Sensors 2008, 8, 2818-2832

sensors
ISSN 1424-8220
© 2008 by MDPI
www.mdpi.org/sensors

Full Research Paper

Assessment of Organophosphate and Carbamate Pesticide

Residues in Cigarette Tobacco with a Novel Cell Biosensor
Sophie Mavrikou 1,2, Kelly Flampouri 1,2, Georgia Moschopoulou 1,2, Olga Mangana 3,
Alexandros Michaelides 2 and Spiridon Kintzios 1,2,*

1 Laboratory of Plant Physiology, Faculty of Agricultural Biotechnology, Agricultural University of

Athens, Greece
2 EMBIO Diagnostics Project, Nicosia, Cyprus
3 Ministry of Agriculture, Centre of Athens Veterinary Institutions, Institute of Infectious and
Parasitic Diseases, Virus Laboratory, Athens, Greece

*Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel. +30 210 529 429;
Fax. +30 210 529 4286

Received: 29 January 2008 / Accepted: 22 April 2008 / Published: 23 April 2008

Abstract: The conventional analysis of pesticide residues in analytical commodities, such

as tobacco and tobacco products is a labor intensive procedure, since it is necessary to
cover a wide range of different chemicals, using a single procedure. Standard analysis
methods include extensive sample pretreatment (with solvent extraction and partitioning
phases) and determination by GC and HPLC to achieve the necessary selectivity and
sensitivity for the different classes of compounds under detection. As a consequence,
current methods of analysis provide a limited sample capacity. In the present study, we
report on the development of a novel cell biosensor for detecting organophosphate and
carbamate pesticide residues in tobacco. The sensor is based on neuroblastoma N2a cells
and the measurement of changes of the cell membrane potential, according to the working
principle of the Bioelectric Recognition Assay (BERA). The presence of pesticide residues
is detected by the degree of inhibition of acetylcholine esterase (AChE). The sensor
instantly responded to both the organophoshate pesticide chlorpyriphos and the carbamate
carbaryl in a concentration-dependent pattern, being able to detect one part per billion (1
ppb). Additionally, tobacco leaf samples (in blended dry form) were analyzed with both the
novel biosensor and conventional methods, according to a double-blind protocol. Pesticide
Sensors 2008, 8 2819

residues in tobacco samples caused a considerable cell membrane hyperpolarization to

neuroblastoma cells immobilized in the sensor, as indicated by the increase of the negative
sensor potential, which was clearly distinguishable from the sensor’s response against
pesticide-free control samples. The observed response was quite reproducible, with an
average variation of +5,6%. Fluorescence microscopy observations showed that treatment
of the cells with either chlorpyrifos or carbaryl was associated with increased [Ca2+]cyt .
The novel biosensor offers fresh perspectives for ultra-rapid, sensitive and low-cost
monitoring of pesticide residues in tobacco as well as other food and agricultural
commodities.

Keywords: Bioelectric Recognition Assay (BERA), carbamates, neuroblastoma,

organophosphates, tobacco.

1. Introduction

More than 130 different pesticides are used worldwide for the production of tobacco, with
approximately 25 compounds used on a regular basis. They belong to different chemical groups, such
as organophosphates, carbamates, organochlorine and heterocyclic pesticides, nitro compounds,
pyrethroids and amides. Many of these compounds can cause moderate to severe respiratory and
neurological damage or act as genotoxic and carcinogenic agents, therefore increasing the health risks
associated with smoking. Organophosphate (such as chlorpyrifos) and carbamate pesticides (such as
carbaryl) in particular, affect the nervous system by inhibiting acetylcholine esterase (AChE), an
enzyme that is important for the transmission of nerve impulses [1, 2]. Furthermore, compared to other
crops, tobacco leaves have more surface to weight ratio [3] and with excessive use of pesticides there is
always a chance for accumulation of pesticide residues in the leaf tissue.
The conventional analysis of pesticide residues in agricultural commodities is a labor-intensive
procedure, since it is necessary to cover a wide range of different chemicals, using a single procedure
[4]. Standard analysis methods include extensive sample pretreatment (with solvent extraction and
partitioning phases) and determination by gas chromatography (GC), high-pressure liquid
chromatography (HPLC) and mass spectrometry (MS) to achieve the necessary selectivity and
sensitivity for the different classes of compounds under detection [5]. As a consequence, current
methods of analysis provide a limited sample analysis capacity, on a day/instrument basis. Therefore,
rapid pesticide residue testing is necessary. In a region-specific pattern, this results to a general lack of
resources for implementation and enforcement of environmental, pesticide and labour regulations in
tobacco-producing countries.
A biosensor is a device that detects, transmits and records information regarding a physiological or
biochemical change. In recent years there has been a rapid increase in the number of diagnostic
applications based on biosensors, including live, intact cells and -in some cases- tissues, organs or
whole organisms. A cell-based sensor design employs the physiological responses of whole living cells
as the sensing component, namely oxygen consumption, surface chemical or electrical potential,
mobility or genetic activity [6].
Sensors 2008, 8 2820

A number of biological methods and protocols for toxicity measurement (including pesticide
residues) have been developed, based on microrganisms such as bacteria and yeast. The advantages of
toxicity bioassays include their low cost, easy detection protocol, reproducibility and rapid response. In
addition, bioassays offer direct toxicity information on tested chemicals or samples that analytical
methods cannot provide. Cairns and Mount [7] stated that chemical concentrations can be measured
with an instrument, but living organisms can measure toxicity only. The number of toxic compounds
that can be assayed by cell-based sensors is virtually endless. Current cell-based sensors for
environmental monitoring can detect heavy metals or their salts, organic compounds, various
carcinogens and pesticides such as diuron, atrazine, organotin compounds, organophosphates,
glyphosate and various antibiotics (for a review see [8, 9]). However, it must be mentioned that cell
sensors commonly are non-selective, not being able to discriminate between individual bioactive
compounds. Also, cell-based sensors must provide for an environment maintaining sufficient cell
viability (e.g. CO2-enriched culture atmosphere).
Commercial environmental cell biosensors utilize luminescence from natural or genetically
modified microbial species, whereas specificity of the response against an analyte is based on the
fusion of reporter genes, with a metabolic operon induced by its respective analyte. Bioluminescence-
based systems have been extensively used to monitor cellular stress responses, either by inducing the
response at low analyte concentrations or by inhibiting the luminescence at higher concentrations [5,
10-15].
Fluorescence-based systems are not always suitable for quantitative determinations. They are also
far more expensive than amperometric techniques [16]. Fluorescent dyes may also have considerable
side-effects on cellular physiology, such as uncoupling or respiratory inhibition and membrane
permeabilization [17, 18] and may interfere with the cell parameter that they assay [19].
Approached from different angles, electrically active cells can be interfaced with microelectrodes
which allow the capture of extracellular spikes or impedance changes associated with cellular or tissue
response. An example is the Bioelectric Recognition Assay (BERA) which has been originally
developed for the detection of viruses on the basis of their specific interaction with appropriately
immobilized, mammalian cells and the measurement of the change of the electric potential that is
caused by the aforementioned interaction [9, 20, 21].
In the present study, we developed a cellular biosensor based on the BERA working principle for
detecting organophosphate and carbamate pesticide residues in tobacco. The sensor is based on
neuroblastoma N2a cells. The presence of pesticidal compounds is detected by the degree of inhibition
of AChE, which is directly associated with changes of the immobilized cell membrane potential after
administration of acetylcholine (ACh).

2. Experimental Section

2.1. Materials

Mouse neuroblastoma (N2a) cell cultures were originally provided from LGC Promochem (UK).
Acetylcholine iodide (ACh) was purchased from Sigma Chem. Co. (USA). Fresh stock solutions of 14
mM S-acetylthiocholine-iodide (ATCi; Alfa Aesar Gmbh & Co KG) were made weekly in distilled
Sensors 2008, 8 2821

water and kept at 4°C in the dark. Dithiobisnitrobenzoic acid (DTNB (Ellman's reagent; Aldrich) was
used as an indicator for ATCi hydrolysis. Fresh stock solutions were made monthly in P-buffer (0.1 M
KH2PO4/K2HPO4; pH = 7,4) and kept at 4°C in the dark. Clorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-
pyridyl phosphorothioate; CAS [2921-88-2]; Mw = 350.59; DowAgroSciences, USA) was used as a
standard organophosphate insecticide and carbaryl (1-naphthyl methylcarbamate; CAS [63-25-2]; Mw
= 201.221 Bayer Cropscience, France) as a standard carbamate insecticide. Pesticide mixes which
contained 10 µM of each pesticide, were prepared daily in acetone solution. All other reagents were
purchased from Fluka (Switzerland).

2.2. Sensor fabrication from N2a cells

Cells were cultured in Dulbecco’s medium with 10% heat-inactivated foetal calf serum (FCS), 10%
antibiotics (streptomycin) and 10% l-glutamine. After cell detachment from the culture vessel by
adding trypsine/ EDTA for 10 min at 37◦C and cell concentration by centrifugation (6 min, 1200 rpm,
25 ◦C), 1 ml of cells (at a density of 2.5x106/ml) were mixed with 2ml of 4% (w/v) sodium alginate
solution and then the mixture was added drop wise, by means of a 22G syringe, in 0.8M CaCl2. Each
of the resulting calcium alginate beads had an approximate diameter of 2mm and contained
approximately 50x103 cells.

2.3. Sample preparation

Tobacco leaves (of the oriental cultivar Basmas) were kindly provided by the local tobacco
processing company SEKE S.A. in blended dry form. Leaves were derived from plants grown at
thirteen different locations in Northern Greece.
Control samples contained strictly organically-grown tobacco, which was free of pesticide residues.
Standard solutions of either chlorpyriphos or carbaryl were prepared by spiking control (pesticide-
free) tobacco samples with the respective pesticide at concentrations of 0.0001, 0.001, 0.01, 0.1, 1 and
2 ppm. This was done in order to avoid the so-called “matrix effect”, i.e. the interference of sample-
specific components on the sensor performance. In this way, the baseline (control) response of the
sensor would reflect the effect of plant constituents on the sensor.
In all samples the solvent used for extraction was water:acetone = 1:3 (v/v). A portion of ground
tobacco (4 g) was placed in a 60 mL conical vial after adding 40 ml of the extraction solvent. The
extract was placed under 37oC in a hot bath for 1 hour in order to allow the acetone evaporation. The
supernatant was then collected and transferred to 1.5 mL eppendorfs.
Each sample solution consisted of 50 µl tobacco extract and 10µl acetylcholine iodide.
Acetylcholine iodide (10 mM) was dissolved in a 50mM Tris-aminomethane buffer (Tris) of pH 8.

2.4. Assay principle

BERA sensors based on N2a cells were used for assaying the inhibition of cellular AChE to
hydrolyze Ach to acetate and choline according to the following equation:

AChE + Ach
Choline + acetate

Sensors 2008, 8 2822

According to the working principle of the method, the presence of organophosphate or carbamate
compounds is detected by the degree of inhibition of cellular AChE, which is inversely associated with
ACh concentration. ACh is an excitatory neurotransmitter [22], the activity of which is regulated by
AChE. Therefore, inhibition of AChE can lead in increased excitatory ACh transmission, which can be
measured by the depolarization of the cell membrane. In other words, inhibition of AChE by pesticide
residues in the sample will result to excessive stimulation of N2a cells by Ach, which will further lead
to membrane depolarization above a pre-determined threshold. Membrane depolarization events and
associated electrolyte influx/efflux will reflect themselves on the sensor’s response as a change of the
sensor’s potential, due to changes in the concentration of electrolytes in the immediate vicinity of the
working electrode.

2.5. Assay procedure.

Each cell-bearing bead (cell sensor) was connected to a working electrode made from pure silver,
electrochemically coated with an Ag/AgCl layer and having a diameter of 0.75 mm. Electrodes were
connected to the recording device, which comprised the PMD-1608FS A/D card (Measurement
Computing, Middleboro, MA). The software responsible for recording the signal and data processing
was InstaCal (Measurement Computing) (Fig. 1).

Figure 1. Schematic representation of the immobilized cell biosensor. The reference

electrode is inserted directly in the liquid sample, while the measuring electrode is
inserted in the immobilized cell-gel bead, which has an approximate diameter of two
mm. Measurement and reference electrodes are connected via wiring to the PMD 1608-
FS data converter. The displayed sensor response refers to a sample response containing
Ach (blue) and a sample containing Ach + chlorpyriphos as the AChE inhibitor (red).

For each assay, the sensor system, comprising of the bead attached to the working electrode and a
reference electrode, was immersed into each sample solution (200 µl). The response of each sensor was
estimated by recording the average change of the sensor potential for a period of 180 sec after sample
application.
Sensors 2008, 8 2823

2.6. Conventional sample analysis

Tobacco samples were also analyzed by GC and LC-MS/MS according to the International Standard
ISO 4389 [23] and by following the protocols of Djordjevic et al. [24] and Haib et al. [4].

2.7. Acetylcholinesterase inhibition assay.

In order to demonstrate that (a) AChE is functionally expressed in N2a cells and (b)
organophosphates and carbamates inhibit N2a-located AChE, we carried out AChE inhibition assays.
AChE activity was determined spectrophotometrically with the Ellman AChE assay [25], using
acetylthiocholine as the substrate. To prepare the cells for the AChE assay, 1 ml of P-buffer (0.1M
sodium phosphate, 1% Triton X-100, pH 7.4) was added to a 15 ml sterile Cellstar® tube containing
the cell pellet (approximately 7,5 × 106 cells) and the mixture was slowly triturated until the pellet was
resuspended. All experiments were performed in P-buffer. S-acetylthiocholine-iodide (ATCi) was used
as a substrate to determine specific cholinesterase activity. ATCi hydrolysis was measured as an
increase in OD405 (50 s time intervals) caused by the reaction of thiocholine with DTNB to produce the
yellow 5-thio-2-nitro-benzoic acid anion [25]. Specific activity is reported as nmol substrate
hydrolyzed/min/mg protein. The protein content was determined by the Bradford method using bovine
serum albumin as the standard [26]. The spectrophotometric assays were carried out with a
spectrophotometer (UV-160A SHIMADZU) at a wavelength of 595nm.

2.8. Assay of pesticide-cell interaction

Changes in cytoplasmic Ca2+ concentration in N2a cells before and after the addition of an
organophosphate (chlorpyrifos) or a carbamate (carbaryl), each at two different concentrations (0.03 or
0.3 µM) were monitored by the uptake of the acetomethyl ester of Fluo3 [27]. After application of five
µl of the dye, the fluorescence of the specimens was recorded for five minutes at 10 s intervals. Slides
with stained cells were mounted on a Zeiss Axiolab fluorescent microscope equipped with a BP-546
excitation filter and an FT-580 chromatic beam splitter. A digital camera (SONY S75 digital still
camera) was attached to the microscope with adjustable BP-546/FT-580 excitation filter/chromatic
beam splitter combinations. In order to control photobleaching, we kept specimen exposure times at a
minimum level. No significant alteration of the intensity of the fluorescence was observed during the
observation of the specimens.

2.9. Experimental design

Both biosensor and conventional sample analysis were conducted according to a double-blind
protocol. Experiments were set up in a completely randomized design and each experiment was
repeated three times. In each application, a set of five biosensors was tested against each individual
sample. Data means among different days were compared using Duncan’s multiple range test (with
significance at p<0.05).
Sensors 2008, 8 2824

Figure 2. Biosensor response against standard solutions containing carbaryl (A) or

chlorpyriphos (B) at various concentrations. Sensor response is expressed as a change in
the membrane potential of immobilized cells. (n = 15 replications (different sensors) for
each sample and error bars represent standard errors of the average value of all
replications with each sample). Columns marked with different letters indicate
statistically different values.
Sensors 2008, 8 2825

3. Results

3.1. Response of the sensor to standard pesticide solutions

The results of the assay of standard pesticide solutions at different concentrations with the BERA
neuroblastoma biosensors are shown in Figure 2. When no pesticide was present in the sample (control
sample), a constant sensor response of -0,019 ± 0.002 mV was observed. The sensors responded to
carbaryl by considerable negative increase of the sensor’s potential (Figure 2A). This response was
concentration-depended in the range of 100 ppt to 1 ppm (r2=0,7432), so that both the qualitative and
the quantitative detection of the pesticide was possible, at least at concentrations lower or closer to the
Minimum Residue Level (MRL) (1,5 ppm). A similar, concentration-dependent pattern (r2=0,8558)
was observed in the sensor’s response against chlorpyriphos (Figure 2B), although the sensor’s
response did not change at the 0,0001-0,01 ppm range. The biosensor response against either pesticide
was quite reproducible, with 8,6/7,8 % variation from the average in the response observed against
carbaryl/chlorpyriphos, respectively.

Figure 3. Biosensor response against thirteen different tobacco leaf samples containing
chlorpyriphos and carbaryl at various concentrations. Samples have been ranked in the
order of ascending cumulative pesticide residue concentration. Sensor response is
expressed as a change in the membrane potential of immobilized cells. (n = 15
replications for each samples and error bars represent standard errors of the average
value of all replications with each sample). Samples 12 and 13, marked with pink,
contain pesticide residues at concentrations equal or above the official Minimal Residue
Levels.
0
0 0,25 0,27 0,27 0,31 0,49 0,53 0,79 1,01 1,08 1,31 1,56 1,87 2,96

-0,02
Biosensor response (V)

-0,04

-0,06

-0,08

-0,1

-0,12 Cumulative pesticide residue concentration (ppm)

Sensors 2008, 8 2826

3.2. Residue profile by conventional analysis of tobacco leaf samples

The organophosphate chlorpyrifos and the carbamate carbaryl were detected by conventional
analysis in the thirteen tobacco leaf samples. The concentrations of each pesticide residue in the
individual samples are presented in Table 1, while the cumulative pesticide residue concentration in
each sample is presented in the x axis of Figure 3. The biosensor response against each sample is also
presented in the same figure. Pesticide residues in tobacco samples caused a considerable cell
membrane hyperpolarization on neuroblastoma cells immobilized in the sensor, as indicated by the
increase of the negative sensor potential, which was clearly distinguishable from the sensor’s response
against pesticide-free control samples. There were no false-negative measurements. The observed
response was quite reproducible, with an average variation of +5,6%. In spite of the observed
correlation, the biosensor-based assay can presently be considered only qualitative.

Table 1. Pesticide residue concentration (ppm) in each assayed tobacco leaf sample.

Sample
Compound 1 2 3 4 5 6 7 8 9 10 11 12 13
chlorpyrifos 0,04 0,07 0,06 0,05 0,06 0,05 0,04 0,16 0,21 0,15 0,14 0,36 0,27
Carbaryl 0,23 0,42 0,21 0,48 0,25 0,2 0,97 1,4 1,1 0,64 0,94 2,60 1,6

Figure 4. Results of the acetylcholinesterase inhibition assay. AChE activity in N2a

cells was reduced after the addition of carbaryl (pink) or chlorpyriphos (yellow),
compared to control cells (blue).
0,35
y = 0,00104x
Specific AChE activity (nmol substrate

2 y = 0,00100x
R = 0,97554 2
0,3 R = 0,97292
hydrolyzed/min/mg protein)

0,25
y = 0,00093x
2
R = 0,98032
0,2

0,15

0,1

0,05

0
0 50 100 150 200 250 300 350
Time (s)
Sensors 2008, 8 2827

Figure 5. Changes (expressed as differences in fluorescence intensity) of the

cytoplasmic calcium ion concentration before (A) and after (B, C, D, E) treatment of
N2a cells with chlorpyrifos or carbaryl (A) Untreated cells (control) (B, C) Cells treated
with 0.03 or 0.3 µM chlorpyrifos, respectively (D, E) treated with 0.03 or 0.3 µM
carbaryl, respectively.
Sensors 2008, 8 2828

3.3. Acetylcholinesterase inhibition assay.

As presented in Figure 4, AChE activity in N2a cells was reduced after the addition of carbaryl or
chlorpyriphos, compared to control cells. Therefore, it was clearly demonstrated that functional AChE
units exist on the surface of N2a cells, which can be inhibited by either pesticide. The inhibition was
greater in the case of carbaryl.

3.4. Effect of pesticides on calcium uptake by N2a cells

Neuroblastoma cells responded to the presence of either chlorpyrifos or carbaryl by a rapid and very
considerable increase of their intracellular Ca2+ stores (Fig. 5), compared to untreated cells. However,
the intensity of this effect was not depended on the type or the concentration of the pesticide.

4. Discussion

The operating principle of a cellular biosensor is entirely different from conventional methods for
pesticide residue detection: cell sensors respond to different molecules according to their bioactivity
rather than their structure or physical chemical properties (as assayed, for instance, by mass
spectrometry or chromatography) [6, 8]. Additionally, biosensors rarely provide a full spectrum of the
compounds present in a sample [1, 5, 24]. One of the traits that make cell biosensors attractive as a
clinical analytical tool is their considerable sensitivity, which is assumed to enable, in some cases, the
possibility to detect just a single target molecule [13, 14]. This theoretical assumption is based on the
intricate interaction, which has emerged as a result of relentless trial-and-error testing through
countless millennia and allows cells to respond to individual molecules and organisms in rather precise
and reproducible ways. This ability, however, comes at a cost, or, rather, a compromise: because cells
can react in roughly the same manner against an amazingly large number of different molecules, cell
sensors can exhibit a very poor selectivity. This is a very common problem in toxicity assays, where
cell sensors successfully detect the presence of a toxic (or genotoxic) compound, but generally fail to
determine the exact nature of the toxic analyte [10, 11, 28].
Despite this, the present biosensor system offers a number of distinct performance characteristics for
pesticide residue detection, including:
• Ultra-high speed: an assay can be completed in less than 3 min.
• High sensitivity, with the ability to detect pesticides at concentrations as low as 1 ppb (for
carbaryl).
• High reproducibility.
• Low cost, not exceeding 30-50 € per assay batch.
• The ability to monitor at real-time conditions.
• Finally, the ability to detect toxicity with complex, “real world” mixtures of toxicants and
interfering compounds.

From a practical point of view, these traits could turn the novel biosensor into an attractive future
technology for routine, field-based pesticide assays. Providing novel solutions for food quality
assurance can be a very critical issue, especially in view of the new EU [29] and international
Sensors 2008, 8 2829

regulations for minimal residue concentration in marketed food and agricultural products. The
development of the novel sensor concept is still at a preliminary stage: this means that further
experiments are required in order to define the linear range of the response and the possible
interference of different pesticides on the selectivity of the sensor. In the same context, it may be worth
mentioning that, within the framework of the present study, standard solutions of pesticides for
calibrating the novel biosensor were prepared by spiking residue-free tobacco, in order to avoid the so-
called “matrix effect”, i.e. the interference of sample-specific components on the sensor performance.
Different samples, different types of extract, different commodities and different “concentrations” of
matrix may exhibit matrix effects of different magnitudes. Matrix effects are variable in occurrence and
intensity and calibration should be matrix-matched routinely [30]. For example, when we prepared
pesticide standard solutions by directly dissolving carbaryl or chlorpyriphos in a solvent, rather than by
spiking them in tobacco, an entirely different pattern of sensor response to either compound was
observed (analytical results not shown).
According to the working hypothesis of our method, inhibition of AChE by either pesticide may
have caused excessive stimulation of N2a cells by Ach, thus resulting to membrane depolarization
[22]. The considerably increased concentration of intracellular Ca2+ stores after treating neuroblastoma
cells with the pesticides provides an indication that this hypothesis may be true, although it is yet
uncertain whether calcium ions are the sole contributors to the observed depolarization effect. In
various cell types (including neural and cancer cells) membrane polarization events are associated with
other stress-induced changes, such as an increase in cytoplasmic free calcium [31]. Whelan and Zare
[27] have previously shown that receptor-like interactions between molecules on the cell surface and
target analytes resulted in a detectable change in the concentration of cytosolic Ca2+, while an
association between conformational changes of electroinserted SOD molecules, calcium ion efflux and
cell membrane potential has been demonstrated by Moschopoulou and Kintzios [32]. In this way, cells
with a particular type of surface-bound receptor serve as recognition platforms for homologous target
molecules, whereas the translation of the recognition reaction into potential changes makes the assay
much more rapid (and, in some cases, more sensitive) than any conventional methods.
Further increase in the sensor sensitivity could arise by increasing the concentration of immobilized
cells per sensor, whereas the selectivity of the sensor’s response against different pesticides could be
increased by developing genetically engineered cell lines expressing different AChE isoforms on their
surface. In addition, the utilization of artificial neural network technology [33] for rapid, automated
pattern recognition of the sensor’s response could transform the method into a powerful, high-
throughput analytical tool. These approaches are being currently tested by our research group.

Acknowledgements

The research project was funded by the EMBIO Project of the Cypriot Ministry of Industry,
Commerce and Tourism. The authors wish to thank Mrs. Angela-Lucy Petrou (Hellenic Ministry of
Foreign Affairs) for proof-reading the manuscript.
Sensors 2008, 8 2830

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