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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 × 10 13 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 to the surface of the modified electrode.

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.

In this work, S-layer proteins were used as an immobilization matrix to link aptamers on different solid supports. In nature, S layers operate amongst others as an immobilization matrix for exoenzymes. Consequently, they provide a biocompatible environment with different kinds of chemical groups perfect for the sequential coupling of any kind of biofunctional molecules. In addition, their nanostructure ensures a regular arrangement of these biomolecules. In biosensors, different biological recognition molecules are used. In this study, aptamers were chosen as bio-receptors. Aptamers are oligonucleotide ligands that are especially selected for high-affinity binding to target molecules. Because of their small size and stability, they exhibit a high potential as biological sensing molecules. By coupling aptamers to different surfaces or combining them with other biofunctional molecules, target binding can be detected for example optically or gravimetrically. In this work, a thrombin-binding aptamer and an ofloxacin-binding aptamer were immobilized by different chemical crosslinkers to surfaces modified with S-layer proteins. To verify the functionality of immobilized aptamers, the aptamer-target-binding was proven by Laser Induced Fluorescence Spectroscopy (LIFS), a Resonant Mirror Sensor (IAsys) and a Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D), respectively. Due to their properties of building up a physiological environment on their surface, their high content of modifiable functional groups on their surface and their ability to crystallize in a nanometer thick monolayer on surfaces, S-layer proteins are suitable as biotemplates for various recognition biomolecules like enzymes, antibodies and aptamers. Hence, this paper presents with S-layer proteins an interesting alternative to existing immobilization matrices for recognition biomolecules.

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.

To enable the analysis of several hundreds to thousands of interactions in parallel, high-throughput systems were developed. We used established thrombin aptamer assays to compare three such high-throughput imaging systems as well as analysis software and user influence. In addition to our own iRIf-system, we applied bscreen and IBIS-MX96. As non-imaging reference systems we used Octet-RED96, Biacore3000, and Monolith-NT.115. In this study we measured 1378 data points. Our results show that all systems are suitable for analyzing binding kinetics, but the kinetic constants as well as the ranking of the selected aptamers depend significantly on the applied system and user. We provide an insight into the signal generation principles, the systems and the results generated for thrombin aptamers. It should contribute to the awareness that binding constants cannot be determined as easily as other constants. Since many parameters like surface chemistry, biosensor type and buffer composition may change binding behavior, the experimenter should be aware that a system and assay dependent KD is determined. Frequently, certain conditions that are best suited for a given biosensing system cannot be transferred to other systems. Therefore, we strongly recommend using at least two different systems in parallel to achieve meaningful results.

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.

In our previous work, we selected aptamers binding to ethanolamine, one of the smallest molecular aptamer targets so far (Mann, D., Reinemann, C., Stoltenburg, R. and Strehlitz, B. Biochem. Biophys. Res. Commun. 2005, 338, 1928-1934). Two representatives of these aptamers (EA#14.3 and EA#9.4) were analyzed regarding their specificity. Ethanolamine is a very small organic molecule (M(w) = 61.08) with biological, medical, and industrial relevance. Its small size represented a challenge for aptamer development, as ethanolamine only consists of a short carbon chain (2C) and two functional groups (amino and hydroxyl group). Related organic molecules, ethanolamine derivatives, and some amino acids were tested to act as potential binding partners for these aptamers. In this way we were able to determine the exact binding domain within the target. The results revealed that both aptamers bind to various molecules, which contain a freely accessible ethyl- or methylamine group. In contrast to the amino group (in a primary, secondary, or tertiary amine) the hydroxyl group was not necessary for the aptamer binding. The aptamers were not able to bind to negatively charged organic molecules, despite containing an ethyl- or methylamine group, nor did they bind to molecules with quaternary amines. The selected ethanolamine binding aptamers are useful for the detection of molecules containing accessible ethyl- or methylamine groups; they can be used as linker elements to immobilize a target molecule of interest on a surface or to purify targets from complex samples.

SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 × 10 16 randomised ssDNA molecules. The dissociation constants of these aptamers range between K D = 6 and K D = 19 nmol L −1 . The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.

Aptamers are ssDNA or RNA oligonucleotides with very high affinity for their target. They bind to the target with high selectivity and specificity because of their specific three-dimensional shape. They are developed by the so-called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. We have modified this method in two steps-use of fluorescent labels for DNA quantification and use of magnetic beads for target immobilization. Thus, radioactive labelling is avoided. Immobilization on magnetic beads enables easy handling, use of very small amounts of target for the aptamer selection, rapid and efficient separation of bound and unbound molecules, and stringent washing steps. We have called this modified SELEX technology FluMag-SELEX. With FluMag-SELEX we have provided a methodological background for our objective of being able to select DNA aptamers for targets with very different properties and size. These aptamers will be applied as new biosensor receptors. In this work selection of streptavidin-specific aptamers by FluMag-SELEX is described. The streptavidin-specific aptamers will be used to check the surface occupancy of streptavidin-coated magnetic beads with biotinylated molecules after immobilization procedures.

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications.