Cranfield University, UK

SENSPOL SURVEY OF SENSOR CAPABILITIES

Sensors for the Abatement of Water Pollution from Contaminated Land, Landfills and Sediment

SENSPOL SURVEY OF SENSOR CAPABILITIES

Sensors for the Abatement of Water Pollution from Contaminated Land, Landfills and Sediment

A Sesay, JD Newman, RO Kadara and SJ Alcock

Cranfield University, UK

SENSPOL is supported by the EC Environment and Sustainable Development Programme (DG Research, Key Action "Management and Quality of Water”) contract EVK1-CT-1999-20001.

For more information about SENSPOL please see: http://www.cranfield.ac.uk/biotech/senspol.htm

LEGAL NOTICE

Neither Cranfield University, the European Commission nor any person acting on behalf of Cranfield University or the European Commission is responsible for the use which might be made of the following information

Cranfield University Press, Cranfield University, Cranfield, Bedfordshire, MK43 OAL, UK

ISBN 1 871315 85 9

Executive Summary I

1 Chemical Sensors and Biosensors 1 1.1 Introduction 1 1.2 Chemical sensors 1 1.2.1 Chromatographic and Spectrometric Sensors 1 1.2.2 Electrochemical Sensors 2 1.2.3 Mass Sensors 2 1.2.4 Optical Sensors 2 1.3 Biosensors 2 1.3.1 Assembling the picture 4 1.4 Sensor development and integration 4 1.4.1 Sensor arrays: looking at the bigger picture 5 1.5 Supporting technologies 5 1.5.1 Membranes and immobilisation 5 1.5.2 Fabrication techniques 5 1.6 Improving performance 6 1.6.1 Sensitivity 6 1.6.2 Stability 7 1.6.3 Selectivity 7 1.7 Target organic and inorganic compounds 8

2 Hydrocarbon Detection Devices 9 2.1 Introduction 9 2.2 Sampling preparation and extraction devices 10 2.2.1 Cranfield University 10 2.2.2 Exposmeter AB 10 2.3 Sensor device: prototype and commercially available sensors 10 2.3.1 Bordeaux 3 University 10 2.3.2 Fugro Milieu Consult 10 2.3.3 IIQAB-CSIC 11 2.3.4 VEGAS 11 2.3.5 RS Dynamic 11 2.3.6 Sensor Tech 11 2.3.7 UFZ 12 2.3.8 Universidad Complutense de Madrid 12 2.3.9 University of Tuebingen 12 2.4 Conclusions 13

3 Heavy and Trace Metal Detection Sensors and Devices 14 3.1 Introduction 14 3.2 Selected Metal Ion Sensing Systems 15 3.2.1 Aboatox Oy (Finland) 15 3.2.2 Ben-Gurion University of the Negev (Israel) 15 3.2.3 Cranfield University (UK) 15 3.2.4 Bohrlocmessungen-dr. Buckup (DBM) (Germany) 15 3.2.5 NITON Europe GMBH (Germany) 15 3.2.6 NMRC (Ireland) 16 3.2.7 University of Coimbria 16 3.2.8 University of Neuchatel/University of Geneva (Switzerland) 16 3.3.9 Vlaa mse Instelling voor Technologisch Onderzoek (VITO) (Belguim) 16 3.3 Conclusions 17

4 Toxicity and Genotoxicity Testing Devices 18 4.1 Introduction 18 4.2 Selected Toxicity Sensors 19 4.2.1 Coventry University (England) 19 4.2.2 Cybersense Biosystems Ltd (England) 19 4.2.3 Delta Consult B.V. (The Netherlands) 19 4.2.4 DLR (Germany) 19 4.2.5 Ecole Nationale Supériere des Mines de St Etienne (France) 19 4.2.6 Genotronix Limited (England) 20 4.2.7 University of Aberdeen / Remedios Ltd (Scotland) 20 4.2.8 University of Nantes (France) 20 4.3 Conclusions 20

5 Sensor Devices for other targets: general parameters, gases, anionic, cationic and phenolic compounds 21 5.1 Introduction 21 5.2 Selected Sensing Devices 21 5.2.1 Barcelona Institute of Microelectronics (IMB-CNB) (Spain) 21 5.2.2 Coventry University (UK) 22 5.2.3 Cranfield University (UK) 22 5.2.4 University of Ulster (UK) 22 5.3 Conclusions 22

6 Bottlenecks and Bridging the Gap 23 6.1 Bottlenecks in sensor technology 23 6.2 Bridging the gap between sensor developers and end-users 24

7 Conclusions and Overview 25

8 Bibliography 28

9 References 30

Appendices 32

1 Questionnaire Bridging gaps between sensor developers and users: sensor capability study 33

2 Questionnaire responses 38 SENSPOL Survey of Sensor Capabilities

Executive Summary

This report is based on the findings arising from a questionnaire designed specifically to identify current sensor development research and the capability of the devices in monitoring groundwater, sediment and contaminated land pollution. The questionnaire was distributed in November 2002 amongst the European research institutions, universities, organisations and companies that are members of the SENSPOL network. The questionnaire was well received, resulting in 48 returned questionnaires by December 2002. The present report analyses all responses from the questionnaire.

The report focuses on:

• heavy metals with specific attention to mercury-related problems; • aromatics and non-chlorinated hydrocarbons; • chlorinated volatile, semi-volatile chlorinated compounds with particular attention to DNAPLs (Dense Non-aqueous Phase Liquids); • General pollutant toxicity testing. • Other targets including: pH, conductivity and redox potential; phenolic compounds; anionic and cationic analytes; nitrogen dioxide gas.

The report features 42 sensors, biosensors and detection kits within the 32 included entries (7 Companies, 15 Universities and 10 Research Institutes), spanning 13 European countries. Many of the sensors featured are biosensors or immunoassay based platforms. However, there are other monitoring systems included that are able to measure the specific analytes addressed by this report.

The report is introduced with a brief account of sensors, instruments and relevant integrated technologies. The compiled inventory study goes on to give an overview of sensors that can potentially be applied to environmental samples. The report contains information on the status of development, the sensor characteristics in terms of sensitivity, detection levels, selectivity, usability and other relevant information.

Extra information was asked of the developer concerning their opinion of how they felt their sensor's development had been impeded and whether there was anything that may have helped alleviate this problem. As much sensor development is co-partnered questions were asked about collaboration and commercial backing. Finally, as the aim of the report is to facilitate information transfer an order to close the gap between research development and end users, the responder was asked how they felt this could be addressed.

A report derived from the present full report has been supplied to the European Network on Industrially Contaminated Land (NICOLE).

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Conclusions

The response to the sensor survey was very good. Of the 32 selected developers featured in the report 7 are companies, 15 were from universities and 10 from research institutes all coming from 13 European countries. The report features 42 sensors, biosensor and detection kits able to detect a wide range of analytes including DNAPLs, LNAPLs, aromatic, and halogenated compounds, heavy metals, toxicity, genotoxicity, gases, and anionic, cationic and phenolic compounds. Also general parameters like pH, conductivity and redox potential can be measured.

The targeted analytes and parameters were divided into four categories: hydrocarbons; heavy and trace metals; toxicity testing; and other targets (general parameters, gases, anionic, cationic and phenolic compounds). The following are the main extrapolated findings.

Sample extraction devices

Detection of hydrocarbons in the field for environmental samples often requires sample extraction and preparation prior to measuring, especially in soil and sediment matrices. The report features two different sample extraction field based approaches:

Cranfield University has developed a field based Supercritical Fluid Extraction (SFE) device and protocol that can be employed in the field for PAHs but can be adapted to other analytes. Exposmeter have developed a semi-permeable membrane that can accumulate the whole range of target analytes of interest such as DNAPLs, LNAPLs, aromatic, chlorinated and PAHs in environmental samples. Both devices can be used in conjunction with a variety of commercially available detection systems.

Hydrocarbons

Devices have been selected that target priority analytes such as DNAPLs, LNAPLs, aromatics and halogenated compounds.

Out of the 15 featured sensors measuring hydrocarbons, six are available as fully commercialised analytical devices or demonstration units. LNAPLs, DNAPLs and PAHs can be detected by six sensors, of which four are commercially available. The RIANA device is a fluorescence-based immunoassay biosensor that is adaptable to many analytes and has demonstration units commercially available. Detection levels of these units are in the sub-ppb range.

Heavy metals and trace metal detection

The sensors described cover the full spectrum of heavy and trace metals that are commonly measured in environmental samples.

Of the 14 featured sensors that can detect metals, there are four devices, from Niton Europe GmbH, the universities of Neuchatel and Geneva and DBM that are fully

II SENSPOL Survey of Sensor Capabilities developed and commercially available. Heavy metals in environmental samples have sub-ppb detection requirements. The devices developed by Aboatox Oy, Ben Gurion University and VITO are all bacteria-based detection systems. The sensors utilise genetically engineered strains of a bacterium. The Ben Gurion device is easily deployable in the field and can also detect genotoxicity levels in the sample. The two (bacteria-based) systems from Aboatox Oy and VITO are commercially available.

Toxicity and genotoxicity

Bio-accumulation and high levels of persistent organic pollutants and heavy metals in the environment can have a toxic effect on many simple organisms, invertebrates and whole cells. By utilizing this phenomenon toxicity test kits have been developed that are able to give an early warning and semi-quantitative detection in environmental samples.

Only two of eight reported toxicity test devices (the Mosselmonitor  by Delta Consult and that produced by the University of Aberdeen/ Remedios Ltd) are available commercially. However, Cybersense Biosystem Ltd has a portable toxicity testing system that will be available commercially in the near future. All of the test kits and biosensors are able to detect toxicity in environmental samples.

Other targets: general parameters, gases, anionic, cationic and phenolic compounds

Sensor devices able to measure general parameters like pH, conductivity, and redox potential as well as analytes like calcium, potassium, nitrate, nitrogen dioxide, chlorine and phenolic compounds are featured here.

All but one of the sensing devices is based on the use of electrochemical principles (IMB-CNM, Cranfield University and University of Ulster) while the one that is not, is based on an optical principle (Coventry University). The sensor devices can be used in variety of sample matrices including freshwaters, gas samples, surface water, groundwater, soils and pore water in clay materials.

Bottlenecks in sensor technology

Bottlenecks that impede sensor technology can be categorised into three sections:

• First stage: sensor and sample experimentation conditions. • Middle stage: design and conversion of a crude laboratory-based device into a prototype demonstration unit. • Last stage: final design, production and sensor marketing.

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Bridging the gap between sensor developers and end-users

There were a number of areas where developers felt improvements could be made to alleviate some of the problems they encountered during device development and commercialisation. These included:

• Greater communication between instrument developers and end-users. • Information transfer and communication links between governmental regulators and technology developers (heightening awareness of available technologies). • Closer collaboration and partnerships with other developers and specialists.

Suggestions that could address these problems were:

• Regular workshops, symposia and networking meetings bringing end-users, government, regulators and developers together for information transfer and networking.

• Funding programmes and collaboration incentives for sensor development, based on end-user needs.

• Making funding application processes more efficient and shorter, with administrative benchmarks and more transparent selection procedures.

Several other devices that had not featured in the questionnaire replies were demonstrated in the SENSPOL Technical Meetings in Sevilla, Spain, in November 2002 and in Koblenz, Germany, in October 2003. These devices are briefly mentioned in the Conclusions and Overview section and some of them can be viewed on DVD/video.

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1. Chemical Sensors and Biosensors

1.1 Introduction

In recent years huge advances in scientific research combining the extensive knowledge in electrochemistry, biochemistry, physics, electronics (particularly silicon technology) and software design has made possible the development of highly specific, sensitive and accurate test kits, chemical sensors and biosensors. These devices offer many promising solutions to on-site, real-time monitoring of target analytes and significant commercial opportunities in the environment, biomedical sciences, veterinary sciences, industrial processing, defence and security.

The sensors and test kits featured in this report have been selected from the responses to the sensor questionnaire that was sent to all developers in the SENSPOL network. European organisations, companies, research institutes and universities have sensing devices at different stages of development that range from first stage prototype to commercially available devices. A summary of each entry is given in the following chapters (whilst detailed individual entries are provided in Appendix 2).

1.2 Chemical sensors

There are many well-developed sensors (e.g. thermometers) that can measure external environmental parameters exploiting the inherent physical or chemical properties of materials. Chemical sensors often require a transducer integrated with a chemically sensitive material. The transducer translates the physico-chemical change of this material into a recognisable physical parameter that can in turn be amplified (if necessary) and read (either as an analogue or digital signal).

A significant number of modern chemical sensors are based on modified metal oxide semi-conductor field effect transistor (MOSFET) devices, often in ion-selective (ISFET) formats. The chemical sensors shown in this report can be categorised into four sections: chromatographic and spectroscopic sensors; electrochemical sensors (of which ISFETs are a type); mass sensors and optical sensors. The following provides a short description of each:

1.2.1 Chromatographic and Spectrometric Devices

Chromatography is a method that is able to separate and analyse complex mixtures of volatile organic and inorganic compounds. It is highly efficient at separating complex mixtures into individual components via a separation column. A detector positioned at the end of the column can then be used to quantify the concentrations of individual components. The liquid or gaseous sample can then be injected into a spectrometer and ionised by laser or radioactive source, resulting in a positively or negatively charged species, which can be accelerated over a short distance and the time of flight determined.

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1.2.2 Electrochemical Sensors

Electrochemical sensors can be categorised into three groups:

• Potentiometric (measurement of voltage at zero current) • Amperometric (measurement of current at a fixed applied voltage) • Conductometic (measurement of conductivity using an AC approach)

Sensors of this type are able to measure ions and molecules directly, using chemically-sensitive layers (potentiometry), but may use membranes to improve selectivity (amperometry).

1.2.3 Mass Sensors

These sensors are often based on acoustic wave devices that vibrate at a certain frequency. When adapted with a chemically-absorbent film on a piezoelectric substrate the sensor can be used to measure bound material, since it changes the mass on the surface and, hence, the resonant frequency. In solution, the behaviour is slightly more complex. In this case, the sensor is sensitive to the viscoelasticity and mass of the thin film on the surface, which also translates to changes in the vibrational frequency.

1.2.4 Optical Sensors

These sensors are often found in a fibre optic arrangement and rely on changes in the various light properties to detect the analyte of interest. Light is generated by a suitable source (typically an LED or laser) and is sent through the fibre. The reflected light can then be measured by a photodetector. There are three general classes of fibre optic sensors:

• Passive spectroscopic • Chemically interacting (thin film deposited on the tip of the fibre) • Approaches requiring reagent injection near the sensor

1.3 Biosensors

Biosensors can be considered to be a sub-section of chemical sensors and can be defined as chemical sensors that use active, biologically-derived components integrated with a suitable transducer.

There are numerous components to any biosensor configuration. Over the years, a great many combinations have been proposed and demonstrated, though far fewer have been commercial successes. A generalised schematic of a biosensor is shown in Figure 1.1. The basic principle is to convert a biologically induced recognition event into a usable signal. In order to achieve this, a transducer is used to convert the (bio) chemical signal into an electronic one, which can be processed in some way, usually with a microprocessor.

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For the purposes of this report a biosensor is defined as:

a compact analytical device incorporating a biological or biologically derived sensing element either integrated within or intimately associated with a physicochemical transducer. The usual aim of a biosensor is to produce either discrete or continuous digital electronic signals, which are proportional to a single analyte or a related group of analytes (Turner et. al., 1987).

Figure 1.1: Generalised Schematic of a Biosensor

The schematic representation shows, in very simple terms, the molecular recognition achieved by a lock and key mechanism. When biological components interact there may be the generation of ions, electrons, gases, heat, a mass in a localised vicinity, or a change in the way in which light interacts in the material close to this layer. These discreet changes can be monitored and converted into digital signals by the transducer used and amplified and displayed computationally in a suitable user-friendly form.

There are many different transducers, which are currently being applied in biosensors, as shown in Table 1.1.

Table1.1 : Examples of Transducers used in Biosensor Construction

Transducer Examples Electrochemical Clark Electrode; Mediated Electrodes; Ion-Selective Electrodes (ISEs); Field-Effect Transistor (FET) based Devices; Light Addressable Potentiometer Sensors (LAPS) Optical Photodiodes; Waveguide Systems; Integrated Optical Devices Piezoelectric Quartz Crystals; Surface Acoustic Wave (SAW) Devices Calorimetric Thermistor; Thermopile Magnetic Bead-based Devices

For further explanation of the transduction principles involved, the interested reader could refer to any of the excellent texts referred to in the Bibliography Section at the end of this report. Several shorter descriptions have also been written, including an article by Newman and Turner (1992).

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1.3.1 Assembling the picture

One of the chief attractions of biosensors is the remarkable specificity that their biological component confers on them. Enzymes are the most commonly used reagents, but many other biologicals and biomimics have also been featured. These include:

• Antibodies • Whole cells including microbial, plant and animal cells • Sub-cellular organelles • Tissue slices • Lectins • Numerous synthetic molecules with affinity or catalytic properties similar to biologicals, extending to those obtained through parallel synthesis and imprinted polymers

Since biological components offer such exquisite selectivity (and often sensitivity), why are synthetic molecules so attractive? The answer is frequently that biological reagents are often poorly stable outside of their normal environment. Thermal stability is usually particularly poor, resulting in short lifetimes and limited ranges of application. In addition, pH sensitivity is often troublesome, as is the need for cofactors and other reagents in some instances. The aim of the biomimicry approach is to utilise the best features of the biological reagent (sensitivity, specificity etc.) in a more stable matrix.

1.4 Sensor Development and Integration

Biosensor technologists strive for the simplest possible solution to measurement in complex matrices. While notable success has been achieved with individual sensors, pragmatic solutions to many problems involve the construction of a sensor system in which the carefully optimised performance of the sensor is supported by associated electronics, fluidics and separation technology.

There are increasing demands for a systems-orientated approach in other sectors; and environmental monitoring places demands on sensor technology that, in many cases, are unlikely to be met by isolated sensors.

The sensor/sampling system bio-interface is a key target for further investigation, involving tools such as evanescent wave techniques, and atomic force and electrochemical microscopy to aid further understanding of interactions between biological molecules or their mimics and surfaces.

Immunosensors offer a general example where microseparations, using for example immuno-chromatographic methods, can be coupled with electrochemical or optical detectors to yield simple dipstick style devices combining the speed and convenience of sensors with the specificity and sensitivity of immunoassays. The advent of micro- machining makes these and other hyphenated techniques amenable to such a high degree of miniaturisation that the distinction between sensor and analytical instrument becomes hazy.

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1.4.1 Sensor Arrays – Looking at the Bigger Picture

The success of single analyte sensors has been followed by the formulation of arrays of sensors to offer menus relevant to particular locations or situations. The most obvious example is in environmental analysis where there is a niche for hand-held instruments to provide industries with real-time information on the concentration of major pollutant parameters at source. These features are well established in clinical field and soon to be a reality in the environmental field.

1.5 Supporting Technologies

The basic components of the biosensor, as outlined above, are only the start of the story. In order to obtain functional devices, which can be manufactured within the necessary performance and cost constraints, numerous other components and technologies are needed.

1.5.1 Membranes and Immobilisation

The very first biosensors relied on membranes for their functionality. These were based on the Clark oxygen electrode, which contains a gas-permeable membrane, which allows oxygen to pass, whilst excluding undesirable, interfering species. Many other membranes have been used and these have a wide variety of purposes. They may be used to retain the biological component, whilst allowing the analyte to pass. Another useful function is their ability to extend the (linear) range of a biosensor by acting as a mass transport barrier (White et al., 1995).

1.5.2 Fabrication Techniques

It has long been realised that advanced fabrication techniques are a key to the successful development of commercially viable biosensors in many applications (Newman, 1998). Fortunately, many technologies have been developed for other applications, such as within the microelectronics industry, and are therefore available with much greater reliability and at a much lower cost than would otherwise be the case, although they obviously require certain modifications and considerable development.

Screen-Printing Screen-printing is a thick-film process which has been used for many years in artistic applications and, more recently, for the production of miniature, robust and cheap electronic circuits. The main developments, from a biosensor viewpoint, have involved ink formulation. There is now a wide array of inks suitable for producing biosensors. Most of the applications to date have involved electrochemical devices, but the technique is applicable to the production of any planar device.

Since the technique has been developed for mass production, it is possible to produce very large numbers of reproducible, inexpensive devices at high speed. The process has been one of the major reasons for the commercial success of many biosensors and

5 SENSPOL Survey of Sensor Capabilities is the process by which MediSense (now Abbott) produce over 1 billion biosensor strips annually.

Photolithography Photolithography is a technique used to define the shape of micromachined structures. It is essentially the same as that used in the microelectronics industry. Silicon wafers coated with an oxide layer are often used. It is desired that some of the oxide is selectively removed so that it only remains in particular areas on the silicon wafer.

Firstly a mask is produced. This is often a chromium pattern on a glass plate. The wafer is then coated with a polymer, which is sensitive to ultraviolet light, called a photoresist. Ultraviolet light is then shone through the mask onto the photoresist, which transfers the pattern on the mask to the photoresist layer. There are two types of photoresist, termed positive and negative. Where the ultraviolet light strikes the positive resist it weakens the polymer, so that when the image is developed the resist is washed away, transferring a positive image of the mask to the resist layer. The opposite occurs with negative resist, so that when developed the resist that was not exposed is washed away. Chemical etching is used to remove the oxide where it is exposed through the openings in the resist. Finally the resist is removed leaving the patterned oxide.

Liquid Handling Techniques The ability to handle small volumes of liquids with high precision is a key area in the development of novel next generation biosensors. As devices become smaller and more sophisticated, it becomes increasingly difficult to handle the analytical reagents involved in production. Some of the latest advances in transducer design, for example, make the production of 1 million measurement points on a 1 cm2 chip a possibility. The most difficult aspect of the production of these devices is, currently, incorporating the biological reagents onto the surface of such arrays.

1.6 Improving Performance

Improvements in the performance of analytical devices are a continuing theme in all areas of their application. Legislators, particularly in environmental applications, change consent levels, often based on what it is possible to detect. The development community itself continuously pushes the boundaries of what is possible.

1.6.1 Sensitivity

Environmentalists have an interest in generally increased sensitivity and limits of detection for a range of analytes. While today’s demands for precision may be modest in these respects, few would contest the longer-term benefits of reliable detection of trace amounts of pollutants or indicators. With the advent of atomic force microscopy we can consider single molecule detection in the research laboratory, but great strides have also been made with conventional sensors. Enzyme electrodes have been designed which pre-concentrate the analyte of interest (Saini and Turner, 1995).

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Advances are not limited to the liquid phase. For example, a gas-phase micro biosensor for phenol, in which polyphenol oxidase was immobilised in a glycerol gel on an interdigitated microelectrode array (Dennison et. al. 1995) has been reported, where phenol vapour partitioned directly into the gel and was oxidised to quinone. Signal amplification was enhanced by redox recycling of the quinone/catechol couple resulting in a sensor able to measure 30 ppb phenol. Detection limits of parts per trillion for volatile organic carbons are feasible with this type of approach.

Ultra-low detection limits are achievable with many affinity sensors and electrochemical detection may be readily integrated with chromatographic techniques to yield user-friendly devices (Alcock et al., 1994), an approach which overcomes the need for multiple sample manipulation steps, which was a major drawback of many early sensors of this type. In an alternative approach, double-stranded DNA may be used as a receptor element. “Sandwich”-type biosensors based on liquid-crystalline dispersions formed from DNA-polycation complexes may find application in the determination of a range of compounds and physical factors that affect the ability of a given polycation molecule to maintain intermolecular crosslinks between neighbouring DNA molecules (Skuridin et al., 1996).

1.6.2 Stability

Arguably the most obvious disadvantage of using complex biological molecules in sensors is their inherent instability. Many strategies may be employed to restrain or modify the structure of biological receptors to enhance their longevity. One way which has been demonstrated as a means of stabilisation is to use sol gels as an immobilisation matrix (Psoma and Turner, 1994).

Some desirable activities, however, remain beyond the reach of current technology. Methane monooxygenase is one such case where, despite reports of enhanced stability in the literature, the demands of hydrocarbon detection require stability far beyond that exhibited by the enzyme. In these cases it is valuable to resort to biomimicry to retain the essence of the biocatalytic activity, but to house this within a smaller and more robust structure. For example, a simple and rapid method for quantifying a range of toxic organohalides based on their electrocatalytic reaction with a metalloporphyrin catalyst has been demonstrated. This approach can be used to measure lindane and carbon tetrachloride (representative of haloalkane compounds), perchloroethylene (a typical haloalkene), 2,4-D and pentachlorophenol (aromatics) and the insecticide DDT (Dobson et al., 1996).

1.6.3 Selectivity

Improvement in the selectivity of biosensors may be sought at two levels; the interface between the transducer and the biological receptor may be made more exclusive thus reducing interference, and new receptors can be developed with improved or new affinities.

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1.7 Target Organic and Inorganic Compounds

The report focuses on heavy metals with specific attention to:

• heavy metals especially mercury-related problems; • aromatics and non-chlorinated hydrocarbons (Polyaromatic Hydrocarbons (PAHs) and Volatile Organic Compounds (VOCs) • volatile, semi-volatile chlorinated compounds with particular attention to DNAPLs (Dense Non-aqueous Phase Liquids) and toxicity testing of pollutants.

Non aqueous phase liquids (NAPLs) are a wide group of hydrocarbon compounds that are not readily dissolved in water and can exist as a separate phase for extended periods of time. They can be subdivided into two groups:

• Lighter than water (LNAPLs) • Denser than water (DNAPLs)

Examples of these are given in table 1.2.

Table 1.2: Common compunds classified as LNAPLs and DNAPLS

LNAPLs DNAPLs Hydrocarbon fuels Chlorinated hydrocarbons Petrol 1.1.1 trichloroethane Heating oil Chlopheneols Kerosene Chlorobenzenes Jet fuel Tetrachoroethylene Aviation gas PCBs

For the purpose of this report the target analytes were divided into four categories: hydrocarbons; heavy and trace metals; toxicity testing; and other targets (general parameters, gases, anionic, cationic and phenolic compounds).

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2. Hydrocarbons Detection Devices

2.1 Introduction

This chapter takes a look at the current sensor technologies and devices designed to detect hydrocarbon-based compounds. This is a broad category but an attempt has been made to select those devices that target particularly for DNAPLs, NAPLs, aromatics and halogenated compounds. There are some general devices included that are able to be easily converted to include those analytes that are of interest in the realm of this report. Table (2.1) gives an outline of the developers and sensors with the range of analytes that can be detected. (Further information on individual sensor entries can be found in Appendix 2, and are cross-referenced in table (2.1).

Table 2.1: List of hydrocarbon sensor devices and their target analytes.

Hydrocarbons

Organisation Name Sensor Device (PAH) Volatile LNAPL General Phenolic DNAPL/ Aromatics Aromatics Compounds Compounds Halogenated Page

Bordeaux 3 University CO2 based sensor X 39

Cranfield University Field based (SFE) soil preparation X 42

Exposmeter Exposmeter-liphophlic X X X X 47

Membrane Interface Probe X X X 51 Fugro Milieu Consult Rapid Optical Screening Tool (ROST) X X X 54

IIQAB-CSIC Portable phenolic biosensor X X 58

Fluorimetry based sensor X 63 Institute of Hydraulic Optical fibre based sensor 65 Engineering- VEGAS X X Acoustic based sensor X X X 66

RS DYNAMICS Ltd Ecoprobe 5 X X 70

Sensor Tech Ltd Potentiometric biosensor X 74 UFZ Centre for Environmental FIA enzyme sensor X 78 Research Universidad Complutense de Mardrid/ Gruppo OPTOSEN X 82 Iinterlab S.A RIANA X 91 University of Tuebingen AWACSS X 96

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2.2 Sample preparation and extraction devices

One of the main problems with detecting hydrocarbons in the field for environmental samples is the need for sample extraction and preparation prior to measuring, especially in soil and sediment matrices. Laboratory extraction techniques are often time consuming and samples cannot be measured on site. Below are two different field based approaches that address this problem.

2.2.1 Cranfield University (UK)

The University has developed a field based Supercritical Fluid Extraction (SFE) device and protocol that can be employed in the field. Used in conjunction with a commercially available Rapid Assay Kit, PAHs can be measured with detection levels of 0.5 to 100ppb. The SFE Extraction system is adaptable to other analytes, and detection limits would depend on the analyte detection system.

2.2.2 Exposmeter AB (Sweden)

Exposmeter is an RandD Company focused on sampling and analysing persistent organic pollutants, metals and pesticides in a whole range of environmental samples. They have developed a semi-permeable membrane that can accumulate the whole range of target analytes of interest such as DNAPLs, LNAPLs, aromatic, chlorinated and PAHs in environmental samples. The membrane device acts as a bioaccumulative filter. It can be used in conjunction with a variety of detection systems.

2.3 Sensor Devices: Prototypes and Commercially Available Sensors

2.3.1 Bordeaux 3 University

The University has developed a sensor system that directly measures biodegradation rate in aquifers. The sensor can detect all degradable organics in groundwater. It will detect the breakdown of hydrocarbons, but not selectively. The measurements are taken in situ as a continuous measurement. Detection levels are degradation of 1mg/l/day. The sensor system is currently in prototype form and is being field-tested.

2.3.2 Fugro Milieu Consult (The Netherlands)

Fugro is a large consultancy firm specialising in geotechnical and environmental soil investigations. The consultancy has two sensor devices, one is able to detect VOCs and the other is able to detect DNAPLs and LNAPLS. The Membrane Interface Probe (MIP) system is integrated in a Cone Penetration Testing (CPT) System. The system works in situ and gives a continuous measurement in soil, sediment and groundwater. Detection limits are in the order of 100ppb. Depending on the detector used, of which there are three available, it can detect three different groups of compounds: (1) all hydrocarbons, (2) all non-saturated hydrocarbons and (3) all hydrocarbons containing chlorine. The Rapid Optical Screening Tool (ROST) can detect all mineral oil products and PAHs as the detection is based on fluorescence

10 SENSPOL Survey of Sensor Capabilities patterns of the PAHs that are present in most oil products. The sensor works in situ and in all phases (soils, sediments, surface and groundwater). The system does not detect exact concentration but is able to map the plume thickness of NAPL or DNAPL in the field. Both systems are commercially available via Fugro and require a skilled operator and experienced personnel for treatment and interpretation of the data.

2.3.3 IIQAB-CSIC (Spain)

CSIC is a public research organisation that is based in an institute that deals with investigations in environmental chemistry. They have developed a portable amperometric biosensor that works with screen-printed electrodes. Currently, the technology has been used to detect pesticides in different complex aqueous samples. The sensor can be adapted to measure phenolic compounds as its detection system is based on enzyme activity. The sensor is in prototype stage and has the ability to detect down to 1µg/ml. It requires minimally skilled personnel and has repeatability error of 5-10%.

2.3.4 VEGAS, Institute of Hydraulics Engineering, University of Stuttgart (Germany)

VEGAS is a research facility for subsurface remediation located at the University of Stuttgart and funded by governmental bodies. The facility has three sensors, that between them can detect a range of hydrocarbons. The first is a fluorimetry-based system that can detect PAHs in the ground with a detection limit of 1ppb. The second sensor is an optical based sensor that can detect DNAPLs and LNAPLs by indicating if they are present or not present in soils and groundwater samples. This is performed as a continuous measurement. The third is an acoustic based sensor that is able to detect DNAPLs, BTEX and chlorinated compounds and distinguished between each contaminant group with detection levels around 100-30000ppm. The sensor can be adapted to measure volatile components. The first two sensors are commercially available prototypes, with the third prototype shortly to be available. Unskilled personnel can use all of the three sensors. No sample preparation is required as it measures in the gaseous phase. The sensors are currently being tested in the field.

2.3.5 RS Dynamics (Czech Republic)

RS Dynamics are a research and development group based in Prague that focus on environmental instrumentation. They have developed the Ecoprobe 5 which is a commercially available device that can measure LNAPL, DNAPL and PAH to detection levels as low as 0.1ppb in soil, gas and atmospheric samples (e.g. soil gas). With minimal training, the instrument can be used by an unskilled person. They are currently trying to market their product more widely in central Europe.

2.3.6 Sensor Tech Ltd (England)

Sensor Tech Ltd are a small start up company that have developed a potentiometric biosensor based on screen-printed electrodes with a conjugated conducting polymer, utilizing enzyme activity as a recognition platform. This allows for all analytes which currently or potentially can be detected by enzyme-linked assays to be measured. The sample is required in free aqueous phase and measurements can be taken in matrices

11 SENSPOL Survey of Sensor Capabilities such as soil, oil, and water. For environmental samples detection levels are often down to the ppb range. The electrodes are multi-arrays, therefore many analytes can be measured simultaneously. The sensor is currently being developed into a commercial system for environmental monitoring of persistent organic pollutants and it is hoped that the system will be available for validation by the end of 2003.

2.3.7 UFZ Centre for Environmental Research (Germany)

The UFZ is a research centre focusing on remediation of contaminated environments. They have developed an amperometric modified sensor that is currently able to measure phenolic compounds in surface, ground water and wastewater. Sample handling is automated with detection levels of 2µg/l phenol. The sensor is still a prototype and UFZ are currently working towards commercialisation of the system.

2.3.8 Universidad Complutense de Mardrid / Grupo Interlab S.A “(SPAIN)

The University and Grupo Interlab have collaborated in developing the OPTOSEN system, which is a fiber optic chemical sensor. It is an environmental/industrial sensing instrument capable of simultaneous monitoring of several parameters that are relevant to controlling water, air and/or soil quality. Dissolved hydrocarbon is one of the eight parameters. Sample handling is automated with detection measurement range of hydrocarbons being 5-15000ppm (in water). The system is commercially available from Grupo Interlab and can be used by unskilled personnel with basic initial training.

2.3.9 University of Tuebingen

The University has a long history in developing optical based immunoassay techniques for detection of analytes in environmental samples. They have two systems, the River Analyser (RIANA) and the automated water analyser computer supported system (AWACSS), which are both based on a fluoresence optical system (TIRF). The sensor is commercially available as a demonstration prototype and can be adapted to measure any analytes that can elicit an immunogenic response. It has been demonstrated in the field and was able to quantify multi-analyte concentrations in real samples with detection levels of below 0.1µg/l. Usability of the sensor requires minimal training. The second system prototype is currently being developed with the hope that it will be operational in 2003. The AWACSS will analyse multiple analytes, one at a time in a quasi-continuous way with each measurement taking around fifteen minutes. Detection levels are similar to the RIANA system.

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2.4 Conclusions

From the featured organisations, research institutes and universities, the state of development and availability of devices or sensor protocols for the detection of hydrocarbons for environmental samples ranges from lab-based prototype to fully commercialised devices. Some of the main points are:

• Cranfield University and Exposmeter have both developed field extraction systems for initial preparation of samples for the measurements of PAHs and LNAPLs/DNAPLs. These systems can potentially be used with other detection system for the measurement of specific analytes.

• Bordeaux 3 University has developed a sensor that detects the degradation of all organic materials in groundwater, using a CO2 sensor. It will detect the breakdown of hydrocarbons, but not selectively.

• LNAPLs and DNAPLs and PAHs can be detected by six featured devices. Three of these, one from RS Dynamics and two from Fugro Milieu Consult, are commercially available devices. Another three, from VEGAS, are commercially available evaluation prototypes. Detection ranges vary from total hydrocarbons in sample present/not present, to 0.1ppb.

• PAH sensors have been developed by two research institutes: IIQAB-CSIC and VEGAS. These sensors are both at a prototype stage. However, the VEGAS system is available for evaluation. Their detection levels are in the ppb range.

• The four systems from Sensor Tech, UFZ and the University of Tuebingen have the ability to be adaptable in measuring a range of analytes, as they are based on immunoassay / enzyme linked bioassays for analyte detection. The RIANA instrument from the University of Tuebingen is a fully developed prototype available for evaluation.

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3. Heavy and trace metal detection sensors and devices

3.1 Introduction

The following chapter features current sensor technology able to detect metal ions in environmental samples. Most of the detection systems are based on electrochemical electrode type probes. However, other transducers such as radiation and organism based recognition systems are also featured. The sensors cover the full spectrum of heavy metals and trace metals that can be measured in environmental samples. Table 3.1. shows a list of sensors and analytes of each device.

Table 3.1 List of heavy and trace metal sensors and devices and their target analytes

TRACE METALS

Organisation Name: Sensor Device Pb As Hg Cd Cu Others Page

Aboatox Oy Luminescence-based biotest XXXX X X 101

Ben-Gurion University Microbial luminescent response sensor X 104

Urease electrode biosensor X XX 108 Cranfield University Anodic stripping voltammetery 112 XXXX X Chronopotentiometry XXX X 116

DBM Pulse neutron-neutron XXXX X X 120

Niton Europe GmbH Pulse neutron XXXX X X 125

NMRC, Ireland Heavy metal analyser HEMA 2002 X XX 129

University of Coimbra Batch analysis injection XXX X 132

Voltammetric probe XXXX X X 136

University of Neuchatel / Voltammetric in situ profiling system XXXX X X 138 University of Geneva Voltammetric in-line analyzer XXXX X X 140

Microfabricated microelectrode array XXXX X X 143 Vlaamse Instelling voor Technologisch Onderzoek Luminometry assay XXXX X X 146 (VITO)

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3.2 Selected Metal Sensing Systems

3.2.1 Aboatox Oy (Finland)

Aboatox Oy is a private company working in the field of luminescent bacterium biosensors. They have developed a luminescence-based biotest for the detection of heavy metals in environmental samples. All bioavailable heavy metals can be detected in an aqueous phase sample. Detection limits are in the ppb range for all measured heavy metals (eg. inorganic mercury: 2ppb, organomercurials: 0.05ppb). The biotest is commercially available and does not require a skilled person to perform the test.

3.2.2 Ben-Gurion University of the Negev (Israel)

The university has an optical fluorescence analyser that utilizes genetically engineered luminescent bacteria for the detection of heavy metals (Hg, As, Cd, Pb and Zn); it can also detect genotoxicity levels. The device is fully portable and can be taken into the field. The device has been developed into a demonstration unit and can be used by an unskilled user. Sample matrices range from soil sediment to any aqueous phase, with detection down to 2ppb.

3.2.3 Cranfield University (UK)

Cranfield University has four disposable screen-printed electrode based sensors for the detection of heavy metals ions in environmental samples. Three of them are based on stripping voltammetric and chronopotentiometric measurements and the fourth is based on amperometric enzyme inhibition. The screen-printed electrodes are produced in house at the university. The sensors can be used in the field for metal detection, using the commercially available PalmSens meter. A 3-minute extraction protocol has been designed to work in conjunction with the sensors. Detection limits are down to ppb.

3.2.4 Bohrlocmessungen-dr. Buckup (DBM) (Germany)

DBM is a small start up company that has developed a pulse neutron based sensor with their partners GTK (Finland), Selor Eeig (Netherlands) and Terramentor (Greece). The device which is commercially available is claimed to detect ppb levels (Hg <0.005mg/l, Ni <0.01mg/l, Cu 0.1 mg/l) and also to detect total organics down to 0.0005%. The instrument works automatically. The system can be adapted to measure all elements and organic compounds with prior calibration in the field. The unit has been used in the field in fifteen countries and the company has over 1000 field measurements.

3.2.5 NITON Europe GMBH (Germany)

The Niton group is an American based private instrumentation company that has offices in Europe. It has two well-developed devices the Niton Xli and XLT 700 Series, both devices are commercially available and are based on x-ray fluorescence. The sensors, which are both hand-held devices, can be used by an unskilled worker with minimal training and can detect heavy metals in soils, solids, air and water.

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They are able to detect 25 elements simultaneously with a testing time ranging from a few seconds to a few minutes, depending on the desired analysis precision. Depending on the customisation of the sensor for specific elements, the detection limit is in the ppm range to percentage concentration, again dependent on the sample matrix and element.

3.2.6 NMRC (Ireland)

NMRC is Ireland's information and communications technologies (ICT) research institute based in Cork. They have developed a heavy metal analyser (HEMA 2002) electrochemical system which can be used to determine heavy metal in aqueous samples. The system is field deployable and requires a semi-skilled technician to operate the system. Target analytes are all heavy metals (copper, lead and mercury) with detection at approx 25ppb-100ppb with detection of analytes in complex matrices.

3.2.7 Instituto Pedro Nunes – University of Coimbra (Portugal)

The insitute has developed an electrochemical batch injection analysis system that allows the direct determination of the free fraction of trace metal ions in raw, untreated water samples. The system is small and portable and still in a prototype stage. The detection limit is 5nmol/l for all measured metals (Zn, Cs, Pb and Cu). Sensor adaptability to other metal elements is possible. The use of the sensor requires semi-skilled personnel with basic knowledge of chemical analysis and computer skills.

3.2.8 University of Neuchatel/University of Geneva (Switzerland)

The universities have developed four electrochemical-based devices able to detect metals in environmental samples. Three of their devices have been developed with a commercial partner, Idronaut SRL, for in-situ, inline and field deployable modes respectively. One of the devices, the voltametric in-situ profiling system, is commercially available with the others in demonstration prototype stage. Based on similar technology and methodology, the devices differ by the use of different microelectrodes and electrode array configuration. Trace metal elements can be measured in aqueous samples in freshwater, salt water and the sediment/water interface. They are also field deployable. Detection is in the ppt-ppb range depending on the target elements. The sensor requires a skilled technician to operate the devices.

3.2.9 Vlaamse Instelling voor technologisch onderzoek (VITO) (Belgium)

VITO is the Flemish institute for technological research, which is an independent research centre. They have developed seven luminometry assays based on genetically engineered bacteria that can detect a whole range of heavy metals in environmental samples. The tests are available from VITO and require a skilled technician. Detection limits are in the ppb range.

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3.3 Conclusions

Of the featured sensors, nine are based on electrochemical devices. Detection limits for heavy metals in environmental samples vary from sub-ppb to ppm range. The two devices from Niton Europe GmbH and the universities of Neuchatel and Geneva have sensors that are fully developed and commercially available.

The sensors developed by DBM and Niton are both commercially available devices that utilize a radiation source for their detection system. Both devices are able to be customer set for a desired elemental analysis and can detect heavy metals down to percent levels. They both require some level of training and for the maintenance of the DBM device a skilled person is required.

The devices developed by Aboatox Oy, Ben Gurion and VITO are all bacteria-based detection systems. The sensors utilise genetically engineered strains of a bacterium. The Ben Gurion device is easily deployable in the field, can be used by an unskilled user and can also detect genotoxicity levels in the sample. The two systems from Aboatox Oy and VITO are commercially available.

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4. Toxicity and Genotoxicity Testing Devices

4.1 Introduction

Bio-accumulation and high concentrations of persistent organic pollutants and heavy metals in the environment can have a toxic effect on many simple organisms, invertebrates and whole cells. By utilizing this phenomenon toxicity test kits have been developed that are able to give early warning signs and semi quantitative detection in environmental samples. The following selected test devices and kits are quite different from each other as they utilise different monitoring techniques. However, what they do have in common is that the all give an indication of the toxicity levels, and in some cases genotoxicity levels, in environmental samples. Although most of them give a measurement of total toxicity there are a few that allow measurement of specific analytes to ppm. Table (4.1) gives an overview of the sensors and organisation with each entry crossed referenced to Appendix 2.

Table 4.1 List of toxicity and genotoxicity sensing devices and their target analytes

ANALYTES

Organisation Name Device or Sensor Page Metals General Hydrocarbons Hydrocarbons

Coventry University Bipotentiostat bilayer lipid membrane X X X 165

Cybersences LtD Portable toxicity testing system X X X 169

Delta Consult B.V. MOSSELMONITOR ® X X X 172

DLR SOS-LUX-Test X X X 178 Ecole Nationale Supérieure des Mines de Optical whole-cell biosensor X X X 183 St Etienne Gentronix Limited GreenScreen EM X X X 186

University of Aberdeen Whole cell toxicity sensors X X X 195

University of Nantes Bioassay and biosensor for TBT and DBT X 199

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4.2 Selected Toxicity Sensors

4.2.1 Coventry University (UK)

The Centre of Molecular and Biomolecular Electronics at the University of Coventry have developed a bilayer lipid membrane on an electrode in a bipotentiostat configuration. A commercial prototype is currently in development and requires a skilled technician. The device measures toxins in all aqueous samples. They are currently working on optimising detection to reach the sub-picomolar (pM) range.

4.2.2 Cybersense Biosystems Ltd (UK)

Cybersense Biosystems Ltd are a small biotechnology company based in Oxford. They have developed a portable system specifically for testing toxicity in solids (soil, sediment and sludge) samples. Based on bioluminescent bacteria, the detection system is non-specific and detects the general toxicity of the sample. The device is still in a prototype stage but is expected to be launched as a commercial system in the UK soon. The target analytes are all organic and inorganic. Examples of detection limits are 100ppb for benzene, 50ppb for phenol and 20 ppm for copper and 125 ppm for PAHs. Although sensitive to ppb, the sensor is not selective.

4.2.3 Delta Consult B.V. (The Netherlands)

Delta Consult is an independent company. They have a well-developed commercially available sensor device called the Mosselmonitor. The system is an early warning system that is designed for continuous on-line monitoring of surface waters, water, effluents and drinking water. The system is based on the behaviour of mussels. They have many systems in deployment and field results available. Although it is a general early warning system, detection limits for certain elements can be down to the sub- ppm range (e.g. 0.005 mg/l for copper).

4.2.4 DLR (Germany)

DLR is the Aerospace Agency of the Federal Republic of Germany. Their system detects the genotoxicty of environmental samples by employing a bacterial bioassay. Measurement is derived by the DNA damage-dependent induction of the bacterial SOS system. The device requires minimal training and the test kit is currently not available commercially. The kit will detect all genotoxic compounds in aqueous phase. The test is based on a 96 well plate format, and allows multiple measurements.

4.2.5 Ecole Nationale Supérieure des Mines de St Etienne (France)

The graduate engineering school of Saint Etienne has an optical whole cell biosensor that is able to detect heavy metals, herbicides and solvents in aqueous phase samples. The device can be both manual and automated and requires a skilled technician to operate it. The biosensor measures toxins in environmental samples at low levels of toxicity (concentrations as low as 0.1nM of pesticide have been measured with the system). The sensor is still in the prototype stage, with work on optimising and improving measurement reproducibility.

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4.2.6 Genotronix Limited (England)

Genotronix Ltd is a small start up technology transfer company. The Green Screen environmental monitoring system (GreenScreen EM) is in mid-developmental stage and is currently undergoing field trials. The system is a yeast cellular biosensor used for the simultaneous detection of both genotoxicity and cytotoxicity. The detection system is based on yeast fluorescence and yeast density is measured on a prototype portable reader known as the YETI (Yeast Environmental Toxicity Indicator). The bioassays performed on the system are designed to be carried out by relatively unskilled personnel. The system detects the whole sample toxicity in aqueous samples. Detection limits are in the ppb-ppm range.

4.2.7 University of Aberdeen / Remedios Ltd (Scotland)

The university, in conjunction with Remedios Ltd, a small technology transfer environmental biosensor consultancy, have developed a luminescence whole cell toxicity sensor. The biosensor is currently being used by Remedios Ltd in its consultancy work for commercial clients. The biosensor requires a skilled technician and it is available commercially via consultation. Target analytes are all acutely toxic chemicals and metals and organics in the bio-available fraction (aqueous samples). The sensor will only provide a primary screen.

4.2.8 University of Nantes (France)

The university has developed a bioassay in a 96 well plate prototype immunoassay kit and an optical based biosensor that can detect organo-tin compounds. These compounds such as TBT and DBT are highly toxic biocides. Measurements are in aqueous samples with detection limits for the plate-based assay for TBT at 0.08µM and DBT at 0.0001µM (sub-ppb range).

4.3 Conclusions

Of the seven devices featured in this chapter, only one of them, the Mosselmonitor from Delta Consult is available commercially. However, Cybersense Biosystem Ltd, with their portable toxicity testing system and University of Aberdeen/ Remedios Ltd have systems that will be available commercially in the near future. All of the test kits and biosensors are able to detect toxicity in environmental samples.

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5. Sensor devices for other targets: general parameters, gases, anionic, cationic and phenolic compounds

5.1 Introduction

Sensor devices able to measure general parameters such as pH, conductivity, and redox potential as well as analytes like calcium, potassium, nitrate, nitrogen dioxide, chlorine and phenolic compounds are looked at in this chapter. Only one of the featured devices is based on optical principles. The rest are electrochemical. Table 5.1 gives an overview of the sensors and the analytes and general parameters that can be measured.

Table 5.1 List of sensors for a variety of analytes and parameters

Analytes General parameters Organisation Sensor Name: Device Page

2 3 2 2+ + pH Cl K NO NO Ca Redox Phenolic potential conductivity compoundsc compoundsc Barcelona ISFET and IDS- Institute of based sensor X X X XX X 205 Microelectronics Coventry Optically- University interrogated X 209 system

Cranfield Chlorine sensor University X 214

University of Electrochemical Ulster sensor X 217

5.2 Selected Sensing Devices

5.2.1 Barcelona Institute of Microelectronics (IMB-CNM) (Spain)

Consejo Superior de Investigaciones Científicas’s IMB-CNM is a non-profit Research and Development Institute that has developed ion selective field effect transistor 2+ + - (ISFET) based sensors for measuring pH, Ca , K and NO3 as well as interdigital structures (IDS) for measuring conductivity and redox potential (Eh). The sensors

21 SENSPOL Survey of Sensor Capabilities are fabricated with standard technology and are encapsulated individually in PCB boards and covered with a special photopolymer to protect electrical parts. To use the sensors a skilled person is required.

5.2.2 Coventry University (UK)

The Centre for Molecular and Biomolecular Electronics at Coventry University have developed an optically-interrogated sensor system for detecting nitrogen dioxide in which a thin film of an active substrate is exposed to a gaseous ambient and the optical changes recorded. The prototype device responds in some seconds at room temperature and allows detection down to 100 ppb.

5.2.3 Cranfield University (UK)

Cranfield University have developed an electrochemical sensor for determining ultra- low concentrations of chlorine within fresh waters. Free, total and combined chlorine can be detected down to 0.002 ppm. The measurements are carried out with a dedicated instrument provided in conjunction with disposable screen-printed electrodes. It can be used by unskilled personnel.

5.2.4 University of Ulster (UK)

The University has developed a biosensor for the determination of flavanols using either plant tissue material (polyphenoloxidasees or commercial tyrosinase) immobilised in either carbon paste electrode or screen-printed in with modified polypyrrole. The method relies on the electrochemical reduction of a quinone produced from the catalysed oxidation of the phenolic compound by ambient oxygen. It will detect a broad range of analytes containing a catechol (1,2-dihydroxybenzene) group and this includes dopamine as well as a range of polyphenols of the catechin type found in flavanols. Detection levels are down to 2.5 µM. To use the sensors, some skill is required initially.

5.3 Conclusions

Of the four featured sensor devices, three developed by IMB-CNM, Cranfield University and University of Ulster are based on an electrochemical approach and the other, by Coventry University, is based on an optical principle. The sensor devices developed by Coventry University and Cranfield University are available as prototypes while that of University of Ulster is not available either commercially or as a prototype. IMB-CNM has one available as a prototype while the others are just developed for research purposes and not available commercially.

The sensors can be used in variety of sample matrices including freshwaters (chlorine sensor by Cranfield University), gas samples (nitrogen dioxide sensor by Coventry University), surface water, groundwater, soils and pore water in clay materials (ISFET 2+ + - and IDS based sensors for pH, Ca , K and NO3 , conductivity and redox potential by IMB-CNM).

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6. Bottlenecks and bridging the Gap

6.1 Bottlenecks in sensor technology

As well as asking developers about their devices, several probing questions were asked about the sensor development, collaborations and partnerships and commercial backing. This section attempts to summarise these responses.

Sensor technologists often have many hurdles that impede the advancement of their devices from concept into fully developed commercialised sensors. These bottlenecks can be categorised in to three stages:

• First Stage: Choice of "bio"-system, sensor, protocol methodology and sample experimentation conditions. Transducers are often chosen as a function of how well the biological sensing system can be integrated into the sensor. Bottlenecks tend to occur due to interference in selectivity for the analytes to be detected, by contaminants or the matrix of the sample. Stability of the biological components and selectivity often need to be addressed if shelf life and reliable reproducible detection of analytes is to be ensured for conversion into a commercialised device.

• Middle stage: The transfer of the developed sensor, methodology, protocols and crude prototype device into a viable demonstration prototype that can be evaluated and tested by other users. This stage requires a wider selection base of expertise, knowledge and collaboration with technologists in other fields such as mechanical, electronic, electrical and instrumentation design engineering. Funding for this stage of development is often quite hard to attract and, commonly, potential commercial partners would be more interested in the device once this stage is completed.

• Third stage: Marketing of sensors. Once a sensor has been developed into a demonstrator device for commercial evaluation the developer comes across the final hurdles which are by no means easy to pass: marketing and selling the system. Marketing new technologies in the sensing and biosensing field is difficult, as end users are often unaware of the technologies that are available and reluctant to invest in these novel devices unless legislation and directives are imposed on them. They need to be aware of the possibility to detect certain pollutants using new regimes, that is not achievable with their current detection methods. Often, there is the “better the devil you know, than the devil you don't” attitude. Without legislative pressure it is difficult for new technology to be widely implemented.

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6.2 Bridging the gap between sensor developers and end- users

Sensor developers are in agreement on many points concerning what would aid the development of their devices. These include:

• Greater communication between instrument developers and end-users.

• Information transfer and communication links between governmental regulators and technology developers (heightening awareness of available technologies).

• Closer collaboration and partnerships with other developers and specialists.

Suggestion that could address these problems were:

• Providing a question survey, similar to the SENSPOL survey, designed for industrial and potential end-users, with the results published (anonymously if need be) for perusal by the developer and other interested parties.

• Regular workshops, symposia and networking meetings bringing together end- users, government, regulators, venture capitalists and developers for information transfer and networking.

• Funding programmes and collaboration incentives for developers and venture capitalists.

• Making funding application processes more efficient and shorter, with administrative benchmarks and more transparent selection procedures.

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7. Conclusions and Overview

The response to the sensor survey was very good. Of the 32 selected developers featured in the report, 7 are companies, 15 were from universities and 10 from research institutes, all coming from 13 European countries. The report features 42 sensors, biosensor and detection kits able to detect a wide range of analytes including DNAPLs, LNAPLs, aromatic, and halogenated compounds, heavy metals, toxicity, genotoxicity, gases, and anionic, cationic and phenolic compounds. Also general parameters like pH, conductivity and redox potential can be measured.

The report features two different field based sample extraction approaches. Cranfield University has developed a field based Supercritical Fluid Extraction (SFE) device and protocol that can be employed in the field for PAHs and also adapted to other analytes. Exposmeter have developed a semi-permeable membrane that can accumulate the whole range of target analytes of interest such as DNAPLs, LNAPLs, aromatic, chlorinated and PAHs in environmental samples. Both devices can be used in conjunction with a variety of commercially available detection systems.

An attempt has been made to select those devices that target particularly the hydrocarbons DNAPLs, NAPLs, aromatics and halogenated compounds. Out of the 13 featured sensors measuring hydrocarbon based compounds, six of them are available as fully commercialised analytical devices or demonstration units. LNAPLs, DNAPLs and PAHs can be detected by six sensors, of which four are commercially available. A device called the RIANA is a fluoro-immunoassay based biosensor that is adaptable to a full range of analytes and has demonstration units commercially available. Detection limits of these units are sub-ppb.

The featured sensors cover the full spectrum of heavy metals and trace metals that can be measured in environmental samples. Of the 14 featured sensors that can detect metals there are four devices from Niton Europe GmbH, the universities of Neuchatel and Geneva, and DBM that are fully developed and commercially available. Heavy metals in environmental samples have detection limits below ppb. The devices developed by Aboatox Oy, Ben-Gurion University and VITO are all bacteria-based detection systems. The sensors utilize genetically engineered strains of a bacterium. The Ben-Gurion device is easily deployable in the field and can also detect genotoxicity levels in the sample. The two systems from Aboatox Oy and VITO are commercially available.

Bio-accumulation and high concentrations of persistent organic pollutants and heavy metals in the environment can have a toxic effect on many simple organisms, invertebrates and whole cells. By utilizing this phenomenon toxicity test kits have been developed that are able to give and early warning signs and semi-quantitative detection in environmental samples. Only two of eight toxicity test devices, the Mosselmonitor  by Delta Consult and the University of Aberdeen/ Remedios Ltd have systems that are available commercially. However, the Cybersense Biosystem Ltd portable toxicity testing system will be available commercially in the near future. All of the test kits and biosensors are able to detect toxicity in environmental samples.

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There are many other developers of detection systems known to SENSPOL that did not respond to the questionnaire and are worth a brief mention in this report. Merck have developed a series of well-developed commercialised kits. Between the three available test kits Spectroquant , Bioquant  (enzymatic methods) and Toxalert  (toxicity testing) a vast battery of analytes can be detected. Spectoquant test kits can measure Pb, Hg, Ni, Zn, Cd at ppm levels. Toxalert Test kits (10 and 100) have been used to measured toxicity in wastewater and surface water environmental sample. Millipore Envogard™ has a rapid test kit EnSys ™ for polynuclear aromatic hydrocarbon (PAH). Checklight Ltd have a commercially available toxicity test kit (Toxscreen) for Cd, Mn and Pb with detection of toxicity at 60-200µg/l. Furthermore, Hg, Cu and Co can be determined at concentrations down to 7-30µg/l. The Laboratory for Environmental Toxicology and Aquatic Ecology LETAL at the University of Ghent, Belgium have developed miniaturised mirobiotests (Microtest, commercially available) for toxicity testing. All of the aforesaid test kits have successfully been used in the field and used by many research groups in the SENSPOL network.

Sensor Tech are close to commercialisation of a sensor for persistent organic pollutants (POPs) in soil and water and possibly oil. Oceanor’s Soilwatch is a system for automatic, continuous monitoring of pollutants in soil and groundwater. Their Trace Metal Monitor can determine various metals onsite having resolutions down to 1 ppb: as examples in continuous monitoring of effluent water one sensor can measure Zn, Pb, Cd, Ni, Co, Tl and Cu, while another can measure Hg and Cu.

Other sensing devices have been presented and discussed at the SENSPOL workshops, details can be found in the Proceedings (Domínguez and Alcock, 2001; Alcock and Kadara, 2003; Alcock, 2004). The present state of commercial development of some of these devices is not known.

Devices demonstrated in the SENSPOL Technical Meeting on ‘Sensors for Characterization and Monitoring of a Contaminated Site’, Seville, Spain, 6-9 November 2002 can be viewed on DVD/video (Alcock and Valiente, 2003) and are described in the Proceedings (Valiente et al, 2003). Several sensor technologies that were demonstrated on site in Seville do not appear in the present report. Universidade Nova de Lisboa and the Geological Survey of Portugal used the hand-held field instrument HANNA Tester COMBO for pH, electrical conductivity, total dissolved solids and temperature. The Autonomous University of Barcelona, Spain, tested a laboratory-based prototype automated lead analyser (AQUAMET) incorporating a potentiometric sensor coupled with FIA. The University of Florence, Italy, demonstrated detection of Cd, Cu and Pb using a disposable electrochemical sensor which avoids the need to use mercury solutions in the field. They also verified a disposable DNA-based biosensor for toxicity screening.

A number of other devices that had not featured in the questionnaire replies were demonstrated in the SENSPOL Technical Meeting on ‘Problems Related to Diffuse Pollution Sources: Characterization of Sediment, Dredged Material and Groundwater’, Koblenz, Germany, 28 - 31 October 2003. Warsaw University, Poland used potentiometric sensors for Ca, Mg and Pb ions and free Cl ions. MTI Marketing and Trading AG, Switzerland, used the PDV 6000 portable analyser that incorporates reusable electrodes to test sediment samples for heavy metals. Jenway, UK, and University of Cambridge, UK, tested out a device for detecting high levels of toxicity.

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King's College London, UK, verified their rapid immunoassay protocols for estrone and total estrogens. Biosense Laboratories AS, Norway, used bioassays for estrogens for dioxin/PCBs. A new immunoassay for MTBE developed by Cranfield University, UK, and Diaclone, France was demonstrated. The Technical University of Berlin, Germany tested for endocrine effects using the enzyme linked receptor assay (ELRA). In addition, Kiwa Water Research, The Netherlands, took samples to their own laboratory for rapid analysis for organic micropollutants using a non-portable online HPLC-UV fingerprint monitor. Information about publication of the Technical Meeting results will be made available at the site: http://www.cranfield.ac.uk/biotech/senspol.

Bottlenecks that impede sensor technology can be categorised into three sections:

Areas where developers felt action would alleviate some of the problems they encountered while developing their devices were: greater communication between instrument developers and end-users; information transfer and communication links with governmental regulators and available technologies; closer collaboration and partnerships with other developers and specialists.

Suggestions that could address these problems were:

• Providing a complementary question survey designed for industrial and potential end-users, with the results published (anonymously if need be) for perusal by the developer and other interested parties. • Regular workshops, symposia and networking meetings bringing together end- users, government, regulators and developers under the same roof for information transfer and networking. • Funding programmes and collaboration incentives for sensor development. • Making funding application processes more efficient and shorter, with administrative benchmarks and more transparent selection procedures.

A report (Sesay et al, 2003) derived from the present full report has been supplied to the European Network on Industrially Contaminated Land (NICOLE).

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8. Bibliography

Further general information about sensors and related issues can be obtained from any of the following texts and reports:

Alcock S.J., and Newman, J.D. (Eds.) (2000). Biosensors for Environmental Technology: State Of The Art 2000. Cranfield University, 47pp. http://www.cranfield.ac.uk/biotech/bioset/bioset_brochurev2.pdf.

Alcock, S.J. and Turner, A.P.F. (Eds.) (1993). In Vivo Chemical Sensors: Recent Developments. Cranfield Press, Bedfordshire, UK, 199pp, ISBN-1-871315-49-2.

Bilitewski, U. and Turner, A.P.F. (2000). Biosensors for Environmental Monitoring. Harwood City. 421pp.

Cass, A.E.G. (Ed.) (1990). Biosensors: A Practical Approach. Oxford University Press, New York.

Cunningham, A.J. (1998). Introduction to Bioanalytical Sensors. John Wiley and Sons, New York.

Freitag, R. (1996). Biosensors in Analytical Biotechnology. Academic Press, San Diego, CA.

Hall, E.A.H. (1991). Biosensors. Prentice Hall, Englewood Cliffs, NJ.

Laschi, S., Mascini, M and Turner, A.P.F (2002). Biosensors. Kirk-Othmer Encyclopedia of Chemical Technology (on-line edition).

Newman, J.D., Tigwell, L.J. and Warner, P.J. (1998). Biotechnology Strategies in Healthcare: A Transatlantic Perspective. Financial Times Professional Limited, London.

Newman, J.D., Tigwell, L.J., Warner, P.J and Turner, A.P.F (2001). Biosensors: boldly going into the new millennium. Sensor Review. Vol 21 No.4, pp 268-271.

Rogers, K.R., Mulchandani, A. and Zhou, W. (1995). Biosensor and Chemical Sensor Technology: Process Monitoring and Control (ACS Symposium Series, No 613).

Tran-Minh, C. (1993). Biosensors. Chapman and Hall, London.

Turner, A.P.F. (1991-). Advances in Biosensors. Multivolume. JAI Press, Greenwich, CT.

Turner, A.P.F. (1999). In: McGraw-Hill Encyclopedia of Science and Technology, 8th edition (Ed. S. P. Parker).

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Turner, A.P.F (2000). Science. 5495, 290pp, 1315-1317.

Turner, A.P.F., Karube, I. and Wilson, G.S. (Eds). (1989). Biosensors: Fundamentals and Applications. Oxford University Press, New York.

Usmani, A.M. and Akmal, N. (1994). Diagnostic Biosensor Polymers. American Chemical Society, Washington, DC.

Wang, J. (1997). Biosensor Design, Technology and Applications. Technomic Publishing Company.

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9. References

1) Sesay, A., Newman, J.D., and Alcock S.J. (2003). Bridging Gaps Between Sensor Developers and Users in a Pragmatic Approach: Sensor Capability Study 2002. Cranfield University Press, Bedfordshire, UK. CD, 212pp.

2) Newman, J.D. and Turner, A.P.F. In “Essays in Biochemistry” (Ed. K.F. Tipton). Portland Press, London, UK, 27, 147, 1992.

3) White, S.F., Tothill, I.E., Newman, J.D. and Turner, A.P.F. Anal. Chim. Acta., 321, 165, 1995.

4) Tothill, I.E., Newman, J.D., White, S.F. and Turner, A.P.F. Enzyme and Microbial Technol., 1996.

5) Newman, J.D. “Advanced Manufacturing Processes for the Production of Biosensors”, PhD Thesis, Cranfield University, 1998.

6) Saini, S. and Turner, A.P.F. Trends Anal. Chem., 14, 304 (1995).

7) Dennison, M.J., Hall, J.M. and Turner, A.P.F. Anal. Chem., 67, 3922 (1995).

8) Alcock, S.J., White, S.F., Turner, A.P.F., Setford, S., Tothill, I.E., Dicks, J.M., Stephens, S., Hall, J.M. and Warner, P.J. British Patent Application 9416002.5, 1994.

9) Skuridin, S.G., Yevdokimov, Y.M., Efimov, V.S., Hall, J.M. and Turner, A.P.F. Biosensors Bioelectron., 11, 903 (1996).

10) Psoma, S. and Turner, A.P.F. "3rd World Congress on Biosensors", 1-3 June1994, New Orleans, USA. Elsevier Applied Science, Oxford, UK.

11) Dobson, D.J. and Saini, S. (1997). Anal. Chem., 69 (17): 3532-3538.

12) Domínguez, E. and Alcock, S. (Eds.) (2001). Proceedings of the 1st SENSPOL Workshop: Sensing Technologies for Contaminated Sites and Groundwater. University of Alcalá, 237pp, ISBN 84-8138-482-8.

13) Alcock, S.J. and Kadara, R. (Eds.) (2003). Proceedings 2nd SENSPOL Workshop: Response to New Pollution Challenges. Cranfield University Press, Bedfordshire, UK. ISBN 1 871315 86 7.

14) Alcock, S.J. (Ed.) (2004). Proceedings 3rd SENSPOL Workshop: Monitoring in Polluted Environments for Integrated Water-Soil Management. Cranfield University Press, Bedfordshire, UK. In preparation.

15) Alcock, S. and Valiente, M. (Eds.) (2003). SENSPOL Technical Meeting on Sensors for Characterization and Monitoring of a Contaminated Site. Sevilla, Spain, 6-9 November 2002. DVD/video, Cranfield University Press, Bedfordshire, UK. 33 min. ISBN 1 871315 78 6.

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16) Valiente, M, Perez, G; Gaona, X; van Ree, D and Alcock, S J. (2003). SENSPOL Technical Meeting, Sevilla. Proceedings. In preparation.

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APPENDICES

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APPENDIX 1

Questionnaire Bridging gaps between sensor developers and users: sensor capability study

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Bridging gaps between sensor developers and users

SENSPOL EU network: sensor capability study

This questionnaire is aimed at identifying current sensor development research and the capability of existing devices (including prototypes) for monitoring groundwater, sediment and contaminated land pollution.

Your responses to these questions will be used to compile an innovative study on sensors and instruments that can potentially be applied to soils, sediments, surface and groundwater. The final sensor catalogue will be made widely available. It will provide information on the status of sensor development, sensor characteristics in terms of sensitivity (detection levels), selectivity and usability and other relevant information that can be used to bridge the gap between research, development and end-users.

Some of the results will be used as part of a collaborative study with the Network for Industrially Contaminated Land in Europe (NICOLE) on ‘Bridging gaps between sensor developers and (end) users in a pragmatic approach’. For the study with NICOLE, targeted pollutants will focus on heavy metals especially mercury; aromatics and non-halogenated hydrocarbons; chlorinated volatile and semi-volatile compounds with particular attention to DNAPL’s and toxicity testing. The final report will be made widely available. Please note that this SENSPOL questionnaire is intended to gather information on all sensors that can potentially be applied to soils, sediments, surface and groundwater and is not limited to this list of target pollutants.

University / Institute / organisation* Name* Address* Department/ Research group Address*

Respondents Name* Position Email address* * Essential information

Please note: if you have more than one sensor system, please complete the sections on pages 2-5 for each separate system.

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SENSOR SYSTEM

Description of sensor system Please include at least one diagram or picture as a clearly labelled attachment. A representative graph and its legend would also be useful. If necessary pictures and graphs can be supplied at a later date.

Environmental relevance

Commercial relevance

Usability (i.e skilled, unskilled)

Availability (eg. commercial, prototype, etc..)

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…)

Sample matrix ( e.g. soils, sediments, surface and groundwater )

Sample phase (i.e. residual, free, dissolved or absorbed)

Sample preparation (e.g. filtering, SPE, derivatisation etc…)

Sample handling (i.e. manual, automated etc,,,)

Sample size

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Sensor Measurements

Assay protocol

Continuous measurement YES NO

Present measurement performance: Detection levels -

Selectivity

Repeatability

Potential interference

Adaptability to other pollutants

Further work developments

Envisaged performance and optimisation

IF POSSIBLE PLEASE INCLUDE ANY DIAGRAM, RESULTS OR REPORTS AS AN ATTACHMENT

BOTTLENECKS

Please comment on any area of sensor development that has impeded practical implementation of your sensor device:

Do you have any suggestions on how this could be addressed?

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Do you have partners helping with the development of this sensor YES NO Please comment:

Do you have any commercial backing? YES NO Please comment: (partner’s names can be left undisclosed if bound by confidentiality)

Are there any other type of collaboration that would help in sensor development? Please comment:

FURTHER INFORMATION AND COMMENTS

Do you have any further information or comments?:

Please give any additional comment you may have on how the gap between sensor developers and end users can be bridged

PAPERS PUBLISHED

Please list any publications/ posters or presentations related to this sensor

PROFILE OF ORGANISATION

Please give a short description of your organisation and research group

Would you be prepared to take part in an interview to explore your responses in greater depth? YES NO

Thank you for taking the time to complete this questionnaire

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APPENDIX 2

Questionnaire responses

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BORDEAUX 3 UNIVERSITY

University Name: Bordeaux 3 University

Department: EGID Address* 1 allée Daguin 33607 Pessac cedex

Respondents Name: Atteia Olivier Position: Professor Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

The sensor system is a technique to measure directly biodegradation rate in aquifers. It is based on a continuous measurement of CO2 in the air at equilibrium with the aquifer fluid. The analysed fluid is recirculated within the aquifer by the use of 3 packers. This recirculation enables the characterisation of a fixed volume around the well. (description file CO2, not included here).

Environmental relevance This sensor technique may be helpful to assess natural attenuation efficiency at sites, and if necessary, to test bioremediation techniques at the well scale.

Commercial relevance This technique is devoted to be sold to PME working as consultant for site assessment or developing biodegradation technology. It can replace the highly time and money consuming microcosms.

Usability (i.e skilled, unskilled) Skilled people is necessary

Availability (eg. commercial, prototype, etc..) prototype

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) All degradable organics

Sample matrix ( e.g. soils, sediments, surface and groundwater ) groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) water

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Sample preparation (e.g. filtering, SPE, derivatisation etc…) No

Sample handling (i.e. manual, automated etc,,,) No handling

Sample size Continuous measurement

Sensor Measurements

Assay protocol No protocol, just branching to the pump

Continuous measurement Yes

Present measurement performance:

1. Detection levels : Degradation of 1 mg/L/day

2. Selectivity Yes if injection of one specific compound, otherwise no selectivity

3. Repeatability : not relevant

4. Potential interference : all degradable organics interfere

5. Adaptability to other pollutants : not relevant

Further work developments Presently the apparatus is tested under field conditions to provide advises for field work

Envisaged performance and optimisation Will be a result of field tests

BOTTLENECKS

The major difficulty is to find the location in the plume where degradation is significant owing to the generally small number of wells existing at one site. If wells are absent the measurement cannot be done

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

This sensor is developed in collaboration with a PME manufacturing classical ion probes and sensors dedicated to water quality.

Collaboration on tests on real plumes in other countries could be helpful. A joint work on real sites may be helpful

PAPERS PUBLISHED

O. Atteia, (2001) Kinetics of natural attenuation of BTEX : review of the critical chemical conditions and measurements at bore scale. Présentation orale, workshop :"Analysis, toxicity and biodegradation of organic pollutants in groundwater from contaminated land, landfills and sediments" Barcelona, 8-10 November 2001. O. Atteia (2002) In situ measurement of biodegradation and its use for natural attenuation study or geochemical modelling. Poster, colloque Response to new pollution challenges, 4-7 Juin 2002 London.

PROFILE OF ORGANISATION

EGID Institute is a group working on several aspects of environment, particularly related to geological objects. The hydrogeology team work on both hydrodynamics and geochemical aspects, on deep and surface aquifers.

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CRANFIELD UNIVERSITY

Institute Name: Institute of BioScience and Technology Research group: Cranfield Centre For Analytical Sciences Address: Cranfield University Silsoe, Bedfordshire MK45 4DT, UK Respondents Name: Peggy Rigou Position PhD student Email address: [email protected]

SENSOR SYSTEM

Description of sensor system Field-based Supercritical Fluid Extraction (SFE) device for hydrocarbons monitoring from contaminated soils.

SFE device consists of - a small CO2/Methanol gas cylinder connected to a CO2 pump equipped with an electronic Peltier cooling system. The premixed CO2/MeOH is pumped from the cylinder and taken to a pressure above the supercritical pressure. - a heating unit to heat the extraction vessel (EV) containing the matrix to extract to a temperature above the supercritical temperature. The supercritical fluid passes through this EV from the CO2 pump and interacts with the matrix to extract the analytes of interest - a back pressure regulator and a back pressure regulator controller to keep a constant pressure and flow rate through the system. The SF solubilising the analytes passes through this BPR, and by depressurisation of the SF at atmospheric pressure, analytes are recovered in methanol by transfer from the SF to the liquid methanol (10ml).

Environmental relevance Hydrocarbons monitoring from contaminated lands. Follow of bioremediation on contaminated sites.

Usability (i.e skilled, unskilled) Must be used by a trained person. No special skills required.

Availability (eg. commercial, prototype, etc..) Developed at Cranfield Centre for Analytical Sciences, Cranfield University,

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Mainly PAHs, but other compound such as pesticides can be extracted under other optimised conditions.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils

Sample phase (i.e. total, residual, free, dissolved or absorbed) Total

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Sample can be extracted as received without any pre-treatment. For wet sample, it is mixed with an absorbent like hydromatrix (generally 1g), and with copper powder (1 or 2g) for samples containing sulphur compounds. The mixture is manually homogenised before loading in the extraction vessel.

Sample handling (i.e. manual, automated etc,,,) Manual: can extract one sample after another but possibility for standing two extractions vessels in the heating unit (on development).

Sample size From 3-5g for wet sample to 10g for dry sample.

Sensor Measurements

Assay protocol

Sample preparation: Soil or mixture of soil, hydromatrix, and cooper powder is loaded into the EV and screwed in the heating unit. EV is then heated to the temperature of extraction.

Extraction procedure: CO2/MeOH is automatically pumped and pressurised in the system to the pressure of extraction. When the pressure and temperature of extraction are reached, CO2 pump is stopped in order to allow a static flow for 5-10 minutes, for the SF to interact with the matrix. After this static period, CO2 pump is started again, allowing a dynamic flow rate for a period of 60-90 minutes. Analytes are recovered in a vial in 10ml of methanol.

Extraction conditions for PAHs: (note that other methods are available, depending on the analyte of interest) Pressure: 13.8/32.5Mpa Temperature : 80/120°C Flow rate : 0.8ml/min Time of extraction: 60 to 90 minutes.

After extraction: CO2 pump is stopped and methanol extract with solubilised analytes stored in appropriate conditions for subsequent analysis such as immunoassay. System is cleaned by passing CO2/MeOH through, with a cleaned EV in the heater unit.

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Cleaning conditions: Pressure: 13.8Mpa Temperature: 80°C Flow rate: 0.8ml/min Time of cleaning: from 15 to 30minutes, depending on the dirt of the sample previously extracted.

Adaptability to other pollutants SFE system is adaptable to other pollutants such as pesticides, total hydrocarbons, by varying pressure and temperature of extraction. Methods are available through the EPA.

BOTTLENECKS

Water and matrix is one of the major problems. Soil samples too wet, or too compact are difficult to extract for SF penetration reasons.

SENSOR SYSTEM

Description of sensor system Field-based analysis of Polynuclear Aromatic Hydrocarbons (PAHs) from soils extracts.

PAHs RaPID Assay test kit can be used as a semi-quantitative enzyme immunoassay for reliable and rapid screening analysis of PAH from soils extracts (SFE extracts). It applies the principles of enzyme linked immunosorbent assay (ELISA) and is based on the recognition between an antibody (enzyme) and an antigen (analytes). Colour developed by the substrate is read by spectrophotometry and is inversely proportional to the concentration of PAH in the sample. PAH concentration is interpreted relative to standard curve generated from kit standards (as phenanthrene).

Environmental relevance Field-based PAHs analysis from soil extracts. Used as a screening method.

Usability (i.e skilled, unskilled) Unskilled

Availability (eg. commercial, prototype, etc..) Commercially available

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Polynuclear Aromatic Hydrocarbons

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Water, methanol

Sample phase (i.e. total, residual, free, dissolved or absorbed) Total

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Sample preparation (e.g. filtering, SPE, derivatisation etc…) Sample has to be diluted in 10% methanol/Phosphate Buffer.

Sample handling (i.e. manual, automated etc,,,) Manual

Sample size 250µL

Sensor Measurements

Assay protocol The sample to be tested is added, along with an enzyme conjugate, to a disposable test tube, followed by paramagnetic particles with antibody specific to PAH attached. At the end of an incubation period, a magnetic field is applied to hold the paramagnetic particles (with PAH and labelled PAH analogue (enzyme conjugate) bound to the antibodies on the particles, in proportion to their original concentration) in the tube and allow the unbound reagents to be decanted. After decanting , the particles are washed with washing solution. The presence of PAH is detected by adding the enzyme substrate and the chromogen. The enzyme labelled PAH analogue bound to the PAH antibody catalyses the conversion of the substrate/chromogen mixture to a coloured product. After an incubation period, the reaction is stopped and stabilised by the addition of acid. Colour development is read by spectophotometry and is inversely proportional to PAHs concentration in the unknown sample. More detailed protocol is available with the kit.

Continuous measurement No

Present measurement performance:

1. Detection levels – 0.5 to 100ppb

2. Repeatability: % coefficient of variation between standard duplicates of 10% or less

3. Potential interference Co-extracted hydrocarbons

4. Adaptability to other pollutants Different immunoassay test kits available. Further work developments

BOTTLENECKS

Sample matrix effect: co-extracted compounds. Maximum dilution of the sample, but has to be within the limit of detection.

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Ther are no partners or commercial partners.

PAPERS PUBLISHED

Publication: Rigou P., Saini S., Setford SJ. Field-Based Supercritical Fluid Extraction of Hydrocarbons at Industrially contaminated sites. ScientificWorld Journal, 2002, vol.2, p.1063-1069

Poster presented:

“Field-based Supercritical Fluid Extraction for BTEX and PAHs Monitoring at Industrially Contaminated Sites” - Harrogate workshop on “Contaminated Sites, Landfills, sediments, Diffuse Pollution. Workshop Co-organised by the EU funded Concerted Action on Environmental Technologies (ETCA), The Environmental and Climate Programme of the European Commission and the School of Civil Engineering, University of Leeds, Uk, 21-23 May 2001 - Barcelona workshop on “Analysis, toxicity and biodegradation of organic pollutants in groundwater from contaminated land, landfills and sediments”. Workshop co-organised by Gpoll of European Science Foundation (ESF) and Consejo Superior de Investigaciones Cientificas (CSIC). 8-10 november 2001 . -Second Senspol Workshop. London: 4-7 June 2002. Presentation: “Field-Based Supercritical Fluid Extraction for Hydrocarbons in soils.”

Trace analysis, bio products and quality of the environment. Marrakech, Morocco: May 2002

PROFILE OF ORGANISATION

Cranfield Centre for Analytical Science (CCAS) is an integral part of the Institute of BioScience and Technology (IBST), within Cranfield University (Silsoe, UK). The CCAS mission is to advance current knowledge and technology in analytical science and exploit the resulting research products across a wide range of industrial sectors. The centre houses a multi-disciplinary team of physicists, chemists, engineers, biotechnologists, medical consultants and software + hardware engineers. The centre is active in research and development for both industry and academia, post-graduate education, professional training. IBST is the UK’s foremost centre for biotechnology and is a centre of excellence in post- graduate education, training and industrially funded research. The centre houses a wide variety of research groups including cell and molecular biology, combinatorial chemistry, polymer imprinting, mycology and environmental research as well as housing a state of the art fabrication facility for the pre-production of sensor devices. IBST has a flourishing industrial contracts division and has been at the forefront of diagnostics development since its inception in 1981, creating a wide variety of products for industrial clients, including the Exac-tech blood glucose monitor, the world’s most successful biosensor to date.

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EXPOSMETER

Company Name: Exposmeter Address: Trehorningen 34, SE-922 66 Tavelsjo, Sweden Respondents Name: Audrone Zaliauskiene Position: Consultant Email address: [email protected]

SENSOR SYSTEM

Description of sensor system :

Exposmeter-liphophilic

Is a method for quantitative measurement of lipophilic compounds in different environments (air, water, soil, industrial processes) employing SPMD (Semipermeable membrane devices). The SPMD technique is based on a simple device which accumulates the compounds in a lipid phase after passage through a diffusion membrane layer. The compounds can subsequently be analysed employing toxicity tests or GC-MS or other instruments. This accurate, quantitative measurement can be for either ultra trace of high concentrations and can be related to free water concentrations.

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Environmental relevance:

HIGH, since sampling of bioavaible, bioaccumulative compounds.

Commercial relevance: Already on the market and used in many European countries by governments and companies as well as universities

Usability (i.e skilled, unskilled) Non-mechanical, passive device which is easy to deploy and requires no maintenance

Availability (eg. commercial, prototype, etc..)

A commercially available membrane sampler together with stainless steel deployment apparatus, which has a capacity for 5 standard membranes.

ANAYLTES

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Examples of contaminants that are significantly concentrated in triolein-containing SPMDs (not all-inclusive):

1. Polychlorinated dioxins and furans 2. Polycyclic aromatic hydrocarbons (PAHs) 3. Polychlorinated biphenyls (PCBs) 4. Organochlorine insecticides 5. Pyrethroid insecticides 6. Several herbicides and many industrial chemicals 7. Alkylated selenides 8. Tributyl tin 9. Potentially bioaccumulative organic compounds

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Air, surface and groundwater, sediments, soil, industrial processes

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable free fraction of pollutants

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Extraction and GPC (depending on analytes also including cleanup steps)

Sample handling (i.e. manual, automated etc,,,) Manual, storage in freezer.

Sample size Sampling of 10-300 litre of water or cubic meter of air during a 20-day integrative sampling period depending of analytes.

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Sensor Measurements

Assay protocol Consists of a 2.5-cm wide (layflat) by 91.4-cm-long SPM (70-95 µm wall thickness and surface area is 450 cm² or cm²/g SPMD) containing 1 mL (0.915 g) of triolein as a thin film.

(See SOP available on homepage www. exposmeter.com).

Continuous measurement Yes

Present measurement performance:

1. Detection levels Depends on detection method. For PCB isomers sub-pg/L range

2. Selectivity Samples : only dissolved fraction of a multitude of compounds

3. Repeatability Sampling less than 10%

4. Potential interference Depends on detection method

5. Adaptability to other pollutants No

Further work developments Have to be certified as standard measurement method

BOTTLENECKS

The acceptance by the scientific community and end users that bioavaibility parameter of detected anayltes is an important factor in risk assessment. This could be addressed by conducting demonstration projects

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

The research and development of the sensor device has partners and commercial backing. However collaboration in different countries and discussion on the bioavailablity issue is required along with the query on certification procedures relevant to each country would be beneficial to the application and development of the sensor to the wider community.

FURTHER INFORMATION AND COMMENTS

The participation in demonstration projects, with financing from external partner (EU), would mark the possibility of the future use of the sensor.

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PAPERS PUBLISHED

See homepage of Exposmeter AB www.exposmeter.com

PROFILE OF ORGANISATION

Exposmeter is RandD company mainly dealing with a supply of products and services for sampling and analysis of persistent organic pollutants, metals and phosphorus. Company provide customers with sampling technologies for detection of bioavailable, time-weighted average concentrations of pollutants in air, water, soil and industry processes. Company can offer design of investigation strategy, chemical analysis of pollutants, interpretation and reporting of data.

During the last decade Exposmeter involved in development and implementation of new or modified sampling technologies such as sampling system of organic hydrophilic compounds in water.

Company is representative of number of sampling technologies in Europe, Asia, South America and Afrika.

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FUGRO MILIEU CONSULT

Organisation Name: Furgo Milieu Consult Address: P.O.Box 5122 6802 EC Arnhem, The Netherlands

Respondents Name: B.L. Schalk Position Senior consultant Email address* [email protected]

SENSOR SYSTEM 1

Description of sensor system Membrane Interface Probe (MIP) system integrated in a Cone Penetration Testing (CPT) System On the out side of the cone a heated, permeable membrane is embedded, The inside face of this membrane is swept with a carrier gas. During the penetration of the cone into the soil, VOC's in the soil diffuse across the membrane and partition into the carrier gas stream. The VOC's are carried to detectors in the CPT-truck on ground level. Because tip resistance and sleeve friction of the cone are also measured, the correlation between soil litology and contamination can be frond.

Environmental relevance Investigation of all more or less volatile hydrocarbons. The system provides detailed profiling of contaminant against a detailed soil profile provide by the CPT system. To the system can be added integrated measurement of oil conductivity and soil pore water pressure.

Commercial relevance The MIP/CPT combination delivers much more detailed information than obtained by borehole drilling and sampling from monitoring wells. The cost are comparable or lower for soil investigation at depth of 10 to 40m-GL/

Usability (i.e skilled, unskilled) The system is only available with some specialised contractors. It requires a skilled operator and experienced personnel for interpretation of the data.

Availability (eg. commercial, prototype, etc..) The system is commercially available with Fugro in several West-European countries

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) All Volatile Hydrocarbons

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soil, Sediments and Groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) All Phases are measured depending on the ease in which they become available for transfer through the membrane in gaseous phase. In order to promote transfer the membranes is heated to about 80 degree Celsius.

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None, It is an in-situ measurement techniques

Sample handling (i.e. manual, automated etc,,,) n.a.

Sample size n.a.

Sensor Measurements

Assay protocol After set up the system and stabilisation of the detector response, a flash test is carried out to check the proper functioning of the membrane, the gas flow and the detectors. The results are recorded. Each test consists of pushing the MIP/CPT probe into the soil up to the required depth. The system records maximum and minimum detector response and membrane temperature over each 1.5 cm of profile. In order to maintain the membrane temperature in the optimal range of 80 to 90 degrees Celsius, we stop pushing the probe every 0.3 to 0.5m. After each test the quality of the membrane, which is subject to ware and tear, is tested with a bubble test. Membranes are replaced when worn, which occurs generally every few days.

Continuous measurement Yes

Present measurement performance:

1. Detection levels For the most relevant contamints detection levels are in the order of 100µg/l. Several parameters such as membrane quality and acutual membrane temperature differ somewhat from site to site form date to date. Exact concentration levels are not measured. MIP is a screening technique

2. Selectivity The System uses three different detectors (FID, PID and DELCD), each detecting a different group of compounds: FID: all hydrocarbons PID: all non-saturated hydrocarbons DELCD: all chlorine containing hydrocarbons

52 SENSPOL Survey of Sensor Capabilities

It is hardly possible to distinguish which component form a certain group is present, For example when both PID and DELCD respond, this may indicate tera, tri,di- or monochloroethylene (vinylvhloride). Or it may indicate a mixture of benzene and trichloroethylene.

3. Repeatability The measurements are approximately repeatable. However, the quality of the membrane and the exact membrane temperature vary somewhat, resulting in somewhat higher of lower detector response. Further, the volatile compounds measured come form a thin slice of oil profile. In many cases the sol is rather heterogeneous, especially when non- dissolved phase is concerned; Therefore the results of two tests at almost the "same place" are not always very well comparable.

4. Potential interference Other volatile compounds in the subsoil may be detected such as methane or ethylene. Proper interpretation (often based on some knowledge of the compounds present at the site) can largely distinguish these compounds however.

5. Adaptability to other pollutant Not only the well-known compounds in certain mixtures of volatile contamination are measured. All compounds that can detected by one of the detectors used, show up in the results.

Further work developments It is possible to divert (part of) the gas to other instruments or to a sample container. This makes it possible to identify individual compounds by for example GCMS in the CPT truck (has already successfully been tested at a site with MTBE contamination) or allows sending of samples to a laboratory.

Envisaged performance and optimisation Quite a number of technical improvements have been made in the last few years to increase the stability and reliability of the measurements. Fugro is still working on further improvements that should make the system more rugged and increase daily production form about 60m of profile per day to about 100m of profile per day.

BOTTLENECKS

Use of a mass spectrometer allows direct identification for individual compounds, it is rather expensive however. MS detection would lead to about doubling the cost for screening, which are now comparable to the cost of drilling sampling and analysing a few samples per profile. Most clients do not seem to be prepared to pay extra costs of MS identification

On way to address this is to wait for the availability of more affordable sensors that would allow further identification/ speciation of the volatile compounds collected in the gas flow

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

The MIP system (block, membrane and detector array) is commercially available from Geoprobe. Fugro works together with Geoprobe on technical improvements. Fugro is commercially using the system as part of the consultancy services it provides. The system uses existing sensor allowing for satisfactory screening of the most frequently encountered contaminants

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FURTHER INFORMATION AND COMMENTS

A scheme of the system and an example of the presentation of the results are to be found in another document (description file MIP, not included here).

SENSOR SYSTEM 2

Description of sensor system

Rapid Optical Screning Tool (ROST) system integrated in a cone Pentration Testing (CPT) system The system consist of a laser producing a pulsating light at 290nm, a sapphire window through which the pulsating laser light is sent for excitation. The result fluorescence is captured behind the window and transferred to the detection system that measures the fluorescence signal at four different wavelengths in a certain timeframe. Fibre optics are used to transfer the excitation light to the window in the cone and the fluorescence signal from the window back to the truck at ground level.

The height of the signal thus obtained is a measured for the amount of product. The wavelength/ time pattern obtained is typical for the product (or mixture) found. Because tip resistance and sleeve friction of the cone are also measured, the correlation between soil litology and contamination can easily be found

Environmental relevance Investigation of almost all mineral oil products The system provides detailed profiling of contaminant against a detailed soil profile provide by the CPT system. To the system can be added integrated measurement of oil conductivity and soil pore water pressure.

Commercial relevance The ROST/CPT combination delivers much more detailed information than obtained by borehole drilling and sampling from monitoring wells. The cost are comparable or lower for soil investigation at depth of 10 to 40m-GL/

Usability (i.e skilled, unskilled) The system is only available with Fugro. It requires a skilled operator and experienced personnel for teatment and interpretation of the data.

Availability (eg. commercial, prototype, etc..) The system is commercially available with Fugro in several West-European countries and United States.

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) All mineral oil products. In fact the detection is based on the fluorescence pattern of the PAH that are present in most oil products. Even a very small amount of PAH in the oil product can be sufficient detection.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soil, Sediments and Groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) All phases are measured

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None, It is an in-situ measurement techniques

Sample handling (i.e. manual, automated etc,,,) n.a.

Sample size n.a.

Sensor Measurements

Assay protocol After set up the system and stabilisation of the fluorescence response, a standard test sample in a frame is exposed to the window in the cone. The test sample is used for calibration of the system.

Each test consists of pushing the Rost/CPT probe into the soil up to the required depth. The system records the fluorescence signal over each 1.5cm of profile.

Continuous measurement Yes

Present measurement performance:

1. Detection levels The Fluorescence response can strongly differ from one product to the other. The strength of the fluorescence depends on the type and amount of PAH present on one hand and on the amount of quenching of fluorescence (absorption of the fluorescence signal) mainly by heavy PAH molecules. Exact concentration levels are not measured. ROST is a screening technique. The system does produce highly detailed data over the profile however and does not suffer form disturbances like tailing effects. The system is therefore well suited to provide detailed and reliable measurements of the thickness of NAPL or DNAPL

2. Selectivity The system measures fluorescence at four different wavelengths that have been selected to obtain optimal results for a wide range of oil products. The waveform graphs that can be identify the product by comparison with the wave form graphs of known products stored in the systems database. In many cases the waveform does not lead to

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automated identification of the product, because biological degradation of mixing leads to a somewhat different waveform. It is possible however to increase the tolerance for the automated matching process, this also increases the risk of false correlation however.

3. Repeatability The measurements are well repeatable. However, the compounds measured come form a thin slice of soil profile. In many cases the soil is rather heterogeneous, especially when non-dissolved phased is concerned; therefore the results of two test at almost the same place are not always very comparable.

4. Potential interference Other PAH containing compounds in the subsoil may be detected. Proper interpretation can distinguish different products encountered however. The four wavelengths at which the system measures fluoresce have been selected such that fluorescence by humic acids does not interfere.

5. Adaptability to other pollutant Not only the well known (compounds in) products are measured. All PAH-containing compounds show up in the results. It should be realised that apart from the 16 PAH compounds of the EPA series that are reported in laboratory analysis, hundreds of anonymous PAH compounds often are resent in a product. All of these PAH compounds contribute to the fluorescence.

Further work developments The system collects massive amounts of data. Further developments will mainly be focussed on improving the presentation and interpretation of these data.

Envisaged performance and optimisation Fugro is still working on further improvements that should make the system more rugged.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

As far as we know no further technical development is taking place or foreseen in the near future. Any further developments would be done together with patent holder. Fugro is commercially using the system as part of the consultancy services it provides.

FURTHER INFORMATION AND COMMENTS

A scheme of the system and an example of the presentation of the results are to be found in another document (description file ROST, not included here).

PAPERS PUBLISHED

The US EPA produced a verification report (EPA/600/R-97/019) and a verification statement in January 1997 for the ROST system. In " Bodem" of August 1998 and article titled" de Rapid Optical Screening Tool Voor Detectie Van Bodemverontreiniging" was published Several Posters where presented at different conferences (among other Consoil 2000)

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PROFILE OF ORGANISATION

Fugro is large consultancy firm of which an important part of its business is in the field of geotechnical and environmental soil investigation. The company is active both on-shore and off-shore and has offices in more than 50 countries.

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IIQAB-CSIC

Institute Name: IIQAB-CSIC Department: Environmental chemistry Address: Jordi girona 18-26, 08034 Barcelona, Spain Respondents name: Silvia Lacorte Position: Scientific researcher Email address: [email protected]

SENSOR SYSTEM

Description of sensor system A portable amperometric biosensor working with screen-printed electrodes has been developed to analyze pesticides in different matrices. It includes a home made potentiostat and a current to voltage converter that yields an output voltage proportional to the sensor current. This output voltage is converted to a digital format using an analog to digital converter (ADC), and then this information is sent to the PC, using a standard serial interface. Moreover, a program in C for Virtual Instrumentation (Labwindows) has been designed to control the biosensor: the user can have a graph of the current plotted, autoscale the graph, make calibrations, and store data and information about current waveforms in files. The applicability of the portable biosensor has been performed by analysing blind solutions containing or not organophosphorus and carbamate pesticides in the following matrices: surface and waste water, fruit extracts, vegetable extracts, fruit juices, tea, champagne and milk.

Figure: Whole work station

Environmental relevance The biosensor permits to detect the total pesticide concentration at the mM range (ppb concentration) with a very high reproducibility (max 12%), good accuracy (detects presence or absence of pesticides) and therefore is suitable to be used as screening method for pesticide analysis.

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Commercial relevance Since the system can discern between positive and negative samples, it would be very useful to discard those samples with negative response (not toxic) so that the chromatographic analysis, much more time consuming, sophisticated and expensive, could be minimized. An analysis with the biosensor may take 20 min without any need for sample preparation. In addition, since it includes a battery and a portable PC, it can be used in the field.

Usability (i.e skilled, unskilled) Not highly skilled people are needed to run the biosensor in a routine basis.

Availability (eg. commercial, prototype, etc..) Prototype

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Organophosphorus and carbamate pesticides if using the enzyme acethylcholinesterase in the screen printed electrode. Other compound families could be used, if the enzyme in the electrode was changed. E.g. tyrosinase to detect phenolic compounds.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) All types of waters, fruit juice, etc. .

Sample phase (i.e. total, residual, free, dissolved or absorbed) dissolved

Sample preparation: (e.g. filtering, SPE, derivatisation etc…) Filtering water only in the case of very charged samples. For fruits, solvent extraction.

Sample handling (i.e. manual, automated etc,,,) Manual, but it consists only of inserting the electrode in the sample.

Sample size 10 x 8 cm , a stirrer and a portable PC

Sensor Measurements

Assay protocol Determination of the percent inhibition of acetylcholinesterase when placing the electrodes in contact with different solutions of buffer (PO4, pH=8, 0.05 M) that contained known concentrations (in the range of 0.5·10-9 M to 4·10-9 M) of chlorpyrifos-oxon, and the obtained calibration curve is approximately linear in this range, with inhibitions from 10% to 80%. These measurements for the calibration curve were made using electrodes that contained 2 mU of deposed acetylcholinesterase enzyme over the working electrode. Measurments were also made using electrodes containing 5 mU of enzyme, and the obtained results were that with this kind of electrodes the sensitivity was lower, but the saturation threshold was higher.

Continuous measurement No

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Present measurement performance:

1. Detection levels - 1 ug/L

2. Selectivity Only for total concentration of organophosphorus compounds and carbamates.

3. Repeatability 5-10%

4. Potential interference Surfactants

5. Adaptability to other pollutants Good, by changing the enzyme immobilized upon the screen printed electrode

Further work developments Develop an array of electrodes for the determination of a wide number of chemical classes of pollutants

Envisaged performance and optimisation

Since the system is very reproducible, sensitive and selectivity can be enhanced by some sample pre-treatment (e.g. filtration), it is envisaged that an array of biosensors could be very useful for site measurements, since the portability is very good (20 g, connected to a portable PC)

BOTTLENECKS

We are still developing more applications. However, since there is no project behind, it goes very slowly.

We plan to ask for a project to develop and enlarge the electronic part and then we will again test the performance and applicability.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

The biosensor has been developed by the Polytechnical University of Catalunya, under the supervision of Dr. Ramon Bragos. The screen-printed electrode design and optimisation has been performed in the Centre de Phytopharmacie, of the University of Perpignan, under the supervision of Dr. Jean Louis Marty.

No commercial backing. An enterprise which could prove the real applicability.

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FURTHER INFORMATION AND COMMENTS

The sensor could be specially suitable for screening processes, in both environmental and food safety and quality controls.

PAPERS PUBLISHED

1. J.L. Marty, N. Mionetto, S. Lacorte and D. Barceló. Validation of an enzymatic biosensor with various liquid-chromatographic techniques for determining organophosphorus pesticides and carbaryl in freeze-dried waters. Analytica Chimica Acta, 1995, 311, 265- 271. 2. D. Barceló, S. Lacorte and J.L. Marty. Validation of an enzymatic biosensor with liquid- chromatography for pesticide monitoring. Trends in Analytical Chemistry, 1995, 14 (7), 334-340. 3. S. Lacorte, G. Jeany, J.L. Marty and D. Barceló. Identification of fenthion and temephos and their transformation products in water by high performance liquid chromatography with diode array detection and atmospheric presure chemical ionization mass spectrometric detection, Journal of Chromatography A, 1997, 777, 99-114. 4. Conference “Methods of detection of chlorpyrifos and transformation products based on biosensors and immunosensors” in Biosensors for Environmental Monitoring, Menorca, España, December 1999. 5. Coordination of master studies: Proyecto Final de Carrera de Jordi Ribas, "Development of a portable biosensor for the determination of pesticides in the environment", Universidad Politécnica de Catalunya, july 2000. 6. Master Experimental de Alain Hildebrandt “Optimization and validation of a portable biosensor for the detection of organophosphorus and carbamate pesticides in environmental samples”, presentado en el Departamento de Química Analítica de la Facultad de Químicas de la Universidad de Barcelona, Septiembre 2002. 7. Poster: Validation of an enzymatic biosensor with liquid-chromatography for pesticide monitoring in 3rd European Workshop on Biosensors for Environmental Monitoring organizado por la "European Commission, Directorate General XII for Science Research and Developement. Environmental Research Program", Florencia, Italia, February 1995. 8. Poster: New biosensor development and existing sensors improvement by means of “smart sensor”, Fourth European Workshop on Biosensors for Environmental Monitoring, Barcelona, España, February 1996.

PROFILE OF ORGANISATION

CSIC is a public research organization with a multi-disciplined and multi-sectorial character. It is composed by around 130 research institutes and 9000 people including scientific, technical and administration personnel. Two institutes will participate in this project, the Instituto de Ciencia de Materiales de Barcelona (ICMAB) and the Instituto de Investigaciones Químicas y Ambientales de Barcelona (IIQAB). The Department of Environmental Chemistry is part of the IIQAB and has a total budget of around 800.000 Euros per year (excluding salaries of the permanent staff) in different projects, being approximately 50% financed by the local and state budget and the other 50% by the EU. Within the Department, S. Lacorte has been involved in different scientific activities related to water and sediments quality in the European Union. The group is devoted to the development of novel mass spectrometric analytical methods for the analysis of organic pollutants in environmental matrices. Among others, compounds of environmental interest are pesticides, phenols, dyes, detergents, PAHs and endocrine disrupting compounds

61 SENSPOL Survey of Sensor Capabilities like 17-B-estradiol and polybromodiphenylethers. In addition, pharmaceuticals have also been analyzed and detected in urban effluents as a consequence of their widespread use in human and veterinary medicine. Techniques available are gas chromatography coupled to mass spectrometry (GC-MS) and coupled to high resolution mass spectrometry (GC-HRMS) for exact mass determination and liquid chromatography coupled with mass spectrometry with electrospray and atmospheric pressure chemical ionization interfaces (LC-MS). Other activities are related to toxicity measurements, using the Tox-alert system, CellSense, biosensors and ELISA tests and yeast recombinant assay.

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INSITUTE OF HYDRAULIC ENGINEERING

Institute Name: Institute of Hydraulic Engineering Research group: VEGAS– Research Facility for Subsurface Remediation Address: University of Stuttgart, Pfaffenwaldring 61 D- 70550 Stuttgart, Germany Respondents Name: PD Dr. B. Barczewski Position: Scientific Director Email address: [email protected]

SENSOR SYSTEM 1

Description of sensor system Detection system for contaminants in the groundwater based on fluorimetry

Environmental relevance Detection of in the groundwater

Usability (i.e skilled, unskilled) unskilled

Availability (eg. commercial, prototype, etc..) - measurements available - prototype available

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) PAHs

Sample matrix ( e.g. soils, sediments, surface and groundwater ) groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) dissolved

Sample preparation (e.g. filtering, SPE, derivatisation etc…) filtering

Sample handling (i.e. manual, automated etc,,,) automated

Sample size 100ml

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Sensor Measurements

Assay protocol Sensor signals

Continuous measurement Yes

Present measurement performance:

1. Detection levels - currently down to approx. 1ppb

2. Selectivity Currently not

3. Repeatability Very good

4. Potential interference Humic substances

5. Adaptability to other pollutants Currently not planned

Further work developments Miniaturisation, ruggedization

Envisaged performance and optimisation New components, commercially available prototype

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

The sensor device is conducted under the DBU-Project „High-Tech-Methods for the Probing of the Subsurface” wee there are 12 Partners. There is no commercial backing. However, since applying the system in the field we have partners from engineering companies, which supply the field sites

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SENSOR SYSTEM 2

Description of sensor system Sensor for detecting NAPL (liquid phase contaminants) based on optical fibers

Environmental relevance Detection of BTXE, CHC, Mineral oil

Usability (i.e skilled, unskilled) unskilled

Availability (eg. commercial, prototype, etc..) - measurements available - prototype available

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Detection of mineral oil, BTEX and chlorinated hydrocarbons in liquid phase (NAPL). No distinction possible

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils, groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) Free

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None

Sample handling (i.e. manual, automated etc,,,) None

Sample size Few drops

Sensor Measurements

Assay protocol Sensor signal, digital Signal: NAPL present/absent

Continuous measurement Yes

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Present measurement performance:

1. Selectivity None

2. Repeatability Depends on the contaminant

3. Potential interference None

Further work developments More rugged design, other optical components, miniaturisation

BOTTLENECKS

After usage the sensor might be contaminated and has to be changed

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

There are no partners helping in developing this sensor and there are no commerical backers

SENSOR SYSTEM 3

Description of sensor system - Detection system for contaminants in the soil gas - Consists of metal oxide sensors and quartz oscillators

Environmental relevance Detection of mineral oil, BTEX and chlorinated solvents

Usability (i.e skilled, unskilled) unskilled

Availability (eg. commercial, prototype, etc..) - measurments available - prototype soon available

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Detection of mineral oil, BTEX and chlorinated hydrocarbons, distinction between different contaminant groups possible

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Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soil gas

Sample phase (i.e. total, residual, free, dissolved or absorbed) Gas phase

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None

Sample handling (i.e. manual, automated etc,,,) Automated

Sample size 1-2 liter gas

Sensor Measurements

Assay protocol Sensor signals, temperature, humidity

Continuous measurement Yes

Present measurement performance:

1. Detection levels - Ca. 100ppm until 30.000ppm

2. Selectivity Between different contaminant groups

3. Repeatability Not possible with one sample

4. Adaptability to other pollutants To volatile components possible

Further work developments Prototype should be commercially available in near future

BOTTLENECKS

Due to different subsurface situation, enough soil gas should be available

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

There are 12 partners, the sensor has been developed under the DBU-Project „High-Tech- Methods for the Probing of the Subsurface” (12 Partners). There are no commercial backers. Since we are applying the system in the field we have partners from engineering companies which supply the field sites

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PAPERS PUBLISHED

Please list any publications/ posters or presentations related to this sensor Barczewski, B., Batereau, K., Klaas, N., Schmid, G. (2000): Sensors for on-site assessment of contaminated sites, Abstracts of lecture group Environmental Technology, Achema 2000, Frankfurt am Main, 22.-27.05.2000 Batereau, K., Klaas, N., Barczewski, B. (2001): On-Site Assessment of Contaminated Sites: Application of Sensors for On-Site Instrumentation, Field Screening Europe 2001, ISBN 1- 4020-0739-6, Kluwer Academic Publishers, 2002 Klaas, N., Batereau, K., Barczewski, B. (2001): Assessment of Contaminated Sites: Development of Field Screening Instruments, Field Screening Europe 2001, ISBN 1-4020- 0739-6, Kluwer Academic Publishers, 2002 Batereau, K., Barczewski, B., Klaas, N. (2002): On-site measurement of VOC: Application of sensors for site characterization, IN: Halm, D. and Grathwohl, P. (2002): Proceedings of the 1st International Workshop on Groundwater Risk Assessment at Contaminated Sites (GRACOS), Tübinger Geowissenschaftliche Arbeiten (TGA), S. 265 – 269 Batereau, K. (2002) : Application of sensors for site characterization, NICOLE Meeting “Cost-effective site characterization – Dealing with uncertainties, innovation, legislation constrains, Pisa, 2002 Batereau, K., Barczewski, B., Müller, M. (2002) : Application of sensors for site characterization, Response to New Pollution Challenges, 2nd SENSPOL Workshop, June 2002

PROFILE OF ORGANISATION

The VEGAS Concept and Facility

VEGAS is a research facility for subsurface remediation located at the University of Stuttgart. It was funded by both the German Federal Ministry of Education and Research and the Baden-Württemberg Ministry of Environment and Transport. Use and access to the facility are regulated by the VEGAS Statute which was established in a cooperation agreement between the university and the above mentioned ministries. Criteria and priorities for the use of the VEGAS facility are decided upon by an independent scientific advisory board.

The VEGAS Profile

Available to on and off-campus research institutions, industry, consulting companies and cooperation partners abroad

• Facility for Research and Development (R+D) projects at the technical scale • Link between laboratory and field research • R+D+A principle: Research + Development + Application • Transfer of knowledge and technology • Interdisciplinary research • International co-operation The main feature of the VEGAS facility is its laboratory with its large containers (max. capacity: 750m3). This facilitates the investigation of remediation technologies with realistic 3-D subsurface structures at both medium and large scales. In contrast to field investigations VEGAS offers controllable and reproducible conditions which are essential for numerical modelling as well as mass balancing. The main advantage of

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VEGAS experiments in comparison to more traditional laboratory research is the ability to simulate natural subsurface conditions more realistically.

VEGAS Research and Development efficient, cost effective, environmentally suitable measures

Research topics - Remediation of Hot Spots (in-situ extraction and degradation of contaminants) - Modification of Subsurface Properties (in-situ methods of containment and remediation) - Characterization and Evaluation Procedures (field screening, risk assessment, monitoring) - Brownfields Restoration, Soil Protection (overall concept, combination of methods) - Transfer of Technology from pilot studies to commercial use

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RS DYNAMICS LTD

Company Name: RS DYNAMICS Ltd. Address* BOCNI II/1401, 141 31 Prague 4, Czech Republic Respondents Name: George BLAHA, Lada Kouklik Position Director, Chief Geophysicist Email address* [email protected] [email protected]

SENSOR SYSTEM

Description of sensor system RS DYNAMICS has developed a new methodology and instrumentation -- Ecoprobe 5, which is especially useful for the in-situ mapping of contamination spread caused by various environmental disasters, tank or tube leakage, various residuals from leaky storage-containers or from other surface contamination. Ecoprobe 5 comprises two autonomous analyzers -- PID and four channel IR -- in one container. This combination provides interpretation for a variety of contamination survey tasks.

PID analyzer measures total concentration of volatile organic compounds and other toxic gases including chlorinated hydrocarbons, detection limit 0.1 ppb IR analyzer consists of three independent channels (plus one reference channel), which simultaneously measure the following parameters: a) SV concentration of the most common group of petroleum products – detection limit 20 ppm b) Methane - detection limit 20 ppm c) CO2 - detection limit 20 ppm

Environmental relevance Ecoprobe 5 is widely used by remediation companies, Universities, methane monitoring companies and others. Ecoprobe 5 offers fast in-situ contamination mapping and thus helps to prevent contaminant to be spread and reach the underground water table.

Commercial relevance Ecoprobe 5 provides cost-effective, definitive soil-contaminant surveys. Very fast and reliable in–situ mapping of contaminated sites secures fast return of the investment.

Usability (i.e skilled, unskilled) Unskilled users can operate the instrument after the training.

Availability (eg. commercial, prototype, etc..) Ecoprobe 5 represents the last model in the Ecoprobe development line based upon extensive experience from the previous versions. Ecoprobe 5 is a completed system commercially available, nevertheless RandD program continues on the project.

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) LNAPL, DNAPL , PAH

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soil; Air in special applications.

Sample phase (i.e. total, residual, free, dissolved or absorbed) vapour phase (soil gas) or air

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil gas is sucked directly and automatically into the instrument without any preparation

Sample handling (i.e. manual, automated etc,,,) As above

Sample size Inner pump speed is adjustable (from 0.25 to 4 litres of soil gas/minute)

Sensor Measurements

Continuous measurement : Yes, together with a typical measurement during a pre-settable interval. Automatic monitoring mode is also available

Present measurement performance:

1. Detection levels PID analyser – 0.1 ppb IR analyser – Hydrocarbon Channel – 20 ppm Methane Channel - 20 ppm CO2 Channel - 20 ppm

2. Selectivity PID analyser – measures the total amount of organic compounds with no selectivity IR analyser – selective reading of methane, CO2 and a group of petroleum hydrocarbon compounds

3. Repeatability – about 1%

4. Potential interference PID: as per ion potential detection table (see www.rsdynamics.com IR: Methane channel – phenols (0.1%); other channels – various compounds < 1%.

5. Adaptability to other pollutants Yes

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Further work developments RandD on: - different analytical technologies (laser florescence, hot PID) - Internet communication between the remote ECOPROBE 5 and the main office (data download, calibration, firmware upgrading…) - almost finished; - ECOPROBE 5 connection to various sampling machines (AMS…); - Development of PID analyser not influenced by moisture. - Introducing and improving new and already existing sensors to the practice is a matter of coming research. (From other fields RS DYNAMICS is involved in RandD for medical and security purposes)

Envisaged performance and optimisation RS DYNAMICS has developed advanced signal processing system, enabling to gain signal for very small concentration of contamination. This featurer allows to catch even very small values coming from deep contaminations or from contaminations caused by almost no volatile substances like cutting and transformer oils. The PID sensor is especially constructed to suppress moisture. Our RandD is still focused on moisture suppression, based on a new approach.

For IR analyser the advanced signal processing system enables to catch exceptional low contamination level of 20 ppm (methane, hydrocarbon compounds, CO2), which is unique for earth science instruments.

BOTTLENECKS

1. Design and testing of mechanical part of the instrumentation 2. Administrative and various paperwork 3. Better government arrangement

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

InfraTec BmbH, Dresden/Germany participated in the development of infrared sensors. No commercial backing

Any commercial help would be appreciated. Extensive RandD has been based only upon marketing the ECOPROBE instruments that still does not cover the possible RandD expansion and capability of the company research team.

PAPERS PUBLISHED

RS DYNAMICS has attended several environmental exhibitions: International Symposium and Exhibition on Environmental Contamination in Prague in 2000, the same conference in 2001 in Orlando, Florida – both with oral presentation and the booth; Environmental exhibition POLLUTEC in Paris in 2001 and the same exhibition in 2002 in Lyon, France. We attended several smaller exhibition and conferences, and many instrument presentation around the world.

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PROFILE OF ORGANISATION

RS DYNAMICS was established in 1990 in Prague, based on a research group previously developing geophysical instruments. Since 1990 the RandD was focused on environmental instruments - ECOPROBE, using close collaboration with universities and remediation companies to fulfil their requirements for the in-situ instrumentation and methodology for the soil contamination survey. Together with environmental instruments, RS DYNAMICS RandD group is engaged in development of other instruments for medical and security purposes.

RS DYNAMICS research group has 5 electronic and software engineers. RS DYNAMICS collaborates with several companies and Universities to carry out the research and production of the instruments. About 30 people are engaged in the ECOPROBE 5 production.

In 2001 RS DYNAMICS has established daughter companies in France and Japan (2002) which are specialized in selling and marketing the instruments.

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SENSOR TECH LTD

Company Name: Sensor Tech Ltd Address: C/o Harston Mill, Harston, Cambridge, CB2 5GG, UK Respondents Name*: Dr Duncan Purvis Position Technology Development Manager Email address* [email protected]

SENSOR SYSTEM

Description of sensor system Potentiometric Biosensor based on screen-printed electrodes coated with a conjugated conducting polymer and using an ELISA or enzyme biosystem as the recognition part of the sensor platform. This platform technology was initial developed to work on clinical diagnostic assays.

80 70 60 50 40 30

Response (mV) 20 10 0 ) ( 0.001 0.0 0.01 0.1 1 10 Detection[TNF], of TNF ng/ml in 50% serum, using potentiometric immunoassay.

Current Sensor Formats

(Descriptive flier available for more details)

Environmental relevance Currently transferring PCB, Aflatoxin M1 and Estradiol assays to the system as further proof of application. All of which are of concern to the environment.

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Commercial relevance Works with complex fluids, inexpensive to manufacture. Interest from food quality control an environmental market sectors, Fast, sensitive and quantitative. Ability to transfer current ELISA systems directly onto the platform

Usability (i.e skilled, unskilled) Currently skilled but being reduced to unskilled for commercialisation

Availability (eg. commercial, prototype, etc..) Prototype, with a commercial system being developed for environmental applications, initially for persistent organic pollutants. (POPs). This should be available for validation late 2003.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…)

All analytes, which can currently, or potentially, be detected using enzyme, linked assays, and assays which produce a redox, pH or ionic change during reaction or recognition. These include DNA assays for GMO detection

Sample matrix ( e.g. soils, sediments, surface and groundwater )

All matrices where sample preparation can provide a aqueous phase containing contaminant. This includes soil, oil, food, water.

Sample phase (i.e. total, residual, free, dissolved or absorbed)

Free aqueous

Sample preparation (e.g. filtering, SPE, derivatisation etc…)

Whatever is required to give an aqueous phase.

Sample handling (i.e. manual, automated etc,,,)

Currently manual and possibly intensive depending on analyte and matrix, but will be reduced to manageable widgets and/or automated.

Sample size

Currently require100µl extracted sample for ease of use but can use less than 10µl if necessary. Usually sample size is not an issue.

Sensor Measurements

Assay protocol

Same as an ELISA, but can use more complex fluids without much difficulty. If it is just an enzyme assay such as can be used for nitrates, this could possibly be used directly.

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Continuous measurement No

Present measurement performance:

1. Detection levels - For our clinical models we were getting pg/ml sensitivity (fM). We expect to get similar results with our environmental models (ppb). We have always improved the sensitivity of a commercial sandwich immunoassays and reduced the assay time significantly. We are now working on improving competitive assay performance.

2. Selectivity: Depends on the antibodies or DNA used

3. Repeatability: CV’s of <10% at

4. Potential interference: Crossreactivity, overhead powerlines, oil contamination of samples.

5. Adaptability to other pollutants Very adaptable.

Further work developments Multianalyte for testing panels of analytes.

Envisaged performance and optimisation Multianalyte, ppb quantitative sensitivity in less than 15minutes. Electronic analysis and data storage.

BOTTLENECKS

We need access to fluidics expertise and assays for transfer to our system. But it is mainly resource limited. Networking, knowing the right people. More money and partners. Would help the development of the sensor.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Yes we have a collaboration with an environmental analysis company for PCB detection. A small sum for doing feasibility in both environment and Food quality control. Hopefully leading to more when complete. We are looking for more partners and have several under discussion.

There are many other type of collaboration that would help in sensor development including Corporate, Universities, JV’s and also acquisition.

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FURTHER INFORMATION AND COMMENTS

See website: www.sensortech-uts.com

The gap between sensor developers and end users can be bridged by working together.

PAPERS PUBLISHED

Biosensors 2002 (Kyoto). See website for copy of the poster. Paper submitted at this conference, currently with referees

PROFILE OF ORGANISATION

We are a small start-up company, currently incubating within the incubation facilities at Scientific Generics Ltd. Looking for corporate partners in many market sectors including environmental. We are also looking for synergistic technologies and expertise in assay development and fluidics.

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UFZ CENTRE OF ENVIRONMENTAL RESEARCH

Organisation Name: UFZ Centre for Environmental Research Department: UbZ Environmental Biotechnology Centre Research group: Applied biosensor development Address: Permoserstr. 15, D-04318 Leipzig, Germany Respondents Name: Dr. Beate Strehlitz Position: Deputy head of the UbZ, Team leader Applied Biosensor Development Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

Phenol biosensor Amperometric mediator-modified enzyme sensor; Technology: thick film printing Enzyme: Tyrosinase, PCS-matrix immobilized Transducer: amperometric three electrode arrangement (SensLab) Application with FIA-system (IMT Dresden) Measurement of phenolic compounds

Environmental relevance Control of ground and surface waters with phenol contamination Control of waste water treatment plants

Commercial relevance Measurement is more easy and cheaper than standard determination procedures (e.g. DIN), avoidance of usage of toxic chemicals for measurement Method application for specific sample is necessary

Usability (i.e skilled, unskilled) Daily use by unskilled, weekly supervision and solving of problems by skilled employees

Availability (eg. commercial, prototype, etc..) prototype

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Phenolic compounds

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Surface and ground water, waste water,

Sample phase (i.e. total, residual, free, dissolved or absorbed) total

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Filtering (if necessary)

Sample handling (i.e. manual, automated etc,,,) automated

Sample size Ca. 50 ml

Sensor Measurements

Assay protocol Sample and calibration solution have to be provided Measuring protocol of the device: calibration procedure – measuring procedure – measuring break with cooling down and reducing speed of buffer flow Display of the measuring result in µg/l

Continuous measurement Yes

Present measurement performance:

1. Detection levels - 2 µg/l phenol

2. Selectivity Depending from the selectivity of tyrosinase for phenolic compounds, measurement of a sum parameter

3. Repeatability Standard deviation 5 %

4. Potential interference Sulfide, sulfite (as far as known)

Further work developments If customers are interested: Revision of sensor technology and FIA-device for commercialisation, application for specific measuring task

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BOTTLENECKS

The potential market is too small for successful commercialisation. Potential users need comparability to standard method results Search for new applications (food industries?). Give information and examples for application to convince the potential users.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Basic sensor manufacture: SensLab GmbH, Leipzig FIA device development and prototype: Institute for Medical Technology, Dresden

Do have commercial backing on joint research projects

FURTHER INFORMATION AND COMMENTS

Users of biosensor systems need intensive guidance and careful application of the sensor for the specific measuring task.

PAPERS PUBLISHED

1) Gründig, B.; Strehlitz, B.; Krabisch, C.; Thielemann, H.; Kotte, H.; Gomoll, M.; Kopinke, H.; Pitzler, J.: The use of redox mediators for amperometric biosensors. In: R.D. Schmid, F. Scheller (Eds.): Biosensors: Fundamentals, Technologies and Applications. GBF-Monographs, Volume 17. VCH: Weinheim, 1992, S. 275-285 2) Kotte, H.; Gründig, B.; Vorlop, K.-D.; Strehlitz, B.; Stottmeister, U.: NMP+-modified enzyme sensors based on polymer thick films for subnanomolar detection of phenols. Analytical Chemistry 67 (1995) 65-70. 3) Kopinke, H.; Gründig, B.; Strehlitz, B.: Durchflußmeßzelle für Biosensoren. DE 195 37 506; 26.9.1995. 4) Strehlitz, B.; Gründig, B.; Kopinke, H.; Hegewald, B.; Riis, U.: Planar structured phenol sensor for long-term application in FIA-systems. Poster: 2nd Workshop on Biosensors and Biological Techniques in Environmental Analysis. 11.-13.9.1996, Lund. 5) Strehlitz, B.; Gründig, B.; Kopinke, H.; Hegewald, B.; Riis, U.: Planar structured phenol sensors for long - term application in FIA - systems. Vortrag zum Fifth European Workshop on Biosensors for Environmental Monitoring and Stability of Biosensors. 28. - 30.5.1997, Freising. 6) Strehlitz, B.; Gründig, B.; Kopinke, H.; Böhland, C.: Field measurement of phenol, nitrate, and nitrite with amperometric enzyme sensors (S. 46-50) und 7) B. Strehlitz: Field measurement of phenol with hand hold device and amperometric enzyme sensors (S. 87-90) in: EU-Bericht: Two European Technical Meetings Biosensors for Environmental Monitoring. Ed.: Hansen, P.D.; Köhler, J.; Nowak, D.; ENV 4 CT 97 6154/ ENV 4 CT 98 6137; 1999. 8) Strehlitz, B: (Bio)Sensors for Environmental Analysis of Phenols and Ammonia. Vortrag auf dem Fourth Workshop on Biosensors and Biological Techniques in Environmental Analysis, 1.- 3.12.1999, Maó, Menorca, Spain. 9) Strehlitz, B.: Angewandte Biosensorik – FIA-Monitor für die Phenolbestimmung. Poster zur Analytica 2002 (UFZ-Stand), 23.-26.04.2002, München.

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10) Strehlitz, B.; Bauer, H.-J.; Böhland, C.; Kröber, H.; Müller, R.A.; Hanke, G.: Application of FIA-Monitor with biosensor for phenol monitoring in a waste water treatment plant. Poster zum SENSPOL Workshop: Response to new pollution challenges, 04.-07.06.02, London.

PROFILE OF ORGANISATION

The major tasks of the UFZ Centre for Environmental Research are remediation and renaturation of contaminated landscapes, as well as the preservation of natural landscapes. The aim is to develop sustainable land use systems for impacted landscapes (e.g. agricultural, industrial, and mining areas), their terrestrial and aquatic ecosystems, and their typical flora and fauna. The UbZ Centre for Environmental Biotechnology at the UFZ has competence in development, application and translation of biotechnological methods into practice. The UbZ closes the gap between applied research and industry. The UFZ's scientific spectrum is expanded by the UbZ's skills in active, project-based technology transfer: commercial companies are integrated into co-operation projects at an early stage to ensure the smooth transfer of research work. The Applied Biosensor Development is one of the UbZ topics with several projects dealing with development, validation and application of biosensors. Modern methods are used for sensor development, especially new recognition elements based on DNA structures (e.g.aptamers).

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UNIVERSIDAD COMPLUTENSE DE MADRID GRUPO INTERLAB, S.A.

University Name: UNIVERSIDAD COMPLUTENSE DE MADRID

Department: Faculty of Chemistry, Dpt.Organic Chemistry Laboratory of Applied Photochemistry

Address* Departamento de Quimica Organica Facultad de Quimica Universidad Complutense de Madrid 28040 MADRID (Spain)

Respondents Name: Guillermo Orellana Position Professor Email address* [email protected]

SENSOR SYSTEM

Description of sensor system From the successful combination of chemical and technological advances in molecular engineering, photochemistry, analytical spectroscopy, material science, optoelectronics and information technology, the current fiberoptic chemical sensors have been born. Such tools are versatile rugged devices capable of real time, continuous, in-situ, multiplexed monitoring of concentration levels of chemical species in harsh environments, unaccesible or constrained spaces, explosive atmospheres, or strong radiation (electromagnetic, nuclear) fields. Moreover, sensors may be developed for quantification of analytes for which no alternative monitoring devices are currently available. The instrumentation that uses such type of sensors (Figure 1) consists of a light source, the radiation of which is launched into the core of an optical fibre. The waveguide carries the light to the sensitive tip, —the true 'heart' of the device—, that contains a specific indicator molecule (reagent material) immobilised onto a polymer support. Upon (selective) interaction with the target analyte species, the luminescence of the reagent material undergoes a measurable change. In this way, the emitted light leaves the sensitive tip and is guided back through the optical fibre to a photonic transducer, the electrical signal of which is conveniently amplified and digitally recorded. An appropriate calibration using standard samples allows carrying out in situ, continuously the measurement of the target chemical species, since the sensor-analyte interaction is reversible. Fiberoptic chemical sensing based on luminescence measurements has become the technique of choice due to the clear advantages it confers to the resulting devices (higher sensitivity, selectivity, versatility and operational lifetime). Our challenge has been to design and prepare a homogeneous family of luminescent indicator dyes, all of them being excitable by blue light and emitting red light. In this way, it is easy to separate the strong excitation light from the luminescence. Moreover, it allows the use of a single optoelectronic device to interrogate multiple sensitive terminals for different chemical parameters (Figure 2).

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The OPTOSEN® system, developed by Grupo INTERLAB in collaboration with the Laboratory of Applied Photochemistry and Optical Sensors Groups at Complutense University of Madrid (UCM), is an environmental/industrial sensing instrument capable of simultaneously monitoring several parameters (see below) that are relevant to control water, air and/or soil quality. The optoelectronic system is based on fibre optics and measures the luminescence of specific indicator dyes immobilised at the distal end of the light guide. In this way, a family of novel interchangeable sensors has been developed to monitor up to 8 different parameters (or a single parameter in multiple locations) due to the multichannel configuration of the OPTOSEN® system. Moreover, the optical nature of the monitoring principle yields a longer operational lifetime of the sensitive terminals compared to many competing sensors currently used for environmental or industrial control.

Figure 1. Basic components of fiberoptic chemical sensors developed at UCM (F: wavelength selectors; D: photonic detector). An oxygen-sensitive fluorescent indicator material is depicted in the microphotography. A scheme depicting how dissolved oxygen is measured using fluorescence quenching of the (immobilised) indicator molecule is included on the left of the figure.

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Figure 2. Fiberoptic luminescent sensor developed at UCM and multichannel optoelectronic system OPTOSEN® for in-situ environmental chemical measurements and process control developed, manufactured and marketed by Grupo INTERLAB (Madrid).

Figure 3. (clockwise from left) Examples of several UCM’s fiberoptic oxygen sensors for on-line measurements in a water treatment plant, a pilot composting unit and a laboratory fermentor.

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Figure 4. Examples of several Grupo INTERLAB-UCM’s OPTOSEN® multiparametric fiberoptic systems installed at a riverside automatic water-monitoring booth (dissolved oxygen, temperature, pH), in a a water treatment plant (BOD, dissolved oxygen), and an atmospheric monitoring station (relative humidity, temperature).

Environmental relevance

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Commercial relevance

♦ Multichannel system: Capable of measuring and logging up to 24 target parameters (8 optical channels + 16 electrical inputs) ♦ Versatility: Each instrument can be configured to operate either independently or in slave mode within a distributed environmental monitoring network ♦ Local processing features and signal outputs to control the monitored site ♦ Connectivity: The measured parameters can be sent to a remote base station through different communication options (wire, radio, GSM,...) ♦ Long-life optical sensors to minimize maintenance and facilitate remote distributed deployment ♦ Cost-effective: A single instrument may substitute several different monitors to perform all measurements required for environmental compliance or process control

Usability (i.e skilled, unskilled) Usable by unskilled operators. Basic initial training is required.

Availability (eg. commercial, prototype, etc..) Commercially available from Grupo INTERLAB (Madrid, Spain) (www.interlab.es; c/o Jesus Delgado). A wide number of features are customisable (i.e. number of channels, sensor tips, materials in contact with sample, flow-through cells, sampling co-systems,...)

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…)

♦ O2 (both dissolved and gas phase) ♦ Temperature ♦ pH ♦ BOD (in situ, almost real time)

♦ CO2 (dissolved and gas phase) ♦ Humidity ♦ Hydrocarbons (dissolved)

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Surface/ground water, atmosphere

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None (relative “clean” waters or atmosphere; suspended matter or coloured substances do not affect measurements) or pre-filtering (“dirty” waters, industrial effluents)

Sample handling (i.e. manual, automated etc,,,) Automated (data logged by OPTOSEN system); local connection (RS-232) provided. Remote instrument control and data dumping are available by basic telephone line or GSM.

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Sample size Depends on sensor tip (customisable; fibre optics allow miniaturisation)

Sensor Measurements

Assay protocol Introduce sensor tip into water or expose to air (directly or through piping). Some analytes may require pre-filtering or water conditioning steps. Sensor cleaning or off-line calibration are required from time to time depending on the specific analyte.

Continuous measurement Yes

Present measurement performance:

1. Detection levels – (typical values)

Molecular OXYGEN Measurement range 0.03 - 100% (in air) 0.04 - 35 mg L-1 (in water) Precision 0.02 mg L-1 Response time < 6 s (in air) Sample temp. range -10 to 60 ºC

TEMPERATURE Measurement range -5 to 70 ºC Precision 0.1 ºC Response time < 1 min Sample temp. range -30 to 90 ºC

pH Measurement range 4 - 9 pH units Precision 0.05 pH units Response time < 60 s Sample temp. range up to 50 ºC

BOD Measurement range 1 - 2000 mg L-1 Precision 0.5 mg L-1 Response time < 30 min Sample temp. range 10 to 40 ºC

CO2 Measurement range 0.1 - 30% (in air) Precision 1% of the measured value Response time < 20 s Sample temp. range 0 - 40 ºC

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Humidity Measurement range 1 - 100% RH (at 30 ºC) Precision 0.3% Response time < 6 s Sample temp. range -10 to 50 ºC

Hydrocarbons Measurement range 5 – 1500 ppm (in water) Precision 5% Response time < 120 s (water flow dependent) Sample temp. range up to 50 ºC

2. Selectivity Temperature correction required. Some analytes require additional atmospheric pressure or oxygen level correction.

3. Potential interference Depends on sensor type

4. Adaptability to other pollutants Customised developments available for other analytes.

Further work developments Additional in situ validation for some of the chemical parameters listed above.

Envisaged performance and optimisation Extensive customisation according to operational requirements of environmental measurements or industrial control.

BOTTLENECKS

Extensive in-situ validation is very expensive. Nevertheless, it is absolutely essential to jump the gap between sensor development and commercial success. Actual environmental conditions are harsher than laboratory experiments. Sensors are often fooled by biological contamination, physical damage, indicator leaching, etc. and such tests can not be performed in the lab. Different monitoring sites must be selected to test sensor performance.

Do you have any suggestions on how this could be addressed?

National governments and the EU should encourage and support in-situ validation stages. It is probably not as flash as basic laboratory discoveries, but it is the only way to bring sensor technologies to environmental monitoring. Normally, public funding is just provided for basic research but not for fabrication of small series of instruments to perform adequate field validation of the prototype sensors and optoelectronic equipment.

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Grupo INTERLAB (Madrid, Spain) supports our research at UCM under contracts since 1996. Chemical optosensors research at UCM is possible thanks to the tight collaboration between the Laboratory of Applied Photochemistry (G. Orellana) and the Optical Sensors Group (Maria C. Moreno-Bondi), from the Departments of Organic Chemistry and Analytical Chemistry, respectively. Funding for basic research (including doctoral grants) has been provided by the Spanish Ministry of Science and Technology (formerly the research agency CICYT), the European Union, the Madrid Autonomous Community, and the Complutense University.

Collaboration and input from end-users is one of the most valuable for our sensor research.

FURTHER INFORMATION AND COMMENTS

Extensive in-situ validation to show performance and usability of novel sensors developed in the lab is essential (see above). Customer needs cannot be created but they should be addressed cost-effectively with any new sensing instrument.

Disolved oxygen pH Temperature 7 8.5 30

6 28 8.0 5 -1 26

4 C 7.5 24 o pH ] / mg] L 3 T / 2 22 [O 2 7.0 1 20

0 6.5 18 0123456 time / days Figure 1. Representative in situ, continuous, parallel dissolved oxygen, pH and temperature measurements with three of the 8 fiberoptic channels of the OPTOSEN® monitor in a small water course during one week (12 week overall monitoring test without re-calibration).

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1800 1600

1400 -1 1200

1000

800

600

400 [DBO] / mg L [DBO] / mg 200

0 jue 17 vie 18 sáb 19 dom 20 lun 21 mar 22 mié 23 jue 24 t / days

Figure 2. Representative on-line BOD measurements at the entrance of a water treatment plant with OPTOSEN® system and the optical biosensor fabricated with a luminescent oxygen optode and an immobilised biomass (8 week overall monitoring test). The higher the amount of organic matter in the water, the lower the dissolved oxygen contents monitored by the oxygen transducer.

0 10 Optode Hair hygrometer 20 Capacitive sensor 30 40 50

RH / % / RH 60 70

80 HR Optodo HR Capacitive sensor 90 100 25 26 27 28 29 30 31 time / days Figure 3. Representative in situ, continuous relative humidity measurements at an atmospheric monitoring station with the luminescent fiberoptic sensor of the OPTOSEN® monitor (inset) during one week (8 week overall monitoring test without re-calibration). The optical data are compared against measurements by a commercial capacitive sensor and a recording hair hygrometer

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UNIVERSITY OF TUEBINGEN

University Name: University of Tuebingen Department: Institute for Physical Chemistry Address: Auf der Morgenstelle 8 72076 Tuebingen, Germany Respondents Name: Prof. Guenter Gauglitz Position: Professor Email address: [email protected]

SENSOR SYSTEM 1

Description of sensor system : The goal of the RIANA (River Analyser) project was to develop a sensitive and cost-effective analytical system rugged enough for field use and powerful enough 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 RIANA project was based on immunochemistry technology coupled with detection via Total Internal Reflection Fluorescence (TIRF). RIANA was a successful project that was completed in 1999. 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 (1997, 1998). 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 µg/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.

Environmental relevance The European Community Water Directives have been implemented to review and establish strategies and measures to control pollution from diverse sources. Guidelines are meant to standardise water throughout Europe. Water Directives, such as Directive 2000/60/EC, demand greater measurement for an ever-expanding list of pollutants. True enforcement demands more frequent monitoring of water catchments. These new demands require new analytical instrumentation that is easier to use, faster in analysis, and more cost-effective. If possible, instrumentation should be automated and rugged, capable of operating in the field unattended.

Commercial relevance Even though water managers are armed with conventional analytical techniques, such as HPLC, GC, and MS, greater demands on water monitoring is occurring. True enforcement demands more frequent monitoring of water catchments. Also, industrial plants need greater control of their waste water to meet the demands of increased regulation. By developing these instrument prototypes, the project has delivered a new technology that water monitors, both public and private, can utilise to meet tomorrow’s sustainable development requirements.

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Usability (i.e skilled, unskilled) The instruments are easy to use, requiring only minimal training and maintenance.

Availability (eg. commercial, prototype, etc..) Demonstration units are available through ProLiquid GmbH. Uwe Guenther Managing Director ProLiquid GmbH Heiligenbreite 19 D-88662 Überlingen Tel: +49 (0) 7551-916260 Fax: +49 (0) 7551-916261 [email protected] www.proliquid.com

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Small Organic Water Pollutants: Herbicides, Fungicides, Insecticides, and Endocrine Disrupting Compounds.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Surface and groundwater sources

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved pollutants in water

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Water samples filtered with 1µm filters and reagent solutions are presented to instrument. Otherwise operation is fully automated.

Sample handling (i.e. manual, automated etc,,,) Water samples filtered with 1µm filters and reagent solutions are presented to instrument. Otherwise operation is fully automated.

Sample size 1 ml

Sensor Measurements

Assay protocol Analyte detection is based on Total Internal Reflection Fluorescence (TIRF) technology. Laser light is coupled into an optical transducer and 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. Upon binding to the transducer surface the fluorescent markers are excited in the evanescent field. The emitted light is then collected for detection. The design allows for the simultaneous measurement of multi-analyte spots.

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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 analyte samples. After this incubation period, the analyte solution flows over the transducer. Only analyte-specific antibodies with free paratopes will bind to the transducer surface while antibodies inhibited with bound analytes will pass without surface interaction. The surface-bound labelled antibodies are excited in the evanescent field and the fluorescence is detected. Measurements are made in 15 min. or less. Before water samples are measured, the instrument must first be calibrated with standard water solutions containing known analyte concentrations.

River analyser (RIANA )

Continuous measurement Quasi-Continuous: Yes

Present measurement performance:

1. Detection levels - <0.1 ppb typically possible depending on the analyte of interest

2. Selectivity Excellent to good depending on the analytes of interest.

3. Repeatability Very good.

4. Potential interference Very high turbidity, Very high and low pH

5. Adaptability to other pollutants Any pollutant that can produce an immuno-response in livestock.

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Further work developments The Gauglitz Group continues to expand the capabilities of the instrument by testing new analytes and analyte combinations for multi-analyte analysis. We also hope to adapt the technology for easier sample handling, new labelling techniques, and new assay formats that expand the multi-analyte platform.

BOTTLENECKS

Demonstration units are available. Justifying the creation of a customer service infrastructure to support the sale of commercially available units has been difficult. Familiarising water monitor users with the product has been problematic. Users are traditionally skilled with HPLC, GC, and MS technologies and are unfamiliar with immuno-assay platforms. In addition, potential industrial polluters are interested in the technology but are willing to only invest the initial capital in new equipment when directly required by government regulation or public pressure.

Greater communication between instrument developers and water monitoring end-users. In addition, communication needs to exist between government regulators and the available technology.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

The instrument design was a collaboration between the University of Tübingen, Perkin Elmer GmbH, and Optoelectronics Research Centre at the University of Southampton, with consultation from Anjou Recherche, France and Centro de Investigacion y Desarrollo, Spain.

Demonstration units are available through ProLiquid GmbH. Uwe Guenther Managing Director ProLiquid GmbH Heiligenbreite 19 D-88662 Überlingen Tel: +49 (0) 7551-916260 Fax: +49 (0) 7551-916261 [email protected] www.proliquid.com

FURTHER INFORMATION AND COMMENTS

The work reported here was funded under the European Union 5th Framework Programme (AWACSS, Automated Water Analyser Computer Supported System: Contract #EVK1-CT- 2000-00045).

SENSPOL and SENSPOL meetings are clearly a step in the right direction.

PAPERS PUBLISHED

Brecht A, Klotz A, Barzen C, Gauglitz G, Harris RD, Quigley GR, Wilkenson JS, Sztajnbok P, Abuknesha R, Gascón J, Oubina A, Barceló D, (1998) Optical

94 SENSPOL Survey of Sensor Capabilities immunoprobe development for multiresidue monitoring in water. Anal. Chim. Acta. 362: 69-79.

Klotz A, Brecht A, Barzen C, Gauglitz G, Harris RD, Quigley GR, Wilkinson JS, Abuknesha RA, (1998) Immunofluorescence sensor for water analysis. Sens. Actuators. B 51: 181-187.

Barzen C, Brecht A, Gauglitz G, (2002) Optical multiple-analyte immunosensor for water pollution control. Biosens. Bioelectron. 17: 289-295.

CoilleI, Reder S, Bucher S, Gauglitz G, (2002) Comparison of two fluorescence immunoassay methods for the detection of endocrine disrupting chemicals in water. Biomolecular Engineering 18(6): 273-280.

Kroeger K, Jung A, Reder S, Gauglitz G, (2002) Versatile biosensor surface based on peptide nucleic acid with label free and total internal reflection fluorescence detection for quantification of endocrine disruptors. Anal. Chim. Acta. 469: 37-48.

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SENSOR SYSTEM 2

Description of sensor system The goal of the Automated Water Analyzer Computer Supported System (AWACSS) project is to develop a cost-effective, on-line, water-monitoring device that will measure a variety of pollutants quickly with remote control and surveillance. Prototype instruments are being developed in a three year EU funded project beginning in 2001. The instruments are based on immunochemistry technology coupled with detection via Total Internal Reflection Fluorescence (TIRF). The project is focusing on 1) expanded multi-analyte analysis capability allowing for up to 30 simultaneous analyte measurements, 2) novel design approaches to the optical detection and fluidics including miniturized integrated optics and microfluidics, and 3) intelligent remote surveillance and control that will allow for unattended continuous monitoring. Immunochemical reagents are being developed for simultaneous measurement of a host of small organic water pollutants.

Automated Water Analyser Computer Supported System (AWACSS).

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Commercial relevance Even though water managers are armed with conventional analytical techniques, such as HPLC, GC, and MS, greater demands on water monitoring is occurring. True enforcement demands more frequent monitoring of water catchments. Also, industrial plants need greater control of their waste water to meet the demands of increased regulation. By developing these instrument prototypes, the project is delivering a new technology that water monitors, both public and private, can utilise to meet tomorrows sustainable development requirements.

Usability (i.e skilled, unskilled) The instruments will be easy to use, requiring only minimal training and maintenance.

Availability (eg. commercial, prototype, etc..) Prototype instruments are being developed within the project and will be operational in 2003.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Small organic water pollutants: herbicides, fungicides, insecticides, endocrine disrupting compounds, antibiotics, suspected carcinogins, and industrial wastes/chemical markers

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Surface and groundwater sources

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved pollutants in water

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Water samples filtered with 1µm filters and reagent solutions are presented to instrument. Otherwise operation is fully automated.

Sample handling (i.e. manual, automated etc,,,) Water samples filtered with 1µm filters and reagent solutions are presented to instrument. Otherwise operation is fully automated.

Sample size <1 mL volumes expected

Sensor Measurements

Assay protocol Analyte detection is based on Total Internal Reflection Fluorescence (TIRF) technology. Laser light is coupled into an optical transducer with a fibre pigtail assembly and guided down the transducer through micron sized channels illuminating 32 analysis windows simultaneously. The transducer surface is chemically modified with analyte derivatives. Analyte-specific antibodies are labelled with a fluorescent marker. Upon binding to the transducer surface the fluorescent markers are excited in the evanescent field. The emitted light is then collected for detection. The design allows for the simultaneous measurement of multi-analyte spots.

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

97 SENSPOL Survey of Sensor Capabilities labelled with fluorescent markers are incubated with the analyte samples. After this incubation period, the analyte solution flows over the transducer. Only analyte-specific antibodies with free paratopes will bind to the transducer surface while antibodies inhibited with bound analytes will pass without surface interaction. The surface bound labelled antibodies are excited in the evanescent filed and the fluorescence is detected. Measurements are made in 15 min. or less. Before water samples are measured, the instrument must first be calibrated with standard water solutions containing known analyte concentrations.

Continuous measurement (Quasi-Continuous: Yes)

Present measurement performance:

1. Detection levels - Typically <0.1 ppb (expected)

2. Selectivity Excellent to good depending on the analytes of interest. (expected)

3. Repeatability Very good. (expected)

4. Potential interference Very high turbidity, Very high and low pH (expected)

5. Adaptability to other pollutants Any pollutant that can produce an immuno-response in livestock.

Further work developments The instruments are currently being assembled. The first performance tests will be performed at the beginning of 2003.

Envisaged performance and optimisation Instruments will be fully automated field deployable units. The instruments will be connected via modem call-ups and receive measurement instructions and deliver results to and from a central remotely located computer. Up to 30 analytes will be measured simultaneously. Once calibrated (~30 min), the instrument will be capable of delivering measurement results in ~15 min.

BOTTLENECKS

N/A. Still in the development stage.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

This project gathers expertise from 8 groups in 4 different European nations: 1) The University of Tuebingen, Germany is co-ordinatoring the project and leading the biosensor surface technology, 2) IIQAB-Instituto de Investigaciones Quimicas y Ambientales de Barcelona, Spain leads the field testing partners who are developing the instruments for field use, 3) Environmental Institute, Slovak Republic is the 2nd field testing partner, 4) DVGW- Technologiezentrum Wasser, Germany is the 3rd field testing partner, 5) King’s College of London (KCL), UK is developing the immunochemistry, 6) Optoelectronics Research Centre

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(ORC), UK leads the design and construction of the instruments, 7) Siemens AG, Germany leads the data processing, software communications, and exploitation strategies, and 8) Central Research Laboratory, Ltd. (CRL), UK is designing and assembling the final instruments.

Siemens AG and Central Research Laboratory, Ltd. are the commercial project partners.

FURTHER INFORMATION AND COMMENTS

The work reported here was funded under the European Union 4th Framework Programme (RIver ANAlyser, RIANA: Contract #ENV4-CT95-0066. SANDRINE: Contract #ENV4- CT98-0801 ). SENSPOL and SENSPOL meetings are clearly a step in the right direction.

PAPERS PUBLISHED

Lopez de Alda, Maria, J. and Barcelo, D. “Use of solid-phase extraction in various of its modalities for sample preparation in the determination of estrogens and progestogens in sediment and water”. J. Chromatogr. A, 938, (2001), 145-53.

Lopez de Alda, Maria, J. and Barcelo, D. “Review of analytical methods for the determination of estrogens and progestogens in waste waters”. Fresenius J. Anal. Chem., 371, (2001), 437-47.

Lopez de Alda, Maria, J.; Gil, A.; Paz, E.; and Barcelo, D. “Occurrence and analysis of estrogens and progestogens in river sediments by liquid chromatography-electrospray-mass spectrometry”. Analyst, 297, (2002), 1279-398.

Willard, Dale; Proll, Guenther; Reder, Sabine; and Gauglitz, Guenter. “New and versatile optical-immunoassay instrumentation for water monitoring”. Environ. Sci Poll. Res., In press.

PROFILE OF ORGANISATION

The Institute for Physical and Theoretical Chemistry at the University of Tuebingen has for many years emphasized research in the spectroscopy of surface layers, analytics at surfaces, and the development and characterisation of sensors. Numerous scientific results have brought increased interest from industry. Because of this, the institute has actively attempted to transfer research knowledge to practical applications partnered with industry. Within the institute, the Gauglitz Group works in the areas of optical spectroscopy, detection problems in the chromatography, photokinetics, the investigation of photochromic systems, i.e. photoresists, and chemical and biochemical sensors. Research projects are ongoing that address: • UV/Vis, absorption and fluorescence spectroscopy, • Photo-stability of laser coloring materials,

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• Photopolymerization of siloxane oligomeres with directed cross-linking, in order to produce, special characteristics, such as suitably for sensitive layers, • Photo-kinetic processes in photochromic systems and liquid crystals, • Spectral refractrometry in micro volumes for HPLC applications, • Microrefractrometry with integrated-optical components, • Sensor development based on principles of multi-wavelength reflection and interference, • Software development for experimental control, data acquisition, and data evaluation.

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ABOATOX OY

University Name: Aboatox Oy Address: Lemminkäisenkatu 36 FIN-20520 TURKU FINLAND Respondent Name: Juha Lappalainen Position: Managing Director Email address: [email protected]

SENSOR SYSTEM

Description of sensor system Luminescence based biotest for the detection of heavy metals in environmental samples. Heavy metal in the sample induces the production of luciferase enzyme in a bacterial cell. The increase in the luminescence is measured after adding the substrate.

Environmental relevance Bioavailable heavy metal can be measured with very good sensitivity.

Commercial relevance Very cost effective (reagent), simple luminometer (portable), easy to upscale for screening purposes (plate luminometer).

Usability (i.e skilled, unskilled) Unskilled.

Availability (eg. commercial, prototype, etc..) Available.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Heavy metals: mercury, organomercurials, arsenic (both III and V, speciation), cadmium, lead, copper.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soil, sediment, water, effluent.

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable (the test is much more sensitive than traditional toxicity tests).

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Water or water suspension (solid samples).

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Sample handling (i.e. manual, automated etc,,,) Both.

Sample size During the measurement 100 µl.

Sensor Measurements

Assay protocol

Measurement protocol

1. Pipette 100 µl of sample water or sample suspension into the luminometer cuvette 2. add 100 µl reconstituted sensor bacteria 3. incubate 2 h at 37 °C 4. add 200 µl substrate D-luciferin 5. incubate 20 min at RT 6. measure luminescence

Continuous measurement No

Present measurement performance:

1. Detection levels - Inorganic mercury: 2 ppb Arsenic: 2 ppb (III) and 4 ppb (V) Cadmium: 0,4 ppb Organomercurials: 0,05 ppb Chromate: 2 ppb Cadmium: 1 ppb Lead: 10 ppb

2. Selectivity Some cross reactivity, normally not relevant.

3. Repeatability CV better than 10 % both interassay and intra-assay.

4. Potential interference Very toxic samples: avoided with dilution series.

5. Adaptability to other pollutants Yes, possible.

Further work developments More sensors for different metals. New applications.

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BOTTLENECKS

Bioavailability beyond toxicity a new parameter.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Dr. Marko Virta, University of Turku, Turku, Finland is our partner helping with the development of this sensor. No commercial backing Diagnostics companies for screening purposes. Collaboration that would help in sensor development.

PAPERS PUBLISHED

Turpeinen, R., M. Virta and M. M. Häggblom (2003). Analysis of arsenic bioavailability in contaminated soils. Environmental Toxicology and Chemistry 22(1):1–6. Ivask, A., M. Virta and A. Kahru (2002). Construction and use of specific luminescent recombinant bacterial sensors for the assessment of bioavailable fraction of cadmium, zinc, and mercury in the soil. Soil Biology and Biochemistry 34(10): 1439-1447. Ivask, A., K. Hakkila and M. Virta (2001). Detection of organomercurials with sensor bacteria. Analytical Chemistry 73(21): 5168-5171. Petänen, T., M. Virta, M. Karp and M. Romantschuk (2001). Construction and Use of Broad Host Range Mercury and Arsenite Sensor Plasmids in the Soil Bacterium Pseudomonas fluorescens OS8. Microbial Ecology 41(4): 360-368. Lappalainen, J. O., M. T. Karp, J. Nurmi, R. Juvonen and M. P. J. Virta (2000). Comparison of the total mercury content in sediment samples with a mercury sensor bacteria test and Vibrio harveyi toxicity test. Environmental Toxicology 15: 443-445. Tauriainen, S., M. Virta, W. Chang and M. Karp (2000). Detecting bioavailable toxic metals and metalloides from natural water samples using luminescent sensor bacteria. Water Research 34: 2661-2666. Tauriainen, S., M. Karp, W. Chang and M. Virta (1998). Luminescent bacterial sensor for cadmium and lead. Biosensors and Bioelectronics 13: 931-938. Tauriainen, S., M. Karp, W. Chang and M. Virta (1997). Recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite. Applied and Environmental Microbiology 63(11): 4456-4461. Virta, M., J. Lampinen and M. Karp (1995). A luminescence-based mercury biosensor. Analytical Chemistry 67(3): 667-669 .

PROFILE OF ORGANISATION

Aboatox Oy is a private company working in the field of luminescent sensor bacteria (V. fischeri and also genetically modified organisms) to make it easy and cost effective to use biotests in the environmental monitoring. The aim is to utilise modern instrumentation for better quality results with complicated samples (The Flash test) and simple instruments for simple tasks. Aboatox works in a close cooperation with the Biotest group at the University of Turku to introduce the life outside university to the students working in the group.

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BEN-GURION UNIVERSITY OF THE NEGEV

University Name: Ben-Gurion University of the Negev Department: Institute for Applied Biosciences Address: PO Box 653, Beer-Sheva, 84105 Respondents Name: Robert Marks Position: Lecturer Email address: [email protected]

SENSOR SYSTEM

Description of sensor system Genetically engineered luminescent bacteria Escherichia coli MC1061 containing the mercury responding promoter to control the expression of luxCDABE from Photorhabdus luminescens as the reporter produce bioluminescent light upon induction and therefore was used as a biological sensing element. Optical fiber tip cores were covered with adlayer films consisting of the mercury and arsenite sensitive luminescent bacteria immobilied in 1% sodium alginate mixture. Microbial luminescent response was measured by a customized photon-counting photomultiplier tube based instrument.

Environmental relevance On-field-Detection of heavy metals On-field-Detection of genotoxicity

Commercial relevance At prototype stage

Usability (i.e skilled, unskilled) Unskilled

Availability (eg. commercial, prototype, etc..) Prototype stage

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SAMPLE

Targeted analyte(s) Mercury, arsenite, cadmium, lead, zinc, mitomycin c, nalidixic acid, N-Methyl-N-nitro-N- nitroso guanidine, ethanol, hydrogen peroxide, methyl viologen.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soil, sediments, groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) As is, or if soil, in suspension

Sample preparation (e.g. filtering, SPE, derivatisation etc…) If soil, place in suspension

Sample handling (i.e. manual, automated etc,,,) Manual

Sample size 500 µl or more

Sensor Measurements

Assay protocol Multimode optical fibers, SFS400/440 (Fiberguide Industries, Inc., USA), were used in these experiments. Their black nylon jackets were stripped away from a 1-cm long optical fiber tip, which was then used for the immobilization of bioluminescent cells. The harvested cells were mixed 1:1 with a filter-sterilized 2% (w/v) low viscosity sodium alginate solution. The 1-cm optical fiber tip was first exposed (for a few seconds) to the bacterial alginate suspension, and then dipped (for a few seconds) into a sterile 0.5M calcium chloride solution, thus entrapping the bacteria onto the fiber within a hardened calcium alginate matrix. Repeating these steps thickened the adlayer, thus, increasing the number of bacterial sensor cells attached to the optical fiber transducer. At this step, the optical fibers, with their immobilized bioluminescent bacteria at their end face tip, were than ready for the experimental monitoring of unknown samples. A portable photon counting system was designed and built in our laboratory. To receive and treat data, a specific driver was developed using LabView (version 3.1, National Instrument Corporation), which allowed the combined monitoring of the bioluminescent signal and data handling in real time.

Continuous measurement Yes

Present measurement performance:

1. Detection levels 2 ppb (mercury)

2. Selectivity- Yes

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3. Repeatability- Yes

4. Potential interference- Possible synergistic or antagonistic effects from other contaminants solution

5. Adaptability to other pollutants Wide range

Further work developments Add GPS system, timer, bar-code reader, storage studies of probe fibres

BOTTLENECKS

Storage studies of the biological components have yet to be completed and further work and research on alternative immobilisation strategies.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

There are two partners Marko Virta (Turku, Finland) and Shimshon Belkin (Jerusalem, Israel) who provided bioluminescent bioreporter bacteria. There are no commerical backers.

FURTHER INFORMATION AND COMMENTS

Investment and adaptation of the system by a commercial firm would benefit the development and testing of the sensor

PAPERS PUBLISHED

Polyak, B., E. Bassis, A. Novodvorets, S. Belkin and R. S. Marks (2000). Optical fiber bioluminescent whole-cell microbial biosensor to genotoxicants. Water Science and Technology, 42: 305 – 311. Polyak, B., E. Bassis, A. Novodvorets, S. Belkin and R. S. Marks (2001). Bioluminescent whole cell optical fiber sensor to genotoxicants: system optimization. Sensors and Actuators B., 74, 18-26.

Premkumar R. J., O. Lev, R. R. Marks, B. Polyak, R. Rosen and S. Belkin (2001). Antibody- based immobilization of bioluminescent bacterial sensor cells. Talanta, 55(5), 1029-1038.

PROFILE OF ORGANISATION

Ben-Gurion University of the Negev is an internationally recognized institution of higher learning that attracts outstanding faculty and researchers from around the world. The University actively promotes hi-tech industry, agriculture, health services and education in the region. BGU is home to more than 15,000 students and has campuses in Beer-Sheva and S’de Boker. The University includes four faculties: Engineering Sciences, Health Sciences, Natural Sciences and Humanities and Social Sciences and two schools: the School of Management

106 SENSPOL Survey of Sensor Capabilities and the Kreitman School of Advanced Graduate Studies. The University also includes the Jacob Blaustein Institute for Desert Research, the Institute for Applied Research, and The Institute for Applied Biosciences. The role of the Institute for Applied Biosciences is to serve as Ben-Gurion University's Center for biotechnology. The main goals of the Institute are twofold: To establish a center of know-how and to apply research results towards commercialization.

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CRANFIELD UNIVERSITY

Institute: Institute of Bioscience and Technology Research group: Cranfield Biotechnology Centre Address: Institute of Bioscience and Technology Cranfield University Silsoe MK45 4DT Bedfordshire United Kingdom Respondents’ Name: Belen Bello Rodriguez Position: PhD student Email address: [email protected]

SENSOR SYSTEM

Description of sensor system The urease biosensor for the determination of heavy metals is based on the amperometric analysis of NADH with screen-printed electrodes at 0.3 V. Urease was chosen because of its sensitivity to metals inhibition, stability and inexpensive cost. Because the urease substrate, urea, is not electrochemically active, this enzyme is coupled to glutamic dehydrogenase, which catalyses the oxidation of NADH into NAD. This reaction only takes place when there is ammonia present in the medium, supplied from the urea breakdown catalysed by urease. When the system is incubated with samples that contain metal ions, these metals bind to the urease suflhydryl groups producing a high inhibition in the enzyme activity. Thus, the production of ammonia as a result of the urea breakdown is dramatically decreased, resulting also in a much lower NADH oxidation rate. This decrease in the NADH oxidation is measured and related to the presence of metals in the samples.

Urease

Urea + H2O CO2 + 2NH3

GLDH NH3 + α-ketoglutarate + NADH + H+ L-Glutamate + NAD+

The analysis are performed with screen-printed electrodes (rhodinised carbon as working, silver/ silver chloride as reference and carbon as counter electrodes). A fixed potential of 0.3 V is applied and the NADH reading taken.

Environmental relevance Fast and reliable toxicity assay to determine the presence of heavy metals in given samples. It can be used in polluted sites as a first scan to check whether further tests or cleaning should be made in those areas, regarding the assay shows positive results for metals presence.

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Commercial relevance Useful as a fast test to determine the presence of heavy metals in samples.

Usability (i.e skilled, unskilled) Skilled in the use of biological substances (enzymes) and substances as toxic as heavy metals.

Availability (eg. commercial, prototype, etc..) Designed and developed at Cranfield University, based on a protocol established to measure urease activity.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Heavy metals, specially sensitive to mercury (II) and copper (II)

Sample matrix (e.g. soils, sediments, surface and groundwater ) Water, extracted soils or sediments.

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Extraction of the analytes in order to avoid any contamination or interferences from the original matrix

Sample handling (i.e. manual, automated etc,,,) Manual

Sample size No more than 5 mL

Sensor Measurements

Assay protocol Samples are diluted in Tris-HCl buffer (pH = 8.0) containing KCl as electrolyte in order to perform the electrochemical measurements. NADH and α-ketoglutaric acid are then added to the resulting solution, followed by both enzymes, glutamic dehydrogenase and urease. An incubation for at least ten minutes is performed, followed by the addition of urea. The final solution with all the compounds is then placed onto the electrode and the NADH signal is read. This signal should follow a decreasing rate, higher when the activity is at its peak. The urease inhibition will result in a low NADH oxidation rate. By comparing the readings obtained for a solution incubated in presence of buffer to the readings given for each sample, the percentage of urease inhibition can be calculated and related to the metal content.

Continuous measurement Yes

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Present measurement performance:

1. Detection levels Down to µg/ L for mercury and copper. Detection in the mg/ L for any other metals.

2. Selectivity Because most heavy metals present the same inhibitory effect on the enzyme, the assay is useful as a toxicity assay to determine the presence of metals in samples, but it is not possible to discern which metals the sample contents.

3. Repeatability Based on disposable screen-printed electrodes.

4. Potential interference Urease and/ or glutamic dehydrogenase inhibitors, such as ammonia, suramin or thiourea (for urease), or bromobenzoic acid, chlorobenzoic acid, thyroxine, glycolglycine for GLDH. Since all these substances are pretty unlikely to be found in samples, we can conclude the assay is highly selective for metals.

5. Adaptability to other pollutants Highly specific for metal ions. Some authors have reported inhibition studies with organophosphorus pesticides, but these compounds showed much lower or no effect on the enzyme activity.

Further work developments A more simple assay can be developed by measuring urea potentiometrically. This sensor would only consist of one enzyme, thus making it easier and maybe more convenient for on- field analysis.

Envisaged performance and optimisation The assay exposed has been optimised for continuously measure NADH by just adding a small volume of the final solution containing all the necessary components. This step makes it faster than the reading based on the electrode immersion on a cell.

BOTTLENECKS

The only problem that can be faced when doing on-field analysis is the quality of the samples. Matrix effects, or low pHs, might affect the sensitivity or reproducibility of the sensor. Extracted samples will minimise the risk of “false” inhibition readings due to the nature of the matrix. A proper dilution of the buffer will eliminate the decrease in the enzyme activity at pHs outside the optimum range.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Funded by the EC, as part of a European project, the project is complemented by other partners who provide extracted samples along with the reference analysis of these samples contents.

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FURTHER INFORMATION AND COMMENTS

The biosensor exposed can only determine the presence of metals. The contents, i.e. which metals are present, are difficult to establish.

PAPERS PUBLISHED

“Biosensors for the determination of heavy metals in waters”. Bello Rodriguez, B. and Tothill I.E. Fifth Workshop on Biosensors and Biological Techniques in Environmental Analysis. Ithaca, NY, USA, 31 May-04 June 2002. “Biosensors for the determination of heavy metals in waters”. Bello Rodriguez, B. and Tothill, I.E. Biosensors 2002, Kyoto, Japan, 15-17 May 2002. “Electrochemical sensor development for toxic heavy metals”. Kadara, R.O., Bello Rodriguez, B., Newman, J.D. and Tothill, I.E. Senspol, University of Alcala, Alcala de Henares, Spain, 9-11 May 2001.

PROFILE OF ORGANISATION

The Institute of BioScience and Technology (IBST), within Cranfield University (Silsoe, UK) is active in research and development for both industry and academia, post-graduate education, professional training. IBST is the UK’s foremost centre for biotechnology and is a centre of excellence in post-graduate education, training and industrially funded research. The centre houses a wide variety of research groups including cell and molecular biology, combinatorial chemistry, polymer imprinting, mycology and environmental research as well as housing a state of the art fabrication facility for the pre-production of sensor devices. IBST has a flourishing industrial contracts division and has been at the forefront of diagnostics development since its inception in 1981, creating a wide variety of products for industrial clients, including the Exac-tech blood glucose monitor, the world’s most successful biosensor to date.

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Cranfield University

Institute Name: Institute of BioScience and Technology Research group: Cranfield Centre for Analytical Science Address: Cranfield University Silsoe, Bedfordshire, MK45 4DT, UK Respondents Name: Miss Joanne Cooper Position: Research Assistant Email address: [email protected]

SENSOR SYSTEM

Description of sensor system Carbon screen-printed electrodes coupled to Palmsens Potentiostat for the simultanenous determination of Cd2+ Pb2+ Cu2+ using differential pulse anodic stripping voltammetry.

Gold screen-printed electrodes coupled to Palmsens Potentiostat for the determination of As3+ and Hg2+ using differential pulse anodic stripping voltammetry.

In DPASV, the sample is first subjected to a deposition step, during which the metal ions in solution are plated down onto a working electrode surface, followed by a stripping step where the metal ions are re-released (stripped) back into solution. The deposition step is performed in the presence of mercuric ions (for the analysis of Cd Pb Cu on Carbon, not required using gold for Hg and As) at a suitably negative potential. Next, the electrode potential is linearly ramped with time to a less negative potential, during which the metal ions are re-oxidised and stripped back into solution at characteristic potentials. The oxidation potential and the size of oxidation current provide both qualitative and quantitative information on the metals of interest.

Environmental relevance Assessment of contaminated land, monitoring of remediation processes

Commercial relevance Provide a rapid, cost effective screening method of analysis for end-users – regulators, site owners, remediation companies

Usability (i.e skilled, unskilled) Semi-skilled operative for use on-site

Availability (eg. commercial, prototype, etc..) Non-commercial – Screen-printed electrodes, assay procedure Commercially available – Palmsens Instrument

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Pb, Cu, Cd – carbon screen-printed electrodes Hg As – gold screen-printed electrodes

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils, sediments, surface and groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) According to extraction protocol – Depends on what the end-user wants to determine BCR F1-bioavailable F4 total

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Water samples – filteration Soils, sediments – acid digestion

Sample handling (i.e. manual, automated etc,,,) Manual

Sample size 1g

Sensor Measurements

Assay protocol Cd Pb Cu on Carbon

Mixed standards of Pb Cu Cd in 100mM KCL and 1% v/v HNO3 + 10 l of 2 mg L-1 Hg(NO3)2 for direct calibration. DPASV: Deposition potential -1.1V deposition time 165s Sample: 1g soil, 5ml 1M aqua regia (3:1 HCl:HNO3), 3 min. shake, filter, dilute +H2O 50 mL, Add 90 l sample +10 l of 2 mg L-1 Hg(NO3)2 to electrode. Measure using DPASV parameters as standards As and Hg on Gold Hg standards – prepared in 100mM KCL and 1% v/v HNO3 for direct calibration. DPASV: deposition potential 0.2V deposition time 30s As standards – prepared in 2M KCL for direct calibration. DPASV: deposition potential –0.3V deposition time 30s sample: As above (no Hg(NO3)2)

Continuous measurement No

Present measurement performance:

1. Detection levels - Pb – 1.5 µg l-1 Cd - 10µg l-1 Cu - 30µg l-1 As - 50µg l-1 Hg – 2.8µg l-1 using deposition potential of 30s - 0.16µg l-1 using 120s

2. Selectivity Highly selective

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3. Repeatability RSD of under 8%

4. Potential interference Sample matrix components As and Hg - Copper

5. Adaptability to other pollutants Other metal ions

Further work developments Seek accredited validation (EPA, AOAC) Alternative applications: Toxic metals in blood

Envisaged performance and optimisation Performance is satisfactory for intended end-use (rapid screening tool) Device has already been subject to an optimatisation process

BOTTLENECKS

Validated sample preparation methods are available for different soil fractions. However these are laboratory based. Validated field sample preparation methods are required in order to carry out analysis of soils and sediments. A 3 minute field based extraction method has been developed in conjunction with this work. However, this would need to follow a formal validation procedure as with laboratory based extractions.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

There are no partners nor commercial backing, Feed-back from end-users would be beneficial in the development of the sensor.

PAPERS PUBLISHED

Publications: S.J.Setford, J. Cooper, P.Rigou, J.A.Bolbot, S Saini. Field-based analytical methods for monitoring at industrially contaminated sites. Proceedings, 1st SENSPOL workshop, University of Alcala, Alcala de Henares, Spain, 9-11 May 2001, pp76-87.

J.Cooper, S.J. Setford, J.A. Bolbot and S. Saini. Electrochemical characterisation of screen- printed carbon electrodes. Workshop on the Protection of European Water Resources, Concerted Action on Environmental Technologies (ETCA), Harrogate, UK, 21-23 May 2001, p257-258.

Conferences attended: Electrochemical characterisation of screen-printed carbon electrodes for on-site heavy metal monitoring applications. Cooper, J., Bolbot J.A., Setford S.J., and Saini S. Poster, New Directions in Electroanalysis. Royal Society of Chemistry Electroanalytical Group, University of Salford, UK. 22-25 April 2001.

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Electrochemical characterisation of screen-printed carbon electrodes for on-site heavy metal monitoring applications. Cooper, J., Bolbot J.A., Setford S.J., and Saini S. Poster, ETCA Workshop – On the protection of European Water Resources. ETCA Science Panel, Harrogate, UK. 21-23 May 2001.

Electrochemical method for the rapid on-site screening of heavy metals in soil, water and plant samples. J Cooper, S Setford, J Bolbot, S Saini. Poster, Fifth Workshop on Biosensors and Biological Techniques in Environmental Analysis. Cornell University, Ithaca, NY. May 31-June 4 2002.

Electrochemical method for the rapid on-site screening of heavy metals in soil, water and plant samples. J Cooper, S Setford, J Bolbot, S Saini. Poster, SENSPOL, Response to New Pollution Challenges Workshop, Kings College, London. 4-7 June 2002.

Electrochemical method for the rapid on-site screening of heavy metals at industrially contaminated sites. J Cooper, S Setford, J Bolbot, S Saini. Oral, Senspol Technical Meeting on Sensors for Characterisation and Monitoring of Contaminated Sites, Seville. 6-9 November 2002.

Electrochemical method for the rapid on-site screening of heavy metals at industrially contaminated sites. J Cooper, S Setford, J Bolbot, S Saini. Abstract submitted, Consoil 2003.

Monitoring Industrially Contaminated Sites. Steven Setford, Hans van Duijne, Peggy Rigou, Joanne Cooper, John Bolbot, Selly Saini, Ken Killham, David Mardlin, Rolf Henckler Alfredo Battistelli, Linet Ozdamar , Melek Demirhan . Response to New Pollution Challenges, 2nd SENSPOL workshop, King’s College London, 4-7 June 2002.

Organism and Whole Cell-based Biosensors: Comparison of electrochemical and biosensor methods for the rapid on-site screening of heavy metals in soil, water and plant samples. J Cooper, S Setford, J Bolbot, S Saini Poster Biosensors 2002, 7th World Congress on Biosensors, Kyoto, Japan, 15-17 May 2002.

Protection of groundwater resources at industrially contaminated sites. J Cooper, S Setford, J Bolbot, S Saini. Oral, Silsoe Conference, Cranfield University, Silsoe, Bedfordshire. 15 June 2002.

PROFILE OF ORGANISATION

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CRANFIELD UNIVERSITY

Institute Name: Institute of Bioscience and Technology Research group: Cranfield Biotechnology Centre Address: Cranfield University Silsoe, Bedfordshire MK45 4DT, UK Respondents Name: Rashid Kadara, Ibtisam Tothill Position: Research student, Reader in Biochemistry Email address: r.o>[email protected]

SENSOR SYSTEM

Description of sensor system The sensor system is based on disposable screen-printed electrodes (incorporating the three- electrode system) coupled to an electroanalytical stripping technique (constant current stripping chronopotentiometry). The screen-printed electrodes (with graphite-carbon working electrode, silver/silver chloride reference electrode and carbon counter electrode) are fabricated in-house onto a plastic substrate. Measurements are made on the graphite-carbon working electrode or with the working electrode modified in-situ with bismuth (III) ions (bismuth film electrode) and mercury (II) ions (mercury film electrode) or by incorporation of modifiers (2,5 dimercapto-1,3,4 thiadiazole and bismuth (II) oxide) onto the surface of the electrode. With this system, information about an analyte is derived from the measurement of potential as a function of time.

PRINCIPLE OF MEASUREMENT TECHNIQUE The measurement is performed on the electrode’s electrochemical cell under polarizing conditions on the working electrode. For this purpose, a deposition potential Ed is applied to the working electrode; this potential should be in the region of the metal ion or ions being deposited. In the subsequent stripping step, the potentiostat is transformed into a galvanostat and the reduced analytes are re-oxidised electrochemically by means of an applied constant oxidative current. The change of potential of the working electrode with time during the stripping of the deposited analyte is recorded and processed. The response is digitically processed into dt/dE (the inverse potential derivative with time) versus potential (E), with the amount of deposited analyte determined by integrating the corresponding stripping peak.

Environmental relevance Monitoring of contaminated sites with toxic heavy metals is important both for identification of trends of pollution and to control the efficiency of remediation activities.

Usability (i.e skilled, unskilled) Both

Availability (eg. commercial, prototype, etc..) The PalmSens potentiostat is commercially available

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Cadmium, copper and lead

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils, sediments, surface and groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) Exchangeable fraction, easily reducible fraction, oxidisable fraction and residual fraction

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Sequential extractions with acetic acid, water and nitric acid/hydrochloric acid

Sample handling (i.e. manual, automated etc,,,) manual

Sensor Measurements

Assay protocol The integrated three electrode strips are connected to the potentiostat/galvanostat with a specially adapted electrical edge connector from Maplin (Milton Keynes, UK) and measurements carried out by placing a 100 µl sample drop on the electrochemical cell. Each experiment is carried out with a new electrode strip in non-deaerated and unstirred solution. Stripping chronopotentiometric measurements are carried out by co-deposition of 500 ppb bismuth (III) (for formation of bismuth film on working electrode surface) with target analyte (lead and cadmium) at a potential of – 1.05 V (vs. screen-printed Ag/AgCl) for 120 s. The deposition time, 120 s, is sufficient for attaining favourable stripping response for parts per billion (µg l-1 or µg kg-1) concentrations of metal ions. After the deposition period, a constant oxidative current (0.5 µA - 1 µA) is applied to strip the deposited target analyte with the stripping process terminated when the electrode has reached a potential of 0 V (vs. screen- printed Ag/AgCl). The background signal resulting from the supporting medium is also measured in the same manner.

Reagents for sample analysis Electrolyte: 0.1 M HCl + 0.5 M NH4AC (final concentration) Metal ion (lead (II) and/or cadmium (II)) co-deposited with 500 ppb Bi3+ (for formation of a bismuth film on SPE) or 10 ppm Hg2+ (for formation of a mercury film on SPE) The concentration of the bismuth/mercury ion solution added is the final concentration in the test solution. For copper and lead on plain-SPE, 0.1 M HCl is used as electrolyte (no mercury or bismuth ions added).

Conditions for analysis Deposition potential = - 1.0 V (cadmium (II)) or – 0.9 V (lead (II), copper (II)) Final potential = 0 V Deposition time = 120 s Stripping current = 1 – 3 µA Max. time of measurement = 3 s

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A standard addition method was adopted to evaluate the metal contents in samples. The samples were diluted with the electrolyte and bismuth ion solutions before evaluation. Sample measurements were performed, by applying the deposition potential for a selected time (usually 2 minutes), using a quiescent non-deaerated solution. Repetitive measurements were carried out on different electrode strips.

Continuous measurement No

Present measurement performance:

1. Detection levels – Bismuth film electrode (8 ppb – lead, 8 ppb – cadmium) Plain carbon electrode (20 ppb – lead, 30 ppb – copper) Mercury film electrode (1 ppb – lead, 1 ppb – cadmium)

2. Selectivity good

3. Repeatability 3 - 10 %

4. Potential interference copper on Bismuth film electrode

5. Adaptability to other pollutants Possible

Further work developments Testing out more samples

Envisaged performance and optimisation The sensor system works well. The evaluation of the accuracy obtained for samples already analysed, shows that the sensor results validated against results generated with ICP-MS are always within this range 70 – 120 % (for bismuth film electrode if the interference problem from copper is low), 80 – 120 % (for mercury film electrode) and 60 – 140 % (for bare carbon electrode). Samples can be analysed using direct calibration or standard addition method

BOTTLENECKS

The type of graphite-carbon printing ink to use because the manufacturers of these inks withhold important information about the ink.

Matrix effect (The presence of Copper in samples at high concentration) – Bismuth film electrode. Copper interferes

Do you have any suggestions on how this could be addressed?

By finding a manufacturer that will produce printing inks that would suit whatever purpose you want to use them for.

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Due to the fact that the development of this sensor is part of an EU project (DIMDESMOTOM) dealing with ‘the development of improved detection systems for monitoring of toxic heavy metals in contaminated groundwater and soils’, the samples used in testing out the sensor system has been provided by our partners. Funded by EU

Working with screen-printing ink manufacturer would help in sensor development

FURTHER INFORMATION AND COMMENTS

More meetings between both sensor developers and end users so as to know what is actually needed by the end user.

PAPERS PUBLISHED

Electrochemical Sensor Development for Toxic Heavy Metals in Various Matrices of Contaminated Groundwater and Soils R.O. Kadara, J.D. Newman and I.E. Tothill New Directions in Electroanalysis, University of Salford, 22-25, April 2001

Electrochemical Sensor Development for Toxic Heavy Metal Kadara, R.O, Bello-Rodriquez, B, Newman, J.D and Tothill, I.E Senspol, University of Alcala, 9-11, May 2001

Electrochemical Sensor Development for Toxic Heavy Metals in Groundwater and Soils R.O. Kadara and I.E. Tothill Senspol Workshop, Kings College, University of London, 4-6, June 2002

Surface Modified Screen-printed Sensor for Lead and Cadmium R.O. Kadara, Tothill I.E Electrochem2002, Preston, Lancs, 1-4, September 2002

PROFILE OF ORGANISATION

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BOHRLOCHMESSUNGEN-DR. BUCKUP (DBM)

Company Name: Bohrlochmessungen-Dr. Buckup (DBM) Department: Address: PF 26 D-39032 Magdeburg Respondents Name: Dr. Klaus Buckup Position: President Email address: [email protected]

SENSOR SYSTEM

Description of sensor system The sensor system in the actual device consists of 2 He3-detectors of different type on different distances (spacings) to record thermal neutrons along the whole neutron lifetime. The neutrons are emitted by help of a controlled neutron source, no radioactivity, if the tool is switched off).

Surface panel for the PNN-monitoring device with GSM-transmission antenna (probe seen in the background)

Environmental relevance The method allows to analyse water and soil samples under in-situ conditions in a flexible selected monitoring period continuously.

Commercial relevance The equipment is commercially available, it needs for absolute output a sufficient calibration.

Usability (i.e skilled, unskilled) The operation is easy, even automatically, but keep it running needs a periodic service, which must be performed by a skilled worker.

Availability (eg. commercial, prototype, etc..) It is available on conditions to be agreed in view of the target and the local conditions.

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) All elements and organics, total mineralisation with sufficient calibration. On-site monitoring with high sensitivity to changes of any kind.

Sample matrix (e.g. soils, sediments, surface and groundwater ) Does not matter.

Sample phase (i.e. total, residual, free, dissolved or absorbed) No influence, because of direct response to the elements.

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Not requested (only in the case of calibration to make the data compatible)

Sample handling (i.e. manual, automated etc,,,) On site measurements, therefore no special requests

Sample size The probe may be adjusted, but for a spacing of 35 cm in fluids coverage of about the same size for optimal measurements is requested.

Sensor Measurements

Assay protocol There are not assay protocols, but references and technical checks for permits. Documents in this sense are available on request. The deviced is approved by the German Technical Society (TUV)

Continuous measurement Yes, periodical switching possible

Present measurement performance:

1. Detection levels Depending on the background. Actually the following numbers are available: Total concentration- 0,01 %, Pb- <0,005 mg/l, Ni< 0,01 mg/l, Cu 0,1 mg/l, organics < 0,005%. Further components not yet established quantitatively.

2. Selectivity No limit, but a higher performance may be reached for continous control of any changings with an automized alarming signal in the case of exceeding a given critical value for an selected element or pollutant.

3. Repeatability 100 %

4. Potential interference Depends on situation and percentage of elements.

5. Adaptability to other pollutants Must be solved along with calibration.

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Further work developments Introducing energy analysis (Gamma-version)

Envisaged performance and optimisation Practical and modelling for quantification of the tool response, methodical development

Monitoring sequence from a site in Simeis/Crimea. There are shown the changess in different recording windows for a 2-weeks period. Changes correlate with rainfall, which causes inflows to the surface waters. Decreases correspond with the appearance of metallic pollutants like Cu, Fe etc., increases correspond with phenol-appearance from traffic along two main roads nearby.

BOTTLENECKS

There may be always something done, but the biggest challenge is always the adjustment to the different sites and the lack of information to take care before starting the onsite monitoring. This could be addressed by better co-operation and information transfer.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

The reserch and devlopment of the system was EU funded project “NuPulse” Contract - G1RD-CT-2002-00714 with Geological Survey of Finland, Selor e.e.e.i.g. , Netherlands, Terramentor e.e.i.g., Greece. The device is commercialised by the company DBM with measurements of different types, especially for the control of chlorine hydrocarbons, there are references available.

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FURTHER INFORMATION AND COMMENTS

The device is applicable as a monitoring network for permanent control of the environmental status and fluctuations. Because of the innovative character the methods needs to be on a wide scale to be introduced to the endusers.

PAPERS PUBLISHED

1. Buckup, K.and P. and M „Neue methodisch-technische Aspekte des Impuls-Neutron- Neutron-Verfahrens“, paper to be presented on the DGG-conference 2003, Jena 2. Buckup, K. andP. „Mulipurpos application of the Pulse Neutron Method (PNM)“, paper presented on the VIII Krajowa Konferencja Naukowo-Techniczna, Szymbark/Poland, Sept., 2002 3. Buckup, K. and P. and M., Yakovlev, K. “ Neue Anwendungen der Impuls-Neutronen- Messungen”, 62. Jahrestagung DGG, March, 2002, Hannover 4. Sideris, G. and Buckup, K. “Common technique for logging and environmental monitoring”, paper presented on the Conference of the Balcan Geophysical society, Sofia, 2002

PROFILE OF ORGANISATION

DBM is a SMS, founded 1990 by Dr. Buckup to provide logging service to SMS s. From an one-man-company it grows up to a meanwhile 8-man enterprise with manufacturing and service facilities mainly for watersupply and exploration. Since 1992 the research is concentrated on the development of pulse neutron technique with sale and monitoring service. Activities are worldwide. Since 1995 DBM is participating in EU-activities. The company has performed almost over 1000 PNN-measurements in 15 countries. Since 1995 the research was concentrated on monitoring application of the PNN-technique. The research is continued. DBM together with GTK/Finland, Selor/Netherlands and Terramentor/Greece participated in the SENSPOL-workshop in Sevilla 2002 with PNN- monitoring.

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SENSPOL Survey of Sensor Capabilities

NITON EUROPE GMBH

Company Name: Niton Europe GmbH Address: Joseph-Dollinger-Bogen 7-9 D-80807 München Germany Respondent’s Name: Bree Allen Position: Director, Sales and Marketing Email address: [email protected]

SENSOR SYSTEM

Description of sensor system The NITON Xli and XLt 700Series Energy Dispersive X-Ray Fluorescence (EDXRF) Analyzers are hand-held instruments used for direct, non-destructive and non-intrusive analysis of soils, ores, air and water filters, paints, and dust wipes for heavy metal concentration. Testing times range from a few seconds to a few minutes, depending on the desired analysis precision. Simultaneous measurement is performed for up to 25 elements (e.g. Hg, Pb, As, Cr, Ba, Cd, Sb, etc.) from low ppm to percent concentrations. The instrument uses rechargeable Li-ion battery packs, can store up to 3000 readings in memory, and may be connected to a PC for easy data downloading and report generation. The instrument may be used directly on-site for site characterization and hazard delineation.

NITON Xli Niton XLt

Environmental relevance Immediate, on-site heavy metal analysis with no necessary sample preparation or tedious instrument calibrations by the operator.

Commercial relevance The ability to offer immediate results for site characterizations, environmental hazards, and detailed remediation control.

Usability (i.e skilled, unskilled) Operation may be performed by unskilled user. Some countries require the user be trained for radiation safety and licensed for operation.

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Availability (eg. commercial, prototype, etc..) Available for purchase on 4 to 5 week delivery time. Available for rental in some countries.

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Heavy Metals. Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Pb, Hg, Rb, Sr, Zr, Mo, Ag, Cd, Sn, Sb, Ba, K, Sc, Ti, V. This list is customisable per the customer’s needs.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils, sediments, ores, concentrates, water filters, air filters, plastics, glasses, dust wipes

Sample phase (i.e. total, residual, free, dissolved or absorbed) Total

Sample preparation (e.g. filtering, SPE, derivatisation etc…) No sample preparation necessary; samples may be analysed in-situ. Optional preparation may be performed to optimise homogeneity and sample conditions to obtain optimal analytical performance.

Sample handling (i.e. manual, automated etc,,,) Manual

Sample size For soil sampling, a minimum of 4 grams is required.

Sensor Measurements

Assay protocol Protocol includes activation of instrument, a 5 minute warm up period, quality control check on certified standard, then direct analysis of unknown samples. Sample preparation is optional per available time of operator, and desired data quality objectives. (Additional sample preparation provides improved analysis accuracy, by reducing “sample sources of error”. These sources of error include water content, contamination heterogeneity, and particle size heterogeneity.) As each test may be done in only a matter of seconds, multiple tests may be performed on one sample and averaged to reduce heterogeneity effects. A bench-top test stand is available for prepared sample analysis in mobile or fixed laboratory. In test stand, instrument may be controlled remotely by a PC.

Continuous measurement No

Present measurement performance:

1. Detection levels ppm to percent level concentrations; varies per matrix, and per element of interest.

2. Selectivity Total metal concentration, not valency selective.

3. Potential interference High concentrations of some elements may interfere with analysis of other elements present only in low concentrations. Instrument automatically compensates by varying individual element LOD’s per sample characteristics.

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4. Adaptability to other pollutants Element list is customisable for the addition of other metals. Calibration is also adaptable to different sample types (e.g. glasses or plastics).

Further work developments Development work is continuously undertaken to improve analysis precision and sensitivities. Customers are always provided with software enhancements and upgrades at no charge.

PAPERS PUBLISHED

United States Environmental Protection Agency, “Environmental Technology Verification Report, Field Portable X-Ray Fluorescence Analyzer, XL Spectrum Analyzer” Washington DC, USA, 1998. link: http://www.epa.gov/etv/pdfs/vrvs/01_vr_niton.pdf

Clark, Scott, Ph.D , William Menrath, Mei Chen, Sandy Roda, and Paul Succop, “Use of a Field Portable X-Ray Fluorescence Analyzer to Determine the Concentration of Lead and Other Metals in Soil Samples” Ann Agric Environ Med, 6, 27-32, 1999.

Hankins, John, Kevin Miller, Robert Kovach, Paul Smart, “The Use of Energy Dispersive X- Ray Fluorescence in Rapid Site Characterization.” Fuss and O’Neill, Manchester, Connecticut.

Kienbusch, Michael R., Don Sackett, Jonathan Bass, Scott Manchester, “Innovative Means of Using XRF for In-Field Lead Analysis of High Volume Air Filters.” Presented at the 1996 Air and Waste Management Association Meeting and Exhibition, Nashville, Tennessee

Ridings, M. AJ Shorter, J. Bawden-Smith, “Strategies for the Investigation of Contaminated Sites Using Field Portable X-Ray Fluorescence (FPXRF) Techniques.” CSIRO Tropical Agriculture, St. Lucia, Australia.

Shefsky, Stephen, “Lead in Soil Analysis Using the NITON XL.” Presented at the Internation Symposium of Field Screening Methods for Hazardous Waste and Toxic Chemicals, Las VEGAS, 1995.

Shefsky, Stephen, “Comparing Field Portable X-Ray Fluorescence (XRF) to Laboratory Analysis of Heavy Metals in Soil.” Presented at the Internation Symposium of Field Screening Methods for Hazardous Waste and Toxic Chemicals, Las VEGAS, 1997.

Spittler, Thomas M. “Assessment of Lead in Soil and Housedust Using Portable XRF Instruments.” USEPA Regional Lab, Lexington, Massachussetts

Sterling, David, Kevin Roegner, Roger Lewis, Douglas Luke, Lynn Wilder, and Sella Burchette, “Evaluation of Four Samplings Methods for Determining Exposure of Children to Lead-Contaminated Household Dust.” Environmental Research, Section A, number 81, pp. 130-141, 1999.

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Sterling, David, Roger Lewis, Douglas Luke, and Brooke Shadel, “A Portable X-Ray Fluorescence Instrument for Analyzing Dust Wipe Sample for Lead: Evaluation with Field Samples.” Environmental Research, Section A, number 83, pp. 174-179, 2000.

Zamurs, John, Jonathan Bass, Bradley Williams, Ruth Fritsch, Donald Sackett, Robert Heman, “Real Time Measurement of Lead in Ambient Air During Bridge Paint Removal.” New York State Department of Transportation, 1997.

Related Readings

Piorek, Stanislaw, “Determination of Metals in Soils by Field-Portable XRF Spectrometry.” Current Protocols In Field Analytical Chemistry, 3B.1.1 – 3B.1.18, 1998.

US EPA Method 6200 “Field Portable X-Ray Spectrometry for The Determination of Elemental Concentrations in Soil and Sediment” January 1998. link: http://www.epa.gov/epaoswer/hazwaste/test/pdfs/6200.pdf

PROFILE OF ORGANISATION

NITON LLC is a privately-held firm headquartered in Billerica, MA USA, comprised of approximately 65 employees worldwide. Niton Europe GmbH is a subsidiary providing sales, marketing, and service support for NITON products in Europe and Africa. NITON LLC develops, manufactures and markets x-ray fluorescence (XRF) instrumentation for a wide variety of testing applications, including the analysis of metal and precious metal alloys, mining samples, paint, soil, powders, filters and coatings. The company’s customers, in dozens of countries around the world, include national, state, and local governments and governmental agencies, Fortune-500 and other large corporations, small businesses, and leading universities and research institutions. NITON’s hand-held XRF analyzers offer laboratory instrument performance you can take with you anywhere; they are easy to use, economical, and priced to provide unsurpassed value. NITON’s first handheld XRF was developed under a grant from the US Department of Energy (DOE) and was released to market in 1994. NITON has earned numerous distinctions, including obtaining multiple patents for XRF analysis and for radon gas detection; successfully completing innovative research and development projects funded by US DOE, US Environmental Protection Agency (EPA), US Housing and Urban Development (HUD), and others; and winning the prestigious RandD 100 Award in 1995 for "One of the 100 Most Technologically Significant New Products of the Year".

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NMRC, IRELAND

Insitute Name: NMRC, Ireland Research group: Transducers Group Address: University College, Lee Maltings, Prospect Row, Cork, Ireland. Respondents Name: Dr Damien Arrigan Position: Senior research scientist Email address: [email protected]

SENSOR SYSTEM

Description of sensor system Heavy Metal Analyser HEMA 2002. Electrochemical system consisting of hardware, software and electrode devices, which can be applied to the determination of toxic heavy metals in the environment, including soil extracts.

Environmental relevance Determination of toxic heavy metals by stripping analysis techniques at solid electrodes. Sample types include waters as well as polluted soil extracts.

Commercial relevance Low-cost, field deployable device with software based calibration and data output. Still at developmental prototype stage. We seek partners with whom to continue the development and examine new applications.

Usability (i.e skilled, unskilled) Semi-skilled (technician)

Availability (eg. commercial, prototype, etc..) Prototype.

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Copper, lead, mercury, etc.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Waters and soil extracts.

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved and solvated extracts

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Addition of suitable electrolyte for water analysis; soil extraction procedure for soil analysis.

Sample handling (i.e. manual, automated etc,,,) Manual

Sample size Approx. 2.5 mL

Sensor Measurements

Assay protocol Defined by the software, the user does not need to follow any detailed methodology. User dilution of sample and addition of standards additions to matrix are required.

Continuous measurement NO

Present measurement performance:

1. Detection levels Approx. 25 ppb up to ppm levels Linear calibration range 25-1000 ppb

2. Selectivity Detection of heavy metals in complex matrix is possible.

3. Repeatability ca. 10 %.

4. Potential interference Organic matter in samples

5. Adaptability to other pollutants Yes, by using electrode modification strategies.

Further work developments Further development of instrumentation and electrode devices is required to get beyond the prototype stage.

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Envisaged performance and optimisation Will be the best “in-the-field” system for toxic heavy metals analysis.

BOTTLENECKS

Lack of information and definition of requirements for the devices/system by potential end- user. This could be addressed by getting the end-users to fill out a questionnaire stating what measurement problems they have – analyte(s), concentration levels, matrix, required response time; screening or detailed analysis? Etc etc.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

There are no partner helping in the development of the sensor and would welcome commercial interest.

FURTHER INFORMATION AND COMMENTS

By asking the end-users a series of questions similar to the Senspol questionnaire – that outline areas such as; what are their measurement problems? What analytes(s), concentration ranges and matrix? Etc etc. These answer could then be published (anonymously if desirable) on the Senspol website.

PROFILE OF ORGANISATION

NMRC Ireland is Ireland’s Information and Communications Technologies (ICT) research institute. It was set up over 20 years ago and is a constituent part of National University of Ireland – Cork (NUI-CORK). It has over 250 staff and students working at ICT/Bio interface, nanotechnology, photonics and microelectronics. The Transducers Group is concerned with the development of systems and devices for chemical, biological and physical measurements and has been an active participant in EU sensor research for over 10 years. Silsoe

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UNIVERSITY OF COIMBRA

University Name: Instituto Pedro Nunes – University of Coimbra

Research group: Laboratory for Electroanalysis and Corrosion (LEC) Address: Instituto Pedro Nunes Rua Pedro Nunes 3030-199 Coimbra, Portugal

Respondents Name: Christopher Brett Position: Director of LEC / Professor of Chemistry Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

Electrochemical batch injection analysis sensor system for trace metal ions Electrochemical batch injection analysis allows the direct determination of the free fraction of trace metal ions in raw, untreated samples of waters at mercury thin film electrodes protected with cation-exchange polymer coatings, using square wave anodic stripping voltammetry. The system is small and portable. In natural waters and effluents trace metal ions can be present in free form or strongly complexed, the free fraction being that which is toxic. It is important to know both of these parameters. The electrode is immersed in a cell filled with electrolyte and injections of analyte sample, volume 50µl, are made directly over the electrode from a motorized electronically-controlled micropipette. Reduction of the metal ions leads to accumulation of the metals; the subsequent square wave redissolution scan leads to current peaks which are quantified. Detection limits are of the order of 5 nM. The standard addition method is used to determine the concentrations of components in analyte samples. The method can be applied to raw and samples digested in different ways, which leads to the free fraction and total amount of the metal present in the sample.

Sample injection Aux. electrode Ref. electrode

Working electrode

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Environmental relevance Rapid diagnostic of toxic trace metals in the environmentally toxic form (chemically labile species)

Commercial relevance Provides rapid diagnostic in the field and can also provide accurate data in the laboratory

Usability (i.e skilled, unskilled) A basic knowledge of chemical analysis and computers is necessary.

Availability (eg. commercial, prototype, etc..) Prototype stage

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Toxic trace metals : zinc, cadmium, lead, copper

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Surface and groundwater, effluents

Sample phase (i.e. total, residual, free, dissolved or absorbed) Free (i.e. chemically labile) fraction in liquid solutions

Sample preparation (e.g. filtering, SPE, derivatisation etc…) No necessity for sample preparation if in the liquid state

Sample handling (i.e. manual, automated etc,,,) Liquid analyte samples manipulated by motorised, electronic micropipettes

Sample size 10 – 100 microlitres

Sensor Measurements

Assay protocol The Nafion coated mercury thin film electrode is previously formed: (1) 3 µl of 0.25% Nafion + 2 µl dimethylformamide dropped onto horizontally held glassy carbon electrode in air. After drying curing is done by a hot air stream at ~70ºC. (2) 10 µl of 0.1 M mercury ions are injected over Nafion coated electrode at -1.0V to form mercury film between Nafion and glassy carbon. Strategy: Screening data, i.e. estimates, are first acquired to choose the deposition potential, any dilution factors and appropriate concentrations for spiking analyte samples for quantification by standard addition. Assay protocol – square wave anodic stripping voltammetry (SWASV): - A volume of 50 µL of analyte sample (without any pre-treatment or electrolyte addition) is injected into the BIA system at beginning of 30 s accumulation step of SWASV. If necessary, dilution of original sample can be done to operate in the linear range of the sensor (< 1 µ mol L-1). - A square wave stripping voltammogram is recorded: the peak potential identifies the metal ions and the peak current is proportional to concentration. - 50µL of electrolyte solution is injected between determinations to remove memory effects.

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Continuous measurement No

Present measurement performance:

1. Detection levels - 5 nmol L-1 for all metal ions

2. Selectivity Good – provided by applied potential

3. Repeatability Good

4. Potential interference In principle, all of these are solved

5. Adaptability to other pollutants Yes, with appropriate electrochemical applied potential protocol

Further work developments Improvement of protocols and software. More compact cell to increase portability.

Envisaged performance and optimisation Performance evaluated in Senspol workshop report (Sevilla meeting, November 2002)

BOTTLENECKS

Good portable electrode control and data analysis instrumentation (now largely solved)

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing

Possibly electronic engineers and software developers collaboration for automation purposes would help with sesnor development.

FURTHER INFORMATION AND COMMENTS

Demonstrations, participation in exhibitions and industrial fairs

PAPERS PUBLISHED

C.M.A. Brett, A.M. Oliveira Brett, F.-M. Matysik, S. Matysik, S. Kumbhat, Talanta, 43 (1996) 2015. C.M.A. Brett, D.A. Fungaro, J.M. Morgado, M.H. Gil, J. Electroanal. Chem., 468 (1999) 150. D.A. Fungaro, C.M.A. Brett, Talanta, 50 (2000) 1223. C.M.A. Brett, D.A. Fungaro, J. Braz. Chem. Soc., 11 (2000) 298. C.M.A. Brett, J.M. Morgado, J. Appl. Toxicol., 20 (2000) 477.

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PROFILE OF ORGANISATION

Instituto Pedro Nunes (IPN) (Internet page: http://www.ipn.pt) is a non-profit-making association created in 1991 for postgraduate training, research and development in science and technology, and technology transfer to industry, as well as providing support services. As the main link between the University of Coimbra and industry, IPN conducts its research activity using its own laboratories in its Centre for Innovation and Technology Transfer and in the laboratories of the University of Coimbra. It also promotes the incubation of new technologically-based companies. The staff includes around 50 research collaborators who are professors or researchers from the University of Coimbra, technical staff and 5 administrative personnel. IPN is developing an active research programme in a number of aspects relating to environment and health. The recently-created Laboratory for Electroanalysis and Corrosion is carrying out an active programme of sensor development at national and international levels, with particular attention to environmental problems and monitoring. There is a strong link with the Electrochemistry and Corrosion Research Group in the University of Coimbra.

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UNIVERSITY OF NEUCHATEL /UNIVERSITY OF GENEVA IDRONAUT SRL

University/ Institute Name: University of Neuchatel/University of Geneva Idronaut Srl Department/ Research group SAMLAB; CABE; Idronaut Address: Jaquet-Droz 1, 2007 Neuchatel, Switzerland; Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland; Via Monte Amiata 10, 20047 Brugherio (MI), Italy Respondents Name: M. Koudelka-Hep (SAMLAB) M.-L- Tercier Waeber (CABE) F. Graziottin (Idronaut) Email address: [email protected] / [email protected] [email protected]

SENSOR SYSTEM 1

Description of sensor system Voltammetric probe with microfabricated individually addressable gel-integrated microsensor arrays

Environmental relevance Real-time, high spatial resolution, concentration profile measurements at liquid-liquid and liquid-solide interfaces

Commercial relevance Mass production of various geometry under well controlled condition First system allowing simultaneous complete voltammogram measurements over a large number of individual microelectrodes using fast dynamic techniques.

Usability (i.e skilled, unskilled) Skilled trained people

Availability (eg. commercial, prototype, etc..) Prototype

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Trace elements

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Sample matrix ( e.g. soils, sediments, surface and groundwater ) Anoxic fresh and anoxic/oxic sea waters / sediments interface

Sample phase (i.e. total, residual, free, dissolved or absorbed) Free + labile trace metal species

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None

Sample handling (i.e. manual, automated etc,,,) None

Sensor Measurements

Assay protocol - Controlled deployment at the interface to be studied using a micromanipulator - Equilibration of the sensor antifouling gel with the sample (typically 5 min); - Simultaneous voltammetric measurements over the 63 sensor lines (measuring time in a range 10 to 30 min as a function of trace elements concentrations)

Continuous measurement Yes

Present measurement performance:

1. Detection levels – ppt to ppb depending of the target elements

2. Selectivity Yes

3. Repeatability RSD max. 10% at ppt level

4. Potential interference - S(-II) with can also be an analyte

5. Adaptability to other pollutants Yes

Further work developments - Optimisation of the sensor geometry, i.e. number of microelectrodes on each individual lines and spacing distance between the sensor lines. - Optimisation of the simultaneous control of the signal applied and detected on each individually addressable microsensor lines.

Envisaged performance and optimisation In situ autonomous measurements of trace element concentration profiles as a function of time at the sediment-water interface

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Idronaut are commerical backers.

FURTHER INFORMATION AND COMMENTS

Meeting involving academic, governmental and industrials people would help with bridging the gap between developers and endusers.

PAPERS PUBLISHED

Electroanalysis ,12, 27, 2000 Anal. Chem., 73, 2273, 2001.

SENSOR SYSTEM 2

Description of probe (sensor) system Voltammetric In situ Profiling System (VIP system) based on gel-integrated microsensor array specifically developed to allow direct measurements in complex media.

Environmental relevance Remote in situ monitoring/profiling of trace elements in natural waters (fresh and sea waters) down to 500 m

Commercial relevance Autonomous environmental and pollution control monitoring

Usability (i.e skilled, unskilled) Trained people

Availability (eg. commercial, prototype, etc..) Commercial

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Trace elements

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Fresh / sea waters (surface and groundwater)

Sample phase (i.e. total, residual, free, dissolved or absorbed) Free + labile metal species, i.e. fraction the most easily available, without pre-treatment of the sample

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Sample preparation (e.g. filtering, SPE, derivatisation etc…) None

Sample handling (i.e. manual, automated etc,,,) In situ automated

Sample size 7 ml 8

Probe (Sensor) Measurements

Assay protocol - Controlled deployment at a given depth via an Ocean Seven 316 multiparameter probe coupled to the VIP voltammetric probe - Flushing of the flow-trough voltammetric cell with the sample (typically 1 min) - Equilibration of the sensor antifouling gel with the sample (typically 5 min); - Voltammetric measurement (measuring time in a range 10 to 30 min as a function of trace elements concentrations)

Continuous measurement Yes

Present measurement performance:

1. Detection levels Simultaneous measurements of Cu(II), Pb(II), Cd(II) and Zn(II) at the ppt level as well as Mn(II) and Fe(II) at the ppb level

2. Selectivity Yes

3. Repeatability RSD max. 10% at ppt level

4. Potential interference - S(-II) with can also be an analyte

5. Adaptability to other pollutants Yes

Further work developments - Development of new voltammetric microsensors - Development of a submersible mini-flow injection system for in situ automated sample pre- treatment before voltammetric detection - Development of a new probe based on 3 individual flow-through voltammetric cells and potentiostats - Improvement of firmware and software Envisaged performance and optimisation - In situ autonomous measurements of other environmental relevant elements and/or trace metal species - In situ simultaneous measurements of specific trace metal species, i.e in situ speciation

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- Automatic data analysis and transmission to land station.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

- SAMLAB, University of Neuchatel – Switzerland (Milena Koudelka-Hep - [email protected]): production of the microelectrode arrays used to prepare the gel- integrated microsensor of the VIP voltammetric probe

Idronaut Srl (MI) are commercial backers

FURTHER INFORMATION AND COMMENTS

Meeting involving academic, governmental and industrials people could help to bridge the gap between developers and end users.

PAPERS PUBLISHED

- Electroanalysis,10, 355, 1998 - Env. Sci. Technol. , 32, 1515, 1998 - Sea Technol. , 40, 74, 1999 - J. Geochem. Expl. 75, 17, 2002 - Ch. 2 in :“Environmental Electrochemistry: Analysis of Trace Element Biogeochemistry“ American Chemical Society, Symposium Series No. 811, Washington DC, 2002.

SENSOR SYSTEM 3

Description of probe (sensor) system Compact portable Voltammetric In-line Analyzer for on-Field monitoring (VIA-Field system). This system has been developed based on the technical and analytical advances of the VIP system.

Environmental relevance Remote/screening on-site trace element measurements in e.g. shallow rivers, runoff waters or for hazardous-waste site investigation.

Commercial relevance Spot or autonomous long-term environmental and pollution control monitoring

Usability (i.e skilled, unskilled) Trained people

Availability (eg. commercial, prototype, etc..)

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SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Trace elements

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Fresh / sea waters

Sample phase (i.e. total, residual, free, dissolved or absorbed) Free + labile metal species, total extractable metal concentrations.

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None or simple preparation (i.e. on-line acidification, on-line filtration followed by acidification) depending of the trace element species to be analysed

Sample handling (i.e. manual, automated etc,,,) Automated

Sample size 5 ml

Probe (Sensor) Measurements

Assay protocol - Flushing of the flow-trough voltammetric cell with the sample (typically 1 min) - Equilibration of the sensor gel with the sample (typically 5 min); - Voltammetric measurement (measuring time in a range 10 to 30 min as a function of trace elements concentrations)

Continuous measurement Yes

Present measurement performance:

1. Detection levels Simultaneous measurements of Cu(II), Pb(II), Cd(II) and Zn(II) at the ppt level as well as Mn(II) and Fe(II) at the ppb level

2. Selectivity Yes

3. Repeatability RSD max. 10% at ppt level

4. Potential interference - S(-II) with can also be an analyte

5. Adaptability to other pollutants Yes

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Further work developments - Development of new voltammetric microsensors - Improvement of the integrated flow injection system and analytical procedure for on-line sample pre-treatment. - Improvement of firmware and software

Envisaged performance and optimisation - on-line measurements of other environmental relevant elements and/or trace metal species - Automatic data analysis and transmission to land station.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Idronaut Srl (MI) are commercial backers

FURTHER INFORMATION AND COMMENTS

Meeting involving academic, governmental and industrials people would help bridge the gap between developers and endusers.

PAPERS PUBLISHED

″In situ, real-time monitoring of specific trace element species in aquatic systems using sophisticated voltammetric probes ″. M.-L. Tercier-Waeber XVI Italian National Meeting on Analytical Chemistry; Plenary lecture of the Division of Analytical Chemistry and Marine Science. Portonovo (Ancona), Italy, September 24-28, 2001.

″Submersible/portable voltammetric probes for real-time continuous trace element monitoring and speciation in aquatic systems : State of the art and futur prospects″. M.-L. Tercier-Waeber, J. Buffle, M. Koudelka-Hep, F. Graziottin. American Chemical Society 221th National Meeting ; Divisions of Industrial, Engineering Chemistry and Environmental Chemistry ; Invited lecture for the session: Process Analytical Chemistry in Support of Green Chemistry. San-Diego, CA, April 1-5, 2001.

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SENSOR SYSTEM 4

Description of sensor system Micro-fabricated C, Au and Ir microelectrodes and microelectrode arrays coated or not with Hg layers and anti-fouling protective gel Volta-metric flow-trough microcells

Environmental relevance Monitoring of various trace elements species in aquatic ecosystems

Commercial relevance Mass production at low cost under well controlled condition and various geometry

Usability (i.e skilled, unskilled) Trained people

Availability (eg. commercial, prototype, etc..) Prototype : C and Au microelectrodes ; voltammetric flow-trough microcells Commercial : Ir single and array microelectrodes

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Trace elements

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Fresh and sea waters

Sample phase (i.e. total, residual, free, dissolved or absorbed) Free + labile, colloidal and particulate species depending on the sample preparation

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None or simple preparation (i.e. acidification or filtration followed by acidification) depending of the trace element species to be analysed

Sample handling (i.e. manual, automated etc,,,) Manual or automated

Sample size Sample volume as low as few tens of µl.

Sensor Measurements

Assay protocol Direct measurements or measurements in two steps for antifouling gel covered sensors, i.e. steps 1: equilibration of the gel with the sample (typically 5 min); step 2: direct voltammetric measurement inside the gel

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Continuous measurement Yes

Present measurement performance:

1. Detection levels - ppt to ppb depending of the target elements

2. Selectivity Yes

3. Repeatability RSD of max. 10% for measurements at the ppt level

4. Potential interference S(-II) with can also be an analyte

5. Adaptability to other pollutants Yes

Further work developments Nano-sized microelectrode arrays. Design optimisation of sensors and cells

Envisaged performance and optimisation Extension to other limits, i.e. sensitivity, measurements of different metal species and target elements.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

We do you have commercial backing Idronaut Srl

FURTHER INFORMATION AND COMMENTS

Meetings with interested academic, governmental and industrial people

PAPERS PUBLISHED

Anal. Chim. Acta, 329, 203, 1996 Anal. Chem., 70, 2949, 1998 Meas. Sci. Technol., 10, 1202, 1999 Electroanalysis, 12, 27, 2000 Anal. Chem., 73, 2273, 2001 US Patent number : 5,865,972, 1999

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PROFILE OF ORGANISATION

SAMLAB – research activities of SAMLAB cover chemical, biochemical, physical sensors and microsystems. A flexible and fully equipped microfabrication facility allows the execution of all necessary technological steps for thin-film devices. The laboratory comprises also various electrochemical equipments and surface characterisation methods (ESEM, AFM).

CABE – research interests of CABE lie in the study of environmental microstructures and microprocesses to better understand the macroscopic structures and behaviour of environmental systems, in particular surface waters, sediments and soils. Research activities of CABE are divided into four subgroups: i) development and application of analytical sensors/techniques and environmental probes for laboratory, on-field and in situ measurements of specific trace elements species; ii) studies of natural microstructures and dynamic processes; iii) experimental studies of model colloidal systems; iv) numerical modelling of physicochemical structures and processes.

IDRONAUT is a research-manufacturing company specialized in: CTDs, sensors, hardware and software for profiling and monitoring the chemical and physical properties of waters (depth, temperature, conductivity, salinity, oxygen, pH, redox, chlorophyll, transmission, turbidity, suspended solids, radiance, etc.) and boreholes. Our main products are: - OCEAN SEVEN 303 Probe, diameter 43 mm, - OCEAN SEVEN 316 Probe, diameter 75/100 mm, - “On-line” module for monitoring chemical and physical parameters, - VIP (Voltammetric In-situ Profiler) System, to profile and monitor trace metals (copper, lead, cadmium and zinc), Conductivity-Temperature Transfer Standard CT 01.

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VLAAMSE INSTELLING VOOR TECHNOLOGISCH ONDERZOEK (VITO)

Organisation Name: Vlaamse Instelling voor technologisch onderzoek (Vito)

Research group: Environmental Technology Address : Boeretang 200 2400 Mol Belgium

Respondent Name: Ludo Diels Position: Head of the department Email address: [email protected]

SENSOR SYSTEM 1

Description of sensor system The construction of E. coli CM1569 is a derivative of E. coli DH10B containing pC202. The construction of CM1569 is based on pC202 in which the regulatory gene arsR and ∆arsB gene, of the ars arsenic resistance determinant are transcriptionally fused upstream of the lux- CDABE genes of pMOL877. Both genes arsR and arsB were cloned from plasmid pI258 containing the 2.7-kb ars operon (consisting of 3 genes arsRBC) from Staphylococcus aureus. This resulted in inducible light production controlled by the ars regulon.

Environmental relevance - 3- A test to quantify bioavailable levels of AsO2 and AsO4 in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives, …

Commercial relevance Useful to determine the bioavailable metal fraction in soils/sediments and assess their potential risk in a very efficient and rapid way. No requirement of complex and labour- intensive handling of samples (unlike chemical analyses like sequential extractions).

Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) - 3- Arsenite (AsO2 ) and arsenate (AsO4 )

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Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

Sample handling (i.e. manual, automated etc,,,) Manual preparation

Sample size 5.00 g sample

Sensor Measurements

Assay protocol Luminometry assays were carried out using an ANTHOS LUCY1 luminometer at 23°C. For metal standards, duplicate samples were set-up per microtitre plate. As negative controls, 8 reaction samples containing bidistilled water were included in the tests. The reconstituted soil/sediment samples were tested in dilution series: typically 1:1, 1:2 and 1:4 and in duplicate. The bioluminescence emitted (ALU) and the optical density (OD620nm) of the cultures were measured over 16h at 30 min. intervals, and processed using the MIKROWIN software, as described by Corbisier et al. (1999). Further data processing was carried out using EXCEL 7.0.

Continuous measurement Yes

Present measurement performance:

1. Detection levels : - 3- ?AsO2 and AsO4

2. Selectivity: Highly selective

3. Repeatability: Highly reproducible

4. Potential interference: Inhibition at too high metal levels

5. Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strains R. eutropha AE2790 or AE864 or AE2279 Further work developments

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BOTTLENECKS

E.coli as biosensor is not an optimal tool to determine the amount of bioavailable As in contaminated soils, … E. coli is a bacterium from the human intestine whereas the soil bacterium R. eutropha is a metal-resistant environmental isolate and consequently much more appropriate for our purposes. However, attempts to construct such a biosensor did not succed yet. Do you have any suggestions on how this could be addressed? Some new constructs in R. eutropha CH34 were made and will be tested in the near future.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing Students- PhD students would help in sensor development

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

PAPERS PUBLISHED

Corbisier,P, G Ji, G Nuyts, M Mergeay, and S Silver.1993. luxAB gene fusions with the arsenic and cadmium resistance operons of Staphylococcus aureus plasmid pI258. FEMS Microbiol Lett, 110(2): 231-238 Corbisier, P., E. Thiry, A. Masolijn, and L. Diels. 1994. Construction and development of metal ion biosensors. In: Bioluminescence and chemoluminescence fundamentals and applied aspects. Campbell, Kricka, and Stanley (eds). John Wiley and Sons.

SENSOR SYSTEM 2

Description of sensor system The construction of R. eutropha AE2440, a derivative of R. eutropha CH34, containing pMOL1531 (20.23 kb) is schematically depicted in the included picture (Figure 1). The construction of AE2440 is based on pMOL1531, in which the genes, chrB∆chrA, of the chr chromaat resistance determinant of R. eutropha CH34 are transcriptionally fused upstream of the lux-CDABE genes of pMOL877. This resulted in inducible light production controlled by the chrB and ∆chrA genes.

Environmental relevance 2- A test to quantify bioavailable levels of CrO4 in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives, …

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Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) 2- Chromate (CrO4 )

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

Sample handling (i.e. manual, automated etc,,,) Manual preparation

Sample size 5.00 g sample

Sensor Measurements

Continuous measurement Yes

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Present measurement performance:

1. Detection levels : 1 µM Cr(VI) (DR : 1 – 40 µM) 2- 1.21 mg/kg dm CrO4

2. Selectivity: 2- High selectivity for CrO4

3. Repeatability: Highly reproducible

Potential interference: Inhibition at too high metal levels

Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strains R. eutropha AE2790 or AE864 or AE2279

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing Students- PhD students would help in sensor development

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

PAPERS PUBLISHED

Nies, A, DH Nies, and S Silver. 1990. Nucleotide sequence and expression of a plasmid- encoded chromate resistance determinant from Alcaligenes eutrophus. J. Biol. Chem, 265: 5648 - 5653. Corbisier, P., et al. 1999. Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Anal. Chim. Acta, 378: 235-244.

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SENSOR SYSTEM 3

Description of sensor system

The construction of R. metallidurans AE1239 is a derivative of R. metallidurans AE866 containing only pMOL90::Tn4431. Strain AE866 itself resulted from biparental mating between R. metallidurans DS185 (pMOL90, pMOL85 and pMOL86) and an E. coli strain (CM601) containing the Tn4431 on the suicide plasmid pUCD623. This tetracycline resistant strain AE866 was selected for its copper-induced light emission. Strain AE984 was derived from strain AE866 after curing plasmids pMOL86 and pMOL85. A cupC-luxCDABE transcriptional fusion was obtained in the strain AE866 after random Tn4431 insertion into the cupC gene on pMOL90 and resulting in the biosensor AE1239 after curing plasmids pMOL86 and pMOL85 and triparental mating with rifampicine resistant R. metallidurans AE104 (CH34 wild type strain without pMOL30 and pMOL28).

The Tn4421 transposon contains a promotorless luxCDABE operon of Vibrio fischeri.

Environmental relevance A test to quantify bioavailable levels of Cu2+ in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives, in situ bioprecipitation, …Furthermore, potential heavy metal bioleaching activity can be determined as well.

Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Copper (Cu2+)

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater/ waste/ solids

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

Sample handling (i.e. manual, automated etc,,,) Manual preparation

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Sample size 5.00 g sample

Sensor Measurements

Continuous measurement Yes

Present measurement performance:

1. Detection levels : 2 µM Cu2+ (DR : 2 – 40 µM)

2. Selectivity: Highly selectivity for copper; slight response with cadmium or at high zinc concentrations.

3. Repeatability: Highly reproducible

4. Potential interference: Inhibition at too high metal levels

5. Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strains R. metallidurans AE2790 or AE864 or AE2279

BOTTLENECKS

The presence of amounts of metal ions higher than e.g. 20µM Cu greatly reduces the use of the biosensor. The plasmid pMOL90::Tn4451 could be transferred into a strain that is resistant to a greater range of heavy metals such as R. metallidurans CH34 to improve the Cu-selectivity.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing Students- PhD students would help in sensor development

152 SENSPOL Survey of Sensor Capabilities

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

PAPERS PUBLISHED

Corbisier, P., E. Thiry, A. Masolijn, and L. Diels. 1994. Construction and development of metal ion biosensors. In: Bioluminescence and chemoluminescence fundamentals and applied aspects. Campbell, Kricka, and Stanley (eds). John Wiley and Sons. Corbisier, P., E. Thiry, and L. Diels. 1996. Bacterial biosensors for the toxicity assessment of solid wastes. Environmental Toxicoloy and Water Quality, 11: 171-177.

SENSOR SYSTEM 4

Description of sensor system The construction of E. coli CM2624 is a derivative of E. coli DH10B containing pMOL1636 (19kb). The construction of CM2624 is based on pMOL1636 in which the regulatory gene merR of the mercury resistance determinant is transcriptionally fused upstream of the lux- CDABE genes of pMOL877. The merR gene was isolated from AE104 (wild type R. eutropha CH34 without pMOL28 and pMOL30). This resulted in inducible light production controlled by the mer regulon.

Environmental relevance A test to quantify bioavailable levels of Hg2+ in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives, in situ bioprecipitation, …

Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Mercury (Hg2+)

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Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

Sample handling (i.e. manual, automated etc,,,) Manual preparation

Sample size 5.00 g sample

Sensor Measurements

Continuous measurement Yes

Present measurement performance:

1. Detection levels : µM Hg2+ (DR: 0.01 – 1 µM)

2. Selectivity: Highly selective

3. Repeatability: Highly reproducible

4. Potential interference: Inhibition at too high metal levels

5. Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strains R. eutropha AE2790 or AE864 or AE2279

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BOTTLENECKS

E.coli as biosensor is not an optimal tool to determine the amount of bioavailable Hg in contaminated soils, … E. coli is a bacterium from the human intestine whereas the soil bacterium R. eutropha is a metal-resistant environmental isolate and consequently much more appropriate for our purposes. However, attempts to construct such a biosensor did not succeed yet.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Student Peter Brien is helping with the development of this sensor No commercial backing

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

PAPERS PUBLISHED

Brien, P. 2000. Construction and characterisation of a bacterial biosensor for mercury. KHL, Thesis. Corbisier, P., et al. 1999. Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Anal. Chim. Acta, 378: 235-244.

SENSOR SYSTEM 5

Description of sensor system The construction of R. metallidurans AE2515, a derivative of R. metallidurans CH34 containing pMOL1150 (20.8kb), is schematically depicted in the included picture (Figure 1). The construction of AE2515 is based on pMOL1550, in which the regulatory genes, cnrYXH, of the cnr cobalt and nickel resistance determinants of R. metallidurans CH34 are transcriptionally fused upstream of the lux-CDABE genes of pMOL877 (Van der lelie et al.,1997). This resulted in inducible light production controlled by the cnr regulon.

Environmental relevance A test to quantify bioavailable levels of Ni2+ and Co2+ in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives, in situ bioprecipitation, …

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Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Nickel (Ni2+) and Cobalt (Co2+)

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

Sample handling (i.e. manual, automated etc,,,) Manual preparation

Sample size 5.00 g sample

Sensor Measurements

Continuous measurement Yes

Present measurement performance: 1. Detection levels : 0.08 µM Ni2+ en 9µM Co2+ (DR : 0.08 – 100 µM)

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2. Selectivity: Highly selectivity for nickel in mixed metal assay.

3. Repeatability: Highly reproducible

4. Potential interference: Inhibition at too high metal levels (> 400µM cobalt)

5. Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strains R. eutropha AE2790 or AE864 or AE2279

BOTTLENECKS

Not able to predict effects of treatment with chemical chelators like metal-EDTA complexes.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing Students- PhD students would help in sensor development

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

PAPERS PUBLISHED

Tibazarwa, C., P. Corbisier, M. Mensch, A. bossus, P. Solda, M. Mergeay, L. Wyns, and D. van der lelie. 2001. A microbial biosensor to predict bioavailable nickel in soil and its transfer to plants. Environmental Pollution, 113: 19-26. Van der Lelie, D., L. Regniers, B. Borremans, A. Provoost, and L. Verschaeve. 1997. The VITOTOX test, a SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics. Mut Res., 2-3:279-290 Corbisier, P., et al. 1999. Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Anal. Chim. Acta, 378: 235-244.

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SENSOR SYSTEM 6

Description of sensor system The construction of R. eutropha AE2450 is a derivative of R. eutropha CH34 containing pMOL1539 (19.1 kb). The construction of AE2450 is based on pMOL1539, in which the regulatory gene, pbrR, of the pbr lead resistance determinant of R. eutropha CH34 is transcriptionally fused upstream of the lux-CDABE genes of pMOL877 (Van der lelie et al.,1997). This resulted in inducible light production controlled by the pbr regulon.

Environmental relevance A test to quantify bioavailable levels of Pb2+ in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives, in situ bioprecipitation, …

Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Lead (Pb2+)

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater/ waste/ solids

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

Sample handling (i.e. manual, automated etc,,,) Manual preparation

Sample size 5.00 g sample

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Sensor Measurements

Continuous measurement Yes

Present measurement performance:

1. Detection levels : 0.5 µM Pb2+ (DR : 0.5 – 18 µM)

2. Selectivity: Highly selectivity for lead; no induction with Hg or Zn; very small induction with Cd.

3. Repeatability: Highly reproducible

4. Potential interference: Inhibition at too high metal levels

5. Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strains R. eutropha AE2790 or AE864 or AE2279

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing Students- PhD students would help in sensor development

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

PAPERS PUBLISHED

Borremans, B., J.L. Hobman, A. Provoost, N.L. Brown, and D. van der Lelie. 2001. Cloning and functional analysis of the pbr Lead resistance determinant of Ralstonia metallidurans CH34. J. Bacteriol., 183: 5651-5658.

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Corbisier, P., et al. 1999. Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Anal. Chim. Acta, 378: 235-244.

SENSOR SYSTEM 7

Description of sensor system The construction of R. eutropha AE1101 is the result of a biparental mating between R. oxalatica A5.3 (rifampicine resistant mutant of A5) and A. eutropha strain AE1050. This strain AE1050 itself containing pMOL28::Tn4431 is the result of a biparental mating between strain AE126 (a derivative of the wild type strain CH34 containing only pMOL28) and an E. coli strain (CM601) containing the Tn4431 on the suicide plasmid pUCD623. A tllA::lux transcriptional fusion was obtained in the derivative strain AE1050 which after conferring rifampicine resistance resulted in the biosensor AE1101. This resulted in inducible light production controlled by tllA genes.

The Tn4431 transposon contains a promotorless luxCDABE operon of Vibrio fischeri.

Environmental relevance A test to quantify bioavailable levels of Tl in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives.

Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Thallium (Tl+)

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater/waste/solids

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

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Sample handling (i.e. manual, automated etc,,,) Manual preparation

Sample size g sample

Sensor Measurements

Continuous measurement Yes

Present measurement performance:

1. Detection levels : 1 µM Tl1+ (DR: 1 – 50 µM)

2. Selectivity: Selective for thallium.

3. Repeatability: Highly reproducible

4. Potential interference: Inhibition at too high metal levels

5. Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strains R. eutropha AE2790 or AE864 or AE2279

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing Students- PhD students would help in sensor development

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

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PAPERS PUBLISHED

SENSOR SYSTEM 8

Description of sensor system The construction of R. metallidurans AE1433 is a derivative of R. metallidurans CH34 containing pMOL30::Tn4431 and pMOL28. A czcS::lux transcriptional fusion was obtained in the wild type strain CH34 after random Tn4431 insertion into the 5’ region of the czcS gene on pMOL30 resulting in the biosensor AE1433. This resulted in inducible light production controlled by czcS genes, which together with the czcR and czcD genes form the downstream regulatory region (DRR) of the czc determinant mediating resistance to cobalt, zinc and cadmium.

The Tn4421 transposon contains a promotorless luxCDABE operon of Vibrio fischeri. Environmental relevance

A test to quantify bioavailable levels of Zn2+, Cd2+, and Pb2+ in environmental samples, to assess the reduction of metal toxicity in contaminated soils following in situ immobilisation treatment e.g. with soil additives, in situ bioprecipitation, …

Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) Available at Vito for testing

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Zinc (Zn2+), and some effects on Cadmium (Cd2+) and lead (Pb2+)

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soils/sediments and surface/groundwater

Sample phase (i.e. total, residual, free, dissolved or absorbed) Bioavailable fraction from soil/sediment extract and surface/groundwater

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Sample preparation (e.g. filtering, SPE, derivatisation etc…) Soil/sediment: extraction by 30 ml RM (reconstitution medium) – dilution soil/sediment extract– biosensor preparation

Sample handling (i.e. manual, automated etc,,,) Manual preparation

Sample size 5.00 g sample

Sensor Measurements

Continuous measurement Yes

Present measurement performance:

1. Detection levels : 5 µM Zn (DR : 5 – 250 µM) ; 5 µM Cd (DR : 5 – 25 µM) 5.9 mg/kg dm Zn2+ and 6.37 mg/kg dm Cd2+

2. Selectivity: Zn, but some influence of Cd and Pb.

3. Repeatability: Highly reproducible

4. Potential interference: Inhibition at too high metal level

5. Adaptability to other pollutants: The presence of other toxic pollutants results in decrease of luminescence of a constitutive control strain R. metallidurans AE864.

Further work developments Envisaged performance and optimisation

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BOTTLENECKS

In the presence of zinc, neither cobalt nor cadmium are probably detected. However, the presence of solely cadmium allows the detection of the bioavailable fraction of this heavy metal.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

No partners nor commercial backing Students- PhD students would help in sensor development

FURTHER INFORMATION AND COMMENTS

Gather information in a central database and distribute it further. This questionnaire really offers a great opportunity to bridge the gap.

PAPERS PUBLISHED

Van der Lelie, D., T. Schwuchow, U. Schwidetzky, S. Wuertz, W. Baeyens, M. Mergeay, and D.H. Nies. 1997. Two-component regulatory system involved in transcriptional control of heavy-metal homeostasis in Alcaligenes eutrophus. Mol. Microbiol., 23: 493-503. Van der Lelie, D., L. Regniers, B. Borremans, A. Provoost, and L. Verschaeve. 1997. The VITOTOX test, a SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics. Mut Res., 2-3:279-290 Corbisier, P., et al. 1999. Whole cell- and protein-based biosensors for the detection of bioavailable heavy metals in environmental samples. Anal. Chim. Acta, 378: 235-244.

PROFILE OF ORGANISATION

Vito, the Flemish institute for technological research, is an independent research centre, a crossroads of knowledge where the latest technologies and practical applications meet. Vito conducts customer oriented contract research and develops innovative products and processes in the fields of energy, environment and materials, and this for both the public and the private sector. Central to all projects are protecting the environment and encouraging sustainable use of energy and raw materials, because we think that everyone, including those to come after us, has the right to a healthy living and working environment.

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COVENTRY UNIVERSITY

University Name Coventry University Address: Coventry University SE-JS Department: CMBE Address: Priory Street Coventry CV1 5FB Respondents Name: Professor Ian R.Peterson Position: Professor Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

Using a bipotentiostat configuration, the sensor monitors the conductance of a bilayer lipid membrane, which incorporates ligand-gated channel proteins specific for the target analyte. We have demonstrated good membrane fluidity and lifetimes in excess of three months. We have engineered a rugged implementation that plugs in to a printed circuit board edge connector. Full scientific details are given on http://www.nes.cov.ac.uk/ research/cmbe/biosens.htm

Environmental relevance

While initial investigations are being limited to high value-added applications, this principle will eventually lead to a generic range of sensors with high sensitivity and high specificity for a wide range of analytes.

Commercial relevance Yes.

Usability (i.e skilled, unskilled) Skilled, unskilled

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Availability (eg. commercial, prototype, etc..) Commercial prototype in development

SAMPLE

Targeted analyte(s) Toxins

Sample matrix (e.g. soils, sediments, surface and groundwater) All

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Extraction into an aqueous phase

Sample handling (i.e. manual, automated etc,,,) Only manual at present

Sample size 10 µL

Sensor Measurements

Assay protocol A fresh membrane element is removed from its package and plugged in to a desktop unit as shown. The liquid sample is dispensed onto the top aperture. After a predetermined time the measured concentration of the target analyte is displayed on the LCD display of the desktop unit.

Continuous measurement Yes

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Present measurement performance:

1. Selectivity Yes

2. Potential interference Yes

3. Adaptability to other pollutants Yes

Further work developments

Further work with the system to evaluate detection levels and reduction of potential interference.

Envisaged performance and optimisation In principle the opening of individual gated channels can be detected, leading to sensitivities in the subpicomolar range coupled with very large dynamic ranges.

BOTTLENECKS

There are still outstanding questions, and there will be plenty of work to develop the principle to detect each new target analyte.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

There is no partner currently helping with the development of the sensor. However there are negotiations in progress in attaining a commercial backer.

PAPERS PUBLISHED

I.R. Peterson and J.A. Beddow, "Systems Aspects of Membrane-based Biosensors", in H.T. Tien, Ed., Planar Lipid Bilayers, Wiley: New York (2002).

J.A. Beddow, I.R. Peterson, J. Heptinstall and D.J. Walton, “Electrical monitoring of gel- protected bilayer lipid membranes using a bipotentiostat”, Proc. SPIE 4414 (2001) 62-69.

R.F. Costello, S.W. Evans, S.D. Evans, I.R. Peterson and J. Heptinstall, "Detection of Complement Activity using a Polysaccharide-Protected Membrane", Enzyme Microbial Technol. 26 (2000) 301-303.

R.F. Costello, I.R. Peterson, J. Heptinstall and D.J. Walton, "Improved gel-protected bilayers", Biosens. Bioelectron. 14 (1999) 265-71.

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R.F. Costello, I.R. Peterson, J. Heptinstall, N.G. Byrne and L.S. Miller, "A Robust Gel- Bilayer Channel Biosensor", Advan. Mater. Opt. Electron. 8 (1998) 47-52.

PROFILE OF ORGANISATION

The Centre for Molecular and Biomolecular Electronics is a collaborative and interdisciplinary Centre of Excellence within the School of Science and the Environment at Coventry University. Our research expertise covers the whole range of physics, chemistry, biology and electronics, and we have strengths in electrochemistry and thin-film self- assembly techniques. We rated 4 in the latest Research Assessment Exercise, up from 3A in the previous one. At the moment the Centre comprises five postgraduate students and a postdoc, led by five senior members. Full details of the Centre can be found on http://www.nes.cov.ac.uk/research/cmbe

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CYBERSENSE BIOSYSTEMS LIMITED

Organisation Name: Cybersense Biosystems Limited Address: Centre for Ecology and Hydrology, Mansfield Road, Oxford. OX1 3SR Respondents Name: Tim Hart Position Managing Director Email address* [email protected]

SENSOR SYSTEM

Description of sensor system We have developed a portable toxicity testing system specifically for solids, e.g. soil, which is based on bioluminescent bacteria. It is non-specific, i.e. it is a generic toxicity sensor.

Environmental relevance The sensor can be used as a generic toxicity test for solids, such as soil.

Commercial relevance The system can be used to more cheaply, quickly and effectively assess contaminated land and be used as an ecotoxicity test.

Usability (i.e skilled, unskilled) As easy-to-use as possible at the moment with no pipetting or weighing of sample as the system is designed for rapid field use.

Availability (eg. commercial, prototype, etc..) Still at the prototype stage, but expecting to launch onto the UK market very soon.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) All organics and inorganics.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Any solid sample, such as soil, sediments and sludge.

Sample phase(i.e. total, residual, free, dissolved or absorbed) Total and free

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Filtration

Sample handling (i.e. manual, automated etc,,,) Manual

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Sample size Few cm3

Sensor Measurements

Assay protocol

Assay takes 1-2 h to perform and measure 20-40 samples. The system is completely portable and easy to use with no pipetting or weighing. As the technology is still under commercial development, further details can be obtained from the company under confidentiality.

Continuous measurement - No

Present measurement performance:

1. Detection levels For example, 100 ppm benzene, 50 ppm phenol, 20 ppm copper, 125 ppm PAH

2. Selectivity No selectivity, general toxicity sensor

3. Repeatability Standard errors between replicates around 1%

4. Potential interference From particulates, pH, temperature and colour of sample

5. Adaptability to other pollutants Highly adaptable as it is a generic sensor

Further work developments Major RandD programmes are under way and the company can discuss these with interested parties under confidentiality.

Envisaged performance and optimisation Can be used for rapid site assessment, monitoring remediation programmes or ecotoxicity testing. Further specific information is available from the company under confidentiality.

BOTTLENECKS

Customer awareness and regulatory acceptance.

Not really, it’s difficult to get the customer more aware, short of doing it yourself. The regulators are very busy and short of time and resources to address all new technologies, so more money is needed I guess for them to devote more time to this.

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Yes, major companies in the UK in a range of sectors within the contaminated land industry. Yes, mostly private investment.

Industry needs to be more committed and provide more resources to fund development work of technology it believes in, or they will never get the benefit of it.

FURTHER INFORMATION AND COMMENTS

My company needs more involvement and interaction with regulators and customers at the European level and I would welcome any opportunities to get involved here.

PROFILE OF ORGANISATION

Cybersense Biosystems Limited is a small biotech company located in Oxford developing bioluminescent-based biosensor technology for industrial applications.

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DELTA CONSULT B.V.

Organisation Name: Delta Consult B.V. Address: Postbus 71, 4420 AC Kapelle. The Netherlands Middenweg 4, 4421 JG, Kapelle The Netherlands Respondents Name: Mr. J.A.G. de Maat Position: Office manager Email address: [email protected]

SENSOR SYSTEM

Description of sensor system The MOSSELMONITOR ® is a biological early warning system for continuous on-line monitoring of surface waters, seawater, effluents, drinking water intakes, even of drinking water (www.mosselmonitor.com )

Environmental relevance The early warning system is based on the behaviour of mussels. In clean water, mussels move their shells according to a characteristic pattern. They remain open for the majority of the time and only close for short periods. A mussel in contaminated water behaves differently. Depending on the type and level of contamination, mussels demonstrate a movement pattern that can differ greatly from normal. Deviation from the normal movement pattern includes a decrease in the average opening, a full closure for a prolonged period or an increase in the valve movement frequency. If a period of (high) contamination continues for too long, death of the mussels will eventually result.

Commercial relevance A fully developed product sold to or tested by: please refer to Reference List 1

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Usability (i.e skilled, unskilled) Medium skilled after instructions by the supplier

Availability (eg. commercial, prototype, etc..) Commercial see www.mosselmonitor.com

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…)

Water quality management is an expensive and time-consuming business. In order to monitor water quality, samples must be taken regularly and chemically analysed. It is decided, on the basis of these results, whether there is a worrying level of contamination. Very often, the result of the analysis will indicate that contamination thresholds have not been exceeded. This early warning system, the MOSSELMONITOR ® is capable of noting contamination and issuing an alarm without external control and without an extensive analysis system. It was developed in order to monitor the water continuously.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Surface waters, seawater, effluents, drinking water intakes and drinking water

Sample phase (i.e. total, residual, free, dissolved or absorbed) Not relevant

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None

Sample handling (i.e. manual, automated etc,,,) 1) in situ: submersible instrument direct into the water Nhandling 2) flow through system: pumping of water flow required.

Sample size In situ: not relevant Flow through: 150-200 lt./hour

Sensor Measurements

Assay protocol Not relevant

Continuous measurement Yes

Present measurement performance:

1. Detection limits Please refer to Table 1

2. Selectivity Biological early warning systems do not identify compounds but react to a wide spectrum of pollutants

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3. Repeatability: In general 20% - 50%

4. Potential interference: Mussels are living organisms and adverse water condition may threaten survival however mussels are hardy because they close their shells so they can temporarily escape their environment (potential treats are too high / low extreme pH, temperature > 28 °C, O2 <3 mg/l.

5.Adaptability to other pollutants: Not relevant

Further work developments Application to chlorinated drinking water by treatment of the water flow (reduction of free chlorine, automated food supply for the mussels). PRESENT IT.net remote operation via intra/internet available Fully developed product. Consultancy and training available

Envisaged performance and optimisation Data evaluation and alarm generating is part of the system; Data handling and presentation program recently developed.

BOTTLENECKS

The system has been fully implemented and is available to the end user

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

The system is Produced by Delta Consult who is supported by Mermayde (www.mermayde.nl) for scientific and technical advise, training and implementation. Delta Consult is the producer with a network of distributors in Europe and the Far East.

Collaboration and integration of several physical/chemicals and biological sensors to develop a dedicated multi sensor system that can improve effectiveness of monitoring operation. Would help in further development of the system

PAPERS PUBLISHED

Jenner, H.A., de Zwart, D., Kramer, K.J.M. Monitoring water quality with bivalves Jenner, H.A., Noppert, F., Sikking T. 1989 A new system for the detection of valve- movement respons of bivalves Kramer K.J.M. , Botterweg J. 1990 Aquatic Biological Early Warning Systems: An Overview Jenner, H,A., 1990 Biomonitoring in Chlorination Anti-Fouling Procedures de Zwart, D., Kramer, K.J.M., Jenner, H.A. 1995 Practical experiences with the Biological Early Warning System MOSSELMONITOR ® ® Baldwin, I.G., Kramer, K.J.M. 1994 Biological Early Warning Systems (BEWS) Kramer, K.J.M., Foekema, E.M. 2000 The Musselmonitor as biological early warning system; The first Decade

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PROFILE OF ORGANISATION

Delta Consult operates as an independent company. Our core business is to support, develop, implement and improve business processes and technical management processes for our clients. Our clients develop, own, operate and maintain complex technical systems and installations, ranging from navy frigates to nuclear power stations. Delta Consult is a 100% subsidiary of SKF Reliability Services Systems.

Reference List 1 Commercial relevance:

1. RIVM, Bilthoven – The Netherlands Location: Study different locations Application: Effluent control 2. Water Research Center, England Location: Laboratory Marlow Application: Comparison with other biological early warning systems. 3. Kema, Arnhem – The Netherlands* Location: Study different location Appliccation: Chlorine reduction in cooling water systems 4. DOW Benelux, Terneuzen – The Netherlands* Location: Cooling water system Application: Chlorine reduction in cooling water systems 5. Rijkswaterstaat DGW, Middelburg – The Netherlands* Location: Jacoba harbour Application: Study on valve movement and contamination respons with heavy metals, PCBS and PAHS 6. RIVM, Bilthoven – The Netherlands* Location: Study different locations Application: Dose-effect relation tests 7. Vlaamse maatschappij voor water voorziening, Belgium* Location: Different locations Application: drinking water influent control 8. Ifremer, France* Location: Laboratory Brest Application: study 9. Bundesanstalt für Gewässerkunde, Germany* Location: Laboratory Koblenz Application: monitoring of river Rhine water quality 10. University of Illinois, U S A* Location: Laboratory Urbana Application: research project 11. Watertransportmij Rijn-Kennemerland WRK, Nieuwegein – The Netherlands Location: Raw water pumpstation Application: drinking water influent control 12. TNO, Den Helder – The Netherlands Location: Different locations Application: research and application

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13. PIDPA, Olmen Belgium Location: Albert Canal Application: drinking water influent control 14. Azienda Acque Metropolitan Location: Torino – Italy Application: drinking water influent control 15. KEMA, Arnhem – The Netherlands Location: Project NAM Application: monitoring effluents of offshore industry 16. Waterleiding maatschappij Gelderland Location: Velp – The Netherlands Application: drinking water influent control 17. Azienda Servizi Municipalizzzata Tortonese Location: Tortona - Italy Application: drinking water influent control 18. Deutscher Forschungsgemeinschaft/ Universität Düsseldorf Location: Düsseldorf - Germany Application: Study on bivalve behaviour 19. Australian Nuclear Science and Technology Organisation Location: Menai - Australia Application: Research 20. Waterleiding maatschappij Limburg Location: Maastricht – The Netherlands Application: drinking water influent control 21. Technische Universität Dresden Location: Dresden – Germany Application: Research 22. Staatliche Umweltbetriebsgesellschaft Zentrallabor Neusörnewitz Location: Meßstation Schmilka – Germany Application: Control surface water 23. TNO Den Helder – The Netherlands Location: Noordzee - The Netherlands Application: Oil and gas exploration 24. Instituto Zooprofilatico Sperimentale dell’ Abruzzo e del Molise Location: Termoli - Italy Application: Research 25. I.A.S. S.p.a. Location: Priolo Gargallo - Italy Application: Industrial waste water control 26. Fővárosi Vízművek Részvénytársaság – Waterworks of Budapest - Hungary Location 1 : Csepel Location 2 : Kőbánya Location 3 : Gellért-hegy Location 4 : Békásmegyer Application : drinking water control 27. WTW meracia a analyticka technika s.r.o. - Waterworks - Slovakia Location 1 : Hencovce Location 2 : Stakcin Application : drinking water control 28. Universitiy of Wisconsin – Center for Water Security – U.S.A. Location: Milwaukee Application: Research

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® Detection limits MOSSELMONITOR

Component Detection limit in mg/l (nominal) fresh water mussels marine mussel Dreissena unio mytilus edulis polymorpha pictorum Ammonia (Unionised) 0.59 Atrazine 0.5 0.5 Bentazone 0.75 0.75

Cadmium (CdCl2) 0.15 0.1 Chloroform 43.0 Chloropyriphos 0.05 0.05

Copper (CuSO4) 0.01 0.01 0.005 Cyanide (KCN) 0.4 1.3 Dichlorobezene 1.4 Dichloromethane 50.0 Formaline 10.0 Hexachlorobutadiene 0.15 Y-Hexachlorocyclohexane 0.06 Hypochlorite (chlorine) 0.037 0.005 Lead 0.25 0.25 Lindane 0.11 Oil (dispersed) 6.0 Pentachlorophenol 0.01 0.01 Phenol 14.0 Selenium (selenite) 0.1 Tetrachloromethane 2.5 Toluene 6.0 Tributyltinoxide (TBTO) 0.006 0.01 Trichloroethylene 8.0 Xylene 16.0 Zinc 0.5 0.5

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DLR

OrganisationName: DLR Research group: Institute of Aerospace Medicine Radiation Biology Address: Linder Höhe 51170 Köln Germany Respondents' Name: Elke Rabbow Position: Research Scientist Email address: [email protected]

SENSOR SYSTEM

Description of sensor system The SOS-LUX-Test is based on the measurement of the DNA damage-dependent induction of the bacterial SOS system. The development of the SOS-LUX-Test for the rapid detection of environmental genotoxins was described previously (Ptitsyn et al. 1997). This bacterial bioassay uses the receptor-reporter principle with the SOS-promoter system as receptor which is sensitive to DNA damage and controls the bioluminescence system as reporter, emitting a bioluminescence light as optical signal which can be recorded by an appropriate detector. The intensity of the emitted light of the bacterial culture is proportional to the concentration of the genotoxic compound. The bioluminescence as a signal for DNA damage is the result of an enzymatic reaction of luciferase with a specific substrate, both encoded by the luxCDABFE operon of Photobacterium leiognathi, and oxygen. Since the bioluminescent light can be measured without destroying the cells, the kinetics of the processing of the DNA damage by the SOS system can be followed. It could already be shown that this reporter system reacts with a high level of light production to certain classes of DNA-damaging agents that have nearly no effect on cell survival in Escherichia coli recA+ strains (Horneck et al. 1998) and Salmonella typhimurium TA1535 cells (Rettberg et al. 1999). When the tested agents are cytotoxic, the determination of the SOS system induction is influenced by the cell death of a part of the whole population. To discriminate between the genotoxic and cytotoxic potency of the tested agent, the cell concentration is monitored by measuring the absorption of the bacterial suspension simultaneously in addition. A test compound is considered not genotoxic if bioluminescence is not increased more than twofold above the untreated control, and not cytotoxic if the cell growth is comparable to that of the untreated control. In the case of decreasing bioluminescence and/or absorption, the compound is suggested to be cytotoxic. Since a constant read-out of absorption does not imply that all cells have died, because cells in the populations may be metabolically active, but not growing, a second parameter to monitor the metabolic activity of the bacteria is introduced in form of the LAC- FLUORO-Test (Baumstark-Khan et al. 2001). The expression and fluorescence of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria gene without any substrates and other cofactors allows the detection and quantification of protein expression in metabolically active cells. Crameri et al. (1996) optimised the GFP for a higher expression in bacteria with a maximum fluorescence output 18times higher than in wtGFP for exciting wavelengths in the near UV-region (360-

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400nm). This GFPuv is inserted in a plasmid in frame with the lacZ initiation codon from pUC19 leading to the expression of a soluble beta-galactosidase-GFPuv fusion protein in transformed bacteria. Because Salmonella typhimurium TA1535 does not produce beta- galactosidase and a functional lacI repressor (Yagil and Hermoni, 1976), the GFPuv expression is not under regulatory constraints, resulting in a constitutive synthesis of GFP in Salmonella typhimurium with the reduction of GFP-fluorescence being a suitable indicator for overall cytotoxicity. This fluorescence can easily be measured, in parallel to bioluminescence in commercially available devices. In the combined bacterial SOS-LUX-LAC-FLUORO-Toxicity-test, Salmonella typhimurium TA1535 transformed either with the SOS-LUX plasmid pPLS-1 or with the LAC-FLUORO plasmid pGFPuv were established to recognize and record in parallel the genotoxic and/or cytotoxic activity of substances or radiation.

Genotoxin Zytotoxin

DNA-Schaden GFP Schaden SOS-abhängiger Promotor konstitutiver lac-Promotor

pPLS-1 Lu x pGFPuv GFPuv

chromosomale DNARi b o so m e n chromosomale DNA

Lu zif e ra se Ald ehyd Fe t t sä ure + Lic h t

Fettsäurereduktasekomplex SO S Lu zi f e ra se

pPLS-1

Fe t t sä u re

Ald ehyd

Fe t t sä u re - Fe t t sä u re Ald ehyd reduktasekomplex konstitutiver lac-Promotor

pGFPuv GFPuv

chromosomale DNA Ri b o so m e n - Rib o so m e n untereinheiten

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125 125 INKUBATION INKUBATION 100 100

75 75

50 50 ZYTOTOXIN GENOTOXIN 25 25 Fluoreszenz/ rel. Einheiten

0 Biolumineszenz / rel.Einheiten 0 0 153045607590105120 0 60 120 180 240 300 360 420 480 540 600 Zeit / min Zeit / min

Environmental relevance With the combined SOS-LUX- and LAC-FLUORO-Test genotoxicity and cytotoxicity of environmental samples like waste water, but also soil or ambient air particulate extracts can be determined within 3 to 8 hours. The test is easy to perform and not labour intensive, making it a fast and cost-effective test with a high sample-throughput to determine genotoxicity and toxicity of environmental samples.

Commercial relevance The test is a fast and cost-effective.

Usability (i.e skilled, unskilled) The test is easy to perform trained unskilled persons after a short training.

Availability (eg. commercial, prototype, etc..) Up to now the SOS-LUX- and LAC-FLUORO-Test is not commercially available.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) All genotoxic compounds tested up to know could be detected by the test.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) all

Sample phase (i.e. total, residual, free, dissolved or absorbed) dissolved

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Filtering

Sample handling (i.e. manual, automated etc,,,) Sample handling can be automated with commercially available instruments. After preparation of the sample, all further measurement steps are already automated.

Sample size microtiter plate formats

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Sensor Measurements

Assay protocol Log phase bacterial cultures of Salmonella typhimurium strains transformed with both plasmids are incubated in pre-warmed 2xNB-medium containing 50 µg/ml ampicillin until the absorption at 600 nm (A600) reached 0.2 (± 0.01). 75 µl of Salmonella typhimurium TA1535- pPLS-1 or Salmonella typhimurium TA1535-pGFPuv bacterial culture are added to each well of the microplate containing 75 µl of the diluted chemical or environmental test compound or controls. The whole plate is placed into the microplate reader (Multilabel Counter 1420 Victor2 form EGandG Wallac, Turku, Finland) with a controlled temperature of the plate of 30°C. The reader is programmed to repeat the measurement cycle of 2 min orbital shaking, luminescence reading without filter for 0.2 s/well, followed by absorption measurement for 0.1 s/well at 490 nm (20 nm band width) and by fluorescence reading for 0.1 s/well at 510 nm after excitation at 405 nm, every 10 min for 50 cycles adding up to 8 hour kinetics.

Continuous measurement x Yes

Present measurement performance:

1. Detection levels - low, comparable to the VITOTOX tes and other SOS-dependent genotoxic assays.

2. Selectivity comparable to the VITOTOX tes and other SOS-dependent genotoxic assays

3. Repeatability comparable to the VITOTOX tes and other SOS-dependent genotoxic assays

4. Potential interference with intensely coloured samples

5. Adaptability to other pollutants to all DNA-damage inducing agents (including ionising and UV radiation)

Further work developments detailed tests with standard substances and direct comparison with other genotoxicity assays

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

In 2000 a project with a commercial partner of the pharmaceutical industry

Workshops with blind tests for the direct comparison of different genotoxicity assays would be useful in the development of the sensor.

FURTHER INFORMATION AND COMMENTS

Test also suitable for High Throughput Screening in chemical/pharmaceutical industry

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PAPERS PUBLISHED

Baumstark-Khan, C., A. Rode, P. Rettberg, et al., Application of the Lux-Fluoro test as bioassay for combined genotoxicity and cytotoxicity measurements by means of recombinant Salmonella typhimurium TA1535 cells, Anal. Chim. Acta 437, 23-30, 2001. Horneck, G., L.R. Ptitsyn, P. Rettberg, et al., Recombinant Escherichia coli cells as biodetector system for genotoxins in: B. Hock, D. Barceló, K. Cammann, P.D. Hansen, A.P.F. Turner (Eds.), Biosensors for Environmental Diagnostics, Teubner, Stuttgart, Germany 215-232, 1998. Ptitsyn, L.R., G. Horneck, O. Komova, et al., A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells, Appl. Environ. Microbiol. 63, 4377-4384, 1997. Rabbow, E., A. Rode, P. Rettberg, et al., Technotox Workshop, Mol, Belgium, 2000. http://www.vito.be/english/environment/environmentaltox5.htm Rabbow E, Rettberg, P, Baumstark-Khan, C and Horneck, G SOS-LUX- and LAC-FLUORO- TEST for the quantification of genotoxic and/or cytotoxic effects of heavy metal salts. Anal. Chim. Acta 456, 31-39, 2002. Rettberg, P., C. Baumstark-Khan, K. Bandel, et al., Microscale application of the SOS-LUX- TEST as biosensor for genotoxic agents, Anal. Chim. Acta, 387, pp. 289-296, 1999. Rettberg, P., K. Bandel, C. Baumstark-Khan, et al., Increased sensitivity of the SOS-LUX- Test for the deetection of hydrophobic genotoxic substances with Salmonella typhimurium TA1535 as host strain, Anal. Chim. Acta, 426, 167-173, 2000.

PROFILE OF ORGANISATION

Please give a short description of your organisation and research group DLR is the aerospace agency of the Federal Republic of Germany. In this function DLR is in charge of a broad scope of research and development involving national and international partnerships. Numerous results of its research and development work go into industrial production process. G. Horneck is the head of the division "Radiation Biology" at the Institute of Aerospace Medicine of DLR in Köln. In this division the group "Photo- and Exobiology" lead by P. Rettberg focuses on the utilisation and application of molecular-biological and biotechnical methods for the quantification of complex biological effects. The reaction mechanisms and the effects of genotoxic agents like radiation and chemicals are investigated. Main subject is the determination of DNA damages at the molecular and cellular level. For the rapid detection of environmental genotoxins the cellular bioassay on the basis of bioluminescence, the SOS- LUX-test (2 patents) in combination with the cytotoxicity assay using gfp was developed to quantify the effects of complex mixtures of genotoxic compounds e.g. in waste water, air and food samples. The ongoing work of this group also includes the development of cellular biosensors on the basis of eukaryotic cells using a fluorescence signal mediated by the gfp gene as reporter for toxic and genotoxic agents.

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FRENCH GRANDES ECOLES

Institute Name: French Grandes Ecoles Research group: SPIN / CP2M / Enzyme engineering Address: Ecole Nationale Supérieure des Mines de St Etienne 158 Cours Fauriel, 42023 Saint Etienne cedex 2, France Respondents Name: Canh Tran-Minh Position: Professor, Research Supervisor Email address: [email protected]

SENSOR SYSTEM

Description of sensor system Optical whole-cell biosensor using micro-algae designed for toxicity assessment

optical fiber guide sheath

O-ring cap algal membrane sample inlet

PVC cell removable support

spring frame

ring 1 cm

Environmental relevance - Easy and fast determination of toxic chemicals - Ecological interest since algae are involved in the primary step of the food chain - Photosynthesis inhibition is an interesting indicator that rapidly reflects the toxic effect of pollutants - compatible with water quality monitoring and its use as an early warning system

Commercial relevance - Large scale production of algal membranes is possible - Easy handling and maintenance (membranes in lots) - Algal membranes are robust and low-cost

Usability (i.e skilled, unskilled) Skilled

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Availability (eg. commercial, prototype, etc..) Prototype

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Herbicides, heavy metals (mercury, cadmium…), solvents (perchlorethylene…)

Sample matrix ( e.g. soils, sediments, surface and groundwater ) surface and groundwater, aerosols

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved or free

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Filtering

Sample handling (i.e. manual, automated etc,,,) Manual, automated

Sample size 20 µL with drop deposition 50 µL and over in batch mode Flow mode is possible

Sensor Measurements

Assay protocol Drop deposition: Twenty microliters of toxic solution at pH 7 were deposited on the surface of a 0.7-cm2 algal membrane. The membrane was then placed in front of the tip of the optical fiber Flow mode: A peristaltic pump was used to allow a continuous flow of solution through the measurement device The fluorescence measurement was carried out immediately after the membrane was illuminated (1-min illumination period with a 470-nm excitation light). The emitted fluorescence was collected at 690 nm.

Continuous measurement Yes

Present measurement performance:

1. Detection levels - Low concentration levels are obtained with herbicides (0.1 nM for diuron).

2. Selectivity Selectivity is not the aim for this biosensor. Therefore, it is sensitive to any toxic chemicals that affect the activity of the microalgae entrapped on the membrane.

3. Repeatability Standard deviations with 10 membranes were 1.9% in the flow mode and 3.2% with the drop deposition technique.

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4. Potential interference The biosensor is designed to have a large spectrum of toxicity measurement.

5. Adaptability to other pollutants Sensitivity varies according to toxicity

Further work developments Improvement of reproducibility

Envisaged performance and optimisation Multimembrane device is being developed

BOTTLENECKS

An industrial partner is needed to develop the biosensor. However, contacts are underway

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Technical advisers have been obtained from academic partners. There are no commercial backers at present.

Engaging closer links with water agency, toxicity services, environmental protection agency

FURTHER INFORMATION AND COMMENTS

The gap can be bridged with a real need for toxicity measurement, laws and regulations on water and air quality.

PAPERS PUBLISHED

-Naessens M. and Tran Minh C., "Biosensor using immobilized Chlorella microalgae for determination of volatile organic compounds", Sensors and Actuators B, 1999, 59, 100-102 -Naessens M., Leclerc J. C. and Tran-Minh C., "Fiber optic biosensor using Chlorella vulgaris for determination of toxic compounds". Ecotoxicol. Environ. Safety, 2000, 46, 181- 185 - Durrieu C. and Tran-Minh C., "Optical Algal Biosensor using Alkaline Phosphatase for Determination of Heavy Metals", Ecotoxicol. Environ. Safety, 2002, 51, 206-209. - Vedrine C., Leclerc J.C., Durrieu C. and Tran-Minh C., "Fiber optic biosensor using Chlorella vulgaris design for herbicides monitoring" Biosensors and Bioelectronics, 2002

PROFILE OF ORGANISATION

The Ecole Nationale Supérieure des Mines de Saint-Etienne is a graduate engineering school with a population of over 360 graduate students and 150 Ph.D. candidates Process Engineering is developed in Centre SPIN: physico-chemical processes with powders, design of sensors for the continuous control of processes, wood-treatment and geochemical problems of natural processes. Our group is involved in enzyme engineering with the study of biosensors and bioprocesses.

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GENTRONIX LIMITED

Company Name: Gentronix Limited Address: c/o Heather Edgar Fairbairn Building PO Box 88 Manchester M60 1QD Respondents Name: Dr. Andrew Knight Position Research Manager - Instrumentation. Email address: [email protected]

SENSOR SYSTEM

Description of sensor system The sensor system is “GreenScreen Environmental Monitoring” or “GreenScreen EM” for short.

The heart of the sensor system is a yeast cellular biosensor used for the simultaneous detection of both genotoxicity (damage to genetic material) and more general cellular toxicity (cytotoxicity). Genotoxicity is evidenced by increasing fluorescence from the yeast cells. The yeast have been genetically modified to express a green fluorescent protein whenever the cells repair DNA damage caused by exposure to a genotoxin. More general cytotoxicity is shown by the degree of inhibition in cell proliferation over the course of the assay, measured as a final cell culture density.

For measurements in the field, or for processing a moderate number of samples in the laboratory, a disposable cuvette based protocol is used. Yeast fluorescence and cell density are measured using a prototype portable reader, known as the YETI (Yeast Environmental Toxicity Indicator). For higher throughput of samples, a microplate protocol using commercially available standard robotic or manual liquid handling techniques, in conjunction with a microplate fluorescence reader, can be used.

Further details of the sensor system and instrumentation are attached.

Environmental relevance The biosensor uses yeast (Saccharomyces cerevisiae) which, unlike bacteria, are eukaryotic organisms, and as such the assessment of toxicity should have much greater relevance to other eukaryotes such as humans and other animals. The yeast’s resilience during handling and exposure to a wide range of pH, temperature and ionic strength conditions, make them a practical organism for analysing environmental samples from a wide variety of sources.

Commercial relevance In a climate of increasing IPPC regulation, there is a growing need for chemical and related industries to monitor the toxicity of their process effluents using methods of rapid, direct toxicity assessment. The current benchmark tests using regulatory species (i.e. algae,

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Daphnia, invertebrates and fish) are generally very expensive and time consuming to perform. GreenScreen EM is being developed as a rapid and relatively inexpensive alternative.

Usability (i.e skilled, unskilled) The assay has been designed to be performed by relatively unskilled personnel.

Availability (eg. commercial, prototype, etc..) GreenScreen EM is currently in its mid-development stage. A protocol and single prototype cuvette reader has been developed, and is currently undergoing field trials in environmental laboratories with a view to obtaining confirmation of the usefulness of the sensor system in the environmental monitoring market, and also to determine what improvements might be needed to the technology before commercial launch.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) GreenScreen EM has been developed for examining “whole effluent toxicity” and as such detects any toxic species capable of inhibiting yeast cell growth and proliferation, or inducing DNA damage. However the sensor has also been characterised using standard chemicals of environmental relevance, including heavy metal ions, pesticides and solvents.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) The sensor has been tested successfully with surface water samples and contaminated aqueous effluents. Environmental samples tested have contained particulate matter, organic matter, significant colour and fluorescence, extremes of pH and contamination by organic solvents.

Sample phase (i.e. total, residual, free, dissolved or absorbed) In order to be detected, the toxic analytes need to be bioavailable to the yeast cell. In general terms this means dissolved in the aqueous phase of the sample.

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Whole effluent and surface water samples have been tested with no sample pre-treatment other than dilution with water for calibration purposes.

Sample handling (i.e. manual, automated etc,,,) For moderate numbers of samples (< 10 for fully characterised quantitative analyses per day, or 10 - 50 single concentration qualitative analyses per day) the sample handling is manual using a simple cuvette based assay. The protocol is not labour intensive and consists of mixing the diluted sample with the yeast cell reagent, leaving to incubate and then reading at a single time point.

For higher throughput (10 - 50 samples per day to be fully characterised) a microplate protocol using commercially available standard robotic or manual liquid handling techniques, in conjunction with a microplate fluorescence reader, can be used.

Sample size Full toxicity characterisation of an effluent for genotoxicity and cytotoxicity requires approximately 8 ml of sample for the cuvette-based assay, and 0.5 ml of sample for the microplate assay.

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Sensor Measurements

Assay protocol The cuvette assay protocol entails mixing 1 ml of yeast cell reagent with 1 ml of a range of dilutions of the effluent in separate disposable, plastic cuvettes, covering with a breathable membrane and then incubating overnight, ideally at 25 - 30 oC, although this is not critical. The contents of the cuvette are then re-suspended by shaking and single fluorescence and cell density measurements simultaneously made using the dedicated portable reader. The data is collected and processed by software on a lap-top computer (or similar) and results for general toxicity produced as an EC50 or LOEC, as well as an assessment of genotoxicity are obtained.

Full details of the protocol are given in the following document.

Continuous measurement No

However, work is currently being carried out under a European Framework Five grant, to examine the feasibility of producing a sequential sampling protocol to enable semi-continuous monitoring.

Present measurement performance:

1. Detection levels The assay has been developed for whole effluent testing, however the sensor and protocol have been characterised with a variety of pure chemicals. Example EC50’s include; 3,5- dichlorophenol (7.5 mg/ml), copper ion (0.052 mg/ml), mercury ion (0.47 mg/ml), 2,4-D (55 mg/l) and cycloheximide (0.015 mg/ml).

2. Selectivity Work in field testing and comparison to standard environmental tests is on-going. The results of these trials will reveal more about the sensor’s selectivity.

3. Repeatability

Trials with repeated analysis of pure chemicals has shown the sensor to achieve good repeatability. Whole effluent samples are by their very nature an unstable, complex cocktail of potentially toxic species. Hence their toxicity profile varies over short periods of time making assessment of repeatability more difficult.

4. Potential interference Highly fluorescent contaminants can interfere with the measurement of yeast cell fluorescence and hence a method of subtraction from “sample only controls” is employed to overcome this. More elegantly a fluorescence polarisation method based on exploiting the high fluorescence anisotropy of GFP, for the discrimination of the GFP signal in the presence of autofluorescent contaminants, has been demonstrated.

Similarly, highly absorbing contaminants can interfere with the measurement of yeast cell density, however this is readily overcome by the method of subtraction from “sample only controls”.

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5. Adaptability to other pollutants GreenScreen EM is designed to examine the toxicity of whole effluents and not be limited to certain groups of pollutants.

Further work developments Work is currently underway to;

• Refine the protocol and instrumentation. • Extend the characterisation by comparison to standard environmental toxicity tests. • Extend the characterisation by testing further standard chemicals, especially pesticides and organics.

Envisaged performance and optimisation It is hoped that as a result of on-going field trials, GreenScreen EM will provide a rapid and relatively inexpensive method for direct toxicity assessment of whole effluents and surface waters, which can be correlated to the results expected from standard regulated tests.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Gentronix Limited is a participant in a BIO-WISE Demonstrator Project involving a collaboration of chemical companies from the Specialised Organic Chemicals Sector Association (SOCSA), three biosensor suppliers (Gentronix Ltd, Euroclone and Vickers Laboratories), the Environment Agency and AstraZeneca Ltd. Based at Brixham Environmental Laboratory, the project involves the use of novel biosensors to detect toxicity in effluents provided by the SOCSA members to comply with impending Direct Toxicity Assessment (DTA) legislation.

Toximon is a research project supported by the European Commission under the Fifth Framework Programme entitled "Development of a Novel, On-line Genotoxicity Monitor Integrated with an Effluent Toxicity Treatment System". The key objective of this project is to provide a solution to the problem of continuous genotoxicity testing for effluent streams and to integrate the results of testing with subsequent waste treatment processes. The project partners are: Gentronix (Project management and biosensor supply); C-Tech Innovation (Chester, UK) (Waste water treatment and analysis of toxicity); BMG Lab-Technologies (Aylesbury, UK) (Microplate reading technology provider); Neoplex (France), AG+ (France) and Lacaze SA (France) (Effluent sample providers).

The GreenScreen technology is owned by Gentronix Ltd, and the GreenScreen EM sensor for environmental monitoring is an important part of the product portfolio being developed and marketed by the Company.

The Company currently has access to the necessary skills and contacts through in-house expertise and existing collaborative projects to pursue the development program for GreenScreen EM effectively; consequently no additional collaborations are being sought at this time.

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PAPERS PUBLISHED

Validation of the Gentronix RAD54-GFP assay with environmental samples. Included in the proceedings of the BIOSET Technical Workshop on Genotoxicity Biosensing - TECHNOTOX held at the VITO Institute in Mol, Belgium - May 2000. Walmsley R.M., Keenan P., Knight A.W. and Schmuckli J. http://www.gentronix.co.uk/keypapers/Proceedings4_03_13.PDF

Development of a Flow-Through Detector for Monitoring Genotoxic Compounds by Quantifying the Expression of Green Fluorescent Protein in Genetically Modified Yeast Cells. Knight A.W., Goddard N.J., Fielden P.R., Barker M.G., Billinton N. and Walmsley, R.M. Measurement Science and Technology 10 (1999) 211-217.

Development of a green Fluorescent Protein Reporter for a yeast genotoxicity biosensor. Billinton N., Barker M.G., Michel C.E., Knight A. W., Heyer W.-D., Goddard N.J., Fielden P.R. and Walmsley, R.M. Biosensors and Bioelectronics 13 (1998) 831-838.

Green Fluorescent Protein as a reporter for the DNA damage-induced gene RAD54 from Saccharomyces cerevisiae. Walmsley R.M., Billinton N. and Heyer W.-D. Yeast 13 (1997) 1535-1545.

PROFILE OF ORGANISATION

Gentronix Ltd was formed in 1999 as a joint venture between UMIST Ventures (The technology transfer company of UMIST-University of Manchester Institute of Science and Technology) and Dr Richard Walmsley, Senior Lecturer in Biomolecular Sciences at UMIST. The company has developed and owns all rights to a new test method for genotoxicity, and also owns commercialisation rights to a range of complementary technologies. This portfolio of technologies is being developed into a range of unique biosensor products for use in the pharmaceutical and other industries. The first product is used to screen pharmaceutical compounds for genotoxicity.

Further information of the company profile can be found at: www.gentronix.co.uk

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BRIEF SUMMARY OF THE GENTRONIX YETI (YEAST ENVIRONMENTAL TOXICITY INDICATOR) ASSAY.

Paddy Keenan / Andrew Knight. Gentronix Limited. August 2002 ______

Overview

Toxic substances can have many different modes of action against cellular organisms. The term “cytotoxic” is used to describe substances which are poisonous to cells in general, by a variety of mechanisms. The term “genotoxic” is used for substances which are capable of causing damage to a cell’s DNA which may have a carcinogenic, teratogenic or mutagenic affect in humans or other animals. The Gentronix cellular biosensor detects simultaneously both gross cytotoxicity and more subtle genotoxicity. The biosensor utilises a strain of the brewer’s yeast Saccharomyces cerevisiae which has been genetically modified to express a green fluorescent protein (GFP) whenever the cell carries out repair of DNA damage. When exposed to a genotoxic chemical which causes DNA damage, the cell’s natural DNA repair mechanisms are triggered and the cells become increasingly fluorescent. Genotoxicity is thus revealed by measuring the induction of this cellular green fluorescence. Cytotoxicity is simultaneously determined by measuring cell density which reflects cell proliferation, i.e. the increase in the number of cells, over the course of the assay, which is lowered by exposure toxic substances. The basic protocol of the assay is illustrated in figure 1.

Figure 1: Schematic of the Gentronix Assay

Yeasts possess many advantages which make them ideal candidates for environmental toxicity sensing. Firstly as microbes they are readily stored, either frozen or freeze dried and are easily and rapidly cultivated on a variety of growth media. Secondly, unlike bacteria, they are eukaryotic organisms, and as such the assessment of toxicity should have greater relevance to other eukaryotes such as ourselves and other animals. Thirdly, the cells are

191 SENSPOL Survey of Sensor Capabilities resilient to a wide range of pH, temperature and ionic strength conditions, qualities desirable when analysing environmental samples from a wide variety of sources.

The Yeast Environmental Toxicity Indicator (YETI) is a portable instrument combining a dedicated fluorescence spectrometer and optical density detector. It was conceived to enable the Gentronix assay for cyto- and genotoxicity, to be performed using highly portable equipment, and as such is especially useful for work in the field with environmental samples.

Figure 2: The YETI reader and close-up of a cuvette placed within the sample chamber.

The assay itself is performed by combining a small volume of the aqueous environmental sample, typically 1 ml, with the test yeast cell culture in a disposable acrylic cuvette, and incubating over several hours. An inherent advantage of this assay is that once the sample and yeast culture volumes have been combined no further reagents or protocol steps are necessary. After incubation the cuvette is simply shaken to re-suspend the cells and read once by placing it into the YETI reader. Measurements are obtained from the YETI reader via a suitable interface, i.e. a laptop computer.

Protocol

Advance Preparation

Prior to the test, cultures of Gen01 (the genotoxicity test strain) and GenCon (corresponding control strain which does not express GFP) are grown up from stock standards (stored in a freezer), over 2 days at 30 oC. The culture density is measured and the culture transferred to sealed tubes for transportation. The stock cultures are stored at approximately 4 oC and can be used for up to a week. In the near future it is envisaged that the yeast cells will be supplied freeze dried, and could be reconstituted ready for testing in matter of hours.

Just prior to the test an aliquot of the yeast culture is added to fresh growth media.

EC50 Determination

For cytotoxicity assessment, 1 ml of each effluent dilution is combined with 1 ml of GenCon yeast strain in a pre-labelled disposable acrylic cuvette. Cuvettes are sealed with an adhesive

192 SENSPOL Survey of Sensor Capabilities breathable membrane and left overnight to incubate, ideally at 25 - 30 oC, although this is not critical.

A simple qualitative (toxic / non-toxic) result can be obtained from a single cuvette measurement, however to obtain a quantitative result an EC50 is determined. To determine an EC50 value the effluent sample is diluted to form a linear range of typically 8 to 10 concentrations and each is tested separately. A parametric curve is fitted to the data describing the variation of final cell density with effluent dilution. From this curve the effluent dilution corresponding to 50 % cell growth (or proliferation) is calculated to be the EC50 (effective concentration giving 50 % response). 50 % cell growth is equivalent to a density reading half way between the culture density at the start of the assay and the density achieved in the non-toxic control.

LOEC Determination

From the data obtained during the cytotoxicity assessment stage, a LOEC (lowest observed effect concentration) value can also be obtained. This is carried out by establishing the concentration at which a toxic effect produces 10% less cell proliferation than observed in the non-toxic control. This figure takes into account the natural variation in proliferation observed when making up replicate control cultures.

Genotoxicity Assessment

For genotoxicity assessment, the process described above is repeated using a linear series of effluent dilutions across the sub-cytotoxic range determined for the effluent sample. Only cell densities greater than that produced by the EC50 level of effluent dilution are used for genotoxicity assessment. Both the GenCon and Gen01 strains are used with each effluent dilution and the induced GFP fluorescence is calculated from the difference between the “brightness” of the Gen01 and GenCon strains, with comparison to a non-toxic control. “Brightness” corresponds to the fluorescence reading normalised for cell density. A positive genotoxicity result is obtained if at least one reading in the series has a brightness > 30 % higher than the blank (i.e. an induction of 1.3). A dose-dependent response provides further evidence of a positive result.

Corrections

For each effluent concentration tested a second cuvette is made up containing 1 ml of the effluent sample and 1 ml of growth media only, without the addition of cells. This cuvette serves as a standard which can be used to correct for the auto-fluorescence and optical density of the sample itself.

Standards

To ensure that the yeast strains are behaving as expected and that the results obtained are both credible and reliable, standard compounds are included in the trial. Two standards of 3,5- dichlorophenol are tested, at 15 and 7.5 mg/L to check for a cytotoxic response, and two standards of methyl methanesulfonate (a known genotoxic alkylating agent) are tested, at 0.005 and 0.00125 % v/v to check for GFP induction and thus a genotoxic response.

Alternative Platforms for High-Throughput

The Gentronix assay for cytotoxicity and genotoxicity has also been optimised for use in a high throughput format using conventional liquid handling robotics and microplate technology. Whilst the equipment used is more extensive and requires a laboratory setting, the technology allows rapid screening and characterisation of 10s to 100s of individual

193 SENSPOL Survey of Sensor Capabilities samples per day. For further information about Gentronix Ltd and the genotoxicity assay see: www.gentronix.co.uk

Example Results

400

300

200

Cell Density / Arbitrary Units / Arbitrary Density Cell 100 LOEC = 3.8 mg/L EC50 = 7.5 mg/L 0 02468101214161820 3,5-Dichlorophenol Concentration / ppm

Figure 3. Cytotoxicity profile of 3,5-dichlorophenol.

1.8

1.6

1.4

1.2 Threshold

1.0

0.8

0.6 Brightness Induction

0.4

0.2

0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Concentration (mg/L)

Figure 4: Variation in Induction in Brightness due to GFP expression with the Concentration of Nickel(II) ions.

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UNIVERSITY OF ABERDEEN/REMEDIOS LTD

University Name: University of Aberdeen/Remedios Ltd Department: School of Biological Sciences Address: Cruickshank Building, St Machar Drive Aberdeen AB24 3UU Address* Remedios Ltd, 581 King Street, Aberdeen AB24 5UA Respondents Name* Ken Killham Position Research Director and Vice Chairman Email address* [email protected]

SENSOR SYSTEM

Luminescence based whole cell toxicity sensors: Bacteria, fungi (yeast and multicellular), algae, nematodes etc.

Bioluminescence

Marine bacteria such as Vibrio fischeri are naturally luminescent

Genes encoding bioluminescence:

luxR luxI luxC luxD luxA luxB luxE

regulation aldehyde luciferase aldehyde

lux reaction:

luciferase FMNH2 + O2 + Aldehyde FMN + fatty acid + LIGHT

UNIVERSITY OF ABERDEEN DEPT. OF PLANT & SOIL SCIENCE

Environmental relevance Sensors are selected and used on the basis of environmental relevance e.g. Soil bacteria are used for routine soil testing and pollutant mineralisers are used to assess toxic constraints to bioremediation; fungi are used for forest soils; aquatic bacteria for water testing; nematodes address trophic issues etc

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Commercial relevance Remedios applies the above biosensors for a wide range of clients commercially, particularly in the contaminated land assessment/remediation market and in the oil and gas industry

Usability (i.e skilled, unskilled) Relatively skilled

Availability (eg. commercial, prototype, etc..) Mixture of commercial and prototype

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) All acutely toxic risk chemicals-metals and organics

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Soil, sediment, water, food, wastes etc (solid phase tests used where appropriate)

Sample phase (i.e. total, residual, free, dissolved or absorbed) Assesses bioavaiable fraction- free and/or other labile fractions

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Generally water extraction, but also used with solid phase tests, and even mild solvent extractions, where appropriate

Sample handling (i.e. manual, automated etc,,,) Manual and automated

Sample size Soil-minimum 10 g representative sample preferred Water-minimum 10 ml representative

Sensor Measurements

Assay protocol Resuscitate freeze-dried cells and expose to water sample, to soil solid phase in solid phase test or to water extract of soil (or other solid matrix). Varying periods of exposure used. Results usually expressed as 100% luminescence of matrix matched control. Minimum of three sample replicates per sample.

Assays performed on-line and in batch.

Continuous measurement Yes

Present measurement performance: 1. Detection levels Toxicity assessed for correlation with other targets

2. Selectivity All acutely toxic chemicals

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3. Repeatability Assays rejected if COV exceeds 10% High QC on sensor production.

4. Potential interference Interference is rare.

5. Adaptability to other pollutants High Further work developments eukaryotic sensors as reliable human surrogates

Envisaged performance and optimisation Will only provide a primary screen but may reduce animal testing

BOTTLENECKS

The sensors are genetically modified and must be used under license by shortening the license approval period would aid the development of such sensors.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Remedios- supply grants to the university. Other industrial partners in the chemical and petrochemical industries. Remedios are commercial backer and we have partners through LINK and TCS grants

Collaboration from regulatory bodies would be helpful in sensor development.

FURTHER INFORMATION AND COMMENTS

GM is an issue important. Workshops involving regulators as well as end-users would be a way forward in bridging the gap.

PAPERS PUBLISHED

Preston, S., Coad, N., Townend, J., Killham, K. and Paton, G. I. 2000. Biosensing the acute toxicity of metal interactions: are they additive, synergistic or antagonistic? Environmental Toxicology and Chemistry, 19, 775-780.

Chaudri, A.M., Lawlor, K., Preston, S., Paton, G.I., Killham, K. and McGrath, S.P. 2000. Response of a Rhizobium-based luminescence biosensor to Zn and Cu in soil solutions from sewage treated soils. Soil Biology and Biochemistry, 32, 383-388.

Shaw, L.J., Sousa, S., Beaton,Y., Glover, L.A., Killham, K., Meharg, A.A. 2000. Lux based biosensing of 2,4-dichlorophenol biodegradation and bioavailability in soil. In: Proceedings of the International In situ and on-site Bioremediation Symposium, San Diego, California, 5, 247-252.

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Hollis, R. P., Killham, K. and Glover, L. A. 1999. Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes. Applied and Environmental Microbiology, 66, 1676-1679.

Knox, O. G. G., Killham, K. and Leifert, C. 2000. Effects of increased nitrate availability on the control of plant pathogenic fungi by the soil bacterium Bacillus subtilis. Applied Soil Ecology, 15, 227-231.

Weitz, H.J., Ritchie, J.M., Bailey, D.A., Horsburgh, A.M., Killham, K. and Glover, L.A. (2001) Construction of a modified mini-Tn5 luxCDABE transposon for the development of bacterial biosensors for ecotoxicity testing. FEMS Microbiology Letters 197, 159-165.

Boyd, E. M., Killham, K. and Meharg, A. A. 2000. Toxicity of mono, di and tri chlorophenols to lux marked terrestrial bacteria, Burkholderia species RASC C2 Tn 4431 and Pseudomonas fluorescens. Chemosphere,43,157-166.

Shaw, L. J., Beaton, Y., Killham, K. and Meharg, A. A. 2000. Soil-bacterial-toxicant interactions during soil-contact lux-based toxicity testing. Environmental Toxicology and Chemistry 19, 1247-1252.

Shaw, L.J., Beaton, Y., Glover, L.A., Killham, K., Osborn, D. and Meharg, A.A. 2000. Bioavailability of 2,4-dichlorophenol associated with soil water-soluble humic material. Environmental Science and Technology, 34, 4721-4726.

PROFILE OF ORGANISATION

The Biosensor group at Aberdeen comprises 4 academic staff, 10 post-doctoral fellows and approximately 30 students. Remedios comprises 12 staff with bases in Aberdeen and Birmingham

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UNIVERSITY OF NANTES

University Name: University of Nantes Research group: Microbiology/grpe CBAC/BSCA (CBAC : capteur bactérien pour l’analyse et le contrôle) (BSCA : bacterial sensor for control and analysis Address: IUT de la Roche sur Yon 18 Boulevard G. Defferre Respondents Name: Thouand Gérald Position : Head of group Email address: [email protected]

SENSOR SYSTEM

Description of sensor system Bioassay using a recombinant bioluminescent Escherichia coli strain harboring the luxAB genes of Vibrio harveyi. See added figure as an example of results (publication currently submitted to Chemosphere)

Environmental relevance Organotin compounds like TBT are higly toxic biocide still detected in marine sediments though they are currently regulated in ther EC. The latter included this compound in the dangerous priority substances in the field of water policy (N°2455/2001/EC)

Commercial relevance Detection in marine sediment and sea water for health purpose and to follow the bioremediation of sediments harbors

Usability (i.e skilled, unskilled) Unskilled

Availability (eg. commercial, prototype, etc..) Prototype (96 microwells plate with lyophilized strains)

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) TBT and DBT

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Freshwater, marine water and sediment

Sample phase (i.e. total, residual, free, dissolved or absorbed) Total fraction if the sample is water and the intersitial liquid fraction for sediment

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Sample preparation (e.g. filtering, SPE, derivatisation etc…) No sample preparation for water. Low speed centrifugation for sediment

Sample handling (i.e. manual, automated etc,,,) Manual

Sample size 50 µL

Sensor Measurements

Assay protocol Two possibilities : Overnight culture from freezed or lyophilized bacterial sensor at 37°C and then, induction with the sample for 1 hour in microplates. Luminescence measurement after addition of decanal. Direct induction of lyophilized bacteria with the sample for 1 hour in microplates. Luminescence measurement after addition of decanal.

Continuous measurement No

Present measurement performance:

1. Detection levels - TBT : 0.08 µM (26 µg.L-1); DBT : 0.0001 µM (0.03 µg.L-1);

2. Selectivity TBT and DBT (with Cl, Br or I as the halogen group)

3. Repeatability : 8%

4. Potential interference : in progress

5. Adaptability to other pollutants Yes, the protocol could be applied to other pollutants

Further work developments We currently developed the test in the overall sediment

BOTTLENECKS

Mainly the difficulty to optimize the growth conditions of the strain.

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

We are backed by a grant of several years from several local authorities and the french ministry for research. Biolumine are our commercial backers

Scientific collaborations would help in sensor development

PAPERS PUBLISHED

Durand MJ, Thouand G, Dancheva-Ivanova T, Vachon P and MS DuBow, Specific detection of organotin compounds with a recombinant luminescent bacteria, submitted to Chemosphere. Durand MJ, Thouand G, Ivanova T, Chahov B and MS DuBow, 2001, Development of a microbial bioassay for the detection of organotin in environmental sample, 10th International Symposium on Toxicology Assessment, 26-31 Aout, Québec, Canada. Thouand G, Durand MJ, Chahov B. and MS DuBow, 2001, Bioassay for the detection of organotin compounds with a recombinant luminescent bacteria, Quatrième colloques Franco-Japonais –Biosensors and Bioelectrochemistry, 23-26 Octobre, Tokyo, Japon.

PROFILE OF ORGANISATION

The BSCA laboratory is a young research laboratory from the University of Nantes. It was founded in 2000, with the aim to develop biosensor for the detection of pollutants in the environment as well as bacteria in the food industry. Both methods involves the use of bioluminescent microorganisms. The BSCA laboratory coordinate a research group from other universities specialized in the development of optic, immobilization and molecuar microbiology.

SENSOR SYSTEM

Description of sensor system Biosensor using the same recombinant bioluminescent Escherichia coli strain as for the bioassay (see figure attached). The strain (bioelement) is cultivated in continuous culture condition in a dedicated (100 mL) bioreactor. The pH, oxygen, temperature are continuously measured. We patented (french patent) a method for the measurement of bioluminescence and cell density in situ, on line and without any sample. Hence, bioluminescence is continuously reported to the cell density, allowing a better interpretation of the luminescence signal behavior (in progress).

Our system was both developed for the detection of TBT with the mentioned strain and mainly for the development of a standard platform. Then, it will become possible to compare and to evaluate several bioluminescent bacteria with comparable criteria.

Environmental relevance Same as for the bioassay

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Commercial relevance Same as for the bioassay

Usability (i.e skilled, unskilled) Skilled for the moment

Availability (eg. commercial, prototype, etc..) Prototype

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) TBT and DBT

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Freshwater, marine water

Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved

Sample preparation (e.g. filtering, SPE, derivatisation etc…) No sample preparation for water

Sample handling (i.e. manual, automated etc,,,) Automatic

Sample size variable (from the microliter to the mililiter)

Sensor Measurements

Assay protocol Batch growth followed by a continuous one for about 7 days

Continuous measurement Yes

Present measurement performance:

1. Detection levels - Not available at this time

2. Selectivity : TBT and DBT (with Cl, Br or I as the halogen group)

3. Repeatability : not available at this time

4. Potential interference : not available at this time

5. Adaptability to other pollutants : Yes, the device is versatile

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Further work developments A miniature biosensor with immobilized bacteria are under development (research stage)

BOTTLENECKS

Mainly the difficulty to optimize the growth conditions of the strain and the optical devices

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

We are backed by a grant of several years from several local authorities and the french ministry for research. Biolumine are commercial backers? Scientific collaborations would help in sensor development

PAPERS PUBLISHED

Thouand G, Horry H, Durand MJ, Picart P, Daniel Ph, MS DuBow, Development of a biosensor for on line detection of organotin compounds with a recombinant bioluminescent Escherichia coli, in preparation. Thouand G, Picart P et Daniel Ph, Jouvaneau L, Bioréacteur pour le contrôle et/ou la culture en milieu liquide d’organismes vivants, déposé le 19 octobre 2001 à l’INPI, N°01.13518. (bioreactor for the monitoring of living cells in liquid media, submitted on 19 october 2001, french patent N°01.13518)

BIOASSAY

MB TBT TPT 80 DBT 70 TBT MBT 60 TBTox DB TPT 50 40

30 TB Induction ratio Induction 20

0 10-5 10-4 10-3 10-2 10-1 100 101 102 Concentration (µM)

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Specificity of strain TBT3 for several organotin compounds. Each point shown are the averages of data from four replicates. Standard errors are bellow 1% and hence are not shown. For comparative assays, two other experiments were repeated and the trends were found to be identical. (TBT : tributyl tin monochloride ; DBT : dibutyl tin dichloride ; MBT : monobutyl tin trichloride ; TBT0x : Bis (tributyltin) oxide; TPT : triphenyl tin monochloride)

BIOSENSOR 2000

1800 D = 0.6 h-1 D = 0.9 h-1 1600

1400

1200 Decanal 300 µM -1 1000 RLU.s 800

600 Decanal 30 µM 400

200 Addition of TBT (2 µM) 0 10 12 14 16 18 20 22 24 26 28 30 Time (h)

Bioluminescence of the strain Ec::luxAB grown in continuous culture after the automatic addition of TBT (2 µM, final concentration). Decanal, added in the feed medium, was continuously supplied to the bacteria either at 300 or 30 µM.

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CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS

Institute Name: Consejo Superior de Investigaciones Científicas Address: Department/ Research group Barcelona Institute of Microelectronics (IMB-CNM)/ Chemical Transducers Group (CTG) Campus UAB, 08193-Bellaterra Respondents Name: Cecilia Jimenez Position: Tenured Scientist Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

Sensor system based on: 2+ + - - ISFET based sensors for measuring pH, Ca , K , NO3 , and ions required for each application. - Interdigital structures (IDS) for measuring conductivity and redox potential (Eh). - Instrumentation adapted for each sensor and interfaces to control sensors with a PC using a LabVIEW software. The programme permits to obtain the signal of the sensor (potential) and also the concentration of species in the sample resulting from the calibration data.

Sensors are fabricated with standard technology in the Institute of Microelectronics of Barcelona. They are encapsulated individually in PCB boards and covered with a special photopolymer to protect electrical parts (see figure 1). ISFET ion selective membranes are also fabricated with photocurable polymers. The adhesion of such membranes to the ISFET substrate is good and permits a long-term stability of sensors of around 6 months (depending on the sample and frequency of measurements). We have developed different systems and probes: - A probe with one ISFET and two IDS (conductivity and redox potential) for measuring the pore water in clay materials (see figure 2). - A probe with four ISFET (pH, Ca, K, NO3-) for measuring in soils (see figure 3) - A flow system for general purposes (see figure 4).

Environmental relevance

The system can be applied directly in the sample. Therefore, data is obtained at time and no sample treatment is required. It can be useful for environmental applications that require a continuous monitoring of the sample or as an alert system. Sensors can also be applied in flow systems for “on-line” measurements. In that case, an automated monitoring analytical system can be achieved and sample treatment is available.

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Commercial relevance The sensors are mass fabricated and low cost. They are robust and can be applied in relatively extreme conditions of temperature, pH and pressure. Depending on the application it is possible to design a multiparametric system with sensors and virtual instrumentation adapter to user requirements.

Usability (i.e skilled, unskilled) Skilled

Availability (eg. commercial, prototype, etc..) All systems and probes developed until now are research developments. The probe for measuring pore waters in bentonite barriers for radioactive waste containers is a prototype.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) pH, calcium, potassium, nitrate, conductivity and redox potential.

Sample matrix (e.g. soils, sediments, surface and groundwater) Surface and groundwater, soils, pore water in clay materials.

Sample phase (i.e. total, residual, free, dissolved or absorbed) Free ions dissolved in water.

Sample preparation (e.g. filtering, SPE, derivatisation etc…) Not required

Sample handling (i.e. manual, automated etc,,,)

Manual or automated (flow system). Sample size Up to 5 ml.

Sensor Measurements

Assay protocol Sensors require, before being applied to the sample, to be calibrated against standard solutions in the range of concentrations appropriated for each application. This calibration has to be carried out with a frequency of 1 per week or less. For special samples (high ion concentration or extreme pH), a calibration with solutions which composition is similar to the matrix of the sample should be carried out. Measurement is performed directly on the sample.

Continuous measurement Yes

Present measurement performance: Detection levels – Ion sensors: see table 1. Conductivity: 400 µS/cm. (see figure 5)

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Selectivity Depending on the ion sensor.

Repeatability pH-ISFET: 0.2% r.s.d. (see figure 6) Conductivity: 1-2% r.s.d. Redox potential: 1-2% r.s.d.

Potential interference

Adaptability to other pollutants

Further work developments

Sensors have been applied to food processes (wine monitoring, ice cream microbiological contamination), as multiparametric systems for quality control in waters (electronic tongues) and in clinical diagnosis.

Envisaged performance and optimisation Optimisation is performed after each application and user demand.

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Do you have partners helping with the development of this sensor Yes

Please comment: Sensor and Biosensor Group, Universitat Autónoma de Barcelona, Spain

Do you have any commercial backing? Yes

Please comment: (partner’s names can be left undisclosed if bound by confidentiality) All these projects were financed under industrial contracts and EU projects.

Are there any other type of collaboration that would help in sensor development? Please comment: Almost all sensors applications require a special probe or cell where sensors are implemented. The design or development of these probes will need always the collaboration of an engineering group.

PAPERS PUBLISHED

Please list any publications/ posters or presentations related to this sensor

J. Artigas, A. Beltran, C. Jiménez, A. Baldi, R. Mas, C. Domínguez, J. Alonso Development of a ISFET based analysis system for “in-situ” monitoring of soils Proceedings of the 8th International Meeting on Chemical Sensors, IMCS8, July 2000, Basel, Switzerland.

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J. Artigas, A. Beltran, C. Jiménez, J. Bartrolí, J. Alonso Development of a photopolymerisable membrane for calcium ion-sensors.application to soil drainage waters. Analytica Chimica Acta, 426(1) (2001) 3-10.

J. Artigas, A. Beltrán, C. Jiménez, A. Baldi, R. Mas, C. Domínguez, J. Alonso. Application of ion sensitive field effect transistor based sensors to soil analysis. Computers and Electronics in Agriculture, Vol. 31 (3) (2001) 281-293.

J.Artigas, A.Beltran, J.Bartroli, J.Alonso, C.Jimenez, J.Muñoz, R.Mas, C.Dominguez Application of ISFET based sensors to soil analysis Proceedings of the Third International Symposium on Sensors in Horticulture, ISHS Publishers, 2001, Belgium, ISBN 9066059540, pp.287-294.

C.Jimenez, A.Bratov, A.Beltran, J.Alonso, C.Dominguez Development of ISFET based sensors for groundwater monitoring Proceedings of the 1st SENSPOL workshop, University of Alcalá, Alcalá de Henares, Spain, 2001, ISBN 84 8138 482 8, pp.196-197.

C. Jimenez , L.Moreno, C. de Haro, F.X.Muñoz, A.Florido, P. Rivas, A.Mª Fernández, P.L. Martín, A.Bratov, C.Dominguez Development of a multiparametric system based on solid-state microsensors for monitoring a nuclear waste repository. Sensors and Actuators, in press

PROFILE OF ORGANISATION

The Centro Nacional de Microelectrónica (CNM) is a non-profit RandD Institute, which belongs to the Spanish Science Research Council (Consejo Superior de Investigaciones Científicas-CSIC). The CNM activities are channeled towards the strengthening of microelectronics capability in Spain by means of scientific and technological research. This task is focused on the design and fabrication of integrated circuits, electronic devices and related materials as well as providing technological support for industry (small and medium- sized enterprises) and university research groups. The Clean Room facilities (1000 m2, class 100/10.000 suitable for VLSI) are placed in the Instituto de Microelectrónica de Barcelona (IMB-CNM), one of the departments of CNM. The activities carried out in this center are dealing with silicon technology for integrated circuits and devices including digital IC design, microsystems, chemical transducers and power devices and systems. The activities on Chemical Transducers started in 1992. These are centred in the design and development of chemical transducers based on semiconductor technology as well as new technologies for sensor development. The current research activities include design and development of ion- and biosensors based on ISFET (Ion Sensitive Field Effect Transistor) devices, microelectrodes based on interdigitated electrode arrays (IDS), amperometric microelectrodes for biosensor applications and integrated opto-chemical transducers. These activities are combined with the development and application of new materials for sensing: ion selective membranes for ISFET sensors abased on photocurable polymers, chalcogenide glasses, conductive materials for amperometric sensors, as ion-selective membrane matrix; and as packaging material.

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COVENTRY UNIVERSITY

University / Institute / organisation: Coventry University Address: Priory Street, Coventry CV1 5FB Department/ Research group Centre for Molecular and Biomolecular Electronics (CMBE) Address: School of Science and the Environment

Respondents Name: Professor DJ Walton Position: Director of CMBE Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

We have an optically-interrogated system in which a thin film of an active substrate is exposed to a gaseous ambient and the optical changes recorded. Originally the film was a Langmuir-Blodgett film, but for nitrogen dioxide sensing (the analyte for our most highly- optimised system) the active substrate can be entrapped in a sol-gel cast matrix, or else in a thin film of polysiloxane elastomer. Our prototype NO2 device responds in some seconds at room temperature and can be reset by a short heating pulse. It can detect in the 100 ppb range. The sensor is not affected by carbon monoxide, or by sulphur dioxide (except at high concentrations). A simple diode light source and detector makes for a small, compact and economical device. We have not fully optimised this device. The optical principle offers benefits of remote sensing by active optical fibres, ambient temperature operation, no sparks or electrical signals in the presence of the analyte gas, and since a range of active substrates can be immobilised in various matrices and these then interrogated by different wavelengths of light there is room for great selectivity. We have shown feasibility for carbon monoxide at ambient temperature (in a system that is safe in the presence of hydrogen) and also for organic vapours, but without attempting to optimise these responses. Our NO2 sensor is not affected by carbon monoxide, or sulphur dioxide Our aim to further this research and to examine further gases, cross-sensitivity, long-term device stability and other important factors, but we have run out of funding.

Environmental relevance The sensing of toxic gases and vapours is of crucial importance in the environment. A multigas detector that has fast response and recovery in ambient conditions, does not require electrical signals in the presence of the gas, has good sensitivity, selectivity and absence of interferents is a desirable environmental aim.

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Commercial relevance We think that a genuine multigas sensor using optical principles (and the advantages thereof) ought to be a desirable commercial goal. Our nitrogen dioxide sensing system has been patented by our University.

Usability (i.e skilled, unskilled) We envisage an eventual device that will not require great skill to operate.

Availability (eg. commercial, prototype, etc..) We have a prototype device available for nitrogen dioxide sensing.

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…)

Sample matrix (e.g. soils, sediments, surface and groundwater) We have targeted the gaseous ambient, but are keen to test our optical interrogation system on liquid phases, if we can obtain funding for this.

Sample phase (i.e. total, residual, free, dissolved or absorbed) See above

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None necessary, apart from filtering foreign bodies.

Sample handling (i.e. manual, automated etc,,,) Simple gas intake system.

Sample size Gas samples

Sensor Measurements

Assay protocol

Gas is passed into the sample chamber, exposed to an active substrate that (in our NO2 prototype device) is entrapped in a suitable matrix, and changes in optical properties are measured. We have tested interference-enhanced reflectivity (a sensitive technique), which may be useful for some different analyte gases, and compared this to surface plasmon resonance (another optical technique). In fact for our NO2 system we have found that simple changes in absorption are sufficiently sensitive to allow detection of NO2 down below 100ppb. However, simultaneous detection via different optical techniques and at different wavelengths is a useful feature of the optical methodology that gives advantage over sensors based on changes in electronic conductivity in terms of potential selectivity.

Continuous measurement --- in principle Yes

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Detection levels - For NO2 down to approx 100 ppb with our existing prototype, but this is not fully optimised. For CO and for toxic organic vapours we have successful feasibility but have not optimised detection limits.

Selectivity Our NO2 sensor is not affected by SO2 or other common gases, nor by humidity within certain values.

Repeatability The prototype device gives a repeatable response over limited numbers of cycles, but has not been tested over eg hundreds of cycles, or given rigorous tests in climatic chambers

Potential interference Chlorine and ozone could interfere in principle. We have not tested these.

Adaptability to other pollutants The optical principle applies to many other gases, vapours and even liquids. We have shown promising feasibility for carbon monoxide and certain toxic organic vapours, using different active materials and interrogation wavelengths. We believe a multigas sensor is feasible.

Further work developments Our aim is to have several different sensing components each ideally targeted to one gas, although some cross-sensitivity is acceptable, because the long-term aim is a multigas sensor that interrogates each active component and then computes the make-up of the gas mixture. Our optical sensing compounds are individually much more selective than the conducting polymer components in the ‘artificial nose’ that operated by change in electronic conductivity. We will also interrogate different components at different wavelengths, using this feature of optical measurement that contributes to improved selectivity.

Envisaged performance and optimisation We hope our NO2 sensor will become yet more sensitive. We have a very responsive active substrate material, but we have not examined all possible entrapment matrices. These will also affect long-term stability, sensitivity to water and other features

BOTTLENECKS

Please comment on any area of sensor development that has impeded practical implementation of your sensor device:

Funding for further research and development. This is a very serious problem.

Do you have any suggestions on how this could be addressed?

This is a major problem in all research at the moment, but our gas sensor work has suffered more than other aspects of our research (we also undertake biosensor research with modified proteins that we are not able to mention here). Despite producing a prototype device for one gas and having demonstrated capabilities to sense two other bioactive gases we have been unable to obtain grant awards to follow this up.

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COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Do you have partners helping with the development of this sensor Yes we do, but these do not provide sufficient research funding to support pairs of hands at the bench, which is what is required.

Are there any other type of collaboration that would help in sensor development?

Anything that provides funding. It is upsetting to have shown successful feasibility yet be unable to take a project any further.

FURTHER INFORMATION AND COMMENTS

Do you have any further information or comments?

My colleague Professor Ian Peterson has a novel gel-protected biosensor that he is currently trying to commercialise.

In the University laboratory here we investigate the electrosynthetic modification of proteins, a novel patented approach which has ramifications in several types of biosensors.

Please give any additional comment you may have on how the gap between sensor developers and end users can be bridged

End-users need to be more proactive in seeking out novel research developments, and also they need to better describe the practical limitations of existing sensor systems. Rather than aim at a specific eg sensitivity or selectivity that we have been given as a target we have had to simply try to optimise our systems and then see if they are useful to the end-users. In other words we are trying to describe the practical benefits of ambient-temperature optical sensors to end-users who ought to be better aware than us of the niches in the market and the desired improvements in performance criteria needed.

PAPERS PUBLISHED

Please list any publications/ posters or presentations related to this sensor

CMBE Publications on Gas sensors 1999-2002 Nb : these are the most recent, we have earlier papers in the literature D.J. Walton, I.R. Peterson, L.S. Miller, A. Bradford, O. Worsfold, J. Scheerder and D.A. Parry, "Organic thin films for optical gas sensing", Surf. Coat. Int. B, 85 #1 (2002) 55-60. A. Bradford, P.L. Drake, O. Worsfold, I.R. Peterson, D.J. Walton and G.J. Price, "An Improved Azo Chromophore for Optical NO2 Sensing ", Phys. Chem. Chem. Phys. 3 (2001) 1750-1754. D.J. Walton, L.S. Miller, I.R. Peterson, A. Bradford, O. Worsfold, J. Scheerder, D.A. Parry, M.G. Forkan, C. Malins, B.D. MacCraith, "Organic thin films for optical gas sensing of nitrogen dioxide: comparison of sol-gel and LB layers", Synth. Metals 109 (2000) 91-96. O. Worsfold, C. Malins, M.G. Forkan, I.R. Peterson, B.D. MacCraith and D.J. Walton, "Optical NO2 Sensing based on Sol-gel Entrapped Azobenzene Dyes", Sensors Actuators B 56 (1999) 15-21.

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PROFILE OF ORGANISATION

Please give a short description of your organisation and research group

The Centre for Molecular and Biomolecular Electronics at Coventry University may be found on the web at http://www.se.coventry.ac.uk/research/cmbe

Our University School has recently changed its name from Natural and Environmental Sciences (NES) to Science and the Environment (SE) and not everything has caught up with this yet. It may be necessary to change ‘se’ in the above web address to ‘nes’.

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CRANFIELD UNIVERSITY

University / Institute / organisation: IBST – Cranfield University Name: S.P.J. Higson Address: Cranfield University at Silsoe Silsoe, Bedfordshire MK45 4DT, UK Respondents Name: S.P.J. Higson Position: Professor Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

Sensors for determining ultra-low concentrations of chlorine within fresh waters as an environmental pollutant, from, for example, power station cooling water outlets.

Environmental relevance Damage to fish stocks especially trout

Commercial relevance There is at present no sensor (or test kit) commercially available that will determine chlorine concentrations down to 0.002ppm Cl

Usability (i.e skilled, unskilled) Unskilled

Availability (eg. commercial, prototype, etc..) Prototype with first orders placed from the Environment Agency of the UK

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) Chlorine – and in particular – Total Chlorine, Combined Chlorinw and Free Chlorine.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Freshwaters

Sample phase (i.e. total, residual, free, dissolved or absorbed) Total Chlorine, Combined Chlorine (eg chloramaines) and Free Chlorine

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Sample preparation (e.g. filtering, SPE, derivatisation etc…) None

Sample handling (i.e. manual, automated etc,,,) Manual – by riverside or other remote locations

Sample size 1ml or less

Sensor Measurements

Assay protocol

A dedicated instrument is provided for use in conjunction with disposable screen-printed based electrodes.

Continuous measurement No

Present measurement performance: Detection levels - Down to 0.002 ppm Cl

Selectivity

Repeatability Better than +/- 5%

Potential interference Other electrochemically reducible solutes

Adaptability to other pollutants Geeric based instrumentation and screen-printed electrode platform

Further work developments Tightening of fabrication protocols, enhanced electronics for improvement of signal / noise ratios

Envisaged performance and optimisation It is envisaged that this instrument will offer advantages in terms of analytical reproducibility as well as ease of use in comparison to conventional photometric DPD chlorine analyses.

BOTTLENECKS

Please comment on any area of sensor development that has impeded practical implementation of your sensor device:

Incorporation / immobilisation of reagents within custom screen printed hydrogels

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Do you have any suggestions on how this could be addressed?

Packaging and associated costs – we have developed further IPR for the sealing of protective covering strips over the surface of sensors to minimise the cost of protective packaging

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Do you have partners helping with the development of this sensor Yes an electronics / instrumentation company (Magus Electronics)

Do you have any commercial backing? Yes Business angels for the funding of a spin-off company.

PROFILE OF ORGANISATION

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UNIVERSITY OF ULSTER

University / Institute / organisation: University of Ulster at Jordanstown Name: Dr Brian R Eggins Department/ Research group: NIBEC/Photocatalysis and Sensors Research Group Address: Shore Road, Newtownabbey, Co Antrim, BT37 0QB Respondents Name: B R Eggins Position: Reader Email address: [email protected]

SENSOR SYSTEM

Description of sensor system

Biosensor for the determination of flavanols using either plant tissue material (polyphenoloxidasees or commercial tyrosinase) immobilised in either carbon paste electrode or screen-printed in with modified polypyrrole. Detection by flow-injection analysis. The method relies on the electrochemical reduction of a quinone produced from the catalysed oxidation of the phenolic compound by ambient oxygen.

Environmental relevance Designed for use with beers and wines, but could have wider applications for anti-oxidant polyphenols

Commercial relevance Potential use in brewing industry.

Usability (i.e skilled, unskilled) Requires some skill intitially.

Availability (eg. commercial, prototype, etc..) No

SAMPLE

Targeted analyte(s) (e.g. Mercury, PAHs, DNAPL’s, algal toxins etc…) It will detect a broad range of analytes containing a catechol (1,2-dihydroxybenzene) group. This includes dopamine as well as a range of polyphenols of the catechin type found in flavanols, which are useful anti-oxidants for minimising heart disease.

Sample matrix ( e.g. soils, sediments, surface and groundwater ) Mainly used for beers so far.

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Sample phase (i.e. total, residual, free, dissolved or absorbed) Dissolved

Sample preparation (e.g. filtering, SPE, derivatisation etc…) None

Sample handling (i.e. manual, automated etc,,,) Either

Sample size

Sensor Measurements

Assay protocol Standard addition method Prepare standard solutions (0.01 M) of calibration standard (catechol or catechin) in phosphate buffer (0.05 M pH 7.4). Run amperometric scan at –0.2 V vs. SCE on base electrolyte buffer alone. Add equal volume of beer sample and repeat scan. Add successively five aliquots (0.1 ml) of standard solution and repeat measurement. Plot multiple standard addition calibration graph of current vs. added concentration. Negative intercept at I = 0 gives conc. Of catechol in sample.

Continuous measurement

Present measurement performance: Detection levels – 2.5 µM 6.9 mA M-1 using (-) epicatechin as reference standard

Selectivity: broad as described above

Repeatability +/- 5%

Potential interference: tyrosine

Adaptability to other pollutants: potentially good

Further work developments None at present as I have formally “retired”.

Envisaged performance and optimisation

With the use of plant polyphenol oxidase there is no interference from tyrosine. The method relies on the electrochemical reduction of a quinone. Monitoring the oxygen uptake would make the method less dependent on the nature of the substrate.

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BOTTLENECKS

Please comment on any area of sensor development that has impeded practical implementation of your sensor device:

Unwillingness of Guinness Ltd to continue to sponsor this work, because of a policy change

COLLABORATIONS AND COMMERCIAL PARTNERSHIPS

Do you have partners helping with the development of this sensor Yes French group at JFU Grenoble: Dr Pascal Mailley and Dr Serge Cosnier. Originally Guinness Ltd, Dublin. (Dr David Madigan)

Do you have any commercial backing? No See above

PAPERS PUBLISHED

Please list any publications/ posters or presentations related to this sensor B R Eggins, Biosensors- an Introduction (Chichester: J Wiley and Sons Ltd 1996), pp. 179 – 183.

B R Eggins, Chemical Sensors and Biosensors (Chichester: J Wiley and Sons Ltd 2002), pp. 227 – 230.

Eggins, B R , Hickey, C, Toft, S A and Zhou, D M, Anal Chim Acta, 347 (1997) 281- 288.

Cummings, E A, Mailley, P, Linquette-Mailley, S, Eggins, B R McAdams, E T and McFadden, S, Analyst 123 (1998) 1975 – 1980. E A Cummings, PhD thesis, University of Ulster 2000.

S Linquette-Mailley, E A Cummings, P Mailley, S Cosnier, B R Eggins and E T McAdams, “Electrode, sérigraphiees pour la transduction ampérométrique: optimisation, characterisation et application à la detection de composées phénoliques”. (Journées d’Electrochimie, Toulouse, 1 – 4 June 1999).

E A Cummings, S Linquette-Mailley, P Mailley, B R Eggins and E T McAdams, “Dosage ampérométrique des flavanols par des ampérométrique pâtes de carbone incorporant des tissues de plante”( Journées d’Electrochimie, Toulouse, 1 – 4 June 1999).

S Linquette-Mailley, E A Cummings, P Mailley, S Cosnier, B R Eggins and E T McAdams, “Biocapteurs ampérométriques à pâte de carbone utilisant un liant polymère electrogénéré” (Journées d’Electrochimie, Toulouse, 1 – 4 June 1999).

E A Cummings, B R Eggins, S Linquette- Mailley, E T McAdams, S Cosnier and P Mailley, “Novel Biosensors based on Polypyrrole modified Carbon Paste Electrodes”, SAC99 (Dublin City University 28th July 99).

219 SENSPOL Survey of Sensor Capabilities

E A Cummings, B R Eggins, E McAdams, C Coleman, M Clements and D Madigan, “Biosensing to ensure the perfect pint”, The Irish Scientist Year Book 1999, 88

. E A Cummings, D Madigan, B R Eggins et al, “Development of a tyrosinase based screen- printed amperometric electrode for the detection of flavanoid polyphenols in lager beers”, J. American Soc. of Brewing Chemists, 2001, 59(2), 84 – 89.

S C Linquette-Mailley, P Mailley, E Cummings, S Cosnier, B R Eggins, and E T McAdams, “A comparison of amperometric screen printed carbon electrodes and their application to the analysis of phenolic compounds in beers”, Talanta, 2001, 55, 1015 – 1027.

PROFILE OF ORGANISATION

Please give a short description of your organisation and research group

Our research group consists of 2 readers, 3 post- docs and 6 post-grads, working on both sensors and photocatalysis for water treatment. Currently we are involved in 2 EU projects. We are now based in the Bioengineering Centre of the University of Ulster at Jordanstown. As well as collaboration with UJF Grenoble, we are a member of the BEST centre (Biomedical, Environmental Sensor Technology), which also includes Dublin City University, University of Limerick and Queens University Belfast.

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