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Bioluminescent Biosensors Based on

Genetically Engineered Living Cells in
Environmental and Food Analysis
a a a a a
E. Michelini , M. Guardigli , M. Magliulo , M. Mirasoli , Prof. A. Roda ,
b c
P. Simoni & M. Baraldini
a
Department of Pharmaceutical Sciences , University of Bologna , Bologna,
Italy
b
Department of Internal Medicine and Gastroenterology , University of
Bologna , Bologna, Italy
c
Department of Metal Sciences, Electrochemistry and Chemical
Techniques , University of Bologna , Bologna, Italy
Published online: 23 Aug 2007.

To cite this article: E. Michelini , M. Guardigli , M. Magliulo , M. Mirasoli , Prof. A. Roda , P. Simoni & M.
Baraldini (2006) Bioluminescent Biosensors Based on Genetically Engineered Living Cells in Environmental and
Food Analysis, Analytical Letters, 39:8, 1503-1515, DOI: 10.1080/00032710600713156

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 Analytical Letters, 39: 1503–1515, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 0003-2719 print/1532-236X online
DOI: 10.1080/00032710600713156

Bioluminescent Biosensors Based

Downloaded by [Universidad Nacional Colombia] at 11:32 15 August 2014

on Genetically Engineered Living Cells

in Environmental and Food Analysis

E. Michelini, M. Guardigli, M. Magliulo, M. Mirasoli, and

A. Roda
Department of Pharmaceutical Sciences, University of Bologna,
Bologna, Italy

P. Simoni
Department of Internal Medicine and Gastroenterology, University of
Bologna, Bologna, Italy

M. Baraldini
Department of Metal Sciences, Electrochemistry and Chemical
Techniques, University of Bologna, Bologna, Italy

Abstract: The development of cost-effective on-site analytical methods is an urgent

need to monitor food safety and detect environmental pollution. Whole-cell biosensors
have shown the potential to complement both laboratory-based and on-field analytical
methods for the detection of general stress conditions, cyto- and genotoxic compounds,
organic xenobiotics and metals, as well as endocrine disrupting compounds.
The sensitive and rapid detection of biochemiluminescence, combined with the
specificity or selectivity of gene regulation in living cells, offers significant advantages
over conventional analytical methods, providing data on the bioavailability and bio-
logical activity of contaminants; this information is very valuable for risk assessment
purposes the identification of suitable remediation systems. Moreover, complex
pretreatment of the environmental sample, as in usual chemical analysis techniques,
is often not necessary for biosensor measurements.

Received 15 December 2005; accepted 20 February 2006

Address correspondence to Prof. A. Roda, Department of Pharmaceutical Sciences,
University of Bologna, Via Belmeloro 6, Bologna 40126, Italy; Tel./Fax: þ39 051
343398; E-mail: [email protected]

1503
 1504 E. Michelini et al.

Keywords: Whole-cell biosensor, bioluminescence, food analysis, heavy metals,

endocrine disruptors

INTRODUCTION

A variety of whole cell –based biosensors was developed using numerous

native and genetically engineered cells (bacteria, yeasts, or mammalian
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cells), the latter representing the real advancement in bioluminescent (BL)

whole-cell biosensors (Gu 2004).
The development of genetically engineered cells led to new low cost,
rapid, and sensitive analytical tools for environmental, medical, and food
analysis.
Diverse BL whole-cell biosensors were obtained by genetically engineer-
ing cells with a reporter protein, whose expression is controlled by regulatory
proteins and promoter sequences (Table 1). In most cases, an existing regulat-
ory system in the bacterial cell is exploited to drive expression of a specific
reporter gene, such as bacterial luciferase, green fluorescent protein, beta-
galactosidase, or others. This is achieved by fusing the DNA for a promoter-
less reporter gene to an extra copy of the selected regulatable promoter and
introducing this construction into the bacterial cell.
The regulatory protein is able to recognize the presence of a specific
analyte or a class of compounds and consequently to activate the expression
of the reporter protein. The reporter protein can then be readily measured
and directly related to the analyte bioavailable concentration in the sample
(Fig. 1). Regulatory systems that have been applied include those for heavy
metal resistances (to obtain heavy metal responsive biosensors), for organic
compound degradation (to obtain organic compound sensors), and for
cellular stress responses (to obtain general toxicity sensors). An important
feature of cell-based assays is that they are suitable to automation for the
development of high-throughput screening procedures. Although whole-cell
biosensors do not always fulfill all the ideal biosensor characteristics, such
as high sensitivity, selectivity, and long-term stability, their main feature is
the ability to measure the bioavailable fraction of a pollutant (i.e., the fraction
able to enter live cells and activate specific response pathways) rather than
its total concentration, providing information that is of particular interest
in environmental and toxicological studies. In addition these bioreporter
systems are cost-effective if compared to conventional methods such as
liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-
mass spectrometry (GC-MS).
However, a major limitation encountered in the use of these sensing
systems is that other factors influence the biosensor signal, including matrix
components, temperature, and pH, leading to high interassay variability and
poor robustness. These stimuli do not directly induce a generation of signal,
but they may affect cell metabolism. As a result, the modulation of the
 Bioluminescent Biosensors in Environmental and Food Analysis
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Table 1. Selected BL biosensors using firefly luciferase (luc) or bacterial luciferase (lux) as luminescent reporter genes

Host strain Reporter gene Analyte Detection limit Reference

E. coli luc Mercury, organomercurials 0.1 f M Virta et al. 1995

S. aureus luc Cadmium, lead, antimony 10 nM (Cd), 33 nM (Pb), 1 nM (Sb) Tauriainen et al. 1998
R. eutropha luc Cobalt, nickel 0.1 mM (Ni) 9 mM (Co) Tibazarwa et al. 2001
A. eutrophus luxCDABE Copper 1 mM Leth et al. 2002
P. putida luxCDABE Naphthalene, salicylate 0.35 mM Heitzer et al. 1994
E. coli luc, luxCDABE Aromatic compounds 10 mM Kobatake et al. 1995;
Kim et al. 2005
E. coli luxAB Alkanes 24 nM Sticher et al. 1997
E. coli luxCDABE Tetracyclines 44 nM Korpela et al. 1998
Human and mouse luc, lux Polyhalogenated aromatic 2.4 pM (TCDD), 0.5 fM (TCDD) Sanderson et al. 1996;
hepatoma cells, hydrocarbons (PAHs) Murk et al. 1996
breast cancer cells
S. cerevisiae luc Androgenic and estrogenic 0.05 nM dihydrotestosterone Michelini et al., 2005a
compounds (DHT) 0.03 nM (E2) Leskinen et al. 2005

1505
 1506 E. Michelini et al.
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Figure 1. Schematic view of a BL whole-cell biosensor showing the steps involved in

gene expression and signaling.

reporter protein expression is the result not only of the specific interaction with
the analyte but also of the overall cell vitality and metabolic activity.
The choice of reporter proteins includes several luminescent ones:
BL reporter proteins (such as bacterial and firefly luciferases, aequorin. . .)
provide the best performance in terms of detectability (attomole amounts of
enzyme can be detected) and broad linear range of the signal, whereas fluor-
escent reporters such as green fluorescent protein (GFP) provide superior
spatial resolution of gene expression but less sensitivity (Roda 2004).
Each reporter protein has particular characteristics that affect its suit-
ability for a given application. For example, a biosensor based on bacterial
luciferase can glow without adding a substrate. Cells can be transformed
by inserting the luxCDABE gene cassette, which encodes for both bacterial
luciferase and the enzymes involved in the synthesis of its substrate.
Also firefly luciferase, which emits yellow-green (557 nm) light, and the
corresponding cDNA have been used successfully as a BL reporter of gene
expression in bacterial or mammalian cells, but they require external
addition of substrates.
The firefly luciferase protein (Luc) catalyzes the formation of luciferyl-
adenylate (LH2-AMP) from substrates luciferin (LH2) and ATP [Eq. (1)].
Through a multi-step oxidative process, LH2-AMP is converted to excited
state oxyluciferin, the light-emitting product [Eqs. (2) and (3)].
Mg2þ
 Luc  LH2  AMP þ PPi
Luc þ LH2 þ ATP ! ð1Þ

Luc  LH2  AMP þ O2 ! Luc  AMP  Oxyluciferin þ CO2 ð2Þ

Luc  AMP  Oxyluciferin ! Luc þ Oxyluciferin þ AMP þ hy ð3Þ

Since the generation of light from LH2 is highly efficient (Seliger and McElroy
1960) affording great sensitivity for the detection of the luciferase protein, the
 Bioluminescent Biosensors in Environmental and Food Analysis 1507

luciferase gene is extremely suitable for reporter gene applications (Gould and
Subramini 1988) and in vivo BL imaging. In eukaryotic cells such as yeasts,
insect luciferase is transported into peroxisomes due to peroxisomal targeting
signal Ser – Lys – Leu (skl) at the C-terminus of the luciferase. To avoid
accumulation of the luciferase into the peroxisomes during cell growth a luci-
ferase truncated version lacking the peroxisome-targeting codons was
produced and used as reporter protein in yeast and mammalian cell biosensors
(Leskinen et al. 2003).
In particular, the ease of cultivation and genetic manipulation of the yeast
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Saccharomyces cerevisiae are among the properties that made it a suitable

and flexible organism for a wide range of applications as a simple eukaryotic
bioreporter system (Baronian 2004).

Whole-Cell Biosensors for Heavy Metals

Heavy metals are ubiquitous in nature and constitute a potential environmental

threat, even at low concentrations because they are nonbiodegradable. Due
to their high toxicity, ultrasensitive analytical methods are required to
detect them at trace levels in environmental, clinical, and food samples
(Verma and Singh 2005). In particular mercury, chromium, cadmium,
copper, and zinc are the most frequently observed metal contaminants and
conventional techniques to analyze them, such as ultraviolet (UV)-visible
spectrophotometry and cold vapor atomic absorption spectrometry, are very
expensive, need trained personnel, and do not allow in situ analysis (Brown
et al. 1998).
As an alternative, biosensors are now being utilized for monitoring heavy
metal contamination in environmental and food samples (Leth et al. 2002).
A number of bacterial biosensors were developed to detect arsenic,
antimony, cadmium, lead, chromium, copper, mercury, zinc, tin, nickel, and
cobalt using specific regulatory proteins that interact with the analyte giving
detection limits as low as 10212 – 10215 M and adequate accuracy and
precision (Verma and Singh 2005). These biosensors are specific to one or a
few metals and may employ mechanisms of resistance including efflux of
the metal, sequestration of the metal, or both (Brown et al. 1998).
A luminescent biosensor was constructed by a gene fusion between a
regulatory region of the mer operon and firefly luciferase to quantitatively
detect Hg2þ (Virta et al. 1995; Ivask et al. 2001). The biosensor showed a
linear relationship between the light signal intensity and the Hg2þ concen-
tration in the range of 1.7  10213 –1.7  1027 mol/L, with a detection
limit of 1.7  10213 mol/L, corresponding to 4.0  10218 mol/tube. The
system proved to be precise, accurate, and highly selective for Hg2þ, with
no light emission induced by other heavy metals. It was used to analyze the
mercury urine samples from subjects with or without dental amalgam restor-
ations to evaluate the release from dental amalgams. There is, in fact, evidence
 1508 E. Michelini et al.

that mercury vapor is slowly released from amalgam fillings and absorbed by
the lungs and, at high levels, this highly toxic heavy metal may damage brain,
kidneys, and the developing fetus. Urinary mercury levels in subjects with
amalgam fillings were slightly significantly higher than those without
amalgam fillings (p , 0.1) (Roda et al. 2001).
In addition, by using 384-well microtiter plates and a CCD-based lumi-
nescence imaging detector a high analytical throughput (more than 5000
samples/h) was achieved, making the assay suitable for screening studies.
A panel of whole-cell biosensors for diverse heavy metals (mercury,
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copper, chromium, zinc, et al.) was also used to analyze sediments and
other environmental samples. The sample analysis was carried out either
directly or after a few pre-analytical steps in 96- or 384-well microtiter
formats, thus being suitable for high throughput screening.

Mammalian Cell Based Biosensor for Polyhalogenated Aromatic

Compounds

Mouse hepatoma cells, which naturally express the aromatic hydrocarbon

receptor (AhR), were transformed by introducing a plasmid bearing the
dioxin responsive gene sequences and the luc reporter gene encoding firefly
luciferase. The biosensor showed a detection limit of 5  10212 mol/L
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), corresponding to 2.5  10215
mol/well. It responded also to other dioxin-like chemicals, even if with
higher detection limits, and to mixtures of different compounds (Murk et al.
1996).
This biosensor, together with bacterial biosensors for heavy metals were
employed to detect heavy metals and dioxin-like compounds in sediments
from Tam Giang-Cau Hai lagoon (Vietnam). During the war in Vietnam
(1961 – 1971) large amounts of the herbicide Agent Orange were used in the
Tam Giang-Cau Hai lagoon area. Only later was it found that this herbicide
contained dioxins, particularly TCDD, as impurities and various severe path-
ologies related to dioxins exposition were diagnosed in the population.
In order to determine the actual degree of dioxin pollution, superficial
samples were collected in 20 sampling locations and 25 sediment cores
from 2 locations, corresponding to the northern and southern areas of the
lagoon.
In particular, the highest dioxin-like compounds concentration values
were found at a depth level that, by taking into account the sediment depo-
sition speed, approximately corresponds to 35– 40 years ago, thus confirming
the release of such compounds into the environment during the Vietnam
war (Fig. 2). The detected values are in agreements with dioxin analysis on
the same sediment samples carried out by HRGC-HRMS (Frignani et al.
2004).
 Bioluminescent Biosensors in Environmental and Food Analysis 1509
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Figure 2. Depth distribution of dioxins in a sampling core of the Tam Giang-Cau Hai
lagoon (Vietnam) as evaluated by the dioxin bioassay.

Yeast-Based Biosensors for Endocrine Disrupting Compounds

Many endocrine disrupting compounds (EDCs), which are molecules able to

interact with hormone receptors with adverse health effects, are continuously
introduced into the environment and need to be monitored (Rodriguez-Mozaz
et al. 2004). Such compounds are, in fact, able to mimic or antagonize the
effects of endogenous hormones such as estrogens and androgens and they
have shown involvement in sex differentiation and reproductive abnormalities
in wildlife, human population and laboratory animals, also determining an
increasing incidence of cancers.
Environmental EDCs are persistent pollutants, thus remaining intact in
the environment for many years, and are subject to bioaccumulation, i.e.,
they can accumulate in the fat tissues of animals and humans. These EDCs
have been found in human blood and breast milk, in marine mammals, and
in marine and freshwater fish; and their levels are continuously increasing
(Guillette et al. 1994; Hotchkiss et al. 2002).
We investigated the applicability of whole-cell biosensors in monitoring
of EDCs by testing samples spiked with known estrogen- and androgen-like
compounds and then we analyzed aqueous samples collected from influents
and effluents of activated sludge sewage treatment plants. Androgenic and
estrogenic activity were monitored in municipal effluents before and after
treatment in sewage treatment plants in the cities of Rome, Florence,
 1510 E. Michelini et al.

Parma, and Bologna; and endocrine activities were appreciably lower after
progression of wastewater through the plants.
We used a luminescent whole-cell biosensor, based on bioengineered
Saccharomyces cerevisiae cells expressing the estrogen receptor and contain-
ing the reporter gene b-gal, which expression being regulated by an estrogen
responsive element (ERE) sequence. These cells synthesize the enzyme
b-galactosidase only when the estrogen receptor is activated by any EDC
present in the sample and are, therefore, suitable for a direct first level screen-
ing of the samples. The b-gal activity was detected using a chemiluminescent
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1,2-dioxetane-b-D-galactopyranoside substrate, which allowed achievement

of a detection limit of 1  10211 mol/L estradiol.
In addition, we have developed a rapid androgen bioassay based on
recombinant yeast cells genetically modified to express human androgen
receptor (hAR) (Michelini et al. 2005a; Leskinen et al. 2005). The
biosensor is based on recombinant S. cerevisiae cells, which express hAR
and contain the sequence androgen response element (ARE) that drives the
expression of the reporter gene luciferase from Photinus pyralis. In the
presence of androgenic compounds, hAR moves into the nucleus and binds
ARE sequences, resulting in luciferase expression, whose activity is
measured by the BL emitted after addition of D-luciferin. The same yeast
strain with a stably expressed luciferase was used as an external control to
take into account variations in cell vitality due to matrix and excess analyte
toxicity. The recombinant assay requires only 2 h 30 min of incubation time
of the cell suspension with 10 mL of sample or the analyte in solution
(Michelini et al. 2005b). Luminescence measurements are then performed
by simple addition of D-luciferin without cell lysis or washing steps thus
improving the assay analytical performance. The yeast biosensor showed a
limit of detection of 0.05 nM for testosterone, with a dynamic range from
0.05 to 1000 nM and the assay is accurate and precise.
In addition, the biosensors proved suitable to measure the hormonal
activity of baby food samples in order to assess the safety of infant
nutrition sources, an important issue recently raised by the National
Research Council.

BRET Whole-cell Bioassays

As an alternative to BL biosensors, new bioassays have been explored based

on bioluminescence resonance energy transfer (BRET) (Boute et al. 2002;
Eidne et al. 2002). As shown in Figure 3B, BRET is a non-radiative energy
transfer, occurring between a luminescent donor and a fluorescent acceptor
that strictly depends on the closeness between the two partners and,
therefore, can be used for studying protein-protein interactions (Issad et al.
2002). An homogeneous assay to evaluate the presence of estrogen-like
compounds has been developed and optimized based on a estrogen receptor
homo-dimerization (Michelini et al. 2004).
 Bioluminescent Biosensors in Environmental and Food Analysis 1511
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Figure 3. New perspectives in BL biosensors. A. Custom-designed microtiter plate

that contains 24 main wells, each divided into 7 subwells (Roda et al. 2005).
B. Schematic view of the BRET technique applied to the study of protein-protein inter-
action. C. Schematic drawing of a bioreporter integrated system (Nivens et al. 2004).

We cloned ERa coding sequence in frame with either a variant of the

green fluorescent protein (enhanced yellow fluorescent protein, EYFP) as
acceptor or Renilla luciferase (Rluc) as donor. Upon ligand induced ERa
homo-dimerization, an energy transfer occurs from Rluc to EYFP, leading
to EYFP fluorescence emission at its characteristic wavelength. Based on
this energy transfer, a quantitative ‘in vivo’ homogeneous assay in HepG2
cells was developed and validated. The developed assay fulfills all the
standard requirements of accuracy and precision with a limit of detection of
as low as 1 nM of 17-b estradiol in a 96-well microplate format, which is as
good as that of analog bioassays, together with the peculiar advantages of
homogeneous assays and short incubation times. The BRET assay, therefore,
could be suitable for high-throughput screening of a large number of compounds
like drugs or environmental chemicals suspected to be xenoestrogens and as a
first level screening of environmental samples.

New Perspectives in Bioluminescent Biosensors

The BL whole-cell biosensors take advantage of the sensitive and rapid

detection of biochemiluminescence coupled with the specificity or selectivity
of gene regulation, originating efficient bioanalytical methods (van der Meer
 1512 E. Michelini et al.

et al. 2004). Unfortunately, up to now, whole-cell biosensors have not been

frequently applied outside research laboratories, although the trend is
growing. Now the main challenge is to move from laboratory studies to
real-life monitoring situations and to develop portable systems suitable for
field testing. The attractiveness of BL reporter genes for remote whole-cell
biosensors originates from the fact that microorganisms use biochemical
energy to originate the signal without any external source of energy, and
they are amenable for the development of integrated formats.
Nivens et al. (2004) reported a system, shown in Fig. 3C, that interfaces a
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BL biosensor with an integrated circuit able to detect, process, and record

the signal produced in response to the analyte. This prototype chip-based
environmental biosensor is based on bioengineered bacteria that express the
luxCDABE gene and glow blue-green in the presence of contaminants such
as ammonia and zinc with possible applications ranging from spacecraft to
antiterrorism (Nivens, 2004).
One of the major concerns about in situ biosensing is the cell suscepti-
bility to changes in external conditions or in the sample matrix composition
that may occur in the field.
Cells containing the reporter system must face field-contaminated
samples, usually consisting of complex mixtures of different compounds,
and the ability to respond to the presence of a specific compound is influenced
by the physiological state of the cell.
The use of two reporter genes that are independently expressed and can be
independently measured within the same cell is raising exciting new perspec-
tives and has been already explored using fluorescent proteins. For example,
a dual-reporter fluorescent biosensor with an internal response correction
system was developed (Mirasoli et al. 2002). Two fluorescent reporter
proteins with different emission spectra were used: one reporter provided
the analytical signal, whereas the second was used as an internal reference.
Improved reagents which identify Photinus pyralis luciferase mutants
that efficiently emit red bioluminescence, were developed. In this way, the
proven advantages of the P. pyralis protein can be combined with the
potential advantages of a red-shifted emitter. Using site-directed mutagenesis
techniques, some mutants emitting red bioluminescence were obtained
(Branchini et al. 2005) with potential application in multiplexed bioassays
and biosensors with vitality internal control.
The recent availability of new BL probes that emit at different wave-
lengths, such as new reporter genes or luciferase mutants, could effectively
allow the introduction of a second BL reporter gene as an vitality internal
control. The use of different BL proteins allows the development of multi-
plexed systems for different analytes; alternatively multiplexed detection
can be achieved using new formats relying on the spatial resolution of BL
signal.
For example, we have developed a custom-designed microtiter plate that
contains 24 main wells, each divided into 7 subwells (Fig. 3A) that has been
 Bioluminescent Biosensors in Environmental and Food Analysis 1513

successfully used to immobilize oligonucleotide probes and bacteria in order

to perform chemiluminescent multianalyte PCR ELISA binding assays for
typing human Papillomavirus (HPV) (Roda et al. 2005) and multiplexed
assays for heavy metals (unpublished results). In this context, cell immobiliz-
ation in suitable media in order to keep them alive and functional for long-
term storage will undoubtedly represent a fieldwork challenge, which in
perspective will lead to simple, low-cost, rapid and, possibly, in situ multi-
plexed analysis.
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