IBP2102_16

RAPID DETECTION OF CORROSION INDUCING

MICROORGANISMS USING ANTIBODIES AS
BIORECEPTORS FOR ITS APPLICATION
INTO MICRODEVICES
Patricia Léo1, Débora C. Linhares2, Maria Filomena A. Rodrigues3,
Kleber L. Guimarães4, Mário R. Gongora-Rubio5, Jonas C. Gomes6,
Antônio F. Montemor7, Douglas H. S. Oliveira8, Beatriz A. A. Silva9,
Luciana W.S.L. Ramos10
Copyright 2016, Instituto Brasileiro de Petróleo, Gás e Biocombustíveis - IBP
Este Trabalho Técnico foi preparado para apresentação na Rio Oil & Gas Expo and Conference 2016, realizado no período de 24 a
27 de outubro de 2016, no Rio de Janeiro. Este Trabalho Técnico foi selecionado para apresentação pelo Comitê Técnico do evento,
seguindo as informações contidas no trabalho completo submetido pelo(s) autor(es). Os organizadores não irão traduzir ou corrigir
os textos recebidos. O material conforme, apresentado, não necessariamente reflete as opiniões do Instituto Brasileiro de Petróleo,
Gás e Biocombustíveis, Sócios e Representantes. É de conhecimento e aprovação do(s) autor(es) que este Trabalho Técnico seja
publicado nos Anais da Rio Oil & Gas Expo and Conference 2016.

Abstract
Biocorrosion or Microbial Induced Corrosion (MIC) is a process caused by anaerobic microorganisms, especially
sulfate-reducing bacteria (SRB). Checking the presence and concentration of these microorganisms is part of the
preventive measures routine against corrosion in several stages of oil and gas exploitation. In this article, the
development process of an impedimetric biosensor to rapidly detect SRB using specific antibody is described. The
target is the APS-reductase enzyme, which is involved in the biocorrosion process and presents common characteristics
into the SRB group, allowing the indirect detection and quantification of SRB. Specific monoclonal and polyclonal
antibodies were successfully obtained after animal immunization using a synthetic peptide as antigen, which is
representative of APS-reductase enzyme. Both monoclonal and polyclonal antibodies (anti APS-reductase) proved to be
SRB specific by Dot Immuno Binding Assay (DIBA). Monoclonal antibodies anti APS-reductase was immobilized in
commercial Screen-Printed Electrodes (SPE), which were first modified at the surface by a self-assembled monolayer
(SAM) technology using cysteamine coupling. Electrochemical Impedance Spectroscopy (EIS) was selected as the
analytical tool for evaluating the biosensor performance. Results have shown that this type of sensor is suitable to detect
different antigen (APS-reductase synthetic peptide) concentration. At this point, anti APS-reductase biosensors are being
prepared to be tested with SRB lysate from pure culture and from SRB field sample. Main challenges are related to
ensure repeatability of results using commercially available SPEs, and also to overcome possible interferences present in
field samples.

Resumo
Biocorrosão ou corrosão induzida por microrganismos (Microbial Induced Corrosion – MIC) é um processo causado
por microrganismos anaeróbios, especialmente bactérias redutoras de sulfato (BRS). O monitoramento da presença e da
concentração destes microrganismos auxilia na determinação de medidas para a prevenção da corrosão em vários
estágios da exploração de petróleo e gás. Neste artigo, o processo de desenvolvimento de um biossensor impedimétrico
para a detecção acelerada de BRS usando anticorpos específicos é descrito. O alvo da detecção é a enzima APS-
redutase, a qual está envolvida no processo de biocorrosão e apresenta características comuns ao grupo das BRS,
permitindo a detecção indireta e até mesmo a quantificação de BRS. Anticorpos monoclonais e policlonais específicos
para a detecção de BRS foram obtidos com sucesso após imunização de animais utilizando como antígeno um peptídeo
sintético representativo da enzima APS-redutase. Ambos os tipos de anticorpos, monoclonal e policlonal (anti APS-
redutase), provaram especificidade para as BRS por meio do teste DIBA (Dot Immuno Binding Assay). Os anticorpos
do tipo monoclonal anti APS-redutase foram imobilizados na superfície de eletrodos impressos (Screen-Printed
Electrodes – SPE), previamente modificada pela tecnologia de formação de monocamadas auto-organizadas (Self-

1
Ph.D., Biologist – Instituto de Pesquisas Tecnológicas – IPT/SP
2
M.Sc., Biologist – Instituto de Pesquisas Tecnológicas – IPT/SP
3
Ph.D., Biochemist – Instituto de Pesquisas Tecnológicas – IPT/SP
4
Ph.D., Materials Engineer – Instituto de Pesquisas Tecnológicas – IPT/SP
5
Ph.D., Electrical Engineer – Instituto de Pesquisas Tecnológicas – IPT/SP
6
Chemical Technician – Instituto de Pesquisas Tecnológicas – IPT/SP
7
Clinical Analysis Technician – Instituto de Pesquisas Tecnológicas – IPT/SP
8
B.Sc., Pharmaceutic – Hospital das Clínicas /SP
9
Undergraduate Student, Pharmaceutic – Fundação do Instituto de Pesquisas Tecnológicas – FIPT/SP
10
Ph.D., Mechanical Engineer – Instituto de Pesquisas Tecnológicas – IPT/SP
 Rio Oil & Gas Expo and Conference 2016

Assembled Monolayer – SAM) usando cisteamina para acoplamento do anticorpo. Espectroscopia eletroquímica de
impedância foi a ferramenta analítica selecionada para avaliação do desempenho do biossensor. Os resultados mostram
que o biossensor é capaz de detectar diferentes concentrações de antígeno (peptídeo sintético da APS-redutase). No
momento, os dispositivos biossensores anti-APS estão sendo preparados para testes com lisados de culturas puras de
BRS e com BRS provenientes de amostras de campo. Os principais desafios estão relacionados à garantia da
repetitividade dos resultados utilizando SPEs disponíveis comercialmente, e também a superar possíveis interferências
em amostras de campo.

1. Introduction
1.1. Problem characterization
Prevention of corrosion processes has fundamental importance to the oil industry, since the costs related to
such events result in additional spending on petroleum exploitation, transportation and refining. Critical problems come
from microbial induced corrosion (MIC), caused by anaerobic microorganisms (Muyzer & Stams; 2008).
An important group of microorganisms involved in most of the biocorrosion cases are the sulfate reducing
prokaryotes (SRP), conventionally known as sulfate reducing bacteria (SRB), which occur in aqueous environments in
the absence of oxygen. The hydrogen sulfide (H2S), produced as a metabolite from the sulfate to sulfide reduction
process, contaminates gas and stored oil, precipitates ferrous sulfide found at injection wells and promotes corrosion.
Therefore, the risk of corrosion can be directly associated with the concentration of these microorganisms (SRB) present
in the environment. SRB detection and quantification during the oil extraction, storage and transportation, as well as
during oil products processing, is an important task, since the result will define the routine of preventive measures,
which includes the use of PIG (Pipeline Inspection Gauge) cleaning and biocides application.
In general, conventional methods for monitoring the SRB population are based on the estimation of the most
probable number (MPN) of microorganisms, determined after a growth step in multiple tubes with selective medium,
which needs about 28 days of incubation. The extended growth time does not allow the application of an immediate
corrective action against the biological corrosion process. Moreover, the method is dependent on the operator ability for
sample processing, inoculation, and analysis of results, which is based on visual observation of a black precipitate,
mainly represented by ferric sulfide resulting from microbial metabolism.
Therefore, more effective, accurate and fast measuring methods are being studied. Technologies involving
immunoassays using antibodies against conserved proteins could allow high sensitivity and selectivity for the detection
of SRB. Such approach has been already explored by some research groups, leading to the development of commercial
kits (Modernwater, 2012, Saniv Check, 2007, Strategic Diagnostics Inc., 2005).
The technique of Electrochemical Impedance Spectroscopy (EIS) is an analytical tool extensively used in
corrosion studies (Wang et al, 2014), characterization of charge transport through membranes (Montalvillo et al, 2014),
development of batteries ( Noack et al, 2014), and its usefulness in detecting interfacial events is known for over two
decades (K'Owino, et al, 2005). Recently, the development of biosensors and the integrated use of EIS are becoming
more common as a potential solution to contribute in these monitoring processes (Wan et al, 2009; 2010).

1.2. Biosensor
A biosensor is an analytical tool used for the detection and/or quantification of biochemical elements, and is
characterized by the presence of a biological component, as a recognition molecule, fixed on the surface of a transducer.
This latter generates a signal proportional to the intensity of the reaction in course on its surface, when in contact to the
sample containing the element to be detected, i.e., the target (Nirsch et al, 2011 & Collings et al, 1997).
The recognition molecules may be enzymes, cellular receptors, antibodies, DNA sequences, microorganisms,
cells/tissues of animals or plants (Daniels et al. 2007).
According to the type of signal transducer, a biosensor may be electrochemical (impedimetric, potentiometric,
potentiometric by stripping, amperometric or conductimetric), acoustic (surface acoustic wave, quartz microbalances
and piezo-electric acoustic elements), optical or colorimetric (absorbance, chemiluminescence, SPR - Surface Plasmon
Resonance, optical fibers) and thermal, among others (Collings et al, 1997 & Sua et al, 2011).

1.3. Proposal for Detection Method

In this work we aim to develop a more efficient, accurate and fast method for monitoring biocorrosion in oil
production systems. We are focused in constructing biosensors for SRB detection based on the use of monoclonal and
polyclonal antibodies as molecules to recognize a highly conserved enzyme present in most of SRB. APS-reductase
enzyme has been selected as a target for the antibody recognition because of its characteristic presence at the SRB
group. This enzyme is widely distributed in different groups of living beings, with a common signature to all of them,
but with specific clusters that mark certain groups. Based on these considerations, the antigen for animal immunization
and antibody production should represent a fragment from the APS-reductase enzyme, consensual to SRB group.

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Commercial Interdigital Electrodes (IDEs) from Dropsens, G-IDEAU5, have been chosen as the transducer.
They are composed of two connection tracks, all made of gold/chrome sputtered on a glass substrate, with bands/gaps of
5 μm, number of digits of 250 x 2 and digit length of 6760 μm.
The IDE is connected to a potentiostat AUTOLAB / PGSTAT302N with frequency analyzer suitable to
perform Electrochemical Impedance Spectroscopy (EIS). Gold electrodes are already used for the immobilization of
enzymes and antibodies. IDEs offer advantages, such as, increased sensitivity, fast dynamic response, high aspect ratio,
and also increased signal to noise ratio for impedimetric measurements using impedance spectroscopy (Wang et al,
2012).

2. Experimental
2.1. Antibody production
Monoclonal and polyclonal antibodies anti APS-reductase were obtained from immunized animals against a
synthetic peptide representing a consensus sequence of APS-reductase from SRB group. The structure of the synthetic
peptide was determined from the characteristics of the target enzyme. Using a preliminary alignment, the antibodies
supplier (Rheabiotch) employed appropriate software and selected the most promising amino acid sequence. The chosen
sequence was synthesized, resulting in a 24 amino acid peptide (Rheabiotch). Polyclonal antibodies were obtained after
IgG purification of serum from immunized rabbits and monoclonal antibodies were obtained by hybridoma technique
using cells from immunized mouse. Monoclonal antibody secreted by hybridoma was purified from culture supernatant.

2.2. Bacteria cultivation and lysate preparation

Bacterial lysates were prepared for application on detection and specificity tests. Pure cultures of two SRB
lineages, as well as a field sample confirmed for SRB, were grown in modified Postgate’s medium at 30°C during 4 days
under anaerobic conditions. Pseudomonas aeruginosa and Escherichia coli were grown in nutrient broth medium. All
bacterial cells were then harvested by centrifugation and pelleted cells were lysated using about 10 volumes of lysis
buffer (1% SDS, 5 mmol/L EDTA, 20 mmol/L TRIS pH 8.0, 1 mmol/L PMSF e 10 mmol/L ditiothreitol). Cell lysate
suspensions were homogenized and incubated at 90°C, 20 min., centrifuged, and supernatant containing proteins was
kept at -20°C.

2.3. Biological Recognition by Immunoassay

The recognition of the synthetic peptide, which represents the key enzyme to biocorrosive process, by
monoclonal and polyclonal antibodies was realized by ELISA tests (Enzyme Linked Immuno Sorbent Assay). Different
antigen concentration, antibodies and conjugate titers were assayed, in order to selected the best antibodies to be tested
for SRB recognition, Pseudomonas aeruginosa and Escherichia coli in qualitative assays (Dot Immuno Binding Assay –
DIBA). Supernatant lysates of pure culture of Pseudomonas aeruginosa, Escherichia coli and two strains of SRB, as
well as supernatant lysates of field samples confirmed for SRB presence, were incubated with the monoclonal and
polyclonal antibodies and revealed using anti-mouse or anti-rabbit HRP (horse radish peroxidase) conjugate,
respectively.

2.4. Electrode preparation

The protocol used for cleaning the IDE gold surface consisted of consecutive steps described as follows: keep
at RCA-1 cleaning solution for 15 min, wash in flowing deionized (DI) water and dry in a filtered nitrogen stream, keep
at oxygen plasma cleaning for 10 min, wash in flowing DI water and dry in a filtered nitrogen stream. Then the
electrodes were immersed in boiling ethanol at a temperature of 70°C to 80°C for 5 min, washed in flowing DI water,
dried in a filtered nitrogen stream, immersed in boiling acetone at a temperature 70 e 80°C for 5 min and exposed to
ultrasound cleaning in isopropanol for 10 min. Afterwards, the electrodes were again washed in flowing DI water and
dried in a filtered nitrogen stream.
Cystamine dihydrochloride (CYS, C4H12N2S2·2HCl) (Sigma–Aldrich), 11-mercaptoundecanoic acid (MUA,
HS(CH2)10CO2H) (Sigma-Aldrich) and 25% glutaraldehyde solution (GA) (Merck) were used for the preparation of the
SAMs. Deionized water was used to prepare a 3·10-2 mol·L-1 cystamine solution and to rinse the electrodes prior to the
electrochemical measurements. A 80% (v/v) ethanol:water solution was used to prepare a 10 -2 mol·L-1
mercaptoundecanoic solution. A 0.1 mol·L-1 phosphate buffer solution at pH 7.4 was used to prepare a 2.4% (v/v)
glutaraldehyde solution and also to prepare the supporting electrolyte containing the redox pair necessary to perform the
EIS measurements (10-3 mol·L-1 Fe(CN)63-/4-). The protocol for preparing the SAM consisted of consecutive steps
described as follows: the electrodes were immersed in cystamine solution, overnight, washed with DI water and
immersed in hydroalcoholic MUA solution during 2 hours. After DI water washing, electrodes were then immersed in
glutaraldehyde solution for 2 hours and afterwards washed with PBS (Phosphate Buffer Saline).
For monoclonal antibody anti APS-reductase immobilization, SAM modified electrodes were immersed in the
antibody solution (1:1000) prepared in PBS during 2 hours under agitation at 37°C and then overnight at 4°C. The

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biosensor was treated with 2% of BSA-PBS solution for 2 hours, under agitation at 37°C, to block the nonspecific sites.
Then, the electrodes were washed with PBS and immediately used.

2.5. Antigen Recognition by Impedance Measurements

The electrodes with the surface already modified and presenting the immobilized antibody were immersed in
suspension with the target APS-reductase synthetic peptide at 1:500 (4 µg/mL), 1:1500 (1,3 µg/mL), and 1:2500 (0,8
µg/mL) dilutions, incubated at 37°C for 2 hours. After the reaction time, the electrode was immersed in electrolyte
(buffer + pair Redox) and subjected to the passage of AC current to obtain the electrochemical impedance spectroscopy
curve.
The electrochemical impedance measurements were performed with a PGSTAT 302-N, AUTOLAB instrument
from Echo Chemie, using a 10 mV sinusoid within the frequency range of 0,05Hz to 50kHz and 10 points per decade of
frequencies at room temperature. The FRA software was used for complex circuit modelling. Potassium ferricyanide
(K3Fe(CN)6) and ferrocyanide (K4Fe(CN)6) (Sigma–Aldrich) were employed for electrochemical characterization of the
SAMs using EIS.

3. Results and Discussions

Recognition of the APS synthetic peptide by monoclonal and polyclonal antibodies was shown through ELISA
tests. In these assays, both monoclonal (1:2000 dilution) and polyclonal (1:200 dilution) antibodies showed positive
reaction against APS synthetic peptide (0,5 µg/well) adsorbed on the plate.
Selected antibodies were applied on qualitative assay DIBA. Monoclonal and polyclonal antibodies anti APS-
reductase were able to recognize APS-reductase whole enzyme present in SRB cell lysates supernatant, both pure
culture and enriched field samples. As indicated in the Figure 1, results of DIBA with different samples showed positive
reaction in the presence of supernatant lysates of SRB cultivation (indicated by dark spots), using both monoclonal and
polyclonal antibodies, while no dark spots were observed against supernatant lysates of Escherichia coli or
Pseudomonas aeruginosa. The positive response of field samples for both antibodies indicates the potential for SRB
detection using an anti APS-reductase sensor device based on the principle of antigen-antibody reaction. Further tests
will be carried out using more strains, including anaerobic microorganisms other than SRB.

Figure 1. Qualitative assay to detect SRB using monoclonal (left)

and polyclonal (right) antibodies at DIBA (dot immunobinding
assay). Positive reaction (dark spots) to cell supernatant lysates of
SRB A and SRB B (pure cultures of SRB) and field (field
sample). Negative reaction (no dark spots) against E. coli and
Pseudomonas cell lysates supernatants.

Polyclonal antibodies usually have greater affinity for the target molecule than monoclonal antibodies. On the
other hand, their production is dependent on the provided immunization of animals, so each new batch produced can be
different from the previous one, resulting in non-reproducibility of the experiments. Despite the restricted recognition of
a small and unique target fragment, monoclonal antibodies have the advantage of being suitable for continuous
production in laboratory through cultivation of antibody secreting hybridoma cells (Roitt &Delves, 2001). Here,
monoclonal antibody anti APS-reductase was applied to detect SRB at impedimetric biosensor. This is the first report of
monoclonal antibodies being explored for SRB recognition and detection as well as used to construct a sensor device.
Impedance results for modified surfaces in the presence of Fe(CN)63-/ Fe(CN)64- redox couple in phosphate
buffer solution, exposed to different antigen (APS-reductase synthetic peptide) concentrations ranging from 0.8 µg/mL
to 4.0 µg/mL, is shown is Figure 2. A significant change in the semicircle diameter in the Nyquist plot, and hence in the
charge transfer resistance (Rct), was observed at each step of the interface fabrication (results not reported here),
considering the bare state conditions, including the SAM formation, and the antibody immobilization step.
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1:2500

1:500
1:1500

Figure 2. Complex plane plots (Nyquist plots) on

SAM modified and antibody coupled Au electrode
for different concentrations of the antigen APS-
reductase synthetic peptide.

The charge transfer resistance corresponding to the situation in which the SAM is already formed and the
antibody is immobilized on the surface is designated as Rct(0), as presented in Figure 3. A further increase in Rct was also
observed after the subsequent exposition and resultant attachment of different concentrations of the antigen APS-
reductase synthetic peptide. This increase of the charge transfer resistance may be justified based on a barrier effect that
hinders the ability of the redox molecule from accessing the electrode surface. The Randles circuit used to fit the
Nyquist plot, includes the solution resistance (Rs), the charge transfer resistance (Rct), the double layer capacitance,
treated as a constant phase element impedance (Cdl) indicating the depressed semi-circle obtained, and the Warburg
impedance element (Zw) which is the consequence of the ionic diffusion from the bulk of the electrolyte to the electrode
interface. Results indicated that the Rct is a suitable signal for sensing the interfacial properties of the prepared biosensor
during all the assembly procedures.

Gold Surface + SAM +

Rct(o) = 27,2 kΩ
Antibody
Gold Surface + SAM +
Rct = 40,8 kΩ
Antibody + Antigen (1:2500)
Gold Surface + SAM +
Rct = 35,4 kΩ
Antibody + Antigen (1:1500)
Gold Surface + SAM +
Rct = 27,4 kΩ
Antibody + Antigen (1:500)

Figure 3. The Randles equivalent circuit for the impedance spectroscopy measurement and the respective
experimentally determined charge transfer resistance (Rct) using APS-reductase synthetic peptide as antigen.

A more diluted sample containing a 0,8 µg/mL (1:2500) antigen concentration resulted in 50% increase of
the experimentally determined charge transfer resistance once compared to the surface modification represented by the
adsorbed SAM and immobilized antibody. Incubating the surface in a solution of 1.3 µg/mL (1:1500) resulted in only
30% increase in reference to Rct(0). These limited results indicate that once increasing the concentration of the antigen, a
progressive reduction of charge transfer resistance was evidenced. Once increasing the concentration to 4,0 µg/mL
(1:500) the relative Rct variation is almost negligible, corresponding approximately to 0.8%.
In order to compare the different electrode performance when exposed to different antigen concentrations in
a quantitative base, the relative values ([(Rct(i)-Rct(0)]/Rct(0)) were used as the argument function for the y axis. Rct(i) is the
charge transfer resistance of the modified surface after reaction with antibodies for predefined period of time. Despite
the limited concentration range studied in the present work and also the limited number of experimental points analyzed,
preliminary results indicate that the sensing interface can detect APS-reductase synthetic peptides with a promising
potential to be used as a quantitative analytical tool for SRB determination.
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Although a linear relationship between the electron transfer resistance and the APS synthetic peptide
concentration was found (R2 factor is approximately 94% in Figure 4), it is highly recommended to expand the
concentration range and also to include other experimental data to ensure that the linear fit is valid.

Figure 4. The calibration plot corresponding to the change of electron

transfer resistance of the biosensor with the concentration of APS-
reductase synthetic peptide.

Several authors, such as Shervedani et al. (2006), cite the equation Rct=RT·(n2·F2·A·kct·[S])-1 to explain the
relation between bulk concentration of the redox probe and charge transfer resistance, where R is the ideal gas constant,
T is the absolute temperature, n is the number of transferred electrons per one molecule of the redox probe, F is the
faraday constant, A is geometric surface area of the electrode (cm2), kct is potential dependent charge transfer rate
constant and [S] corresponds to the concentration of the redox probe (mol/cm3). By applying a simplification to the
presented equation, one may replace [S]=k1·[analyte], where k1 is a constant. If all other parameters are set constant, a
linear relationship as 1/Rct=k·[analyte] is simply found, in which k includes all constants. As a function of this, a
calibration plot represented by 1/Rct=f(concentration) is proposed and widely used. The option to use relative values
([(Rct(i)-Rct(0)]/Rct(0)) as done in this work and supported by Liu et al.(2012) appears as an alternative to overcome
reproducibility problems concerning the variability of Rct(0) typical of commercially available sensing platforms
represented by disposable screen-printed electrodes from a single production batch. This critical issue is severely
discussed by Kadara et al. (2009) and still represents a challenge to enable a laboratory development to turn into a
commercial product.
Further studies will be conducted with a focus on improving repeatability of the developed device. Future
challenges are also related to overcome possible interferences inherent to field samples.

4. Conclusion
In this work monoclonal and polyclonal antibodies (anti-APS) were obtained using as antigen a synthetic
peptide representative of APS-reductase enzyme, common to the target microbial group (SRB). Both types of antibodies
showed reaction against the tested antigen and specificity to SRB group. Impedimetric immunosensor was developed by
immobilizing anti-APS antibodies on self-assembled cysteamine monolayers via covalent coupling on commercial
screen-printed electrodes (SPE). Immunosensor test results are preliminaries but it is possible to show that the antibody
was properly immobilized and impedimetric measures are proportional to different antigen (APS-peptide synthetic)
concentration. This result indicates that the immunosensor can detect APS reductase enzyme present in SRB lysate in a
rapid way. This technique represents a promising alternative to the traditional MPN method for a rapid monitoring of
microbial populations involved in biocorrosion, thus supporting the faster application of preventive measures.

5. Acknowledgments
The authors thank Professor Hideko Yamanaka and Dr. Antônio Pupim Ferreira from the Institute of Chemistry at
Unesp-Araraquara for the contribution on the sensor preparation, and IPT/FIPT/Governo do Estado de São Paulo for the
financial support.

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