TRENDS

Guenther Proll Æ Jens Tschmelak Æ Guenter Gauglitz

Fully automated biosensors for water analysis

Published online: 19 November 2004

� Springer-Verlag 2004

Introduction

Clean water, its secure delivery to consumers, and pro-tection of resources are among the most important tasksfor humans in the future. To protect water resources andcontrol water quality it is necessary to develop fast,sensitive, cost-effective, and easy-to-use analytical sys-tems capable of measuring a variety of small organicpollutants in aqueous samples. This trend in science issupported world-wide, including the 5th and 6thFramework of the European Community, and has re-sulted in numerous national and international researchprojects.

The most prevalent technologies in water analysis areliquid chromatography (LC/HPLC) and gas chroma-tography (GC) in combination with different detectiontechniques. These methods are well established and lookback on decades of development and successful appli-cation but both require enrichment of water samples byseveral orders of magnitude prior to analysis. This stepin sample pre-treatment makes it difficult and ratherexpensive to develop suitable online monitoring systemsand might be the reason why only a few systems arecurrently available [1, 2].

Current developments

Some of the most promising analytical techniques,such as the enzyme linked immunosorbent assay(ELISA), are based on immunochemistry and requireneither clean-up steps nor pre-concentration. ELISA is

the most commonly used environmental immunoassaymethod [3] with a high potential for new applicationsin the future. Just as for classical analytical tech-niques, several groups are working on strategies forELISA automation [4]. To do so, problems arisingfrom the nature of the heterogeneous competitive testformat must be solved.

The use of fluorescence is still one of the most sen-sitive detection principles. Immunoassays with fluores-cence-labeled antibodies can achieve very low limits ofdetection (LOD). If combined with a suitable test format(e.g. sandwich or non-competitive) these immunoassayscan easily be carried out by flow-injection analysis (FIA)[5]. New approaches in array technology, miniaturiza-tion and transduction in sensor signals have led to a newgeneration of immunosensor systems. One of the firstportable and fully automated total-internal-reflectionfluorescence (TIRF) based biosensor systems suitablefor multi-analyte detection was developed by the groupof Ligler [6, 7]. In contrast to the well established ELISAmethod, for example, biosensors enable easy automationof immunoassays and therefore have perfect features formeeting current requirements of water-analysis systems.The key features are the non-pre-treatment strategy andthe capability of multi-analyte detection of small organicpollutants from different compound classes in a singlemeasurement. A summary of research groups currentlyusing TIRF-based biosensors for a variety of applica-tions can be found in [8]. The AWACSS system [9],a fully automated biosensor system capable of remote-control, early warning, and trend monitoring is one ofthe first analytical tools ready to be used for monitoringwater resources and for water-quality control. Thisbiosensor includes an integrated optical waveguide (IO-chip) to produce a spatially resolved pattern of mea-surement windows, each providing an evanescent field toexcite surface-bound dye-labeled antibodies. In contrastto most other TIRF biosensors for water monitoring,the AWACSS system uses the binding inhibition testformat, resulting in a sensor surface which can beregenerated 500 times without significant loss of sensi-

G. Proll (&) Æ J. Tschmelak Æ G. GauglitzInstitute of Physical and Theoretical Chemistry (IPTC),Eberhard-Karls-University of Tuebingen,Auf der Morgenstelle 8, 72076 Tuebingen, GermanyE-mail: guenther.proll@ipc.uni-tuebingen.deURL: http://barolo.ipc.uni-tuebingen.deTel.: +49-7071-2973048Fax: +49-7071-295490

Anal Bioanal Chem (2005) 381: 61–63DOI 10.1007/s00216-004-2897-2

tivity. The accurate multi-analyte analytical perfor-mance is shown in Fig. 1 as a set of six calibration curvesproduced simultaneously. Based on this calibration thesystem was tested with real water samples (tap water andsurface waters) and validated in international inter-lab-oratory tests [10]. In addition, an LOD for estrone in theppq range (180 pg L�1) was demonstrated with the to-tal-internal-reflection fluorescence (TIRF) based bio-sensor RIANA, and was achieved without pre-concentration of the samples [11].

When working with immunoassays there is alwaysthe problem of cross-reactivity between antibodies andcompounds with similar chemical structures. Methods inchemometry provide solutions which take advantage ofthis natural behavior of antibodies. Neural networksand other intelligent data-treatment strategies will beincluded to enable automated calibration and evaluationprocedures for biosensor systems, thus enabling multi-analyte detection [12]. To monitor threshold values orlimit values stipulated by regulations it is not alwaysnecessary to quantify single substances but to sensitivelydetect the sum-concentration of a class of analytes froma list of emerging substances. All this generated infor-mation should then be collected within a data formatwhich is compatible with international databases, toenhance transparency for consumers and to improve thedata situation for scientists and authorities requiring abetter knowledge of the actual quality of water re-sources, and enabling immediate response to pollutionin the future.

In the AWACSS system state-of-the-art technologyin optical sensing and engineering was combined with asoftware module enabling fully automated calibrationand determination of analyte concentrations, includingstatistical tests for accuracy and validation. Networkingcapability was also provided to enable remote controland data exchange with a newly developed AWACSS

database, via the internet, for unattended monitoringand surveillance (Fig. 2).

Outlook

Modern biosensor systems for water analysis are nolonger merely simple transducers with fluidic surround-ings—they are, in fact, high-tech products developedwithin interdisciplinary co-operation between all fieldsof science and engineering. A key task for this research ismicrofluidics. Although companies and research groupshave been working on this topic for years, further re-search is still necessary to gain a better understanding ofhow to create, for example, mixing devices or fluidicstructures for small volumes. In addition, manyimprovements in optical sensing are based on achieve-ments made in the field of technical optics and hardwaredevelopment of in-silica products for excitation lightsources (e.g. laser and LED) and detection (e.g. photodiodes and CCD) [8]. Improvements in detection can beachieved by use of CCD or CMOS cameras. With thesedevices it is possible to detect a very large number ofmeasurement spots simultaneously. Only the price ofhighly sensitive devices (e.g. cooled CCD) still limitsreasonable application.

A new trend for transducers, including waveguidetechnology, is the use of plastics instead of glass. Theadvantage is the cheap mass production of disposablechips. It is, therefore, necessary to develop new strategiesin surface chemistry that can be based on photo-linkingif covalent binding of molecules on to the surface of atransducer is necessary.

To improve existing biosensors, another key featureis the bio-molecules used as recognition elements. Ap-tamers, for example, can be produced which stronglybind to a specific target molecule [13]. Fluorescence-la-beled, these synthetic recognition elements can be used

Fig. 1 Set of calibration curves for atrazine, bisphenol A, estrone,isoproturon, sulfamethizole, and propanil which were measuredsimultaneously on a multi-analyte transducer (IO-chip). For allcompounds the calculated LOD is below 0.020 lg L�1

Fig. 2 Illustration of the capabilities of AWACSS monitoringnetworks with the capability of on-line, unattended, and centralcontrolled monitoring and surveillance

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as an alternative to antibodies in biosensor systems (e.g.AWACSS). Furthermore, the development of morestable receptors (e.g. estrogenic receptors) by use of themethods of molecular biology can lead to a new gener-ation of functional assays that not only detect the con-centration of analytes but also measure the potentialimpact of pollution on the human body [14]. This isespecially of interest if endocrine-disrupting compounds(EDC) are measured in water samples.

Several groups have demonstrated the proof-of-principal of immunochemistry-based optical sensors.The future of these biosensor systems, which weredeveloped for continuous water monitoring, stronglydepends on their market potential. With this in mind it isrelevant that by the end of 2007 EU member states haveto define surface-water-monitoring programs ready forcommencement [15].

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