Detection of Listeria Monocytogenes Using an Electrochemical Biosensor

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Investigation into the development of a
biosensor for the detection of Listeria
Monocytogenes using a Macro-fabricated device,
and Electrochemical Impedance Spectroscopy as
an electrochemical analysis method.
Student name: Brian Keith Muldoon.
Student number: 111329141.
Supervisor: Dr. Karen Twomey.
Course Coordinator: Dr. Miloslav Pravda.
Head of Chemistry: Prof. Martyn Pemble.
BSc. Chemistry with Forensic Science Final Year Project Report.

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Declaration of Originality:
I declare that this is my own work, in partial fulfilment of the requirement of the
degree of BSc in Chemistry with Forensic Science.
Information derived from the published and unpublished of others has been
acknowledged in the text and a list of references is given in the bibliography.
This report is based on research carried out in the Tyndall National Institute, UCC,
Cork, between September 2014 and March 2015.
Date: ___________
2nd
April 2015.
Signature: __________________
BrianKeithMuldoon.

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Acknowledgements:
I would like to express a particular word of thanks to my supervisor Dr. Karen
Twomey for her assistance throughout the completion of the project and her help for
whenever I encountered a problem.
I would like to thank the Tyndall Institute, especially, the facilities and equipment of
the Life Science Interface group and all its members. Their help and guidance was more than
appreciated. Especially, Eileen Hurley, for all the insight and training she provided.
The Chemistry Department, UCC, namely Dr. Miloslav Pravda, for his advice before,
during and after the project.
My gratitude to my parents for putting me through 4 years of college and providing
encouragement and support (and funding) over the years.
For my lab partner, Alison Mahon, I would like to say an extra word of gratitude for
her great work throughout the duration of the experiment and making it enjoyable and
worthwhile.

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Abstract:
Foods contaminated with pathogens that cause serious illnesses (e.g.) Listeria
monocytogenes impacts hugely on the health and economy throughout the world. Due to
this, it is detrimental to find a cure for these problems and illnesses that people worldwide
face on a daily basis.
One such way of preventing the problems from even starting is to develop and
validate methods for the real time and rapid detection of listeria monocytogenes. Current
detection methods including, PCR and ELISA, require 3-4 days for presumptive testing and a
further 5-7 days for confirmation tests. Rapid, state of the art detection techniques are
urgently needed. Miniaturization of biosensors is very useful as they can be incorporated
within a portable device and in-situ measurements can be carried out for on-site
applications particularly in the detection of listeria monocytogenes.
The main aim of this project is to develop a biosensor which can readily detect a
major biological food borne pathogen. In developing the biosensor many different surface
chemistry steps and electro-analytical techniques will be analysed. These techniques include
cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). When CV and
EIS are used together they give a more in depth analysis of the solid/liquid interface surface.
The solid interface is the gold biosensor and the liquid interface is Ferrocence carboxylic acid
(FCA) and this acts as an electro-active probe species. CV reacts to a change in potential
whereas EIS changes with a change in frequency. The surface chemistry that were used to
functionalise the sensor involved treating the gold active site with EDC/NHS (1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide / N-hydroxy succinimide), and MPA, 99% (3-
mercaptopropionic acid). EDC/NHS facilitates the attachment of antibodies to the sensor,
and MPA allows the formation of SAMs (self-assembled monolayers).

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Table of contents:
Cover Page ………………………………………………………………………………………………………………………1
Declaration of Originality …………………………………………………………………………………………………2
Acknowledgements …………………………………………………………………………………………………………3
Abstract …………………………………………………………………………………………………………………………..4
Table of Contents …………………………………………………………………………………………………………….5
List of Abbreviations and Symbols ………………………………………………….……………………………….7
Chapter 1: Introduction and Literature Review…………………………………………………………………9
1.1 Listeria Monocytogenes ………………………………………………………………………………..….9
1.1.1 Current Methods of Detection …………..…………………………………..………12
1.1.2 State of the Art Detection Techniques ……………………………………..…….13
1.2 Biosensors ………………………………………………………………………………………………….….…13
1.3 Electrochemical Immunosensors ……………………………………………………………………..16
1.4 Difference between Macro- and Micro- electrodes ………………………………………….16
1.5 Electrochemical Characterisation……………………………………………………………………19
1.5.1 Cyclic Voltammetry …………………………………………………………………………19
1.5.2 Electrochemical Impedance Spectroscopy ………………………………………21
Chapter 2: Methodologies ……………………………………………………………………………………………..26
2.1 Risk Assessment
…………………………………………………………………………….…………………..26
2.2 Fabrication …………………………………………………………………………………….…………………27
2.3 Resist Removal ……………………………………………………………………………………….………..29
2.3.1 Resist Removal ……………………………………………………………………….………….30

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2.4 Electrochemical Cell Set-up …………………………………………………………………………..32
2.5 Cyclic Voltammetry and Electrochemical Impedance Spectroscopy …….……35
2.6 Cleaning Electrode with Piranha …………………………………………………………….……..37
2.7 Formation of Self Assembled Monolayers (SAMs) ……………………………….………38
2.7.1 Overnight step in MPA …...38
2.7.2 Treatment with 0.4M EDC and 0.1M NHS …………………………………………39
2.8 Antibody attachment ……………………………………………………………………….……………..41
2.8.1 Removal of unbound antibodies ……………………………………………………….41
2.9 Blocking the electrode ………………………………………………………………………….………….42
2.10 Dipping in PBS ……………………………………………………………………………………….……….42
2.11 Attachment of Listeria Innocua Cells …………………………………………………….………..42
Chapter 3: Results and Discussion ………………………………………………………………………………….44
Chapter 4: Conclusion and Future Work ………………………………………………………………………...63
Chapter 5: References ……………………………………………………………………………………………………65

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Table of Abbreviations and Symbols:
Chemicals:
Ag/AgCl Silver/Silver Chloride
Au Gold
EDC 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide
EtOH Ethanol
FCA Ferrocence Carboxylic Acid
H2O2 Hydrogen Peroxide
H2SO4 Sulphuric Acid
IPA Iso Propyl Alcohol
KCl Potassium Chloride
MeOH Methanol
N2 Nitrogen
NaCl Sodium Chloride
NHS N-hydroxy succinimide
Pt Platinum
Si3N4 Silicon Nitride
SiO2 Silicon Oxide
BSA Bovine Serum Albumin
PBS Phosphate Buffered Saline
Acronyms
Ab Antibody
Ab-Ag Antibody-Antigen
CV Cyclic Voltammetry
DI De-Ionised
EIS Electrochemical Impedance Spectroscopy

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ELISA Enzyme Linked Immunosorbent Assay
Hz Hertz
RT Room Temperature
SAM Self-Assembled Monolayers
Symbols
A Maximum Amplitude
A Area of Electrode
Da Diffusion Coefficient
E Electrochemical Potential(V)
F Faradays Constant
I Current
If Faradic Current
N Number of electrons
R Gas Constant
T Temperature
T time
V Scan Rate in CV (V/s)
Z Impedance

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Chapter 1: Introduction and Literature Review
1.1 ListeriaMonocytogenes:
Listeria monocytogenes is a food borne biological gram positive bacteria that can cause
listeriosis[1]. Listeria monocytogenes is a facultative pathogenic saprophyte and is able to
multiply over a wide range of pHs and osmolarity, at low temperatures and both aerobic
and anaerobic conditions. This causes a concern and necessitates control along the food
chain. Listeria monocytogenes can grow over a temperature range of -0.4°C to 45°C, with
the optimum about 4°C, a pH range of 4.0 to 9.6, with the optimum 6-8[2].
Listeria can be seen in food processing environments due to its saprophytic lifestyle[3].
Listeria usually contaminates foods during fermentation, processing, storage and packaging
of foods [2]. Listeria monocytogenes is able to maintain itself in food processing facilities for
several months or even years. This is because it can stick to many various surfaces such as
stainless steel and polystyrene. Also, listeria can survive as biofilms which can protect it
from cleaning agents and environmental agents at high concentrations[2]. Poor hygiene
practices, temporary breakdown in hygiene barrier efficiency and unhygienic equipment
may lead to food plant contamination. Once listeria monocytogenes contaminates a food
processing environment, it can spread through the inappropriate movements of personnel
and food workflows. This type of contamination can be a secondary step of contamination
form the origin of the listeria monocytogenes (e.g.) in biofilms, water and organic soil
residues, to food contact surfaces and food under processing. As part of the sampling
scheme for food business operator’s manufacturing ready to eat foods need to continuously
test processing areas and equipment for the absence and presence of listeria
monocytogenes. Guidelines for the sampling of food processing environments and
equipment have already been published[3].
In Regulation (EC) 2073/2005, amended in 1441/2007 the EU has established
microbiological food safety criteria for listeria monocytogenes in ready to eat foods. At all

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stages of the food chain, business operators must comply with the relevant microbial
criteria. There are two categories of food in regards to listeria, food that can support the
growth of listeria monocytogenes and those that cannot, with different standards applying
in each case. Ready to eat foods that are unable to support the growth of listeria have a
100CFU/g limit during its shelf life. In the Regulation, for ready to eat foods that support
listeria, which poses a health risk the pathogen must be absent in 25g of product, it states
‘’before the food has left the immediate control of the food business operator who has
produced it’’. For the 100 CFU/g limit to be allowed the food business operator must provide
a guarantee that it will observe all limits during processing. The Regulation goes on to state
that ‘’as necessary, the food business operators responsible for the manufacture of the
product shall conduct studies … in order to investigate compliance with the criteria
throughout the shelf life’’. These studies should include ‘’physic-chemical characteristics of
the product such as : pH, aw, salt content, concentration of preservatives and type of
packaging system, taking into account the storage and processing conditions, the
possibilities for contamination and the foreseen shelf-life, and – consultation of available
scientific literature and research data regarding the growth and survival characteristics of
micro-organisms of concern’’[4].
It has been reported that listeria monocytogenes can grow in raw and unpasteurized milk
semi-soft rind washed cheeses when temperature varies from 4 to 15 °C during storage[5].
On certain cheeses (e.g.) Graviera cheese, listeria was unable to grow, regardless of
packaging systems and storage temperatures, but listeria can survive at different
temperatures. Listeria monocytogenes survived better at 4 °C than at 12 °C. such survival at
low temperatures was also seen in other cheeses from, hard processed cheese to semi-hard
raw sheep milk cheese[4]. It is believed that the survival of listeria monocytogenes at such
low refrigeration temperatures is to stress resistance mechanism, its metabolism slowing
down, when lactic acid growth is at its weakest and when aw decreases least (0.96- 0.93)[4].
The first known outbreak of listeria monocytogenes as food borne listeriosis was reported in
Canada in 1983, with coleslaw as the infected food source. The deadliest outbreak that has

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been reported occurred in 2011 in USA. It caused 146 illnesses 30 deaths and one
miscarriage. The food that was associated with the outbreak was cantaloupe. Listeria
monocytogenes can enter the food chain directly or farm animals (e.g.) zoonotic disease.
The dose that is suspected to cause infection is very high. Contamination levels in food
responsible for causing listeriosis cases are typically >104 CFU/g. Consuming food that
contain low levels (e.g.) <102 CFU/g are not suspected of causing infection but levels
between 102 and 104 CFU/g has been known to cause listeriosis in immunocompromised
people. In the European Union, it was reported that there was 1476 confirmed cases of
listeria monocytogenes in 2011. Of all the zoonotic diseases being watched by the EU,
listeriosis caused the most severe human disease with a fatality rate of 12.7%. Listeriosis is
estimated to have an economic cost of up to 2.04x1012$ and each separate case costs
1,282,000 annually. This cost is made up of health care costs, lost productivity and
diminished quality of life[2].
Some symptoms of listeriosis include meningitis, septicaemia, abortion and febrile
gastroenteritis[1]. Listeriosis has a high mortality rate of 30%[1]. Due to its high mortality rate,
the disease is one of the most dangerous food borne infections. Listeriosis results in up to
2000 hospitalizations and 500 deaths in the US annually[6]. Most victims include pregnant
women, new-born children and immunocompromised people especially the elderly. Listeria
monocytogenes contaminates mostly dairy food such as milk, cheese, meats, ice-cream and
raw vegetables[6]. The United States of America adopt a zero tolerance policy for the
presence of listeria monocytogenes in food. Whereas, Canada allow up to 500 CFU/g of
listeria monocytogenes in food [1]. The European Union has also adopted the same zero
tolerance policy as the USA[7]. The main problem with implementing a zero tolerance
programme is the lack of reliable procedures to determine such low values of listeria in
food. This may be difficult to identify pathogens rapidly from clinical and food contaminated
samples[7]. The flora of healthy animals and man does not include listeria.
The strain of listeria monocytogenes that affects humans is highly sensitive to most
antibiotics such as, penicillin, ampicillin, amoxicillin, gentamicin, erythromycin, tetracycline

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and rifampicin. Further to this, listeria monocytogenes also shows natural resistance to
fluoroquinolones and cephalosporin’s. To treat the infection, clinicians use aminopenicillins
(i.e.) ampicillin or amoxicillin with aminoglycoside (i.e.) gentamicin. When treating cases
where there is reduced sensitivity or total resistance to beta-lactams, other medication used
against grampositive bacteria can be used such as cotrimoxazole[8]. Some listeria species
like listeria innocua have become resistant to antibiotics and through various different
mechanisms can transfer genetic material from one species to another. The transfer
happens in vitro for streptomycin, erythromycin and chloramphenicol. Therefore, it has
become of utmost importance to monitor the emergence of antimicrobial-resistant listeria
strains, considering its high mortality rate. Due to this is, it is also highly important to ensure
the effectiveness of antimicrobials for listeriosis[8].
It has become very important to distinguish between listeria monocytogenes and listeria
innocua as both species can co-exist in the same food or processing environment[8]. The
non-pathogenic form listeria innocua is just as abundant in the environment as listeria
monocytogenes. It is found in the same niches as listeria monocytogenes such as ground
and surface water samples, in faecal samples and in the soil. Listeria innocua has a higher
prevalence and incidence over listeria monocytogenes. This high presence of listeria innocua
is owed to its dominance and efficient use of nutrients during enrichment culturing[9].
Listeria innocua may inhibit the growth of listeria monocytogenes by producing
antimicrobial substances, such as phage tails, replicative phage’s, inhibitory proteins and
peptides or through a strain dependent quorum-sensing mechanism[9]. The fact that the
presence of listeria innocua masks the presence of listeria monocytogenes could lead to a
false negative result for the presence of listeria monocytogenes[10].
1.1.1 Current methods of detection.
Conventional methods, including standard ISO and USDA/FSIS methods, for the detection
of LM in food are accurate and reliable. Some methods have been developed and are used
for rapidly detecting or identifying LM in food, including polymerase chain reaction (PCR)
assay and enzyme-linked immunosorbent assay (ELISA). These methods meet the

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requirement for LM detection, but have some disadvantages. For instance, these methods
are labour-intensive and time-consuming. ELISA requires sample enrichments and
processing before analysis, whereas PCR assay requires pre-enrichment, DNA extraction,
amplification, and so on. These techniques take up to 3-4 days presumptive testing and a
further 5-7 days for confirmation tests. Moreover, PCR-based methods are highly
dependent on efficient DNA isolation and limited by its accuracy in detecting live bacterial
cells, leading to false positive identification[1].
1.1.2 State of the art methods of detection.
There are also some reports on biosensors used for the detection of LM, such as quartz
crystal microbalance (QCM) immunosensor, fibre-optic immunosensor and surface plasmon
resonance, the detection limit of them are 107 CFU/ml, 4.3 × 105 CFU/ml and 105 CFU/ml,
respectively[1].
1.2 Biosensors
In 1956, Clark published his definitive paper on the oxygen electrode. Based on this
experience he made a landmark address in 1962 at a New York Academy of Sciences
symposium and described “how to make electrochemical sensors (pH, polarographic,
potentiometric or conductometric) more intelligent” by adding “enzyme transducers as
membrane enclosed sandwiches”. The idea was shown by an experiment in which glucose
oxidase was entrapped at a Clark oxygen electrode using a dialysis membrane. The decline
in measured oxygen concentration was proportional to glucose concentration. In the
published paper, Clark and Lyons coined the term enzyme electrode. [11]
In 1987, Vo-Dinh and co-workers showed that antibodies could be incorporated in situ for
the detection of a chemical carcinogen in a fibre optic-based immunosensor. [12] Antibodies
have since proven their usefulness as powerful tools for electrochemistry applications.
Essentially, the selectivity or specificity of the biosensor is dependent on the bio recognition
element, which is capable of “sensing” the presence of an analyte. [13][14] Immunosensors
utilising antibody-based recognition elements, have been developed on a wide range of

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transduction platforms for a multitude of analytes. The transducer element translates the
selective recognition of the analyte into an easy to detect signal and therefore, has major
influence on sensitivity.[15]Transduction approaches include electrochemical, piezoelectric
and optical systems.[16]
There are currently three generations of biosensors in existence. The term generation was
originally created to describe the stages of integration in biosensors. Biosensors with
mediated response mainly generated by membrane-bound or membrane entrapped bio
components (first generation), were followed by virtually reagent less biosensors where bio
components are fixed directly to the sensor surface and either the reaction of co-
immobilised co-substrates (bound to the electrode, polymer or enzyme itself) or the direct
heterogeneous electron transfer between the prosthetic group and the electrode is
exploited (second generation). While the immobilisation of the receptors directly on an
electronic circuit leads to systems with integrated signal generation, transduction and
processing (third generation) having the potential of considerable miniaturisation.[17] The
devices tested in this project would be considered third generation under this classification
system and this highlights the progress sensing systems since their relatively recent
development
Biosensors monitor bimolecular interactions in real time. Biosensors are analytical devices
composed of a recognition site that is usually biological in origin and a transducer that can
be either: physical, chemical or electrical. The biological element is capable of sensing the
presence, activity or concentration of a chemical analyte in solution. [18][19] In a biosensor
one of the components is immobilised on a solid surface (usually the sensor chip), and the
other component to be detected is present in the solution phase.[20] An electrochemical
biosensor contains working electrode (Gold Macro electrode). Ag/AgCl is usually the
reference electrode and an auxiliary/counter electrode (Pt Wire) makes a three electrode
system.[21]

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A design option for the biosensor is a bio specific membrane that selectively interacts with
the analyte. A physical sensor coupled to the membrane detects this interaction and
generated the resulting electrical signal. The biospceific membrane can be made up of a
material that undergoes electron exchange reactions with the analyte so that there is a
direct electrical output signal consisting of a potential or a current that can be detected by
electrodes and interpreted. [22]
Sensors need to be specific, sensitive, stable, easy to use, portable and inexpensive.
Biosensors are finding increasing application and there is a demand in established areas, but
also in new fields. Some of the more recent areas of biosensor development include the
ability to detect new toxic chemicals, explosives and biological agents.[23]
One of the most ground breaking developmental areas in biomedical sensors in recent
years has been the development of bio analytical sensors. These devices are used for
detecting small amounts of biochemical substances in specimens. These sensors make use
of a selected biologically specific material to form a selective transducer for a specific
analyte, in this case, Listeria Monocytogenes as the analyte or antigen. Since enzymes are
complex proteins, they must be immobilized in such a way that they retain their biochemical
activity. It must also be possible to maintain this immobilized enzyme for a long enough
period of time to make the sensor useful. The shelf life of the sensor must be considered in
to make the sensor viable. This remains a significant stumbling block which limits the

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development of enzyme electrodes for applications requiring a more protracted (long
lasting) enzyme-complex contract time.
1.3 Electrochemical Immunosensors
In recent years, electrochemical immunosensors have attracted great interest because of
their quick response, simplicity, sensitivity, low cost, and high selectivity. It was widely used
for detection of many different substances, such as toxins, hormones, pathogens and,
pesticides and veterinary medicines and so on. According to papers, there are no reports on
electrochemical immunosensors for the detection of L. monocytogenes. While, they are
almost always used for the detection of antibody–antigen interactions because the main
advantages of electrochemical immunosensors are that they are non-invasive and require
little sample pre-treatment. The immobilization of antigens or antibodies is vital in the
construction of immunosensors, particularly in the stability, reproducibility, and sensibility
of the measured signal. With the development of scientific research, many immobilization
processes in immunosensors were found, such as Langmuir–Blodgett films, polymeric
membranes and, pre-modification with the protein A, Au nanoparticles, and thiol self-
assembled monolayers (SAM). Among them, SAM offers the simplest way to generate
reproducible, ultrafine, and well-structured monolayers. The choice of detection system is
also crucial for detection limits. In recent years, water soluble dyes such as, methylene blue,
Meldola blue, thionin, has been used as electron transfer mediator for reduction and
oxidation of hydrogen peroxide. In order to improve the sensitivity and amplified signal,
labelled horseradish peroxidase (HRP) and thionin–H2O2 system has been used in
electrochemical immunosensors[1].
1.4 Difference betweenMacro- andMicro- electrodes
Miniaturization of electrodes offers many practical advantages: reduced resistance (ohmic
drop), reduced sample consumption, ability to incorporate many electrodes in a small area,
and increased ability to facilitate measurements in low-ionic strength water samples.[24] The
reduced size and distance travelled can increase the electrode fluxes, which can enhance

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signal-to-noise ratios in trace metal ion determination making microelectrodes more
efficient.[25] Fabrication of microelectrodes is simple. Photolithography can be used to
construct microelectrodes suitable for simultaneous electrochemistry and atomic force
microscopy measurements.[23] Microelectrodes are produced using thin-film technology
which guarantees high sensitivity and reproducibility of the electrodes due to the clean
materials and chemicals, as well as the conditions during the processing steps.[27]
IUPAC has outlined that a microelectrode has dimensions of tens of micrometres or less,
down to the sub micrometre range while a nanoelectrode is defined as an electrode having
dimensions on the order of a few tens of nanometres.[28][29]Nanotechnology is defined as
the study of synthesis, properties and application of structures and materials having at least
one critical dimension on the scale of < 100 nm.[30] In relation to biosensors, a critical
dimension is one directly related to the measurement function of the biosensor, such as a
dimension that controls the area available for immobilization of a bio recognition element,
or a dimension that controls the magnitude of a signal, such as electrode surface area in the
case of electrochemical biosensors, or area available for detecting formation of complexes
between recognition elements and target analytes in the case of biosensors based on
mechanical transducers. These nanostructures offer unique electrical, optical and magnetic
properties that can be exploited for chemical sensing. The large surface area to volume ratio
available on nanostructures for immobilization of biological recognition elements offers high
signal amplification and improved measurement sensitivity. Biosensors based on
nanocantilevers measure a mechanical property in response to an affinity reaction and offer
the possibility of label free measurements. Recent developments in the field of DNA
nanobiosensors have resulted in molecular detection and amplification schemes, capable of
outperforming PCR in sensitivity and ease of use.[31]
In recent years, the development of biosensors using microelectrodes has gained a lot of
interest. This is due to some major advantages like: fast mass transport, high signal to noise
ratio, short time to reach the steady state and they can also be mass produced. In terms of
practical use the high signal to noise ratio and mass production potential makes

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microelectrode sensing systems desirable. The drawbacks of the use of single
microelectrodes; however are that they produce low current output and are more
susceptible to mains interference. [32] To prevent this occurrence if the collections of
microelectrodes are arranged in an ordered manner with a controlled inter-electrode
spacing they are referred to as arrays; and if the collections are not so ordered and there is
no specific control over the inter-electrode spacing then they are referred to as ensembles.
[33]
Fig 1.4.1 – Sample Microelectrode designs.
A hexagonal array was chosen as it optimised the inter-electrode distance and also ensured
the maximum number of microelectrodes per surface area.

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1.5 Electrochemical Characterisation
1.5.1 Cyclic Voltammetry
In this project a bare gold working Macro electrode is tested, which is a singular component
to a larger biosensor which will form a working biological device, which would be a
considered a working electrode. The characterisation of this device will carried out using the
established Cyclic Voltammetry (CV) method and Electrochemical Impedance Spectroscopy
(EIS) method, which is becoming more commonly used in testing the integrity of on chip
electrodes and ‘on chip technologies’. Electrochemical characterisation methods, such as
electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), are useful
understanding of the mechanisms behind the electrochemical process of the Macro
electrode sensor.[34] Amongst voltammetric methods, CV is quite popular and is generally
used to first investigate the electrochemical behaviour of new redox systems.[35] Rather than
monitoring the response of an electrochemical systemto a small amplitude periodic
potential change is imposed on the system. Many of the relaxation techniques developed
for fast reactions have the disadvantage that small amplitude perturbations are used, and
differences or changes in mechanism are not easily detected as a result. A method which
overcomes this disadvantage and at the same time gives a direct estimate of how
reversibility CV is. The presence of homogeneous reactions in the mechanism is readily
detected, and interpretation of results is usually simple.[36]
In CV the potential is ramped between two chosen limits. It is swept back and forward
between these limits a set number of times and the current is monitored continuously. A
resultant CV plot is then current vs. the (time-dependent potential).xli CV offers a rapid
location of redox potentials of these systems and a convenient evaluation of the effect of
media on the redox process involved. [37]CV is vital as its application in testing redox
potentials can directly detect the interaction between the recognition moiety site and the
electrochemical transducer. If the recognition moiety and electrochemical transducer in a
biosensor site are not coupled properly, problems in selectivity, sensitivity, reproducibility,

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range and stability can occur limiting the sensors applications in a real world environment.
[38]
CV uses a periodic triangular waveform. It consists of first applying a linear sweep potential
to the working electrode from an initial potential (Ei) to a final potential (Ef).
E = E0 + scan rate · t
After reaching Ef, the sweep is reversed and the potential returns to Ei. During this method,
a current resulting from the potential applied at a precise scan rate is recorded. In CV, the
triangular waveform shown in fig 1.5.3 is applied to the working electrode. After applying a
linear voltage ramp between times t0 and t1, usually a few seconds, the ramp is reversed to
bring the potential back to its initial value at time t2. The cycle may be repeated many more
times as defined.
Fig 1.5.3 Triangular waveform used in cyclic voltammetry.
The recorded cyclic Voltamogram is characterized by five main features: the cathodic and
anodic peak potentials (denoted Ec and Ea respectively) which correspond the cathodic and
anodic peak currents (ic and ia), and the half-peak potential (E1/2). The values of these
parameters and the relationship between them provide the basis for classifying CVs as
reversible, irreversible or quasi-reversible systems.

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Fig. 1.5.7 – Macroelctrode planar diffusion.
Fig 1.5.8 – Microelectrode radial diffusion.
1.5.2 Electrochemical Impedance Spectroscopy
The flow of direct current (DC) through an electric circuit is impeded only by the electrical
resistance presented by the circuit. In the case of alternating currents (AC) other
mechanisms impede the flow of current, in addition to the resistance. The physical quantity
that quantifies the opposition of a capacitor to the flow of AC is called electrical impedance.
This is of particular relevance to chemical sensors. This is due to the fact that an
electrochemical reaction is a multistep process involving diffusion, ion migration and

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electron exchange at the electrode/solution interface. Impedance measurements allow for
the characterisation of each of these steps and provide information about the
characteristics of the electrical double layer.
Fig 1.6.1 – Sin wave voltage and current Fig. 1.6.2 –Vector representation of
impedance.
The flow of current through a conductor is governed by Ohm’s law that states that the
current is the ratio of the applied voltage to the conductor resistance (I=V/R). In the case of
AC circuits, some circuit components (such as capacitors and inductors) induce a time lapse
between the voltage and the current this is taken into consideration when quantifying the
opposition the current flow experiences. [39] A sine wave AC quantity is characterised by its
amplitude (Vm or Im), frequency (f, in Hz) and the phase angle (φ) with respect to a
reference signal. In general, the applied voltage results from the superposition of a bias DC
voltage (VDC) and a sine wave voltage
VAC: V = VDC + VAC = VDC + Vm(sinωt)
Although CV is considered a useful technique is coupled with electrochemical impedance
spectroscopy (EIS), which can also be referred to as A.C Impedance spectroscopy.
Impedance spectroscopy is considered one of the most powerful non-destructive methods
of testing the boundaries between materials with varying conductivities. Its main use is in
analysing the interface between electronically conducting solid electrodes and ionically
conducting electrolytes, both electrodes and ionically conducting electrolytes can be either

23 | P a g e
in a solid or liquid state. It is a process that depends on diffusion of reactants toward or
away from the surface which has a particular low frequency character.
Fig 1.6.3 Warburg impedance and sample circuit.
Diagrams shows the Randles equivalent circuit, taking into account the electrolyte(solution)
resistance Rs , the charge transfer resistance Rct , and the double layer capacitance Cdl .
The Warburg impedance is the diffusional impedance for 1D linear diffusion. This
Demonstration shows the Randles equivalent circuit, taking into account the electrolyte
resistance, the charge transfer resistance, and the double layer capacitance. The plot of
the real part of the impedance against the imaginary part gives a Nyquist Plot, as shown in
fig 1.6.3. The advantage if Nyquist representation is that it gives a quick overview of the
data and one can make some qualitative interpretations from this resultant data. While
plotting data in the Nyquist format the real axis Z (Ω Ohm) must be equal to the imaginary
axis so as not to distort the shape of the curve. [40] The complex response of the systemis
usually displayed in Nyquist format, with reactance inverted; this is due to the fact that
most systems are capacitive. The response of the systemas a function of the perturbation
frequency can reveal internal dynamics. The capacitance at the metal/electrolyte interface
always plays an important role. EIS, in its traditional form, is restricted to electrical
perturbation of oltamelectrochemical systems. Electrochemical impedance spectroscopy
allows access to the complete set of kinetic characteristics of electrochemical systems, such
as rate constants, diffusion coefficients, and so on, in single variable-load experiment. It is
restricted to characteristics that describe system behaviour in linear range of electrical

24 | P a g e
excitation, for example, when it can be approximated by linear differential equations. It can
be contrasted with other methods where explicitly nonlinear properties are investigated,
such as cyclic voltammetry.
EIS is now well established as a powerful tool for investigating the mechanisms of
electrochemical reactions, for measuring the dielectric and transport properties of
materials, for exploring the properties of porous electrodes, and for investigating passive
surfaces. The power of the technique arises from: (i) It is a linear technique and hence the
results are readily interpreted in terms of Linear Systems Theory. (ii) If measured over an
infinite frequency range, the impedance contains all of the information that can be gleaned
from the systemby linear electrical perturbation/response techniques. (iii) The
experimental efficiency (amount of information transferred to the observer compared to
the amount produced by the experiment) is extraordinarily high.
Fig 1.6.5 &1.6.6 EIS plots of a micro disc and micro band array respectively highlight
the position of 2πf
The importance of the 2πf on an EIS plot which is the point with the highest
frequency is that will be used to calculate the double layer capacitance of the
2πf
2πf

25 | P a g e
electrode designs and aid in comparing the capacitance after the biological
component has been bound to the electrode surface.

26 | P a g e
Chapter 2: Methodologies
2.1 Risk Assessment:
Supplier Order Code Product Disposal
Sigma Aldrich Hydrogen Peroxide (H2O2) H2O2 waste
Sigma E7750-10G (N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimidehydrochloride
(EDC)
Non-chlorinated
waste
Sigma 130672-25G N-hydroxy-succinimide(NHS) Non-chlorinated
waste
Sigma B4287-25G Bovine serum Albumin (BSA) Autoclaved
Sigma Aldrich Phosphate Buffer saline(PBS) Non-chlorinated
waste
Thermo Scientific PA17230 Listeria Monocytogenes
Monoclonal Antibody
Autoclaved
Teagasc Strains of Listeria Innocua Autoclaved
VLSI Acetone Non-chlorinated
waste
Sigma - Aldrich Ethanol Non-chlorinated
waste
Sigma Ferrocence CarboxylicAcid (FCA) Non-chlorinated
waste
VLSI Isopropyl Alcohol (IPA) Non-chlorinated
waste

27 | P a g e
Aldrich 3-Mercaptopropionic Acid (MPA) Non-chlorinated
waste
Sigma - Aldrich Sulphuric Acid (H2SO2) Non-chlorinated
waste
Fischer Scientific PotassiumChloride(KCl) Chlorinated waste
2.2 Fabrication:
The gold electrode that was used in this project was fabricated in the Tyndall National
Institute Fabrication Unit. The device underwent micro fabricated process stared as a Silicon
wafer 525µm thick:
Fig2.2.1: Silicon Wafer 525µm thick (Red)
A 1 micron layer of Silicon Oxide was thermally grown on the silicon wafer:
Fig2.2.2: 1 micron Silicone Oxide (Blue)

28 | P a g e
Alingmentmarkswere made usingaPhotolitographystepbyUV light:
Fig2.2.3: Alignment marks made by photolithography
Photolithography is a process used to pattern parts of a thin film or the bulk of a substrate.
It uses light to transfer a pattern from a photo mask to a light sensitive chemical,”photo
resist”. After the application of the photo resist, a photo mask is aligned and exposed to UV
light which is then developed and the photo resist is removed. The exposed oxide is
removed along with the photo resist.
Fig2.2.4: Method of photolithography.

29 | P a g e
A 20nm deposition of Ti to facilitate the adhesion of the Gold (Au). A layer of gold layer
(100nm) was then added:
Fig2.2.5: 20nm Ti (White) + 100nm Au (Gold).
This layer of gold was protected by a strong Silicon Nitride polymer:
Fig2.2.6: Silicon Nitride polymer
2.3 Resist Removal:
The silicon nitride protective layer was removed by placing the device in a beaker of hot
Acetone (Boiling Point = 56⁰C). This was done using a hot plate in a fume hood. The
electrodes were placed in the hot acetone for 5 mins, then washed thoroughly with copious
amounts of DI water and dried with a N2 gun.
This process was then repeated using hot Isopropyl Alcohol (IPA). The boiling point of IPA is
82.6⁰C.

30 | P a g e
This sequence was repeated 3 times to make sure the entire resist layer was removed, along
with all contaminants and foreign material. The devices were examined under a compound
microscope to see if any physical defects were observed.
2.3.1 Oxygen Plasma Cleaning:
After treating the devices with acetone and IPA the devices were placed in an Oxygen
Plasma Cleaner.
Oxygen Plasma Cleaning is a dry, solvent free technology which uses highly reactive oxygen
radicals to clean the gold surface of the gold electrode. The oxygen radicals react with
organic contaminants and turn them into volatile species that can desorb. This technique
ensures that there are no hydrocarbons contaminating the surface. Oxygen cleaning allows
further modification of the electrode by making the surface more hydrophobic, allowing for
increased wettability which promotes adhesion and enhances bonding.
The Harrick© Plasma Cleaner is operated by placing the electrodes on a shelf inside the
plasma chamber. The door was closed and the nozzle was turned clockwise. The vacuum
pump was turned on, and then the PlasmaFlo unit. When the meter on the PlasmaFlo unit
dropped to 100 microns, the RF unit was turned on and the dial was set to high. The gas
valve was then opened till a purple colour was observed. The purple colour indicates that
the technique is working properly at high pressure. This was then timed for 10 minutes.
After the 10 mins, the cleaner was turned off. The nozzle was opened slowly to release a
rush of air. The electrodes were then removed and rinsed with copious amounts of DI water
and dried using an N2 gun.

31 | P a g e
Fig2.3.1.1: Plasma cleaner at high pressure
Fig2.3.1.2: Plasma Cleaner

32 | P a g e
2.4 Electrochemical Cell Set-up
Electrochemical measurements analyse the chemical reactions taking place at the electrode-
solution interface. Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy
(EIS) measurements were used to characterise the devices. CV and EIS were performed
using an Autolab potentiostat connected to a PC and a faraday cage is also used. The
faraday cage helps to reduce electromagnetic noise that could affect the electrochemical
measurements.
Fig 2.4.1: Autolab potentiostat connected to PC for electrochemical measurements.

33 | P a g e
Fig2.4.2: Faradays Cage
The electrochemical cell for characterisation of gold macro electrodes is set up as seen in
fig: A reference electrode is placed inside the small beaker along with a few ml of ferrocene
carboxylic acid (FCA) and a counter electrode. The reference electrode is silver/silver
chloride (Ag/AgCl). For characterisation of the gold macro electrodes, the WE is the gold
electrode and the counter electrode is the Pt Wire. FCA is the redox probe and is used for
the characterisation of the gold macro electrode as it has been well researched for working
with gold electrodes. The electrode, where the reaction is taking place, is the working
electrode. The reference electrode has a well-known and stable potential compared to the
working electrode. The counter electrode closes the circuit current so that the circuit flow is

34 | P a g e
as close to zero as possible. Ferrocenecarboxylic acid was the solution used for the
electrochemical characterisation of the electrodes. It was a 1mM Ferrocence carboxylic
acid/ phosphate buffered saline (PBS) solution.
Fig2.4.3: Three electrode set-up.
Mw of FCA = 230.041g/mol
0.001M x 230.041g/mol = 0.230041 g/L
0.230041g/L / 20 = 0.0115g FCA in 50ml PBS
CounterElectrode (PtWire)
Reference Electrode(Ag/AgCl)
Working
Electrode
(GoldMacro
electrode)

35 | P a g e
The reference electrode was stored in a 0.1M KCl (Potassium Chloride) solution.
Mw of KCl = 74.56g/mol
0.1M x 74.56g/mol = 7.456g in 1L
7.456g/L/5= 1.4912g KCl in 200ml of DI Water
2.5 Cyclic Voltammetry and Electrochemical Impedance Spectroscopy
Cyclic Voltammetry (CV) was run at a scan rate of 0.1 Vs-1, between a potential range of 0.0
 +0.6 V and this was run relative to the reference electrode (Ag/AgCl). The system was
then accessed using a Windows XP computer with the GPES program.
Fig 2.5.1: Image of GPSE Computer Programme
Electrochemical Impedance Spectroscopy (EIS) was run at a frequency range between 10-2
and 105 Hz and amplitude of 0.01V. The current at which the EIS measurements were run

36 | P a g e
were obtained from CV. The systemwas then accessed using a Windows XP computer with
the FRA (Frequency Response Analyser) program.
Fig 2.5.2: Image of FRA Computer programme
After each modification to the electrode the CV and EIS processed was run again.

37 | P a g e
2.6 Cleaning with Piranha Solution
Piranha is a 1:3 v/v solution of H202 and H2SO4 respectively. This was made up fresh to 100ml
by adding 25ml H2O2 with 75ml H2SO4 slowly in a fume hood. The devices were placed in
piranha for 5 mins. The device were then washed with DI water and dried with nitrogen. The
piranha solution is used to further clean the electrode by using Cyclic Voltammetry at a scan
rate of 50mV/s and with a potential range of -0.2V to 1.6V until a stable CV was obtained.
The CV was run in 0.5M H2SO4. The 0.5M H2SO4 was made up by:
Mw of H2SO4 = 98.08g/mol
0.5M x 98.08g/mol = 49.04g/L
Density= Mass/Volume
49.04/1.840g/ml = 26.65ml
26.65ml/5 = 5.33ml of H2SO4 in 200ml of DI Water

38 | P a g e
2.7 The formationof Self Assembled Monolayers(SAMs).
2.7.1: Overnight step in MPA
The formation of these Self-Assembled Monolayers (SAMs) was facilitated by immersing the
gold electrodes in an ethanol solution of 1% 3-Mercaptopropionic Acid (MPA). This step
took place overnight and was kept at Room Temperature (RT).
MPA was found in the lab to be 99% but only 1% was needed. This was made up fresh each
time it was used so only 500µL was used. This was to ensure only the active was exposed to
MPA.
-3.00E-04
-2.00E-04
-1.00E-04
0.00E+00
1.00E-04
2.00E-04
3.00E-04
-0.5 0 0.5 1 1.5 2
I(mA)
E(V)
Graph of CV after cleaning with
Pirnaha Solution
pirhana /

39 | P a g e
500µm / 1000 = 0.5ml
1/100 x 0.5ml = 0.005ml MPA
99/100 x 0.5ml = 0.495ml Ethanol
Before examining the electrodes, they were washed with ultrapure water and dried with
nitrogen to eliminate any absorbed sulfhydryl groups. The following day, CV and EIS was run
on the electrodes.
2.7.2: Treatment with 0.4M EDC and 0.1M NHS
1-ethyl-3-(2dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-succinimide (NHS)
were used to build up the surface chemistry on the electrode. These convert the terminal
carboxylic group of the MPA into the NHS ester allowing for more reactivity of electrode.
The following reaction scheme shows just how the carboxylic group is converted into the
NHS ester:

40 | P a g e
The 0.4M EDC was made up by:
Mw of EDC = 155.24g/mol
155.24g/mol x 0.4M = 62.096g/L
62.096g/L / 1000 = 0.062096g EDC in 1ml DI Water
The 0.1M NHS was made up in the same way:
Mw of NHS = 115.09g/mol
115.09g/mol x 0.1M = 11.509g/L
11.509g/L / 1000= 0.011509g NHS in 1ml of DI Water
Both of these solutions were made up fresh on the day they were being used. The electrode
was treated wit EDC/NHS for 30 mins at RT. When mixed with DI water they needed to be
vortexed to ensure all the powder was emalgamated in the water.

41 | P a g e
CV and EIS were carried out after this step also.
2.8 Antibody attachment
The gold surface was treated with 70µL of Listeria Monocytogenes antibody which is
captured by the NHS ester. Only 70µL was used because it was only the active site that was
covered. These were then incubated for 1hr at 37°C and CV and EIS was carried to examine
the structural characterisation.
2.8.1 Removal of the unbound antibodies
The unbound antibodies that remained after the incubation were removed by dipping the
electrodes in Phosphate Buffered Saline (PBS). The electrodes were dipped in the pH 7 PBS
slowly three times.

42 | P a g e
2.9 Blocking the electrode
The electrode was blocked using 1% Bovine Serum Albumin (BSA). The electrode surface
needed to be blocked because there were some non-specific and unreacted sites. By
blocking with BSA the Listeria cells could only be picked up by the reactive sites. BSA was
made up fresh with PBS each time it was needed.
500µL / 1000 = 0.5ml
99/100x0.5ml = 0.495ml PBS
1/100 x 0.5ml = 0.005ml BSA
Density = Mass/Volume
1.32g/ml x 0.005ml = 0.0066g BSA in 0.495ml PBS
2.10 Dipping in PBS
The electrode was then dipped another three times in PBS at pH7. This time they were
dipped for 3 minutes each time. One electrode didn’t have any more involvement in the
modification process and this electrode was the control.
2.11 Attachment of the ListeriaInnocuaCells.
The final step of the procedure was to attach Listeria Innocua cells to the electrode. This is
achieved by the antigen-antibody reactions. To react the Listeria Innocua cells, 70µL of
Listeria Innocua cells were used to cover the electrode surface. Three different dilutions of

43 | P a g e
Listeria Innocua cells were used 1:10, 1:100 and the 1:1000. Once the electrode surface was
covered, they were incubated for 1hr at 37°C. CV and EIS were then performed once again.
The Standard Operating Procedure that I followed was adapted from the following Journal:
Rapid detection of Listeria monocytogenes in milk by self-assembled electrochemical
immunosensor
Chaonan Chenga, 1, Yuan Pengb, 1, Jialei Baib, Xiaoyan Zhangc, Yuanyuan
Liub, Xianjun Fanb, Baoan Ningb, Zhixian Gaoa, b,

44 | P a g e
Chapter 3: Results and Discussions:
The first set up in the overall procedure was to attach a 0.1mg/ml Listeria Monocytogenes
antibody to the gold surface. This should make the electrode selective for Listeria
Monocytogenes only. We then treated the device, via the above procedure, with different
dilutions of Listeria Innocua. The reason for this is to investigate if there was any cross-
selectivity as some antibodies bind to other antigens.
The result that is expected is for all the dilutions to overlap perfectly with the average PBS
data.
The above graph is a Cyclic Voltamogram showing the different dilutions of Listeria Innocua.
Because, an electrochemical response is seen here it can be said that there is a cross
selectivity. This is seen in the 1:10 and the 1:1000 dilutions of Listeria Innocua.

45 | P a g e
The first set up in the overall procedure was to attach a 0.1mg/ml Listeria Monocytogenes
selectivity as some antibodies may bind to other antigens.
data.
The above graph is an Electrochemical Impedance plot showing the different dilutions of
Listeria Innocua. Because, an electrochemical response is seen here it can be said that there
is a cross selectivity. This is seen in the 1:10 and the 1:1000 dilutions of Listeria Innocua.

46 | P a g e
The next set up in the overall procedure was to attach a 0.5mg/ml Listeria Monocytogenes
data.
selectivity. This is seen in the 1:10 and the 1:100 dilutions of Listeria Innocua.

47 | P a g e
The next set up in the overall procedure was to attach a 0.5mg/ml Listeria Monocytogenes
data.
is a cross selectivity. This electrochemical response is visible for all the devices here.

48 | P a g e
The 3rd set up in the overall procedure was to attach a 1.0mg/ml Listeria Monocytogenes
data.
selectivity. This is seen in the 1:1000 dilutions of Listeria Innocua. It is believed that this
device was faulty and the procedure is going to be repeated with this antibody
concentration in the future.

49 | P a g e
The 3rd set up in the overall procedure was to attach a 1.0mg/ml Listeria Monocytogenes
data.
is a cross selectivity. This is seen in the 1:10 and the 1:100 and the 1:1000 dilutions
Of Listeria Innocua. This experiment with the same antibody concentration will be repeated
again in the future.
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
0 100000 200000 300000 400000 500000
Z''(ohm)
Z' (ohm)
Handhold1device with 1 mg/ml antibody
concentrationimmobilizedon the surface and
exposed to three listeria innocuaconcentrations
Average PBS data
Listeria Innocua 1:10

50 | P a g e
This Electrochemical Impedance plot shows the different modifications at the gold surface
for the 0.1 mg/ml Listeria Monocytogenes antibody, without the Listeria Innocua present,
and how the impedance increases with the blocking ability of the Self-Assembled
Monolayers (SAMs), antibody attachment and the antigen. The above plot doesn’t show any
antigen attachment as this is used as a control. The results for each surface chemistry step
0
200000
400000
600000
800000
1000000
1200000
1400000
0 200000 400000 600000 800000
Z"(Ohm)
Z'(Ohm)
Handhold1device with0.1 mg/ml antibodyconcentrationimmobilizedonthe
surface and exposedtothree listeriainnocuaconcentrations
Resist removal
MPA
EDC/NHS
blocking
antibody
PBS

51 | P a g e
should be similar to the plot from a paper above. The results of the theoretical plot Fig :
should be (a) is the gold electrode after resist removal, (b) is the formation of the MPA
monolayer, (c) is the step after activation with EDC/NHS, (d) is after the antibody
immobilization, (e) is after blocking with 1%BSA, (f) is after the antigen binds.
The plot should show an increase in impedance; however the experimental plot is showing a
decrease in impedance.

52 | P a g e
for the 0.1 mg/ml Listeria Monocytogenes antibody, with the Listeria Innocua 1:10 dilutions
present, and how the impedance increases with the blocking ability of the Self-Assembled
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
0 200000 400000 600000 800000 1000000
Z''(Ohm)
Z' (Ohm)
Handhold1device with 0.1 mg/ml antibody
resist removal
MPA
EDCNHS
antibody
after blocking
PBS
lis innocua 1:10

53 | P a g e
for the 0.1 mg/ml Listeria Monocytogenes antibody, with the Listeria Innocua 1:100
dilutions present, and how the impedance increases with the blocking ability of the Self-
Assembled Monolayers (SAMs), antibody attachment and the antigen. The above plot
doesn’t show any antigen attachment as this is used as a control. The results for each
surface chemistry step should be similar to the plot from a paper above. The results of the
theoretical plot Fig : should be (a) is the gold electrode after resist removal, (b) is the
formation of the MPA monolayer, (c) is the step after activation with EDC/NHS, (d) is after
the antibody immobilization, (e) is after blocking with 1%BSA, (f) is after the antigen binds.
0
200000
400000
600000
800000
1000000
1200000
0 100000 200000 300000 400000 500000 600000 700000
Z"(Ohm)
Z'(Ohm)
resist removal
MPA
EDCNHS
antibody
blocking
PBS
lis innocua 1:100

54 | P a g e
dilution present, and how the impedance increases with the blocking ability of the Self-
0
200000
400000
600000
800000
1000000
1200000
0 100000 200000 300000 400000 500000 600000 700000 800000
Z''(Ohm)
Z' (Ohm)
resist removal
MPA
EDCNHS
antibody
blocking
PBS
Lis innocua 1:1000

55 | P a g e
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
0 100000 200000 300000 400000 500000 600000 700000
Z''(Ohm)
Z' (Ohm)
after resist removal
EDC/NHS
after antibody
after blocking
after PBS

56 | P a g e
0
200000
400000
600000
800000
1000000
1200000
0 100000 200000 300000 400000 500000 600000 700000
Z''(Ohm)
Z' (Ohm)
after MPA
after EDC/NHS
after antibody
after blocking
after PBS
after lis innocua 1:10

57 | P a g e
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
0 100000 200000 300000 400000 500000 600000 700000
Z''(Ohm)
Z' (Ohm)
Handhold1 device with 0.5 mg/ml antibody
concentration immobilizedon the surfaceand
exposed to three listeria innocua
concentrations
after MPA
after antibody
after blocking
after PBS
after Lis innocua 1:100

58 | P a g e
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
0 200000 400000 600000 800000 1000000
Z''(Ohm)
Z' (Ohm)
after MPA
after EDC/NHS
after antibody
after blocking
after PBS
after Lis Innocua 1:1000

59 | P a g e
0
100000
200000
300000
400000
500000
600000
700000
800000
0 100000 200000 300000 400000 500000 600000
Z''(Ohm)
Z' (Ohm)
Handhold1 device with 1.0 mg/ml antibody concentration
immobilized on the surfaceand exposed to three listeria
innocua concentrations
after MPA
after EDC/NHS
after antibody
after blocking
after PBS

60 | P a g e
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
1000000
0 100000 200000 300000 400000 500000 600000 700000
Z''(Ohm)
Z' (Ohm)
after MPA
after antibody
after blocking
after PBS

61 | P a g e
This plot shows somewhat of decreased impedance as the Listeria Innocua dilution is
showing the most impedance followed by the antibody, followed by the MPA, then the PBS
and EDC/NHS steps and finally the resist removal. This shows promising results.
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
0 200000 400000 600000 800000 1000000
Z''(Ohm)
Z' (Ohm)
after MPA
after EDC/NHS
after antibody
after blocking
after PBS

62 | P a g e
dilution present, and how the impedance increases with the blocking ability of the Self-
This plot shows somewhat of decreased impedance as the Listeria Innocua dilution is
showing the most impedance followed by the antibody, followed by the resist removal, then
the PBS and EDC/NHS steps.
0
100000
200000
300000
400000
500000
600000
700000
800000
0 100000 200000 300000 400000 500000 600000
Z''(Ohm)
Z' (Ohm)
after MPA
after EDC/NHS
after antibody
after blocking
after PBS

63 | P a g e
Chapter 4: Conclusion and Future Work
The main aim of the experiment was to develop a biosensor for the detection of Listeria
Monocytogenes. The results throughout the experiment show promise that the biosensor
can be developed further. This is seen when the concentration of the Listeria
Monocytogenes antibody was increased to 1mg/ml. Although the biosensor is not optimised
to detect Listeria Monocytogenes, a model system was developed using Listeria Innocua.
Due to time constraints of final year, was an issue for carrying out lab work. Especially when
it came to timed steps as lectures clashed with the timed experiments, etc.
Some sources of error that were present during the experiment were the use of such
miniaturised technology (i.e.) setting up the 3-electrode cell in faradays cage and handling
the devices. Also as seen in the 1mg/ml L.M antibody concentration it can be seen that the
device with 1:1000 dilution of Listeria Innocua was found to be faulty.
Some advantages of the experiments were that, it allows for the label free detection of
antigens, its ease of use, cost effectiveness and real time, rapid results.
It is of paramount importance that the future work outlined below is carried out and
research develops further as there is a real need for the rapid detection of Listeria, as it
causes Listeriosis, a serious disease which is fatal for approximately 30% of those
contracting it. Although this novel immunosensor is in the early stages of research and,
admittedly, future work must be done to reach a point where the sensor is ready to detect
LM.
This future work needs to include, repeating the experiment with the 1mg/ml Listeria
Monocytogenes antibody concentration. This is because; even though it is suspected that
the device was faulty that there may have been some results for cross selectivity. If the
experiment is repeated and the cross selectivity remains, the antibody concentration may
be increased further or the dilutions could be decreased. If the experiment is repeated and

64 | P a g e
there is no electrochemical response for Listeria Innocua then the model systemcan be
used to detect Listeria Monocytogenes.
In the future, different characterization techniques may be incorporated into the
experiment for more accurate results. These techniques may include: Square Wave
Voltammetry, Contact Angle measurements and Linear Sweep Voltammetry.
To further better the experiment and even the results the experiments could be carried out
in a different redox probe (i.e.) Ferrocence cyanide instead of FCA. Also, different surface
chemistry steps could be used like APTES.
The final step to be carried out in the future would be to miniaturize the system from macro
 micro  Nano electrodes as this would increase the sensitivity of the results as the
smaller surface volume allows more signal to noise ratio.

65 | P a g e
Chapter 5: References
1. Cheng,C.,etal., Rapid detection of Listeria monocytogenesin milk by self-assembled
electrochemical immunosensor. SensorsandActuatorsB:Chemical,2014. 190(0): p. 900-
906.
2. Välimaa,A.-L.,A.Tilsala-Timisjärvi,andE.Virtanen, Rapid detection and identification
methodsforListeria monocytogenesin thefood chain – a review. FoodControl,(0).
3. Muhterem-Uyar,M.,et al., Environmentalsampling forListeria monocytogenescontrolin
food processing facilitiesreveals three contamination scenarios. FoodControl,2015. 51(0): p.
94-107.
4. Chatelard-Chauvin,C.,etal., Behaviourof Listeria monocytogenesin raw milk Cantaltype
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