Analytische Chemie
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A vast number of emerging pollutants has been detected in the environment over the last decades. Analytical methods suitable for trace analysis are needed that are desirably also fast, inexpensive and, if possible, robust and portable. Immunoanalytical methods which are available in a broad range of formats, can be profitably used here to analyze for the distribution and the trends of concentration levels of contaminants in the environment. Some of these formats are single-analyte but high-throughput methods. In order to use them wisely, indicator substances, sometimes called anthropogenic markers, should be selected and used in screening approaches. Other methods are suitable to be performed on portable instrumentation in the field (on-site) or in facilities such as wastewater treatment plants for on-line monitoring. Furthermore, there are the socalled array technologies that allow for parallel analysis of several analytes of interest (multiplexing). The microtiter-plate based ELISA (Enzyme-linked Immunosorbent Assay) is the method of choice for the analysis of a large number of samples [1]. ELISA screening data for anthropogenic markers such as the antiepileptic carbamazepine, the analgesic diclofenac, the anti-histaminic cetirizine, the steroid hormone estrone, the antimicrobial sulfamethoxazole, the stimulants caffeine and cocaine, the priority pollutant bisphenol A, and the bile acid isolithocholic acid [2] are presented. For on-site screening and monitoring, simpler formats, like mix-and-read assays, e.g. the Fluorescence Polarization Immunoassay (FPIA) or Lateral-flow Immunoassays (LFIA) are more suitable tools. Electrochemical formats run on portable devices provide additional advantages as no light source is required. Some examples are presented and discussed. The suitability of multi-analyte formats such as immunomicroarrays depends on the choice of a signal-producing system that provides small uncertainties and good reproducibility of the measurements. Biochip (“flat”) arrays read out on slide scanners and bead-based (“suspension”) arrays read out in flow cytometers are two options and show their distinct pros and cons. Altogether these approaches show the great potential immunoanalytical methods provide for the screening for environmental contaminants in the aquatic environment.
Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers.
Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater Treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension Array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer.
For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater Treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.