Silver nanoparticles are widely used for many applications. In this study silver nanoparticles have been tested for their toxic effect on fibroblasts (NIH-3T3), on a human lung adenocarcinoma ...epithelial cell line (A-549), on PC-12-cells, a rat adrenal pheochromocytoma cell line, and on HEP-G2-cells, a human hepatocellular carcinoma cell line. The viability of the cells cultivated with different concentrations of silver was determined by the MTT assay, a photometric method to determine cell metabolism. Dose-response curves were extrapolated and IC50, total lethal concentration (TLC), and no observable adverse effect concentration (NOAEC) values were calculated for each cell line. As another approach, ECIS (electric-cell-substrate-impedance-sensing) an automated method to monitor cellular behavior in real-time was applied to observe cells cultivated with silver nanoparticles. To identify the type of cell death the membrane integrity was analyzed by measurements of the lactate dehydrogenase releases and by determination of the caspase 3/7 activity. To ensure that the cytotoxic effect of silver nanoparticles is not traced back to the presence of Ag+ ions in the suspension, an Ag+ salt (AgNO3) has been examined at the same concentration of Ag+ present in the silver nanoparticle suspension that is assuming that the Ag particles are completely available as Ag+ ions.
This work describes the design of optical aptamer-based porous silicon (PSi) biosensors for the direct capture of Lactobacillus acidophilus. Aptamers are oligonucleotides (single-stranded DNA or RNA) ...that can bind their targets with high affinity and specificity, making them excellent recognition elements for biosensing applications. Herein, aptamer Hemag1P, which specifically targets the important probiotic L. acidophilus, was utilized for direct bacteria capture onto oxidized PSi Fabry-Pérot thin films. Monitoring changes in the reflectivity spectrum (using reflective interferometric Fourier transform spectroscopy) allows for bacteria detection in a label-free, simple and rapid manner. The performance of the biosensor was optimized by tuning the PSi nanostructure, its optical properties, as well as the immobilization density of the aptamer. We demonstrate the high selectivity and specificity of this simple "direct-capture" biosensing scheme and show its ability to distinguish between live and dead bacteria. The resulting biosensor presents a robust and rapid method for the specific detection of live L. acidophilus at concentrations relevant for probiotic products and as low as 10(6) cells per mL. Rapid monitoring of probiotic bacteria is crucial for quality, purity and safety control as the use of probiotics in functional foods and pharmaceuticals is becoming increasingly popular.
•Establishing a bench mark CHO cultivation to test spectrometers.•Monitoring several parameters inline by infrared spectroscopy.•Near infrared for monitoring cell parameters (cell concentration and ...viability).•Mid infrared for monitoring chemical compounds such as glucose, lactate.
Process analytical technology (PAT) is a guide to improve process development in biotech industry. Optical sensors such as near and mid infrared spectrometers fulfill an essential part for PAT. NIRS and MIRS were investigated as non-invasive on line monitoring tools for animal cell cultivations in order to predict critical process parameters, like cell parameters as well as substrate and metabolite concentrations. Eight cultivations were performed with frequent sampling. Variances between cultivations were induced by spiking experiments with intent to break correlations between analytes; to keep causality of the models; and to increase model robustness.
Calibration models were built for each analyte using partial least-squares regression method. Cultivations chosen for validation were not part of the calibration set. Glucose concentration, cell density and viability were predicted by NIRS with a root mean square error of prediction (RMSEP) of 0.36g/L, 3.9 106cells/mL and 3.62% respectively. Based on MIR spectra glucose and lactate concentrations were predicted with a RMSEP of 0.16 and 0.14g/L respectively.
Results show that MIRS has higher accuracy regarding the prediction of single analytes. For prediction of a main course of a cultivation, NIRS is much better suited than MIRS.
Determination of antibodies against ToRCH antigens at the beginning of pregnancy allows assessment of both the maternal immune status and the risks to an adverse pregnancy outcome. Age-standardised ...seroprevalences were determined in sera from 1009 women of childbearing age residing in Mexico, Brazil, Germany, Poland, Turkey or China using a multiparametric immunoblot containing antigen substrates for antibodies against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), herpes simplex viruses (HSV-1, HSV-2), Bordetella pertussis, Chlamydia trachomatis, parvovirus B19, Treponema pallidum and varicella zoster virus (VZV). Seroprevalences for antibodies against HSV-1 were >90% in samples from Brazil and Turkey, whereas the other four countries showed lower mean age-adjusted seroprevalences (range: 62.5–87.9%). Samples from Brazilian women showed elevated seroprevalences of antibodies against HSV-2 (40.1%), C. trachomatis (46.8%) and B. pertussis (56.6%) compared to the other five countries. Seroprevalences of anti-T. gondii antibodies (0.5%) and anti-parvovirus B19 antibodies (7.5%) were low in samples from Chinese women, compared to the other five countries. Samples from German women revealed a low age-standardised seroprevalence of anti-CMV antibodies (28.8%) compared to the other five countries. These global differences in immune status of women in childbearing age advocate country-specific prophylaxis strategies to avoid infection with ToRCH pathogens.
•In situ microscopy monitors online microbial infections of a mammalian cell culture.•A warning system based on imaging is used to alert operators on microbial contaminations in bioreactor ...cultures.•In situ microscopy with imaging processing distinguish between mammalian cells and yeasts.
Microbial contamination in mammalian cell cultures causing rejected batches is costly and highly unwanted. Most methods for detecting a contamination are time-consuming and require extensive off-line sampling. To circumvent these efforts and provide a more convenient alternative, we used an online in situ microscope to estimate the cell diameter of the cellular species in the culture to distinguish mammalian cells from microbial cells depending on their size. A warning system was set up to alert the operator if microbial cells were present in the culture. Hybridoma cells were cultured and infected with either Candida utilis or Pichia stipitis as contaminant. The warning system could successfully detect the introduced contamination and alert the operator. The results suggest that in situ microscopy could be used as an efficient online tool for early detection of contaminations in cell cultures.
In recent years, the occurrence of
Legionella
in wastewater treatment plants (WWTP) has often been reported. However, until now there is limited knowledge about the factors that promote
Legionella’s
...growth in such systems. The aim of this study was to investigate the chemical wastewater parameters that might be correlated to the concentration of
Legionella
spp. in WWTP receiving industrial effluents. For this purpose, samples were collected at different processes in three WWTP. In 100 % of the samples taken from the activated sludge tanks
Legionella
spp. were detected at varying concentrations (4.8 to 5.6 log GU/mL) by the quantitative real-time polymerase chain reaction method, but not by the culture method. Statistical analysis with various parameters yielded positive correlations of
Legionella
spp. concentration with particulate chemical oxygen demand, Kjeldahl nitrogen and protein concentration. Amino acids were quantified in wastewater and activated sludge samples at concentrations that may not support the growth of
Legionella,
suggesting that in activated sludge tanks this bacterium multiplied in protozoan hosts.
•A new assay procedure was developed and evaluated for determination of vitamin B12.•Vitamin B12 was quantified in a range of matrices.•Hippophae rhamnoides was found to be a significant source of ...vitamin B12.•Above 98% of active vitamin B12 was found in Hippophae rhamnoides.
Based on increased demands of strict vegetarians, an investigation of vitamin B12 content in plant sources, was carried out. The vitamin B12 concentration was determined by RP-HPLC with UV detection, after prior matrix isolation by immunoaffinity chromatography (IAC). Vitamin B12 was extracted in the presence of sodium cyanide, to transform all forms of cobalamin into cyanocobalamin. Diode array detector was used to monitor vitamin B12, after its chromatographic separation under gradient elution with a mobile phase consisting of acetonitrile and trifluoroacetic acid 0.025% (w/v). The method demonstrated excellent linearity with a limit of detection 0.004μg/ml. The method precision was evaluated for plant samples and it was below 0.7% (n=6). Significant amounts of vitamin B12 in plants were detected in Hippophae rhamnoides (37μg/100g dry weight), in Elymus (26μg/100g dry weight) and in Inula helenium (11μg/100g dry weight).
•First time application of in situ Microscopy to high density Escherichia coli cultivations.•An efficient image processing algorithm is presented to determine cell density in Escherichia coli ...cultivations.•Online access to cell concentration up 70g/L dry cell mass.
In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Escherichia coli is the most studied and used organism in biotechnology.
In this article the cell density in Escherichia coli cultivations was monitored by applying ISM in these cultivations. The acquired images were analyzed with an image processing algorithm to determine the turbidity of the cultivation medium. In three cultivations the cell density was monitored with the algorithm and offline samples were taken to determine the dry cell mass (DCM). Both results were correlated and concentrations up to 70g/L DCM could be measured via ISM. For higher cell densities a saturation was recognized. The deviation of the calibration lines within three cultivations was 8%.
•Novel senor for noninvasive measurement of cell growth.•Simple inductive permittivity measurements.•Linear variable transformer for permittivity and conductivity measurements through a low ...permittivity reactor wall.•Comparison of different approaches for the realization of an adequate measuring system for noninvasive measurement of cell growth.
To ensure high quality output of biotechnological processes, relevant process parameters need to be monitored. As bioprocesses are increasingly executed in single use bioreactors, there is an increasing demand for new sensors applicable to these processes. In this work, we investigate different approaches for continuous non-invasive cell growth monitoring, especially for single use bioreactor applications. Therefore, the permittivity of the cell culture is used as a measure for the biomass. In a first step, a measuring procedure based on the transmission measurement of an electromagnetic wave is investigated. It appears that the penetration depth of this sensor is not sufficient for a noninvasive measurement through the polymer wall of a single use bioreactor. Therefore, alternative setups based on magnetic induction are investigated. The initial setup is very simple. It consists of a planar coil connected to an impedance analyzer. The coil is attached to the outside of the polymer foil of the single use bioreactor and an impedance spectrum is measured. To evaluate the sensor, E. coli cultivations are performed in a modified cultivation setup, which enables measurements through the polymer foil of a Sartorius BIOSTAT® CultiBag RM, and additionally allows sampling of culture medium for optical density reference measurements. The resonance peak of the coil in the impedance spectrum, is observed as measure for the optical density. Regardless of the simple sensor construction, we found a good correlation between optical density and the damping ratio of the resonance peak. However, the sensor signal shows saturation towards high optical densities. Therefore, an LTCC coil producing a higher magnetic flux density in the culture medium is investigated subsequently. This sensor shows a linear response up to high optical densities, but the sensitivity is reduced compared to the former used coil and therefore scattering of the data is increased. However, to increase the sensitivity, a linear variable differential transformer is realized. Using this setup, the influence of the primary magnetic flux is eliminated from the measuring voltage. This approach delivers the most promising results, as the sensor response is linear up to high optical densities and data scattering is low.
Stem cell therapy as a part of regenerative medicine provides promising approaches for the treatment of injuries and diseases. The increasing use of mesenchymal stem cells in various medical ...treatments created the demand for long-term in vivo cell tracking methods. Therefore, it is necessary to analyze post-transplantational survival, biodistribution, and engraftment of cells. Furthermore, stem cell treatment has been discussed controversially due to possible association with tumor formation in the recipient. For therapeutic purpose, stem cells must undergo substantial manipulation such as differentiation and in vitro expansion, and this can lead to the occurrence of genetic aberrations and altered expression of both tumor suppression and carcinogenic factors. To control therapy, it is necessary to find a reliable and general method to track and identify implanted cells in the recipient. This is especially challenging for autologous transplantations, as standard fingerprinting methods cannot be applied. An optimal technique for in vivo cell monitoring does not yet exist, and its development holds several challenges: small numbers of transplanted cells, possibility of cell number quantification, minimal transfer of the contrast agent to non-transplanted cells, and no genetic modification. This review discusses most of the proposed solutions, including magnetic resonance imaging, magnetic particle imaging, positron emission tomography, single-photon emission computed tomography, and optical imaging methods. Additionally, the recent research on cell labeling for stem cell monitoring after transplantation including in vitro, ex vivo, and in vivo imaging studies is described. Promising future imaging modalities for stem cell monitoring after transplantation are shown.
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