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Biosensors & Bioelectronics 16 (2001) 467– 480

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The application of the bioelectric recognition assay for the


detection of human and plant viruses: definition of operational
parameters
S. Kintzios a,*, E. Pistola a, J. Konstas a, F. Bem b, Th. Matakiadis a,
N. Alexandropoulos c, I. Biselis d, R. Levin e
a
Laboratory of Plant Physiology, Agricultural Uni6ersity of Athens, 75 Iera Odos, 11855 Athens, Greece
b
Benaki Phytophathological Institute, 14561 Athens, Greece
c
Microbiology Department, Hippokration Hospital, 11527 Athens, Greece
d
Laboratory of Animal Husbandry, Agricultural Uni6ersity of Athens, 11855 Athens, Greece
e
Osmotek Ltd., PO Box 550, Kiryat Weizmann, Reho6ot 76120, Israel

Abstract

The bioelectric recognition assay (BERA) is a novel biosensory method based on a unique combination of a group of cells, their
immobilization in a matrix that preserves their physiological functions and the expression of the cell interaction with viruses as
a change in electrical properties. A BERA sensor consists of an electroconductive, tube-like probe containing components of
immobilized cells in a gel matrix. Cells are selected to specifically interact with the virus under detection. In this way, when a
positive sample is added to the probe, a characteristic, ‘signature-like’ change in electrical potential occurs upon contact between
the virus and the gel matrix. In the present study, we demonstrate that BERA can be used for the detection of viruses in humans
(hepatitis C virus) and plants (tobacco and cucumber viruses) in a remarkably specific, rapid (1 – 2 min), reproducible and
cost-efficient fashion. The sensitivity of the virus detection with BERA (0.1 ng) is equal or even better than with advanced
immunological, cytological and molecular techniques, such as the reverse transcription polymerase chain reaction. Moreover, a
good storability of the sensors can be achieved without affecting their performance. The potential use of portable BERA
biosensors in medicine, for mass screening purposes, as well as for the detection of biological warfare agents without prior
knowledge of a specific receptor-molecule interaction is discussed. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Biosensor; Cell immobilization; Electrophysiological response; Virus; Hepatitis C; Plant pathogenic

1. Introduction quinone (Rawson et al., 1989; Marty et al., 1998).


Cell-based biosensors are likely to have improved sta-
A biosensor can be defined as a device incorporating bility, higher biocatalytic activity and low cost in their
a biological sensing element connected to a transducer favour; however, they have poor selectivity and can
(Eggins, 1996). In recent years there has been a rapid suffer from long recovery times compared to enzymic
increase in the number of diagnostic applications based biosensors. In addition, the need for biosensors with a
on biosensors including live, intact cells. Cell-based shelf life of weeks or months precludes the use of fresh
biosensors used for the detection of environmental pol- cells requiring time-consuming harvesting and
lutants (commonly herbicides), are usually based on the preparation.
immobilization of microorganisms on a Clark oxygen Cellular responses to various compounds under de-
electrode or they include an amperometric detection of tection can be evaluated by measuring the cellular
an electron mediator such as ferrycyanide or p-benzo- electric properties, in particular the electric potential,
which reflects changes of a network of inter-related
* Corresponding author. Tel.: + 30-1-529-4292/202-7041; fax: + metabolic reactions. The interior of animal and plant
30-1-529-4286.
E-mail address: skin@aua.gr, spiroskintzios@usa.net (S. Kintzios). cells is electrically negative with respect to the exterior.

0956-5663/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 1 6 1 - 0
468 S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480

The magnitude of this potential difference is generally ing electric signals produced thereof (Koch et al., 1989;
between 5 and 90 mV (in some cases 100– 500 mV) Schutz et al., 1999; Schroth et al., 1999).
(Tsong and Astumian, 1988), with most of the potential However, the measurement of the membrane poten-
being developed across the membrane (Shapiro, 1982). tial, membrane conductance and membrane electromo-
Thus, the cell membrane is polarized between its layers, tive force across a cell surface is usually complicated,
having a nominal capacitance of 1 mF/cm2 and a nomi- due the zoning effects and the ‘cable property’ (Ogata
nal resistance of 104 Siemens/cm2. Rapid changes in the et al., 1983; Smith, 1983), i.e. when current is applied
electrical potential and ion flux across cellular mem- via external electrodes on a cell membrane, an apprecia-
branes occur within minutes of specific ligand binding ble error in the measurement of the change in mem-
to transmembrane receptors (Jaffe, 1976). Iwata et al. brane voltage occurs, due to the effect of additional
(1999) measured membrane currents using the whole- membrane-area specific conductance effects. The paral-
cell patch clamp technique to study effects of rabies lel impedance of the uncovered electrode area and the
virus infection on ion channels in mouse neuroblastoma impedance of cell– cell contact areas also interfere with
cells. By using the same technique, Wang et al. (1994) the accurate measurement of an individual cell’s
demonstrated that the influenza A virus M2 protein impedance characteristics. By using secondary screens,
had an ion channel activity in mammalian cells. In several hours or days may be required for electrophysi-
addition, potentials can result from changes of the lipid ologically assaying compounds interacting with integral
composition of the cell membrane in response to membrane proteins, such as receptors and ion channels.
changes in the proximate cellular environment (‘home- It would, therefore, be desirable to develop a system
ophasic adaptation responses’) (Vigh et al., 1998). In for assaying the cell potential not on an isolated mem-
the absence of any interaction, cells retain a rest poten- brane region but rather as a result of processes associ-
tial, i.e. a time average potential that is sustained by ated with the whole cellular metabolism and preferably
metabolic energy through ion pumps (e.g. H+ -AT- on a tissue level. We have previously reported (Kintzios
Pases) or electron transport chains. et al., 2000) on the development of a novel method for
Membrane interactions are commonly studied in the qualitative and/or quantitative detection of biologi-
their electrophysiological aspect by patch clamp tech- cal and chemical compounds on the basis of their
niques, which rely on the fact that the ionic flow across specific interaction with appropriately immobilized, vi-
a cell membrane can be measured as an electrical able cells and the measurement of the change of the
current if the membrane potential is held constant electric potential that is caused by the aforementioned
(Orwar and Jardemark, 1998; Zhang and Hamill, 2000). interaction. For this purpose, we constructed a biosen-
A cell or part of the cell is firmly attached by suction to sor comprising of an electroconductive gel matrix with
the tip of a glass microelectrode, and a highly sensitive a cellular sensory material (the input transducer) and an
feedback current-to-voltage converter measures sub-pi- appropriate microelectrode/data acquisition system (the
coampere currents. Shapiro (1982) developed a method output transducer). In the present report we demon-
for detecting changes in the membrane potential of strate that the bioelectric recognition assay (BERA) can
individual cells, after incubation with a membrane-per- be used for the qualitative and quantitative detection of
meant, fluorescent ionic dye that becomes distributed viruses in humans (hepatitis C virus) and plants (to-
on opposite sides of the cell membrane as a function of bacco and cucumber viruses) in a remarkably specific
membrane potential. Rosenthal and Shapiro (1983) and reproducible fashion. The sensitivity of the virus
used this method in order to rapidly assay the binding detection with BERA is equal or even better than with
of Epstein-Barr virus on receptor-bearing B advanced immunological, cytological and molecular
lymphocytes. They observed that changes in the mem- techniques, such as the polymerase chain reaction
brane potential differed in time course and direction (PCR), which have also been used in our study.
with respect to the cell’s capacity to internalize the virus
and the cell type. Kovacs and Borkholder (1998)
demonstrated the effective use of electrodes small 2. Experimental
enough (10 mM diameter) to study single cell/electrode
coupling and monitored cellular viability and position 2.1. Biosensor system and working principle
by impedance measurement. Since the immobilization
of a single neuron to the gate of a FET by Fromherz et Each biosensor comprised of cells immobilized in a
al. (1991) (which allowed for the first direct bioelec- matrix of appropriate material and in such a way that
tronic signal transfer) biosensors carrying olfactory re- the viability and functional integrity of the sensory
ceptor proteins have been used for odor detection as material was preserved, along with its specific mode of
electronic noses. Some recent applications rely on the interaction with the agent(s) under detection. The cellu-
use of whole, intact organs (in particular insect anten- lar sensory material was selected to (i) respond specifi-
nae) for the detection of volatile compounds by record- cally to the compound under detection, and (ii) express
S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480 469

this specific response via changes of the electric recording device, which comprised an Advantec PCL-
potential. 711TM PC I/O card. The Analog-to-Digital Converter
Cells and electrodes were arranged in the sensor in (ADC) of this computer card was recording the signal
the main configuration, which is schematically repre- (voltage). The ADC was a 16-bit, 5-scale unipolar/bipo-
sented in Figs. 1 and 2. The sensor consisted of an lar converter. To the shortest scale (+ /− 325 mV
electroconductive, tube-like probe made of polypropy- bipolar voltage) the ADC can accomplish an accuracy
lene (4×0.8 cm2) and containing components of immo- of 0.01 mV (accuracy= range of measurement (325×
bilized cells in a gel matrix. A matrix volume of 1.28 ml 2+ 1)/216 (bit analysis of the ADC)=0.009918 or 0.01
was obtained. A 50 ml syringe was used for applying the mV). The software responsible for the recording of the
sample. The measuring electrode laid in the area of signal and processing of data was a modified version of
sample application (top of the probe, 3 mm below the the Advantec GenieTM v3.0.
matrix fill line) while the reference electrode laid in the At the beginning of each assay, and prior to sample
opposite position (3 mm from the bottom of the probe). application, a minor, non-zero potential exists between
The electrodes were made from pure silver, electro- the electrodes, possibly due to differences in the cellular
chemically coated with an AgCl layer, having a diame- microenvironment (for example, immobilized cells at
ter of 0.2 mm and were inserted into the tube for 7 mm. the are of the measurement electrode are in closer
Both electrodes were connected via coaxial cable to the contact with air, thus an increased respiration can be

Fig. 1. (a) Main outline of the BERA-biosensor configuration. (b) Working principle of the sensor: (A) Before sample application (B) After
application of a positive sample (Vo, sensor rest potential, V(t), biosensor response as a function of time).
470 S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480

Fig. 2. (a) Equivalent electric circuit of the BERA matrix-cell sensor system. 1, the measuring electrode; 2, the reference electrode, Gi,j,
conductance and Ci,j, capacitance of each immobilized cell (i= 1…n and j = 1…m), R, resistance of the matrix (gel and pores with solution). (b)
Outline of dual biosensor system for cancelling out the effects of various properties of the sample solution.

observed). In addition, the data acquisition circuit is the moment of sample application to the top of the
configured in such a way that the reference electrode is probe (Fig. 1(b), point A), the compounds under detec-
maintained at a virtual ground potential. In theory, at tion will interact with the part of the sensory cellular
S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480 471

material in the area of the measuring electrode, causing rpm, 25 °C), cells (at a density of 4× 106/ml) were
a change of its electric properties. At the same time, mixed with a 1% (w/v) low melting point agarose
however, the part of the sensory cellular in the area of solution in PBS-Dulbecco’s medium. This was done at
the reference electrode will retain the initial value of 37 °C in order to avoid agarose solidification. The
these properties (rest potential). In this way, an electric cell-agarose mixture was transferred into an appropri-
potential will be created between the two electrodes. As ately configured box bearing Ag/AgCl electrodes, as
the sample constituents will progressively move towards described in Section 2.1.
the bottom, they will react with increasingly more parts
of the cellular material so that a change in electrical 2.2.2. Detection of plant pathogenic 6iruses
potential is occurring over time until a new ‘steady-
state’ value is obtained (Fig. 1(b), point B). 2.2.2.1. Tobacco biosensors. Biosensors were con-
From another point of view, the BERA matrix-cell structed under sterile conditions by immobilizing proto-
sensor system can be considered as a series of n cell/ma- plasts of the tobacco (Nicotiana tabacum L.) cultivar
trix layers acting both as bipolars and as capacitors ‘Samsun’, which is susceptible to Tobacco rattle 6irus
whereas the potential varies in relation to the total (TRV) but resistant against Cucumber green mottle
impedance between the measuring electrode (Fig. 2(a), mosaic 6irus (CGMMV).
point 1) and the reference electrode (Fig. 2(a), point 2). Protoplasts were isolated from tobacco leaves by
Each immobilized cell has a conductance Gi,j and a preplasmolysing 0.5 g of them in 20 ml of CPW solu-
capacitance Ci,j (where i =1…n and j= 1…m) depend- tion (Reinert and Yeoman, 1982) supplemented with
ing on its position within the probe. In addition, and in 0.7 M mannitol for 1 h and then incubating them in 20
particular at higher gel densities, there is an additional ml solution of the same composition and additionally
resistance R of the matrix (gel and pores with solution). supplemented with 3 mg pectinase (8.5 units/mg, from
Thus, the sensor resembles an RC circuit consisting of Aspergilus niger) and 2 mg cellulase (9.5 units/mg, from
a group of n capacitors serially connected to each other: Trichoderma 6iridae) for 20 h. One microlitre of proto-
upon sample application, the initially reacting part of plast solution (at a density of 6× 106 cells/ml) was
the sensor (the first cell/matrix layers or ‘capacitors’) is centrifuged at 14.000 rpm (20 min, 25 °C) and the
electrically charged until a certain maximum value is pellet was resolved and mixed with a 1% (w/v) low
achieved. The equivalent capacitative time constant of melting point agarose solution in water. This was done
the sensor is characteristic and specific for each bio- at 37 °C in order to avoid agarose solidification. The
chemical agent. Thus, a given virus demonstrates a cell-agarose mixture was transferred into an appropri-
unique pattern of biosensor response over a definite ately configured box bearing Ag/AgCl electrodes, as
range of concentrations, like a ‘signature’. From a described in Section 2.1.
practical point of 6iew, the response of the biosensor In addition, and in order to study the effect of
should be plotted against a series of dilutions of the immobilized cell concentration on the sensor response,
sample under investigation. A second biosensor bearing tobacco biosensors bearing either 3× 106 or 6 ×106
cellular material with a different sensitivity should be cells/ml were constructed and used in the virus assay.
used in order to cancel out the possible presence of
other compounds demonstrating similar response pat- 2.2.2.2. Cucumber biosensors. According to the proce-
terns. In similar fashion, the effects of various proper- dure described above, a set of biosensors was con-
ties of the sample solution, such as pH, conductivity structed by immobilizing protoplasts (at a density of
and ionic strength, on the sensor response were can- 3× 106 cells/ml) of the cucumber (Cucumis sati6us L.)
celled out by using a suitable reference solution in a cultivar ‘20202-G’, which is susceptible against both
dual biosensor system (Fig. 2(b)). It is obvious, how- viruses (CGMMV and TRV) used in this study.
ever, that results can vary with minor changes in the
biosensor configuration, e.g. the distance between the 2.3. Assay
two electrodes.
2.3.1. Detection of the hepatitis C 6irus
2.2. Procedure Twenty microlitre of negative controls (30 individual
HCV-free blood samples) or HCV-positive sample (30
2.2.1. Detection of the hepatitis C 6irus individual clinical blood samples) were applied. Sam-
Biosensors were constructed under sterile conditions ples were clinically obtained from patients at the Hip-
by immobilizing liver cells (a porcine cell line) in vitro pokration Hospital (Athens, Greece) without revealing
selected to have a specific response against the hepatitis their identity prior to assaying. In some cases, control
C virus (HCV). After cell detachment from the culture and sample blood was infected with other kind of
vessel by adding trypsine/EDTA for 10 min at 37 °C viruses, such as the hepatitis B virus (analytical data
and cell concentration by centrifugation (6 min, 1200 not shown). The samples were assayed for HCV with
472 S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480

reverse transcription polymerase chain reaction (RT- well as with ELISA and PCR, as described in follow-
PCR), as essentially described by Garcia et al. (2000). ing: F(ab%)2-ELISA tests for CGMMV were done as
described by Ploeg et al. (1992) using specific antiserum
2.3.2. Effect of an interferon (Betaferon ®) on the against the PL104 isolate of the virus. Total plant RNA
biosensor response was isolated according to Verwoerd et al. (1989). RT-
In an additional experiment, 20 ml of an interferon PCR for CGMMV was performed essentially as de-
solution (Betaferon, Schering AG) (29 million units/mg) scribed by MacFarlane (1996).
has been administered to liver cell-biosensors 30 min
after the application of blood samples from infected 2.3.3.4. Cytological assays. Membrane potential
patients and the sensor response was measured. changes were monitored by the cellular uptake of a
Betaferon is a genetically engineered, modified human lipophilic cationic fluorochrome, the distribution of
interferon beta with significant antiviral activity which is affected by the potential difference across the
(Anonymous, 1995). Prior to application, the protein cell membrane, as described by Rosenthal and Shapiro
(0.75% w/v) was diluted in a 0.54% (w/v) sodium (1983). Briefly, plant (tobacco and cucumber) proto-
chloride solution. plasts were incubated in 3 ml of PBS buffer (pH 7.4,
106 cells/ml) and stained with 3 mg of 3,3 dipropylthiad-
2.3.3. Detection of plant pathogenic 6iruses icarbocyanide iodide. Samples were analysed before
and after the addition of 40 ml buffer containing various
2.3.3.1. Virus purification. Virus isolates were purified quantities of either CGMMV or TRV (6.6, 13.3, 20 and
from infected Nicotiana tabacum cv. Samsun leaves (a 60 ng), after excitation at 580 nm. Fluorescence emis-
Greek isolate of TRV) or infected cucumber (Cucumis sion at 688–690 nm was measured with a Jasco FP-920
sati6us) leaves (the watermelon isolate PL104 of CG- Fluorescence detector.
MMV), as described previously (Lister and Bracker,
1969; Tung and Knight, 1972; Bem, 1987; Bem and
2.4. Storage of the biosensors
Vassilakos, 1996; Brown et al., 1996).
In order to test the effect of storage conditions on the
2.3.3.2. Sample application. (a) Tobacco sensors: For the
performance of BERA biosensors, the following experi-
quantitative determination of the CGMMV or TRV
ments were carried out:
virus in each assay, gradually increasing virus concen-
(a) Biosensors which have been prepared as described
trations (40 ml of buffer containing 1, 3, 6.6, 9, 13.3, 20,
above (Sections 2.2.1 and 2.2.2) were stored for 2
30, 40, 60, 70, 80, 90 and 100 ng purified virus) and the
months at −5 °C and then used for virus assays in
control solution (phosphate buffer pH 7.4) were
exactly the same manner as in Sections 2.3.1 and 2.3.3.
applied.
(b) Biosensors were also prepared as described above
In addition, we tested the response of the biosensors
(Sections 2.2.1 and 2.2.2) but cells were immobilized in
against samples containing both CGMMV and TRV
a modified procedure, which involved covalent bonding
viruses, at a quantity of either 9 or 20 ng.
of membrane fragments to the gel matrix (analytical
(b) Cucumber sensors: For the quantitative determi-
protocol not described). The biosensors were stored for
nation of the CGMMV virus in each assay, gradually
2 months at room temperature and then used for virus
increasing virus concentrations (40 ml of buffer contain-
assays in exactly the same manner as in Sections 2.3.1
ing 1, 3, 6.6, 9 and 13.3 ng purified virus) and the
and 2.3.3.
control solution (phosphate buffer pH 7.4) were
applied.
(c) Effect of immobilized cell density: In order to 2.5. Chemicals
study the effect of immobilized cell concentration on
the tobacco sensor response, biosensors bearing either All solvents and chemicals used were of analytical
3 × 106, 6×106 or 12 ×106 cells/ml were tested against quality (from Sigma Company, St. Louis). Water was
CGMMV or TRV virus solutions (with virus quantities double distilled.
of 6.6, 13.3, 20 and 60 ng)
2.6. Experimental design
2.3.3.3. Assay of crude sap extracts. Crude sap extracts
were prepared from cucumber plants infected with CG- Experiments were set up in a randomized complete
MMV by homogenizing leaves in phosphate buffer (pH block design and each experiment was repeated three
7.2) (1 g leaf tissue: 10 ml buffer). Infected leaves were times. In each application, a set of 15 biosensors was
removed from plants at 2, 3, 4, 6 and 11 days after tested against each individual sample. Different series
inoculation. The extracts were assayed for CGMMV of measurements were compared by means of Student’s
with tobacco sensors prepared as in Section 2.2.2.1, as t-test.
S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480 473

3. Results and (b). There was a significant and fully reproducible


difference between the liver BERA sensor response to
3.1. Detection of the hepatitis C 6irus the HCV-positive samples and the control, even if
control and/or sample blood were infected with other
The results of the detection are presented in Fig. 3(a) kinds of viruses (Fig. 3(a)). The virus elicited a devia

Fig. 3. (a) Results of the detection of the HCV in a human blood sample using a BERA-sensor based on porcine liver cells. The original recording
of the biosensor system is presented. The arrow indicates the moment of the application of the virus. Dashed line indicates biosensor response to
HCV-negative sample (control). (b) Comparative response of a BERA-sensor based on porcine liver cells against the HCV in a human blood
sample before and after the treatment of the sensor with Betaferon®. (1, control (non-infected sample); 2, HCV-infected sample; 3, HCV-infected
sensor treatment with Betaferon®; 4, control treatment with Betaferon®). Variations in the response against the infected samples (bar 2) may be
due to quantitative differences between samples (mean values, 15 biosensors, n =45).
474 S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480

Fig. 4. Results of the response of a tobacco BERA biosensor against the TRV virus (3 ng). The original recording of the biosensor system is
presented. The arrow indicates the moment of the application of the virus.

tion of the biosensor potential from the average rest response than the non-pathogenic strain CGMMV,
value of +4 (control) to + 9 mV (sample) (Fig. 3(b), while at concentrations higher than 0.5 ng/ml this effect
bar 2). was reversed. In every assay, measurements taken by 45
different biosensors did not vary more than 0.00001%
3.2. Effect of an interferon (Betaferon ®) on the of the average response i.e. independent measurements
biosensor response of a virus concentration were virtually identical. Inter-
estingly, at 0.5 ng/ml both viruses caused a very low
On the contrary, the addition of Betaferon® to biosensor response (Fig. 5(a)). Therefore, the response
biosensors previously (30 min) treated with HCV-posi- did not increase with virus concentration in a linear
tive samples caused a reduction of the rest potential to fashion. If, however, recorded data were logarithmically
− 6 mV (Fig. 3(b), bar 3), possibly associated with (− 1/log − 1) transformed, then a negative linear corre-
inhibition of virus replication. The addition of lation could be observed between the sensor response
Betaferon® to non-infected biosensors caused an in- and virus quantities in the range of 1–70 ng (r 2 = −
crease of the potential to 12 mV, indicating an elicita- 0.97132, TRV) or 50–100 ng (r 2 = − 0.9492, CG-
tion of cellular response similar to viral infection (Fig. MMV). However, when we tested the biosensors
3(b), bar 4). Application of a non-functional placebo against samples containing both CGMMV and TRV
(0.54% sodium chloride solution) did not have any viruses (at a quantity of either 9 or 20 ng) the observed
effect. response was virtually identical to the response against
only TRV, which is 6irulent on ‘Samsun’ tobacco (ana-
3.3. Detection of plant pathogenic 6iruses lytical results are not shown).
Under the experimental conditions described in the
3.3.1. Tobacco sensors present study, a detection limit of  0.01 ng/ml was
A remarkable difference was observed between the achieved for both viruses.
sensor response to different viruses and the control.
The biosensor response to each virus solution was 3.3.2. Cucumber sensors
estimated as the difference of the sensor potential be- The cucumber protoplast biosensor response against
fore and after sample application (Fig. 4). For both TRV (i.e. the difference of the sensor potential before
viruses, this response generally declined with increasing and after sample application) generally declined with
virus concentration. However, at low concentrations, increasing virus quantities (Fig. 5(b)), however, a mini-
the tobacco pathogenic strain TRV elicited a higher mum was observed at 6.66 ng. A similar minimum,
S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480 475

within the quantity range of 1– 13.3 ng has been ob- 3.3.4. Assay of crude sap extracts
served in the response of the tobacco sensor against The time course study of cucumber plants inoculated
TRV (Fig. 5(a)). As for the tobacco sensors, the vari- with CGMMV was conducted on a daily basis for a
ability of virus detection with BERA did not exceed total period of 24 days after inoculation.
10 − 5% of the median value i.e. independent measure- (a) By assaying the crude sap extract of cucumber
ments of a virus concentration were virtually identical. plants infected with CGMMV with tobacco BERA
biosensors we have been able to detect the presence of
3.3.3. Effect of immobilized tobacco cell density
the virus even on the second day after inoculation.
Increasing the density of immobilized tobacco proto-
(b) Virus was detected by F(ab%)2-ELISA only after
plasts from 3 to 12 million cells/ml caused varying
changes of the biosensor response against CGMMV or the sixteenth day after inoculation.
TRV (Fig. 6(a) and (b)). Therefore, no linear relation- (c) No virus was detected by RT-PCR in RNA
ship between cell density and the biosensor response extracted from leaves of inoculated plants, sampled in
against either virus could be observed. days 2, 3, 4, 6 and 11 after inoculation. It is possible

Fig. 5. Dose-response curve of (a) the tobacco biosensor to the CGMMV virus (non-virulent on tobacco) or the TRV virus (virulent) (b) the
cucumber biosensor to the CGMMV virus (virulent on cucumber) (mean values, 15 biosensors, n = 45). Error bars represent a variability in
response B0.00001%.
476 S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480

Fig. 6. Dose-response curve of the tobacco biosensor to (a) the CGMMV virus (non-virulent on tobacco) (b) the TRV virus (virulent on tobacco)
in relationship to different immobilized cell densities (open boxes: 3 ×106 cells/ml, shaded boxes: 6 × 106 cells/ml, solid boxes: 12 × 106 cells/ml)
(mean values, 15 biosensors, n=45).

that virus concentration during this period was below the cell membrane potential. For cucumber, this change
the detection threshold of the method. Alternatively, never exceeded 15% of the control value (cells untreated
this could be due to a possible uneven distribution of with virus). Increasing CGMMV quantity from 6.6 to
the virus in the plant. 13.3 ng caused a slight increase of the potential, but
higher virus concentrations did not (Fig. 7(a)). Tobacco
3.4. Assay of plant cell membrane potential with a cell membrane potential fluctuated with virus concen-
fluorescent dye trations, however, an increasing trend was observed
towards higher concentrations (Fig. 7(b)). Any
The addition of either CGMMV or TRV virus to a change, however, did not exceed 1125% of the control
plant protoplast solution caused an instant change of values.
S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480 477

3.5. Effect of storage on biosensor performance 4. Discussion

All assays were also carried out with biosensors In the present study, we investigated the potential
which either (a) contained viable cells and were stored application of the BERA biosensory system for the
for 2 months at −5 °C or (b) contained covalently detection of viruses in humans (HCV) and plants (CG-
bonded cells and were stored for 2 months at room MMV, TRV). For this purpose, BERA sensors were
temperature. In every case, the observed pattern of constructed consisting of an electroconductive probe
response was identical to the pattern obtained with containing immobilized cells in an agarose matrix. Cells
freshly prepared sensors (analytical results not shown). were selected to specifically interact with the virus
Therefore, although the actual half-life of the biosen- under detection. When a positive sample was added to
sors has not been estimated, their storage in either way the probe, a change in electrical potential was measured
did not affect the replicability of measurements. upon contact between the virus and the gel matrix.

Fig. 7. Changes of the membrane potential (expressed as relative fluorescence) of (a) tobacco and (b) cucumber protoplasts incubated in a
fluorescent dye (3,3 dipropylthiadicarbocyanide iodide) before and after the addition of CGMMV or TRV virus. Increasing treatment numbers
(1 –4) indicate increasing virus quantities (6.6, 13.3, 20 and 60 ng).
478 S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480

Although based on a rather simple concept, BERA histochemical methods). However, the time required for
could be used in a remarkably specific and reproducible antibody production may be considerably long (e.g.
fashion. The sensitivity of the virus detection with several months), during which infection will remain
BERA is equal to advanced techniques, such as the undetected. For example, detection of the HCV in
PCR, which have also been used in the present study. blood by means of an antibody assay is not possible
The results of the assay with a fluorescent dye demon- before 15–24 weeks have elapsed since infection (Aach
strated that rapid changes of the plant cell potential are et al., 1991). In addition, identification is particularly
indeed associated with virus interactions. In contrast to troublesome because available antibodies mainly bind
BERA, however, this method was impractical and not to non-specific regions on the protein envelope of the
suitable for quantitative determinations. virus. The application of such assays is further limited
There already exist a considerable number of biosen- by the variability due to differences in reagent specific-
sory methods based on electrophysiological effects (see ity between different manufacturers or testing laborato-
the Section 1 for more details). Some of them use cells, ries, as well as the non-specific elevation of antibodies
or assay electric properties, or use immobilized compo- due to other causes (e.g. chronic liver disease, liver
nents. However, no other method has been previously cirrhosis and hepatocellular carcinoma) (Anonymous,
reported to combine these three elements. In addition, 1989; Houghton et al., 1991). The amount of HCV
most of the electrophysiologically operating biosensors genome in infected patients is too low to be detected by
include microelectrodes in direct contact with a single conventional hybridization techniques and the applica-
cell, which makes the setup impractical and expensive. tion of more advanced techniques such as RT-PCR
Contrary to those methods, a BERA sensor comprises (Dash et al., 2000) is necessary. The sensitivity of
electrodes that are inserted into a matrix bearing a RT-PCR is : 0.1 ng per assay (Markoulatos et al.,
group of cells. In this way, the sensor represents, more 1999; Dash et al., 2000), which is equal to the virus titer
or less, the natural tissue where the interaction takes detectable by BERA in our experiments. However,
place. Although BERA is still in the stage of develop- commercially available PCR tests are usually expensive
ment, we feel that the results are encouraging and this (which prohibits their use on mass sample screening)
approach is advantageous or can efficiently comple- and the method often requires separate laboratory
ment immunological or molecular methods, since: space and equipment in order to avoid sample contam-
(I) Irrespective of the field of desired application, it is ination with exogenous viral DNA (Weiner et al., 1990;
possible to find or isolate (by appropriate means of Chan et al., 1991; Romeo et al., 1993; Maier, 1995).
selection) cells with a differential response to different Contrary to PCR, which detects viral DNA or RNA
chemical or biological compounds, so that the desired sequences, BERA assays the virus in its complete,
specificity is created. virulent form. In this way, an accurate correlation can
(II) The construction and operation of BERA biosen- be established between the assay and the actual status
sors is quite cost-efficient. of infection and virus replication in the host, providing
(III) The biosensor is portable and the assay rapid important information on the viral load during anti-vi-
(almost instant), which makes it suitable for mass ral therapy. BERA could also complement the mass
screening purposes, as well as for the detection of detection of virulent compounds, enabling simplified
biological warfare agents. tests by the lay population and in developing countries.
(IV) Immobilized cells can retain unalterable their Applications also include screening new vaccines and
properties for a comparatively long time (\2– 3 pharmaceuticals, such as using liver cell biosensors in
months), at least under the conditions described in this order to study receptor antagonists used in pharmaceu-
study. ticals meant to affect the liver.
(V) The measured response to a virus is closely linked Further work is in progress in order to improve the
to its mechanism of virulence, particularly when whole, standardization of the method (in particular the biosen-
intact cells are used as the biosensory material. sory material) and to properly evaluate the reliability,
(VI) The method has an outstanding repeatability by performing a very large number of tests in a wide
(variability B 10 − 5). Most biosensory methods usually variety of environmental conditions. There is a need to
have a variability of at least 5% between measurements. improve our understanding of the fundamental pro-
As a biosensory method, BERA could be efficiently cesses taking place during cell-virus interactions within
used in drawing a first conclusion on the presence of a the probe. This interaction has a number of compo-
given virus in an unknown sample. A particularly valu- nents, which can be classified in three groups: (i) inter-
able application of the present method in medicine is action with the cell membrane (ii) interaction with the
the possible detection of viruses and other microorgan- whole cellular physiology and (iii) interaction with
isms before the host develops antibodies against them. other parts of the probe, such as the gel and the
The detection of these antibodies is the basis of stan- solution gathered in to the matrix pores. In addition, it
dard diagnostic methods (such as ELISA and immuno- is necessary to specify the various resistance and capac-
S. Kintzios et al. / Biosensors & Bioelectronics 16 (2001) 467–480 479

itance components present into the probe, before and Anonymous, 1989. Enzyme Immunoassay for the quantitative mea-
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of the signal and factor out noise (which was rather Bem, F., 1987. A serious disease in tobacco fields of Pieria county,
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The authors wish to acknowledge the following per- World Congress on Biosensors, San Diego, USA.
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