B/otec/t Adv. Vol. 13, pp.

1-12,1995
Copyright © 1995 Elsevier Science Ltd
Pergamon Printed in Great Britain.All Rights Reserved.
0734-9750/95 $29.00

0734-9750(94)00020-4

BIOSENSORS FOR ENVIRONMENTAL MONITORING

M. J. DENN/SON and A. P. F. TURNER

Cranfield Biotechnology Centre, Cranffeld University, Cranfield, Bedford MK43 OAL, UK

ABSTRACT

Increasing environmental legislation which controls the release and the levels of certain
chemicals in the environment has created a need for reliable monitoring of these substances in
air, soil and especially water. Conventional analytical techniques, although highly precise,
suffer from the disadvantages of high cost, the need for trained personnel and the fact that they
are mostly laboratory bound. Biosensors because of their specificity, fast response times, low
cost, portability, ease of use and a continuous real time signal, can present distinct advantages
in certain cases. Their biological base makes them ideal for toxicological measurements which
are suited for health and safety applications. Over the last 3-4 years there has been an increase
in the number of publications concerning biosensors for environmental monitoring, especially
in the field of pesticide measurements.
This paper reviews some of the more important developments over the past 3-4 years.

Keywords: biosensors, enzymes, pollutants, pesticides, phenols, heavy metals

INTRODUCTION

Increasing legislation in both the USA and the EU designed to protect the environment
has been a feature of recent years. Strict limits have been placed on the release of certain
chemicals into the environment. Enforcement of this legislation necessitates reliable monitoring
of the environment for the presence of compounds which may adversely affect human health
and local ecosystems. Environmental monitoring in the USA is primarily driven by legislation
passed by Congress. The Resource Conservation and Recovery Act, the Comprehensive
2 M.J. DENNISON and A. P. F. TURNER
Environmental Response, Compensation and Liability Act of 1980 and its 1986 amendment are
all examples of legislation requiring regulation and control of chemicals released into the
environment. The EU commission has also enacted directives concerning the release of
substances into the environment, and is currently trying to tighten standards regarding water
quality throughout the Union.

ENVIRONMENTAL M O N I T O R I N G

The environment around us consists of basically three phases: water, air and soil.
Water, a basic requirement for any population, can be polluted by natural or man-made
chemicals causing changes to aqueous flora and fauna. Soil pollution, like water pollution can
lead to contamination of aquifer systems. Direct uptake of the pollutant by plants and then
animals can have serious consequences for human health. Air pollution can directly affect
human health leading to or aggravating conditions such as bronchitis or asthma. There exists a
continual equilibrium between the air, water and soil phases. Leaching of soil pollutants will
result in water pollution, depending on how strongly bound the pollutant is to the soil phase
while, volitilaztion of pollutants from water and soil phases will result in air pollution. Water,
soil and air pollution are interdependent and pollution in one phase will generally affect
pollution in the other two phases depending on the pollutant in question.

BIOSENSORS

Classical laboratory techniques, although being continually improved are limited by

certain consideration's, being generally expensive, time consuming and laboratory bound.
Biosensor technology lends itself to portable, economical and selective analysis and may
provide some of the solutions for the problems encountered in environmental monitoring.
Biosensors are generally defined as an analytical device, incorporating a biological or
biologically derived sensing element (e.g. enzymes, antibodies, microorganisms or DNA),
either intimately associated with or integrated within a physicochemical transducer (e.g.
electrochemical, optical or piezoelectric transducers) (Turner, 1989). Biosensors, because of
their biological base, can make ideal sensing systems to monitor the effects of pollution on
both flora and fauna, e.g. toxicity sensors. While on the other hand, the exquisite specificity
of some enzymes can even distinguish between stereo isomers of the same compound.
Biosensors can also achieve the high sensitivity necessary for monitoring very low levels of
hazardous chemicals, some immunosensing systems achieving limits of detection of the order
of 10-21 moles (Jenkins et al., 1991 ). The other advantages of biosensors are the ability to
operate in complex matrices, considerably reducing the need for sample preparation, fast
response times, a continuous signal which can provide real time monitoring data, small size
 BIOSENSORSFOR ENVIRONMENTAL MONITORING 3
making the biosensors easily portable and the ability of biosensors to be mass produced
leading to the important consideration of low manufacturing costs.
Much biosensor technology has been developed to meet the demands of the lucrative
biomedical markets and awaits adaptation to environmental applications. The majority of
commercially available biosensors have been specifically designed for the biomedical market
and especially for the determination of blood and urine glucose concentration.
The biological components used in biosensor construction can in general be divided
into two categories: those where the primary sensing event results from, catalysis (e.g.
enzymes, microorganisms) and those which depend on an essentially irreversible binding of
the target molecules (e.g. aff'mity sensors based on antibodies or nucleic acids).
The most commonly used biological components in catalytic biosensors are enzymes.
There is currently a wide selection of enzymes commercially available and advances in enzyme
technology should increase the range of enzymes available. The ability of enzyme based
biosensors to operate in such diverse media as aqueous solutions (Turner et al., 1989),
organic solvents (Hall et al., 1988) and air (Tierney et al., 1993) has been demonstrated. The
component parts of a biosensor must be in close proximity to each other for effective
functioning and immobilisation of the biological component at the surface of the transducer
lead's to more efficient signal transfer, increased stability and the ability to perform continuous
or repeated assays. Methods of immobilisation include chemical or physical adsorption,
physical entrapment within a membrane or gel, cross linking of molecules or covalent binding.
The principal transducers used in biosensor construction are electrochemical,
photometric, thermometric and acoustic devices. Amperometric biosensors have dominated
both research and commercial activity to date largely due to their relative simplicity and
flexibility. This technique generally utilises a three electrode system: a working electrode
through which a potential is applied, a reference electrode and an auxiliary electrode through
which current flows. Any species which undergoes electrochemical oxidation or reduction at
the potential applied are detected at the working electrode. Electroactive species which can be
monitored at the electrode surface include substrates of the biological reaction (e.g. 0 2,
NADH), products (e.g. hydrogen peroxide for oxidase reactions, benzoquinone for phenol
oxidation) and also electrochemical mediators which can directly transfer electrons from the
enzyme to the working electrode surface (e.g. hexacyanoferrate, ferrocene, methylene blue).
Coulometry measures the total charge transferred rather than the current, while
potentiometry measures the difference in potential between the working electrode and a
reference electrode at near zero current flow. Ion-selective electrodes operate on potentiometric
principles and have found many applications in the field of biosensors. Selective membranes
which are preferentially permeable to the ion in question minimise interference from competing
ions.
4 M.J. DENNISONand A. P. F. TURNER
Optical devices are the third most commonly used transducers, after amperometric and
potentiometric approaches. Recent advances in fibre optics mainly due to research in the
telecommunications industry, have increased the potential in this area considerably. Optical
techniques range from simple light absorbance of a reagent layer on binding the substrate in
question, to techniques such as emission spectroscopy, atomic absorption and raman
scattering. This area has been reviewed by Dakin & Culshaw (1989).

Biosensors for Monitoring Biochemical Oxygen Demand

The biochemical oxygen demand (BOD) is one of the most widely used and standard
tests in the measurement of organic pollution. Some conventional BOD tests require long
incubation periods - up to five days. It is here that biosensors have made a significant
commercial impact on environmental monitoring. Research which began in the seventies in
Japan and Germany has resulted in several commercial BOD biosensors, with fast response
times and automated sampling. The "BOD module" produced by Medigen (Prufgerate-Werk
Medigen GmbH, Germany), has a response time of 50 seconds and a BOD measuring range of
2 to 22mg/L BOD. The biosensor system is based on microorganisms which are immobilised
behind a membrane. When organic matter is introduced into the system, it is consumed by the
micro-organisms, which results in a decrease in 0 2 in the solution. This decrease in 02 is
monitored by a Clark electrode, which is then correlated with the amount of organic material
present. Each membrane is viable for 2,000 or more assays.

Biosensors for Monitoring Pesticides

Soil is the basis of agriculture and pesticides have become indispensable tools in the
efficient control of weeds, insects and fungi, which would otherwise have a detrimental effect
on crop production. There are approximately 600 different pesticide active ingredients
registered in the USA alone, with a world market estimated at greater than 20 billion dollars
(Ross & Lembi, 1985). Pesticides can be classified into three different groups: insecticides,
herbicides and fungicides. Insecticides are usually organophosphorous compounds (e.g.
parathion), organochlorine compounds (e.g. DDT) or carbamates (e.g. carbofuran).
Fungicides are either sulphur, copper or organic based compounds, while herbicides can be
either organic or inorganic compounds. Pesticides can be applied as dust or granules, as a
vapour or more commonly applied as, or in the presence of a liquid (water or oil usually).
Pesticides, depending on their water solubility can either remain in the soil to be broken down
by the action of certain organisms (Lewis, 1992), or washed off, eventually washing into
rivers and sometimes water supplies.
 BIOSENSORSFOR ENVIRONMENTAL MONITORING 5
The high toxicity of certain pesticides, such as dinitro-ortho cresol and PCP (Barnes,
1979) has led to their use being restricted (Mayer,1993). Pesticides or their breakdown
products can present hazards to human health, directly as in the spray drift of pesticides,
consumption of contaminated water, or indirectly as accumulated pesticide in edible plant or
animal tissue. Some public supplies of drinking water have been shown to be contaminated
with pesticide concentrations above the minimum EU drinking water levels (Lewis, 1992).
There exists a need for cheap reliable monitoring of pesticides in water and also in air and soil.
Recently there has been a large increase in the number of publications concerning biosensors
for monitoring pesticides, most likely due to increasing environmental awareness, manifested
by increasing regulations concerning pesticides and a greater public awareness concerning
pesticides.
Many pesticide biosensors are based on the inhibition of the enzyme cholinesterase by
organophosphorous compounds. A biosensor for the detection of organophosphate and
carbamate insecticides was based on the action of two enzymes, acetylcholinesterase and
choline oxidase (Marty et al., 1992). Acetylcholinesterase acts on acetylcholine to form
choline, while choline oxidase oxidises choline to betain with the concurrent production of
hydrogen peroxide. The inhibition of acetylcholinesterase by pesticides is measured as
decreased formation of hydrogen peroxide as monitored amperometrically at 700mV Vs
Ag/AgC1 reference. The limit of detection was 10nm paraoxon, with wet stability being two
months at 4°C.
Skladal and Mascini (1992) described an amperometric biosensor for organophosphate
and carbamate pesticides which could detect paraxon levels of 1.5gg/L and heptenophos levels
of 8.4gg/L within 3 minutes. Acetylcholinesterase or butyrlcholinesterase was immobilised on
a nylon net, with their relevant substrates. The inhibition of either acetylcholinesterase or
butyrlcholinesterase by pesticides was measured by the decrease of the product of
acetylthiocholine or butylthiocholine cleavage- thiocholine. Thiocholine production was
monitored by direct electrochemical oxidation at 300mV (Vs Ag/AgC1). The use of a cobalt
phthalocyanine modified composite electrode overcame the problem of needing a high
overvoltage as would be required for a platinum electrode.
An amperometric biosensor which used the two enzymes cholinesterase and choline
oxidase has been used for pesticide detection. Cholinesterase catalyses the cleavage of choline
esters to choline and an organic acid. Choline oxidase oxidises choline to betaine and hydrogen
peroxide. Hydrogen peroxide production was monitored electrochemically at 650mV (Vs
Ag/AgC1). Analysis was performed by monitoring the decrease of cholinesterase activity in the
presence of a pesticide and a substrate for cholinesterase (a choline ester). Limits of detection
were 0.5ppb paraoxon for an incubation time of 30 minutes (Bernabei et al., 1993). One of the
advantages of biosensors over other analytical techniques is the fact that biosensors, because
they are biologically based can measure toxicity, whereas analytical techniques measure only
6 M. l- DENNISONand A. P. F. TURNER
concentration. The magnitude of response from this biosensor correlated directly with the acute
toxicity of the pesticide in question, expressed as LD50 (oral) for rat.
An immunosensor for the herbicide atrazine has been described by Scheper & Muller
(1994). Polyclonal sheep antibodies were used to bind atrazine and alkaline phosphate labeled
anti-antibodies were used to bind atrazine antibodies. After washing, enzyme substrate was
added. The amount of bound antibody could be determined since the substrate was cleaved by
the enzyme resulting in a fluorescing product. The fluorophor was excited at 365nm and the
fluorescence measured at 450nm, atrazine levels as low as 10-2pmol/ml could be detected.
A piezoelectric immunobiosensor for measuring atrazine in drinking water was
described by Guilbault et al., (1992). A piezoelectric crystal was coated with atrazine
antibodies (polyclonal) from sheep. When atrazine molecules came into contact with the
antibodies, they were bound causing a change in the crystal mass, which in turn lead to a shift
in its oscillation frequency. This biosensor could determine concentrations of 0.03-100~tg/L
(ppb) atrazine. The sensor was reversible being reusable for 8/9 assays.
A technique based on the principle of a flow injection system, named flow injection
immunoanalysis was used for the measurement of the herbicide atrazine. The principle was
based on a sequential competition enzyme immunoassay, where the hapten (triazine herbicides)
competed with the corresponding enzyme-labeled hapten (modified atrazine conjugate with
peroxidase) for the limited binding sites of the anti-atrazine antibodies. Those antibodies were
bound onto a membrane within the flow injection immunoanalysis apparatus. A fluorogenic
substrate of peroxidase was used to produce a fluorescent product whose concentration was
proportional to the amount of enzyme labeled hapten. The amount of fluorescence was
determined in a fluorescence detector. The limit of detection was 0.021.tg/L atrazine with a 15
minute determination time (Kramer & Schmid, 1991).

Biosensors for Monitoring Phenols

Phenols are widely used in the manufacture of a wide variety of products: pesticides,
pharmaceuticals, plasticizers, surfactants and explosives. Phenol itself is one of the most
widely used chemicals - annual USA production being some 1.56 million tonnes (Seiber,1990)
and has been identified as a major organic chemical with potential for environmental
contamination (Gilbert, 1994). Industrial by-products from many industries and especially
from the wood pulp industry give rise to the presence of phenols as contaminants of air, soil
and especially water. Some pesticides are based on the phenolic skeleton and degradation of
these pesticides will give rise to phenols, while phenols themselves can be degraded into even
more reactive products. There have been numerous reports of biosensors for monitoring
phenols in aqueous andnon-aqueous media. The abundance of reports in the literature is
largely due to the wide availability of the enzyme tyro sinase (E.C. 1.14.18.1), which has been
 BIOSENSORSFOR ENVIRONMENTAL MONITORING 7
well characterised (Dawson & Magee, 1955, Horowitz et al., 1970). Tyrosinase catalyses the
oxidation of phenols to catechols and then to quinones. Oxidation of phenols is concurrent
with quinone generation and oxygen depletion. Phenolic biosensors either electrochemically
measure the depletion of oxygen using the Clark oxygen electrode or the generation of
quinones using an electrode poised at a negative potential (Vs Ag/AgC1).
The detection of phenols with biosensors using quinone reduction has been used
successfully. Cosnier & Innocent (1992) immobilised tyrosinase in an electrochemically
generated polypyrrole film, obtaining detection limits of 0.47ppb phenol. Kulys & Schmid
(1990) used a mediated system (with tetracyanoquinodimethane) to obtain detection limits of
22ppb. A flow injection system described by Ortega & Dominguez (1993) based on direct
electrochemical reduction of quinone products, could detect phenol levels as low as 0.282ppb.
The tyrosinase was immobilised on the surface of a carbodiimide activated graphite electrode
and the response was stable for up to 50 consecutive injections of 0.01mM phenol.
The use of biosensors in organic solvents for phenol detection presents a number of
advantages. Oxygen is usually more soluble in organic solvents - it is approximately ten times
more soluble in chloroform than in water (Buckland et al., 1975). This has a distinct advantage
for oxidase enzymes. Many enzymes are more stable in certain organic solvents and tyrosinase
does not seem to be inactivated as it is in aqueous solutions by the products of quinone
oxidation (Kazandijan & Klibanov, 1985). The use of an organic phase biosensor for phenol
was In'st described by Hall & Turner (1988), and since then more organic phase biosensors for
phenol have been described. Wang et al. (1993) used a polymer of poly(ester-sulphonic acid)
to entrap tyrosinase for phenol detection in acetonitrile solutions. Lower detection limits were
0.85ppm.
Campanella et al. (1992) used an oxygen electrode as a transducer, and entrapped
tyrosinase with a dialysis membrane. Phenol concentrations of 47ppb could be detected.
Unchiyama et al. (1993) constructed a catechol sensor using tyrosinase in conjunction with an
oxygen electrode, and used substrate cycling to increase the sensitivity of the sensor to
catechol. As catechol was consumed, quinone was produced and oxygen was consumed.
However, introducing ascorbic acid into the solution brought about the non-electrochemical
oxidation of quinone back to catechol. This catechol was then oxidised to quinone with the
further consumption of oxygen. This substrate cycling resulted in larger rates of oxygen
consumption, giving signal amplification. Using ascorbic acid to recycle quinones back to
catechols, Unchiyama obtained detection limits of 5.5ppb catechol.
A tyrosinase based sensor has also been used to detect cyanide in aqueous media. As
tyrosinase is a copper based enzyme, it is very sensitive to cyanide inhibition. Cyanide can be
detected as the decrease in oxygen consumption, as the oxidation of tyrosine by tyrosinase is
inhibited by cyanide (Smit & Rechnitz, 1993).
8 M.J. DENNISONand A. P. F. TURNER
Biosensors for Monitoring Heavy Metals

Heavy metals such as lead, cadmium and mercury present major hazards to ecosystems
and are a serious danger to human populations because of their ability to be accumulated in
livestock (Schlatter, 1994). Biosensors can form ideal sensors for toxicity measurement of
heavy metals because of their biological base, while conventional techniques can only measure
concentration.
Heavy metal biosensors using immobilised oxidases and dehydrogenases have been
demonstrated for the detection of mercury, zinc, silver, cadmium and copper salts based on the
amount of enzyme inactivation. The enzyme was covalenfly bound with gluteraldehyde onto an
affinity membrane and coupled with a Clark oxygen electrode. When incubated with substrate
there was a decline in oxygen levels. Addition of heavy metals inhibited the enzyme and
resulted in lower consumption rates of oxygen. Using L-glycerophosphate, pyruvate oxidase
or L-lactate dehydrogenase the 50% inhibition levels were l~tM for HgC12, 0.1~tM for
AgNO 3, 101aM for CdCI2 and ZnCI2, 50raM for Pb-acetate and 250gM for CuSO 4
respectively (Gayet et al., 1993).
A fibre optic biosensor using carbonic anhydrase could detect nanomolecular
concentrations of zinc. Carbonic anhydrase binds its cofactor, zinc(II), with very high
selectivity. This selective recognition of zinc by carbonic anhydrase was measured as a change
in the fluorescence of an inhibitor which bound to the zinc in the active site. The concentration
of zinc was proportional to the ratio of fluorescence intensity at two wavelengths,
corresponding to the emission from bound and free inhibitor (Thompson & Jones, 1993).

Biosensors for Monitoring Polluting Gases

Increasing regulations concerning control of environmentallyrelevant gases such as

NOx, SOx, CO2, methane and ozone have provided a need for reliable monitoring of air
pollution. This would seem to be an area ripe for biosensor technology as certain current
commercial gas sensors seem to suffer from cross-interferences (Shurmer et al., 1990). Much
work in the eighties on biosensors for monitoring of gases, whether in the gas phase or
dissolved in solution (Guilbault,1983; Turner et al., 1985; Karube & Tamiya, 1987;), has not
been emulated in the past few years. One paper by Suzuki et al. (199t), described a disposable
amperometric carbon dioxide sensor using immobilised S-17 autotrophic bacteria. The bacteria
were immobilised in an alginate gel behind a gas permeable membrane, using an oxygen
electrode as a transducer. As the bacteria metabolised any CO 2 present, using 02 in the
process, there was a concurrent decrease in the oxygen levels in the gel. The response time
was between one to three minutes and the sensor could detect between 0.5mM and 3.5mM
CO2 in aqueous solution.
 BIOSENSORSFOR ENVIRONMENTALMONITORING 9
Tierney et al. (1993) reported a potentiometric biosensor for detection of gaseous
CO 2. Carbonic anhydrase which catalyses the formation of HCO 3" and H +, from CO2 and
H20 was immobilised in a hydrogel polymer electrolyte. When CO 2 was absorbed in the gel
there was an increase in the pH of the gel. The change in pH which was directly proportional
to the amount of CO 2, was measured by using a thin film of iridium oxide which functioned as
a pH electrode. Concentrations between 1% and 10% CO 2 could be measured, the response
time being fast- 90% of the response in 900ms. The sensor was stable for several months at
4°C.

CONCLUSIONS

Over the last few years, there have been an increasing number of publications
concerning the application of biosensors to environmental analysis. Biosensors have been
reported for the commonly used pesticides and industrial chemicals. In some cases there is a
need for sensitivity and lifetime improvements as conventional techniques can outperform
biosensors in these respects at present. The vast majority of biosensors for environmental
analysis have not yet made significant commercial impact. If biosensors are to be made
commercially viable, they need to be at least as sensitive as conventional techniques, cheaper
and easier to operate. Conventional analytical techniques such as gas chromatography and high
pressure liquid chromatography have the advantage of being able to identify a range of
pollutants, while biosensors can usually only detect at most one class of compounds. For
environmental monitoring biosensors would be applicable to situations where the polluting
compound was known, or had been identified. Continuous monitoring of the compound could
then be undertaken cheaply and reliably. Ideal applications would be for monitoring factory
effluents whose waste product(s) had been identified. Discharge above the legal limits could
then be detected on a real time basis. Pesticide levels could be monitored on a real time basis
during periods of pesticide application, once the active ingredients in the pesticides were
known. The health and safety of workers applying pesticides or indeed any other chemical
could be protected by providing them with biosensors for monitoring the levels of pesticides or
chemicals in the air around them. Biosensors could contribute towards monitoring the progress
of clean-up operations after environmental spiUages of certain chemicals. It is clear that the
field of environmental monitoring presents a number of niche opportunities, which biosensors
could profitably exploit.

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