Graphene-Based Biosensor Platform For The Development of An Artificial Nose PDF

DIPLOMARBEIT
Graphene-based Biosensor Platform for

the Development of an Artificial Nose
Die approbierte gedruckte Originalversion dieser Diplomarbeit ist an der TU Wien Bibliothek verfügbar.
Thema
The approved original version of this thesis is available in print at TU Wien Bibliothek.
Ausgeführt am Institut für

E163 Institut für Angewandte Synthesechemie
E164 Institut für Chemische Technologien und Analytik
der Technischen Universität Wien
unter der Anleitung von
Univ.-Prof. Dipl.-Ing. Dr. Peter Ertl
durch
Anja Agneter, BSc
Wien, 30.12.2019
Datum Unterschrift (Student)
Master Thesis
Graphene-based Biosensor Platform for

the Development of an Artificial Nose
ausgeführt unter der Leitung von
Univ.-Prof. Dipl.-Ing. Dr. Peter Ertl

E163 Institut für Angewandte Synthesechemie

E164 Institut für Chemische Technologien und Analytik
der
Technische Universität Wien
von
Anja Agneter, BSc

Masterstudium Biomedical Engineering
e1225291
Schottenfeldgasse 2/20
A-1070 Wien
Wien, 2019
Acknowledgements
I would like to thank Prof. Dr. Ertl and the cellchip group for their support.
I especially thank Prof. Dr. Knoll at the AIT for giving me the opportunity to work
on this project and for all the help and advice.
I would also like to thank Ulrich Ramach for all the shared adventures in Singapore.
My thanks go to Prof. Dr. Lim Chwee Teck and his team of the Biomedical
Engineering Department of NUS for their support and warm welcome to their team.
I am also grateful to Prof. Dr. Slaven and his team at the Centre for advanced
2D materials at NUS for sharing their lab equipment with us and the interesting
discussions, especially Marcos Surmani for his advice on the thermal reduction and
his help with the Raman spectra.
I
Abstract
The sense of smell is the oldest, most versatile and as some biologists claim, the most
important one for living creatures. It is understandable that mimicking this sense
by building an artificial nose would be highly useful in an abundance of applications.

For instance it could be used in food industry, cosmetic production, environmental
monitoring and medical diagnostics, where the detection of volatile biomolecules
plays an important role. However, the sense of smell in humans and animals still
outperforms any analytical device. As the sensitivity and selectivity of the biological
olfactory system is by far not reached yet, new approaches to build a stable and
miniaturised biosensor as an artificial nose are needed.
This study is an attempt to improve and miniaturise a biosensor used for the de-
tection of odorants. A reduced graphene oxide field-effect transistor (rGO-FET)
functionalised with odorant-binding proteins is used as a biosensor. Due to its
unique electrical properties and easy handling procedure, reduced graphene oxide
is utilized as the transducer. To reduce the complexity of the biological olfactory
system, odorant-binding proteins (OBP) are chosen as a biorecognition element.
They are very stable and can bind selectively to odorants. This sensor platform
consisting of an rGO-FET functionalised with odorant-binding proteins can be used
to detect binding events of odorants in real-time. To test the sensor properties and
gain more insights in the function of an odorant binding protein, the binding prop-
erties of the wild type of the OBPs of a moth are compared to an engineered mutant
of this protein. The mutant shifts its affinity from pheromones to plant volatiles.
Therefore, the binding of a plant volatile and a pheromone to these two proteins are
investigated. With the here presented sensor the detection of odorants is possible.
However, a shift in the affinity of the mutant OBPs could not be detected.
This biosensor offers an approach to the easy and safe construction of an electrical
smell sensing device. As the described biosensor could be modified easily for future
investigations using different biomolecules immobilised on the sensor as a biorecog-
nition element, the current study may serve as a basis for further steps towards the
development of an artificial nose.
II
Kurzfassung
Der Geruchssinn ist der älteste, vielfältigste und vielleicht sogar der wichtigste Sinn
aller Lebewesen. Daher wäre eine künstliche Nase, die diesen Sinn nachahmt, für
einer Reihe von Anwendungsgebieten, wie in der Lebensmittelindustrie, der Kos-

metikbranche, der Umwelttechnologie und in der medizinischen Diagnostik, zur
Detektion flüchtiger Biomoleküle von großer Bedeutung. Derzeit jedoch ist der
Geruchssinn von Menschen und Tieren jedem analytischen Gerät bei Weitem überlegen,
sowohl wasdie Sensitivität als auch die Selektivität betrifft. Es werden somit neue
Ansätze für einen stabilen und miniaturisierten Biosensor verfolgt.
In dieser Arbeit wird die Detektion von Geruchsstoffen mit Hilfe eines verbesserten
und miniaturisierten Biosensors getestet. Dazu wird ein Feldeffekt-transistor, der
auf reduziertem Graphen Oxid basiert, mit Geruchsbindeproteinen funktionalisiert.
Aufgrund der außergewöhnlichen elektrischen Eigenschaften von Graphen-Oxid (GO)
und um eine einfache Herstellung des Sensor zu gewährleisten, wird dieses als Trans-
duktor verwendet. Um die Komplexität des biologischen Geruchssinnes in vere-
infachter Weise nachzuahmen, werden Geruchsbindeproteine als biologisches Sys-
tem des Biosensors verwendet. Odoranten binden spezifisch an diese Proteine und
ermöglichen so die Messung der Odorantenbindung in Echtzeit. Um die Sensor-
eigenschaften zu testen und mehr Einblick in die Bindungsweise von Odoranten
an Geruchsbindeproteinen zu erlangen, wurde der Wildtyp des Geruchsbindepro-
teine von Motten (Plutella xylostella) mit mutierten Geruchsbindeproteinen ver-
glichen. Das mutierte Protein wurde mit dem Ziel konstruiert vorzugsweise pflan-
zliche Geruchsstoffe zu binden, wohingegen der Wildtyp eine höhere Affinität zu
Pheromonen aufweist. Somit wird die Bindung von einem pflanzlichen Geruch-
stoff und einem Pheromon an diese beiden Bindungsproteine untersucht. Mit dem
hier vorgestellten Biosensor können flüchtige Geruchstoffe detektiert werden, die
Affinitätsänderung des mutierten Geruchsbindeprotein im Vergleich zum Wildtyp
jedoch nicht gezeigt werden.
Der hier beschriebene Biosensor kann aufgrund seiner einfachen und sicheren Her-
stellung als Ausgangsbasis für einen elektrischen Geruchssensor dienen. Darüber
hinaus kann der Sensor mit unterschiedlichen Bindungsproteinen beladen werden
und somit für Detektion unterschiedlichster Odoranten verwendet werden.
III
Contents
Abstract II
Kurzfassung III
List of abbreviations VI
List of figures VIII
1 Introduction 1
1.1 Overview of the sense of smell . . . . . . . . . . . . . . . . . . . . . . 1
1.2 General concept of biosensors . . . . . . . . . . . . . . . . . . . . . . 3

1.3 Artificial nose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.4 Biosensing with reduced Graphene Oxide Field-Effect Transistors (rGO-
FET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4.1 General characteristics of a field-effect transistor . . . . . . . . 8
1.4.2 Concept of reduced graphene oxide field-effect transistors . . . 8
1.5 Graphene as a biosensing element . . . . . . . . . . . . . . . . . . . . 11
1.5.1 Properties of graphene . . . . . . . . . . . . . . . . . . . . . . 12
1.5.2 Fabrication methods of graphene sheets . . . . . . . . . . . . . 13
1.5.3 Semiconducting properties of an rGO-FET: Ambipolar be-
haviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.6 Functionalisation of the biosensor with odorant binding proteins . . . 16
1.6.1 Physiological role of odorant binding proteins . . . . . . . . . 19
1.6.2 General odorant binding proteins of Plutella xylostella . . . . 20
1.6.3 Binding properties of odorant binding proteins . . . . . . . . . 22
1.7 Signal transduction of the binding events during biosensing measure-
ments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.7.1 Interface solid-liquid: Electrical double layer and Debye length 26
1.8 Theory of data evaluation with the Langmuir model . . . . . . . . . . 28
1.9 Aim of this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2 Materials and Methods 31

2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2 Preparation of the rGO-FET . . . . . . . . . . . . . . . . . . . . . . . 32
2.2.1 Chemical reduction of the graphene oxide . . . . . . . . . . . 33
IV
V
2.2.2 Thermal reduction of the graphene oxide . . . . . . . . . . . . 33

2.3 Immobilization of the proteins . . . . . . . . . . . . . . . . . . . . . . 34
2.4 Preparation of the solution . . . . . . . . . . . . . . . . . . . . . . . . 35
2.5 Measurement set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3 Results 37
3.1 Set-up characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2 Functionality tests of measurement set-up . . . . . . . . . . . . . . . 38
3.3 Measurements with plant volatile (Coniferyl Aldehyde) . . . . . . . . 44
3.3.1 Langmuir isotherm . . . . . . . . . . . . . . . . . . . . . . . . 44
3.3.2 Kinetic evaluation of the data . . . . . . . . . . . . . . . . . . 51
3.4 Measurements with pheromone (cis 11 - Hexadecenal) . . . . . . . . . 58
4 Discussion 60
5 Conclusion and Outlook 63
Bibliography 65
List of abbreviations
Id drain current
Id Vg drain current over gate voltage

Kd dissociation constant
Vth threshold voltage

kobs observation rate constant
APTES (3-Aminopropyl)-triethoxysilane
CSP chemosensory protein
DSC differential scanning calorimetry
EDL electrical double layer
FET field-effect transistor
gFET graphene field-effect transistor
GO graphene oxide
GO-FET graphene-oxide field-effect transistor
GOBP general odorant-binding protein
GOBP2 general odorant-binding protein 2
GOBPM5 mutant 5 of the general odorant-binding protein 2
MOSFET metal oxide semiconductor field-effect transistor
OBP odorant-binding protein
VI
VII
OR olfactory receptor
PBP pheromone-binding protein
PBS phosphate buffer saline
PBSE 1-pyrenebutanoic acid succinimidyl ester
ppm parts per million
rGO reduced graphene oxide
rGO-FET reduced graphene-oxide field-effect transistor
TGA thermogravimetric analysis

THF tetrahydrofuran
TRIS tris(hydroxymethyl)-aminomethan
List of figures
Fig. 1.1 Schematic view of a human nose . . . . . . . . . . . . . . . . . . 2

Fig. 1.2 Schematic illustraion of the olfactory epithelium . . . . . . . . . 2
Fig. 1.3 Schematic illustraion of a biosensor . . . . . . . . . . . . . . . . 3
Fig. 1.4 Comparison of biological olfactory system and electronic nose . . 5
Fig. 1.5 Electronic nose based on silicon nanowire FET . . . . . . . . . . 6

Fig. 1.6 Field-Effect Transistor in liquid gate configuration . . . . . . . . 9
Fig. 1.7 rGO-Field-Effect Transistor in liquid gate configuration . . . . . 9
Fig. 1.8 Structure of graphene . . . . . . . . . . . . . . . . . . . . . . . . 11
Fig. 1.9 Brillioun zone and band structure of graphene . . . . . . . . . . 12
Fig. 1.10 Fermi levels of graphene . . . . . . . . . . . . . . . . . . . . . . . 14

Fig. 1.11 Id Vg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Fig. 1.12 Insect sensillum . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Fig. 1.13 Plutella xylostella . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Fig. 1.14 Structure of general odorant binding protein and the mutant of
the general odorant binding protein . . . . . . . . . . . . . . . . . . . 21
Fig. 1.15 Affinity constants for GOBP2 and their mutants to different ligands 21
Fig. 1.16 Energy funnel of a protein . . . . . . . . . . . . . . . . . . . . . 24
Fig. 1.17 Electric double layer . . . . . . . . . . . . . . . . . . . . . . . . . 27
Fig. 2.1 rGO-FET chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Fig. 2.2 TGA and DSC measurements . . . . . . . . . . . . . . . . . . . 34
Fig. 2.3 Raman spectra of GO before reduction, after chemical and after
thermal reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Fig. 2.4 Open flow cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Fig. 2.5 Flow cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Fig. 3.1 Schematic concept of the sensor and the current changes upon
titration of different ligand concentrations . . . . . . . . . . . . . . . 37
Fig. 3.2 Different PBS buffer concentrations on a chemically reduced GO-
FET (commercial flow-cell top-up) . . . . . . . . . . . . . . . . . . . 40
Fig. 3.3 Different PBS buffer concentrations on a thermally reduced GO-
FET (commercial flow-cell top-up) . . . . . . . . . . . . . . . . . . . 41
Fig. 3.4 Different PBS buffer concentrations on a thermally reduced GO-
FET (custom-made flow-cell top-up) . . . . . . . . . . . . . . . . . . 42
Fig. 3.5 Id Vg curve of PBS-buffer concentration . . . . . . . . . . . . . . 43
VIII
IX
Fig. 3.6 Example of a typical ∆Id t curve of a rGO-FET with Coniferyl

Aldehyde concentrations . . . . . . . . . . . . . . . . . . . . . . . . . 44
Fig. 3.7 Data evaluation with the Langmuir isotherm . . . . . . . . . . . 45
Fig. 3.8 Thermally rGO-FET with only reduced graphene oxide for Coniferyl
Fig. 3.9 Thermally rGO-FET with only hydrolysed PBSE-linker for Coniferyl
Fig. 3.10 Thermally rGO-FET with the wild-type GOBP2 for Coniferyl
Fig. 3.11 Thermally rGO-FET with the mutant GOBPM5 for Coniferyl
Fig. 3.12 Boxplots of the dissociation constants to compare the GOBP2
to its mutant GOBPM5 . . . . . . . . . . . . . . . . . . . . . . . . . 50
Fig. 3.13 Overview of the kinetic evaluation of the data . . . . . . . . . . . 51

Fig. 3.14 Kinetic evaluation of Coniferyl Aldehyde concentrations on rGO-
FET with only reduced graphene oxide . . . . . . . . . . . . . . . . . 52
FET with only hydrolysed PBSE-linker . . . . . . . . . . . . . . . . . 53

FET with the immobilised wild type of GOBP2 . . . . . . . . . . . . 54
FET with the immobilised mutant GOBPM5 . . . . . . . . . . . . . . 55
FET with immobilised GOBPM5 with narrower linear range . . . . . 56
Fig. 3.19 Comparison of the reaction time at 1µM for both proteins . . . . 57
Fig. 3.20 Id t curve of a rGO-FET with GOBPM5 and Hexadecenal con-
centrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Fig. 3.21 ∆Id t curve of a rGO-FET with 100µM of Hexadecenal . . . . . 59
1. Introduction
1.1 Overview of the sense of smell

The sense of smell is a complex system that converts a chemical stimulus into an
electrical signal that is processed by the brain. With this system it is possible to
detect volatile biomolecules, called odorants, with a high sensitivity and selectivity.
Humans can discriminate more than one trillion odorants [1] and can recognise them
down to the nano-molar range [2]. Still the human smell sense is more sensitive than
any analytical instrument, although it is outperformed by the one of insects [2]. This
means that the olfactory system of humans and animals is a powerful tool and is
used, for example, as a warning system, to detect toxins and rancid food or to find
mating partners by detecting pheromones.
The high sensitivity and selectivity of the biological olfactory system makes it de-
sirable to build an analytical instrument with the same properties for the detection
of biomolecules. Therefore, understanding the function of the olfactory system is
of great importance. A major step was the discovery of olfactory receptors and the
encoding of their genes by Buck and Axel [3]. In 2004 they received the Nobel price
for their work on this cellular mechanisms of olfaction. The olfactory receptors are
membrane proteins found on the cilia of the sensory neurons in the olfactory epithe-
lium. This epithelium is found in the upper part of the nose cavity, see Fig. 1.1. In
the olfactory epithelium three types of cells are found, the sensory neurons, basal
cells and supporting cells, see Fig. 1.2. Basal cells are stem cells and by division
new sensory neurons are created every 30-60 days [4]. The sensory neurons have
cilia on their dendrites that are located in the mucus of the olfactory epithelium
[3]. For the olfactory perception odorants bind to the olfactory receptor. This bind-
ing is probably facilitated by odorant binding proteins. These proteins can bind
specifically to volatile biomolecules and tranport the hydrophobic odorants through
the mucus to the receptor [5]. The binding of odorants to the olfactory receptors
triggers a signalling cascade that depolarises the sensory neuron leading to an action
potential. The action potential is then transmitted to the olfactory bulb and further
to the brain, where the complex olfactory information is processed [3, 4, 6].
1
Chapter 1
Figure 1.1: Schematic view of a human nose [4]
Figure 1.2: Schematic illustraion of the olfactory epithelium [4]

2
Chapter 1 3
1.2 General concept of biosensors
A biosensor in general combines a biorecognition element, which is a biological sens-

ing element, like proteins, with a transducer [7]. Basically a biosensor consists of
these two elements and an electronic system, which are basic electronic compo-
nents that amplify and process the signal, see also Fig. 1.3 [8]. Biosensors can be
categorised by their transducer or their biorecognition element. A transducer can
be optical, thermal, piezoelectric or electrochemical, which can be further devided
into an amperometric, pontiometric or impedance sensor element. A biorecognition
element can be a receptor, DNA or proteins, like antibodies or odorant binding
proteins.
Figure 1.3: Schematic illustraion of a biosensor [8]
The biological sensing element or biorecognition element, for example a receptor,

translates the information from a biochemical reaction, usually an analyte concen-
tration, into a chemical or physical output signal with a defined sensitivity and
selectivity. This signal is then converted into an electrical signal by the transducer.
In a biosensor the recognition element is in direct contact with the transducer to
provide a self-contained and small sensor. A biosensor can for example measure
the interaction of an analyte with molecules immobilized on the sensor. Therefore,
antibody-antigen interactions, enzymatic reactions or analytes binding to proteins
can be monitored [9].
A biosensor can be constructed by immobilizing a biological receptor such as pro-

teins, antibodies or cells on the transducer surface, which then acts as a biorecogini-
tion unit. This can be achieved by different methods, either with covalent binding of
Chapter 1 4
the receptor to the surface or by having an additional linker that binds the receptor
to the transducer and also acts as a spacer between them [9].
A cheap and easy way to create biosensors is to use a field effect transistor as a
sensor element. In this work a field-effect transistor with graphene oxide (GO) is
used. Here the GO is the transducer and odorant binding proteins are used as a
biorecognition unit.
Biosensors have gained increasing importance in medical applications, in agriculture

and in the food industry. In 1962 the first biosensor for the amperometic measure-
ment of glucose was developed by Clark et al. [10]. This gave rise to the emergence
and improvement of many electrochemical glucose biosensors [11]. In 2013 glucose
sensors were successfully integrated in a lab-on-a-chip to detect glucose in whole
blood [12].
The development of the glucose biosensor gave an initial impetus for the creation of
a variety of biosensors in medical applications, the food industry and enviornmental
monitoring. In all these fields of application the detection of biomolecules with an
artificial nose, as a sensitive and selective detection system of volatile compounds in
real-time, is more and more desired.

Chapter 1 5
1.3 Artificial nose
An artificial nose, also called electronic nose consists of a sensor array and a signal
processing software, like pattern recognition or artificial intelligence [6, 13]. The first
electronic nose was built in 1982 by Persaud and Dodd. Since then the technology
was further developed and many different approaches of building an artificial nose
by combining various types of biosensors with different signal processing methods
emerged. The basic principle of an electronic nose can be seen in Fig. 1.4, where it
is compared with the biological olfactory system.
Figure 1.4: Comparison of biological olfactory system and electronic nose [14]
The perception of odorants plays an important role in food industry, cosmetic

production, environmental monitoring, medical diagnostics and many more. In
medicine non invasive detction and monitoring of diseases is a challenging need.
The detection of volatile organic compounds in the breath, urine or saliva generated
by different diseases was already achieved [6]. One example is the analysis of the
breath as certain diseases, like cancer, change its composition. For example, She-
hadeh et al. [15] developed a silicon nanowire field-effect transistor. This sensor can
distinguish between lung cancer, respiratory diseases and gastric cancer by analysing
the breath of the patient, see Fig. 1.5. In food industry electronic noses are used to
monitor contamination, like fungi, the shelf life of vegetables, fermentation or meat
spoilage [6, 13]. Electronic noses are even used to control the production of beer and
descriminate between different types of beer [14]. Moreover, electronic noses can be
used to detect toxins in the environment and control air and water pollution [6].
To detect different chemical compounds also sensors based on grahene have been
Chapter 1 6
used [16]. In agriculture electronic noses are a promising candidate to replace the
slow laboratory analysis to monitor for example crop. A recent application that be-
comes more and more important is the detection of explosives in the public security
sector[6].
Figure 1.5: Electronic nose based on silicon nanowire FET to detect diseases via breath analysis
[15]
Sensors used for electronic noses are mainly based on gas sensors and can be field-
effect transistors, quartz crystal microbalances and chemoresistors. Most of the gas
sensors used are chemoresistive. This means that they change their resistance upon
binding of chemicals or changes of the chemical environment of the sensor [6]. In
a field-effect transistors used as a gas sensor the channel conductance is varied due
to environmental changes. Hereby, different FET parameters can change, like the
threshold voltage or the charge carrier mobility. This makes it possible to extract
more parameters for the pattern recognition than chemoresistors [6]. 2D materials,
like phosphorene nanosheets or reduced graphene oxide are favoured for field-effect
transistors due to their fast response and high signal to noise ratio resulting from
their atomic thickness [6].
Although many different approaches for an electronic nose have been developed
recently, some challenges still remain to mimic the good detection characteristics
of the olfactory system of humans and animals. Thereby, an important part of
an artificial nose is the biosensor, which should become more sensitive, especially
for medical applications, like breath analysis, or in public security for the early
detection of explosives. In electronic noses mostly sensor arrays are used to increase
the selectivity of the sensor. Nevertheless, the selectivity of one sensor in this array
plays a not negligible role and needs further improvement. Moreover, a fast response
and fast recovery of the sensor are desired. A strong chemical reaction or strong
adsorption of chemicals to the sensor may prevent its fast recovery, although it
Chapter 1 7
would be beneficial for the sensitivity and selectivity of the sensor. A disadvantage
of an electronic nose based on a gas sensor is that many of them need ultraclean air
carrier gas and the sensing signals need to be calibrated, which makes a detection
outside the lab or controled environments difficult. Moreover, the miniaturization
of the electronic nose is still a challenge [6, 14]. Therefore, an improvement of the
sensor characteristics is still needed to use them for complex applications.
To improve and miniaturise the sensor nanomaterials for the transducer are in-
vestigated. Since the discovery of graphene in 2004 [17] it gained more and more
importance for the development of a biosensor due to its unique electrical properties.
Also, it became representative for 2D-nanomaterials. For an easy fabrication process
of sensors with graphene, a soluble form of it, namely graphene oxide is used. To
further simplify the complex smell sensing process found in nature for a biosensor,
odorant binding proteins are used as a biorecognition element. These proteins are
very stable and bind selectively to a set of odorants (see Chap. 1.6). As the odor-
ant binding proteins, found in the mucus of humans [18] and the sensillar lymph of
insects [19], are in an aqueous solution, the detection of odorants is also in liquid.
Therefore, also the biosensor is in a liquid configuration. This type of biosensor was
already tested by Larisika et al. [20], Kotlowski et al. [21] and Reiner-Rozman et
al. [22] with odorant-binding proteins from the honeybee.
Based on that work a modified sensor is built and tested for this thesis. The modifi-
cation included testing the binding properties of different odorant-binding proteins,
which are from a moth (Plutella xylostella). Moreover, the production process of the
biosensor is shortened by the usage of interdigitated electrodes form the company
Micrux as a basis.
Chapter 1 8
1.4 Biosensing with reduced Graphene Oxide Field-

Effect Transistors (rGO-FET)
1.4.1 General characteristics of a field-effect transistor
Field-effect transistors (FET) are electronic elements favoured for sensors. Sensors
based on FETs have a simple configuration, are of low costs and easy to miniaturize.
Moreover, real-time measurements with a high sensitivity can be performed with
FETs, which is of great advantage in various applications, such as in medical devices
and biosensors [23].
A field-effect transistor consists of three electrodes: source, drain and gate. The
current flows between source and drain over a thin channel and can be manipulate
with the gate electrode. For this channel between source and drain a semiconduct-
ing material can be used, which increases the sensor performance [24]. Typically,
FETs have an external gate electrode, which is perpendicular to the semiconducting
material. The current in a field-effect transistor is controlled by the gate voltage due
to the electric field-effect. So, the channel conductance can be varied capacitively by
changing the gate voltage through a thin dielectric layer. This layer should prevent
leakage current, which is the current that flows directly from the gate electrode to
the drain electrode. For the proper function of a FET the leakage current should be
way smaller than the current between source and drain [25]. By applying voltage
on the gate over a certain threshold voltage the semiconductor becomes conductive.
Below this threshold voltage the semiconductor is not conductive. The properties
of a field-effect transistor depend on the geometry of the channel, i.e. length and
width, and of the semiconducting material. Due to the properties of the semicon-
ductor, FETs can be distinguished into two different types. If the charge carriers
are dominated by electrons, the FET is called n-type and if they are dominated by
holes (absence of electrons), it is called p-type.
1.4.2 Concept of reduced graphene oxide field-effect tran-

sistors
A field-effect transistor in the liquid-gate configuration is used in this work. In this

configuration the dielectric layer is an electrolyte solution, see Fig. 1.6. Reduced
graphene-oxide (rGO) can be optimally used as a transducer for semiconducting de-
vices due to its easy fabrication. Nevertheless, the properties of rGO are similar to
pristine graphene. These properties depend on the special arrangement of the atoms
of graphene in a crystal lattice, see also Chap. 1.5. Due to graphenes high carrier
mobility, low intrinsic noise and high carrier density a sensor with a high signal-
Chapter 1 9
Figure 1.6: General structure of a FET in liquid gate configuration (source and drain are on
opposite sides of the gate contact) [26]
to-noise ratio and high sensitivity can be built. Graphene oxide is already used in
various applications to manufacture sensitive biosensors [27, 28]. Moreover, the elec-
trodes of a sensor covered in rGO have higher conductivity, sensitivity and a higher
signal-to- noise ratio compared to carbon nanotubes. [29]. Therefore, graphene ox-
ide is used as a transducer in the here presented biosensor. The rGO-FET is then
functionalised with odorant binding proteins as a biorecognition element, see Chap.
1.6. The schematic view of the functionalised rGO-FET can be seen in Fig. 1.7.
Figure 1.7: Left: General structure of an rGO-FET in liquid gate configuration, Right:
Schematic view of the functionalised rGO-FET used in this thesis
An rGO-FET as a biosensors is operated with a constant gate voltage and the varia-
tions of the drain current due to environmental changes are measured. The detected
drain current, Id , is a signal for the biochemical process happening in the transistor
channel. This is contrary to the typical use of a FET, where the channel conduc-
tance is adjusted by the gate voltage [30]. By keeping the gate voltage fixed, the
Id is changed as the semiconducting material is modified by different environmental
processes. The changes of the Id can be readout and linked to biochemical process,
Chapter 1 10
like the binding events of ligands to immobilized proteins on the sensor channel.
Due to the ambipolar behaviour of an rGO-FET (see also Chap. 1.5.3), it can be
operated with a positive as well as a negative gate voltage. Therefore, an optimal
working point can be set. Moreover, biorecognition events influence the electrical
characteristics of the FET, which can be used as an analytical signal [31, 32].
Reduced graphene oxide field-effect transistors can be used to measure the ion
strenght and the pH-value easily [33]. The changes in pH can be measured with
the rGO-FET because the fuctional groups, such as hydroxyl (OH) and carboxyl
(COOH) groups, of the rGO-surface interact with the H + atoms of the electrolyte
and change the charge density of the surface. Also, the electrical double layer (see
Chap. 1.7.1) is changed causing electrostatic gating effects. This means a FET with
pristine pure graphene would be less sensitive to pH changes than the rGO-FET,
because with no fuctional groups on the surface there would be no available bonding
sites for the H + ions [34].

Chapter 1 11
1.5 Graphene as a biosensing element
In the last decade graphene and carbon nanomaterials in general gained more and
more importance for the development of biosensors. The interest to further study
and use graphene for various applications lies in its unique electrical, optical and
mechanical properties.
The unique properties of graphene are related to its structure. Graphene is defined as
a monolayer of pure carbon atoms [35, 36]. The sp2 -hybridized carbon atoms form
a hexagonal lattice by covalent σ-bonds, see Fig. 1.8. The carbon atoms, which
have a distance of 1.42 Ångström, form a σ-band [37]. Due to this band the lattice
structure has his robustness. Ideally graphene should be completely 2-dimensional,
but normally graphene contains a different number of layers and it tends to fold, so
it is never completely flat [17].

Figure 1.8: Hexagonal structure of graphene[38]
The electrical properties, like the high conductivity, and the band structure of
graphene were published already in 1947 by Wallace et al. [39]. Already in 1962
Boehm et al. [40] fabricated the first graphene-oxide (GO). GO can be seen as sheets
of graphene with oxygen functional groups on edges and on the basal planes. By
reducing in sodium hydroxide or heating this GO, they even fabricated thin carbon-
films. Hummers et al. [41] introduced a method to produce graphene oxide with
wet-chemistry, which is still used today and known as the Hummers method. In
2004 Novosevlov and Geim [17] mechanically exfoliated graphite. With this method
they were able to prepare thin films of graphene with only one or two atomic layers.
Chapter 1 12
1.5.1 Properties of graphene
The structure of graphene has an effect on its electrical properties. The trigonal
planar structure is formed by the sp2 -hybridization between one s-orbital and two
p-orbitals (px , py ). The carbon atoms bond covalently and form σ-bonds. As men-
tioned above this so formed σ-band is responsible for the robustness of graphenes
structure. Moreover, this band has a filled shell due to the Pauli principle and
therefore forms a valence band. The electrons of the unaffected pz -orbital bind
more weakly to neighbouring carbon atoms and form a π-band. This p-orbital is
perpendicular to the planar structure. The π-band is only half-filled, because the
p-orbital has one extra electron. Due to the mobility of the electrons of the π-band,
graphene is conductive. These electrons are delocalized over the whole surface and
are therefore called delocalized electrons [37].
In the lattice of graphene each carbon atom has a π-orbital that contributes to
the delocalized electrons. Due to the structure of graphene the first Brillouin zone
′
(Brillouin zone: unit cell of the reciprocal lattice) has two points K and K called
Dirac points, where the band crossing occurs. In graphene the first Brillouin zone is a
hexagon (see Fig. 1.9, left) [42]. At this Dirac points the charge carries change from
electrons to holes (see Fig. 1.9, right). Meaning that the cone-shaped conduction and
valence band meet at the K points and there is no bandgap [43]. Therefore graphene
has an ambipolar behaviour. The electronic spectra of mono- and bilayer graphene
is a zero-gap semiconductor with only one type of electrons and holes. For more than
three layers the conducting and the valence band start overlapping and more types
of charge carriers appear [44]. The charge carriers of graphene have a unique nature
and can be seen as massless Dirac fermions, as they behave relativistically. They
behave as if they have no mass, which results from the interaction of the electrons
with the special structure of graphene [35].
Figure 1.9: Left: hexagonal structure of graphene, Right: one Brillouin zone with band structure
(conical energy spectrum), conductance band and valence band touch at the Dirac points K and
K’ [45]
Moreover, graphene has a very high charge carrier mobility µ, that is little affected
by chemical doping and temperature. At room temperature mobilites from 3000
cm2
to 10 000 were measured in graphene with only a few layers and even higher
Vs
Chapter 1 13
cm2
mobilities with around 15 000 at multilayer graphene [17, 42]. These mobilities
Vs
are one order of magnitude higher than those of silicon transistors [45]. This high
mobilities are an effect of delocalized electrons and the quality of the lattice of
graphene [29]. Graphene’s mobility also depends on the number of impurities in
a layer and on structural defects. At a high crystal quality the charge carries can
travel thousands of interatomic distances without scattering [44].
1.5.2 Fabrication methods of graphene sheets
Graphene sheets can be fabricated with wet-chemistry, with chemical vapor deposi-
tion or by exfoliation of graphite. By exfoliation of graphite, for example, with the
scotch tape method, mono-layer graphene can be produced with the best quality
and a large size. The problem, hereby, is to identify the number of layers and size
[44]. Also the fabrication process is complicated and with this method graphene
sheets can only be produced in a very small amount and not in a reproducable way
[30].
An easier fabrication process, which achieves also a higher yield can be achieved
with wet chemistry, for example, with the Hummers method [41]. With this method
graphite is oxidized with strong oxidizing agents in water and converted into graphene
oxide. Graphene oxide (GO) consists of saturated sp3 -carbon atoms, which are
bound to oxygen. So, GO contains functional groups, such as carboxyl, epoxide and
hydroxyl groups, due to the oxidation process that increase the distance between
the graphene layers and make it hydrophilic [29]. This makes it easy to disperse the
graphene oxide mechanically into single sheets in water. Even the highly oxidized
GO keeps the aromatic ring structure of pristine graphene [46]. On the one hand the
functionalization of different sensors with GO-solution can be simply achieved by
drop-casting, spin-coating or dip coating. On the other hand graphene in the form
of graphene oxide loses its high conductivity and becomes an insulator due to the
functional groups. To regain the unique properties, GO is reduced. The reduction
can be accomplished with chemicals, such as hydrazine or thermally by heating it
under reducing atmosphere. Although the reduction flattens the graphene and it
gains a higher conductivity there are still carbon-oxygen bonds left. By thermally
reducing the GO the hydroxyl and epoxy groups are transformed into carbonyl and
ether groups. The hydroxyl groups, mainly oxygen that is double bonded to car-
bon, are reduced above 200◦ C [46, 47]. By investigating the mass loss with TGA
(Thermogravimetric analysis), see also Chap. 2.2.2, it is seen that already at 100◦ C
GO starts to lose adsorbed water and gas molecules [46].
Raman spectra (see also Chap. 2.2.2) can be used to find out how defective graphene
is. In a spectrum two main peaks, the G and D peak, are observed, which occur by
laser excitation and are vibrational modes. At 1580 cm−1 , the G-peak, a primary in-
plane vibrational mode can be found. At ∼ 1350 cm−1 , depending on the excitation
Chapter 1 14
energy of the laser, the D-peak can be found in defective graphene. The D-peak
appears due to scattering of excited charge carriers at defects or zone boundaries
[48]. The ratio of the intensities of the D and G-peak gives an estimation of the
number of defects and is inversely proportional to the crystallite size [49].
1.5.3 Semiconducting properties of an rGO-FET: Ambipo-

lar behaviour
Graphene is not a good semiconductor as it has no bandgap. Therefore, a graphene

field-effect transistor (gFET) is always conductive and can never be turned off. In
a zero-bandgap graphene field-effect transistor (gFET) the gate voltage can vary
the Fermi level. (The Fermi level is the top energy level, which is fully filled with
electrones in the ground state [50].) This means that depending on the gate voltage
the charge carriers can either be electrones or holes, see Fig. 1.10. This results
in an ambipolar behaviour of the gFET, which can be seen by plotting the drain
current over the gate voltage (Id Vg ), see also Fig. 1.11. The current decreases for
negative gate voltage until it reaches its minimum, called Dirac point and increases
for gate voltages higher then the Dirac point. The branch at negative gate voltage,
cathodic scan, resembles the hole mobility and the branch at positive gate voltage,
anodic scan, the electron mobility [20, 51]. Due to this ambipolar behaviour and the
absence of an electronic band gap, the sensor is always turned on in contrast to a
typical MOSFETs. MOSFETs, therefore, can be turned on and off by changing the
gate voltage. The turning point is defined as the threshold voltage (Vth ). However,
a Vth can also be determined for gFETs. By linear fitting the two branches of the
Id Vg curve, Vth is the interception of these lines with the voltage axis [22].
Figure 1.10: Band structure near the Dirac point and the Fermi level depending on the charge
carrier concentration [52]
Chapter 1 15
Figure 1.11: Drain current over gate voltage [30]
Nevertheless, graphene, at least bilayer graphene, with a tunable energy gap is found
as a semiconducting material. An electric field can change the band structure of

bilayer graphene in a way that the gap between the valence and the conduction band
can be varied between zero and midinfrared energies [53].
The advantage of the ambipolar behaviour of graphene is that it allows for the sensor
to be operated at negative and positive gate voltage. Due to this fact, an optimal
working point for each measurement can be chosen very easily [42].
Chapter 1 16
1.6 Functionalisation of the biosensor with odor-

ant binding proteins
For animals the olfactory system is very important for the detection of food, toxic
substances and pheromones. Two types of proteins are involved in the perception
of odorants, the odorant-binding proteins and the olfactory receptors, which are
membrane proteins. Both of them bind specifically to odorants and would be suit-
able for a biorecognition element in a biosensor. Although olfactory receptors are
known to detect odors, they are less stable than odorant-binding proteins. Moreover,
odorant-binding proteins are easier to overexpress than olfactory receptors and can
also be expressed in bacterial systems with good yield [54]. These advantages make
odorant-binding proteins an optimal choice for the usage in a biosensor. Therefore,
they are used as a biorecognition element in the here presented biosensor.
In general an odorant-binding protein (OBP) is defined as a small soluble protein

that binds to several odorant molecules. They were discovered by Pelsoi et al.
[55] in 1981 as they searched for olfactory receptors. Since then they gained great
importance for the research in olfaction. Moreover, OBPs are the only class of
olfactory proteins, for which binding of odorants has been proven experimentally
[56]. At the same time also Vogt et al. [57] discovered pheromone-binding proteins
PBPs in moth antennae. These proteins interact with the pheromone of the silk
moth and are located in the pheromone-sensitive sensilla.
Odorant-binding proteins are water-soluable and are extracellular proteins of low

molecular weight, that are located between the odour stimuli and the neuronal
sensory membrane [5, 19]. In vertebrates and in insects the olfactory system is used
for the detection of odorants. The perception of odorants is a series of complex
events. Although it is certain that OBPs play an important role in the olfactory
system, the physiological function is not yet understood. Due to the fact that OBPs
can be found in the olfactory organs in a large amount, it is thought that they are
involved in the olfactory perception. Especially in insects the antennal sensillum is
a specialized organ, which functions only as a perception system for odorants and
pheromones [58].
Amino acid sequences of insect OBPs are completely different to OBPs of vertebrates
and also their 3D-structure differs. Despite this, OBPs may have similar functions,
because they share similar properties, for example, their molecular weight, small
size and their low isoelectric point [59, 60]. Additionally, general odorants of insects
as well as vertebrates belong to the same chemical class. Moreover, in vertebrates,
as well as, in insects, OBPs are found in high concentrations near the olfactory
neurons.
Odorant-binding proteins in insects do not share any similarities with other protein
Chapter 1 17
classes. The olfactory system in insects can detect, on the one hand, specific sex
pheromones with a high sensitivity and specificity and, on the other hand, general
odorants. The latter system is less sensitive and reacts to all different kinds of
odorants. Therefore, odorant-binding proteins in insects can be divided into differ-
ent subfamilies based on their amino acid sequences: pheromone-binding proteins
(PBP), general odorant-binding proteins (GOBP) and antennal binding protein X
(ABPX) [58]. Another class of soluble proteins can also be found in insects, the
chemosensory proteins (CSPs). CSPs have a different 3D structure than insect
OBPs. Furthermore, CSP can be found in different parts of the body and often
have a non-specific function.
GOBPs of the Lepidoptera (order of insects which includes moths and butterflies)
can be found in the male as well as the female antennae, whereas PBPs are highly
concentrated in the male antennae and bind specifically to pheromones. Interestingly
PBPs can also be found in the female antennae and in the male sensilla, which
are not pheromone sensitive [19]. Due to their amino acid sequence GOBPs of
Lepidotera can be further divided into two classes: GOBP1 and GOBP2 [59]. The
proteins in these classes share very similar sequences. GOBPs are more similar
within their groups than PBPs. This is a result of their sex pheromones, which
are specific for each species, while general odorants are able to bind to GOBPs in
different species. For example, medium chain alcohols and aldehydes bind to GOBPs
of different species. Of course, also the chemical structure of sex pheromones and
general odorants differs.
OBPs in insects can be found in the antennae, where they are highly concentrated in
the sensillar lymph. The concentration is in the millimolar range. In the sensillum
there are dendrites of at least one olfactory neuron. The membrane of these dendrites
contain the olfactory receptors (OR) and around the dendrite is a lymph, which
provides the aqueous medium for the dendrite and protects it. The OBPs are found
in this aqueous medium [19].
The amino acid sequence in most of the insect OBPs has six cysteines [19]. A cystein
is one of the 20 amino acids with a sulphur atom in its side chain. The sulphur atoms
of two cysteines can bond covalently and form bridges. These disulfide bridges are
formed in an OBP to stabilize the 3D-structure. Moreover, there are always three
amino acids between the second and the third cystein, and eight between the fifth
and sixth. This arrangement became representative for an insect OBP. Besides the
six-cysteine signature, insect OBPs have a molecular weight of about 15-20kDa, the
3D structure contains α-helices and they are water-soluble. However, the amino
acid sequence of OBPs varies extremely [19].
OBPs also have a hydrophobic binding pocket. This cavity is formed by five α-
helices. All insect OBPs have therefore a similar structural pattern, but they differ
in position, length and angle of the α-helices and in the conformation of the C-
termini. These differences have the effect that the binding pocket varies and as a
Chapter 1 18
result becomes accessible to different odorants.
OBPs as well as CSPs have a very stable and compact structure and they both have
hydrophobic binding pockets. Due to these properties, it is suggested that they are
used in various organs for the transport of hydrophobic compounds through aqueous
solution[60]. But it is also proposed, that insect OBPs cannot alter their functions
easily, because of the three interlocked bridges that stabilise the protein. CSPs
on the other hand can change more easily, because they have only two separated
bridges.
Chapter 1 19
1.6.1 Physiological role of odorant binding proteins
Although the physiological role of OBPs is still unknown, several models have been
introduced that suggest the part OBPs could play in the olfactory perception [5].
The first hypothesis suggests that the proteins could act as carries. They would bind
an odorant and bring it to membrane receptors, although the odorant can also bind
directly to the olfactory receptor (OR). This hypothesis is built on the circumstance
that odorants are generally hydrophobic, which makes the diffusion in the aqueous
barrier of the sensillum very slow and inefficient. Therefore, OBPs would increase
the solubility, so that molecules are able to reach the receptor. Another possibility
would be, that the odorants enter the sensillum lymph through pores connected
to channels, where they are able to reach the receptors. OBPs in this case would
bind the ligand after activating the receptors to hinder them on reactivating the
receptor. The third model suggests a combination of the two models above, which
would give the proteins two roles, as can be seen in Fig 1.12. Another possible part
of OBPs, in the perception of odorants, is based on the fact that several different
types of odorant-binding proteins were found. These proteins can bind specifically to
different kinds of ligands. Therefore they could also be part of the odour recognition
and can discriminate between different molecules. In this model the protein-ligand
complex is then bound to the receptor [5].
Figure 1.12: Schematic drawing of an insect sensillum and

one suggested role OBPs could play in the olfactory perception [61]
However, experiments showed that OBPs in some insect species are necessary for
the detection of molecules. For example Xu et al. displayed that the Drosophila
OBP lush is necessary for the detection of pheromones [62]. This suggests that
insect OBPs could discriminate odorants [60]. Studies also proved that PBPs are
very important for the pheromonal signal transduction [19]. So, they are most likely
carrying the molecules to the olfactory receptors to increase the solubility of the
pheromones and nonpheromones, the so called semiochemicals.
Chapter 1 20
1.6.2 General odorant binding proteins of Plutella xylostella
To test the proper function of the measurement setup, General odorant binding-
proteins (GOBPs) of the diamondback moth (Plutella xylostella) provided by P.
Pelosi have been chosen. The diamondback moth (see Fig 1.13) is mostly found as a
pest in agriculture, which eats for example cabbage [63]. Still only little information
can be found on the OBPs of this moth. GOBPs can bind specifically to different
odorant molecules. Furthermore, by studying this protein a better understanding of
how OBPs bind to different ligands can be achieved.
Figure 1.13: Plutella xylostella [64]
In the case of GOBPs the name does not reflect their function, but the name results
in finding the proteins equally in the female and male antenna of the diamondback
moth [63].
Moreover, GOBPs can also bind to sex pheromones. Already the larvae can detect
sex pheromones, but only in combination with food. Their specific sex pheromones
on food influence their food choice. The larvae is more attracted to food with
pheromones. The reason for this may be that they know that also their mother
has chosen this food, because of the pheromones. In contrast to Pheromone-binding
Proteins, GOBP2 binds (Z)-11-hexadecenal highly specific, while PBPs bind all sex
pheromones with similar affinities [63, 65].
Zhu et al. [63] also showed that they can shift the affinities from pheromones to-
wards plant volatiles, such as coniferyl aldehyde, by mutation of the GOBP2. In
the mutants only one or two amino acid residues in the binding pocket have been
replaced. The structure of the GOBP2 and its mutant can be seen in Fig. 1.14.
Moreover, it was shown that an additional His-residue does not change the affinities
significantly [63]. There was no change because the additional polypeptide, which
bonds the His-tag to the N-terminus of the protein is hydrophilic and therefore
prevents an interaction with the hydrophobic binding pocket.
Zhu et al. [63] investigated the ligand-binding experiments with fluorescence mea-
surements. According to their studies GOBP2 is highly specific for their sex pheromone
Z-11-Hexadecenal with a dissociation constant Kd of 3,3µM [63]. In contrary, the
Chapter 1 21
Figure 1.14: Structure of general odorant binding protein and the mutant of the general odorant
binding protein [63]
mutant GOBPM5 has a much higher dissociation constant to Z-11-Hexadecenal

(∼ 70µM ). Their affinity was shifted towards plant volatiles, like Coniferyl Alde-
hyde, with a Kd of about 7µM. Before the mutation the Kd of GOBP2 to Coniferyl
Aldehyde was about 17 µM. All the different affinities can be seen in the graph
below 1.15 [63].
Figure 1.15: Affinitie constants for GOBP2 and

1
their mutants to different ligands (x-axis: wild-type(wt) and different mutants, y-axis: ) [63]
Kd
Chapter 1 22
1.6.3 Binding properties of odorant binding proteins
The interaction of odorant binding proteins with odorants can be decribed by a

simple protein-ligand binding. In general proteins require interaction with different
molecules for their proper function. One of the functions of a protein is reversible
binding to other molecules. These molecules or other proteins are then called lig-
ands. The interaction between a protein and a ligand is important to change the
environmental and metabolic circumstances of an organism [66]. The structure of
a protein affects how ligands can bind and the interaction also has an effect on the
protein’s structure in terms of a conformational change.
A ligand binds only to a specific site of the protein, the binding site. This binding site
is complementary to the ligand in size, shape, charge and hydrophobic or hydrophilic
character. Proteins bind very specific and can distinguish between thousands of
different molecules to selectively bind only a few. A protein can also have more
than one binding site to be able to separately bind to a variety of ligands. During
a protein-ligand interaction it comes to a conformational change to tighten the
binding. This structural adjustment is also called induced fit, because it can make
the binding site more complementary to the ligand. In a protein with different
subunits, a ligand binding to one subunit can therefore also affect the conformation
of other subunits. Due to the interaction between the subunits, a highly sensitive
response to small changes in the ligand concentration can be achieved.
The binding event of ligands is associated with different affinities. That means that
a protein is more likely to bind a specific ligand than others. This affinities can be
described with an equilibrium expression [66]:
[P L] ka
Ka = = (1.1)
[P ] [L] kd
P + L ⇋ PL (1.2)
Hereby, Ka is the association constant, that describes the equilibrium between the
1
bound and the unbound state. The unit is and therefore a higher Ka means
M olar
higher affinity. [P ] is the number of unbound proteins, [L] number of ligands and
[P L] describes the number of ligands bound to proteins. ka and kd are rate constants,
whereby ka describes the binding of a ligand to the protein and kd the reverse process,
so the release of the ligand. The binding equilibrium is described by the ratio of
binding sites occupied to the total binding sites. This ratio ϑ can be rearranged
with the equations above:
Chapter 1 23
[P L] [L]
ϑ= = (1.3)
[P L] + [P ] 1
[L] +
Ka
ϑ([L]) is therefore a function of the concentration of free ligands and describes

a hyperbola. This means by increasing the free ligands, the concentration ϑ is
saturating asymptotically. This relation is described by the Langmuir model [67],
see also Chap. 1.8.
1
is defined as the dissociation constant Kd .
Ka
1
Kd = (1.4)
Ka
When Kd equals the free ligand concentration, where half of the binding sites are
occupied. The lower the dissociation constant the higher is the affinity. That means
that at lower Kd the ligand binds more tightly to a protein and a lower ligand
concentration is needed to occupy half of the binding sites. The association between
protein and ligands is driven by various interactions and energy exchanges between
the protein and the ligand, as well as the ions in the buffer solution.
To describe how proteins fold into their stable native conformation an energy funnel
can be used (see Fig 1.16). The energy funnel shows the folding process thermody-
namically. Hereby, the width of the tunnel shows the entropy whereas the height
shows the energy. As the funnel narrows the entropy decreases and the protein can
fold into semistable conformations, which are described by the small energy minima
on the side of the funnel. At the bottom of the funnel only one conformation is left,
the native state with the smallest energy. By calculating the difference between the
energy of the native state and the energy of any other minima in the energy funnel,
the amount of energy that a protein needs to fold into a certain conformation can be
obtained [66]. This is very useful for the description of the conformational change a
protein undergoes during the binding process of a ligand (see Eq. 1.5).
Chapter 1 24
Figure 1.16: Energy funnel of protein folding into its native state
(width is the entropy and hight describes the energy needed) ([66], S.143)
With the model of the energy funnel (see Fig. 1.16), a connection between the
dissociation constant Kd and the Gibbs free energy, that is needed for the confor-
mational change, can be drawn. Hereby ∆G(pH) is the hight of the energy funnel
between the bottom and the conformation of the protein-ligand complex and can be
described with the Nernst equation [30, 68].
∆G(pH) = −RT ln(Ka ) (1.5)
∆G(pH)
−
Ka = e RT (1.6)
∆G(pH) describes the difference between the Gibbs free energy of the protein-ligand
complex and the native state, where R is the gas constant and T the temperature.
The Gibbs free energy depends on the pressure p and the enthalpy H. By rearranging
the Nernst equation, the association constant Ka can be directly calculated with
∆G(pH). So, the changes in the Gibbs free energy determine how stable a protein-
ligand complex is.
To describe the binding process at any time the equation 1.5 is adjusted to [68, 69]:
∆G(pH) = −RT ln(Ka ) + RT lnQ (1.7)

Chapter 1 25
Q hereby, is the reaction quotient, which is defined as the following relation (1.8) at
any moment in time [68].
[P L]
Q= (1.8)
[P ] [L]
So, if the reaction is in equilibrium, which means Q = Ka , then ∆G(pH) = 0.

Moreover, a binding event only happens when the Gibbs free energy is negativ for
constant pressure and temperature. As seen above the magnitude of ∆G depends
on the binding affinity Ka and therefore determines the stability of a protein-ligand
complex [68].
Chapter 1 26
1.7 Signal transduction of the binding events dur-

ing biosensing measurements
The signal transduction in a biosensor in general can be achieved via different mech-
anisms. They can be for example voltammetric, potentiometric or conductometric.
In an rGO-FET the binding events can be detected by measuring the drain current.
The conductance of the sensor is changed by a series of events triggered by the
conformational change of the protein due to the binding of a ligand. As the ligand
binds to the protein the conformation of the protein changes, which results in a
displacement of the charged areas in the protein. The binding event also influences
the Gibbs free energy given by the energy landscape of a protein. (see Eq. 1.5).
This induces a local energy transfer to the neighbouring ions from the buffer solu-
tion. Consequently, the electrical double layer is displaced and therefore the surface
potential changes (see also Chap. 1.7.1). Due to the changes of the surface potential
the charge carrier mobility of the rGO is varied, which modifies the resistance of the
FET. So, the change in the Gibbs free energy of the protein also changes the energy
of the sensor surface [30].
1.7.1 Interface solid-liquid: Electrical double layer and De-

bye length
This section should give a short introduction in one phenomenon of the interface
between solid and liquid. Extensive research in this topic has been done [70–73].
An electrical double layer (EDL) occurs on interfaces of an electrode (solid) and an

electrolyte. This double layer induces a large electric field.
To describe the EDL different models can be used. The Helmoltz theory describes
this phenomenon in the most basic way. Thereby, it is assumed that the solid
surface is charged and a single ion layer of opposite charges in solution is formed at
the surface (see Fig. 1.17a). These discrete charges can be idealised with a double
layer and can be described mathematically with a simple capacitor.
The diffusion model is described by the Gouy-Chapman model, where the charge
distribution and therefore, also the electrical potential depend on the distance from
the surface. This diffusion is the equilibrium state of the electrostatic attraction of
the ions to the surface and the thermal motion, which equalizes the ion concentration
in the solution. Thereby, the electric potential decreases exponentially with the
distance from the surface (see Fig. 1.17b) [70, 72].
These two theories are combined in the Gouy-Chapman-Stern model. Hereby, a

Chapter 1 27
fixed Stern layer on the surface is introduced, the Helmholtz layer, and combined
with an outer diffuse layer (see Fig. 1.17c). Grahame further modified this model.
One modification is the inclusion of adsorbed ions, which are closer to the surface
then unadsorbed ions, that are described in the Helmholtz theory. So the Helmholtz
layer is divided in an inner and an outer Helmholtz layer. A detailed explanation of
this model can be found in Grahame et al. [74].
Figure 1.17: Models of the three different EDL theories [70]
Another important parameter for biosensing in liquid is the Debye length. With this
parameter it is possible to calculate the interaction radius of molecules in solution.
Therefore, the response signal of the liquid-gate transistor also depends on the Debye
length. This parameter depends on the properties and ion strength of the ionic
liquids and the thickness of the electrical double layer.
Moreover, the surface potential of a FET depends on the Debye length. This means
that a binding event can only be detected when the distance between this event and
the sensor surface is less than the Debye length. If this is not the case the event will
not have a significant effect on the surface potential, as it decreases exponentially
with the Debye length. Also, the debye length decreases with increasing buffer
concentration [30]. This has to be taken into account when choosing the ion-strength
of the PBS-buffer, which should not exceed 17mM when working with proteins [75].
Chapter 1 28
1.8 Theory of data evaluation with the Langmuir

model
The Langmuir theory originally describes the adsorbtion of a gas or a solution to a

solid surface [76, 77]. For this model some assumptions have been made. Firstly,
only monolayer adsorption occures and secondly, the adsorption can only take place
at a definite number of localized sites. This means that for this model the number
of binding sites has to be known and constant [30, 78]. Moreover, in his model
an homogenous solution is assumed, which states that each molecule has the same
enthalpy and all sites have equal affinities.
Langmuir proposes that a kinetic equilibrium is established between the molecules
that come from the surface to the interior and the molecules that arrive at the
surface from the interior. The rate at which the molecules reach the surface is pro-
portional to the concentration. To this extend the surface coverage also depends
on the concentration of the analyte of the solution. At a certain concentration all
binding sites are occupied, this is then called the saturation concentration. Graph-
ically this can be described by the Langmuir adsorbtion isotherm (see Eq. 1.9).
At low concentrations the surface coverage increases linearly and it gets saturated
non-linearly at higher concentrations describing a hyperbola. For the equilibrium
saturation it is stated that once a molecule occupies a site no further adsorption can
take place. The Langmuir binding isotherm therefore describes the adsorption on a
plane surface with only one kind of space that can only adsorb one molecule [67]:
cKa
ϑ= [30] (1.9)
1 + cKa
Hereby ϑ is the number of occupied sites, so it describes the surface coverage. c is the
concentration of the solution and Ka is the association constant at equilibrium (see
also Chap. 1.6.3). And again the dissociation constant is given as Kd = K1a . To asses
the binding constants the surface coverage ϑ can be plotted over the concentration,
which is normally logarithmically scaled. The surface coverage is determined by
searching the minimum values at equilibrium of the Id t curve for each concentration.
The saturation concentration, where the response signal does not change any more,
is set to 100% of surface coverage. The other values are all normalized to this
response signal and then plotted over the concentration and fitted with the Langmuir
isotherm (Eq. 1.9). The dissociation constant Kd can be read out from this plot at
50% surface covarge.
Another way to determine the binding constants is by calculating the kinetic asso-
ciation and dissociation constants. Hereby, the binding of a ligand to a protein can
correspond to a second order process (Eq. 1.10), whereas the dissociation of the
protein-ligand complex is a first order process (Eq. 1.11).
Chapter 1 29
association : (1.10)
P + L −→ P L
rate = ka [P ] [L]
dissociation : (1.11)
P L −→ P + L
rate = kd [P L]
But for simplification purposes the binding event can be seen as a pseudo-first order
process with the presumption that the concentration of the ligand is available in
excess. This means that the reaction rate depends linearly on the concentration
[66]. So the change in the concentration of unbound proteins [P ] on the surface is
equal to the rate constant k times [P ]:
d [P ]
− = k [P ] (1.12)
dt
By integrating Eq. 1.12 the following is obtained:
[P ] = [P ]0 e−kt (1.13)
[P ]0 is the concentration of unbound proteins at the beginning (t=0). This equation

describes the behaviour of the binding process. Therefore, different values for k
can be observed by fitting this exponential curve to every signal obtained for each
concentration c. These values are then called kobs and are plotted over the corre-
sponding concentration. This curve can be fitted linearly. The relation is described
as: [22]
kobs = ka · c + kd (1.14)
Hereby the slope of the linear fit is ka and the offset of it is kd . The equilibrium
constant is estimated with the following equation [22]
kd
Kd = (1.15)
ka
For the evaluation of the data in this thesis the Langmuir isotherm as well as the
binding kinetics are investigated.
Chapter 1 30
1.9 Aim of this study
The aim of this study is to modify the biosensor presented in Larisika et al. [20],
Kotlowski et al. [21] and Reiner-Rozman et al. [22] to miniaturise the sensor, to
improve its sensitivity and to simplify its manufacturing process. Electrochemical
sensors with interdigitated electrodes from the company Micrux are tested as a basis
for the here presented biosensor. By using this electrodes the fabrication process
could be shortened and the resistance of the field-effect transistor reduced. More-
over, the fabrication of the sensor should be as safe as possible sparing the use of
toxic chemicals in the reduction process of graphene oxide.
The sensor is functionalised with odorant binding proteins to characterise the bind-
ing properties of certain odorants to these proteins. Hereby, the general odorant
binding protein from the moth (wild-type) is compared to a mutant of this protein.
The mutant is engineered in a way that it shifts its affinity from pheromones to
plant volatiles. Moreover, the improvements of this sensor should facilitate pro-
duction and enhance the usability of this biosensor as a step towards an artificial
nose.
2. Materials and Methods
2.1 Materials
Chemicals
• Ethanol 99% (Sigma Aldrich)

• (3-Aminopropyl)-triethoxysilane (APTES) (Sigma Aldrich)
• Graphene oxide solution (University of Bayreuth)
• DI water
• Phosphate buffer saline (PBS) tablets (Sigma Aldrich)
• Coniferyl Aldehyde (Sigma Aldrich)
• cis-11-Hexadecenal (Sigma Aldrich)
• Methanol (Sigma Aldrich)
• Acetonitrile 99.8% (Sigma Aldrich)
• TRIS buffer saline (Sigma Aldrich)
• Hydrazine monohydrate 80% (Sigma Aldrich)
• Tetrahydrofuran 99% (Sigma Aldrich)
• 1-Pyrenebutyric acid N-hydroxysuccinimide ester (PBSE) (Sigma Aldrich)
• Odorant binding proteins of Plutella xylostella (GOBP2 wild-type with His

tag and GOBP M5 with His tag, both MW: ∼ 25kDa) (from Prof. Paolo
Pelosi)
31
Chapter 2 32
Equipment
• Keithley 4200 SCS

• Peristaltic Pump P-720 (Instech)
• Thin-film Gold InterDigitated Electrode (10/10 µm) ED-IDE1-Au (Micrux
Technologies)
• Electrochemical flow cell (Micrux Technologies)
Figure 2.1: rGO-FET chip from Micrux with a schmatic drawing of the interdigitated electrodes
[79]
2.2 Preparation of the rGO-FET
All the chemicals are purchased from Sigma Aldrich unless stated otherwise. For
the field-effect transistor (FET) electrochemical sensors from the company ”MicruX”
[79] are purchased (see Fig. 2.1). Chips with a glass substrate and 150nm inter-
digidated thin-film gold electrodes are used [79]. For the preparation of the chip a
slightly altered protocol as in Reiner-Rozman and Piccinini et al [30, 33] is used. For
the Silanisation a two percent (3-Aminopropyl)-triethoxysilane (APTES) solution in
ethanol is prepared. The chips are submerged in a 2 % APTES in ethanol solution
and incubated for one hour. The silane forms a self-assembled monolayer on the
chips which increases the adsorption of graphene [30]. After one hour the chips are
rinsed with absolute ethanol and are blown dry. Then the chips are baked at 120◦
degrees Celsius for one hour. After they are cooled down at room temperature,
about 20µl of diluted (1:80) Graphene oxide (GO) solution is drop-casted on the
sensing area of each chip. After one hour the chips are rinsed with deionised water
and are blown dry. Then the graphene oxide has to be reduced to regain the elec-
trical properties, similar to pristine graphene and most importantly the reduction
process decreases the resistance of the chips. Therefore, two different methods have
been used, the chemical and the thermal reduction.
Chapter 2 33
2.2.1 Chemical reduction of the graphene oxide
For the chemical reduction 1ml of Hydrazine is placed next to the chips in a sealed
(with vacuum tape) glass petri dish and put into the oven at 70◦ degrees Celsius
overnight. When the chips have reached room temperature again, they are washed
with deionised water. To prove that the reduction was successful a simple resistance
measurement with a multimeter is sufficient. All chips with a resistance below 100
Ohm are used for the following measurements to be able to compare them better.
2.2.2 Thermal reduction of the graphene oxide
For the thermal reduction the chips are placed in the furnace. Different reducing
parameters have been tested, see Tab. 2.1. The best results are gained by reducing
the rGO-chips under an Argon/Hydrogen atmosphere at 450◦ degrees Celsius for six
hours. Higher temperatures are not possible as the chip is not stable above 500◦ C.
The success is again tested with a multimeter. The resistance of the thermally
reduced chips is higher than that of the chemically reduced ones.
reduction temperature (degrees Celcius) reduction time (hours) resistance (Ohm)

400◦ C 2 h (490 ± 230)Ω
500◦ C 2 h (280 ± 23)Ω
400◦ C 6 h (250 ± 25)Ω
450◦ C 6 h (107 ± 12)Ω
Table 2.1: Resistance measurement at different reducing parameters
To investigate the reducation of graphene further the weight loss of the GO is mea-
sured by TGA and DSC (see Chap. 1.5.2). As can be seen in Fig. 2.2 a weight loss
of more than 20% at around 200◦ C and at 600 − 800◦ C is observed. This means that
in this temperature range functional groups are removed from GO. These results fit
to the hypothesis of Bagri et al. [47], where the hydroxyl groups are desorbed at
around 200◦ C and the epoxy groups are only removed at higher temperatures.
Chapter 2 34
(a) First derivative of TGA (b) TGA and DSC
Figure 2.2: TGA and DSC measurements

Moreover, Raman spectra have been done to compare the chemical and thermal
reduction process (see Fig. 2.3). The ratio of the intensity of the D and the G peak,
ID /IG , gives a rough estimation on how defective the GO is compared to pristine
graphene, where a ratio below one is desired. The ratio ID /IG was nearly one for GO
before the reduction. After thermal reduction the ratio is lower than after chemical
reduction. This means that thermally reduced GO is less defective than chemically
reduced one.
(a) GO (b) chemical reduced GO (c) thermal reduced GO
Figure 2.3: Raman spectra of GO before reduction, after chemical and after thermal reduction
2.3 Immobilization of the proteins
After the successful reduction of the chips, odorant binding proteins (OBP) are im-
mobilised on the chip. For this process 1-Pyrenebutyric acid N-hydroxysuccinimide
ester (PBSE) is used as a linker. The linker is solved in tetrahydrofuran (THF) and
diluted to a 2,5µM solution. PBSE has a pyrene group on one end, which nonco-
valently binds to the graphene surface and on the other end the succinimidyl ester
group, which can bind to the amines of the protein through π − π interaction.
Chapter 2 35
10 µl of diluted PBSE linker are then put on the sensing area of the chip three times
with drying steps in between. Then the chips are rinsed with pure THF. After the
THF is evaporated completely, 10 µl of proteins are put on the sensing area and
are incubated for two hours. The OBPs have been extracted and diluted to a 0,1X
concentration in Phosphate buffer saline (PBS) by Prof. Pelosi.
2.4 Preparation of the solution
For all measurements phosphate buffered saline (PBS) with an ion concentration of
17mM and a ph-value of 7,4 is used. The ligands that are used for this measure-
ment are all hydrophobic. Therefore, Coniferyl Aldehyde is dissolved in Ethanol
or Acetonnitrile and then diluted in PBS buffer. Cis-11-Hexadecenal on the other

hand is only solvable in methanol. So, it is dissolved in methanol before the dilu-
tion in PBS buffer. This solutions are further diluted into different concentrations.
All the concentrations are kept with one percent of either Ethanol, Acetonnitrile or
Methanol.
2.5 Measurement set-up
Electrical measurements are run with a ”Keithley 4200”. For the experiments chips
from the company ”Micrux” [79] are used as field-effect transistors. The round area
of the 150 nm thin film, interdigitated, gold electrodes (with a diameter of 3,5 mm)
is used as the sensing area, see Fig. 2.1. This sensing area has about 90 pairs of
electrodes. It is functionalized with graphene oxide (GO), reduced and proteins are
immobilized on this area with a PBSE linker (see also 2.3). A chlorinated silver wire
(Ag/AgCl) was used as an external gate electrode.
The chips are then mounted into a commerical available flow cell, also from ”Mi-
cruX” [79]. The chip placement in the flow cell can be seen in figure 2.4. The
top-up, to close the flow cell, is custom-made and manufactured by ”MicruX” Tech-
nologies, see Fig. 2.5. A significant advantage of this custom-made flow cell top-up
over the commercial available ones is, that inlet, outlet and the gate electrode are
all mounted separately. This has the benefit that the electric field is not disturbed
by irregularities of the flow. Moreover, the distance between the sensing area of
the chip and the gate electrode can be easily fixed to keep it constant. This makes
the measurements more stable and reproducible. Also the water chamber over the
sensing area is slightly bigger than that of the commercially available ones. There-
fore, small irregularities of the pump have less influence on the signal. The liquid
is pumped over the sensing area with a peristaltic pump at a speed of 200µl/min
which is kept constant for all measurements.
Chapter 2 36
Figure 2.4: Flow cell from Micrux without the top-up to see the chip placement
Figure 2.5: Custom-made flow cell from Micrux with the external gate electrode, inlet and outlet
and USB-connection to the computer
3. Results
3.1 Set-up characteristics

During all electrical measurements the gate voltage and the drain voltage is kept
constant. Prior to each kinetic measurement an Id Vg curve (drain current over
sweeping gate voltage) is obtained to prove the semiconducting properties of the
rGO-FET (see Chap. 1.4). First the baseline is obtained by running 17mM PBS-
buffer through the flow cell. Then different concentrations of the ligand are washed
over the sensing area of the chip, which results in a current change upon the ligand
binding to the odorant binding protein (OBP), see Fig. 3.1. Each concentration
is measured until equilibrium is reached, which is a prerequisite for the Langmuir
model. After the different concentrations, the chip is again washed with PBS-buffer.
This results in a release of the ligands from the protein-ligand complex and therefore
in a complete recovery of the current. Due to the fact that the current reaches its
original level after the buffer wash, the measurements are completely reversible. This
is also a prerequisite for the Langmuir model.
Figure 3.1: Schematic concept of the sensor and the current changes upon titration of different
ligand concentrations
37
Chapter 3 38
During every measurement a drift of the electric current can be observed. Therefore,
a baseline correction is used prior to the evaluation of the data. It turns out that the
baseline for all the measurements is a second order exponential curve. This takes
the drift of the transistor and the formation of the electric double layer into account.
As PBS-buffer is used, the ions from the buffer form an electric double layer on the
sensing area of the transistor, whereas no layer is formed on the gate electrode as
it is chlorinated. Firstly, measurements with only blank reduced graphene-oxide
field-effect transistors, functionalised only with reduced graphene-oxide (rGO), are
done with different PBS-buffer ion concentrations to test the functionality of the
measurement set-up and to compare the two different reduction processes. Sec-
ondly, measurements with the ligand Coniferyl Aldehyde, which is the plant volatile,
are done. Hereby, the data is evaluated kinetically as well as with the Langmuir
isotherm. These evaluations are done with blank chips, only rGO and only function-
alised with the linker to test the unspecific binding, in the first step. In the second
step, the chips are functionalised with the odorant binding proteins, either with
the wild-type of the general odorant binding protein of the moth (GOBP2) or the
mutant of this protein, GOBPM5. Then the properties of the rGO-FET with these
two different OBPs are compared. Thirdly, measurements with the pheromone, cis
11-Hexadecenal, are taken.
3.2 Functionality tests of measurement set-up
Firstly, measurements with only PBS-buffer with different ion concentrations are
done to test the functionality of the measurement set-up. PBS-buffers with con-
centrations ranging from 170mM to 17mM are washed through the flow-cell until
equilibrium is reached for every concentration. It can be observed that the chips are
sensitive to changes of the ionic strength. With increasing ionic strength the resis-
tance increases, which leads to a decreasing current. To evaluate the performance
characteristics of the rGO-FET fabricated with the thermal reduction process com-
parative measurements are done. Therefore, chemically reduced chips are measured
with increasing (17-170mM) and decreasing (170-17mM) ion concentration, see Fig.
3.2. The minimum value of the current for each ion concentration is divided by the
current value, which is taken directly at the beginning of the titration curve and
before the baseline correction, Id,max . Then these minima are plotted over the ion
concentration and fitted linearly, see Fig. 3.2b,c. The slope of the linear fit indi-
cates the response of the current to the concentration changes. For the chemically
reduced chips the current changed by 90,7 ppm (increasing concentrations) and 178
ppm (decreasing concentrations) for each milli-Molar of ion concentration. More-
over, all the following measurements with the odorant binding proteins are done with
a new custom-made flow-cell top-up (see Chap. 2.5). But the tests with the chem-
ically reduced chips are still taken with the commercial available flow cell top-up.
Chapter 3 39
Therefore, to better compare the different reduction processes, the ion concentration
measurements are done with both flow-cell top-ups for the thermally reduced chips.
The response to the ion concentration changes for the thermally reduced chips is
146 ppm per mM in the commercial flow-cell top-up, see Fig. 3.3 and 186 ppm per
mM in the custom-made flow-cell, see Fig. 3.4. In both cases these responsivities
are in the same order of magnitude then the chemically reduced ones. Moreover, it
is found that the noise level for the chemically reduced GO-FETs (300nA) is higher
than for the thermally reduced ones (100 nA).
Chapter 3 40
(a) Id t of chemically rGO-FET for PBS buffer concentrations

(b) Change of Id over increasing concentrations (17mM -

170mM)
(c) Change of Id over decreasing concentrations (170mM -

17mM)
Figure 3.2: Different PBS buffer concentrations on a chemically reduced GO-FET

(commercial flow-cell top-up)
Chapter 3 41
(a) Id t of thermally rGO-FET for PBS buffer concentrations
(b) Change of Id over concentrations (17mM - 170mM)
Figure 3.3: Different PBS buffer concentrations on a thermally reduced GO-FET

(commercial flow-cell top-up)
Chapter 3 42
(a) Id t of thermally rGO-FET for PBS buffer concentrations
(b) Change of Id over concentrations (17mM - 170mM)
Figure 3.4: Different PBS buffer concentrations on a thermally reduced GO-FET

(custom-made flow-cell top-up)
Chapter 3 43
To characterise the transistor further the current (Id ) was measured with sweeping
gate voltages (from -0,9V to 0,9V). Here the ambipolar characteristics of the FET
could be seen. By measuring this Id Vg for different ionic strengths of the PBS-buffer,
one could see that the Dirac point (minimum of the Id Vg ) shifts as a response to the
ion concentration changes. Moreover, it can be seen that the graphene for negative
voltages is p-doped. This means that the current decreases for increasing ionic
strength at negative gate voltages (see Fig. 3.5). For positive gate voltages the
current would increase for increasing buffer concentration.
Figure 3.5: Id Vg curve of PBS-buffer concentration on a thermally rGO chip
For all measurements a constant negative gate voltage (-0,4; -0,6 or -0,7 V) has been
used, because at these points the Id Vg -curves are most linear.
Chapter 3 44
3.3 Measurements with plant volatile (Coniferyl

Aldehyde)
Measurements with different concentrations of the ligand Coniferyl Aldehyde have

been done on rGO-FETs with immobilised GOBP2, the wild type of the general
odorant binding proteins, or GOBPM5, their mutant. As Coniferyl Aldehyde so-
lutions seemed more stable when solved in Ethanol, most measurements are done
in 1% Ethanol solutions. The current is measured over time and each ligand con-
centration is measured until equilibrium is reached (see Fig. 3.6). It can be seen
that the binding sites are mostly saturated with less than 100µM of Coniferyl Alde-
hyde. Firstly, the measurements are evaluate using the Langmuir binding isotherm.
Secondly, a kinetic evaluation of the data is done.
Figure 3.6: Example of a typical ∆Id t curve of a rGO-FET with Coniferyl Aldehyde
concentrations
3.3.1 Langmuir isotherm
The Langmuir isotherm, see Eq. 3.1, is used to calculate the dissociation constant
and the detection limit. Firstly, the minima of each concentration are found. Then,
the minima are normalized to 1 and plotted over the concentration. The Langmuir
isotherm is then fitted through this points, see Fig. 3.7. The dissociation constant,
Kd , is the concentration, where 50% of the ligand is bound to the protein and can
Chapter 3 45
be readout directly from the graph. The detection limit is calculated by using the
signal-to-noise ratio. Therefore, the concentration where the signal is still three
times higher than the noise is calculated.
cKa
ϑ= [30] (3.1)
1 + cKa
Figure 3.7: Data evaluation with the Langmuir isotherm
Unspecific binding
Unspecific binding is a major challenge for these measurements. Due to the aro-
maticity of the ligands, they can easily bind to graphene instead of the proteins.
Therefore, measurements with different concentrations of the plant volatile, Coniferyl
Aldehyde, are done with blank chips (only rGO on the chip), see Fig.3.8, and with
just the linker on the chip (see Fig. 3.9). For the latter measurements the PBSE
linker is hydrolysed with TRIS-buffer to prevent the direct binding of the ligand to
the linker. As can be seen in Fig. 3.8, Coniferyl Aldehyde binds to the rGO-surface
with a Kd of 36µM. The detection limit for rGO-FETs without proteins is about
100nM. The Kd is 5µM for Coniferyl Aldehyde with the hydrolysed PBSE-linker,
seen in the Fig. 3.9, and the detection limit is also 100nM.
Chapter 3
concentrations
Figure 3.8: Thermally rGO-FET with only reduced graphene oxide for Coniferyl Aldehyde
46
Chapter 3
concentrations
Figure 3.9: Thermally rGO-FET with only hydrolysed PBSE-linker for Coniferyl Aldehyde
47
Chapter 3 48
Measurements with the odorant binding proteins and the plant volatile
The binding of the plant volatile, Coniferyl Aldehyde, to the wild type of the general
odorant binding protein (GOBP2) is investigated, see Fig. 3.10. The dissociation
constant (Kd ) for this protein is calculated using the Langmuir isotherm. Hereby, the
Kd obtained from the Langmuir isotherm is Kd = 3,6 µM . For all measurements with
GOBP2 and Coniferyl Aldehyde the Kd -values are in the same order of magnitude.
Moreover, the calculated detection limit is 50nM for this kind of measurements.
Figure 3.10: Thermally rGO-FET with the wild-type GOBP2 for Coniferyl Aldehyde
concentrations
Chapter 3 49
The binding of the mutant (GOBPM5) to the plant volatile, Coniferyl Aldehyde,
is shown in Fig. 3.11. The Kd for the mutant should be lower than for the wild-type
GOBP2, as the affinity was shifted towards plant volatiles. But the Kd is in the
same order of magnitude. In this measurement the Kd is 4µM and the calculated
detction limit is also 50nM, which is the same as for the wild-type GOBP2.
Figure 3.11: Thermally rGO-FET with the mutant GOBPM5 for Coniferyl Aldehyde
concentrations
Chapter 3 50
A comparison of the dissociation constants for both odorant bindnig proteins can
be seen in Fig. 3.12. On the left side the boxplot for the mutant, GOBPM5 can be
found and on the right side for the wild-type GOBP2. The median is given by the
line in the middle of the boxplot and the mean is given by the dot in the boxplot.
For both proteins the mean Kd is nearly the same, which is 3,6µM for GOBP2 and
3,4µM for the mutant GOBPM5. The variance of the different Kd is higher for the
mutant than for the wild-type GOBP2. The difference mentioned in the paper of
Zhu et al. [63] could not be found with these sensors.
Figure 3.12: Boxplots of the dissociation constants for Coniferyl Aldehyde to compare the
GOBP2 to its mutant GOBPM5
Chapter 3 51
3.3.2 Kinetic evaluation of the data
The reaction rate for each concentration (kobs ) is determined by fitting the reaction of
each concentration exponentially. Then, all kobs are plotted over the concentration
and fitted linearly, see Fig. 3.13. Out of this linear fit, the linear range and the
reaction time can be readout. The reaction time is:
1
t= (3.2)
kobs
Figure 3.13: Overview of the kinetic evaluation of the data
Unspecific binding
The kinetics of the binding of the plant volatile, Coniferyl Aldehyde, to reduced
graphene oxide is investigated, see Fig. 3.14. The reaction times range from 161
seconds for the lowest concentration to 6 seconds for 100 µM. Also, all concen-
trations are in the linear range. Furthermore, the unspecific binding of Coniferyl
Aldehyde to the hydrolysed PBSE-linker is shown in Fig. 3.15. For this measure-
ment the reaction time is 52s for 1µM and 29s for 100µM. So the reaction time
for Coniferyl Aldehyde binding to the linker is one order of magnitude faster for
the lowest concentration, but slower for the highest concentration compared to the
binding of reduced graphene oxide.
Chapter 3
reduced graphene oxide

Figure 3.14: Kinetic evaluation of Coniferyl Aldehyde concentrations on rGO-FET with only
52
Chapter 3
hydrolysed PBSE-linker
Figure 3.15: Kinetic evaluation of Coniferyl Aldehyde concentrations on rGO-FET with only
53
Chapter 3 54
Measurements with the odorant binding proteins and the plant volatile
In Fig. 3.16 the kinetic evaluations can be seen for the binding of the plant
volatile, Coniferyl Aldehyde, to the wild type of the general odorant binding protein
(GOBP2). Here, the concentrations 1-50µM are in the linear range. The reaction
time is 85 seconds for the lowest and 34s for the highest concentration.
Figure 3.16: Kinetic evaluation of Coniferyl Aldehyde concentrations on rGO-FET with the
immobilised wild type of GOBP2
Chapter 3 55
In Fig. 3.17 the kinetic evaluations can be seen for the binding of the plant
volatile, Coniferyl Aldehyde, to the mutant of the general odorant binding protein
(GOBPM5). The linear range for this measurement covers the whole concentra-
tion range (1-100µM). The reaction time is 67 seconds for the lowest and 20s for the
highest concentration.
Figure 3.17: Kinetic evaluation of Coniferyl Aldehyde concentrations on rGO-FET with the
immobilised mutant GOBPM5
Chapter 3 56
The reaction times for both proteins is in the same order of magnitude. Furthermore,
the kinetic measurements differ from chip to chip, as in some of them the current for
the higher concentrations increases so much that an exponential fit is hardly possible.
Therefore, the linear range is in some cases limited to the lower concentrations up
to 25µM, see Fig. 3.18. It seems that the linear range depends mostly on the sensor
parameters and not on the binding properties of the protein to the ligand. This
is, because the chip-to-chip differences under the same conditions are greater than
between the two proteins.
Figure 3.18: Kinetic evaluation of Coniferyl Aldehyde concentrations on rGO-FET with

immobilised GOBPM5 with narrower linear range
Chapter 3 57
A comparison of the reaction time at 1µM can be seen in Fig. 3.19. On the left
side the boxplot for the mutantGOBPM5 can be found and on the right side for the
wild-type GOBP2. The median is given by the line in the middle of the boxplot and
the mean is given by the dot in the boxplot. The mean reaction time for GOBP2 is
70 seconds and for the mutant GOBPM5 it is 66s.
Figure 3.19: Comparison of the reaction time at 1µM for both proteins, left: the mutant
GOBPM5, right: wild-type GOBP
Chapter 3 58
3.4 Measurements with pheromone (cis 11 - Hex-

adecenal)
According to Zhu et al. [63] the wild-type GOBP2 should bind specifically to the
pheromone, cis 11-Hexadecenal, wheras the Kd for the mutant, GOBPM5, should
be higher by one order of magnitude. Unfortunately, this could not been proven as
the reaction curve to the different concentrations of the pheromone is not exponen-
tial, see Fig. 3.20. A nearly exponential reaction could only be obtained for 100µM
Hexadecenal with the wild-type GOBP2, see Fig. 3.21a. Therefore, no binding coef-
ficients can be obtained for this ligand. The different reaction of cis-11-Hexadecenal
could be due to additional chemical reactions happening on the chip. However, no
unspecific binding is observed as there is no reaction between Hexadecenal and the
hydolyzed PBSE-linker, see Fig. 3.21b.

Figure 3.20: Id t curve of a rGO-FET with GOBPM5 and Hexadecenal concentrations

Chapter 3 59
(a) ∆Id t curve of a rGO-FET with GOBP2 and

100 µM of Hexadecenal
(b) ∆Id t curve of a rGO-FET with only hydrolysed

PBSE-linker and 100µM of Hexadecenal
Figure 3.21: ∆Id t curve of a rGO-FET with 100µM of Hexadecenal

4. Discussion
During this study a cheap and easy to build smell sensor is manufactured and
tested with odorant binding proteins. One aim of this study was to find a safe
and easy reduction process for the graphene oxide (GO). The thermal reduction
process is safer than the use of the chemical reduction as no toxic chemicals are used.
Depending on the reduction process the properties of GO changes. So, thermally
reduced graphene has less defects (see Chap. 1.5), which can be also seen in the
Raman spectra (see also Fig. 2.3). This is an advantage as its properties are
closer to pristine graphene than chemically reduced GO. However, the challenge for
thermally reduced GO is that it is best reduced at very high temperatures, but the
chips are not designed to withstand more then 450◦ degrees [79]. It proved difficult
to decrease the resistance at this temperature. As the resistance of the rGO-FET
corresponds to the sensitivity of the sensor, the resistance is desired to be as low as
possible. In general, the resistance for thermally reduced rGO-FETs is one order of
magnitude higher than for chemical reduced ones, which typically have a resistance
of less than 50 Ω. The right reduction parameters could be found to get rGO-FETs
with a resistance of about 100 Ω (see Chap. 2.2.2), which is sufficiently low for our
measurements. However, the properties of the thermally reduced GO-FETs varied
in terms of noise level and baseline stability. Moreover, a stronger drift occurred in
the thermally reduced ones compared to the chemically reduced chips. Despite the
chip-to-chip variation being greater due to the altered fabrication method, generally
a lower noise in the chips used for the measurements is achieved (∼ 100nA) compared
to the chemically reduced ones (∼300nA).
The sensor described above was then investigated using the odorant binding pro-
teines (OBPs) form the moth, in form of the wild type and an atrificial mutant. The
OBPs are extracted from the moth antennae and the mutant is engineered by just
changing one or to amino acid residues by Prof. Pelosi [63]. The mutant shifted
its affinity from pheromones to plant volatiles, which was shown by Zhu et al. [63].
The here presented rGO-FET was functionalised with these two types of proteins
and the binding properties of these OBPs were also investigated with this sensor.
Therefore, the binding properties of the GOBP2 to the plant volatile, Coniferyl
Aldehyde, is compared to the binding of the mutant to this ligand, but did not
show a detecable difference in binding behaviour. The dissociation constant, Kd , as
60
Chapter 4 61
well as the reaction rates are in the same order of magnitude for both OBPs and
Coniferyl Aldehyde. Nevertheless, the dissociation constants are in the same order
of magnitude as the ones in the scientific literature [63]. Moreover, calculating the
dissociation constant of the second ligand, the pheromone cis 11-Hexadecenal, to
the two different odorant binding proteins have not been possible as the reaction
did not follow exponential curves (see Chap. 3.4). This suggests additional chemical
reactions and warrants further investigations.
An electrical signal could be observed by measuring the reaction between Coniferyl

Aldehyde and rGO-chips without proteins. Also, a reaction of the ligand and the
PBSE-linker is measured. Although the obtained Kd for rGO is one order of mag-
nitude higher than with proteins, nearly the same Kd can be observed with only
the hydrolyzed PBSE-linker. Also, the reaction times of the binding of the plant
volatile to the OBPs are in the same order of magnitude as the binding to the
linker. The reaction times for the ligand binding directly to rGO are faster for the
higher concentration and lower for the lower ones, both by one order of magnitude.
Several papers investigated the binding of aromatic rings to graphene [80–83]. Due
to the aromaticity of graphene and the oxygen-containg functional groups of GO,
the aromatic ring of Coniferyl Aldehyde can noncovalently bind. The adsorption
of aromatic compounds on GO is mainly regulated through π − π interaction. [84]
This can be seen in the unspecific binding of Coniferyl Aldehyde to rGO and the
PBSE-linker (see Fig. 3.8, 3.9). Unspecific binding for cis 11-Hexadecenal, which
has no aromatic ring, could not be observed, which encourages this theory. So, es-
pecially for odorants that contain aromatic rings a suitable passivation layer has to
be found. A challenge, hereby, is the signal decrease due to the passivation layer.
Despite the binding reaction between graphene and Coniferyl Aldehyde as well as
PBSE-linker and the ligand, the Kd -values obtained with the Langmuir isotherm
are in the same order of magnitude as found in Zhu et al. [63]. To investigate the
difference between the two proteins another ligand instead of 11-Hexadecenal may
be useful. The affinity of both proteins to Coniferyl Aldehyde is too similar and
therefore not distinguishable with this type of sensor. Further investigations of the
surface properties and of the reaction of rGO to different chemical compounds will
probably identify a ligand, which binds only to one of these proteins and does not
interfere with the sensor.
To evaluate the data the Langmuir isotherm is used, which did not fit perfectly for
most of the measurements. This is because the reaction curve for higher concentra-
tions increased instead of equilibriating. This is relevant, because, for the Langmuir
isotherm the minimum of each reaction is searched and then normalized to 1. The
minima for each concentration resemble the surface coverage of the ligand, which
means the percentage of bonded ligands to proteins assuming the odorant binding
proteins cover the whole sensing area. At higher concentrations of Coniferyl Ald-
heyde (above 75µM) the minimum Id t-values of each reaction curve started to in-
Chapter 4 62
crease again, suggesting that the saturation concentration is below that. Therefore,
the fitting of the Langmuir isotherm to the minima plotted over the concentration
was not always perfect. Moreover, the binding seems to be asymmetric and not sym-
metric as the Langmuir model suggests. Interestingly, it could be observed that the
Langmuir isotherm fitted best for the binding of Coniferyl Aldehyde to graphene.
To investigate the kinetics the reaction curves of each concentration are fitted expo-
nentially, see also 1.8. By investigating the kinetic reactions of different concentra-
tions of Coniferyl Aldehyde it could be seen that the current of smaller concentra-
tions changes exponentially as expected. But for higher concentrations the current
started to increase after the initial reaction suggesting additional chemical reactions.
Therefore, an exponential fit for higher concentrations is not optimal to obtain the
reaction rates, kobs . In this cases the kobs plotted over the concentration do not fol-
low a perfect linear fit. This leads to a narrower linear range of the measurements.
By investigating this reactions in more detail in the future a proper model could be
found to increase the linear range.
5. Conclusion and Outlook
The present study has shown a way to detect volatile odorants electrically. The
electrical detection of biomolecules is the first step for a wide range of sensor ap-
plications, for the use in agriculture, the food industry and medical applications.
The study demonstrates a first approach towards designing an improved and minia-
turised biosensor to work as an artificial nose. In this study a reduced graphene
oxide field-effect transistor was fuctionalised with odorant binding proteins to cre-
ate a biosensor that combines cheap and easy fabrication together with a simplified
perception of odorants.
To achieve an easy fabrication process an alternative reduction method for the

graphene oxide was tested and the best parameters for this type of sensor were cho-
sen. It was found that by reducing at 450◦ C for six hours in an Argon/Hydrogen
atmosphere, a resistance of about 100 Ohm could be achieved. Also, it is shown that
the thermally reduced graphene oxide is less defective than the chemically reduced
graphene oxide. The rGO-FETs produced this way also have a lower noise level
than the chemically reduced ones.
The functionalisation of the rGO-FET with odorant binding proteins is achieved.

The binding properties of the OBP found in the moth are compared to an engi-
neered mutant of this protein. Therefore, the dissociation constants as well as the
reaction rates were calculated for the binding of the two odorant binding proteins
with the plant volatile, Coniferyl Aldehyde. Whereas the characterisation of the
binding properties of the plant volatile was achieved, this was not possible for the
pheromone, cis 11-Hexadecenal. Therefore, new evaluation methods for this type
of measurements have to be found in the future. The dissociation constants and
the rate constants of the plant volatile, Coniferyl Aldehyde, to both OBPs are in
the same order of magnitude. Therefore, the difference of the affinities found in the
paper by Zhu et al. [63] could not be measured with this sensor. Nevertheless, the
dissociation constants are in the same order of magnitude as in the paper.
However, unspecific binding proved a major challenge that has not been resolved so
far. Therefore, the main focus of future work should be on making the sensor more
specific, as the use of odorant binding proteins (OBPs) alone does not seem suffi-
63
Chapter 5 64
cient. New methods to suppress unspecific binding, mainly the binding of odorants
to graphene, such as passivation layers [22], have to be investigated. Although a
passivation layer may solve the problem of unspecific binding to graphene, it also
has disadvantages. The main one being the signal decrease, and that the sensor is
therefore not as sensitive to small changes (see also [30]).
The sensor described in this study is an approach to a biosensor that is easy and safe
to build. It allows various improvements and alterations of the sensor properties, for
example by simply exchanging the OBPs for some of the other insects or vertebrates.
As the sensor is already very small and could be further miniaturised, this detection
method could be integrated in a lab-on-a-chip sensor. The results presented in
this thesis warrant further investigation to improve the specificity of the rGO-FET.
The next step would be to create a sensor array with different proteins that can
distinguish between small biomolecules also in a mixture.
Bibliography
[1] C. Bushdid, M. O. Magnasco, L. B. Vosshall, and A. Keller, “Humans can

discriminate more than 1 trillion olfactory stimuli,” Science, vol. 343, pp. 1370–
1372, Mar. 2014.
[2] P. Pelosi, J. Zhu, and W. Knoll, “From gas sensors to biomimetic artificial
noses,” Chemosensors, vol. 6, p. 32, Aug. 2018.
[3] L. Buck and R. Axel, “A novel multigene family may encode odorant receptors:
A molecular basis for odor recognition,” Cell, vol. 65, pp. 175–187, Apr. 1991.
[4] K. W. Jens Huppelsberg, Physiologie 3. Georg Thieme Verlag KG, 2017. Kapitel
Geruchs-und Geschmackssinn.
[5] P. Pelosi, “Odorant-Binding Proteins,” Critical Reviews in Biochemistry and

Molecular Biology, vol. 29, pp. 199–228, Jan. 1994.
[6] W. Hu, L. Wan, Y. Jian, C. Ren, K. Jin, X. Su, X. Bai, H. Haick, M. Yao, and
W. Wu, “Electronic noses: From advanced materials to sensors aided with data
processing,” Advanced Materials Technologies, p. 1800488, Dec. 2018.
[7] S. Higson, ed., Biosensors for Medical Applications (Woodhead Publishing Se-
ries in Biomaterials). Woodhead Publishing, 2012.
[8] P. Suvarnaphaet and S. Pechprasarn, “Graphene-based materials for biosensors:

A review,” Sensors, vol. 17, p. 2161, Sept. 2017.
[9] D. R. Thévenot, K. Toth, R. A. Durst, and G. S. Wilson, “Electrochemical

Biosensors: Recommended Definitions and Classification,” Analytical Letters,
vol. 34, pp. 635–659, Mar. 2001.
[10] L. C. Clark and C. Lyons, “ELECTRODE SYSTEMS FOR CONTINUOUS

MONITORING IN CARDIOVASCULAR SURGERY,” Annals of the New
York Academy of Sciences, vol. 102, pp. 29–45, Oct. 1962.
[11] J. Wang, “Glucose biosensors: 40 years of advances and challenges,” Sensors

Update, vol. 10, pp. 107–119, Nov. 2000.
65
Chapter 5 66
[12] M. M. Picher, S. Küpcü, C.-J. Huang, J. Dostalek, D. Pum, U. B. Sleytr, and

P. Ertl, “Nanobiotechnology advanced antifouling surfaces for the continuous
electrochemical monitoring of glucose in whole blood using a lab-on-a-chip,”
Lab on a Chip, vol. 13, p. 1780, Feb. 2013.
[13] E. Mahmoudi, “Electronic Nose Technology and its Applications,” Sensors &
Transducers Journal, vol. 107, pp. 17–25, Aug. 2009.
[14] J. P. Santos, J. Lozano, and M. Aleixandre, “Electronic noses applications in

beer technology,” in Brewing Technology, InTech, July 2017.
[15] N. Shehada, J. C. Cancilla, J. S. Torrecilla, E. S. Pariente, G. Brnstrup,

S. Christiansen, D. W. Johnson, M. Leja, M. P. A. Davies, O. Liran, N. Peled,
and H. Haick, “Silicon nanowire sensors enable diagnosis of patients via exhaled
breath,” ACS Nano, vol. 10, pp. 7047–7057, July 2016.
[16] E. C. Nallon, V. P. Schnee, C. Bright, M. P. Polcha, and Q. Li, “Chemical

discrimination with an unmodified graphene chemical sensor,” ACS Sensors,
vol. 1, pp. 26–31, Sept. 2015.
[17] K. S. Novoselov, “Electric Field Effect in Atomically Thin Carbon Films,”

Science, vol. 306, pp. 666–669, Oct. 2004.
[18] L. Briand, C. Eloit, C. Nespoulous, V. Bézirard, J.-C. Huet, C. Henry, F. Blon,

D. Trotier, and J.-C. Pernollet, “Evidence of an odorant-binding protein in the
human olfactory mucus: location, structural characterization, and odorant-
binding properties†,” Biochemistry, vol. 41, pp. 7241–7252, June 2002.
[19] J.-J. Zhou, “Odorant-Binding Proteins in Insects,” in Vitamins & Hormones,

pp. 241–272, Elsevier, 2010.
[20] M. Larisika, C. Kotlowski, C. Steininger, R. Mastrogiacomo, P. Pelosi,

S. Schütz, S. F. Peteu, C. Kleber, C. Reiner-Rozman, C. Nowak, and W. Knoll,
“Electronic Olfactory Sensor Based on A. mellifera Odorant-Binding Protein
14 on a Reduced Graphene Oxide Field-Effect Transistor,” Angewandte Chemie
International Edition, vol. 54, pp. 13245–13248, Nov. 2015.
[21] C. Kotlowski, M. Larisika, P. M. Guerin, C. Kleber, T. Krber, R. Mastro-

giacomo, C. Nowak, P. Pelosi, S. Schtz, A. Schwaighofer, and W. Knoll, “Fine
discrimination of volatile compounds by graphene-immobilized odorant-binding
proteins,” Sensors and Actuators B: Chemical, vol. 256, pp. 564–572, Mar. 2018.
[22] C. Reiner-Rozman, C. Kotlowski, and W. Knoll, “Electronic Biosensing with

Functionalized rGO FETs,” Biosensors, vol. 6, p. 17, Apr. 2016.
[23] Q. He, S. Wu, Z. Yin, and H. Zhang, “Graphene-based electronic sensors,”

Chemical Science, vol. 3, p. 1764, Mar. 2012.
Chapter 5 67
[24] J. Bardeen and W. H. Brattain, “The Transistor, A Semi-Conductor Triode,”

Physical Review, vol. 74, pp. 230–231, July 1948.
[25] C. H. Ahn, J. M. Triscone, and J. Mannhart, “Electric field effect in correlated

oxide systems,” Nature, vol. 424, p. 1015, Aug. 2003.
[26] T. Yanase, T. Hasegawa, T. Nagahama, and T. Shimada, “Solvent effects on

the transient characteristics of liquid-gate field effect transistors with silicon
substrate,” Japanese Journal of Applied Physics, vol. 51, p. 111803, Nov. 2012.
[27] A. M. Ng, Kenry, C. T. Lim, H. Y. Low, and K. P. Loh, “Highly sensitive

reduced graphene oxide microelectrode array sensor,” Biosensors and Bioelec-
tronics, vol. 65, pp. 265–273, Mar. 2015.
[28] K. Kenry, K. P. Loh, and C. T. Lim, “Molecular interactions of graphene oxide

with human blood plasma proteins,” Nanoscale, vol. 8, pp. 9425–9441, Mar.
2016.
[29] W. Yang, K. Ratinac, S. Ringer, P. Thordarson, J. Gooding, and F. Braet,

“Carbon Nanomaterials in Biosensors: Should You Use Nanotubes or
Graphene?,” Angewandte Chemie International Edition, vol. 49, pp. 2114–2138,

Mar. 2010.
[30] C. Reiner-Rozman, Graphene-based field effect transistors for the biosensing of

toxins. PhD thesis, Johannes Gutenberg Universität Mainz, 2016.
[31] M. Pumera, “Graphene in biosensing,” Materials Today, vol. 14, pp. 308–315,
July 2011.
[32] P. K. Ang, A. Li, M. Jaiswal, Y. Wang, H. W. Hou, J. T. L. Thong, C. T.

Lim, and K. P. Loh, “Flow sensing of single cell by graphene transistor in a
microfluidic channel,” Nano Letters, vol. 11, pp. 5240–5246, Dec. 2011.
[33] E. Piccinini, C. Bliem, C. Reiner-Rozman, F. Battaglini, O. Azzaroni, and

W. Knoll, “Enzyme-polyelectrolyte multilayer assemblies on reduced graphene
oxide field-effect transistors for biosensing applications,” Biosensors and Bio-
electronics, Oct. 2016.
[34] I.-Y. Sohn, D.-J. Kim, J.-H. Jung, O. J. Yoon, T. N. Thanh, T. T. Quang, and
N.-E. Lee, “pH sensing characteristics and biosensing application of solution-
gated reduced graphene oxide field-effect transistors,” Biosensors and Bioelec-
tronics, vol. 45, pp. 70–76, July 2013.
[35] A. C. Neto, F. Guinea, and N. M. Peres, “Drawing conclusions from graphene,”

Physics World, vol. 19, pp. 33–37, Nov. 2006.
Chapter 5 68
[36] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, M. I. Katsnelson, I. V.

Grigorieva, S. V. Dubonos, and A. A. Firsov, “Two-dimensional gas of massless
Dirac fermions in graphene,” Nature, vol. 438, pp. 197–200, Nov. 2005.
[37] A. H. Castro Neto, F. Guinea, N. M. R. Peres, K. S. Novoselov, and A. K. Geim,

“The electronic properties of graphene,” Reviews of Modern Physics, vol. 81,
pp. 109–162, Jan. 2009.
[38] K. S. Novoselov, “Nobel lecture: Graphene: Materials in the flatland,” Reviews

of Modern Physics, vol. 83, pp. 837–849, Aug. 2011.
[39] P. R. Wallace, “The Band Theory of Graphite,” Physical Review, vol. 71,
pp. 622–634, May 1947.
[40] H. P. Boehm, A. Clauss, G. O. Fischer, and U. Hofmann, “Das Adsorptionsver-

halten sehr dünner Kohlenstoff-Folien,” Zeitschrift fr anorganische und allge-

meine Chemie, vol. 316, pp. 119–127, July 1962.
[41] W. S. Hummers and R. E. Offeman, “Preparation of Graphitic Oxide,” Journal

of the American Chemical Society, vol. 80, pp. 1339–1339, Mar. 1958.
[42] Y. Zhu, S. Murali, W. Cai, X. Li, J. W. Suk, J. R. Potts, and R. S. Ruoff,

“Graphene and Graphene Oxide: Synthesis, Properties, and Applications,”
Advanced Materials, vol. 22, pp. 3906–3924, June 2010.
[43] F. Schwierz, “Graphene transistors,” Nature Nanotechnology, vol. 5, pp. 487–

496, May 2010.
[44] G. A. K. and N. K. S., “The rise of graphene,” Nature Materials, vol. 6,

p. 183191, Mar. 2007.
[45] M. Katsnelson, “Graphene: carbon in two dimensions,” Materials Today,

vol. 10, pp. 20–27, Dec. 2006.
[46] A. Ganguly, S. Sharma, P. Papakonstantinou, and J. Hamilton, “Probing the

Thermal Deoxygenation of Graphene Oxide Using High-Resolution In Situ X-
ray-Based Spectroscopies,” The Journal of Physical Chemistry C, vol. 115,
pp. 17009–17019, Sept. 2011.
[47] A. Bagri, C. Mattevi, M. Acik, Y. J. Chabal, M. Chhowalla, and V. B. Shenoy,

“Structural evolution during the reduction of chemically derived graphene ox-
ide,” Nature Chemistry, vol. 2, pp. 581–587, June 2010.
[48] I. Childres, L. A. Jauregui, W. Park, H. Cao, and Y. P. Chen, “Raman spec-

troscopy of graphene and related materials,” New developments in photon and
materials research, pp. 1–20, 2013.
Chapter 5 69
[49] L. G. Cancado, A. Jorio, E. H. Martins Ferreira, F. Stavale, C. A. Achete,

R. B. Capaz, M. V. O. Moutinho, A. Lombardo, T. S. Kulmala, and A. C.
Ferrari, “Quantifying Defects in Graphene via Raman Spectroscopy at Different
Excitation Energies,” Nano Letters, vol. 11, pp. 3190–3196, Aug. 2011.
[50] C. Kittel, Introduction to Solid State Physics. Wiley, 8 ed., 2004.
[51] C. Reiner-Rozman, M. Larisika, C. Nowak, and W. Knoll, “Graphene-based

liquid-gated field effect transistor for biosensing: Theory and experiments,”
Biosensors and Bioelectronics, vol. 70, pp. 21–27, Aug. 2015.
[52] L. Kong, A. Enders, T. S. Rahman, and P. A. Dowben, “Molecular adsorption

on graphene,” Journal of Physics: Condensed Matter, vol. 26, p. 443001, Oct.
2014.
[53] E. V. Castro, K. S. Novoselov, S. V. Morozov, N. M. R. Peres, J. M. B. L. dos

Santos, J. Nilsson, F. Guinea, A. K. Geim, and A. H. C. Neto, “Biased Bilayer
Graphene: Semiconductor with a Gap Tunable by the Electric Field Effect,”
Physical Review Letters, vol. 99, Nov. 2007.
[54] P. Pelosi, J. Zhu, and W. Knoll, “Odorant-binding proteins as sensing elements

for odour monitoring,” Sensors, vol. 18, p. 3248, Sept. 2018.
[55] P. Pelosi, A. M. Pisanelli, N. E. Baldaccini, and A. Gagliardo, “Binding of [

3
H]-2-isobutyl-3-methoxypyrazine to cow olfactory mucosa,” Chemical Senses,
vol. 6, pp. 77–85, Apr. 1981.
[56] P. Pelosi, N. E. Baldaccini, and A. M. Pisanelli, “Identification of a specific

olfactory receptor for 2-isobutyl-3-methoxypyrazine,” The Biochemical Journal,
vol. 201, pp. 245–248, Jan. 1982.
[57] R. G. Vogt and L. M. Riddiford, “Pheromone binding and inactivation by moth

antennae,” Nature, vol. 293, pp. 161–163, Sept. 1981.
[58] P. Pelosi, “Odorant-Binding Proteins: Structural Aspects,” Annals of the New

York Academy of Sciences, vol. 855, pp. 281–293, Nov. 1998.
[59] P. Pelosi and R. Maida, “Odorant-binding proteins in insects,” Comparative

Biochemistry and Physiology Part B: Biochemistry and Molecular Biology,
vol. 111, pp. 503–514, July 1995.
[60] P. Pelosi, I. Iovinella, A. Felicioli, and F. R. Dani, “Soluble proteins of chemical

communication: an overview across arthropods,” Frontiers in Physiology, vol. 5,
Aug. 2014.
[61] A. Sanchez-Gracia, F. G. Vieira, and J. Rozas, “Molecular evolution of the

major chemosensory gene families in insects,” Heredity, vol. 103, pp. 208–216,
Sept. 2009.
Chapter 5 70
[62] P. Xu, R. Atkinson, D. N. Jones, and D. P. Smith, “Drosophila OBP LUSH

Is Required for Activity of Pheromone-Sensitive Neurons,” Neuron, vol. 45,
pp. 193–200, Jan. 2005.
[63] J. Zhu, L. Ban, L.-M. Song, Y. Liu, P. Pelosi, and G. Wang, “General odorant-
binding proteins and sex pheromone guide larvae of Plutella xylostella to better
food,” Insect Biochemistry and Molecular Biology, vol. 72, pp. 10–19, May 2016.
[64] “Plutella xylostella (Insecta: Lepidoptera: Plutellidae).” http://www2.nrm.

se/en/svenska_fjarilar/p/plutella_xylostella.html.
[65] J. Zhu, P. Pelosi, Y. Liu, K.-j. Lin, H.-b. Yuan, and G.-r. Wang, “Ligand-
binding properties of three odorant-binding proteins of the diamondback moth
Plutella xylostella,” Journal of Integrative Agriculture, vol. 15, pp. 580–590,
Mar. 2016.
[66] A. L. Lehninger, D. L. Nelson, and M. M. Cox, Principles of biochemistry. New

York: W.H. Freeman, 5th ed ed., 2008.
[67] I. Langmuir, “THE ADSORPTION OF GASES ON PLANE SURFACES OF

GLASS, MICA AND PLATINUM.,” Journal of the American Chemical Society,

vol. 40, pp. 1361–1403, Sept. 1918.
[68] X. Du, Y. Li, Y.-L. Xia, S.-M. Ai, J. Liang, P. Sang, X.-L. Ji, and S.-Q. Liu, “In-
sights into Protein–Ligand Interactions: Mechanisms, Models, and Methods,”
International Journal of Molecular Sciences, vol. 17, p. 144, Jan. 2016.
[69] J. Koolman and K.-H. Rhm, Color Atlas of Biochemistry. Thieme, 2012.
[70] T. Fujimoto and K. Awaga, “Electric-double-layer field-effect transistors with

ionic liquids,” Phys. Chem. Chem. Phys., vol. 15, pp. 8983–9006, Apr. 2013.
[71] O. Stern, “Zur Theorie Der Elektrolytischen Doppelschicht,” Zeitschrift für

Elektrochemie und angewandte physikalische Chemie, vol. 30, pp. 508–516, Nov.
1924.
[72] R. van Hal, J. Eijkel, and P. Bergveld, “A general model to describe the elec-
trostatic potential at electrolyte oxide interfaces,” Advances in Colloid and
Interface Science, vol. 69, pp. 31–62, Dec. 1996.
[73] J. Steinkuehler, V. Charwat, L. Richter, and P. Ertl, “Characterization of dou-

ble layer alterations induced by charged particles and proteinmembrane inter-
actions using contactless impedance spectroscopy,” The Journal of Physical
Chemistry B, vol. 116, pp. 10461–10469, May 2012. PMID: 22594659.
[74] D. C. Grahame, “The Electrical Double Layer and the Theory of Electrocapil-
larity.,” Chemical Reviews, vol. 41, pp. 441–501, Dec. 1945.
Chapter 71
[75] C. Rozman, “Konstruktion und Optimierung eines auf Graphen basierenden

Feld-Effekt-Transistor-Devices,” Master’s thesis, Universitien, 2013.
[76] I. Langmuir, “The Constitution and Fundamental Properties of Solids and

Liquids. PART I. SOLIDS.,” Journal of the American Chemical Society, vol. 38,
pp. 2221–2295, Nov. 1916.
[77] I. Langmuir, “The Constitution and Fundamental Properties of Solids and

Liquids II. LIQUIDS.,” Journal of the American Chemical Society, vol. 39,
pp. 1848–1906, Sept. 1917.
[78] K. Foo and B. Hameed, “Insights into the modeling of adsorption isotherm
systems,” Chemical Engineering Journal, vol. 156, pp. 2–10, Jan. 2010.
[79] “MicruX Electrochemical Sensors,” 2017.

[80] H. Vovusha, S. Sanyal, and B. Sanyal, “Interaction of nucleobases and aromatic

amino acids with graphene oxide and graphene flakes,” The Journal of Physical
Chemistry Letters, vol. 4, pp. 3710–3718, Oct. 2013.
[81] J. G. Woo, L.-H. Tran, S.-H. Jang, C. Lee, and T. J. Kang, “Molecular interac-
tions of graphene oxide with aromatic amino acids tyrosine and tryptophan,”
Bulletin of the Korean Chemical Society, vol. 36, pp. 2959–2961, Nov. 2015.
[82] X.-F. Zhang and X. Shao, “π–π binding ability of different carbon nano-
materials with aromatic phthalocyanine molecules: Comparison between
graphene, graphene oxide and carbon nanotubes,” Journal of Photochemistry
and Photobiology A: Chemistry, vol. 278, pp. 69–74, Mar. 2014.
[83] C. Rajesh, C. Majumder, H. Mizuseki, and Y. Kawazoe, “A theoretical study

on the interaction of aromatic amino acids with graphene and single walled
carbon nanotube,” The Journal of Chemical Physics, vol. 130, p. 124911, Mar.
2009.
[84] H. Tang, Y. Zhao, S. Shan, X. Yang, D. Liu, F. Cui, and B. Xing, “Theo-
retical insight into the adsorption of aromatic compounds on graphene oxide,”
Environmental Science: Nano, vol. 5, pp. 2357–2367, Aug. 2018.

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DIPLOMARBEIT

Graphene-based Biosensor Platform for

Ausgeführt am Institut für

unter der Anleitung von

Univ.-Prof. Dipl.-Ing. Dr. Peter Ertl

Anja Agneter, BSc

Graphene-based Biosensor Platform for

ausgeführt unter der Leitung von

Univ.-Prof. Dipl.-Ing. Dr. Peter Ertl

E163 Institut für Angewandte Synthesechemie

Anja Agneter, BSc

on this project and for all the help and advice.

by building an artificial nose would be highly useful in an abundance of applications.

einer Reihe von Anwendungsgebieten, wie in der Lebensmittelindustrie, der Kos-

Ansätze für einen stabilen und miniaturisierten Biosensor verfolgt.

List of figures VIII

1.2 General concept of biosensors . . . . . . . . . . . . . . . . . . . . . . 3

2 Materials and Methods 31

2.2.2 Thermal reduction of the graphene oxide . . . . . . . . . . . . 33

5 Conclusion and Outlook 63

Id Vg drain current over gate voltage

Vth threshold voltage

kobs observation rate constant

CSP chemosensory protein

DSC differential scanning calorimetry

EDL electrical double layer

FET field-effect transistor

gFET graphene field-effect transistor

GO-FET graphene-oxide field-effect transistor

GOBP general odorant-binding protein

GOBP2 general odorant-binding protein 2

GOBPM5 mutant 5 of the general odorant-binding protein 2

MOSFET metal oxide semiconductor field-effect transistor

OBP odorant-binding protein

PBP pheromone-binding protein

PBS phosphate buffer saline

PBSE 1-pyrenebutanoic acid succinimidyl ester

ppm parts per million

rGO reduced graphene oxide

rGO-FET reduced graphene-oxide field-effect transistor

TGA thermogravimetric analysis

Fig. 1.1 Schematic view of a human nose . . . . . . . . . . . . . . . . . . 2

Fig. 1.5 Electronic nose based on silicon nanowire FET . . . . . . . . . . 6

Fig. 1.10 Fermi levels of graphene . . . . . . . . . . . . . . . . . . . . . . . 14

Fig. 2.1 rGO-FET chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Fig. 3.6 Example of a typical ∆Id t curve of a rGO-FET with Coniferyl

Fig. 3.13 Overview of the kinetic evaluation of the data . . . . . . . . . . . 51

Fig. 3.16 Kinetic evaluation of Coniferyl Aldehyde concentrations on rGO-

1.1 Overview of the sense of smell

Figure 1.1: Schematic view of a human nose [4]

Figure 1.2: Schematic illustraion of the olfactory epithelium [4]

1.2 General concept of biosensors

A biosensor in general combines a biorecognition element, which is a biological sens-

Figure 1.3: Schematic illustraion of a biosensor [8]

The biological sensing element or biorecognition element, for example a receptor,

A biosensor can be constructed by immobilizing a biological receptor such as pro-

Biosensors have gained increasing importance in medical applications, in agriculture