Filtern
Dokumenttyp
Sprache
- Englisch (14)
Schlagworte
- Cell (4)
- ICP-MS (4)
- ELISA (3)
- Laser ablation (3)
- Nanoparticle (3)
- Imaging (2)
- Konjugate (2)
- LC-MS/MS (2)
- Sulfamethoxazole (2)
- X-Ray diffraction (2)
- 3T3 cells (1)
- 6xHis (1)
- Affinity chromatography (1)
- Aflatoxin (1)
- Albumin (1)
- Aluminum oxide (1)
- Amperometry (1)
- Bacterial lysates (1)
- Biolabels (1)
- Bioseparation (1)
- Bovine serum albumin (1)
- CLSM (1)
- Carrier (1)
- Conjugates (1)
- Cyclic voltammetry (1)
- Cytoplasm (1)
- D-glucose (1)
- Digoxigenin (1)
- Downstream processing (1)
- EDTAD (1)
- EPR spectroscopy (1)
- Einzelzellanalyse (1)
- Electrochemistry (1)
- Escherichia coli (1)
- Estradiol (1)
- Estrone (1)
- Ethylenediaminetetraacetic acid (1)
- FTIR spectroscopy (1)
- Flow cytometry (1)
- Gold nanoparticles (1)
- Hapten (1)
- HexaHis-Tag (1)
- High throughput biosensing (1)
- His6 (1)
- His8 (1)
- Hybrid nanoprobe (1)
- Hybridoma (1)
- IMAC purification (1)
- Immunization (1)
- Immunoassay (1)
- Immunocapture (1)
- L-alanine (1)
- Lebensmittel (1)
- MALDI-ToF-MS (1)
- Maillard reaction (1)
- Melanoidin (1)
- Melanoidine (1)
- Monoclonal antibodies (1)
- NMR spectroscopy (1)
- Nanosensor (1)
- Nickel (1)
- Nickel chelate (1)
- Ochratoxin A (1)
- Polishing (1)
- Recombinant protein (1)
- Rinderserumalbumin (1)
- SEIRA Technique (1)
- SEIRA methodology (1)
- SERS (1)
- SERS multiplexing (1)
- Sapphire (1)
- Silbernanopartikel (1)
- Speroid (1)
- Spheroid (1)
- Synchrotron radiation (1)
- Zearalenone (1)
- Zelle (1)
- bio-diagnostic (1)
- nanotechnology (1)
Organisationseinheit der BAM
We have efficiently produced collagen-rich microstructures in fibroblast multicellular spheroids (MCSs) as a three-dimensional in vitro tissue analog to investigate silver (Ag) nanoparticle (NP) penetration. The MCS production was examined by changing the seeding cell number (500 to 40,000 cells) and the growth period (1 to 10 days). MCSs were incubated with Ag NP suspensions with a concentration of 5 μg/mL for 24 h. For this study, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to visualize Ag NP localization quantitatively. Thin sections of MCSs were analyzed by LA-ICP-MS with a laser spot size of 8 μm to image distributions of 109Ag, 31P, 63Cu, 66Zn, and 79Br. A calibration using a NP suspension was applied to convert the measured Ag intensity into the number of NPs present. The determined numbers of NPs ranged from 30 to 7200 particles in an outer rim of MCS. The particle distribution was clearly correlated with the presence of 31P and 66Zn and was localized in the outer rim of proliferating cells with a width that was equal to about twice the diameter of single cells. Moreover, abundant collagens were found in the outer rim of MCSs. For only the highest seeding cell number, NPs were completely captured at the outer rim, in a natural barrier reducing particle transport, whereas Eosin (79Br) used as a probe of small molecules penetrated into the core of MCSs already after 1 min of exposure.
Electrochemical methods offer great promise in meeting the demand for user-friendly on-site devices for Monitoring important parameters. The food industry often runs own lab procedures, for example, for mycotoxin analysis, but it is a major goal to simplify analysis, linking analytical methods with smart technologies. Enzyme-linked immunosorbent assays, with photometric detection of 3,3’,5,5’-tetramethylbenzidine (TMB),form a good basis for sensitive detection. To provide a straightforward approach for the miniaturization of the detectionstep, we have studied the pitfalls of the electrochemical TMB detection. By cyclic voltammetry it was found that the TMB electrochemistry is strongly dependent on the pH and the electrode material. A stable electrode response to TMB could be achieved at pH 1 on gold electrodes. We created a smartphonebased, electrochemical, immunomagnetic assay for the detection of ochratoxin A in real samples, providing a solid basis forsensing of further analytes.
Immobilized metal affinity chromatography (IMAC) is a widely used technique for purifying polyhistidine-tagged recombinant proteins. However, it often has practical limitations that require complex optimizations and additional steps for purification. In this study, we introduce functionalized corundum particles as a novel, efficient, and economical method for purifying recombinant proteins in a column-free format. The corundum surface is modified with amino silane APTES, followed by EDTA dianhydride, and then loaded with nickel ions. We used the Kaiser test to monitor the modification process and ICP-MS to quantify the metal-binding capacity. To evaluate the system, we used His-tagged protein A/G (PAG) mixed with bovine serum albumin (BSA). The corundum particles exhibited a binding capacity of approximately 3 mg of protein per gram of corundum or 2.4 mg per 1 mL of corundum suspension. We also examined cytoplasm obtained from different E. coli strains as an example of a complex matrix. Varying the imidazole concentration in the loading and washing buffers showed that higher concentrations during loading improved purity. Even with sample volumes as large as one liter, we successfully isolated recombinant proteins down to a concentration of 1 µg/mL. We found higher purity levels with corundum when comparing the corundum material to standard Ni–NTA agarose beads. We successfully purified His6-MBP-mSA2, a fusion protein comprising monomeric streptavidin and maltose-binding protein, from E. coli cytoplasm, demonstrating the method's applicability. We also purified SARS-CoV-2-S-RBD-His8 expressed in human Expi293F cells, confirming its suitability for mammalian cell culture supernatants. The material cost of the nickel-loaded corundum material (without regeneration) is estimated to be less than 30 cents per gram of functionalized support or 10 cents per milligram of isolated protein. Another advantage of this system is the exceptional physical and chemical stability of corundum particles. Overall, we have demonstrated that this novel material offers an efficient, robust, and cost-effective purification platform for His-tagged proteins, even in challenging, complex matrices and large sample volumes with low product concentrations. This method has potential applications in both small laboratories and large-scale industrial settings.
A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity.