Aptamers for pharmaceuticals and their application in environmental analytics

Beate Strehlitz Christine Reinemann Soeren Linkorn Regina Stoltenburg 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. - Even though, until now, aptamers have been developed mainly for medical applications or clinical diagnostics, they are well suited as novel biological recognition elements for the detection of pharmaceutical residues in the environment because of their specific properties. Aptamers are short single-stranded oligomers (ssDNA or RNA), which are able to bind their target molecules with high specificity and selectivity. Binding occurs because of their specific and complex three-dimensional shape characterized by stems, loops, bulges, hairpins, pseudoknots, triplexes, or quadruplexes. The aptamer-target binding results from structure compatibility, stacking of aromatic rings, electrostatic and van der Waals interactions, and hydrogen bondings, or from a combination of these effects [1, 2]. Initially, RNA aptamer development was described for bacteriophage T4 DNA polymerase [3] and organic dyes Cibacron blue and Reactive blue 4 [4]. These publications described for the first time the evolutionary process to select aptamers starting with of a big variety of oligonucleotides in a so-called library (ca. 1015 different structures) by repeated rounds consisting of the steps (a) binding between target molecule and library, (b) elution of the bound oligonucleotides, and (c) their amplification. The resulting pool of pre-selected oligonucleotides forms the starting pool of the following round. This process is called systematic evolution of ligands by exponential enrichment (SELEX) and mimicks the Darwinian principle. The first DNA aptamers were described only 2 years later for Cibacron blue, Reactive blue 4, and Reactive green 19 [5]. Since then, aptamers for very diverse targets of different molecule classes and sizes were developed. Proteins are the predominant aptamer selection targets, but aptamers are also described for larger targets like whole cells, viruses, and tissues, or smaller targets like small organic molecules [6]. The SELEX principle was modified with a lot of variations, and most of them have their own names. The process of aptamer selection with its variants is not in the focus of this review. To get an overview, the reader is referred to our former review article [2] and similar articles [710]. One of the biggest advantages of aptamers in comparison to other biological recognition elements is the possibility to develop them for toxic substances as the frequently used biological recognition elements enzymes and antibodies cannot be developed for toxic targets. Pharmaceuticals are shown to have poisonous effects at least when used in high doses. Therefore, the development of antibodies for pharmaceuticals is a difficult thing to deal with. Aptamers are described for a great variety of pharmaceuticals with medical application. Some of them are used in detection systems but are mostly utilized for the measurement in blood or other body fluids. A new application field for aptamers is the detection of pharmaceutical residues in the environment, which have to be determined in a fast and simple way. On the other hand, aptamers can be pharmaceuticals by themselves. These aptamer therapeutics are used because of the high affinities to their target and specificities comparable to those of monoclonal antibodies for therapeutical treatments [11]. The most successful therapeutic application of an aptamer has been the adaptation of an antivascular endothelial growth factor aptamer [12]. The PEGylated form of this aptamer (called pegaptanib) is used as the medicinal active component in a drug for treatment of age-related wet macular degeneration. The pharmaceutical product Macugen (pegaptanib sodium injection) from Pfizer Inc./OSI Pharmaceuticals was approved in December 2004 (USA) and January 2006 (Europe) [11, 1315]. Aptamer therapeutics will not be reviewed in this paper. The focus of our examination lies on aptamers able to bind to pharmaceuticals, which are used for human and animal treatment and can be found in surface and ground waters as well as in drinking water. Pesticides are another group of water pollutants, identified as an environmental problem much earlier than pharmaceuticals. They are not considered in this review. Pharmaceuticals in the environment Pharmaceuticals belong to the trace contaminants in water which are large in number, low in quantity, huge in interference, and high in toxicity and presenting high challenges for detection on site and in real time [16]. The enormous amount of about 95% of the pharmaceuticals administered to humans is excreted unchanged or as decomposition or conversion product in urine or stool. Additionally, the disposal of leftover pharmaceuticals via toilet or sink is still going strong. By this way, human pharmaceuticals reach the wastewater treatment plants over the path of the wastewater. Common wastewater filter technologies do not remove all of the pharmaceutical residues, which finally arrive at the surface water bodies. A total of 95% of the pharmaceuticals found in the environment derive from the treatment of humans and 5% of animals [17]. The pharmaceuticals for animal treatment often go directly to the soil with the urine of grazing animals or by fertilization using stall manure and liquid manure and drain away into the ground water or by surface runoff into conterminous water bodies. Although the pharmaceutical residues in the water cycle are mostly in the range of nanograms or micrograms per liter, the implications of this presence are mainly unknown. Anyhow, aquatic plants and animals are exposed to the pharmaceutical residues during their whole life time. Endocrine-disrupting substances in lakes and rivers, for instance, lead to feminization of male fish [18]. Drinking water is often made from ground and surface water (in Germany 76.2% ground water, 13.3% surface water, (...truncated)