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In der Natur sind zahlreiche biologische Prozesse mit der Übertragung von Elektronen verbunden, insbesondere die Stoffwechselprozesse zur Energiegewinnung aller Organismen. Technische Systeme zur elektrischen Energiegewinnung, zur Synthese bestimmter Produkte mittels elektrischen Stroms oder zur elektrischen Signalbildung in Messsystemen basieren auf denselben Grundprinzipien der Elektrochemie. Daher ist die Kopplung von Elektronenflüssen in biologischen und technischen Systemen nur folgerichtig und steht im Mittelpunkt der Elektrobiotechnologie.

In der Natur sind zahlreiche biologische Prozesse mit der Übertragung von Elektronen verbunden, insbesondere die Stoffwechselprozesse zur Energiegewinnung aller Organismen. Technische Systeme zur elektrischen Energiegewinnung, zur Synthese bestimmter Produkte mittels elektrischen Stroms oder zur elektrischen Signalbildung in Messsystemen basieren auf denselben Grundprinzipien der Elektrochemie. Daher ist die Kopplung von Elektronenflüssen in biologischen und technischen Systemen nur folgerichtig und steht im Mittelpunkt der Elektrobiotechnologie.

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM.

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 1013 aptamer molecules per cm2. As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus, the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL−1 and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis. This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

A joint action of ssDNA aptamers and electrochemistry is a key element in developing successful biosensing platforms, since aptamers are capable of binding various targets with high specificity, and electrochemistry is one of the most sensitive techniques for on-site detections. A continuous search for improved immobilization and sensing strategies of aptamers on transducer surfaces resulted in the strategy presented in this article. The strategy is based on the covalent attachment of gold nanoparticles on the surface of glassy carbon electrodes through sulfhydryl-terminated monolayer, acting as a glue to connect AuNPs on the electrode. The covalently attached gold nanoparticles modified glassy carbon electrodes have been applied for the efficient immobilization of thiolated ssDNA probes, with a surface coverage of about 8.54 × 1013 molecules cm−2 which was 7-fold higher than that on the electrochemically deposited gold nanoparticles. Consequently, improved sensitivity, good reproducibility and stability are achieved for electrochemical aptasensor. Combined with the high affinity and specificity of an aptamer, a simple, novel, rapid, sensitive and label-free electrochemical aptasensor was successfully fabricated for ofloxacin (OFL) detection. The linear dynamic range of the sensor varies between 5 × 10−8 to 2 × 10−5 M OFL with a detection limit of 1 × 10−9 M OFL. A potential application in environmental monitoring was demonstrated by using this sensing strategy for the determination of OFL in (experimentally spiked) real samples such as tap water and effluent of sewage treatment plant. The proposed nanoaptasensor combines the advantages of the covalent attachment of neatly arranged AuNPs (enlarged active surface area and strengthened electrochemical signal) and the elimination of labels for the amplified detection of OFL, with the covalent attachment of highly specific aptamers

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function.

Quinolones are antibiotics that are accredited in human and veterinary medicine but are regularly used in high quantities also in industrial livestock farming. Since these compounds are often only incompletely metabolized, significant amounts contaminate the aquatic environment and negatively impact on a variety of different ecosystems. Although there is increasing awareness of problems caused by pharmaceutical pollution, available methods for the detection and elimination of numerous pharmaceutical residues are currently inefficient or expensive. While this also applies to antibiotics that may lead to multi-drug resistance in pathogenic bacteria, aptamer-based technologies potentially offer alternative approaches for sensitive and efficient monitoring of pharmaceutical micropollutants. Using the Capture-SELEX procedure, we here describe the selection of an aptamer pool with enhanced binding qualities for fluoroquinolones, a widely used group of antibiotics in both human and veterinary medicine. The selected aptamers were shown to detect various quinolones with high specificity, while specific binding activities to structurally unrelated drugs were not detectable. The quinolone-specific aptamers bound to ofloxacin, one of the most frequently prescribed fluoroquinolone, with high affinity (KD=0.1–56.9 nM). The functionality of quinolone-specific aptamers in real water samples was demonstrated in local tap water and in effluents of sewage plants. Together, our data suggest that these aptamers may be applicable as molecular receptors in biosensors or as catcher molecules in filter systems for improved monitoring and treatment of polluted water.

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

S-layer proteins provide a biocompatible environment with different kinds of functional groups, perfect for the sequential coupling of any kind of biofunctional molecule. In addition, their nanostructure and their ability to crystallize on surfaces in a nanometer-thick monolayer ensures a regular arrangement of these molecules on solid supports. In this work, a thrombin-binding aptamer and an ofloxacin-binding aptamer were coupled with different chemical crosslinkers to S-layer proteins using them for defined immobilization. S-layer protein monomers and paracrystalline S-layers were successfully modified with the thrombin-binding aptamer. However, S-layer protein monomers were not able to crystallize after aptamer modification and showed no thrombin binding during random surface attachment. In contrast, aptamers linked to an intact S-layer in suspension or an S-layer coating were still functional. The modification rate of S-layers with the thrombin-binding aptamer was estimated with one aptamer to two unit cells (unit cell = four monomers). Verification of the functionality of both aptamers through target-binding after S-layer-immobilization on solid supports was proven by laser-induced fluorescence spectroscopy (LIFS), resonant mirror sensor (IAsys) and quartz crystal microbalance with dissipation monitoring (QCM-D), respectively. Hence, this study presents S-layer proteins as an interesting alternative to existing immobilization matrices for recognition biomolecules.

The International Water Research Alliance Saxony (IWAS) is addressing the global challenges concerning water quality in the areas of drinking water and sanitation, agricultural irrigation and the quality of surface and ground waters, as well as developing specific ecosystem-relevant services to be implemented on an exemplary basis in selected model regions. Locations (model regions) have been selected in Eastern Europe (R1), Central and Southeast Asia (R2 and R3), the Middle East (R4) and Latin America (R5) that are representative international regions with respect to climate, land use and demographic change (Ibisch et al., Helmholtz Centre for Environmental Research—UFZ, Department of Aquatic Ecosystem analysis and management, 2013). The causes of water problems and the relevant boundary conditions vary from region to region (Borchardt and Ibisch, Integrated water resources management in a changing world : lessons learnt and innovative perspectives, pp 225, 2013). Mongolia and Vietnam were selected model regions in the first IWAS phase; the research was transferred and continued in one of the cross-cutting projects in IWAS II (Vietnam → capacity development) or in the frame of related project activities in Central Asia (R2 Mongolia, Karthe et al., Environ Earth Sci, doi:10.1007/s12665-014-3789-1, 2014). The IWAS consortium exists on scientific institutions like the Technische Universität Dresden and the Helmholtz Centre for Environmental Research—UFZ, Leipzig, as well as partners from industry like the Stadtentwässerung Dresden GmbH (SE-DD), DREBERIS—Dresden consulting for international strategies, and itwh—institute for technical-scientific Hydrology, Hanover. This thematic issue compiles the most important scientific results of the second phase of the IWAS project. The project itself and findings of the first phase were already introduced in a previous special issue by Kalbus et al. (Environ Earth Sci 65:1363–1366, 2012). Main results: The IWAS

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. Based on 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.

Aptamers are single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) oligonucleotides, which are able to bind their target with high selectivity and affinity. Owing to their multiple talents, aptamers combined with nanoparticles are nanosystems well qualified for the development of new biomedical devices for analytical, imaging, drug delivery and many other medical applications.Because of their target affinity, aptamers can direct the transport of aptamer-nanoparticle conjugates. The binding of the aptamers to the target “anchors” the nanoparticleaptamer conjugates at their site of action. In this way, nanoparticle-based bioimaging and smart drug delivery are enabled, especially by use of systematically developed aptamers for cancer-associated biomarkers. This review article gives a brief overview of recent relevant research into aptamers and trends in their use in cancer diagnosticsand therapy. A concise description of aptamers, their development and functionalities relating to nanoparticle modification is given. The main part of the article is dedicated to current developments of aptamer-modified nanoparticles and their use in cancer diagnostics and treatment.

Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

Due to their multiple attributes, aptamers are the most qualied nanosystems for the development of new biomedical devices for analytical, imaging, drug delivery, and many other medical applications. For instance, the common use of nanoparticles for drug delivering and bioimaging can be substantially improved by modication with aptamers to enhance the specic binding of the nanoparticles via the specic aptamer binding to their target molecule. Aptamers can serve as “drive propulsion system” for the transport of the nanoparticles to their site of action caused by the target afnity of the aptamers.

The genetically modified yeast strain Yarrowia lipolytica H222-S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild-type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose-peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose-peptone. Furthermore, two process strategies (fed-batch and repeated fed-batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed-batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.

Small organic molecules are challenging targets for an aptamer selection using the SELEX technology (SELEX—Systematic Evolution of Ligans by EXponential enrichment). Often they are not suitable for immobilization on solid surfaces, which is a common procedure in known aptamer selection methods. The Capture-SELEX procedure allows the selection of DNA aptamers for solute targets. A special SELEX library was constructed with the aim to immobilize this library on magnetic beads or other surfaces. For this purpose a docking sequence was incorporated into the random region of the library enabling hybridization to a complementary oligo fixed on magnetic beads. Oligonucleotides of the library which exhibit high affinity to the target and a secondary structure fitting to the target are released from the beads for binding to the target during the aptamer selection process. The oligonucleotides of these binding complexes were amplified, purified, and immobilized via the docking sequence to the magnetic beads as the starting point of the following selection round. Based on this Capture-SELEX procedure, the successful DNA aptamer selection for the aminoglycoside antibiotic kanamycin A as a small molecule target is described.

Aptamers are oligonucleotide ligands that are selected for high-affinity binding to molecular targets. Only limited knowledge relating to relations between structural and kinetic properties that define aptamer–target interactions is available. To this end, streptavidin-binding aptamers were isolated and characterised by distinct analytical techniques. Binding kinetics of five broadly similar aptamers were determined by surface plasmon resonance (SPR); affinities ranged from 35–375 nM with large differences in association and dissociation rates. Native mass spectrometry showed that streptavidin can accommodate up to two aptamer units. In a 3D model of one aptamer, conserved regions are exposed, strongly suggesting that they directly interact with the biotin-binding pockets of streptavidin. Mutational studies confirmed both conserved regions to be crucial for binding. An important result is the observation that the most abundant aptamer in our selections is not the tightest binder, emphasising the importance of having insight into the kinetics of complex formation. To find the tightest binder it might be better to perform fewer selection rounds and to focus on post-selection characterisation, through the use of complementary approaches as described in this study.

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed-batch, repeated batch and repeated fed-batch cultivation were tested. The best results were achieved during repeated fed-batch cultivation: Within 3 days of cycle duration, approximately 100?g/L citric acid were produced. The yields reached values between 0.51 and 0.65?g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth-decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.

Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors) will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers.