower right pane, a large diamond aggregate (occupying almost the entire area of the pane) can be seen precipitating on the cell (as it is surrounded by the actin filaments of the cytoskeleton) (DAPI was omitted to make the diamond more visible)