Large arrays of femtoliter-sized chambers are important tools for single molecule research as well as bioanalytical applications. We have optimized the design and fabrication of two array types consisting of 250 × 250 (62 500) femtoliter chambers either by surface etching of fused silica slides or by polydimethylsiloxane (PDMS) molding. Highly diluted solutions of β-galactosidase were enclosed in such arrays to monitor the fluorogenic reactions of hundreds of individual enzyme molecules in parallel by wide-field fluorescence microscopy. An efficient mechanical sealing procedure was developed to prevent diffusion of the fluorescent reaction product out of the chambers. Different approaches for minimizing non-specific surface adsorption were explored. The signal acquisition was optimized to grant both a large field of view and an efficient signal acquisition from each femtoliter chamber. The optimized femtoliter array has enabled a three-in-one enzyme assay system: First, the concentration of active enzyme can be determined in a digital way by counting fluorescent chambers in the array. Second, the activity of the enzyme bulk solution is given by averaging many individual substrate turnover rates without the need for knowing the exact enzyme concentration. Third-unlike conventional enzyme assays-the distribution of individual substrate turnover rates yields insight into the conformational heterogeneity in an enzyme population. The substrate turnover rates of single β-galactosidase molecules were found to be broadly distributed and independent of the type of femtoliter array. In general, both types of femtoliter arrays are highly sensitive platforms for enzyme analysis at the single molecule level and yield consistent results. Graphical Abstract Isolation and analysis of individual enzyme molecules in large arrays of femtoliter-sized chambers.
Experimental Acceleration of Fracture HealingIII. Ribonucleic Acids Summary. The influence of heterologous organ-specific RIqA on fracture healing was examined in rabbits. To obtain an objective measurement of callus stability the bones were bent mechanically. The force applied and the degree of bending were recorded continuously. The slope of the curve was taken as a parameter of callus stability. In the first series of experiments, different dosages of RNA solution were injected into the fractures on different days. In the second series RNA was given intramuscularly at a site remote from the fracture site. The optimal dosage was found to be 1.8 mg when injected into the fracture on the 2nd day and a total of 7.2 mg when injected intramuscularly from the 2nd to the 10th day. It was possible to reduce the time of fracture healing by 3 and 2z/2 days respectively (p < 0.00001 and p < 0.001) by the means.In den friihen Phasen der Callusbildung tritt eine bindegewebige Matrix auf, die in sp/iteren Stadien mineralisiert und dann zum endgfiltigen Knochen umgebaut wird. Die Bildung der organischen Matrix ist yon der Fi~higkeit der Zellen zar Proteinsynthese abh/~ngig. Die Grundlagen der Proteinsynthese in lebenden Zellen sind in den letzten Jahren weitgehend erkannt worden [i3]. Die Bedeutung der Nucleins/iuren ffir den Eiwei~aufbau ist dabei gekl~trt worden [t, 7, ii]. Die Organspezifit/~t der Ribonucleins/iuren (RNS) konnten Kalb [t0] und Emanuellson [8] nachweisen. In mehreren Versuehen mit Regenerationsgewebe bei der Wundheilung konnten GuscMbauer und Williamson [14, i5] zeigen, dab sich die Zusammensetzung der versehiedenen RNS w/~hrend der Wundheilung/indert. Es bestand eine deutliche ErhShung des RNS-Gehalts im Regenerationsgewebe, aul3erdem konnte der fSrdernde EinfluB der RI~S auf die Proteinsynthese nachgewiesen werden.
A semiconductor die typically undergoes a variety of assembly processes, each of them influencing the mechanical stress environment of the chip. We report on a stress-sensitive testchip and its application to measure and characterize the respective stress distributions across the die. Particularly in the field of MEMS and sensor devices, even low stresses might deteriorate the electrical performance of a device significantly, while higher stress levels may even cause cracks and irreversible damage. This is why the present methodology has a high potential to develop into a useful tool in the field of sensor package development. After a brief introduction to the sensing principle, we investigate the stress distribution in various sample assemblies – all of them targeting pressure sensors. Typical low stress packages use silicone based soft elastic encapsulations. Changing to a harder material with a young’s modulus comparable to standard mold compounds, we demonstrate an increase of the average stress level by more than one order of magnitude.
In high-tech processing even smallest concentrations of metal ions in process media are of the utmost significance because they cause expensive production failures. Currently, cost-intensive equipment, special trained staff and time consuming analyses are necessary to detect these contaminations in order to avoid failures. The Centers of Excellence Nanochem and Sensorics at the University of Applied Sciences Regensburg (owner of patent PCT/EP2010/064833) and their industrial partner Micro-Epsilon GmbH are developing a new miniaturized measurement device which allows cost-effective real-time analysis of fluidic media for the first time. The system is fully automated and can be directly connected to wet-etch benches. Hence it allows continuous real-time surveillance of metal contaminations in the ppb-range through absorption spectroscopy in process media. For this purpose a very small sample amount of the process medium and a specific complexing agent are mixed together. This leads to an increase in the molar extinction coefficients and though even smallest contaminations become visible. The main parts of our development are the simulation of the different system components, their production and chemical analyses with the evaluation model.
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