Biochemical Assays

As mentioned above, on a biochemical assay, the biorecognition element has been isolated. The biorecognition element consists in a biomolecule such as an enzyme, a nuclear or membrane receptor or an antibody that recognizes selectively or specifically the analyte of interest. The mode of action of each biomolecule depends on different mechanism. In the case of enzymes, the mechanism involves the catalytic transformation of the pollutants. Regarding the nuclear receptors, their affinity versus particular endogenous and exogenous substances is exploited. For instance, the affinity of the estrogen receptor (ER) for estrogenic compounds such as estradiol, estrone and ethynylestradiol has been used to develop a variety of methods. One of the most important biorecognition elements are the antibodies. Because of the broad variety of specificities that can be achieved, several immunochemical assays have been developed for a great variety of substances, such as pharmaceuticals. However, the use of these biochemical assays for the detection of pharmaceuticals in the environment has not been frequently reported. Following we will describe some of the most frequently described biochemical assays available for the detection of pharmaceuticals with a great potential for environmental analysis. Biochemical assays based on receptors

Many biochemical processes, essential for the functioning and survival of cells (and the organism), are regulated by hormones, neurotrans-mitters, cytokines and other ''messenger'' molecules. This regulation proceeds by interaction of these naturally occurring molecules with receptors that are either embedded in the cell membrane (membrane-bound) or present in the cytoplasm (soluble receptor) or the nucleus of the cell. The membrane-bound receptors can be subdivided into G-protein coupled receptors (GPCRs), ion channels and receptors with a single transmembrane segment. Nuclear or soluble receptors are represented by the group of steroid receptors (i.e. the estrogen receptor) and the non-steroidal receptors (i.e. Vitamin D receptor) that regulate biological functions by controlling gene expression. This class of receptors consists of a DNA-binding and a ligand-binding domain.

Receptor-screening methodologies can be based on either the determination of a functional response (i.e. cell proliferation), the production of second messengers (i.e. Ca2+) or the interaction of a ligand with its receptor. While in the first two cases, we would consider those methods as bioassays, the third case can readily be considered as a biochemical assay. Moreover, it is sometimes still costly and difficult to obtain stable eukaryotic cell lines to perform these types of functional measurements, for which reason receptor biochemical assays can be contemplated as excellent alternatives. Binding of a ligand (agonist or antagonist) to its cognate receptor is the initial and indispensable step in the cascade of reactions that finally cause a pharmacological effect and many successful and widely used techniques are thus based on measuring ligand binding.

As with the well-known immunochemical assays (see below), recep-tor-ligand binding assays may be classified according to the need for separation of bound from free ligand or the detection technique. According to the first criterion, the assay types can be heterogeneous (use of a solid phase for separation) or homogeneous (no need of separation steps). Regarding detection methods, receptor assay formats usually require labelling of either the ligand or the receptor. Radio-isotopic labels such as 3H, 125I and 32P have been used (RRA, radio receptor assay; SPA, scintillation proximity assay), however because of the disadvantages of disposal of radioactive waste, costs, health hazards, the requirement for special licenses, etc., efforts have increased to develop new technologies based on either colorimetric (ELRA, enzyme-linked receptor assay), fluorescence (i.e. FRET, fluorescence resonance energy transfer; FP, fluorescent polarization, etc.) or (chemo-/bio-) luminescence detection systems. The ideal assay should be specific, sensitive, easy to perform, reliable and reproducible, unexpensive, rapid and suitable for automation. Moreover, the possibility to quantify multiple analytes in a single assay (multiplexed assays) is becoming one of the important goals in this area. For more information on these types of assays the reader is addressed to recent reviews [36].

RRA assays have been reported for the determination of benzodiazepines [37,38], neuroleptics [39,40], opioids [41], antipsychotic [42] and antihypertensive drugs [43,44]. SPA has been developed for a range of receptors including the a1- and a2-adrenergic receptors (a-AR) [45,46].

The first receptor assays that made use of fluorescence was described by McCabe et al. [47] for the benzodiazepine receptor using a fluo-rescein-labelled ligand. The significant background signal presented was reduced in the assay developed by Takeuchi et al. [48], who made use of time-resolved fluorescence (TRF), by labelling the benzodiaze-pine ligand with a europium chelate. Neuroactive drugs have been determined by means of a FRET assay through their competitive binding to the labelled human M1 muscarinic receptor (hM1-R) in the presence of a labelled antagonist [49] or also by measuring the biding to the ligand-gated ion channel GABAa receptor a1-subunit, using the same assay format [50]. The luminescent variant of FRET, where energy transfer occurs between a luminescent donor and a fluorescent acceptor, is called bioluminescence resonance energy transfer (BRET). The enzymatic oxidation of a substrate results in the emission of energy from the donor, which means that no excitation light is needed in contrast to FRET. Moreover, the enzyme reaction does not produce a background signal and the assay is therefore more sensitive than

FRET [51]. Because of the fact that there is no requirement of a light source, the instrumentation for BRET assays is simpler and cheaper [52] which makes these assays very valuable in high-throughput screening. BRET has been mainly used in protein-protein interaction research, for example in studying the b2-adrenergic/b-arrestin interaction [53] and the determination of insulin receptor activity [54,55], where the latter is governed by a conformational change in the b-subunits of the receptor, bringing them into close proximity. The FP technology has been applied to, i.e. the soluble estrogen receptor [56], the G-protein coupled delta-opioid receptor [57] and the ligand-gated ion channel serotonin 5HT3 receptor [57,58]. This receptor is involved in rapid signal transduction in the central nervous system and the peripheral nervous system. Strong interest for this receptor has been provoked by the ability of 5HT3 receptor antagonists to treat emesis caused by anticancer chemotherapy. Moreover, antagonists for this receptor show promise for the treatment of colonal dysfunction. Fluorometric microvolume assay technology (FMAT) has also been used on few cases to set up receptor assays. This technology makes use of a scanner that measures multiwell plates. It is a mix-and-measure assay where the small molecule ligand is labelled with a fluorophore and the receptor is immobilized on beads or in the bottom of special multiwell plates (either 96-, 384- or 864-well with a clear bottom and black sidewalls). The FMAT scans a 1mm2 area at the bottom of the multiwell plate where the generated images indicate the size and amount of bound fluorescence. The capillary-based scanner uses as an excitation source a Helium-Neon (He-Ne) red laser (Ex — 633 nm) and makes simultaneous detection of two independent red dye emissions, i.e. Cy5 and Cy5.5, possible via two photomultiplier tubes with bandpass filters for the respective labels (multiplexing). Multiplexing minimizes reagent consumption and increases the throughput [59]. A different format of a homogeneous bead-based assay, called Alpha-ScreenTM (Amplified Luminescence Proximity Homogeneous Assay [60]) makes use of singlet oxygen (1O2, half-life 4 ms) production on donor beads, and a chemiluminescent reaction on the acceptor beads is observed. This assay allows probing interactions over longer distances than FRET and BRET. An example of a receptor-ligand-binding assay, which made use of the AlphaScreenTM methodology, was described for the estrogen (ERJ-receptor by Rouleau et al. [61]. Flow cytometry has also been used to discriminate between agonist and antagonist binding using the solubilized ^-adrenergic receptor fused to green fluorescent protein (b2AR-GFP) [62,63].

Finally, a variety of ELRA assays have been established in parallelism with the well-known ELISAs (see below). The amplification provided by the enzyme allows reaching excellent detection limits for a variety of drugs and environmental contaminants with specific activity on the isolated protein receptor. In some examples, the detectability has been increased by substituting the chromogenic substrate by a luminescent one (ELBRA, enzyme-linked bioluminescent receptor assay). As a result of the increasing concern regarding the hormonal effects of a wide variety of pollutants (endocrine disrupter compounds, EDC), several research groups have invested great efforts on developing biochemical assays based on the use of the nuclear receptors responsible of such type of bioactivity. Steroid hormone receptors are the members of the nuclear receptors family, which are ligand-dependent transcriptional modulators. These kind of receptors can be produced by genetic engineered bacteria, for instance by fusion of glutathione-S-transferase (GST) with the D, E and F domains of native receptors in E. Coli [64]. With this technology human receptors for estrogens (hER), androgens (hAR) and progestagens (hPR) have been produced. These receptors have been used to set up ELRAs with excellent detectability limits. As an example, the androgenic receptor has been used assess binding of a variety of pesticides and industrial pollutants [65-67]. Similarly, hER has been used to detect 17^-estradiol (E2), tamoxifen, bisphenol-A and resveratrol. A detection limit of 20ng/L has been found for E2 [68-70]. Recently, an ELRA and a yeast estrogen screen (YES) assay has been applied to determine whether automobile tires immersed in fresh water can leach chemicals, which display estrogenic activity [71]. Table 2.8.2 shows some of the most frequently used biochemical assays for the detection of hormones using nuclear receptors.

Other receptor assays have been established for the determination of antibiotics. One of the most well known is based on the use of a specific ^-lactam receptor (^-StarTM). By using appropriate labels, the assay can detect a wide range of penicillins and cephalosporins with excellent detectability. For instance, the limit of detection for the determination of Penicillin G and Amoxicilin is between 2-4 ppb in milk samples [72]. The assay that is commercialized as a test strip is commonly used to determine antibiotic contamination of dairy products. However, to our knowledge the application to the analysis of environmental samples has not been described.


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