Anal. Chem. 2008, 80, 1169-1175
Development of a Faradic Impedimetric
Immunosensor for the Detection of Salmonella
typhimurium in Milk
Aikaterini G. Mantzila,† Vassiliki Maipa,‡ and Mamas I. Prodromidis*,†
Laboratory of Analytical Chemistry, Department of Chemistry, and Department of Hygiene, Medical School, University of
Ioannina, 45 110 Ioannina, Greece
The development of a faradic impedimetric immunosensor for the detection of S. typhimurium in milk is
described for first time. Polyclonal anti-Salmonella was
cross-linked, in the presence of glutaraldehyde, on gold
electrodes modified with a single 11-amino-1-undecanethiol (MUAM) self-assembled monolayer (SAM) or a mixed
SAM of MUAM and 6-mercapto-1-hexanol at a constant
1 + 3 proportion, respectively. The mixed SAM was also
deposited in the presence of triethylamine, which was
used to prevent the formation of interplane hydrogen
bonds among amine-terminated thiols. The effect of the
different surface modifications on both the sensitivity and
the selectivity of the immunosensors was investigated. The
alteration of the interfacial features of the electrodes due
to different modification or recognition steps, was measured by faradic electrochemical impedance spectroscopy
in the presence of a hexacyanoferrate(II)/(III) redox
couple. A substantial amplification of the measuring signal
was achieved by performing the immunoreaction directly
in culture samples. This resulted in immunosensors with
great analytical features, as follows: (i) high sensitivity;
the response of the immunosensors increases with respect to the detection time as a consequence of the
simultaneous proliferation of the viable bacteria cells in
the tested samples; (ii) validity; the response of the
immunosensors is practically insensitive to the presence
of dead cells; (iii) working simplicity; elimination of
various centrifugation and washing steps, which are used
for the isolation of bacteria cells from the culture. The
proposed immunosensors were successfully used for the
detection of S. typhimurium in experimentally inoculated
milk samples. The effect of different postblocking agents
on the performance of the immunosensors in real samples
was also examined.
Salmonella typhimurium is a Gram-negative foodborne pathogen that affects the abdomen causing infection, diarrhea, and pain,
and it is recognized as the second most common serotype (after
* To whom correspondence should be addressed. E-mail: mprodrom@
cc.uoi.gr, Tel: +30-26510-98301/412. Fax: +30-26510-44831.
†
Laboratory of Analytical Chemistry, Department of Chemistry.
‡
Department of Hygiene.
10.1021/ac071570l CCC: $40.75
Published on Web 01/25/2008
© 2008 American Chemical Society
Salmonella enteritidis) of Salmonella found in humans.1 Beyond
the inadvertent contamination of foods and water, public concern
also arises from cases of adulteration, such as the contamination
of salad bars with S. typhimurium in Oregon.2 Hence, the detection
of this pathogen is extremely important and highly desirable for
the safety of food products and biosecurity. Bearing in mind that
the pathogen has a very high proliferation rate and exists in very
complex matrixes, there is a need for advanced monitoring
methods and techniques.
Although the standard techniques in pathogen detection are
sensitive and selective enough, involving enrichment of samples
and culture plating procedures, are usually elaborate, and need
several days for presumptive results and confirmation.3,4 Many
types of Salmonella detection tests have been developed during
the last decades, involving enzyme-linked immunosorbent assay
(ELISA)5-8 and nucleic acid-based polymerase chain reaction
(PCR) technology.9-11 However, ELISA requires extra enzymelabeled antibodies, while the PCR-based assay is often more laborintensive than ELISA.12 In addition, some components of food and
chemicals required for selective enrichment of cells may influence
the effectiveness of the PCR and cause inhibitory effects.4
A variety of alternative methods have been also reported for
the detection of Salmonella including the monitoring of oxygen
(1) Mahon, C. R.; Manuselis, G. Textbook of Diagnostic Microbiology, 2nd ed.;
Saunders: Philadelphia, PA, 2004; Chapter 6.
(2) Terrorist Threats to Food; Guidance for Establishing and Strengtheninng
Prevention and Response Systems; WHO: Geneva, Switzerland, 2002; pp
1-46.
(3) Andrews, W. H.; June, G. A.; Sherrod, P.; Hammack, T. S.; Amaguana, R.
M. Food and Drug Administration Bacteriological Analytical Manual, 8th
ed.; AOAC International; Gaithersburg, MD, 1995; pp 5.01-5.20.
(4) Jofre, A.; Martin, B.; Garriga, M.; Hugas, M.; Pla, M.; Rodriguez-Lazaro,
D.; Aymerich, T. Food Microbiol. 2005, 22, 109-115.
(5) Cudjoe, K. S.; Hagtvedt, T.; Dainty, R. Int. J. Food Microbiol. 1995, 27,
11-25.
(6) Tian, H.; Miyamoto, T.; Okabe, T.; Kuramitsu, Y.; Honjoh, K. C.; Hatano, S.
J. Food Prot. 1996, 59, 1158-1163.
(7) Beckers, H. J.; Tips, P. D.; Soentoro, P. S. S.; Delfgou-Van Asch, E. H. M.;
Peters, R. Food Microbiol. 1998, 5, 147-156.
(8) Mansfield, L. P.; Forsythe, S. J. Food Microbiol. 2001, 18, 361-366.
(9) Luk, J. M. C. Biotechnology 1994, 17, 1038-1042.
(10) Bennett, A. R.; Greenwood, D.; Tennant, C.; Banks, J. G.; Betts, R. P. Lett.
Appl. Microbiol. 1998. 26, 437-441.
(11) Chen, W.; Martinez, G.; Mulchandani, A. Anal. Biochem. 2000, 280, 166172.
(12) Wray, C.; Wray, A. Salmonella in Domestic Animals; Oxford University
Press: New York, 2000.
Analytical Chemistry, Vol. 80, No. 4, February 15, 2008 1169
consumption with cyclic voltammetry,13 acoustic wave sensors
based on filamentous bacteriophages,14 and indirect fiber-optic
biosensors.15 Other approaches, which are based on quartz crystal
microbalance piezoimmunosensors,16-20 conductometric immunosensors,21 magnetoelastic resonance biosensors,22 and electrochemical ELISA immunosensors23-25 have been also proposed.
Of the methods available for the detection of Salmonella,
impedance microbiology has been successfully used in both
standard and real samples by measuring either the impedance of
selective growth media or the interfacial impedance of the
electrodes.26-28 However, these methods do not provide the
analytical simplifications of biosensors.
Electrochemical impedance spectroscopy (EIS)-based immunosensors are particularly attractive compared with the aforementioned approaches, due to their ability for miniaturization, the low
cost of electrode mass production, and the cost-effective instrumentation.29 In addition, they exhibit low detection limits, especially when the main antibody-antigen interaction signal is being
enhanced by case-specific amplification schemes.30-32 In general,
these schemes follow the main immunoreaction step and include
the application of extra enzyme-labeled antibodies,32 biotinstrept)avidin complex,30 or enzyme labels that precipitate an
insoluble compound on the sensing interface.31 However, the
majority of the published works have not been tested in real
matrixes, since fundamental issues still exist, and this in turn has
brought the reliability of impedimetric immunosensors into
question.
The aim of this work was the selection of a suitable immobilization platform for anchoring anti-Salmonella, so that one
can preserve its biological activity and selectivity of the target
analyte. In order to amplify the antibody-bacteria cell interaction
signal, antibody functionalized electrodes-bacteria cells immunoreaction step was performed directly in the culture samples.
This led to considerable increasing signals compared with those
which would have been obtained if the immunoreaction was
(13) Ruan, C.; Yang, L.; Li, Y. J. Electroanal. Chem. 2002, 519, 33-38.
(14) Olsen, E V.; Sorokulova, I. B.; Petrenko, V. A.; Chen, I-H.; Barbaree, J. M.;
Vodyanoy, V. J. Biosens. Bioelectron. 2006, 21, 1434-1442.
(15) Ko, S.; Grant, S. A. Biosens. Bioelectron. 2006, 21, 1283-1290.
(16) Babacan, S.; Pivarnik, P.; Letcher, S.; Rand, A G. Biosens. Bioelectron. 2000,
15, 615-621.
(17) Fung, Y. S.; Wong, Y. Y. Anal. Chem. 2001, 73, 5302-5309.
(18) Wong, Y. Y.; Ng, S. P.; Ng, M. H.; Si, S. H.; Yao, S. Z.; Fung, Y. S. Biosens.
Bioelectron. 2002, 17, 676-684.
(19) Kim, G-H.; Rand, A. G.; Letcher, S. V. Biosens. Bioelectron. 2003, 18, 9199.
(20) Su, X-L.; Li, Y. Biosens. Bioelectron. 2005, 21, 840-848.
(21) Muhammad-Tahir, Z.; Alocilja, E. C. Biosens. Bioelectron. 2003, 18, 813819.
(22) Guntupalli, R.; Hu, J.; Lakshmanan, R. S.; Huang, T. S.; Barbaree, J. M.;
Chin, B. A. Biosens. Bioelectron. 2007, 22, 1474-1479.
(23) Croci, L.; Delibato, E.; Volpe, G.; Palleschi, G. Anal. Lett. 2001, 34, 25972607.
(24) Delibato, E.; Volpe, G.; Stangalini, D.; De Medici, D.; Moscone, D.; Palleschi,
G. Anal. Lett. 2006, 39, 1611-1625.
(25) Croci, L.; Delibato, E.; Volpe, G.; De Medici, D.; Palleschi, G. Appl. Environ.
Microbiol. 2004, 70, 1393-1396.
(26) Yang, L.; Ruan, C.; Li, Y. Biosens. Bioelectron. 2003, 19, 495-502.
(27) K’Owino, I. O.; Sadik, O. A. Electroanalysis 2005, 17, 2101-2113.
(28) Yang, L.; Li, Y.; Griffis, C. L.; Johnson, M. G. Biosens. Bioelectron. 2004,
19, 1139-1147.
(29) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913-947
(30) Pei, R.; Cheng, Z.; Wang, E.; Yang, X. Biosens. Bioelectron. 2001, 16, 355361.
(31) Ruan, C.; Yang, L.; Li, Y. Anal. Chem. 2002, 74, 4814-4820.
(32) Balkenhohl, T.; Lisdat, F. Analyst 2007, 132, 314-322.
1170
Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
carried out in a buffer solution, where the population of the cells
remains unchangeable during the course of the immunoreaction.
It is also important to point out that the proposed method is
insensitive to dead cells as, in contrast to viable cells, they cannot
proliferate and thus their presence has almost no contribution to
the measuring signal. An additional advantage of this method is
the elimination of time-consuming cleanup sample procedures in
pathogen detection (centrifugation and washing steps), which may
also reduce the original concentration of the viable cells in the
tested samples leading to false negative results.
Finally, the proposed immunosensors were successfully used
for the detection of S. typhimurium in experimentally inoculated
milk samples. The effect of different postblocking agents on the
performance of the immunosensors in real samples was examined
and discussed.
EXPERIMENTAL SECTION
Chemicals and Solutions. 11-Amino-1-undecanethiol hydrochloride (MUAM) was from Dojindo . 6-Mercapto-1-hexanol (MH),
glutaraldehyde (GA) (∼25% in water; kept in sealed vials under
argon at +4 °C), and bovine serum albumin (BSA) were purchased from Sigma. Absolute ethanol, denaturated ethanol, potassium ferrocyanide, potassium ferricyanide, and triethylamine were
from Merck. Tryptone soy broth (TSB) and affinity-purified rabbit
polyclonal antibodies against Salmonella (∼4 mg mL-1 in 10 mM
PBS pH 7.2) were purchased from LAB M and Biodesign,
respectively. Milk powder (Regilait) and fresh, skimmed, pasteurized milk were purchased from the local market and used as
received. All other chemicals were from Merck and Sigma, and
double-distilled water (DDW) was used throughout.
A 10 mM phosphate-buffered saline (PBS) solution containing
140 mM NaCl, pH 7.2, was used for the dilution of the antibody
stock solution and postblocking steps. A 50 mM phosphate buffer
(PB) solution, pH 7, was used for the activation of amine groups
with glutaraldehyde and in various washing steps, whereas a 50
mM PBS solution containing 100 mM KCl, pH 7, was used for
the removal of nonbound antibodies from the surface of the
immunosensors and as electrolyte of the redox couple in EIS
measurements. All buffer solutions were filtered through a 0.2µm pore size membrane (Millipore) and stored at +4 °C for up
to 2 weeks.
Bacteria and Colony Forming Unit (cfu) Determinations.
The tested bacteria were S. typhimurium (4,5:i:1,2) (from National
School of Public Health, Laboratory of Microbiology, Athens,
Greece) and Escherichia coli NCTC 9001 (from Public Health
Laboratory Service-North, Newcastle, England). The bacteria
cultures were grown in TSB at 37 ( 1 oC for 24 h before use, and
the number of viable cells was determined by a microbial plate
count method. Antigen samples were then prepared by serial
dilutions of the stock culture with the broth solution.
Apparatus. EIS experiments were performed with the electrochemical Analyzer PGSTAT12/FRA2 (Eco Chemie) in a onecompartment three-electrode cell. Gold electrodes were used as
working electrodes, and a platinum wire (diameter of 1 mm and
length of 3 cm) served as the auxiliary electrode. The reference
electrode was a Ag/AgCl/3 M KCl (IJ Cambria, UK) electrode,
and all potentials reported hereafter refer to the potential of this
electrode. The impedance spectra were recorded over a frequency
range of 10-1-105 Hz, using a sinusoidal excitation signal,
superimposed on a dc potential of +0.200 V. Excitation amplitude
of 10 mV (rms) was used throughout. All measurements were
performed in a solution of 5 mM hexacyanoferrate(II)/(III) (1 +
1 mixture) in PBS solution (pH 7) at room temperature.
Formation of SAMs and Immobilization of the Antibodies.
Gold electrodes were constructed by using the commercial kit
EasyCon (EasyCon Hellas, provided by Eco Chemie). Before use,
gold electrodes of 2-mm active surface were polished with Al2O3
(0.01-mm grain size) and sonicated for 3 min in DDW. After
polishing, gold surfaces were cleaned by dipping into a solution
of 1 + 1 + 5 (v/v), NH4OH + 30% H2O2 + H2O for 10 min, washed
thoroughly with DDW and absolute ethanol, dried under argon,
and immersed immediately in a mixture of 0.5 mM MUAM and
1.5 mM MH in absolute ethanol or in a solution of 0.5 mM MUAM
in the same solvent for 16 h at room temperature in the dark.
Deposition of a mixed self-assembled monolayer SAM was also
performed in an ethanolic mixture of 0.5 mM MUAM and 1.5 mM
MH with 3% (v/v) triethylamine (TEA).33 The thiol SAM-modified
electrodes were then thoroughly rinsed in fresh baths of absolute
ethanol, 4 × 5 min (electrodes that had been modified in the
presence of TEA were additionally washed with 10% (v/v) acetic
acid in ethanol after the first washing step in absolute ethanol),
dried under argon, and immersed immediately in well-stoppered
vials containing a degassed solution of 2.5% glutaraldehyde in PB
solution for 1 h under mild stirring. Then electrodes were rinsed
several times with the same buffer solution to remove the
physically absorbed glutaraldehyde. The Salmonella antibodies
(anti-SA) were introduced onto the Au/thiol/GA-activated electrodes by dropping 10 µL of ∼2 mg mL-1 antibody in PBS (pH
7.2) and allowed to incubate at 4 °C for at least 12 h in a humidified
glass chamber. After the immobilization of the antibody, the
electrodes were rinsed with PBS solution (pH 7) to remove the
excess of physically bound antibody and immersed in the solution
of the blocking agent [TSB, or BSA (1.6 or 3.2 mg mL-1) or 1.6
mg mL-1 milk powder in PBS solution (pH 7.2)] for 2 h at room
temperature to block nonspecific binding sites.
Procedures. Optimization Studies. The stock culture was
serially diluted with TSB, and samples of different initial concentrations of S. typhimurium (typically in the range of ∼10-106 cfu
mL-1) were thus prepared. The 2-mL portions of them were
introduced in sterilized glass vials. Ready-to-use immunosensors
[fully functionalized (Au/thiol/GA/anti-SA) electrodes after their
incubation in the solution of the blocking agent] were immersed
in the standard culture samples for a specified time interval at
room temperature under mild stirring. Immunosensors were then
thoroughly rinsed with PB solution and transferred to the
measuring cell. During the immunoreaction, the initial transparent
or semitransparent (depending on the initial cell number of the
bacteria) samples became turbid as a result of the proliferation
of the bacteria cells.
Application to Milk Samples. The 0.9-mL portions of fresh
pasteurized milk were inoculated with 100 µL of the bacteria
culture containing 107 or 2 × 104 cfu mL-1 S. typhimurium (positive
controls) or E. coli (negative controls) and kept under natural
conditions for 1 h. Then aliquots (100 µL) of inoculated and
uninoculated (used as blank to evaluate the effect of the matrix
components on the measuring signal) milk samples were mixed
(33) Wang, H.; Chen, S.; Li, L.; Jiang, S. Langmuir 2005, 21, 2633-2636.
with 1.9 mL of TSB in sterilized glass vials. The rest of the
procedure was similar to that followed during the optimization
studies.
Throughout this study, the relative change of the signal of the
ready-to-use immunosensors before and after the immunoreaction,
∆Rct (%), which is expressed as ∆Rct ) {[Rct (after the immunoreaction) - Rct (before the immunoreaction)]/Rct (before the
immunoreaction)} × 100 was taken as a measure of the concentration of bacterial cells in standard and real samples. Correspondingly, ∆Rct (%) values before and after each modification step were
used as a measure to evaluate the effect of the different modification steps on the measuring signal.
Safety Considerations. All glassware in contact with bacteria
must be sterilized for 1.5 h at 115 °C before and after use. Before
heating, glassware was immersed sequentially in baths of 0.1 M
HCl and denatured ethanol. Waste solutions containing bacteria
must be sterilized before disposal.
RESULTS AND DISCUSSION
Evaluation of Different Surface Modifications. A determining factor in the performance of biosensors is the design of the
immobilization platform, as it greatly affects both the sensitivity
and the specificity of the biointerfaces. Our aim was to immobilize
the anti-Salmonella onto the surface of gold electrodes after their
modification with an amine-terminated SAM, and glutaraldehyde
cross-linking. MUAM was chosen among other amine-terminated
thiols, as it provides stable coverage over time.34,35 In order to
define the most suitable protocol for the immobilization of
antibodies, three different electrode assemblies were examined:
(i) Au/MUAM, (ii) Au/MUAM-MH, and (iii) Au/MUAM-MH
in the presence of TEA. MH was used at a 3-fold higher
concentration compared with that of MUAM, in order to be served
as a “dilutor” of the main thiol (MUAM) that brings the functional
headgroup. In general, the use of two-thiol mixed SAMs has been
shown to improve the bioactivity of a protein immobilized on such
layers compared with that on a single thiol-based SAM.36-38 The
second thiol reduces the surface concentration of the functional
groups and thus minimizes steric hindrances and can also lead
to different conformation of the immobilized molecules.36,39
TEA was used to prevent the formation of interplane hydrogen
bonds between amine groups of MUAM on gold and free MUAM
molecules in the bulk that could lead to a partial second layer of
MUAM atop of the gold-bound monolayer (Scheme 1). More
details and scientific evidence of this behavior are provided in
the excellent work of Wang et al.33
Fully functionalized immunosensors that are based on Au/
MUAM, Au/MUAM-MH, and Au/MUAM-MH(TEA) electrodes
were blocked with the TSB solution and then incubated in both
positive and negative control samples containing the same initial
cell number of bacteria. Comparative results (Table 1) reveal that
(34) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12,
977-989.
(35) Lasseter, T. L.; Cai, W.; Hamers, R. J. Analyst 2004, 129, 3-8.
(36) Briand, E.; Salmain, M.; Herry, J. M.; Perrot, H.; Compère, C.; Pradier, C.
M. Biosens. Bioelectron. 2006, 22, 440-448.
(37) Frederix, F.; Bonroy, K.; Laureyn, W.; Reekmans, G.; Campitelli, A.; Dehean,
W.; Maes, G. Langmuir 2003, 19, 4351-4357.
(38) Ge, B.; Lisdat, F. Anal. Chim. Acta 2002, 454, 53-64.
(39) Guiomar, A. J.; Guthrie, J. T.; Evans, S. D. Langmuir 1999, 15, 11981207.
Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
1171
Scheme 1. Tentative View of the Different Modification and Recognition Steps of the BSA-Blocked
Au/MUAM-MH/GA/Anti-SA Immunosensorsa
a
Drawings are not to scale.
Table 1. Buildup of Different Au/Thiol/GA/Anti-SA Electrode Assemblies and Comparative Results of Their Response
(after Postblocking with TSB) to Positive and Negative Controls Containing an Initial Concentration of 106 CFU
mL-1 Bacteriaa
electrode assembly
a
MUAM
MUAM-MH
MUAM-MH TEA
Au, Rct
Au/thiol, Rct
Au/thiol/GA, Rct , (θ)
∆Rct (%) after glutaraldehyde
Au/thiol/GA/anti-Salmonella, Rct
∆Rct (%) after anti-Salmonella
Buildup of Different Electrode Assemblies
0.20
0.21
1.96
0.15
9.71 (0.98)
0.46 (0.54)
+395
+210
18.7
1.1
+93
+135
0.19
0.16
1.86 (0.89)
+1089
3.21
+73
∆Rct (%) S. typhimurium (P)
∆Rct (%) E. coli (N)
∆Rct (P)/∆Rct (N)
After Immunoreaction. Detection Time 2 h
+33
+213
+22
+16
1.5
13.3
+125
+29
4.3
Rct in kΩ. The values in parentheses are of the surface coverage, calculated as θ ) 1 - [(Rct(Au)/ Rct(Au/thiol/GA)].
MH has a beneficial effect on the analytical features of the
immunosensors. Indeed, in the absence of it, the resulted
immunosensors exhibit a poor performance. On the other hand,
the presence of TEA seems to have a rather negative effect on
the performance of the mixed SAM-based immunosensors.
Trying to elucidate these results, the tested electrodes assemblies were repeatedly examined and faradic impedimetric
measurements after each step of the sensor buildup were
performed. Impedance data were fitted to a Randles equivalent
circuit (Figure 1A, side panel), and electrical parameters were
determined using the FRA2 software (Eco Chemie). To ensure
the best reproducible conditions, every series of the tested
electrodes was run in parallel and various surface modification
and immunoreaction steps were made from the same stock
solutions of thiols, glutaraldehyde, antibody, and bacteria. Received results can be rationalized as follows:
(i) The presence of TEA seems to have no effect on the Rct
values of Au/MUAM-MH electrodes indicating that Rct values
are mostly determined on the presence of the “dilutor” (MH).
The fact that the Rct values of Au/MUAM-MH electrodes are
lower than those of bare gold electrodes (Figure 1A and Table 1)
is attributed to the observed enhancement of the electrocatalytic
1172 Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
efficiency of short-chain thiol-modified electrodes to the used
redox probe, in accordance with previous studies.40
On the other hand, the increase of Rct from 0.20 to 1.96 kΩ at
Au/MUAM electrodes indicates the formation of Au/MUAM and
Au/MUAM‚‚‚MUAM layers,33 which provide an effective barrier
to electron transfer of the redox species in solution.
(ii) In the next step of the sensor buildup, free terminal amine
groups react with glutaraldehyde to create amine-reactive aldehyde ends that will function as binding sites for the immobilization
of the antibody molecules (Scheme 1). The extremely high relative
signal changes (1089%) that were observed at Au/MUAM-MH
(TEA) electrodes constitutes great evidence for the high population of free amine groups in these electrode assemblies. On the
other hand, in the absence of TEA, the considerably lower values
of ∆Rct, (395 and 210% at Au/MUAM and Au/MUAM-MH
electrodes, respectively) are probably attributable to the low
percentage of free amine ends due to the partial formation of
interplane MUAM layers. The value of ∆Rct at Au/MUAM-MH
electrodes (210%) is lower compared with that observed at Au/
MUAM electrodes, as the presence of the “dilutor” in the former
(40) Mantzila, A. G.; Strongylis, C.; Tsikaris, V.; Prodromidis, M. I. Biosens.
Bioelectron. 2007, 23, 362-369.
Figure 2. Response and specificity of BSA-blocked Au/MUAMMH/GA/anti-SA electrodes. BSA, 1.6 mg mL-1 protein in 10 mM PBS
solution, pH 7.2. Detection time, 2 h. Measuring conditions, 10-1105 Hz at +0.200 V bias (10 mV rms). Electrolyte, 5 mM hexacyanoferrate(II)/(III) (1 + 1 mixture) in PBS solution, pH 7.
ones reduces the population of the free amine groups. At this
point, it is important to state that the observed increase of the
absolute values of Rct(Au/thiol/GA) in all the tested assemblies is not
solely attributable to the attachment of glutaraldehyde (a relative
small molecule of MW 100.1 Da) but also to the spontaneous
polymerization of it,41 although the necessary measures for the
minimization of this effect have been taken (the type of the stock
material and performance of the reaction in degassed conditions).
(iii) An interesting finding, which is revealed by this study, is
that the ∆Rct(%) values after the immobilization of the antibodies
are irreversibly proportional to those calculated after the treatment
with glutaraldehyde (Table 1). In this regard, factors that control
the glutaraldehyde loadings are rather determinant of the analytical performance of the resulted immunosensors. High glutaral-
dehyde loadings (as they are also defined by the corresponding
values of the surface coverage, Table 1) may result in the
immobilization of antibodies through multiple binding sites, thus
reducing the flexibility (rigidity of binding) and the binding
capacity of the antibodies as well as the accessibility of the target
analyte to them. The experimental results support this assumption,
as it revealed from the comparison of the selectivity ratios (Table
1), expressed as, P/N ) [∆Rct (%) S. typhimurium (P)/∆Rct (%)
E. coli (N)], that have been calculated for each tested electrode
assembly. The buildup of the optimum electrode assembly and
the selectivity of the resulted immunosensors to both positive and
negative control samples are illustrated in Nyquist plots in Figure
1A,B, respectively.
Antibody Loading. Bioreceptor loading for anti-Salmonella
was tested in order to define the antibody loading to obtain the
maximum response. A saturation study was made using the
glutaraldehyde-activated Au/MUAM-MH electrodes and four
discrete antibody concentrations, that is, 0.5, 1, 2, and 4 mg mL-1
in PBS solution (pH 7.2). The sensitivity of the corresponding
immunosensors, after their blocking, to a standard sample
containing an initial concentration of 106 cfu mL-1 S. typhimurium
(data not shown) was taken as the criterion for this study. The
profile of the system sensitivity reaches a plateau at an antibody
concentration of 2 mg mL-1, and therefore, further experiments
were performed, applying the useful antibody content onto the
Au/MUAM-MH/GA electrodes.
Performance of the Immunosensors. The recognition
properties of Au/MUAM-MH/GA/anti-SA electrodes were examined after their incubation for 2 h in 1.6 mg mL-1 BSA solution
(blocking agent) in PBS, pH 7.2. The efficiency of the resulted
immunosensors in a series of standard culture samples over the
(initial) concentration range 102-106 cfu mL-1 S. typhimurium and
to a discrete concentration of E. coli are shown in Figure 2. The
data obtained indicate that, for a detection time of 2 h, it is possible
to detect S. typhimurium at a concentration level 3 orders of
magnitude lower than the infectious dosage, that is, 105 CFU
mL-1.42
(41) Gillet, R.; Gull, K. Histochemie 1972, 30, 162-167. Technical Data Sheet
No.124, Polysciences Inc., 1999; pp 1-3.
(42) Murray, P. R.; Rosenthal, K. S.; Kobayashi, G. S.; Pfaller, M. A. Medical
Microbiology, 3rd ed.; Mosby: St. Louis, MO, 1998; Chapter 29.
Figure 1. (A) Nyquist plots showing the buildup of the immunosensors: (a) Au/MUAM-MH, (b) Au bare, (c) Au/MUAM-MH/GA, and
(d) Au/MUAM-MH/GA/anti-SA electrodes. Side panel, Randles
equivalent circuit Rs(Qdl[RctW]), where Rs is the ohmic resistance of
the electrolyte, Rct is the charge-transfer resistance, due to electron
transfer of the redox probe to the electrode, W the Warbung
impedance resulting from the diffusion of the redox couple toward
the electrode surface and Qdl is the double layer capacitance, which
is represented by a constant phase element [CPE ) (1/Q)-1(jω)-n].
(B) Impedimetric spectra of TSB-blocked Au/MUAM-MH/GA/anti-SA
electrodes (a) before and after their incubation with (b) E. coli and
(c) S. typhimurium culture samples for 1 h. Initial concentration of
bacteria, 106 cfu mL-1. Measuring conditions, 10-1-105 Hz at +0.200
V bias (10 mV rms). Electrolyte, 5 mM hexacyanoferrate(II)/(III) (1 +
1 mixture) in PBS solution, pH 7.
Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
1173
Figure 3. Nyquist plots of BSA-blocked Au/MUAM-MH/GA/antiSA electrodes (a) before and (b) after their incubation for 2, (c) 6,
and (d) 20 h in a culture containing an initial concentration of 102 cfu
mL-1 S. typhimurium. Measuring conditions, 10-1-105 Hz at +0.200
V bias (10 mV rms) Electrolyte, 5 mM hexacyanoferrate(II)/(III) (1 +
1 mixture) in PBS solution, pH 7.
Figure 4. Nyquist plots of BSA-blocked Au/MUAM-MH/GA/antiSA electrodes (a) before and after (b) 2-h incubation in a culture
sample containing an initial concentration of 106 cfu mL-1 S.
typhimurium and (c) 2-h incubation in the culture supernatant solution
(see text for details). Measuring conditions, 10-1-105 Hz at +0.200
V bias (10 mV rms). Electrolyte, 5 mM hexacyanoferrate(II)/(III) (1 +
1 mixture) in PBS solution, pH 7.
The effectiveness of the proposed amplification scheme is
proven in Figure 3. The lowest signal response that was received
(Figure 2, 102 cfu mL-1 S. typhimurium, ∆Rct ) 14% for a detection
time of 2 h) can be substantially increased to 89 and 665% for
detection time intervals of 6 and 20 h, respectively. Correspondingly, ∆Rct values of 33 and 131% were achieved at a sample
containing an initial concentration of 10 cfu mL-1 of the target
pathogen for detection time intervals of 6 and 10 h, respectively.
The nature of the proposed measuring methodology raised a
concern regarding the mechanism that produces the observed
signal changes. During the proliferation of Gram-negative bacteria,
a number of exotoxins (enterotoxins and cytotoxins with a MW
∼70-88 kDa) are also produced and liberated in the culture
medium.43 In order to evaluate signal contributions originated from
the attachment of the exotoxins onto the surface of the BSAblocked immunosensors, the following diagnostic experiment was
performed. A culture sample of S. typhimurium was divided into
two equal parts, one of which was used to perform the proposed
methodology as it has been described so far (2-h incubation time),
while the other was kept aside for 2 h and then was centrifuged
to separate the cells from the culture medium. The supernatant
solution was then transferred to a sterilized vial and a new BSAblocked immunosensor was immersed in it for 2 h. The Nyquist
plots in Figure 4 (curves b and c) indicate that any contribution
to the measuring signal due to exotoxins, is lower than 4% and
the overwhelming contribution is due to the specific antibodyantigen interactions. This also justifies the high selectivity of the
proposed immunosensors to other Gram-negative bacteria that
also produce exotoxins.
Application to Milk Samples. This part of the study was
focused on the qualitative discrimination of S. typhimurium and
E. coli inoculated milk samples, which are used as positive and
negative controls, respectively.
As can be seen from the results presented in Table 2, TSBblocked immunosensors succeed in the discrimination of positive
Table 2. Application of Au/Thiol/GA/Anti-SA Electrodes
in Inoculated Milk Samples (5 × 104 and 102 CFU mL-1
Initial Concentration of Bacteria Cells)a
(43) Hirst, T. R. Assembly and secretion of oligomeric toxins. In Sourcebook of
Bacterial Protein Toxins; Alouf, J. E., Freer, J. H., Eds.; Academic Press:
London, 1991; pp 75-100.
1174 Analytical Chemistry, Vol. 80, No. 4, February 15, 2008
∆Rct (%)
E. coli
inoculated
milk
samplesb
P/N
ratio
blocking agent/
detection time
milk
samples
S. typhimurium
inoculated
milk samplesb
TSB/5 h
milk powder/5 h
BSA (mg mL-1)
3.2/5 h
1.6/5 h
1.6/2 h
1.6/10 h
70
28
320
224
52
239
∼6
∼1
29
44
34
59
506
1449
117
254c
14
17
25
40c
∼36
∼85
∼5
∼6
a The standard deviation of the mean ranges from 12 and 18%, n )
5. b 5 × 104 cfu mL-1. C 102 cfu mL-1.
and negative milk samples with a calculated P/N ratio of 6. This
ratio should not be compared with that calculated in standard
culture samples (P/N 13.3, Table 1), as the magnitude of ∆Rct(%)
values in real samples is affected to a certain degree by background signals (Table 2). The lower signal response, which was
observed at E. coli inoculated real samples, compared with that
observed in milk samples (blank), could probably be explained
by a screening effect of the nonspecifically bound E. coli cells on
the various components of the matrix, thus reducing the surface
coverage of the immunosensors.
Aiming to achieve higher P/N ratios, thus allowing the
successful application of the method in shorter detection times,
the effect of various blocking agents on the background signal
was also examined.
Unexpected results were obtained for milk powder-blocked
immunosensors. Bulk micelles of casein probably block the
specific binding sites as well, thus resulting in practically inactive
(P/N ∼ 1) immunosensors. The observed ∆Rct(%) values in both
inoculated samples could possibly be attributed to nonspecific
electrostatic interactions between the casein micelles and the
bacterial cells. However, the exact mechanism of this behavior
Finally, it is important to point out that none of the analytical
trials we run gave false negative (S. typhimurium) or positive (E.
coli) results, thus constituting strong evidence for the validity of
the proposed immunosensors for the detection of S. typhimurium
in milk samples.
Figure 5. Bode plots illustrating the response of (a) BSA-blocked
Au/MUAM-MH/GA/anti-SA electrodes before and after their incubation for 5 h in milk samples inoculated with 5 ×104 cfu mL-1 (b) E.
coli, and (c) S. typhimurium. Filled symbols, impedance profile; empty
symbols, phase profile. Measuring conditions, 10-1-105 Hz at +0.200
V bias (10 mV rms). Electrolyte, 5 mM hexacyanoferrate(II)/(III) (1 +
1 mixture) in PBS solution, pH 7.
cannot be explained with the given data, and further studies should
be performed.
On the other hand, BSA-blocked immunosensors exhibited an
excellent performance. As can be seen in Table 2, for low- and
high-concentration BSA-blocked immunosensors, the background
signal dropped from 70 (blocking with TSB) to 44 and 29%,
respectively. This effect in combination with the higher sensitivity
provided by the BSA-blocked immunosensors (see values in Table
1 and Figure 2), results in a remarkable increase of the corresponding P/N ratios (85 and 36 for the low and high BSA-blocked
immunosensors, respectively), as can be seen in Table 2. The
calculated P/N ratios ensure safe results and certify the applicability of the method in milk samples (Figure 5). Sufficiently high
P/N ratios of 5-6 were also observed for a detection time interval
of 2 h (Table 2), or at milk samples being inoculated with a low
concentration of bacteria (100 cfu mL-1) for a detection time
interval of 10 h.
CONCLUSION
This study employs functional immunosensors, based on gold
electrodes modified with a mixed thiol-based SAM, as probes to
detect S. typhimurium in milk samples for first time.
Immunosensors that were developed on a mixed SAM of
MUAM-MH at a 1 + 3 proportion were found to exhibit better
sensitivity and selectivity compared with those developed on the
same SAM in the presence of TEA or on a single SAM of MUAM.
A very simple strategy for the amplification of the signal, based
on the performance of the immunoreaction directly in the culture
media, was successfully tested in both standard and real samples.
Due to this amplification scheme, low detection limits can be
achieved at the cost of course of prolonged times of detection.
For a detection time of 2 h, BSA-blocked immunosensors can
provide reliable analytical signals for a concentration of 3 orders
of magnitude lower to the infectious dosage of S. typhimurium.
In addition, the proposed method is insensitive to dead cells.
In our opinion, the present work offers a true alternative to
the existing ELISA and PCR methods, incorporating the simplicity
and advantages of biosensors.
ACKNOWLEDGMENT
The research project is cofunded by the European Unions
European Social Fund (ESF) & National Sources, in the framework of the program “Pythagoras II” of the “Operational Program
for Education and Initial Vocational Training” of the third Community Support Framework of the Hellenic Ministry of Education.
The authors thank Dr. Alexandra Koutsotoli for her valuable
assistance.
Received for review July 25, 2007. Accepted December
15, 2007.
AC071570L
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