Chemische Charakterisierung und Spurenanalytik
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SWIR luminescent nanomaterials – key chemical parameters for bright probes for in vivo bioimaging
(2024)
A current challenge for studying physio-pathological phenomena and diseaserelated processes in living organisms with non-invasive optical bioimaging is the development of bright optical reporters that enable deep tissue penetration, a high detection sensitivity, and a high spatial and temporal resolution. The focus of this project are nanomaterials, which absorb and emit in the shortwave infrared (SWIR) between ~900–2500 nm where scattering, absorption, and autofluorescence of the tissue are strongly reduced compared to the visible and NIR.
Fluorescent sensory MIP (molecularly imprinted polymer) particles were combined with a droplet-based 3D microfluidic system for the selective determination of a prototype small-molecule analyte of environmental concern, 2,4-dichlorophenoxyacetic acid or 2,4-D, at nanomolar concentration directly in water samples. A tailor-made fluorescent indicator cross-linker was thus designed that translates the binding event directly into an enhanced fluorescence signal. The phenoxazinone-type cross-linker was co-polymerized into a thin MIP layer grafted from the surface of silica microparticles following a RAFT (reversible addition-fragmentation chain transfer) polymerization protocol. While the indicator cross-linker outperformed its corresponding monomer twin, establishment of a phase-transfer protocol was essential to guarantee that the hydrogen bond-mediated signalling mechanism between the urea binding site on the indicator cross-linker and the carboxylate group of the analyte was still operative upon real sample analysis. The latter was achieved by integration of the fluorescent core-shell MIP sensor particles into a modular microfluidic platform that allows for an in-line phasetransfer assay, extracting the analyte from aqueous sample droplets into the organic phase that contains the sensor particles. Real-time fluorescence determination of 2,4-D down to 20 nM was realized with the system and applied for the analysis of various surface water samples collected from different parts of the world.
Chlorination procedures are commonly applied in swimming pool water and wastewater treatment, yet also in food, pharmaceutical, and paper production. The amount of chlorine in water needs to be strictly controlled to ensure efficient killing of pathogens but avoid the induction of negative health effects. Miniaturized microfluidic fluorescence sensors are an appealing approach here when aiming at online or at-site measurements. Two meso-enamine-substituted boron dipyrromethene (BODIPY) dyes were found to exhibit favorable indication properties, their reaction with hypochlorite leading to strong fluorescence enhancement. Real-time assays became possible after integration of these fluorescent probes with designed two-dimensional (2D) and three-dimensional (3D) microfluidic chips, incorporating a passive sinusoidal mixer and a microhydrocyclone, respectively. A comparison of the two microfluidic systems, including their abilities to prevent accumulation or circulation of microbubbles produced by the chemical indication reaction, showed excellent fluidic behavior for the microhydrocyclone-based device. After coupling to a miniaturized optical reader for fluorescence detection, the 2D microfluidic system showed a promising detection range of 0.04−0.5 mg L−1 while still being prone to bubble-induced fluctuations and suffering from considerably low signal gain. The microhydrocyclone-based system was distinctly more robust against gas bubbles, showed a higher signal gain, and allowed us to halve the limit of detection to 0.02 mg L−1. The use of the 3D system to quantify the chlorine content of swimming pool water samples for sensitive and quantitative chlorine monitoring was demonstrated.
One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10–15 μm in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH–biotin bond was temperature-sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 °C. When oligonucleotides were bis-biotinylated, all SA/SAH–biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability.
Molecularly Imprinted Polymers with Integrated Fluorescence as Versatile Biomimetic Sensing Matrices
(2018)
Molecularly imprinted polymers (MIPs) are an established, versatile and high-performance matrix for the selective separation or enrichment of (bio)chemical species, especially small molecules of biochemical or environmental relevance. MIPs are prepared through the polymerization of a mixture of functional monomers and cross-linkers in the presence of the template with subsequent extraction of the latter. Conceptionally, this process can be seen as mimicking in a strongly accelerated, though single-step manner a biological process such as antibody formation. Because the resulting MIPs contain cavities in their matrix that are complementary in size, shape and electronic/ electrostatic or hydrogen bonding demand to the imprinted target molecule or template, these polymers are frequently termed “artificial antibodies”. Compared to natural antibodies, they are chemically and physically much more robust. Regarding sensitivity and selectivity, however, there is still a gap to bridge before MIPs can fully compete with antibodies.
Another favorable aspect that distinguishes MIPs from antibodies is that they can be endowed with an explicit function, allowing the use of MIPs in applications that require more than only an efficient binder. For instance, if specifically designed and polymerizable fluorescent indicators are integrated as functional monomers into a MIP, direct fluorescence sensing can be accomplished. Because MIPs can be prepared in a variety of different formats, their combination with miniaturized or other specific analytical techniques or sensory devices is possible, especially when the transduction mode is light. This presentation will introduce basic design considerations, challenges, limitations and the potential that lies with such sensor materials with some recent examples of our group, targeting various organic oxoanions as analytes.
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.
The recently reported luminescent chromium(III) complex 13+ ([Cr(ddpd)2]3+; ddpd=N,N’-dimethyl-N,N’-dipyridine-2-yl-pyridine-2,6-diamine) shows exceptionally strong near-IR emission at 775 nm in water under ambient conditions (F=11%) with a microsecond lifetime as the ligand design in 13+ effectively eliminates non-radiative decay pathways, such as photosubstitution, back-intersystem crossing, and trigonal twists. In the absence of energy acceptors, such as dioxygen, the remaining decay pathways are energy transfer to high energy solvent and ligand oscillators, namely OH and CH stretching vibrations. Selective deuteration of the solvents and the ddpd ligands probes the efficiency of these oscillators in the excited state deactivation. Addressing these energytransfer pathways in the first and second coordination sphere furnishes a record 30% quantum yield and a 2.3 millisecond lifetime for a metal complex with an earth-abundant metal ion in solution at room temperature.
A brief perspective of BAM on nanocarriers is presented including examples with special emphasis on the characterization of such materials and underlying challenges. In this respect, also ongoing activities at BAM on different types of core/shell nanomaterials and related systems are briefly summarized.
Correlating the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) and upconversion nanocrystals (UCNPs) assessed in ensemble studies and at the single particle level is increasingly relevant for applications of these nanomaterials in the life sciences like bioimaging studies or their use as reporters in microfluidic assays. Here we present a comparison of the spectroscopic properties of ensembles and single emitters for QDs like II/VI QDs and cadmium-free AIS/ZnS QDs as well as different UCNPs. The overall goal of this study was to derive particle architectures well suited for spectroscopic and microscopic applications.
Surface functionalization of nanomaterials is nowadays at the core of many applications of functional materials in the life and material sciences. Examples range from membranes and microarrays over bead-based assays, and next generation sequencing to nanometer-sized optical reporters, nanosensors, and magnetic and optical contrast agents. Typical function-nalization steps include silanization and grafting reactions with reactive monomers to introduce functional groups like amino or carboxylic acid groups or the attachment of ligands like polyethylene glycol (PEG) molecules and biomolecules. [1-3] This enables to tune e.g., dis-persibility, hydrophilicity and biocompatibility, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times and allows for the use of nanomaterials as reporters in assays or the design of targeted probes for bioimaging.
At the core of all functionalization strategies are reliable and validated methods for surface group and ligand quantification that can be preferably performed with routine laboratory instrumentation, require only small amounts of substances, and are suitable for many different types of nanomaterials. [3] There is meanwhile a considerable need to make these methods traceable. We present here versatile and simple concepts for the quantification of common functional groups, ligands, and biomolecules on different types of organic and inorganic nanomaterials, using conventional and newly developed cleavable and multimodal reporters, that can be detected with optical spectroscopy. [4-7] These reporters are chosen to enable method validation with the aid of method comparisons and mass balances. Also, strategies how to make these simple assays traceable to SI units using quantitative nuclear resonance spectroscopy (qNMR) and X-ray photoelectron spectroscopy (XPS) are derived.