758

Fast Amperometric Assay for E. coli O157:H7 Using Partially

Immersed Immunoelectrodes
Ihab Abdel-Hamid, Dmitri Ivnitski, Plamen Atanasov, and Ebtisam Wilkins*
Chemical and Nuclear Engineering Department, University of New Mexico, Albuquerque, NM 87131
Received: April 3, 1998
Final version: May 14, 1998
Abstract
A novel amperometric immunoelectrode for fast and sensitive assay of E. coli O157:H7 is presented. Antibodies against E. coli O157:H7 were
immobilized on the surface of carbon rods, which acted as both working electrode and sorbent surface. A sandwich scheme of immunoassay was
used and 5-aminosalicylic acid was employed as a redox mediator for the amperometric detection of the enzyme-label (horseradish peroxidase).
The immunoelectrodes were operated while being partly immersed in the detection cell which resulted in the acceleration of the diffusioncontrolled rates of immunological, enzymatic and electrochemical reactions. The amperometric immunoelectrode allows the achievement of
significantly lower detection limits (40 times lower) than that achievable with standard spectrophotometric detection ELISA method using the same
immunochemicals. The immunoelectrode allows determination of E. coli cell concentrations in the range from 200 to 7000 cells/mL with an overall
analysis time of 40 min. This immunoelectrode can be easily adapted for assay of other microorganisms and may be a basis for creating a new class
of highly sensitive and rapid immunosensors.
Keywords: Escherichia coli O157:H7, Bacteria immunoassay, Immunoelectrode, Amperometric detection

1. Introduction
Bacteria, and other microorganisms are among the most common
pathogens in the environment [1, 2]. An average person carries
more than 150 kinds of bacteria which exist both inside and outside
the body [3]. Bacteria can spread easily and rapidly, requiring food,
moisture and a favorable temperature. E. coli O157:H7 is one of the
most dangerous food-borne pathogens [4, 5]. The O157:H7 is a rare
strain of E. coli that produces large quantities of a potent toxin that
can be accumulated in the lining of the intestine and causes severe
damage to it, resulting in hemorrhagic colitis or hemolytic uremic
syndrome and may lead to death especially in children [5, 6]. E. coli
can easily contaminate ground beef, raw milk and poultry,
therefore, careful control of E. coli is extremely important both
for food quality control and food safety. The effective testing of
food products requires methods of analysis that meet a number of
challenging criteria. Very low detection limits are required, since an
infectious dose may be as little as few hundreds of organisms.
Traditional methods for enumerating coliform bacteria (colony
counts) are often slow (from 24 to 72 h are required to obtain
confirmable results [7]) and may vary in reliability. Therefore,
existing techniques are unable to meet modern requirements of
instantaneous food quality control and considerable effort has been
made on the development of rapid, sensitive and accurate analytical
tools and methods for determining microorganisms concentration in
foods [8, 9].
Immunosensors based on highly specific immunological reactions combined with different amplification systems offer an
attractive approach towards solving this problem [9]. Various
types of immunoassay techniques have been developed for E. coli
determination [912]. However, these methods still require
significant time for analysis (several hours) and are greatly lacking
sensitivity. It has been demonstrated that diffusion to the interface
of the solid phase is a rate-limiting step during heterogeneous
electrochemical immunoassays [13]. Due to this diffusion control,
the time for achieving equilibrium of reaction between the
immobilized antibody and the antigen in solution is usually on
the order of several tens of minutes. The result is a fairly lengthy
overall assay. The general way to achieve significantly short
Electroanalysis 1998, 10, No. 11

immunoassay times is to reduce transport limitations across the

unstirred layer of solvent (the layer of immobilized electrolyte) to
the solid surface. The acceleration of the diffusion-controlled rate of
immunological and enzymatic reactions on the solid-solution
interface has been accomplished by: intensive mixing of the
solution (liquid phase) [14]; conducting immunoreactions in an
ultranarrow microcapillary immunoreactor or porous material [15];
the utilization of highly dispersed carbon-based immunosorbents as
electrode materials [16]; or by using an enzyme-channeling
approach which allows in situ generation of the substrates of the
enzyme-label [17].
In this work and for the first time, we present the possibility of
using of partially immersed immunoelectrodes for enhancing the
speed and sensitivity of immunoassays. A schematic of such a
partially immersed immunoelectrode is illustrated in Figure 1. The
fully immersed immunoelectrode (h 0; where h is the length of
the liquid meniscus) is covered by the liquid and thus contacts the
bulk electrolyte with all of its surface; there is no meniscus or thin
film of electrolyte formed around the electrode. As the electrode is
raised from this fully immersed position (h > 0), a liquid meniscus is
formed at the gas/liquid/solid interface. In addition to this meniscus
the hydrophilic protein layer (antibody or enzyme modifying the
electrode surface) facilitates formation of an additional thin liquid
film, the supermeniscus, on the electrode surface (placed above the
meniscus). This supermeniscus is characterized by a thickness of
approximately 0.40.8 mm. The meniscus and supermeniscus play
an important role in providing hydrodynamic conditions of the thin
film in the process of current signal generation.
It has been shown that for capillary systems (including partially
immersed and gas-diffusion electrodes), the magnitude of the
current obtained from a partially immersed electrode is 5 to 10
times greater than that obtained from a fully immersed electrode
[1820]. Since the magnitude of the diffusionally limited current is
inversely proportional to the thickness of the diffusion layer, the
effects observed with the partially immersed electrodes may be
explained by facilitated diffusion of reagents (antigen, conjugate,
substrate and product of enzymatic reaction) to the electrode
surface in the upper part of the meniscus and in the supermeniscus
where the electrode, the electrolyte, and the gas phase meet. Also,
the thickness of the supermeniscus is usually much less than the

q WILEY-VCH Verlag GmbH, D-69469 Weinheim, 1998

1040-0397/98/1109-0758 $ 17.50.50/0

Fast Amperometric Assay for E. coli O157:H7

759
coli O157:H7 (positive control), affinity purified antibody to E. coli
O157:H7 (anti-E. coli antibody, specific to a flagellum protein of
this particular strain of the bacteria), and peroxidase-labeled affinity
purified antibody to E. coli O157:H7 (conjugate) were obtained
from Kirkegaard & Perry Laboratories (KPL, Inc., Gaithersburg,
MD). Mortalyzed bacteria were used for safety reasons and to
ensure a static (nonproliferating) sample population for quantitative
purposes. Reference quantifications of moralyzed bacteria samples
were performed by hemacytometer counts and used to prepare stock
bacterial suspensions in phosphate-buffered saline. All other
chemicals (including buffer solutions components) were of the
highest purity available and were supplied from Sigma.

2.2. ELISA Procedure

Fig. 1. Schematic of the fully (a) and partially (b) immersed protein-coated
electrodes.

characteristic thickness of the stagnated diffusion layer in the bulk

solution, thus the transport of antigen, conjugate and substrate
molecules through such a film on the electrode surface is facilitated
compared to that through the bulk solution. The effective volume of
the electrolyte participating in the measurement is very small
(concentric cylinder of liquid around the electrode). This may result
in increased local concentration of the product of the enzymatic
reaction due to transport hindrances for the product formed at the
electrode surface to diffuse back to the bulk electrolyte. This
retaining of the product in close proximity of the electrode may
result in higher amperometric signal values. Therefore, the
analytical characteristics of a partially immersed immunoelectrode
are expected to be better than those of a fully immersed one and
result in increasing the signal values and reducing the response
times. This will potentially lead to improvement in the assay
sensitivity and reducing the overall time of analysis. This work
illustrates this approach, while presenting the development of a
heterogeneous enzyme-linked immunoassay for E. coli O157:H7
utilizing a partly immersed immunoelectrode.

2. Experimental
2.1. Materials
Carbon rods used for the fabrication of immunoelectrodes were a
courtesy of DFI Inc. (Erlanger, KY). Horseradish peroxidase (HRP,
E.C.1.11.1.7, activity 90 EU.mg1), its substrates: 5-aminosalicylic
acid (5-ASA), ortho-phenylenediamine (OPD), and hydrogen
peroxide (30 % v/v aqueous solution), as well as: bovine serum
albumin (BSA), N-ethyl-5-phenyl-isoxazolium-30 -sulfonate (Woodwards reagent K), poly-oxyethylene-sorbitan monolaurate (Tween
20) were obtained from Sigma Chem. Co. (St. Louis, MO).
Mortalyzed (by heat treatment) lyophilized pathogenic Escherichia

Sandwich scheme ELISA procedure was performed with

standard polystyrene 96-well plates (Corning Co.). In order to
modify each individual well with the primary antibody, 100 mL of a
10 mg/mL solution of antibody against E. coli O157:H7 diluted in
0.1 M phosphate buffer solution, pH 7.2 (PBS) was passively
adsorbed to the polystyrene cuvettes (wells) at 37 8C for 1 h, and the
cuvettes were rinsed three times (3 min each) with 0.1 M phosphate
buffer solution (pH 7.2) containing 0.5 M NaCl and 0.1 % Tween
20, (washing buffer). Aliquot of 100 mL of E. coli cells suspension
(positive control) was incubated in the cuvettes at room temperature
for 2 h and the cuvettes were rinsed three times (3 min each) with
washing buffer. Then 100 mL of the conjugate solution was added
and incubated for 1 h. The cuvettes were again rinsed as previously
described. For the detection stage 100 mL the enzyme substrate
solution containing 0.66 mg/ml of OPD in 0.1 M citric acidphosphate buffer (pH 5.0), was mixed with 5 mL of 30 % (v/v)
H2O2. The reagents were incubated for 15 min. The enzymatic
reaction was stopped by adding 150 mL of 1 M H2SO4 to each well.
The results of ELISA were measured by an spectrophotometric
ELISA-reader at a wavelength of l 492 nm.

2.3. Immunoelectrode Preparation

Carbon rods (2 mm diameter) were cut into 5 cm pieces. The rods
were insulated by multiple coating with cellulose acetate (from a
45 % w/v acetone solution) except for 1 cm at both ends (one end
designated as a working electrode surface and the other left for
current collecting) and left to dry for 30 min at room temperature.
Then each rod was inserted into a vessel containing 100 mL of
Woodwards reagent K solution (20 mg/mL) and incubated while
being shaken for two hours. This was followed by washing the rods
three times (3 min each) in 20 mM PBS solution (pH 7.4). Then, the
rods were inserted into a vessel containing 100 mL of a solution
containing antibodies against E. coli O157:H7 (0.4 mg/mL) and
shaken for 2 h. This was followed by another washing procedure.
Each rod with immobilized antibody was then inserted into a vessel
containing 200 mL of 1 % w/v BSA in 20 mM PBS (pH 7.8) and
again incubated under shaking conditons for two hours. The
electrodes were then washed and stored at 4 8C until measurement.
Typically 25 to 30 immunoelectrodes were simultaneously prepared.
Some studies of the amperometric assay system were performed
when carbon rod electrodes were unmodified (bare) or modified
directly by horseradish peroxidase by a procedure similar to that
described above, where peroxidase enzyme was immobilized
directly on the electrode surface, instead of incubating the
electrodes with primary antibodies. In this case, carbon rods were
activated by Woodwards reagent K (20 mg/mL) and then
immersed in a 20 mM phosphate buffer solution (pH 7.4) containing peroxidase (1 mg/mL) under shaking conditions for two hours.
Electroanalysis 1998, 10, No. 11

760

I. Abdel-Hamid et al.

Carbon rods with immobilized peroxidase was then incubated in

solution containing 200 mL of 1 % w/v BSA in 20 mM PBS
(pH 7.8) under shaking for two more hours. The electrodes were
washed and stored at 4 8C until measurement. Peroxidase activity
was assayed spectrophotometrically.

2.4. Amperometric Immunoassay Protocol

A sandwich scheme of immunoassay was employed and the
measurement procedure was carried out according to the following
steps. The immunoelectrodes (carbon rods with immobilized antiE. coli antibodies) were incubated for 26 min in 1 mL of 20 mM
acetate buffer (pH 5.0, 1 % w/v BSA) containing the desired
concentration of cells (first stage incubation). Immediately after the
incubation with the bacteria suspension (sample), the immunoelectrodes were washed 2 times (1 min each) in 1 mL of 20 mM acetate
buffer (pH 5.0, 1 % w/v BSA). The next step was incubating the
immunoelectrodes for 7 min in 0.3 mL of 20 mM acetate buffer
(pH 5.0, 1 % w/v BSA) containing 10 mL of 50 mg/mL of
peroxidase conjugated anti-E. coli antibodies (second stage
incubation). Then, the immunoelectrodes were washed, as before,
2 times (1 min each) in 1 mL of 20 mM acetate buffer (pH 5.0, 1 %
w/v BSA). The last step was the electrochemical measurement
which was carried out in a three-electrode cell (volume 0.3 mL)
with the immunoelectrode connected as a working electrode, a
carbon rod auxiliary electrode, and a standard Ag/AgCl reference
electrode. The electrolyte was 20 mM acetate buffer solution,
pH 5.0, containing 0.15 M NaCl. This electrolyte contained
0.6 mM of 5-aminosalicylic acid (5-ASA) and a polarization
potential of 0.0 V (vs. Ag/AgCl) was applied between the working
and reference electrodes using a bipotentiostat AFCBP1 (Pine
Instruments Co., Grove City, PA). The mixing of the electrolyte
was achieved by rotating of the immunoelectrode at a controlled
speed (usually at 300 rpm) by the Pine Instr. rotor/controller unit. In
the case of fully immersed electrode, the working electrode was
introduced in the cell so the electrolyte/air interface was in the
insulated zone of the carbon rod. Partially immersed state was
achieved, starting from the fully immersed condition, by gradually
elevating the electrode (using the rotor/controller unit lifting
mechanism). The length of the liquid meniscus formed was
estimated by observation (under a magnifying glass) and visual
comparison of it to a millimeter graduated standard. Immunoassay
steps: incubation, washing and electrochemical measurements were
performed utilizing both positions of the electrode: the fully
immersed position and the partially immersed position.
The background signal was measured while establishing its
steady-state value for 2 min. At this point 10 mL of 9 mM solution of
H2O2 was added to the cell and the amperometric signal was
monitored continuously employing an analog XY recorder
Ominigraphic 100 (Houston Instruments, Austin, TX). The time
for the amperometric signal to reach a steady-state value never
exceeded 3 minutes. Negative control experiments were carried out
when the first incubation stage was performed with no E. coli cells
in the sample. All experiments were carried out at room temperature
(approximately 20 8C).

Fig. 2. Mechanism of oxidation of 5-aminosalicylic acid in the presence of

hydrogen peroxide and peroxidase.

substrate (5-ASA) is widely used as a chromogenic noncarcinogenic substrate for horseradish peroxidase in enzyme immunoassay
with optical [21, 22] and potentiometric detection [23]. The reaction
scheme of the oxidation of 5-ASA in the presence of horseradish
peroxidase is shown in Figure 2 [24]. The oxidation of 5-ASA is a
two-electron process and is accompanied by the formation of the
electrochemically active species: 5-ASA-quinoneimine (5-ASA-QI).
The quinoneimine can further hydrolyze to benzoquinone which
forms a quinone/ hydroquinone redox couple. Cyclic voltammetry
of 5-ASA on the carbon rod electrodes indicated presence of the
5-ASA-QI form at potentials close to 0.0 V (vs. Ag/AgCl). Constant
potential amperometry experiments in the potential range from
0.1 to 0.1 V were conducted and it was found that for 5-ASAQI, an electro-reduction potential of 0.0 V (vs. Ag/AgCl) was
optimal from the point of view of signal-to-noise ratio and
sensitivity of peroxidase assay. At this potential a minimal (close
to zero) background current was observed.
The effect of pH on the rate of the peroxidase oxidation of 5-ASA
was also investigated. Figure 3 presents the steady-state values of
the amperometric response of a fully immersed carbon rod
electrode to the addition of 60 ng/mL peroxidase (in the solution)
at different pH. It can be seen that the activity of peroxidase was
highest between pH 4 and 5, and it gradually decreased in neutral
solutions (for pH greater than 5). Hence, a pH of 5 was selected as
standard for subsequent experiments of amperometric detection of
the peroxidase catalyzed reaction in the immunoassay.
Figure 4 shows typical response and a comparison of a fully
(h 0) and partially immersed (h > 0) peroxidase modified carbon
electrode in the presence of 0.15 mM hydrogen peroxide and

3. Results and Discussion

The amperometric immunoassay of E. coli was based on using
5-aminosalicylic acid (5-ASA) as a mediator for the measurement
of the activity of peroxidase-label in the immunocomplex
(sandwich) formed on the electrode surface. This peroxidase
Electroanalysis 1998, 10, No. 11

Fig. 3. Effect of pH on the response of the immersed carbon rod electrode to

addition of 60 ng/mL peroxidase. Conditions: potential applied 0.0 V (vs. Ag/
AgCl); 0.6 mM 5-ASA in 0.15 M NaCl; room temperature of 20 8C; rotating
speed 300 rpm. Points are means of three independent determinations.

Fast Amperometric Assay for E. coli O157:H7

761

Fig. 4. The response of the peroxidase modified electrode in 20 mM acetate buffer solution (pH 5) containing 0.15 mM H2O2, 0.6 mM 5-ASA and 0.15 M NaCl
as a function of the electrode position when raising from h 0 mm: current transients after injection of H2O2 in the solution (a) and dependence of the steady
state value of the signal on the electrode raising height (b).

0.6 mM 5-ASA. A constant potential of 0.0 V (vs. Ag/AgCl) was

applied to the electrode and the current transients were measured
following the introduction of H2O2 into the measuring cell at a
constant rotating speed of the cylindrical rod electrode. Figure 4a
presents the current transients obtained with the peroxidase
modified carbon rod electrode at different raising heights. It can
be seen that a fully immersed electrode (h 0) yields the lowest
amperometric response. Raising the electrode from the initial
totally immersed position (h 0) in the electrolyte solution results
in an increase in the amperometric response, reaching a maximum
at h 5 mm. Further raising of the electrode (h > 5) did not result in
further increase of the amperometric response. Figure 4b presents
the steady state signal values (plotted as means of three independent
measurements) vs. the raising height of the rod electrode. It can be
seen that the sensitivity of the partially immersed electrode
(h $ 5 mm) to hydrogen peroxide was 6 folds higher than for the
fully immersed one. The time required to reach 95 % of the steadystate current (after addition of the H2O2 sample) was less than 10 s
for this partially immersed electrode. Under the same conditions,
the response time of the fully immersed electrode is significantly
longer combined with a lower sensitivity. It can be hypothesized,
that the reason for the fast response and high sensitivity of the
partially immersed electrode are due to the attractive features of
the ultrathin electrolyte film in the supermeniscus region on the
electrode surface (see Fig. 1). The transport conditions for
electrochemically active species across the supermeniscus to the
electrode surface permit a rapid enzyme-substrate interaction and
therefore significantly reduce the assay time. This can be explained
by an effect of a small volume effective measuring cell formed by
the thin liquid film. One can think that the product of peroxidase
catalyzed reaction is retained in close proximity of the electrode,
thus resulting in higher values of amperometric response. On the
other hand, concentration of the electrochemically active species in
the supermeniscus might be increased by their surfactant properties,
which may lead to enhancement in the electrode sensitivity.
Figure 5 presents the dependence of the steady-state amperometric signal on increasing concentrations of horseradish peroxidase (varied from 10 to 100 ng/mL) for two 5-ASA concentrations
(0.6 mM and 1.3 mM) at a constant H2O2 concentration of 0.15 mM
for the partially immersed unmodified carbon rod electrode. All
measurements were conducted in a dynamic mode, while recording
the transient amperometric response of the electrode until reaching
the steady-state current plateau. It can be seen from Figure 5 that
for both 5-ASA concentrations, the amperometric signal magnitude
increases with increasing peroxidase concentrations and that
nanogram quantities (in the order of 1012 M) of peroxidase

enzyme can be detected in terms of current transients. It can also be

seen that the amperometric signal value is relatively higher at
0.6 mM 5-ASA concentration and decreases with increasing 5-ASA
concentrations to 1.3 mM. The signal value decreases further when
high concentrations of 5-ASA (> 1.3 mM) were used (not shown in
the figure). This can be explained by the fact that at high
concentration of 5-ASA the direction of the peroxidase oxidation
of 5-ASA changes (see Fig. 2). A polymer film is formed on the
electrode surface as a result of polymerization of the free radicals
formed during the enzymatic oxidation of 5-ASA at high
concentrations of this substrate [24]. This results in fouling of the
electrode surface. Therefore, all subsequent experiments were
performed with 0.6 mM 5-ASA.

3.1. ELISA of E. coli O157:H7

The E. coli samples were assayed by a standard sandwich ELISA
method with spectrophotometric detection in order to compare
ELISA performance with the electrochemical method of assay.
Polystyrene wells were coated with three dilutions of anti-E. coli
antibodies: 1/100, 1/1000, and 1/5000. The calibration plots for
E. coli obtained with the ELISA reader are shown in Figure 6. It
can be seen that the plots obtained with 1/100 and 1/1000 dilution of

Fig. 5. Calibration plots for peroxidase determination obtained with partially

immersed electrode in 20 mM acetate buffer solution (pH 5) containing
0.15 mM H2O2, 0.6 mM 5-ASA and 0.15 M NaCl.
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762

Fig. 6. Calibration plots of an Enzyme-linked Immunosorbent Assay

(ELISA) for determination of E. coli O157:H7 in phosphate buffer solution
with different dilutions of anti-E. coli antibodies (semilogarithmic plot).
Curves: 1) dilution 1:100; 2) dilution 1:1000; 3) dilution 1:5000.

the primary anti-E. coli antibodies practically coincide. When the

primary antibody dilution was increased to 1/5000, a significantly
lower response was obtained. Figure 6 shows that the standard
ELISA technique was able to detect E. coli O157:H7 in the range of
suspension concentrations from 104 to 107 cells/mL. The dynamic
range of the calibration plot (the fraction with a higher sensitivity)
lies within 105 107 cells/mL and the lower detection limit was
approximately 104 cells/mL. Thus, the sensitivity of this standard
ELISA assay is inadequate for practical application in direct assay
of food samples. The overall duration of this assay procedure is
approximately 4 h. The limiting factors of a heterogenic enzyme
immunoassay are diffusion transport conditions and the problem of
nonspecific binding of the conjugate to the solid surface.

3.2. Amperometric Immunoassay

Nonspecific binding of the conjugate to the solid surfaces is
among the most important factors influencing the achievable low
detection limit in immunoassays [25]. Nonspecific binding of antiE. coli-peroxidase conjugate in the format of the immunoelectrode
assay was studied using different blocking reagents: 0.1 M glycine,
1 % w/v BSA, and Tween-20. It was found that addition of 1 % w/v
BSA to the 20 mM acetate buffer (pH 5) markedly decreased
nonspecific adsorption of conjugate without altering the specific
binding. Low nonspecific binding of conjugate to the electrode
surface was observed when 1 % w/v BSA was used in all the steps
of immunoassay, including washing and measurement.
Figure 7 presents the calibration plots obtained for the assay of
E. coli O157:H7 cells using a fully (Fig. 7a) and partly immersed
(Fig. 7b) immunoelectrodes. It can be see from Figure 7a that the
lower detection limit of a fully immersed immunoelectrode was
7000 cells/mL and the dynamic range this calibration plot was
7 103 7 107 cells/mL. These characteristics of the calibration
plot of the fully immersed immunoelectrode were advantageous
while still comparable with that of the standard ELISA assay (see
Fig. 6). A significantly lower detection limit of 200 cells/mL was
obtained when using partially immersed immunoelectrodes
(Fig. 7b). The dynamic range of the calibration plot obtained
with partially immersed electrode was from 150 to 7000 cells/mL
(Fig. 7b). At bacteria suspension concentrations higher than 7 103
cells/mL the calibration plot reaches saturation. Along with the
Electroanalysis 1998, 10, No. 11

I. Abdel-Hamid et al.

Fig. 7. Calibration plots for determination of E. coli O157:H7 by means of a

sandwich scheme immunoassay using the fully (a) and partially (b) immersed
immunoelectrodes (semilogarithmic plot). Background signal level is shown
by a dashed line.

possible effect of concentration of the product of the enzymatic

reaction in the thin film, it can be hypothesized that the lowering of
the detection limit in the case of partially immersed immunoelectrode is associated also with some concentrating of the target
analytes (bacteria) and probably the immunoconjugate in the
meniscus and supermeniscus areas. This is probably due to their
surfactant properties. The presumption of such preconcentration
can explain the significantly lower saturation limit of the calibration
plot. On the other hand absolute signal values (steady-state current)
on both calibration plots, Fig. 7a and Fig. 7b can not be adequately
compared. Current densities are probably quite different, because
the experimental set-up did not allow exact determination of the wet
electrode area in the case of partially immersed electrode.
Contributions to the signal value of both immuno-interaction and
enzymatic reaction may be complex and are not well separated in
this system. By all means the physical chemistry behind the
phenomenon needs further investigation. From a practical analytical point of view, however, the system with fully/partially
immersed immunoelectrodes can be successfully applied for
detection of very low bacterial suspension concentrations (in a
partially immersed format) as well as for quantification of sufficiently
high concentrations (while using the totally immersed electrode). The
immunoassay detection stage demonstrates a fast response time of
20 s and an overall analysis time (including incubation and washing
stages) that does not exceed 40 min. All the assay procedures can be
automated and integrated in a dedicated immunoassay system for
detection of bacterial contamination in aqueous samples.
Immunoelectrodes demonstrated good reproducibility of their
analytical characteristics. In this format of the assay the carbon rod
electrodes were used as disposable sensing elements. Immunoelectrode response to each individual cell suspension concentrations
(presented in Fig. 7) was obtained as an average of four
measurements. Thus, sets of 20 to 30 individual electrodes were
used for obtaining the calibration plots (Fig. 7a and b).

4. Conclusions
This work shows that a partially immersed immunoelectrode can
find application in a rapid immunoassay of low concentrations of

Fast Amperometric Assay for E. coli O157:H7

aqueous suspensions of E. coli. Possible role of the conditions
formed with the partially immersed immunoelectrode in acceleration of the diffusion-controlled rates of immunological, enzymatic
and electrochemical reactions is yet to be studied in detail. The
capillary phenomena which are probably associated with the
observed effects could be explored also in other system formats
such as porous electrodes or hollow capillary tubes. This technique
employs surface reactions and therefore is not strictly dependent
on the reaction volume so that small sample volumes may be used.
E. coli concentrations could be detected in the range of 200 to 7000
cells/mL with an overall analysis time of 40 min. This immunoassay
approach can be easily extended to the detection of other bacterial
cells and may be a basis for creating a new type of highly sensitive
and rapid immunosensors.

5. Acknowledgements
This research was supported in part by a grant from the WasteManagement Education and Research Consortium of New Mexico.
Dr. Dmitri Ivnitski is thankful to the University of New Mexico,
School of Engineering for financial support.

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