Nghiên cứu tổng hợp vật liệu polymer in dấu phân tử MIP bằng phương pháp điện hóa định hướng ứng dụng phát hiện dư lượng chất lượng kháng sinh

MINISTRY OF EDUCATION AND TRAINING
HANOI UNIVERSITY OF SCIENCE AND TECHNOLOGY
INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE

---------------------------------------

VU VAN PHU

ELECTROCHEMICAL SYNTHESIS OF MOLECULARLY
IMPRINTED POLYMER (MIP), TOWARDS THE APPLICATION IN
ANTIBIOTIC RESIDUE DETECTION

MASTER THESIS OF MATERIALS SCIENCE

Hanoi – 2018


MINISTRY OF EDUCATION AND TRAINING
HANOI UNIVERSITY OF SCIENCE AND TECHNOLOGY
INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE

---------------------------------------

VU VAN PHU

ELECTROCHEMICAL SYNTHESIS OF MOLECULARLY
IMPRINTED POLYMER (MIP), TOWARDS THE APPLICATION IN
ANTIBIOTIC RESIDUE DETECTION

MASTER THESIS OF MATERIALS SCIENCE
Batch ITIMS-2016

SUPERVISOR
DR. PHAM DUC THANH

Hanoi – 2018


ACKNOWLEDGMENTS
It is a pleasure to thank the many people who made this thesis possible.
First of all, I would like to express my sincere gratitude to my advisor Dr.
Pham Duc Thanh of the ITIMS Institute for the continuous support of my M.Sc.
study and research, for his patience, motivation, and immense knowledge.
I would also like to express my deepest thanks and sincere appreciation to
Assoc. Prof. Mai Anh Tuan, MEMS/NEMS Laboratory – NACENTECH, for his
valuable advice, constructive criticism, and his extensive discussions around my
work, and lots of good ideas. Especially, for the fantastic internship time he offered
me at Tokyo University of Science - Japan, which brought me valuable experience
and knowledge.
My sincere thanks also go to Dr. Chu Thi Xuan, ITIMS for her untired help,
and generous advice, and MSc. Tran Quang Thinh of the MEMS/NEMS Laboratory
– NACENTECH, for his support during my experimentation in the laboratory and
contributed positively in the analysis and interpretation of data in my study.
I also thank MSc. Nguyen Minh Hieu of the Nano and Energy Center for his
devoted support and guidance during the fabrication process of electrochemical
sensors in cleanroom, which helped me improve my skills very well.
I thank my fellow members in BIOMAT Group for the stimulating
discussions, have made valuable comment suggestions on this thesis which gave me
the inspiration to improve my assignment.
Last but not the least, I must express my very profound gratitude to members
in my family for providing me with unfailing support and continuous encouragement

throughout my years of study and through the process of researching this thesis. This
accomplishment would not have been possible without them. Thank you!

Hanoi, October 2018
i


DECLARATION
I hereby declare that matter embodied in this thesis entitled, “Electrochemical
synthesis of molecularly imprinted polymer (MIP), towards the application in
antibiotic residue detection” is a result of investigations carried out by me under the
supervision and guidance of Dr. Pham Duc Thanh of the International Training
Institute for Materials Science (ITIMS), Hanoi University of Science and
Technology. I further declare that this thesis, to the best of my knowledge and belief,
does not contain any data previously published by another person or submitted for
the award of any degree or diploma at any other university or institute except where
due reference is made in the text.
Author

Vu Van Phu

ii


TABLE OF CONTENTS
ACKNOWLEDGMENTS ........................................................................................ i
DECLARATION ...................................................................................................... ii
LIST OF ABBREVIATIONS ................................................................................ vi
LIST OF TABLES ................................................................................................ viii
LIST OF FIGURES ................................................................................................ ix

INTRODUCTION .....................................................................................................1
CHAPTER 1: THEORY & FUNDAMENTALS ...................................................5
1.1. Molecularly imprinted polymers ......................................................................5
1.1.1. Types of imprinting mechanism .................................................................7
1.1.2. Removal of the imprinted template ............................................................8
1.1.3. Applications of MIPs .................................................................................8
1.2. Polyaniline ........................................................................................................9
1.2.1. Properties of PANi ...................................................................................12
1.2.1.1. Chemical properties ...........................................................................12
1.2.1.2. Optical properties ...............................................................................12
1.2.1.3. Thermal stability ................................................................................12
1.2.1.4. Conductivity properties of PANi .......................................................12
1.2.2. Synthesis of PANi ....................................................................................14
1.3. Antibiotics: Chloramphenicol .........................................................................16
1.4. Methods of electrochemical analysis ..............................................................17
1.4.1. Cyclic voltammetry (CV) .........................................................................17
1.4.2. Chronoamperometry (CA) .......................................................................18

iii


1.4.3. Differential pulse voltammetry (DPV) .....................................................19
1.4.4. Fourier transform infrared spectroscopy (FT-IR) ....................................19
CHAPTER 2: FABRICATION OF MIP-BASED ELECTROCHEMICAL
SENSOR ...................................................................................................................21
2.1. Fabrication of electrochemical sensor ............................................................21
2.1.1. Photo-mask design ...................................................................................21
2.1.2. Fabrication processes of electrochemical sensors ....................................22
2.1.3. Electrode surface pretreatment .................................................................27
2.2. Synthesis of CAP-imprinted PANi NWs........................................................27

2.2.1. Chemical and instrumentation ..................................................................27
2.2.2. Experimental schema ...............................................................................29
2.2.3. Electrochemical synthesis of PANi NWs.................................................30
2.2.3.1. Synthesis of PANi NWs by CV method ............................................30
2.2.3.2. Synthesis of PANi NWs by CA method ............................................30
2.2.4. Synthesis of CAP-imprinted PANi NWs .................................................30
2.2.5. Removal of CAP from CAP-imprinted PANi NWs ................................31
2.3. Detection of CAP by using MIP-based electrochemical sensor .....................31
CHAPTER 3: RESULTS AND DISCUSSIONS ..................................................32
3.1. Au electrode-based electrochemical sensor ....................................................32
3.2. Synthesis of PANI NWs .................................................................................33
3.2.1. Synthesis of PANi NWs using CV technique ..........................................33
3.2.1.1. Effect of scanning potential ranges on CV-based polymerization ....33
3.2.1.2. CV behavior of polymerization of aniline .........................................35

iv


3.2.1.3. The effect of number of sweeps on the morphology of PANi ...........37
3.2.2. Synthesis of PANi NWs using CA technique ..........................................41
3.2.2.1. CA behavior of the polymerization of aniline ...................................41
3.2.2.2. Effect of CA scanning time on the morphology of PANi .................42
3.2.3. Choice of CA or CV for fabrication CAP-imprinted PANi NWs ............45
3.3. MIP-based electrochemical sensor .................................................................47
3.3.1. CA behavior of synthesis of CAP-imprinted PANi NWs ........................47
3.3.2. The morphology of CAP-imprinted PANi ...............................................48
3.3.3. FT-IR spectrum of CAP-imprinted PANi ................................................50
3.3.4. Removal of CAP from CAP-imprinted PANi ..........................................54
3.3.5. Electrochemical characteristics of the MIP-based sensor ........................56
3.3.5.1. CV behavior of the MIP-based sensor ...............................................57

3.3.5.2. DPV behavior of the MIP-based sensor ............................................59
3.4. Detection of CAP-based on MIP sensor .........................................................61
CONCLUSIONS .....................................................................................................64
REFERENCES ........................................................................................................66

v


LIST OF ABBREVIATIONS
ANi

Aniline

CA

Chronoamperometry

CAP

Chloramphenicol

CE

Counter electrode

CPs

Conductive polymers

CV

Cyclic voltammetry

DPV

Differential pulse voltammetry

EB

Emeraldine base

ES

Emeraldine salt

FT-IR

Fourier transform infrared microscopes

GC-MS

Gas chromatography-mass spectrometry

HA

Arbitrary acid

HPLC

High performance liquid chromatography

LB

Leucoemeraldine base

LC-MS

Liquid chromatography-mass spectrometry

MIPs

Molecularly imprinted polymers

MRPL

Minimum required performance limit

NWs

Nanowires

PANi

Polyaniline

PANi NWs

Polyaniline nanowires

PB

Pernigraniline base

PR

Photoresist

vi


PVD

Physical vapor deposition

RE

Reference electrode

SEM

Scanning electron microscope

TMAH

Tetramethylammonium hydroxide

UV

Ultra violet

WE

Working electrode

vii


LIST OF TABLES
Table 3.1. Data of the average diameter and length of PANi NWs by using CV. ...40
Table 3.2. Data of the average diameter and length of PANi NWs by using CA. ...45
Table 3.3. The CV parameters obtained from Figure 3.23. .....................................58

viii


LIST OF FIGURES
Figure 1.1. Number of papers and patents published on the topic of molecularly
imprinted polymers from 1994 to September 2018 [94]. ...........................................5
Figure 1.2. Schematic for synthesis and imprinting process of molecularly imprinted
polymer. ......................................................................................................................6
Figure 1.3. Chemical structure of PANi...................................................................10
Figure 1.4. The structural formula of the redox states of PANi a) fully reduced
leucoemeraldine, b) half oxidized emeraldine, and c) fully oxidized pernigraniline
[57]. ...........................................................................................................................11
Figure 1.5. Chemical structure of CAP. ...................................................................16
Figure 1.6. Cyclic potential sweep. ..........................................................................17
Figure 1.7. The relationship between voltage and current in the CV. .....................18
Figure 2.1. The detailed structure of the integrated electrode. ................................21
Figure 2.2. Photo-mask design of electrode sensor. ................................................22
Figure 2.3. Fabrication processes of the electrochemical sensor. ............................23

Figure 2.4. Photolithography system at Nano and Energy Center. ..........................24
Figure 2.5. The sputtering system at Nano and Energy Center. ..............................25
Figure 2.6. The dicing system. .................................................................................26
Figure 2.7. Autolab PGSTAT302N measurement system at MEMS/NEMS
laboratory – NACENTECH. .....................................................................................28
Figure 2.8. Experimental setup of the electrochemical measurements. ...................28
Figure 2.9. Schematic illustration of the experimental section. ...............................29
Figure 3.1. Image of electrochemical sensors with the configuration of three
integrated Au electrodes on a complete wafer. .........................................................32

ix


Figure 3.2. Cyclic voltammograms of Au electrodes at different scanning potential
ranges with the same of monomer solution (1 M H2SO4 and 0.1 M Aniline) and 25
mV.s-1 scanning rate. .................................................................................................34
Figure 3.3. Cyclic voltammogram of polymerization of aniline in 1 M H2SO4 for 10
cycles at 25 mV.s-1. ...................................................................................................35
Figure 3.4. The transformation of redox states of PANi. .........................................36
Figure 3.5. Schematic representation of (a) the reaction of the ANi nitrenium cation
with a PANi chain to give phenazine structures, (b) the cross-linking reactions
between PANi chains caused by nitrenium cation. ...................................................37
Figure 3.6. SEM images of PANi nanowires at scan rate of 25 mV.s-1 for (a) 4 cycles,
(b) 5 cycles, (c) 6 cycles, and (d) 10 cycles. Scale bar is 1 µm. ...............................38
Figure 3.7. Histograms of diameters of PANi NWs for (a) 4 cycles, (b) 5 cycles, (c)
6 cycles, and (d) 10 cycles. .......................................................................................39
Figure 3.8. Image of PANi NWs synthesized at 12 cycles. .....................................40
Figure 3.9. CA characterization of polymerization of 0.1 M ANi in 1 M H2SO4 at
+0.9 V. .......................................................................................................................41
Figure 3.10. Images of PANi-covered sensors, CA scanning time for (a) 240 s, (b)

360 s, (c) 480 s, and (d) 600 s. ..................................................................................42
Figure 3.11. SEM images of PANi nanowires, CA scanning time for (a) 240 s, (b)
360 s, (c) 480 s, and (d) 600 s. Scale bar is 500 nm. ................................................43
Figure 3.12. Histograms of diameters of PANi NWs for (a) 240 s, (b) 360 s, (c) 480 s,
and (d) 600 s. .............................................................................................................44
Figure 3.13. SEM images of PANi NWs synthesized by CV for 10 cycles (a), and
CA at +0.9 V during 360 s (b). Scale bar is 5 µm. ...................................................46
Figure 3.14. Chronoamperomograms of (a) synthesis of PANi and (b) MIP (CAPimprinted PANi) at +0.9 V for 360 s. .......................................................................47
x


Figure 3.15. The protonation of CAP.......................................................................48
Figure 3.16. SEM images of MIP with the scale bars of (a) 500 nm, (b) 1 µm, and
(c) 4 µm. ....................................................................................................................49
Figure 3.17. Histograms of diameters of CAP-imprinted PANi NWs for 240 s. ....49
Figure 3.18. Hydrogen bonding of CAP and PANi in CAP-imprinted PANi NWs.
...................................................................................................................................51
Figure 3.19. FT-IR spectra of PANi NWs and CAP. ...............................................52
Figure 3.20. FTIR spectrum of CAP-imprinted PANi NWs....................................53
Figure 3.21. Chronoamperomogram of removal of CAP in the solution of 0.5 M
H2SO4 at +0.6 V for 240 s. ........................................................................................55
Figure 3.22. Schematic representation of CAP-imprinted MIP before (a) and after
(b) removal of CAP. ..................................................................................................56
Figure 3.23. Cyclic voltammograms of (a) bare Au electrode and each step of the
modification with (b) PANi, (c) MIP, and (d) after the removal of the template in 0.1
M [Fe(CN)6]3-/4- and 0.25 M KCl. .............................................................................57
Figure 3.24. Differential pulse voltammograms of (a) bare Au electrode and each
step of the modification with (b) PANi, (c) MIP, and (d) after the removal of the
template in [Fe(CN)6]3-/4-...........................................................................................59
Figure 3.25. Differential pulse voltammograms of various CAP concentrations in the

range 0 to 10-3 M. ......................................................................................................62
Figure 3.26. The calibration curve of the Ipeak with the CAP concentration. ...........63

xi


INTRODUCTION
The food market, in general, and seafood, in particular, are increasingly
expanding with fierce competition. In order to better serve the needs of consumers, it
is also necessary for producers to realize and develop applications of science and
technology in production, animal husbandry, quality and supply chain management.
On the one hand, the research and development of new products ensure the quality,
on the other hand they convey the scientific knowledge to farmers in aquaculture and
seafood. In large markets around the world, to ensure the quality of marine and
aquatic products, there are often standards for aquaculture which determine residues
of restricted substances, and lists of banned substances. For example, the ban on
aquaculture includes compounds containing chloramphenicol (CAP) and restricted
group residues including compounds in the tetracycline group (doxycycline,
oxytetracycline, and tetracycline). So as to evaluate and track these products, accurate
and rapid analysis tools are needed.
Most of the current research focuses on increasing the specificity and
sensitivity of traditional analytical methods to determine residues of banned or
restricted substances in foodstuffs and seafood. The LC-MS/MS (liquid
chromatography-mass spectrometry) is the most commonly used technique, not only
by research groups in general but also by regulatory decisions (Commission Decision
2002/657/EC) of the EU [9]. Recent developments of antibiotic residue analysis using
spectroscopic techniques are described in [64]. A variety of compounds, such as oxytetracycline, tetracycline, chlortetracycline, and doxycycline, can be identified by
Thin Layer Chromatography (TLC), CE and HPLC. H. Oka's report also introduced
a sample preparation process that included the steps of separating and cleaning the
sample to match the techniques described [64]. Trace sulfonamides and tetracyclines

can also be found by combining immunoassay and LC-MS/MS [11]. A number of
approaches and developments in LC-MS are described in detail in the report by
Dickson et al. [68]. The application of LC-MS/MS analysis in electrospray ionization
(ESI) mode in solid phase separation (SPE) to determine the antibiotic residues
1


described in the report includes lincomycin, clindamycin, tilmicosin, erythromycin
and tylosin [69].
Traditional analytical techniques that dominate the analytical industry as well
as analysis of antibiotic residues in food also have some certain limitations, such as
long time analysis and carefully trained technicians. Therefore, scientists are still
searching for additional solutions, or even alternatives. Immunological testing is a
semi-quantitative method with high sensitivity, simplicity, and cost-effectiveness.
Immunological assays used in the determination of antibiotic residues are classified
based on labels (markers) detected as ELISA – enzyme -linked immunosorbent assay,
on fluorescent markers - FIA, Fluorescent Markers - TRFIA. ELISA is a catalytic
enzyme-based method of operation plus UV/VIS detection. Antibody conjugates –
secondary enzymes are heated followed by addition of buffers such as ophenylenediamine dihydrochloride (OPD) or tetramethylbenzidine (TMB) [2, 30, 43,
91].
Among advanced screening methods, biosensors (short for biological sensors)
are promising due to their compact size, rapid analysis time and fully automation. It
is an analytical instrument consisting of a part of the biological sensing elements
(enzyme, protein, nucleic acid, cell...) combined with a transducer. Currently,
biological sensors are still in the research and development phase due to some
obstacles such as the degradation of biological components under the influence of the
environment such as pH, temperature… However, there are many types of
transducers that can be used in biosensors such as electrochemical sensors,
mechanical sensors, photochemistry, heat, etc. Thai scientists have used immunity
sensors to detect banned substances in food [18]. Antigen-specific interaction enables

highly selective analysis and extremely low determination limit of 10-16 M. Special
customized electrodes can be reused up to 45 times with deviation standard lower
than 4%. Donglei Jiang and colleagues [36] have developed immune sensors and
magnetic luminous particles to detect allergens in food. The combination of magnetic
and optical properties makes the sensor extremely sensitive to 3.3×10-4 ng/mL. Tested
2


with real samples, the results showed high detection accuracy with a detection limit
of 0.03 μg/mL (in shrimp samples) and 0.16 ng/mL (in fish samples). Among the
biosensors under investigation, molecularly imprinted polymer (MIP) based
biomedical sensors are being promoted due to their selectivity and high specificity.
The term "molecular imprinting" has been used since the 1960s to describe the notion
of molecular recognition by "molecular" marking into the polymer network as
"negative images" of the sample molecules in a process [16, 37, 55, 79, 93, 97]. The
broader and more complete definition of the concept of molecular markers can be
described as follows: the process of forming a prototype of a molecule, including not
only the structural shape of the molding molecule but also the links of that molecule
to the substrate structures. Research on molecular imprinting processes have been
carried out in various fields of materials and biology, and have been used in
separation [3, 14, 15, 51, 75, 82], adsorption [46, 56], sensors [10, 26, 39, 49, 74, 99],
analysis [41, 47], leads [48, 66, 72].
Especially, chloramphenicol (CAP) based on MIP material have also been
studied extensively by scientists around the world with different approaches such as
magnetic detection [12], optical method [84], electroplating [24] or the use of carbon
nanotubes [98]… Chloramphenicol (CAP) is an effective antibiotic with broadspectrum activity which is used in both human and veterinary medicine. However, in
view of the toxic effects, the use of CAP was banned in food-producing animals.
Thus, monitoring of CAP levels in patients’ blood and its detection in food products
are highly important. With this aim, a sensor for CAP using a molecularly imprinted
polymer as the recognition element has been devised.

With the inheritance of know-hows, promising research on conductive
polymer materials (polyaniline and polypyrrole), coupled with the availability of
facilities, and previous results on electrochemical biosensors from our group, I have
been given the task to create molecular imprinting materials for use in biological
sensors under the name of my thesis:

3


“Electrochemical synthesis of molecularly imprinted polymer (MIP),
towards the application in antibiotic residue detection”.
In my work, polyaniline is used for the synthesis of molecularly imprinted
polymer with the template as antibiotics (chloramphenicol) by electrochemical
methods. At the same time, we optimize the fabrication of polyaniline nanowires
through the synthesis and comparison of two technologies, namely cyclic
voltammetry and chronoamperometry. This thesis consists of three chapters:
In chapter 1, the basic concepts of MIP, and properties of polyaniline as well
as antibiotic data will be also introduced.
In chapter 2, the experiment setup and synthesis of MIP will be described in
detail.
In chapter 3, the result of the experiment is presented and discussed.
This work in my thesis is mainly carried out at Nano and Energy Center (NEC)
for electrochemical sensor fabrication process, the National center for Technological
progress (NACENTECH) and the EC301 electrochemical measurement equipment
of Vijases company that is located at ITIMS Institute for material synthesis, and
analytical measurement process.

4

CHAPTER 1: THEORY & FUNDAMENTALS
This chapter reviews the fundamental theory of molecularly imprinted
polymers including history, types of imprinting mechanism, removal of the imprinted
template, and applications of MIP. The structure, properties, and synthesis methods
of polyaniline, as well as antibiotic data and electrochemical analysis are also
presented in this chapter.
1.1. Molecularly imprinted polymers
The term “molecular imprinting” is a generic technology which enables us to
synthesize materials with highly specific receptor sites towards the target molecules.
Molecularly imprinted polymers (MIPs) are a class of highly cross-linked polymer
that can bind certain target compound with high specificity. The polymers are
prepared in the presence of the target molecule itself as the template.

Figure 1.1. Number of papers and patents published on the topic of molecularly
imprinted polymers from 1994 to September 2018 [94].
Molecular imprinting was first introduced in the early 1930s by M. V.
Polyakov, who investigated on silica for use in chromatography [87]. In 1972, the

5


molecular imprinting was applied to organic polymers by the groups of Gunter Wulff,
Takagishi and Klotz [81]. In the early 1980s, the most important development in the
area of molecular imprinting of non-covalent approach was facilitated by the group
of K. Mosbach [5].
Among the alternatives to natural recognition systems, MIPs have shown high
promise. There has been a steady annual growth, increasing amount of papers
published and the patents filed per year over the past two decades which referenced
this discipline (Figure 1.1). This can be taken as a clear indication of expansive
potential and interest it has garnered.

Figure 1.2. Schematic for synthesis and imprinting process of molecularly
imprinted polymer.
The concept behind the formation of the selective binding sites is
schematically shown in Figure 1.2. The mechanism of the imprinting is simple to
understand: a template molecule is entrapped in a polymer substrate during
polymerization, so that the molecular information is traced in polymeric material in
the molecule shape, then its complementary chemical functionality persists in the
substrate network after the complete extraction of the template from the substrate as
cavities [90]. These cavities are the recognition sites for the same template molecules

6


or similar: these molecules bind to the polymer substrate with a very high specificity
[88]. Finally, the template is removed and a polymeric substrate with specific cavities
complementary in size, shape, and bonds are produced. The advantages of MIP are
ease of preparation, low cost, and high chemical and mechanical stability.
1.1.1. Types of imprinting mechanism
Functional monomers interact with the template in the solutions. The
interaction between template and the imprinting polymer includes covalent bonding,
non-covalent bonding, and semi-covalent imprinting.
 Covalent imprinting:
Molecular imprinting via covalent forces is one of the oldest formats used in
artificial receptor design. It involves the formation of a covalent bond between the
template and functional monomer molecules which is then secured in place via
polymerization with a copolymer. The template may then be removed via hydrolysis
or a similar treatment to cleave the chemical bonds, securing it to a newly formed
polymer to create the imprinted receptor.
 Non-covalent imprinting:

The non-covalent imprinting method was first reported by Masbach and his
colleagues, inspired by the diverse range of non-covalent interactions found in
biochemistry, and final imprinting approach is, by far, the most widely used and
referenced in the literature. This is because of the ease with which it can be performed
in a non-specialized laboratory and also, as is the case with the semi-covalent
imprinting approach, the facile and versatile manner in which these MIPs can be
implemented thanks to their ability to rapidly re-bind or re-dock their template
molecule in non-specialized solutions. The major interaction in non-covalent
imprinting is hydrogen-bonding.
 Semi-covalent imprinting:

7


The semi-covalent approach to molecular imprinting, first reported by
Whitcombe et al. in 1995 [95], is a manifestation of the effort made to meld the
advantages of both the covalent and non-covalent imprinting approaches whilst
excluding their respective failings and disadvantages. This method involves the
covalent attachment during the polymerization and hydrogen-bond formation during
the recognition, which could overcome the disadvantages of covalent imprinting.
1.1.2. Removal of the imprinted template
Removal of the imprinted template is an important step in the preparation of
most molecularly imprinted polymers. A combination of physical combination and a
suitable solvent are typically necessary for the removal, in which the choice of
removing procedure should consider two parameters, including: (1) efficiency of the
template removing, and (2) organic solvents or conditions like high-temperature
which cause degradation of MIP, so that keeping of the imprinted structure and the
whole substrates should be ensured.
However, the polymer substrate itself and the affinity of the cavities are
printed out for template making it difficult to remove. If there are remaining template

molecules in the MIP, there will be fewer cavities to rebinding, reducing the
productivity. Thus, in order for MIP to reach its full potential in analytical and
biotechnological applications, an efficient removal process must be demonstrated.
There are several different methods of extraction which are currently being used for
template removal, described in [6, 27, 50].
1.1.3. Applications of MIPs
MIPs has found applications in several different areas, such as purification and
separation, catalysis, sensors, drug delivery systems. In this thesis, we focus only on
the application of MIPs as electrochemical sensors and biosensors.
Electrochemical sensors and biosensors are of increasing interest in the field
of modern analytical chemistry, as can be seen from the growing number of published
papers. This is due to new demands that are appearing particularly in clinical
8


diagnostics, environmental analysis and also in food analysis. Recently, a big effort
to synthesize artificial receptors capable of binding a target analyte with comparable
affinity and selectivity to natural antibodies or enzymes has been done.
MIPs technology can be used as antibody-like materials with high selectivity
and sensitivity, owing to their long-term stability, chemical inertness and insolubility
in water and most organic solvents [34]. To date, MIPs have been successfully used
with different types of transducers and several methods have been used to achieve a
close integration of the transduction platform with the polymer [65].
MIP-based recognition elements were also prepared as layers or thin films by
deposition or grafting onto the transducer platform [31, 80]. The thickness of the film
deposited on the transducer is important to obtain a good time response of the sensor.
This approach was first used with acoustic [22], optical transducers [33], and then
with electrochemical sensors [8].
1.2. Polyaniline
Polyaniline (PANi) was discovered more than 156 years ago [45, 92], which

is one of the typical conductive polymers, where its peculiar electrical conductivity
being explained more and more fully and it has become a well-studied polymer in the
scientific community. In conductive polymers (CPs), PANi is a special case due to
its low cost of preparing monomers, high conductivity, and easy synthesis by
chemical or electrochemical methods, and stability in the environment. The great
advantage of PANi is the ability to denature the doped protons and the environmental
impact. This makes PANi attractive material in many applications such as sensors,
electronic devices, rechargeable batteries [29, 89], corrosion protection [42],
transmission, electromagnetic shielding [4, 7, 78].
PANi is mainly synthesized by oxidation or electrochemical polymerization
from monomer aniline. Monomers aniline (ANi) is an organic compound having the
formula C6H5NH2, molecular weight 93.13 g/mole, density 1.022 g/cm3, viscosity at

9


20 °C is 4.35 mPa.s, dissolve 3.7 g in 100 g of water, specific heat 2.06 J/g.K, boiling
temperature 101.3 kPa at 184 ℃, melt temperature is 6 ℃.

Figure 1.3. Chemical structure of PANi.
PANi is a conducting polymer based on phenylene ring (C 6H4) and has an
-NH- group in the polymer chain on both sides of the phenylene ring. It is a polymer
that can exist in a variety of structures depending on the value (1-y) in the PANi
generic formula (as shown in Figure 1.3), where y represents the fraction of the
reduced units in the chain (0 ≤ y ≤ 1). The electrical properties of PANi can be
reversed by protonation by oxidation-reduction. Thus, with PANi, it is possible to
visualize the redox polymerization state for each unit consisting of the reduction
process (–NH–B–NH–) and the oxidation process (–N=Q=N–), where B and Q
denote a unit of benzenoid and quinoid units. The average oxidation state in the PANi
series is given by the value of (1-y), which can have three redox states:

First state: Fully reduced leucoemeraldine base state (LB, yellow), where 1–y
= 0 as in Figure 1.4a.
Second state: Half oxidized emeraldine base state (EB, blue), where 1–y = 0.5
(Figure 1.4b). The EB is the main form of polyaniline due to its high stability at room
temperature. It is known to be a semiconductor and is composed of the alternating
sequence of two benzenoid units and one quinoid unit [54].
Third state: Fully oxidized pernigraniline base state (PB, purple) where 1–y
= 1 [76] as illustrated in Figure 1.4c.

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Figure 1.4. The structural formula of the redox states of PANi a) fully reduced
leucoemeraldine, b) half oxidized emeraldine, and c) fully oxidized pernigraniline
[57].
Furthermore, emeraldine base can be doped in a non-redox reaction with a
protonic acid (HA) which results in an emeraldine salt (ES). This non-redox doping
process differs from redox doping in that it does not involve the addition or removal
of electrons from the polymer backbone. Instead, the imine nitrogen atoms of the
polymer are protonated to give a polaronic form where both spin and charge are
delocalized along the entire polymer backbone. This process is reversible, as the
conductive ES state can be converted back to the insulating EB state through
treatment with a base. Thus, the emeraldine salt is the most potent conductivity and
oxidation state among PANi’s redox states. On the other hand, LB is easily oxidized
while the PB is easily degraded [54]. The diagram shows the process of transforming
from emeraldine base to emeraldine salt when doped with HA, as described in Figure
1.4.

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1.2.1. Properties of PANi
Polyaniline has chemical, optical, and electrochemical properties that
distinguish it from other conducting polymers.
1.2.1.1. Chemical properties
Some studies have shown that the most energetic chemical properties of
polyaniline are the anionic exchange properties and are distinct from those of
conventional ion exchange polymers [19]. This can be due to the dispersion of charge
on the polyaniline. The effect of the electronic structure has also been demonstrated
in studies of the occurrence of amino acid interactions on PANi. For example, in two
amino acids with similar charge density but different molecular configurations, the
ability to interact with PANi differs significantly.
1.2.1.2. Optical properties
PANi has an electrochromic property because its color changes due to the
redox reaction of the film. It has been demonstrated that PANi exhibits a variety of
colors from light yellow to green, dark blue and dark purple depending on the redox
state at different voltages. Transitions of PANi between different oxidation states and
upon protonation/deprotonation are associated with color changes. ES and EB forms
are green and blue, respectively. LB form is colorless to pale yellow. PB form is violet
and turns to blue by protonation to PS form.
1.2.1.3. Thermal stability
It is another attractive property of PANi. It has been observed that PANi has
good temperature stability up to 400 ℃ in N2 [21, 23]. Counterions in the PANi chain
influence the thermal properties of PANi. The introduction of -SO3H in aniline ring
can weaken the thermal stability of aniline [13].
1.2.1.4. Conductivity properties of PANi
PANi can exist both in the insulating and conducting state depending on the
oxidation state. The emeraldine of PANi as a semiconductor with a conductivity of
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