Aptamers are promising recognition elements for sensitive and specific detection of small molecules. We have previously selected ssDNA aptamers for ethanolamine, one of the smallest aptamer targets so far. The work presented here focuses on the determination of the binding region within the aptamer structure and its exploitation for the development of an aptamer-based assay for detection of ethanolamine. Sequence analysis of the aptamers resulted in the identification of a G-rich consensus sequence, which was able to fold in a typical two- or three-layered G-quartet structure. Experiments with stepwise truncated variants of the aptamers revealed that the consensus sequence is responsible and sufficient for binding to the target. On the basis of the knowledge of the aptamers binding site, we developed an aptamer-based microarray assay relying on competition between ethanolamine and an oligonucleotide complementary to the consensus sequence. Competitive binding of ethanolamine and fluorescently labeled complementary oligonucleotides resulted in fluorescence intensities dependent on ethanolamine concentration with a limit of detection of 10 pM. This method enables detection of small molecules without any labeling of analytes. The competitive assay could potentially be transferred to other aptamers and thus provides a promising system for aptamer-based detection of diverse small molecules.
Dedicated to Professor Heribert Offermanns on the occasion of his 75 th birthdayAptamers are single-stranded oligonucleotides composed of RNA or DNA that are able to bind their corresponding targets via molecular recognition. Thus, aptamers can be thought of as nucleic acidbased alternatives to antibodies and have attracted attention as receptors in biosensors. Aptamers seem to be ideal biological recognition elements, since they enable the design of intelligent sensors based on their specific properties. Especially the fact that most aptamers undergo conformational changes during the binding of the target and their oligonucleotide nature can be used to rationally design novel sensing strategies. This review focuses on aptasensors for the detection of small molecules. In the first part, aptamers, their generation and their properties are briefly described. In the second part, different design strategies for aptasensors are reviewed, and examples for the detection of small molecules are given.
Recruitment of mesenchymal stromal cells (MSC) into the field of tissue engineering is a promising development since these cells can be expanded ex vivo to clinically relevant numbers and, after expansion, retain their ability to differentiate into various cell lineages. Safety requirements and the necessity to obtain high cell numbers without frequent subcultivation of cells raised the question of the possibility of expanding MSC in one-way (single-use) disposable bioreactors. In this study, umbilical cord-derived MSC (UC-MSC) were expanded in a disposable Z ® RP 2000 H bioreactor under dynamic conditions. Z ® RP was characterized regarding residence time and mixing in order to evaluate the optimal bioreactor settings, enabling optimal mass transfer in the absence of shear stress, allowing an reproducible expansion of MSC, while maintaining their stemness properties. Culture of the UC-MSC in disposable Z ® RP 2000 H bioreactor resulted in a reproducible 8-fold increase of cell numbers after 5 days. Cells were shown to maintain specific MSC surface marker expression as well as trilineage differentiation potential and lack stress-induced premature senescence.Bioengineering 2014, 1 232
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