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Nanoparticle suspensions were microprinted onto TEM grids for subsequent analysis by SEM/TSEM and evaluation of particle numbers using Image J software. Various nanoparticle types, concentrations and printing conditions (temperature, rel. humidity) were evaluated in order to determine the optimal conditions for producing a uniform distribution of particles on the substrate and eliminating the coffee ring effect.
Projekt Nano-TG110
(2017)
Die OECD-TG110 mit dem Titel "Particle Size Distribution Fibre Length and Diameter Distribution" wurde 1981 erstellt und seither nicht überarbeitet. Nanomaterialien, wie Nanopartikel oder Nanofasern werden nicht adressiert, genausowenig wie moderne Messverfahren. Das hier vorgestellte Projekt hat zur Aufgabe, die Größenmessung von Nanopartikeln und Nanofasern in einer neuen Technical Guideline der OECD zu standardisieren. Es wird die Projekthistorie dargestellt, die Anforderungen, der aktuelle Projekt-Bearbeitungsstand wird beleuchtet und die weiteren Arbeiten werden kurz skizziert.
Nanomaterials are used in many different applications in the material and life sciences. Examples are optical reporters, barcodes, and nanosensors, magnetic and optical contrast agents, and catalysts. Due to their small size and large surface area, there are also concerns about their interaction with and uptake by biological systems. This has initiated an ever increasing number of cyctoxicity studies of nanomaterials of different chemical composition and surface chemistry, but until now, the toxicological results presented by different research groups often do not address or differ regarding a potential genotoxicity of these nanomaterials. This underlines the need for a standardized test procedure to detect genotoxicity.1,2
Aiming at the development of fast, easy to use, and automatable microscopic methods for the determination of the genotoxicity of different types of nanoparticles, we assess the potential of the fluorometric γH2AX assay for this purpose. This assay, which can be run on an automated microscopic detection system, relies on the determination of DNA double strand breaks as a sign for genotoxicity.3 Here, we present first results obtained with broadly used nanomaterials like CdSe/CdS and InP/ZnS quantum dots as well as iron oxide, gold, and polymer particles of different surface chemistry with previously tested colloidal stability. These studies will be also used to establish nanomaterials as positive and negative genotoxicity controls or standards for assay performance validation for users of this fluorometric genotoxicity assay. In the future, after proper validation, this microscopic platform technology will be expanded to other typical toxicity assays.
References. (1) Landsiedel, R.; Kapp, M. D.; Schulz, M.; Wiench, K.; Oesch, F., Reviews in Mutation Research 2009, 681, 241-258. (2) Henriksen-Lacey, M.; Carregal-Romero, S.; Liz-Marzán, L. M., Bioconjugate Chem. 2016, 28, 212-221. (3) Willitzki, A.; Lorenz, S.; Hiemann, R.; Guttek, K.; Goihl, A.; Hartig, R.; Conrad, K.; Feist, E.; Sack, U.; Schierack, P., Cytometry Part A 2013, 83, 1017-1026.
Immunoassays are an important field of in vitro diagnostics, as they allow for a fast and highly sensitive detection of many biologically and diagnostically relevant analytes such as proteins, hormones, and pharmaceuticals. Fluorescence immunoassays (FIA), where the antibodies and/or antigens are labeled with luminescent reporters, can be easily read out directly by measuring the intensity, decay time, or polarization of the emitted light. Moreover, FIA enable the simultaneous detection of different analytes within a single sample (multiplexing) and are particularly suited for point-of-care (POC) diagnostics and high throughput screening (HTS). The application of luminescent nanoparticles as reporters in FIA could further improve assay sensitivity, as several 100 to 1000 luminophores can be incorporated or attached to such nanoscale carriers, thereby amplifying their absorption and/or emission signals simply by increasing the number of dye molecules.
Although dye-loaded polymeric and silica nanoparticles have been increasingly used as reporters in immunoassays, achievable signal amplification factors related to the use of particle reporters are still difficult to predict and quantify, which also hampers the comparability of different nanoscale reporters. To overcome this challenge, we performed a systematic comparison of spectroscopically and analytically well characterized particle labels in a homogeneous sandwich immunoassay format for the detection of the common inflammation biomarker C-reactive protein (CRP). Hereby, we studied the influence of particle parameters like size, surface chemistry, and dye loading concentration for different dye classes, i.e. organic dyes and metal ligand complexes, varying in their signal-relevant spectroscopic properties (molar absorption coefficients, photoluminescence quantum yields, Stokes shifts, and emission decay times), for different detection schemes (direct read-out vs. dye extraction). The emitters applied were chosen to be commercially available for a reasonable price, to absorb between 400 nm and 450 nm, and to emit in the visible region, as these parameters are accessible with most established microplate readers. Based upon our findings, we highlight the advantages and limitations of nanoscale reporters with respect to the choice of suitable particles, encoding dyes, and detection strategies, and compare the achievable sensitivities and dynamic ranges for our CRP model immunoassay.
Simulational tools are applied to investigate the physical properties of nanoparticles.
For the description of radioactive gold nanoparticles, particles scattering simulations are performed with the Geant4 monte carlo simulation toolkit.
The temperature dependent behaviour of the magnetization dynamics of different magnetic nanoparticles are simulated with the object oriented micormagnetic framework (OOMMF).
New Approaches for the Quantification of Functional Groups on Micro- and Nanoparticle Surfaces
(2017)
Nanometer- and micrometer-sized particles are increasingly used as tolls in (bio)analytics with typical applications being carriers for e.g., drugs or dye molecules for use as multichromophoric reporters for signal amplification in optical assays, platforms for DNA sequencing as well as nanosensors and targeted probes for bioimaging studies.
The application of such particles in the material and life sciences is closely linked to their size (and size distribution), shape, colloidal stability, biocompatibility, and ease of subsequent functionalization, e.g., with linkers, targeting ligands, and sensor molecules. The latter requires knowledge of the number of groups effectively accessible for subsequent coupling reactions and hence, selective and sensitive methods of analysis. Ideally, these methods are be robust, reliable, fast, performable with inexpensive equipment, and can be employed for the characterization of a broad variety of particle systems independent of their optical properties, i.e., scattering or the presence of encoding dyes.
In this respect, we studied a variety of conventional and newly developed labels for optical readout on self-made particles with varying surface group density, utilizing e.g., changes in intensity and/or color of absorption and/or emission. We focus here on the development of a platform of cleavable and multimodal labels for optical assays which consist of a cleavable linker and an optically active reporter moiety. In contrast to conventional reporters measured directly at the particle surface, which are prone to signal distortions by scattering and encoding dyes, these cleavable labels can be detected colorimetrically or fluorometrically both bound at the particle surface and after quantitative cleavage of the linker in the transparent supernatant. Moreover, for heteroatom-containing reporters, they enable straightforward validation by method comparison with elemental analysis, ICP-OES or ICP-MS.
Here, we present representative examples of our newly synthesized cleavable and multimodal labels and their application as reporters for the quantification of amino, thiol and carboxy surface groups on different nanomaterials and compare these results with measurements using conventional optical labels and results from measurements providing the total number of surface groups.
Polymer nanoparticles are of increasing importance for a wide range of applications in the material and life sciences. This includes their application as carriers for e.g., analyte-responsive ligands for DNA sequencing platforms, drugs as well as dye molecules for use as multichromophoric reporters for signal enhancement in optical assays or the fabrication of nanosensors and targeted probes in bioimaging studies.
Application-relevant properties of nanometer- and micrometer-sized particles (NP) include their size (and size distribution), colloidal stability, biocompatibility, and ease of subsequent functionalization, e.g., with linkers, sensor molecules, and targeting ligands. In this respect, the knowledge of the chemical nature, the total number of surface groups and the number of groups accessible for subsequent coupling reactions with differently sized optical labels or biomolecules is mandatory. This requires robust, reliable and validated methods, which can be employed for the characterization of a broad variety of particle systems independent of their optical properties, i.e., scattering or the presence of encoding dyes, and can be preferably performed specifically, sensitively, and fast with inexpensive equipment. Particularly attractive methods are here straightforward colorimetric, and fluorometric assays. In this respect, we studied a variety of conventional labels for optical readout, utilizing e.g., a change in intensity and/or color of absorption and/or emission. While in common assays, most reporters are measured directly at the particle surface, which can easily lead to signal distortions by scattering and encoding dyes, we focus on the development of cleavable and multimodal labels. These labels are detectable both bound at the particle surface and after cleavage of a linker unit in the supernatant with different analytical methods like fluorometry together with elemental analysis, ICP-OES or ICP-MS for straightforward method validation by method comparison. Here, we present our newly-synthesized cleavable labels and their application for photometric quantification of amino, thiol and carboxy surface groups on different types of nanomaterials and compare the results obtained from surface group analysis relying on conventional labels.
Carboxy, amino, and thiol groups play a critical role in a variety of physiological and biological processes and are frequently used for bioconjugation reactions. Moreover, they enable size control and tuning of the surface during the synthesis of particle systems. Especially, thiols have a high binding affinity to noble metals and semiconductors (SC). Thus, simple, inexpensive, robust, and fast methods for the quantification of surface groups and the monitoring of reactions involving ligands are of considerable importance for the characterization of modified or stabilized nanomaterials including polymers.
We studied the potential of the Ellman’s assay, recently used for the quantification of thiol ligands on SC nanocrystals by us1 and the 4-aldrithiol assay for the determination of thiol groups in molecular systems and on polymeric, noble and SC nanomaterials. The results were validated with ICP-OES and reaction mechanisms of both methods were studied photometrically and with ESI-TOF-MS.
The investigation of the reaction mechanisms of both methods revealed the influence of different thiols on the stoichiometry of the reactions2, yielding different mixed disulfides and the thiol-specific products spectroscopically detected. The used methods can quantify freely accessible surface groups on nanoparticles, e.g., modified polystyrene nanoparticles. For thiol ligands coordinatively bound to surface atoms of, e.g., noble or SC nanomaterials, depending on the strength of the thiol-surface bonds, particle dissolution prior to assay performance can be necessary.
We could demonstrate the reliability of the Ellman’s and aldrithiol assay for the quantification of surface groups on nanomaterials by ICP-OES and derived assay-specific requirements and limitations. Generally, it is strongly recommended to carefully control assay performance for new samples, components, and sample ingredients to timely identify possible interferences distorting quantification.
The overall interest in nanotoxicity, triggered by the increasing use of nanomaterials in the material and life sciences, and the synthesis of an ever increasing number of new functional nanoparticles calls not only for standardized test procedures1,2 and for efficient approaches to screen the potential genotoxicity of these materials, but also for standardized and validated methods for surface analysis.
4,5 The analysis and quantification of surface chemistry is hence in the focus of an increasing number of standardization organizations and interlaboratory comparisons with different analytical methods are being done.5 For the monitoring of nanomaterial synthesis and the fast assessment of the number of functional groups such as carboxyl and amino functionalities, which are very commonly used functionalities in the life sciences, simple and validated methods are needed that can be performed with common laboratory instrumentation. 5,6 Here we provide a brief overview of the ongoing research in division Biophotonics employing quantitative NMR (qNMR), conductometry, and colorimetric and fluorometric optical assays for the determination of the total and the accessible number of carboxyl and amino groups on differently sized polymer and silica nano- and microparticles.5-7
The surface chemistry / functionalization of nanomaterials and microparticles largely controls the stability of these materials as well as their solubility and subsequent biofunctionalization and their interactions with biological systems. Moreover, in the case of some nanomaterials like semiconductor quantum dots or lanthanide-based upconversion nanocrystals, the ligand shell strongly affects their optical properties, e.g., via passivation of surface states and traps that favor luminescence quenching or the protection of surface atoms from quenching water molecules. This renders analytical methods for the quantification of surface groups like functionalities very important. Targets of broad interest are here amino, carboxyl, alkine and maleimide groups used for common bioconjugation reactions and typical ligands like thiols and polyethylene glycol (PEG) molecules of varying length, used for the tuning of material hydrophilicity and biocompatibility, minimization of unspecific interactions, prevention of biofouling, and enhancement of blood circulation times as well as surface-bound biomolecules like streptavidin or other biomolecules relevant e.g., for diagnostic assays. Here, we focus on simple optical methods relying on standard laboratory instrumentation, validated by method comparison and/or mass balances and present examples for their use for the characterization of different types of nanomaterials and microparticles.