Analytische Chemie
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Nano- and microparticles are of increasing importance for a wide range of applications in the material and life sciences. Examples are their use as carriers for dye molecules and drugs, multichromophoric reporters for signal enhancement strategies in optical assays, targeted probes for bioimaging, and biosensors. All these applications require surface functionalization of the particles with e.g., ligands (to tune the dispersibility and prevent unspecific interactions), crosslinkers, sensor dyes, or analyte recognition moieties like biomolecules, and subsequently, the knowledge of the chemical nature and total number of surface groups as well as the number of groups accessible for coupling reactions. Particularly attractive for the latter are sensitive and fast photometric or fluorometric assays, which can be read out with simple, inexpensive instrumentation. Here, we present a novel family of cleavable photometric and multimodal reporters for the quantification of conjugatable amino and carboxyl surface groups on nano- and microparticles. These probes allow for the determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface as well as the remaining thiol groups on the particles by photometry and inductively coupled optical emission spectrometry (32S ICP-OES).3 Comparison of the performance of these cleavable reporters with conventional labels, utilizing changes in intensity and/or colour of absorption and/or emission, underlines the analytical potential of this versatile concept which elegantly circumvents signal distortions by light scattering and encoding dyes, and enables straightforward validation by method comparison.
Polymeric nanoparticles (NP) are of increasing importance for a wide range of applications in the material and life sciences, including their use as drug carriers, fluorescent sensors and multimodal reporters in a large variety of bioassays and bioimaging studies. Application-relevant properties of NP include their size (and size distribution), shape, optical properties, and ease of subsequent functionalization, e.g. with linkers, sensor molecules, and bioligands. In this respect, knowledge of the chemical nature, the total number of surface groups and the number of groups accessible for subsequent coupling reactions is mandatory.1 Commercially available polystyrene NP often contain different additives like stabilizers, radical starters and crosslinkers, which can influence the quantification of surface functionalities. Moreover, they often have unknown surface group densities that may vary from batch to batch, which complicates or even hampers their reliable use in many (bio)applications. To circumvent these issues, we synthesized amino- and carboxy-functionalized, monodisperse 100 nm polystyrene NP with three different, well-defined surface group densities. Using a recently developed approach for the quantification of functional groups on nano- and microparticles with cleavable reporters,2 we quantified the assessible functional groups on the self-synthesised PS particles via optical spectroscopy (photometry) and inductively coupled optical emission spectrometry (32S ICP-OES). In addition, we developed a fluorimetric approach for the quantification of surface functional groups on nanoparticles based on the labelling with luminescent lanthanide complexes (LLC). In contrast to common organic dyes, LLC are not prone to photo¬luminescence quenching arising from reabsorption or dye aggregation, and thus, enable a reliable fluorometric quantification of the assessible functional groups on NP surfaces. Moreover, lanthanide tags can be detected with high specificity and sensitivity with analytical techniques such as XPS and ICP-MS, which allow for the multimodal validation of the fluorometric quantification approach.
Non-invasive Cereal Analysis by GC-MS detection of Trichodiene as a Volatile Mycotoxin Biomarker
(2018)
Due to the increasing consumption of cereals worldwide, the monitoring of growth, storage and processing is becoming more and more crucial. Particularly when stored, infested grains breed fungal clusters (“hot spots”) in which mycotoxins greatly exceed allowed maximum levels. Because of their unpredictable presence, current sample drawing and procedures for mycotoxin analysis represent a complex challenge for operators, involving invasive and cost intensive steps.
Therefore, new time- and labour-saving mycotoxin control methods including sampling and analysis steps are needed. A possible approach is the non-invasive analysis of the homogeneous gas phase above the crops, instead of analyzing random samples. However, this procedure requires microbial volatile organic compounds (MVOC´s) being released by the samples and representing the present mycotoxins. Previous investigations revealed trichodiene to be a precursor in trichothecenes biosynthesis – one of the largest mycotoxin groups with over 180 compounds. Due to its non-functionalized sesquiterpene structure, trichodiene has already been quantified using Headspace GC-MS methods (for instance]). Thereby, it can possibly be used as a biomarker for trichothecene contamination in foodstuff.
However, further investigations are necessary. The correlation between trichodiene concentration in the gas phase and trichothecenes mass fraction in the sample must be examined closely to draw conclusions about the exact trichothecene content within samples. Realizing this idea, would widely extend the applicability of trichodiene and enormously simplify trichothecene quantification. Hence, this first step of an ongoing study aims to develop a laboratory reference method using trichodiene as volatile biomarker to quantify trichothecenes in cereals. Static headspace and SPME-enrichment coupled to gas chromatography with mass spectrometry (GC-MS) were employed. In a second step, this reference method is intended to validate new approaches for fast on-site screening of trichodiene in cereals.
Zearalenone (ZEN) and its sulfate and glucoside conjugates have been detected in (a broad variety of) food and feed commodities1. Both conjugated derivatives are formed as part of fungal or plant secondary metabolism and thus, belong to the group of modified mycotoxins2. After consumption of contaminated foodstuff, the conjugates can be hydrolyzed by human intestinal microbiota leading to liberation of ZEN that implies an underestimation of the true ZEN exposure. In order to include ZEN conjugates in routine analysis, as well as for toxicological investigation reliable standards are needed.
The objective of the present study was to develop a simple and economic method for biosynthesis of ZEN conjugates. Preceding experiments on the biotransformation of ZEN by Rhizopus and Aspergillus species showed a mixed metabolite formation3. Therefore, these known ZEN conjugating fungal strains were screened for their potential to selectively synthesize the ZEN derivatives ZEN-14-sulfate (Z14S), ZEN-14-glucoside (Z14G) and ZEN-16-glucoside (Z16G). The screening was conducted by adding ZEN to liquid fungal cultures. Cultivation conditions and ZEN incubation time were varied. All media samples were analyzed for metabolite formation by HPLC-MS/MS. Z14S was exclusively formed by A. oryzae. Under optimized conditions a specific biosynthesis of Z14G by R. oryzae and Z16G by R. oligosporus was achieved. After liquid-liquid-extraction and preparative chromatographic cleanup 1H-NMR purities of ≥ 73% for Z14S, ≥ 82% for Z14G and ≥ 50% for Z16G were obtained. In addition, a consecutive biosynthesis was developed by first using Fusarium graminearum for ZEN biosynthesis on rice based liquid medium. After inactivation of Fusarium the subsequent conjugation reaction was conducted utilizing Aspergillus and Rhizopus species under the various optimized conditions.
In this study an easy and cost-efficient biosynthesis for Z14S, Z14G and Z16G was developed. The developed biosynthesis could be also used for other metabolites like ZEL conjugates. Our results of the in vitro screening indicate also the formation of a ZEL-glucoside and α ZEL-sulfate as major metabolites by R. oryzae. In sum, under optimized cultivation conditions fungi can be easily utilized for a targeted and stereospecific synthesis of ZEN conjugates.
The improvement of immunoanalytical methods for the determination of pharmaceuticals in wastewaters is a crucial yet challenging endeavor. In this work, the development of an automated miniaturized ELISA based on micro-Bead Injection Spectroscopy (μ-BIS) [1] for the determination of carbamazepine, a widely employed anti-epileptic drug and emergent pollutant [2], was pursued.
The experimental workflow comprised the offline functionalization of Sepharose beads with specific anti-CBZ antibodies via affinity immobilization using protein G, and 3 online steps inside the microfluidic analyzer lab-on-valve (LOV): I) packing of the bead column into the detection unit; II) sequential percolation of sample and a CBZ competitor- labeled with horseradish peroxidase (tracer) through the bead column; and III) on-column colorimetric detection employing the enzyme substrate 3,3’,5,5’-tetramethylbenzidine. After each analysis, the bead column was discarded, and the flow cell was washed before receiving new beads.
The elimination of manual washing steps is a novel feature compared to batch-wise ELISA, making the method less error-prone and therefore more robust. The replacement of the solid support prevents memory effects and cross-contamination between runs. The use of microparticles as solid support for the molecular recognition elements accounts for high area-to-volume ratios, and low molecular diffusion distances. For that reason, time-to-result was reduced from several hours to less than 10 min. The consumption of reagents was also very low. For instance, only ca. 200 μg of solid support and 900 ng of anti-CBZ antibody were required per determination. At last, the versatility of the LOV platform offers the possibility of adapting the assay to other relevant pharmaceuticals and anthropogenic markers in water.
Acknowledgements: Inês I. Ramos thanks FCT (Fundação para a Ciência e a Tecnologia) and POPH (Programa Operacional Potencial Humano) for her grant (SFRH/BD/97540/2013). This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC - Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013. Financial support from Deutscher Akademischer Austauschdienst and from Fundação das Universidades Portuguesas under the protocol CRUP-DAAD (Ações Integradas Luso-Alemãs nºE-20/16) is also acknowledged.
[1] Gutzman, Y.; Carrol, A. D. Analyst 2006, 131, 809.
[2] Murray, K. E.; Thomas, S. M.; Bodour, A. A. Environ. Pollut. 2010, 158, 3462.
Imaging of elemental distributions in single cell assays as well as tissue sections can be performed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). This powerful technique offers precise spatially resolved measurements at the trace and ultratrace level and has been established as an excellent tool to answer analytical, biological and biomedical questions. To date, imaging mass cytometry is already able to simultaneously detect up to 40 cellular targets due to conjugation of isotopically pure lanthanides to affinity binders, e.g. antibodies.
To further enhance the ability of multiparametric analysis to more than 100 analytes at once, we investigated lanthanide nanocrystals as new, highly sensitive metal tags for identification of targets in clinical cell assays and tissue samples. Multiparametric analysis will be possible by encoding the lanthanide composition of nanocrystals associated to the affinity binders. Nanocrystals showed remarkable potential for sensitive detection in MS due to high stability and signal amplification compared to e.g. polymer tags, carrying fewer metal atoms.
Synthesis of functionalized lanthanide nanocrystals for further bioconjugation was performed with high reproducibility and monodisperse size distribution. For proof of principle, the uptake and distribution of these nanocrystals within the monolayered cell line A549 were investigated by mapping the intensities at subcellular resolution using LA-ICP-MS. It could be shown, that the cells were efficiently labeled with the nanocrystals and mostly accumulate near the nucleus. Additionally, the bioconjugation of the nanocrystals to antibodies and particularly the preservation of the antibody specificity was investigated using Dot Blot experiments. All in all, the results imply high sensitivity and the possibility of multiparametric analysis by doting various lanthanides into the nanocrystals.
Due to their toxicity and widespread application for mining and industrial purposes, cyanides are ranking among the most important inorganic pollutants which should be tested and monitored not only in the aquatic environment, but also in soils and soil-like materials. Reference materials of soils with relevant contents of cyanide to ensure reliable test results of laboratories are rare today.
New certified reference material (CRM) BAM-U116/CGL306 “Cyanide in soil” was produced within a framework of cooperation between Central Geological Laboratory (CGL) of Mongolia and Federal Institute for Materials Research and Testing (BAM) of Germany in 2013-2017.
The CRM BAM-U116/CGL306 represents a mixture of a sandy soil collected from a contaminated former gasworks area in the Berlin region (Germany) and an unpolluted sandy soil from Nalaikh region (Mongolia). The bulk candidate material for this reference material was prepared at CGL CRM Laboratory exclusively destined to the preparation of reference materials and equipped with modern technical equipment. Homogeneity, stability and shelf life were studied in full compliance with ISO Guide 35. The CRM was evaluated as sufficiently homogeneous. Statistical evaluation of certification analysis was performed using software packages SoftCRM and ProLab Plus. Certified value of total cyanide of the CRM is 12.0 mg/kg and expanded uncertainty was assigned as 0.8 mg/kg.
The intended purpose of this material is the verification of analytical results obtained for the mass fraction of total cyanide in soils and soil-like materials applying the standardized procedure ISO 11262:2011. As any reference material, it can also be used for routine performance checks (quality control charts) or validation studies.
Cancer is a leading cause of death worldwide, and its early detection and resultant treatment contributes significantly to patient recovery and survival. Detection is currently based on magnetic resonance imaging and computed tomography, methods that are expensive, while processing of the results is time consuming. There is a need for low-cost cancer-detection techniques that give conclusive results in the shortest time possible. Molecularly imprinted polymers (MIPs) targeting tumor markers on cancerous cells may provide a cheaper solution for cancer detection. Thin MIP layers immobilized on particle platforms are known to give faster response times and increased selectivity in comparison to bulk MIPs. It has been reported that a fluorescent monomer can be incorporated into the MIP layer, allowing for faster detection of the target group, thus significantly shortening the turn-around time for biopsies.
Changes in sialylation patterns of cell surface glycoproteins indicate malignancy. Here, we present the development of MIPs that target sialic acid-terminated glycoproteins (SA MIPs), prepared as a thin layer on a silica nanoparticle platform. A fluorescent monomer is incorporated into the MIP layer, and upon binding of the target group to the specific binding pockets in the MIP, the fluorescence signal is enhanced. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used for structural characterization. To validate the specificity, fluorescence changes of MIPs in the presence and absence of template are compared to their corresponding non-imprinted polymer particles (NIP). Initial binding experiments with tumor cells using fluorescence microscopy demonstrate that the presented technique shows promise as a cheaper alternative to current detection methods, while allowing for relatively shorter analysis of biopsy results.
Due to the increasing consumption of cereals worldwide, monitoring of growth, storage and processing is becoming more crucial. Particularly when stored, infested grains breed fungal clusters (“hot spots”) in which mycotoxins greatly exceed allowed maximum levels. Because of their unpredictable presence, current sampling and procedures for mycotoxin analysis are complex and contain invasive and cost intensive steps.
A possible approach to avoid sampling inaccuracies is the non-invasive analysis of the homogeneous gas phase above the crops, instead of analyzing random samples. However, this procedure requires microbial volatile organic compounds (MVOC´s) being released by the samples. Previous investigations revealed trichodiene to be a precursor in trichothecenes biosynthesis – one of the largest mycotoxin groups with more than 180 compounds. Due to its non-functionalized sesquiterpene structure, quantification of trichodiene using Headspace GC-MS methods is possible (e.g. [1]). Thereby, it could be used as biomarker for trichothecene contamination in foodstuff.
However, further studies are necessary. The correlation between trichodiene concentration in the gas phase and trichothecenes mass fraction in the sample must be examined closely to draw conclusions about the trichothecene content in samples. Realizing this idea, would widely extend the applicability of trichodiene and enormously simplify trichothecene quantification. Hence, this first step of an ongoing study aims to develop a laboratory reference method using trichodiene as volatile biomarker to quantify trichothecenes in cereals. Static headspace and SPME-enrichment coupled to gas chromatography with mass spectrometry (GC-MS) were employed. In a second step, this reference method is intended to validate new approaches for fast on-site screening of trichodiene in cereals.
IAPR participates in the EMPIR 16SRT02, entitled ‘Certified forensic alcohol reference materials - ALCOREF’ funded by EURAMET as an external funded partner. The Research in this project will address the needs for better enforcement of drink-driving legislation in several European countries, by building up long-term capacities for the production and certification of forensic alcohol reference materials, suitable for the calibration of evidential breath alcohol analyzers. The certification procedure includes characterization of the materials, assessment of homogeneity, stability, and uncertainty estimation. An interlaboratory comparison will be conducted to test the materials and capabilities developed. EMPIR 16SRT02-ALCOREF project is coordinated by BAM (DE).