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Surface-enhanced Raman scattering (SERS) hybrid probes are characterized by the typical spectrum of a reporter molecule. In addition, they deliver information from their biological environment. Here, we report SERS hybrid probes generated by conjugating different reporter molecules to bovine serum albumin (BSA) and using gold nanoparticles as plasmonic core. Advantages of the BSA-conjugate hybrid nanoprobes over other SERS nanoprobes are a high biocompatibility, stabilization of the gold nanoparticles in the biological environment, stable reporter signals, and easy preparation. The coupling efficiencies of the BSA–reporter conjugates were determined by MALDI-TOF-MS. The conjugates' characteristic SERS spectra differ from the spectra of unbound reporter molecules. This is a consequence of the covalent coupling, which leads to altered SERS enhancement and changes in the chemical structures of the reporter and of BSA. The application of the BSA–reporter conjugate hybrid probes in 3T3 cells, including duplex imaging, is demonstrated. Hierarchical cluster analysis and principal components analysis were applied for multivariate imaging using the SERS signatures of the incorporated SERS hybrid nanoprobes along with the spectral information from biomolecules in endosomal structures of cells. The results suggest more successful applications of the SERS hybrid probes in cellular imaging and other unordered high-density bioanalytical sensing.
By adding a gold core to silica nanoparticles (BrightSilica), silica-like nanoparticles are generated that, unlike unmodified silica nanoparticles, provide three types of complementary information to investigate the silica nano-biointeraction inside eukaryotic cells in situ. Firstly, organic molecules in proximity of and penetrating into the silica shell in live cells are monitored by surface-enhanced Raman scattering (SERS). The SERS data show interaction of the hybrid silica particles with tyrosine, cysteine and phenylalanine side chains of adsorbed proteins. Composition of the biomolecular corona of BrightSilica nanoparticles differs in fibroblast and macrophage cells. Secondly, quantification of the BrightSilica nanoparticles using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping indicates a different interaction of silica nanoparticles compared to gold nanoparticles under the same experimental conditions. Thirdly, the metal cores allow the investigation of particle distribution and interaction in the cellular ultrastructure by cryo nanoscale X-ray tomography (cryo-XT). In 3D reconstructions the assumption is confirmed that BrightSilica nanoparticles enter cells by an endocytotic mechanism. The high SERS intensities are explained by the beneficial plasmonic properties due to agglomeration of BrightSilica. The results have implications for the development of multi-modal qualitative and quantitative characterization in comparative nanotoxicology and bionanotechnology.
The analysis of single cells is a growing research field in many disciplines such as toxicology, medical diagnosis, drug and cancer research or metallomics, and different methods based on microscopic, mass spectrometric, and spectroscopic techniques are under investigation. This review focuses on the most recent trends in which inductively coupled plasma mass spectrometry (ICP-MS) and ICP optical emission spectrometry (ICP-OES) are applied for single-cell analysis using metal atoms being intrinsically present in cells, taken up by cells (e.g., nanoparticles), or which are artificially bound to a cell. For the latter, especially element tagged antibodies are of high interest and are discussed in the review. The application of different sample introduction systems for liquid analysis (pneumatic nebulization, droplet generation) and elemental imaging by laser ablation ICP-MS (LA-ICP-MS) of single cells are highlighted. Because of the high complexity of biological systems and for a better understanding of processes and dynamics of biologically or medically relevant cells, the authors discuss the idea of 'multimodal spectroscopies.'
The cellular response to nanoparticle exposure is essential in various contexts, especially in nanotoxicity and nanomedicine. Here, 14-nm gold nanoparticles in 3T3 fibroblast cells are investigated in a series of pulse-chase experiments with a 30-min incubation pulse and chase times ranging from 15 min to 48 h. The gold nanoparticles and their aggregates are quantified inside the cellular ultrastructure by laser ablation inductively coupled plasma mass spectrometry micromapping and evaluated regarding the surface-enhanced Raman scattering (SERS) signals. In this way, both information about their localization at the micrometre scale and their molecular nanoenvironment, respectively, is obtained and can be related. Thus, the nanoparticle pathway from endocytotic uptake, intracellular processing, to cell division can be followed. It is shown that the ability of the intracellular nanoparticles and their accumulations and aggregates to support high SERS signals is neither directly related to nanoparticle amount nor to high local nanoparticle densities. The SERS data indicate that aggregate geometry and interparticle distances in the cell must change in the course of endosomal maturation and play a critical role for a specific gold nanoparticle type in order to act as efficient SERS nanoprobe. This finding is supported by TEM images, showing only a minor portion of aggregates that present small interparticle spacing. The SERS spectra obtained after different chase times show a changing composition and/or structure of the biomolecule corona of the gold nanoparticles as a consequence of endosomal processing.
The interaction of nanomaterials with biomolecules, cells, and organisms plays an important role in cell biology, toxicology, and nanotechnology. Spontaneous Raman scattering can be used to probe biomolecules, cells, whole animals, and nanomaterials alike, opening interesting avenues to study the interaction of nanoparticles with complex biological systems. In this review we discuss work in biomedical Raman spectroscopy that has either been concerned directly with nanostructures and biosystems, or that indicates important directions for successful future studies on processes associated with nano-bio-interactions.
A new laser ablation (LA)-ICP-MS method for single cell and cell nucleus imaging was developed. Therein, iodine was employed as an elemental dye for fibroblast cells and for thin tissue sections. At an incubation time of 60 s, iodine is located mainly within the cell nuclei. This effect was illustrated in fibroblast cells, and iodine signal within the cell nucleus was as high as 5 × 104 cps at 4 µm laser spot size. The surrounding cytoplasm was iodinated as well, but to a lesser extent. The spatial resolution attained was sufficient to detect even smaller cell nuclei within a liver biopsy tissue. Furthermore, iodine was successfully employed for biomolecule labeling and we demonstrated that iodine signal increased with increasing thickness of a palatine tonsil tissue. Thus, the use of iodine as an internal standard to correct for tissue inhomogeneities in LA-ICP-MS was investigated for the simultaneous detection of two tumor markers (Her 2 and CK 7) in breast cancer tissue. Additionally, lanthanide background resulting from glass ablation can be corrected for by Eu standardization.