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Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag–Magnetite and Au–Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surfaceenhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag–Magnetite and Au–Magnetite nanostructures that is very similar to that of other Composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.
This study reports on the development of a single-particle (sp) inductively coupled plasma mass spectrometry (ICP-MS) technique suitable for the multi-mode determination of nanoparticle (NP) metal mass fraction and number concentration. The described technique, which is based on a dual inlet System consisting of a pneumatic nebulizer (PN) and a microdroplet generator (MDG), allows for the sequential introduction of ionic metal calibrant solutions and nanoparticle suspensions via all combinations of the two inlets; thus allowing for a combination of three independent modes of analysis. A novel interface, assembled using standard analytical components (a demountable quartz ICP-MS torch, flexible nonconducting silicon tubing and various connectors), was used to interface the dual inlet system to an ICP-MS. The interface provided improved functionality, compared to a previous design. It is now possible to conveniently exchange and introduce standard solutions and samples via all inlet combinations, analyze them, and also wash the sample inlet systems while the whole setup is still connected to an operating ICP-MS. This setup provided seamless and robust operation in a total of three analysis modes, i.e. three ways to independently determine the metal mass fraction and NP number concentration. All three analyses modes could be carried out within a single analytical run lasting approximately 20 min.
The unique feature of the described approach is that each analysis mode is based on a different.
In this work, a routinely applicable approach is presented to characterize metal NPs. Individual droplets generated from a microdroplet generator (MDG) were merged into an aerosol generated by a pneumatic nebulizer (PN) and introduced into an ICPMS. The MDG offers high transport efficiency of individual and discrete droplets and was therefore used to establish a calibration function for mass quantification of NPs which were introduced through the PN following the single particle procedure as described elsewhere. The major advantages of such a combined configuration include fast processing of large sample volumes, fast exchanges of different sample matrixes, and the calibration of the NP signal using traceable elemental standards, thus avoiding the need to use NP reference materials or other, not always thoroughly characterized, commercially available NPs. The transport efficiency of the sample introduction is calculated based on the fact that 100% of the calibrant reaches the plasma through the MDG, whereas for the PN a NP suspension containing a known number concentration is used. Alternatively, bulk analysis of the NP material allows transport efficiency determination without any additional information from reference NPs. With this method, we could determine the size of standard silver NPs at 60.4 ± 1.0 nm and 80.0 ± 1.4 nm, respectively, which agrees with the size ranges given by the supplier (60.8 ± 6.6 nm and 79.8 ± 5.4 nm). Furthermore, we were also able to determine the NPs number concentration of the sample (Ag/Au) with a deviation of 3.2% the expected value.
We are using laser ablation (LA)-ICP-MS to image the local distribution of elements (metals and hetero-elements) directly or (metallo-)proteins by metal-tagged antibodies in cells and tissue indirectly. Different applications will be discussed to demonstrate the state of the art and to visualize the elemental distribution pattern in soft bio-materials (tissue, single cells). In the first application Pt-containing drugs for cancer treatment are investigated and elemental distribution pattern are shown for tissue samples from animal experiments. Different standardization and quantification schemes including isotope dilution analysis will be discussed. In the second application, which is dedicated to toxicological research, the up-take of nano-particles by single cells are discussed and metal containing stains are used to visualize the distribution of nano-particles, proteins and DNA in a single cell simultaneously. This information is correlated with the distribution of the nanoparticles to identify the cell compartments where nano-particles are enriched. Quantification schemes have been developed to transform the measured intensities into number of particles up taken by the cells. In the third and last application LA-ICP-MS is applied to visualize the local distribution of proteins, which are used as bio-markers for prostate cancer. For this purpose, biopsy samples from patients have been simultaneously stained by eight differently metal-tagged antibodies in a multiplex approach. Detection of house-keeping proteins serves as internal standards to overcome differences in protein expression. Additionally ink-jet printing of metal doped inks onto the surface of these tissue samples has been applied for internal standardization and drift corrections.
Finally future trends to develop an “elemental microscope” will be discussed.
An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
An overview about different analytical approaches will be presented of how to detect metals in individual biological cells by use of ICP-MS. For this purpose, we are using different sample introduction systems for ICP-MS for detection, imaging and quantification of metals at cellular levels.
By use of laser ablation, we have studied the up-take by and distribution of nanoparticles in single cells. Recently we have developed staining techniques to measure protein and DNA content of cells and identifying the cell status by immunoassays using metal-tagging of antibodies. New research based on cell arrays will be shortly discussed.
Using pneumatic nebulization and microdroplet generation, we have also studied the up-take of nanoparticles and toxic metals as well as essential elements in single cells using different ICP-MS mass spectrometric concepts (sector field instrument, triple-quad instrument, time of flight (CyTOF) instrument).
The different ICP-MS based methods will be compared concerning their analytical figures of merit and their strengths and weaknesses will be evaluated.
Gold nanostructures that serve as probes for nanospectroscopic analysis of eukaryotic cell cultures can be obtained by the in situ reduction of tetrachloroauric acid (HAuCl4). To understand the formation process of such intracellularly grown particles depending on the incubation medium, the reaction was carried out with 3T3 fibroblast cells in three different incubation media, phosphate buffer, Dulbecco's Modified Eagle Medium (DMEM), and standard cell culture medium (DMEM with fetal calf serum). The size, the optical properties, the biomolecular corona, and the localization of the gold nanoparticles formed in situ vary for the different conditions. The combination of surface-enhanced Raman scattering (SERS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) microscopic mapping and transmission electron microscopy (TEM) provides complementary perspectives on plasmonic nanoparticles and non-plasmonic gold compounds inside the cells. While for the incubation with HAuCl4 in PBS, gold particles provide optical signals from the nucleus, the incubation in standard cell culture medium leads to scavenging of the toxic molecules and the formation of spots of high gold concentration in the cytoplasm without formation of SERS-active particles inside the cells. The biomolecular corona of nanoparticles formed in situ after incubation in buffer and DMEM differs, suggesting that different intracellular molecular species serve for reduction and stabilization. Comparison with data obtained from ready-made gold nanoparticles suggests complementary application of in situ and ex situ generated nanostructures for optical probing.
Safety assessment of nanoparticles (NPs) requires techniques that are suitable to quantify tissue and cellular uptake of NPs. The most commonly applied techniques for this purpose are based on inductively coupled plasma mass spectrometry (ICP-MS). Here we apply and compare three different ICP-MS methods to investigate the cellular uptake of TiO2 (diameter 7 or 20 nm, respectively) and Ag (diameter 50 or 75 nm, respectively) NPs into differentiated mouse neuroblastoma cells (Neuro-2a cells). Cells were incubated with different amounts of the NPs. Thereafter they were either directly analyzed by laser ablation ICP-MS (LA-ICP-MS) or were lysed and lysates were analyzed by ICP-MS and by single particle ICP-MS (SP-ICP-MS).