Whole cell- and protein-based biosensors for the detection ofbioavailable heavy metals in environmental samples

Philippe Corbisiera,*, Daniel van der Leliea, Brigitte Borremansa, Ann Provoosta,Victor de Lorenzob, Nigel L. Brownc, Jonathan R. Lloydc, Jonathan L. Hobmanc,

Elisabeth CsoÈregid, Gillis Johanssond, Bo Mattiassond

aVlaamse Instelling voor Technologisch Onderzoek (VITO), Boeretang 200, B-2400 Mol, BelgiumbCentro Nacional de BiotecnologõÂa, Campus de Cantoblanco, Universidad AutoÂnoma, Spain

cThe University of Birmingham, School of Biological Sciences, Edgbaston, Birmingham B15 2TT, UKdDepartment of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, PO Box 124, S-221 00 Lund, Sweden

Received 8 July 1998; received in revised form 9 October 1998; accepted 11 October 1998

Abstract

The principal goal of this work was to establish the feasibility of two biosensor technologies with enhanced speci®city and

selectivity for the detection of several bioavailable heavy metals in environmental samples. Two parallel strategies have been

followed. The ®rst approach was to construct whole cell bacterial biosensors that emit a bioluminescent or ¯uorescent signal

in the presence of a biologically available heavy metal. The molecular basis of s-54 promoters as sensing elements of

environmental pollutants has been determined and a number of metal-induced promoter regions have been identi®ed,

sequenced and cloned as promoter cassettes. The speci®city of the promoter cassettes has been determined using luxCDABE

reporter systems. Whole cell-biosensors containing metal-induced lux reporter systems have been incorporated into different

matrices for their later immobilisation on optic ®bres and characterised in terms of their sensitivity and storage capacity. The

second type of sensors was based on the direct interaction between metal-binding proteins and heavy metal ions. In this case,

the capacitance changes of the proteins, such as synechoccocal metallothionein (as a GST-SmtA fusion protein) and the

mercury regulatory protein, MerR, were detected in the presence of femtomolar to millimolar metal ion concentrations.

# 1999 Elsevier Science B.V. All rights reserved.

Keywords: Heavy metals; Bioavailability; Biosensors; Af®nity sensors; Capacitance measurements

1. Introduction

Most metal ions can be detected in environmental

samples using classical analytical methods such as

inductively coupled plasma atomic electron spectro-

metry (ICP/AES) or mass spectrometry (ICP/MS),

¯ow injection atomic absorption (FIAAS) or electro-

chemical methods that include ion selective electro-

des, polarography and other voltammetric electrodes.

The environmental samples need to be digested under

high temperature, pressure and acidic conditions to

free the metal ions in solution as a prerequisite for all

those methods. However, the total amount of metal

Analytica Chimica Acta 387 (1999) 235±244

*Corresponding author. Tel.: +32-14-335-112; fax: +32-14-580-

523; e-mail: corbisip@vito.be

0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

P I I : S 0 0 0 3 - 2 6 7 0 ( 9 8 ) 0 0 7 2 5 - 9

detected after such an extraction may not always be

related to the toxicity of the samples because the

original biological availability of metal ions is not

taken into account. To quantify the biologically avail-

able fraction of metal in environmental samples,

different approaches have been followed. The ®rst

approach is based on the use of soil bacteria that

are genetically engineered so that a quanti®able signal

is produced when the bacteria are in contact with

bioavailable metal ions. A number of fundamental

aspects of the regulation of sensing elements have

been studied, and new metal-induced regulators have

been discovered and characterised. Soil bacteria able

to produce a bioluminescent signal in the presence of

speci®c metal ions have been constructed by genetic

manipulation and immobilised in solid matrices for

use with optic ®bres.

The second approach followed in this work was to

follow directly the interaction between metal ions and

speci®c puri®ed metal-binding proteins by recording

the capacitance changes on metal binding. Both tech-

niques are described below.

2. Experimental

2.1. Construction of heavy metal-induced promoter

cassettes

Two independent clones containing the copSRA

promoter region of the Alcaligenes eutrophus CH34

cop operon and the tetracycline marker (as an EcoRI

fragment from miniTn5-luxAB [1]) were constructed

in plasmid pUC18/S®I. An s®I cassette containing the

mercury responsive elements from Tn501 [2] was

constructed as follows: the E. coli merR gene and

mer promoter (PmerTPAD) of Tn501 was cloned as a

0.7 kb Xba-EcoRI fragment in pUC18/S®I and the

tetracycline resistance marker was cloned into the

HindIII site. An s®I cassette containing the chromate

responsive element from the A. eutrophus pMOL28

plasmid [3] was constructed by cloning the chr pro-

moter/operator region and the chrB gene of plasmid

pMOL28 as a 1.2 kb XbaI-PstI fragment in pUC18/

S®I together with the EcoRI fragment containing the

tetracycline resistance gene. The A. eutrophus pbrR

promoter/operator region and the partially deleted

pbrA gene were cloned as a 3.8 kb EcoRI fragment

in PUC18/S®I, and the tetracycline marker was cloned

into the HindIII site.

2.2. Immobilisation of the whole cell copper

biosensor

To immobilise the strain AE1239 in sodium algi-

nate (BDH Supplies Poole, Dorset, UK), 50 ml of

fresh bacterial culture was harvested by centrifugation

at 6000 rpm for 10 min in a JA-20 Beckman rotor. The

cell pellet was suspended in 20 ml 0.9% (w/w) NaCl,

and 20 ml 4% (w/w) sodium alginate was added to the

bacterial suspension. Next, a syringe with a needle (ID

1.2 mm) was used to create droplets added to 200 ml

0.2 M CaCl2 to form the alginate beads. The beads

were washed twice with 0.9% NaCl and kept in 0.9%

NaCl at 48C until use. To immobilise the whole cell

copper biosensor in seaplaque agarose, the same

procedure was followed but the cell pellet was sus-

pended in 9 ml 0.9% NaCl. Next, 2.22 g agarose was

dissolved in 100 ml 0.9% NaCl with heating, cooled to

308C in a water bath, and 5 ml bacterial suspension

was added. A 2 mm thick biogel was produced on

setting, and discs of 4 mm diameter were cut from

the gel. The biogel was kept at 48C in 0.9% NaCl

until use.

2.3. Bioluminescence measurements

For the copper whole cell-sensor AE1239, the

chromate whole cell-sensor AE2440, or the lead

whole cell-sensor AE2448, an overnight 25 ml LB

bacterial culture was centrifuged for 10 min at

10 000 rpm in a JA-20 rotor and resuspended in cool

cryoprotectants (1 ml/0.1 g wet wt). A volume of

150 ml cell suspension was dispensed in sterile lyo-

philisation glass vials and cooled in ice water before

being frozen at ÿ408C and lyophilised in a vacuum

chamber. The vacuum was released under nitrogen gas

and the vials were sealed. About 107±108 viable cells

were obtained after reconstitution in a reconstitution

medium (RM). This medium is similar to the Tris

medium described previously [4] but Tris buffer was

replaced by MOPS buffer and sodium phosphate by

sodium-b-glycerophosphate to minimise metal chela-

tion and precipitation. The RM medium contained

4.68 g of NaCl, 1.49 g of KCl, 1.07 g of NH4Cl,

430 mg of Na2SO4, 200 mg of MgCl2�6H20, 30 mg

236 P. Corbisier et al. / Analytica Chimica Acta 387 (1999) 235±244

of CaCl2�2H20, 294 mg of Na-b-glycerophosphate,

2 g of Na-acetate or 0.1 g of gluconate, 20 mM of

MOPS, pH 7.0 and one 1 ml of trace element solution

SL7 of Biebl and Pfennig [4] in 1 l of distilled water.

The metal salt solutions (20 ml) were added to 180 ml

cell suspension and the bioluminescence was recorded

over 5 h at 238C in a Lucy1 microtitre plate lumino-

meter.

2.4. Overexpression and purification of heavy-metal

binding proteins

The mercuric ion-binding regulatory protein, MerR

from transposon Tn501, has been puri®ed in large

amounts. The protein was overexpressed in E. coli and

an extraction protocol using sonication, salt-extrac-

tion, and liquid chromatography (LC), modi®ed from

[5], was used to purify 10 mg of MerR. The cloned

merR gene from Bacillus [6] was expressed from the

bacteriophage T7 promoter on plasmid pBS�, via

temperature induction (at 428C) of T7 RNA polymer-

ase from plasmid pGP1-2, co-transformed into the

E. coli host. After 3 h induction, the cells were

sonicated and the MerR protein puri®ed by LC af®nity

chromatography followed by size exclusion chroma-

tography. Yields were low (less than 100 mg l per

culture) but 0.6 mg puri®ed protein was produced

for immobilisation.

The fusion protein GST-SmtA, containing glu-

tathione-S-transferase linked to the synechococcal

metallothionein protein, was overexpressed in E. coli

from an expression vector pGEX3X (Pharmacia) con-

taining smtA, and puri®ed using glutathione sepharose

4B by published methods [7].

2.5. Protein immobilisation and capacitance

measurements

The fusion proteins GST-SmtA and MerR were

produced as described above and dissolved in phos-

phate buffered saline (70 mM NaCl, 1.3 mM KCl,

5 mM Na2HPO4, 0.9 mM KH2PO4, pH 7.3) contain-

ing 50% glycerol to a ®nal concentration of 1 mg/ml

protein. Thioctic acid and glutaraldehyde (GA) were

purchased from Sigma and 1-(3-dimethylaminopro-

pyl)-3ethyl-carbodiimide hydrochloride (EDC) was

obtained from Fluka. 1-Dodecanethiol and the gold

rods used for the electrodes came from Aldrich. Heavy

metal salts CuCl2�2H20, ZnCl2, HgCl2 and

Cd(NO3)2�4H20 were from Merck (Darmstadt, Ger-

many). Polyethyleneglycol diglycidyl ether

(PEGDGE) was obtained from Polysciences (USA).

All other reagents were of analytical grade.

The biosensors were prepared by immobilising

fusion proteins on the gold surface by EDC-mediated

coupling, PEGDGE entrapment or GA cooling. In all

cases 20 ml of the dissolved fusion proteins were

diluted with 480 ml 100 mM borate buffer, pH 8.75

and the solution was ®ltered through a micro-®lter

(Amicon, USA) with a molecular cut-off of 3000 D.

After ultra®ltration, the fusion protein concentration

was adjusted to 0.04 mg/ml in borate buffer. Gold

electrodes were cleaned and pretreated with thioctic

acid, as described elsewhere [8].

The biosensor was arranged as the working elec-

trode in a three-electrode system connected to a fast

potentiostat. It was placed in a ¯ow cell with a dead

volume of 10 ml which was built in-house [8]. A

platinum foil served as the auxiliary and a platinum

wire as the reference electrode. An extra reference

electrode (Ag/AgCl) was placed in the outlet stream,

as the platinum does not have a de®ned potential. The

buffer solution was pumped by a peristaltic pump with

a ¯ow rate of 0.5 ml/min through the ¯ow cell. Sam-

ples were injected into the ¯ow via a 250 ml sample

loop. The buffer was 10 mM borate pH 8.75, ®ltered

through a 0.22 mm millipore ®lter and degassed before

use.

3. Results and discussion

3.1. Development of new genetic tools

A number of genetic tools and the background

knowledge necessary for the successful production

of metal-responsive strains have been developed. Sub-

stantial progress in understanding the molecular basis

of s-54 promoters as sensors of environmental pollu-

tants has been described earlier [9,10]. Understanding

the mechanism of activation of this regulator was

important in designing the gene-based sensors, which

are based on the activation of metal-induced promoter/

operator regions. The mechanisms of metalloregula-

tion of two promoters of E. coli based on the Fur

protein has also been dissected [11]. Finally, the

P. Corbisier et al. / Analytica Chimica Acta 387 (1999) 235±244 237

exploitation of the outer membrane protein LamB as

an anchor of heterologous metal-binding peptides

such as the yeast (CUP1) and mammalian (HMT-

1A) metallothioneins on the surface of Gram-negative

bacteria has been explored [12].

A. eutrophus was chosen as a reference organism

due to its ability to survive in harsh environments [13].

Four different metal-induced promoter cassettes were

constructed (Fig. 1). Promoters responsive to Cu2�,

Hg2�, Cr6�/3� and Pb2� ions were chosen to demon-

strate the feasibility of this approach.

The pMOL30 copper resistance operon from A.

eutrophus was located, sequenced and analysed

[14]. At least eight open reading frames were identi-

®ed and designated copSRABCDGF. The copSR genes

encode for a two-component regulatory system that

was needed as the copper responsive element [15].

The copABCD genes encode the structural copper

resistance genes similar to the pcoABCD and

copABCD genes of the E. coli and Pseudomonas

syringae, respectively [16,17]. In contrast to the E.

coli and P. syringae copper resistance operons, the

regulatory genes copSR are transcribed in the opposite

orientation of the structural genes copABCD. The

copF gene, transcribed in the opposite direction to

copABCD, encodes a Cu-ef¯ux ATPase similar to the

PacS protein of Synechococcus [18].

The copSRA promoter region of the A. eutrophus

CH34 cop operon and the mer regulatory region (merR

and mer promoter, PmerTPAD) of Tn501 were cloned in

S®I cassettes for the detection of copper and mercury

ions, respectively. Once cloned in A. eutrophus to

express the LamB [19] or the GFP [20] reporter

systems, concentrations of 1 mM Cu2� or 0.01 mM

HgCl2 should be easily detected.

When cloned in front of a Vibrio ®scheri luxCDABE

promoterless expression vector such as pMOL877

[21], the chrA promoter was mainly induced by

Cr6� compared to Cr3� ions as shown in Fig. 2. Ni,

Zn, Co, Al, Cd, Mn, AsO4, MoO4, WoO4, SeO3 and

SeO4 in their respective ionic forms did not induce any

bioluminescence and were not toxic up to 100 mM

(data not shown). The use of this fusion to assess the

bioavailability and toxicity of chromium in soil sam-

Fig. 1. Schematic organisation of the heavy metal-induced

promoter SfiI cassettes. (A) Copper promoter region from plasmid

pMOL30 from A. eutrophus CH34 in both orientations; (B)

mercury promoter region from transposon Tn501; (C) chromate

promoter region from A. eutrophus CH34 plasmid pMOL28; (D)

lead promoter region from plasmid pMOL30. Not all restriction

sites are shown.

Fig. 2. Bioluminescence induction of A. eutrophus AE2440

containing the chrB�chrA::luxCDABE fusion in the presence of

increasing concentration of (*) K2CrO4 and (~) CrCl3; (�) other

tested ions: Ni, Zn, Co, Al, Cd, Mn, AsO4, MoO4, WoO4, SeO3 and

SeO4. The bioluminescence, expressed as a signal to noise ratio,

was measured 3 h after induction in the reconstitution medium

(RM) with 0.1% gluconate as C-source medium.

238 P. Corbisier et al. / Analytica Chimica Acta 387 (1999) 235±244

ples has large potentials since it can easily differenti-

ate the trivalent from hexavalent chromium.

The lead resistance operon of A. eutrophus

pMOL30 plasmid has also been cloned and

sequenced. It contains two genes, pbrRA, required

for regulation and lead resistance, respectively. The

pbrR regulator was very similar to the MerR protein

(regulator of the mercury resistance operon). The

PbrA protein has all characteristics of an ef¯ux

ATPase. When cloned in into pMOL877, the pbrR

promoter appeared to be speci®cally induced by lead

ions as shown in Fig. 3. Those four different metal-

induced promoter cassettes can now be further

assembled in the LamB or GFP reporter systems.

The metal-induced bacterial biosensors [22] that

were already available have been further immobilised

in different solid matrices.

3.2. Immobilisation of the bacterial sensors

The bacterial Cu-sensor AE1239 [22] has been

immobilised in alginate and agarose gels and chal-

lenged to increasing concentrations of Cu2� ions in the

LB medium. In both immobilisation matrices, the

relative bioluminescent signals were perfectly linear

between 0 and 200 mM Cu2� and ®tted the linear

equations were y�1.88x�9.51 (R2�0.99) and

y�9.55x�221 (R2�0.99) for agarose and alginate

beads, respectively. As seen from Table 1, both immo-

bilisation matrices showed similar characteristics,

except that the relative signal was higher in agarose

gels. The lower sensitivity of the Cu-biosensor in

alginate was probably due to an overgrown bacterial

culture, since the optical density of the culture was

above the optimal value of 0.4±0.5. The detection limit

was similar to that obtained when cells were in solu-

tion, but remained too high for practical applications.

Therefore the reaction medium (RM) has been opti-

mised to provide suf®cient nutrient supply to the

bacterial cells and to avoid heavy-metal chelation

with the culture medium [4]. When the immobilised

cells were tested in this RM medium, the detection

limit could be reduced to 1 mM Cu and the signal was

kept linear up to 200 mM Cu according to the equation

y�14.93x�32 (R2�0.99) (see also Table 1).

Fig. 3. Specificity of the A. eutrophus CH34 containing the

pbrR�pbrA::luxCDABE fusion in the presence of increasing

concentration of metal ions. The bioluminescence, expressed as a

signal to noise ratio was measured 3 h after induction in the

reconstitution medium (RM) with 0.1% gluconate as C-source

medium.

Table 1

Characteristics of the whole cell Cu-biosensor AE1239 in solution and immobilised in alginate

AE1239 Absorbance

at ��600 nm

Relative

signal (S0.5/S0)

Detection

limit (mM)

Linear range

(mM)

Induction

time (h)

RSD 0.5 mM Cu

after 4 h (%)

Solution 0.34 16.1 20 up to 1000 1.5 19.5

LB/alginate 1.6a 6.96 20 up to 250 1.5 18.4

LB/agarose 0.7a 13.1 40 up to 250 1.5 6.48

RM/solution 0.10a 6.3 1 up to 25 1.5 0.2

RM/alginate 0.49a 11.6 1 up to 200 1.5 7.5

Measurements were performed in Luria-broth medium (LB) and in a mineral salts medium (0.1% acetate, 20 mM MOPS, pH 7) (RM).

RSD: relative standard deviation, the detection limit is calculated as twice the signal to noise ratio.a Calculated figures.

P. Corbisier et al. / Analytica Chimica Acta 387 (1999) 235±244 239

The storage stability of immobilised cells was

assessed by measuring the emitted bioluminescence

for six days (see Fig. 4(A) and (B)). Agarose discs lost

84% of their activity within six days, while alginate

beads were found to be stable. As seen from these

®gures the alginate immobilised cells displayed much

better stability than the cells immobilised in agarose.

3.3. Metal ion-specific capacity affinity sensors

Protein engineering has opened up the possibility of

designing and producing new proteins with, for exam-

ple, a higher selectivity than natural ones [23]. Also,

recent progress in the construction of thin self-

assembled monolayer (SAM)-forming molecules [8]

has allowed us to construct very sensitive and speci®c

metal ion capacity af®nity sensors. The reactive bio-

logical elements have been chosen from proteins

known to bind metal ions in a reversible manner.

The ®rst step for this technology was to overproduce

and purify proteins.

We have made the constructs for overexpression of

the broader-spectrum MerR from Bacillus [6]. Unlike

the Tn501 protein, which responds only to mercuric

ions, this protein also responds in vivo to the presence

of organomercurials and it is know that changes in the

very C-terminal amino acid sequence are responsible

for this speci®city [24]. This broader-spectrum MerR

protein from Bacillus, which responds to both mer-

curic ions and organomercurials, was overexpressed

and puri®ed by af®nity chromatography.

The fusion protein synechococcal GST-SmtA

metallothione protein has been produced by protein

engineering. The ®nal yield was 4 mg protein per l.

The Zn-binding characteristics of the fusion protein

were con®rmed using atomic adsorption spectroscopy.

The fusion protein also binds Cd, Cu and Hg, with pH

half dissociation values similar to those reported for

commercially available equine metallothionein [23].

Future studies can be done with other metal-binding

proteins from bacterial resistance determinants, such

as MerP and PbrR, which we have recently puri®ed

(unpublished data).

In order to develop a sensitive af®nity sensor based

on capacitance measurement, the immobilisation layer

has to be as thin as possible and well-ordered. First,

experiments were focused on ®nding the optimal

immobilisation procedure (EDC-coupling,

PEGDGE-entrapment and GA-crosslinking). So far,

EDC coupling has resulted in the highest sensitivities,

although experiments are planned with <10%

PEGDGE as well.

The effect of blocking the electrode surface with

insulators was studied. The importance of good insu-

lation of the electrode surface has been demonstrated

earlier [8]. The degree of insulation can be studied by

having a small permeable redox couple in solution.

Fig. 5 shows how the blocking increases for each

additional layer. For a clean gold surface the redox

couple K3[Fe(CN)6] was oxidised and reduced at the

metal surface. A surface covered with self-assembled

thiotic acid reduced access to the surface. Immobilisa-

tion of the GST-SmtA protein further insulated the

surface, but it was not until the treatment with 1-

Fig. 4. (A) Storage stability of immobilised AE1239 in (!)

alginate or (*) agarose matrices measured after 4 h incubation

with 0.5 mM Cu2�; (B) relative storage stability expressed in

percentage related to the initial bioluminescence value.

240 P. Corbisier et al. / Analytica Chimica Acta 387 (1999) 235±244

dodecanthiol that the oxidation/reduction peaks com-

pletely disappeared. The activity of the optimised

GST-SmtA electrodes was studied with copper, cad-

mium, mercury and zinc ions and gave a selectivity in

the named order (data not shown). These results are

consistent with the literature, which states that the

GST-SmtA protein has a broad selectivity towards

heavy metals [7].

Fig. 5. Cyclic voltammetry responses recorded in an Fe�CN�3ÿ6 solution when the measuring electrode was (a) unmodified gold, (b) gold

modified with thioctic acid, (c) as in (b) with additional modification with immobilised fusion protein GST-SmtA and (d) as in (c) with

additional modification with 1-dodecanethiol.

Fig. 6. Storage stability of the GST-SmtA electrodes. A calibration curve from 10ÿ15 to 10ÿ10 M Cu2� was recorded each day and the total

capacitance change vs. time was plotted. Between measurements the sensor was stored at 48C in 10ÿ1 M borate buffer 8.75.

P. Corbisier et al. / Analytica Chimica Acta 387 (1999) 235±244 241

The GST-SmtA electrodes could be regenerated by

injection of 1 mM EDTA. It was found that if EDTA

was injected just before storage, the biosensor lost

activity on overnight storage. However, if the elec-

trode was stored in the presence of heavy metal ions

and regenerated immediately before taking measure-

ments on the following day, no activity loss was

observed. This was probably due to the protein being

protected from denaturation (possibly oxidation) by

binding of the heavy metal to cysteine sulphydryl

groups. The stability of these electrodes over 16 days

is shown in Fig. 6.

The activity of the GST-SmtA electrodes was stu-

died for an extended concentration range (see Fig. 7).

As shown three distinct parts can be identi®ed, the ®rst

(up to 10ÿ5 M) being attributed to the capacitance

change upon relative non-speci®c binding of the heavy

metal to sulphydryl groups, the second (up to 10ÿ2 M)

Fig. 7. Capacitance change of the GST-SmtA electrode vs. Cu2� concentration in the range 10ÿ15±10ÿ1 M. The measurements were

performed in 10ÿ1 M borate buffer, pH 8.75 and a flow rate of 0.5 ml/min. Samples with a volume of 250 ml were injected.

Fig. 8. Capacitance change vs. heavy metal ion concentration for MerR immobilised electrode: (a) in the presence of Cd2�, (b) in the presence

of Cu2� and (c) in the presence of Hg2�. The measurements were performed in 100 mM borate, pH 8.75 and a flow rate of 0.5 ml/min.

Samples with a volume of 250 ml were injected.

242 P. Corbisier et al. / Analytica Chimica Acta 387 (1999) 235±244

probably due to the formation of a closed metallothio-

nein `cage' containing the metal ions, and the third due

to saturation (levelling off). Preliminary experiments

were also carried out with an MerR electrode, immo-

bilised by the same method found to be optimal for

GST-SmtA protein. This protein is known to be highly

speci®c towards Hg2� ions in vivo and in vitro [23].

The protein was immobilised on the sensor surface by

the EDC method. The sensitivity was studied for the

three different heavy metals; mercury, copper and

cadmium. It was found, as expected, that the sensi-

tivity was the highest for mercury, and that this

electrode showed higher selectivity than the GST-

SmtA electrode at low metal ion concentrations

(Fig. 8).

4. Conclusions

This work has shown the feasibility of technologies

for the detection of heavy metal ions based on whole

cell-biosensors and on protein-based sensors. A num-

ber of metal-induced promoter regions have been

identi®ed and arranged in cassettes that can be easily

used to activated reporter system such as the lux or

GFP reporter genes or the expression of outer mem-

brane epitopes that can be easily detected by immu-

nochemistry. The high speci®city of such an induced

gene expression has been shown for the lead and

chromate ions. Preliminary results for immobilised

whole cell-sensors have been obtained and demon-

strated the applicability of this technology.

Af®nity sensors based on proteins were also demon-

strated as suitable for monitoring heavy metal ions at

trace levels. The metal ion-speci®c capacitance sen-

sors have an exceptional sensitivity and a wide oper-

ating range. They are also versatile systems because

different kinds of recognition elements can be immo-

bilised directly in a self-assembling monolayer com-

pletely covering the surface of the measuring noble

metal electrode. The electrode then becomes selective

to those metal ions in the solution that show af®nity to

the recognition element on the surface. Compared to

previously described electrochemical sensors, the pro-

tein-based sensor shows many orders of magnitude

better sensitivity [25].

The whole cell-sensor and the protein-based sensor

now need to be tested on real environmental samples.

The main advantage of the whole cell-sensors will

remain their ability to react only to the bioavailable

fraction of metal ions, whereas the protein-based

sensor's potential application remains in its high

sensitivity towards metals ions.

Acknowledgements

We thank Professor Nigel Robinson (Newcastle) for

supplying the pGEX3X-smtA fusion plasmid. NB

thanks Kenneth J. Jakeman for technical assistance.

PC thanks S. Leth, S. Maltoni and A. Bossus for

technical assistance and L. Diels and M. Mergeay

for the constructive discussions. This work was sup-

ported by the European Commission as part of the

contract ENV4-CT95-0141.

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