Analytische Chemie
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Luminescence-based detection methods, ranging from fluorescence spectroscopy for photophysical and mechanistic studies over sensing applications, chromatographic separation techniques and the microarray technology with fluorescence detection to fluorescence microscopy, flow cytometry, single molecule spectroscopy, and molecular imaging to integrating sphere spectroscopy, are among the most widely used methods in the life and material sciences. This is due to e.g., their unique sensitivity enabling the detection of single molecules, potential for multiplexing, ease of combination with spatial resolution, and suitability for remote sensing. Many of these advantages are closely linked to the choice of suitable molecular and nanoscale fluorescent reporters, typically required for signal generation. This includes organic dyes without and with sensor function, fluorophore-encoded polymeric and silica nanoparticles as well as nanocrystalline systems like semiconductor quantum dots and upconversion phosphors, emitting in the visible (vis), near-infrared (NIR), and IR (infrared). Current challenges present the environment sensitivity of most fluorophores, rendering fluorescence spectra, measured intensities/fluorescence quantum yields, and fluorescence decay kinetics matrix-dependent, and instrument-specific distortions of measured fluorescence signals that need to be considered for quantification and comparability of data, particularly fluorescence spectra.
Here, current applications of luminescence-based methods and different types of reporters will be presented. In this context, suitable spectroscopic tools for the characteri-zation of the optical properties of fluorescent reporters and fluorophore-encoded microparticles, analytical tools for the determination of the surface chemistry of different types of particles, and different multiplexing strategies will be discussed.
Elemental imaging of biological samples (bio-imaging) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) provides spatially resolved information on element distribution (qualitative and quantitative) in thin sections of biological samples. By rastering with a laser across the sample, a two-dimensional image of the elemental distribution can be reconstructed that shows the relative intensities of the respective elements. However the method is hampered by a lack of internal standards and quantification concepts, which will be discussed in this lecture in more detail.
In liquid analysis the internal standard is used for drift correction and calibration and thus it is required that the standard should have similar physical and chemical properties similar to the analyte element during the pneumatic nebulization process, the transport, ionization and transmission into the ICP-MS. In laser ablation it should correct additionally for differences in the ablation process by laser instabilities or changes of sample properties to compensate variations or drift effects during the LA process.
X-ray fluorescence (XRF) analysis is a well suited methodology for thin film analysis. With XRF the mass deposition of elements can be non-destructively determined. By knowledge of the density of the thin film as well the thickness can be given. In the field of industrial thin film manufacturing various material systems and layer structures are used. the analysis relies on well-known calibration samples or even reference materials to determine absolute analytical results from the measurement values recorded. Within this concept of chemical traceability the calibration sample has to be as similar as possible to the industrial thin film sample with respect to the spatial distribution of elemental composition in order to minimize matrix effects or analysis related uncertainties. The procurement of such certified similar calibration samples or reference materials including their required traceability is a challenge due to limited number of available calibration samples or reference materials, in particular at the nanoscale. There are only few providers of calibration standards for layer or coating thicknesses which are suitable for XRF analysis. The limited number of available certified reference materials (CRMs) for XRF thin film analysis and in parallel the growing market of novel thin film materials induces a growing gap of required calibration samples for XRF analysis.
The exploitation of process-near samples benefits from a leverage effect: on one hand reference materials are needed for calibration and alignment procedures for X-ray fluorescence devices. They are customized for the special need of the end-user, e.g. a company producing thin film solar cells. On the other hand the market for EDXRF devices develops positively by providing novel thin film calibration samples for industry and end user-related production processes or application. Two aspects are hereby being addressed; the improvement of product quality which in certain sectors like aerospace and automotive industry directly translates to product safety as well as the expansion of the sales potential of EDXRF measuring devices.
Comparison of fluorescence measurements performed on different fluorescence instruments, analyte quantification from fluorescence intensities as well as the determination of fluorescence quantum yields require instrument calibration and consideration of the wavelength-dependent instrument-specific quantities spectral photon flux reaching the sample and spectral responsivity. Here, we present guidelines and recommendations for the qualification of fluorescence instruments and introduce suitable chromophore-based reference materials. Moreover, the design concepts of the different BAM fluorescence standards are discussed.
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 for standardized test procedures1,2 and for efficient approaches to screen the potential genotoxicity of these materials. Aiming at the development of fast and easy to use, automated 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 detection of DNA double strand breaks as a sign for genotoxicity3. Here, we provide 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 and different cell lines like Hep-2 and 8E11 cells, which reveal a dependence of the genotoxicity on the chemical composition as well as the surface chemistry of these nanomaterials. 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.
Optical spectroscopy – Techniques, instrumentation, and typical molecular and nanoscale reporters
(2018)
Different types of optical spectroscopies are introduced with special emphasis on method-inherent limitations and reliable instrument calibration and performance validation. In addition, different classes of molecular and nanocrystalline emitters are presented and the underlying photophysical processes are briefly described.
The rational synthesis and use of nanomaterials require the characterization of many different properties, ranging from particle size and size distribution over surface chemistry to more applicationrelevant features like optical, electrochemical, and magnetic properties. In the following, several methods for the characterization of functional groups on nanomaterials, like polymer and silica nanoparticles, semiconductor quantum dots, and lanthanide-based upconversion nanocrystals are presented. Additionally, procedures for the measurement of the key spectroscopic performance parameters of nanomaterials with linear and nonlinear photoluminescence, such as the photoluminescence quantum yield, are presented for the UV/vis/NIR/SWIR.
The comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters require accurate and quantitative photo-luminescence measurements. This is of special importance for all photoluminescence applications in the life and material sciences and nanobiophotonics. In the following, procedures for the determination of the spectroscopic key parameter photoluminescence quantum yield, i.e., the number of emitted per absorbed photons, in the UV/vis/NIR/SWIR are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed.