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Supervisor: Dr. Karen Twomey.
Investigation into the development of a biosensor for the
detection of Listeria Monocytogenes using a Macro
fabricated device and Electrochemical Impedance
Spectroscopy as an analysis method.
By Brian Muldoon.
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Listeria Monocytogenes:
• Aim of the experiment is to develop a rapid electrochemical immunosensor for Listeria
Monocytogenes(LM).
• LM is a gram-positive food-borne pathogen.
• L.M causes listerosis.
• Symptoms include meningitis, septicaemia, abortion and febrile gastroenteritis.
• Mortality rates = 30%.
• No reports on electrochemical immunosensor for the detection of L.monocytogenes.
• Electrochemical immunosensor used for the detection of other antibody-antigen interactions.
• Biosensors are non-invasive, and require little sample pre-treatment. [1]
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Motivation and Real-World Applications
• The biosensor that I designed needed to be non-invasive, accurate and give real time results.
• Current methods of detection (i.e.) ELISA and PCR, are time-consuming, 3-4 days presumptive testing
and 5-7 days for confirmation. [1]
• State of the art detection techniques include QCM (Quartz Crystal Microbalance)[3] fibre-optic
immunosensor [4] and surface plasmon resonance[5].
State of the Art Techniques:
QCM
SPR
PCR
ELISA
Assay Kit
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Biosensor
• A biosensor is an analytical device incorporating a specific sensing system, which is based
on biological materials.
• The response that is observed is converted into an easy to detect form by the transducer.
• The response is the biological component.
• The recognition component, a bio-receptor, uses biomolecules from organisms or receptors
modelled after biological systems to interact with the analyte of interest.
[6]
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Electrochemical Immunosensors
• Immunosensors detect an immune response from biological species (i.e.) Bacteria.
• The immune response to contaminants will produce antibodies in the body.
• These antibodies will capture the antigen and produce a signal.
• This antibody-antigen signal can be measured.
Comparison of electrochemical immunosensors compared to other analytical techniques.[6]
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The Project
• This project was undertaken in collaboration with Tyndall National Institute and Teagasc.
• Dr. Phil Kelly and Dr. Kieran Jordan (both from Teagasc) were involved in this project.
• Their research interests involve tolerance of food pathogens.
• The project was designed to develop a real time, rapid biosensor for the detection of
harmful pathogens in food.
• The project is looking at the development of a Macro Micro Nano gold electrode.
• The end-users of these devices would be Teagasc and Moorepark.
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My Role
• My role in this project was to look at the macro electrode.
• I was involved in modifying the gold surface and making it selective to Listeria
Monocytogenes(L.M).
• Before analysing L.M, I investigated the modified surface’s selectivity for Listeria Innocua
(provided by Teagasc).
• I varied the L.M antibody concentration when determining the cross selectivity to Listeria
Innocua.
• I looked at the structural characterisation of the macro electrode by Electrochemical
Impedance Spectroscopy.
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Cyclic Voltammetry
• The potential of the working electrode is measured against a reference electrode which
maintains a constant potential.
• The voltage, at the potential from t0 to t1 (forward scan) and t2 to t3 (2nd cycle), is enough to
cause reduction or oxidation of the electrode.
• The voltammogram is obtained by measuring the current at the working electrode.
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Cyclic Voltammetry
• Epa is known as the anodic peak potential.
• Oxidation occurs here and the potential is run positively until all of the solution is oxidised.
• When the potential is run positively, the current is known as the anodic current peak, ipa.
• Epc is known as the cathodic peak potential.
• Reduction occurs here and the potential is run negatively until all of the solution is
reduced.
• When the potential is run negatively, the current is known as the cathodic current peak, ipc.
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Electrochemical Impedance Spectroscopy
• The potential, measured at the surface working electrode from CV, is used in
Electrochemical Impedance Spectroscopy (EIS).
• EIS describes the response of an electrochemical cell to a sinusoidal voltage as a frequency.
• The sine wave differs in phase shift (φ).
• The difference between voltage and current is defined as Impedance (Z).
• Impedance detects the changes in electrochemical properties that are caused by the
blocking ability of Self Assembled Monolayers(SAMs).
[7]
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Electrochemical Impedance Spectroscopy
Double Layer Capacitance:
• An electrical double layer exists on the interface between an electrode and its surrounding
electrolyte. This double layer is formed as ions from the solution adsorb onto the electrode
surface.
Double Layer Capacitance [7]
• Charges separated by an insulator form a capacitor so a bare metal immersed in an
electrolyte will behave like a capacitor.
• Double layer capacitance depends on many variables: electrode potential, temperature,
ionic concentrations, types of ions, oxide layers, electrode roughness, impurity adsorption.
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Electrochemical Impedance Spectroscopy
Charge-Transfer Resistance:
• Charge is transferred when electrons enter the metal and metal ions diffuse into the
electrolyte.
• The charge transfer leads to both faradaic and non-faradaic components.
• As the electrode surface is progressively modified by more than one step, the charge
transfer across the electrode-electrolyte interface to a redox probe, present in the
solution, becomes increasingly difficult due to the blocking effect of the SAM, antibodies,
and antigens.
• The general relation between the potential and the current is directly related with the
amount of electrons and so the charge transfer via Faradays law.
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Electrochemical Impedance Spectroscopy
Diffusion:
• Diffusion can also create an impedance called a Warburg impedance.
• The impedance depends on the frequency of the potential.
• High frequencies: the Warburg impedance is small. Reactants don't have to move very far.
• Low frequencies: the reactants have to diffuse farther, increasing the Warburg-impedance.
• On a Nyquist Plot the Warburg impedance appears as a diagonal line with an slope of 45°.
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Electrochemical Impedance Spectroscopy
• Electrical resistance is determined by Ohms Law:
R=E/I
• EIS can be expressed as a complex function. Potential is:
Et = Eo exp(jωt)
• Current is:
It = I0 exp(jωt - Φ)
• Impedance is then represented as :
Z(ω) = E/I = exp(jΦ) = Zo (cosΦ + jsinΦ)
[7]
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Surface Chemistry
• The electrode was covered
with 70µL of the listeria
monocytogenes antibodies.
• Unbound antibodies were
removed by dipping in PBS.
• Electrode was blocked with
1% BSA.
• Exposed to listeria innocua
antigen.
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Future Work
Repeat with the
same antibody
concentration.
Use different
characterisation
techniques.
Change the
redox probe.
Use different
surface
chemistry
steps
Treat with LM Macro Micro
Nano
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Acknowledgements and References
• I would like to express a sincere thanks to my supervisor, Dr. Karen Twomey.
• Thanks to Tyndall National Institute and Teagasc for giving me the opportunity to complete my Final
Year Project.
• Eileen Hurley, the lab manager of the LSI Lab, for all her training and help throughout the project.
• Dr. Miloslav Pravda for his guidance before, during and after the project.
[1] Cheng, C., Y. Peng, J. Bai, X. Zhang, Y. Liu, X. Fan, B. Ning and Z. Gao (2014). "Rapid detection of Listeria
monocytogenes in milk by self-assembled electrochemical immunosensor." Sensors and Actuators B: Chemical 190(0): 900-
906.
[2] Peng, H. and L. A. Shelef (2000). "Rapid detection of low levels of Listeria in foods and next-day confirmation of L.
monocytogenes." Journal of Microbiological Methods 41(2): 113-120.
[3] Vaughan, R. D., C. K. O’Sullivan and G. G. Guilbault (2001). "Development of a quartz crystal microbalance (QCM)
immunosensor for the detection of Listeria monocytogenes." Enzyme and Microbial Technology 29(10): 635-638.
[4] T. Geng, M.T. Morgan, A.K. Bhunia Detecion of low levels of Listeria monocytogenes cells by using a fiber-optic
immunosensor Appl. Environ. Microbiol., 70 (2004), pp. 6138–6146
[5] Leonard, P., S. Hearty, J. Quinn and R. O’Kennedy (2004). "A generic approach for the detection of whole Listeria
monocytogenes cells in contaminated samples using surface plasmon resonance." Biosensors and Bioelectronics 19(10):
1331-1335.
[6] Dr. Eric Moore.
[7] Dr. Miloslav Pravda.
![www.tyndall.ie
Listeria Monocytogenes:
• Aim of the experiment is to develop a rapid electrochemical immunosensor for Listeria
Monocytogenes(LM).
• LM is a gram-positive food-borne pathogen.
• L.M causes listerosis.
• Symptoms include meningitis, septicaemia, abortion and febrile gastroenteritis.
• Mortality rates = 30%.
• No reports on electrochemical immunosensor for the detection of L.monocytogenes.
• Electrochemical immunosensor used for the detection of other antibody-antigen interactions.
• Biosensors are non-invasive, and require little sample pre-treatment. [1]](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)