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The goal of the RIANA (River Analyser) project has been to develop a sensitive and cost-effective analytical system capable for field use and for the simultaneous detection of multiple-analyte samples in real-world water samples. Three portable prototype instruments, comprising fluid handling and signal transduction, have been manufactured and have been successfully demonstrated both in laboratory and field environments. The instrument design was a collaboration between the Gauglitz Group, Perkin Elmer GmbH, and Optoelectronics Research Centre at the University of Southhampton, with consultation from Anjou Recherche and Centro de Investigación y Desarrollo, ES. Immunoassays have been implemented for the detection of a whole range of pollutants with detection limits in a majority of cases being below 0.1 mg/L. Extensive sample pre-treatment steps required for reference techniques are not required for the RIANA device and test cycles are completed within 15 min. The device has been designed so that remote control of the instrument is amendable.
Analyte detection is based on Total Internal Reflection Fluorescence (TIRF) technology. Light from a laser diode (635nm, 12mW) is coupled into an optical transducer. The transducer is a specially cut glass slide of about 1.5 mm thickness. The laser light is guided down the transducer by total internal reflection. The transducer surface is chemically modified with analyte derivatives. Analyte-specific antibodies are labelled with a fluorescent marker (Cy5.5). Upon binding to the transducer surface the fluorescent markers are excited in the evanescent field. The emitted light is collected by means of high numerical aperture polymer fibres located opposite to the excitation spot. Before detection, pump radiation light is rejected from the fluorescence with an optical filter. Photon intensity is measured with silicon photodiodes. The device utilizes lock-in amplification. Therefore, the laser operates in pulsed mode and the diode signals are converted with an analog-to-digital board. The design allows for the simultaneous measurement of six different analyte spots. Fluid handling, transducer and detectors are fully integrated in the instrument. Measurement cycles, data acquisition, and data evaluation are fully automated and an autosampler can be easily added.
Analyte recognition is based on a binding inhibition assay. Analyte derivatives are immobilised onto the transducer surface prior to the assay. Next, analyte-specific antibodies labelled with fluorescent markers are incubated with the unknown analyte samples. After this incubation period, the device flows the analyte solution over the transducer. Analyte-specific antibodies will bind to the transducer surface according to the law of mass action. Subsequently, antibodies that are bound to analyte molecules during the incubation period will not bind to the surface. Therefore, we measure an inverse analyte signal, with samples having a low analyte concentration giving rise to a large fluorescence signal and samples with a high analyte concentration giving little fluorescence. The signal is evaluated as the difference between the signal averaged for 15 s after binding and the background signal averaged before binding. Before measuring analyte samples, the instrument is calibrated with solutions of known concentration. Integrated optical transducers have been developed to allow for efficient fluorescence excitation and for reduced sample volumes. We have shown that single transducers can be regenerated greater than 100 times. Required sample volumes are below 1 ml.
The RIANA device has proven its utility with many exciting results. RIANA has performed immunoassays on the pollutants atrazine, simazine, 2,4-dichlorophenoxyacetic acid, pentachlorophenol, paraquat and alachlor. We have shown that RIANA is also capable of several multi-component analyses. In addition, RIANA was one of the few biosensors to successfully quantify multi-analyte real-world water samples during field-tests in Berlin at the 1st and 2nd European Technical Meeting of Biosensors for Environmental Monitoring.
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