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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 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.