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Campos C.D.M.,University of Campinas | Reyes F.G.R.,University of Campinas | Manz A.,KIST Europe | Da Silva J.A.F.,University of Campinas
20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016 | Year: 2016

We present a device coupling aqueous two-phase liquid-liquid extraction with capillary electrophoresis, for the purpose of depleting the sodium content in food matrices and enabling in situ amino acid detection. The device was employed for the analysis of soy sauce, but could be adapted for use with other samples containing high levels of sodium. The limit of detection was 1.4 mmol L-1.

Ahrberg C.D.,KIST Europe | Ahrberg C.D.,Sogang University | Manz A.,KIST Europe | Neuzil P.,Northwestern Polytechnical University | Neuzil P.,Brno University of Technology
Analytical Chemistry | Year: 2016

We show the utilization of a recently developed cellphone-sized real-time polymerase chain reaction (PCR) device to detect Ebola virus RNA using single-step reverse transcription PCR (RT-PCR). The device was shown to concurrently perform four PCRs, each with a sample volume of 100 nL: one positive control with both Ebola and GAPDH RNA and one negative control. The last two positions were used to measure the GAPDH and the Ebola content of a sample. A comparison of threshold cycles (CT) from the two samples provided relative quantification. The entire process, which consisted of reverse transcription, PCR amplification, and melting curve analysis (MCA), was conducted in less than 37 min. The next step will be integration with a sample preparation unit to form an integrated sample-to-answer system for point-of-care infectious disease diagnostics. © 2016 American Chemical Society.

PubMed | KTH Royal Institute of Technology, Saarland University, KIST Europe and Science for Life Laboratory
Type: Journal Article | Journal: Cells | Year: 2017

Metastasizing tumor cells show increased expression of the intermediate filament (IF) protein vimentin, which has been used to diagnose invasive tumors for decades. Recent observations indicate that vimentin is not only a passive marker for carcinoma, but may also induce tumor cell invasion. To clarify how vimentin IFs control cell adhesions and migration, we analyzed the nanoscale (30-50 nm) spatial organization of vimentin IFs and cell-matrix adhesions in metastatic fibroblast cells, using three-color stimulated emission depletion (STED) microscopy. We also studied whether wild-type and phospho-deficient or -mimicking mutants of vimentin changed the size and lifetime of focal adhesions (FAs), cell shape, and cell migration, using live-cell total internal reflection imaging and confocal microscopy. We observed that vimentin exists in fragments of different lengths. Short fragments were mostly the size of a unit-length filament and were mainly localized close to small cell-matrix adhesions. Long vimentin filaments were found in the proximity of large FAs. Vimentin expression in these cells caused a reduction in FAs size and an elongated cell shape, but did not affect FA lifetime, or the speed or directionality of cell migration. Expression of a phospho-mimicking mutant (S71D) of vimentin increased the speed of cell migration. Taken together, our results suggest that in highly migratory, transformed mesenchymal cells, vimentin levels control the cell shape and FA size, but not cell migration, which instead is linked to the phosphorylation status of S71 vimentin. These observations are consistent with the possibility that not only levels, but also the assembly status of vimentin control cell migration.

Bodeker B.,B and S Analytik | Davies A.N.,University of South Wales | Maddula S.,KIST Europe | Baumbach J.I.,B and S Analytik
International Journal for Ion Mobility Spectrometry | Year: 2010

The engineering of ion mobility spectrometers has made significant advances in recent years with new levels of sensitivity being achieved which facilitate their use, for example, in the medical analysis of trace components in human breath. In the study reported here direct measurements of 10 mL of exhaled air were carried out using ion mobility spectrometry (IMS) and the influence of the ambient air composition on the validation of potential biomarkers carefully considered. Changes in the IMS-signals observed within a room used for clinical studies were analysed in the absence of clinical staff. A time series of one measurement every hour of ambient air was referenced against clean bottled synthetic air for comparison using two different IMS instruments equipped with multi-capillary columns (MCC). 26 different peaks were identified and selected for the signal intensity monitoring during 1 week. Some peaks show no changes, while certain peaks varied with time in a seemlingly random manner. Correlated peak intensity changes were observed as well as changes apparently relating to variations of the ventilation system during day and night cycling. It is important to fully understand these variation if incorrect conclusions are to be avoided during, for example, the important hunt for validatable biomarkers. These results will be applicable not only to IMS studies but to all experimental design strategies in this fields regardless of the selected instrumentation. As an example it is possible to cite a particular time series where a particular peak observed within the IMS-Chromatogram shows an analyte intensity decrease within the room air between the evening and the next morning of 75%. On a subsequent day the increase between the morning and the evening was observed by a factor of about 3. During 4 days, setting the mean value to 100%, an increase of up to 173% and the decrease of up to 21% were observed. In case of measurements near to the detection limit of the method of investigation, the influence of the surrounding inhaled air must be considered carefully. The daily changes of the concentrations of specific analytes in the ambient air should be taken into account in addition to the measurement of the analyte concentrations taken from the exhaled air of a patient. Recommendations will be made covering more robust validation strategies which include near-simultaneous background ambient air measurements alongside synthetic air measurements in clinical studies. Care should be taken to avoid unnecessary changes to the ambient room air during measurements such as caused by doors opening during the entire measurement time. Ambient air measurements should be included with sampling on the patient and the surrounding air, before and after measurement of the patient breath. © 2010 Springer-Verlag.

Darwiche K.,University of Duisburg - Essen | Baumbach J.I.,KIST Europe | Sommerwerck U.,University of Duisburg - Essen | Teschler H.,University of Duisburg - Essen | Freitag L.,University of Duisburg - Essen
Lung | Year: 2011

Background: The exhaled breath of lung cancer patients contains volatile organic compounds (VOCs) that differ from those in healthy individuals. These VOCs can be detected with methods such as ion mobility spectrometry (IMS); their origin remains unknown. Methods: In 19 patients with lung cancer, exhaled breath was aspirated via the working channel of a flexible bronchoscope from both the tumor-bearing and the opposite lung and analyzed with IMS. Results: IMS measurement through the working channel of a bronchoscope was feasible and safe. In comparison to the opposite lung, we found two peaks that were significantly higher and three peaks that were significantly lower on the IMS of the tumor-bearing site. VOCs differ in concentration depending on the histologic subtype. Conclusion: Our results indicate that VOCs in lung cancer patients are produced locally in or around the tumor, and it is most likely that these VOCs represent underlying metabolic processes of the tumor. © 2011 Springer Science+Business Media, LLC.

Bessa V.,University of Duisburg - Essen | Darwiche K.,University of Duisburg - Essen | Teschler H.,University of Duisburg - Essen | Sommerwerck U.,University of Duisburg - Essen | And 3 more authors.
International Journal for Ion Mobility Spectrometry | Year: 2011

COPD is a disease characterised by a chronic inflammation of the airways and a not fully reversible airway obstruction. The spirometry is considered as gold-standard to diagnose the disease and to grade its severity. In this study we used the methodology of Ion Mobility Spectometry in order to detect Volatile Organic Compounds (VOCs) in exhaled breath of patients with COPD. The purpose of this study was to investigate if the VOCs detected in patients with COPD were different from the VOCs detected in exhaled breath of healthy controls. 13 COPD patients and 33 healthy controls were included in the study. Breath samples were collected via a side-steam Teflon tube and directly measured by an ion mobility spectrometer coupled to a multi capillary column (MCC/IMS). One peak was identified only in the patients group compared to the healthy control group. Consequently, the analysis of exhaled breath could be a useful tool to diagnose COPD. © 2011 Springer-Verlag.

Neuzil P.,Brno University of Technology | Neuzil P.,Institute Of Bioengineering And Nanotechnology, Singapore | Sun W.,Institute Of Bioengineering And Nanotechnology, Singapore | Sun W.,Bruker | And 2 more authors.
Applied Physics Letters | Year: 2015

We report on a microfluidic system formed by 200 nl water droplets, encapsulated by a 600 nl mineral oil placed on a hydrophobically coated glass microscope cover slip. The micromachined heater underneath the glass was able to heat up the sample at a heating rate of 650°C/s, heating the water sample up to 200°C in less than 2s. The sample/glass and the sample/oil interface did not have nucleation centers, showing that the sample reached a superheated stage without the necessity of being pressurized to suppress boiling. This method can be utilized for various applications currently being conducted in autoclaves. © 2015 Author(s).

Ahrberg C.D.,KIST Europe | Manz A.,KIST Europe
MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences | Year: 2015

In this contribution we demonstrate a method to investigate the thermal induced unfolding of proteins. The method is based on virtual reaction chambers compromised out of sample droplet suspended inside of an oil droplet on a hydrophobic surface. This prevents any contact of sample to the glass surface, thus no nucleation sites are present, providing a simple method to superheat the sample under ambient pressure. The unfolding of the protein is observed through the addition of a hydrophobic dye which becomes fluorescent when bound to the hydrophobic domains of the protein uncovered during unfolding. © 15CBMS-0001.

Ahrberg C.D.,KIST Europe | Manz A.,KIST Europe
RSC Advances | Year: 2016

Here we describe a novel method for the study of protein thermal stability using superheated aqueous samples within virtual reaction chambers. Virtual reaction chambers consist of an aqueous sample droplet encapsulated by an oil droplet on a hydrophobic surface. Such samples can be superheated due to the lack of nucleation sites. The thermal denaturation of proteins is induced through the application of a temperature gradient using a bespoke silicon heating chip. The unfolding of proteins is followed through the addition of a hydrophobic dye that attaches to protein hydrophobic domains that become exposed during denaturation. Using this method, we investigated the thermal stability of green fluorescence protein and Taq-polymerase. A possible screening application of the method was demonstrated by evaluating the effect of ionic concentration on the thermal stability of bovine serum albumin. © 2016 The Royal Society of Chemistry.

Westhoff M.,Lung Clinic Hemer | Litterst P.,Lung Clinic Hemer | Maddula S.,KIST Europe | Bodeker B.,BandS Analytik GmbH | Baumbach J.I.,KIST Europe
International Journal for Ion Mobility Spectrometry | Year: 2011

Human breath analysis is a powerful and especially a non-invasive technique for the monitoring and hopefully also for the diagnosis of respiratory diseases, including chronic obstructive pulmonary disease (COPD). The exhaled breath of 95 patients suffering COPD and of 35 healthy controls was investigated using an Ion Mobility Spectrometer (IMS) coupled to a Multi-Capillary Column (MCC) without any pre-separation or pre-enrichment. Starting with the results from a Mann-Whitney-Wilcoxon rank sum test to find analytes with the highest potential with respect to differentiation, box and whisker plots, metabolic maps and probability charts were introduced and compared. In addition, the sensitivity, specificity, positive and negative predictive values and the accuracy of the relation were also summarized. The findings were compared to the results of a principal component analysis. Finally, decision trees were introduced to visualize the interdependencies between the analytes and the classifications. The application of these biostatistical methods with simultaneous inclusion of several VOCs for disease classification by ion mobility spectrometry of human breath will provide much more information than using single peaks and single concentration dependencies for disease classification and discrimination of various groups. Towards the future application of potential biomarkers for clinical diagnostic procedures, complex analytical methods, such as ion mobility spectrometry, need statistical and bioinformatical tools which are simple in application, visualize the results and support decisions on the basis of the data obtained from measurements of analytes in exhaled human breath. © 2011 Springer-Verlag.

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