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CHAPTER II
MOLECULAR IMPRINTED POLYMERS:
A REVIEW
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
15
MOLECULAR IMPRINTED POLYMERS: A REVIEW
2.1. The concept of molecular imprinting
One of the most basic principles in life is molecular recognition. Interest
in it has grown substantially during the last three decades, as life uses this
strategy to detect both desired and unwanted compounds, which thus makes it
the basis of such different phenomena as the immune system or cell
detoxification. In any case, the key to recognition is non-covalent interactions
and self-organization. Both are necessary to ensure reversible binding of a
target compound to a receptor site. When aiming at artificial, highly functional
materials to mimic these natural processes, molecular imprinting has become a
highly interesting strategy to achieve such functionality in man-made polymers.
Imprinting techniques rely on polymerizing a highly cross-linked substrate in
the presence of a structure-directing agent, which is either a model compound or
the analyte-to-be itself. This template determines a porous structure with
predefined non-covalent binding sites for the analyte within the polymer by
self-organization processes between the growing backbone and the moulding
species. This leads to the generation of geometrically and sterically well defined
cavities being stabilized owing to the high amount of cross-linker. The resulting
materials have turned out to be capable of reversibly reincorporating the analyte
of interest. Such properties have made molecularly imprinted polymers (MIPs)
a highly interesting tool for different scientific fields, including separation
sciences, chemical sensor design, purification and catalysis. The method has
gained a strong hold especially in different fields of analytical science, although
applications in catalysis and filtering have also been reported. In analysis,
molecularly imprinted polymers can predominantly be found in separation
science and chemical sensing as well as to a much lesser extent in sample
Chapter 2Chapter 2Chapter 2Chapter 2
16
preparation techniques such as solid phase extraction. Applications in different
chromatographic techniques especially in chiral separations give fundamental
insight into the physicochemical properties of the bulk materials used. In sensor
science on the other hand very high sensitivity and selectivity of the receptor
layer is required, because in sensing only one theoretical plate can be utilized to
separate the desired analyte from its matrix.
2.2. History of molecular imprinting
In reviewing the historical origins of molecular imprinting as a
technique, it is noted that imprinting was first introduced in the early 1930s by a
Soviet chemist M. V. Polykov1 who was performed a series of investigations on
silica for use in chromatography. It was observed that when silica gels were
prepared in the presence of a solvent additive the resulting silica demonstrated
preferential binding capacity for that solvent. It was first time that experiments
of this kind were accompanied by explanations of this nature. The mechanism
proposed by Polykov was largely overlooked by the scientific community. In
1931 the group of Polyakov, reported some unusual adsorption properties in
silica particles prepared using a novel synthesis procedure. Sodium silicate had
been polymerized in water using ammonium carbonate as the chelating agent.
After two weeks, additives (benzene, toluene or xylene) had been added. The
silica was subsequently allowed to dry for 20-30 days, after which the additive
was removed by extensive washing in hot water. Subsequent adsorption studies
revealed a higher capacity for uptake of the additive by the silica than for
structurally related ligands, i.e. some kind of memory for the additive was
apparent, at least in the cases of benzene and toluene.
Attempts were subsequently made to apply the principles of the
instructional theory in an inorganic system, silica. In 1949 a study was
performed by a student of Linus Pauling; Frank Dickey, which involved the
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
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development of molecular imprinting in silica matrices in the presence of dyes2.
The methodology used was very similar to that of Polyakov, but in this case
methyl orange (and other alkyl orange dyes) was used as the template. The
template was present from the start of the reaction process, and acetic acid (and
some other organic acids) was used as the acidifying agent. Dickey observed
that after removal of the “patterning” dye the silica would rebind the same dye
in the presence of others. Dicky’s silica can be considered to be the first
imprinted materials. Dicky’s approach to introduce the template in the sodium
silicate pre-polymerisation mixture produced a more definite influence on the
structure of the silica, whereas Polykov introduced the template after the silica
frame work. Dicky’s work is similar to present methodologies, thus, this
method become the most widely used in subsequent studies.
Silica imprinting continued during 1950s and 1960s, but the number of
publications in this area remained low. Applications were being considered
already at an early stage. In the early 1950’s, chiral selectivity for mandelic acid
and camphor sulfonic acid enantiomers had been demonstrated by Curti et al.
using imprinted silica as stationary phases in column chromatography3-5. Work
in this area involved attempting to use imprinted materials for practical
separations such as solid phase in chromatography and in thin layer
chromatography. The reasons for the limited interests were resulted to
limitations in the stability and reproducibility of the imprinted silica materials.
However, the re-emergence of silica based MIP research has been occurred.
Pinel et al. examined the imprinting of silica gels and showed that
regiospecificity for cresol was successfully imprinted using o-cresol as the
template6. Hunnius et al.7 prepared porous silica through a sol-gel process,
which were developed for the generations of selective adsorption sites by
molecular imprinting. Depending on the preparation conditions, microporous
silica show surprising adsorption selectivity. This selectivity is not related to
Chapter 2Chapter 2Chapter 2Chapter 2
18
imprinting effects but must be attributed to unpredictable changes in surface
polarity of the fine porous materials.
Perhaps most spectacular papers of the early imprinting era came from
the group of Patrikeev. In one case, a bacteria species was incubated with the
chelated silica under the drying process and later heated to dryness. This
‘imprinted’ silica was found to promote the growth of the template bacterial
species better than several different reference silicas8, and in another case the
imprinted silica was shown to exhibit enantioselectivity9.
This group also reported an early synthetic enzyme imprinted polymer,
using the term in a generous sense. Silica imprinted with a tripeptide or
diketopiperazine respectively, would direct product formation during the
polycondensation of amino acids to favour formation of the imprinted species.
After two decades of rather intense research in the area, a decline of molecular
imprinting in silica appears to have coincided with the introduction of molecular
imprinting in organic polymers made independently by Wulff and Klotz in
197210,11. In 1979, however, Sagiv introduced a novel approach for making
imprints in silica12-15. The template was adsorbed onto the surface of silica
particles, while octadecyltrimethoxy siloxanes were chemically connected to the
silica surface. This led to patches of non-derivative silica, with areas
complementary to the template.
More recently the potential use of titanium alkoxide as precursors to
imprinted media has been illustrated by Kunitake and co-workers16. A study
published in 2005 by T. R. Ling involved the recognition of catecholamine
using molecular imprinted silica-alumina gel17. Shiomi et al.18 tested a new
molecular imprinted technique to synthesis protein imprinted silica using
covalently immobilised template haemoglobin for biological applications.
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
19
Although the original method has been abandoned, the Polyakov-Dickey
approach in its developed form represents a most active and highly promising
area of molecular imprinting. However, the early debate on the mechanisms
underlying molecular recognition in these systems, in parallel to that of organic
MIPs, is still ongoing, as reflected by several recent reports.
2.3. Molecular imprinted polymers: General introduction
The area of molecular imprinting technology, enlarged by Wulff and
Sarhan10, is a new technology for introducing molecular recognition properties
to the functional polymer which was synthesized in the presence of the template
molecules. Before the polymer preparation, the template-monomer complex
was formed attributed to the simultaneous multiple interactions between the
template and the functional monomer. During the polymerization of the
polymer, the template molecules were stabilized in the polymer and the
following extraction of the template made the specific recognition sites in the
polymer both from the shape complementary to the template and the strategic
arrangement of the functional groups between the template and the monomer19-21.
There are two types of interactions between the template and the monomer:
covalent interaction and non-covalent interaction. Accordingly, there are three
approaches to prepare the molecularly imprinted polymers: covalent, non-
covalent and semi-covalent methods (Scheme II. 1). Because of pre-treatment
of the template and the low recovery ratio of the template22, the covalent
method is seldom used today. The non-covalent method, introduced by
Mosbach and Ramstrom23, relying on the weak interactions between the
monomer and the template, is the most widely used technique recently. The
covalent and non-covalent imprinting can be distinguished based on the type of
interaction between the template and functional monomer during the synthetic
process and rebinding events. They are (i) in covalent imprinting24 the template
and functional monomer are covalently bound together and incorporated as a
Chapter 2Chapter 2Chapter 2Chapter 2
20
unit into the polymer. This approach is only useful if the covalent bond is
reversible; it must form rapidly but it must also be weak enough to allow easy
extraction of the template to leave behind a polymer with imprinted cavities,
(ii) non-covalent imprinting25 is the most versatile of the approaches. The
template and functional monomer form a complex through non-covalent
intermolecular interactions which can be rapidly formed and easily disrupted.
The drawback to the non-covalent approach is the random nature of complex
formation which may lead to different orientations of the two species and
therefore different types of imprinted cavities and (iii) semi-covalent imprinting
merges the two previous approaches. The imprinted cavities are formed from a
template which is covalently bound to a functional monomer. This minimises
the types of template-monomer orientations incorporated into the imprinted
polymer. The chosen covalent bond should be easily cleaved but should not
form easily under the rebinding conditions. The resulting imprinted polymers
rebinds to the analyte through intermolecular interactions.
Scheme II. 1. Schematic representation of molecular imprinting procedure
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
21
In covalent imprinting, typically the templates are bound to appropriate
monomers by covalent bonds. After polymerization, the covalent linkage is
cleaved and the template is removed from the polymer. Upon rebinding of the
guest molecule by imprinted polymers, the same covalent linkage is formed.
Owing to the greater stability of covalent bonds, covalent imprinting protocols
yield a more homogeneous binding sites distribution. However, covalent
imprinting is also considered as a less flexible method since the formation of
identical rebinding linkages requires rapidly reversible covalent interactions
between templates and functional monomers. Therefore, templates suitable for
covalent imprinting are limited. Moreover, it is very difficult to reach
thermodynamic equilibrium due to the strong nature of the covalent interactions
and consequently it results in slow binding and dissociation. In contrast, non-
covalent imprinting has no such restrictions. In an appropriate solvent,
template-monomer complexes are formed relying on various interactions, such
as hydrogen bonding, ionic interactions, van der Waals forces, π-π interactions,
etc. After polymerization and removal of the template, the functionalized
polymeric matrix can rebind the target (template) via the same non-covalent
interactions, so the range of applicative compounds which can be imprinted is
greatly expanded. Besides the above mentioned advantages, a further factor is
simplicity in operation since only mixing of templates and monomers in a
suitable solvent is required26. Currently, non-covalent imprinting has become
the most popular and general synthetic strategy for molecular imprinting
technology. Interestingly, after a covalently bonded template is removed, non-
covalent rebinding can also be achieved27 which is defined as a semi-covalent
approach, attributed to Whitcombe et al28. This method offers an intermediate
alternative in which the template is bound covalently to functional monomer as
in the covalent approach, (Scheme II. 2) but the template rebinding is based on
Chapter 2Chapter 2Chapter 2Chapter 2
non-covalent interactions. It is characterized by both the high affinity of
covalent bonding and mild operation conditions of non
Scheme II. 2. The overall process of forming molecular imprinted polymers
Compared to other recognition systems,
possess many promising characteristics, such as low cost and easy synthesis,
high stability to harsh chemical and physical conditions, and excellent
reusability. Consequently, molecular imprinted polymers have become
increasingly attractive in many fields, particularly as selective adsorbents for
solid-phase extraction (SPE)
sensors34-37.
Molecular imprinting is widely employed to produce robust, stable, and
cheap materials with s
co-polymerizing functional and crosslinking monomers in the presence of a
molecular template. After removal of the template, complementary cavities are
22
ractions. It is characterized by both the high affinity of
covalent bonding and mild operation conditions of non-covalent rebinding.
The overall process of forming molecular imprinted polymers
Compared to other recognition systems, molecular imprinted polymers
possess many promising characteristics, such as low cost and easy synthesis,
high stability to harsh chemical and physical conditions, and excellent
reusability. Consequently, molecular imprinted polymers have become
ly attractive in many fields, particularly as selective adsorbents for
phase extraction (SPE)29-32, chromatographic separation33
Molecular imprinting is widely employed to produce robust, stable, and
cheap materials with specific binding sites38. This is achieved by
polymerizing functional and crosslinking monomers in the presence of a
molecular template. After removal of the template, complementary cavities are
ractions. It is characterized by both the high affinity of
covalent rebinding.
The overall process of forming molecular imprinted
molecular imprinted polymers
possess many promising characteristics, such as low cost and easy synthesis,
high stability to harsh chemical and physical conditions, and excellent
reusability. Consequently, molecular imprinted polymers have become
ly attractive in many fields, particularly as selective adsorbents for
and chemical
Molecular imprinting is widely employed to produce robust, stable, and
This is achieved by
polymerizing functional and crosslinking monomers in the presence of a
molecular template. After removal of the template, complementary cavities are
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
23
obtained that allow rebinding of the template with very high specificity,
comparable to that of natural receptors. These materials have been widely used
as affinity matrices for sample preparation and selective extraction of
analytes39-41.
The affinity for the target molecule to pockets left by template molecule
suggests that they can be used in applications of advanced separations and as
biosensors, in which the mechanism being similar to antibodies and enzymes.
Molecular imprinting can also be considered as the selective manipulation of
the shape, size and chemical functionality of a polymer matrix by a template
molecule.
Imprinting may be achieved by two approaches: polymerization and
phase inversion42. The polymerization is conducted in a solvent (porogen)
which facilitate the formation of template-monomer complex by stabilization of
interactions. This complex is then fixed into a spatial arrangement by the
inclusion of a high proportion of crosslinking monomer, which confers rigidity
to the polymer network. Removal of the template species affords nano-cavities,
which are complementary in size, shape and chemical functionality to the
template species. These cavities have the ability to selectively rebind the
template. In the phase inversion approach the template is incorporated into the
polymer matrix by phase inversion43-45. Removal of the template affords a
cavity, which is complementary in size, shape and functionality to the template
molecule. The phase inversion method has the advantage that it starts from an
already prepared polymer. The main problems that have to be solved in this
case are finding a good solvent common for the matrix copolymer and for the
imprint and finding an optimum composition for the coagulation bath, so that
the imprint diffusion in the bath or the chemical alteration would not take place.
Chapter 2Chapter 2Chapter 2Chapter 2
24
The most common method for preparing molecularly imprinted
polymers suitable for molecularly imprinted solid phase extraction (MISPE)
consists in bulk thermal or photo polymerization that produces a monolithic
polymer that has to be crushed and sieved to obtain particles of the desired size
distribution. This method, by far the most popular, presents several attractive
properties. It is fast and simple in its practical execution, it does not require
particular skills of the operator, it is widely reported in literature for many
different templates and it does not require sophisticated or expensive
instrumentation46. However, the procedure of grinding and sieving is difficult,
and it causes a substantial loss of useful polymer. Most of the lost polymer is a
very fine sub-micrometric powder, which could adhere to the bigger particles
and cause excessively high back pressures in a SPE column during the
extraction procedure, especially with online devices. Moreover, the bulk
polymerization cannot be scaled-up.
Apart from the more obvious recognition properties of molecularly
imprinted polymers, their physical and chemical characteristics are highly
appealing. These materials exhibit high physical and chemical resistance
towards various external degrading factors. Thus, molecularly imprinted
polymers are remarkably stable against mechanical stress, elevated temperatures
and high pressures, resistant against treatment with acid, base or metal ions and
stable in a wide range of solvents. The storage endurance of the polymers is also
very high. Storage for several years at ambient temperature leads to no apparent
reduction in performance. Further, the polymers can be used repeatedly, in
excess of 100 times during periods of years without the loss of “memory
effect”. In comparison with natural, biological recognition sites, which are often
proteins, these properties are highly advantageous47.
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
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2.3.1. Covalent imprinting
i) Advantages
Monomer-template conjugate are stable and stoichiometric, and thus the
molecular imprinting process (as well as the guest-binding sites in the polymer)
are relatively clear. A wide variety of polymerisation conditions (eg: high
temperature, high or low pH and highly polar solvents) can be employed, since
the conjugate are formed by covalent linkages and are sufficiently stable.
ii) Disadvantages
Synthesis of monomer - template conjugate is often troublesome and
less economical. The numbers of reversible covalent linkages available are
limited. The imprinting effect is some case diminished in the step of cleavage of
covalent linkages, which requires rather severe conditions. Guest-binding and
guest-release are slow, since they involve the formation and the breakdown of a
covalent linkage.
2.3.2. Non-covalent imprinting
i) Advantages
Synthesis of covalent monomer - template conjugate is unnecessary.
Template is easily removed from the polymer under very mild conditions, since
it is only weakly bound by non-covalent interactions. Guest-binding and guest-
release, which take advantage on non-covalent interactions, are fast.
ii) Disadvantages
Imprinting process is less clear (monomer-template adduct is labile and
not strictly stoichiometric). Polymerisation condition must be carefully chosen
to maximise the formation of non-covalent adduct in the mixtures. The
functional monomers existing in large excess (in the order to displace the
Chapter 2Chapter 2Chapter 2Chapter 2
26
equilibrium for adduct formation) often provide non-specific binding sites, thus
diminishing the binding selectivity.
2.3.3. Reagents for molecular imprinting
Polymerization reaction is known as a very complex process, which
could be affected by many factors, such as type and concentration of the
monomer, cross-linking agent, initiator, temperature, time of polymerization,
the presence or absence of magnetic field, and volume of the polymerization
mixture. In order to obtain the ideal imprinted polymer, a variety of factors
should be optimized. Thus synthesis of molecular imprinted polymers is a time-
consuming process. In order to prepare imprinted polymers with perfect
properties, numerous attempts have been made to investigate such effects on the
recognition properties of the polymeric materials48. The selection of appropriate
reagents is a crucial step in the molecular imprinting process. Generally,
template molecules are target compounds in analytical processes. An ideal
template molecule should satisfy the following three requirements. The
template should not contain groups involved in or preventing polymerization,
should exhibit excellent chemical stability during the polymerization reaction
and it should contain functional groups well adapted to assemble with
functional monomers.
2.3.4. Basic composition of molecularly imprinted polymers
The mixture for an imprinted polymer contains a template (the target
analyte), functional monomer and crosslinking monomer (or functionalised
crosslinking monomer), porogen and initiator. A comparison of the performance
of the molecular imprinted polymer and the reference polymer indicates
whether the imprinted polymer has memory for the template.
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
27
i) Template
The template molecule ideally should contain at least one functional
group through which it can interact with the functional monomer as well a
distinctive three-dimensional structure. The type of functional group controls
the imprinting approach that can be utilised. Not all templates will readily form
a covalent bond with a functional monomer that is easily cleaved. On the other
hand the number of functional groups affects the affinity of the template for the
molecular imprinted polymer. Increasing the number of interactions between
the template and functional monomer may increase the affinity with which the
molecular imprinted polymer rebinds the template49. However it also increases
the non-specific binding of the template to the polymer. Removal of the
template after polymerisation is necessary to reveal the imprinted cavities. If
residual template remains, these can leak out while performing tests on the
polymers. This is called template bleed and it is a problem if the molecular
imprinted polymers are used for analytical chemistry applications. This has
been circumvented by using a method called analogue imprinting in which a
structural analogue of the target compound is used as the template.
ii) Functional monomer
The role of the monomer is to provide functional groups which can form
a complex with the template by covalent or non-covalent interactions. The
strength of the interactions between template and monomer affects the affinity
of molecular imprinted polymers50 and determines the accuracy and selectivity
of recognition sites51. The stronger the interaction is, the more stable the
complex is, resulting in high binding capacity of the imprinted polymers, and
therefore, correct selection of the functional monomers is very important.
Tedious trial and error tests are often required to select a suitable monomer.
Chapter 2Chapter 2Chapter 2Chapter 2
28
Commonly used monomers for molecular imprinting include methacrylic
acid (MAA), acrylic acid (AA), 2- or 4-vinylpyridine (2- or 4-VP), acrylamide,
trifluoromethacrylic acid and 2-hydroxyethyl methacrylate (HEMA). MAA has
been used as a ‘‘universal’’ functional monomer due to its unique characteristics,
being capable to act as a hydrogen-bond donor and acceptor, and showing good
suitability for ionic interactions.
The functional monomer contains at least a vinyl group and another
functional group, through which it can interact with the template. Non-covalent
imprinting requires the selection of an appropriate functional monomer that will
form strong intermolecular interactions with the template.
Table II 1. Common functional monomers for non-covalent imprinting
Functional monomers Structure
Methacrylic acid O
OH
Itaconic acid
O
HO
O
OH
4-Vinylbenzoic acid
O
OH
4-Vinylpyridine N
Acrylamide
O
NH2
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
29
The selection of functional monomers for covalent and semi-covalent
imprinting is more restricted because of the antagonistic requirements of
polymerisation and template extraction. The functional monomer must form a
covalent bond with the template that is stable under polymerisation conditions.
iii) Crosslinking agent
Molecular imprinted polymers are solid and porous because the multiple
vinyl groups in the crosslinking monomer can co-polymerise with the functional
monomer and thus interconnect two radical centres from different polymer
chains. Their porous nature allows the template molecules to diffuse into and
from the imprinted cavities. Their solid state allows the molecular imprinted
polymer to maintain structural integrity of the imprinted cavities. The degree of
cross-linking determines the rigidity of the polymer and may affect the
selectivity of a molecular imprinted polymer. A selection of commonly used
cross-linking monomers can be found in Table II.2.
An alternative method of creating molecular imprinted polymers is to
use a single type of monomer that has the characteristics of both a functional
monomer and a crosslinking monomer. An example of a dual-role monomer
reported which has been used to create a molecular imprinted polymer was
N,o-bismethacryloyl ethanolamine. This contains a functional group which can
interact with the template and two vinyl groups for crosslinking polymer chains.
The functional amide group is covalently bound to the polymer backbone at two
points and therefore would restrict its conformational freedom. This minimises
the number of possible interactions between the template and the functionalised
cavities and therefore increases the selectivity of the molecular imprinted
polymer. Another example of single monomer type molecular imprinted
polymers, although not crosslinked are polypyrroles. These may be the interface
between polymers and electronics because the highly conjugated π-electron
Chapter 2Chapter 2Chapter 2Chapter 2
30
system may transduce the recognition event taking place in the molecular
imprinted polymer.
Table II. 2. Commonly used cross-linking monomers
Crosslinking monomer Structure
Ethylene glycol dimethacrylate (EGDMA)
O
O
OO
Divinylbenzene (DVB)
Trimethylolpropane trimethacrylate
(TRIM)
O
O
O
O
O
O
N,N-ethylenebismethacrylamide
(EBMAA)
O
HN
ONH
N,N-1,3-phenylenebis(2-methyl-2-propenamide)
(PBMP) ON
H
O
NH
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31
iv) Porogen
Porogenic solvent plays an important role in polymerization. It acts as
not only a porogen but also solvent in preparation process. Besides, it also
influences the bonding strength between functional monomers and templates,
the property and morphology of polymer, especially in non-covalent interaction
system. Aprotic and low polar organic solvents, such as toluene, acetonitrile and
chloroform are often used in non-covalent polymerization processes in order to
obtain good imprinting efficiency. It is notable that MIPs prepared in organic
solvent work poorly in aqueous media because of the ‘‘solvent memory’’. The
influence of solvents has different roles such as it solubilises all the monomers
in the pre-polymerisation mixture before polymerisation. It stabilises template-
monomer pre-polymerisation complex and it acts as a ‘porogen’ helping to
control the porosity of the resulting polymer.
The range of suitable porogen for a particular molecular imprinted
polymer system is limited by the type of interaction between the template and
functional monomer because the strength of interaction is affected by the
environment. The porogen also plays a role in forming a porous polymer by
solvating the template and monomers during the polymerisation process thus
acting as a space-filler. It is eventually removed, just like the template, to create
channels within the polymer which increases the accessibility of the imprinted
cavities.
v) Initiator
The initiator starts the polymerisation process by providing a source of
free radicals. These can be generated by thermal or photolytic decomposition of
azobis(nitriles) or peroxides. The temperature of initiation can affect the
strength of the complex formed by the reactants before polymerisation. This
depends on the interactions between the template and monomer. It is important
Chapter 2Chapter 2Chapter 2Chapter 2
32
that the temperature of initiation is lower than the boiling point of the porogen.
Some of the commonly used initiators are given in the table II. 3.
Table II. 3. Initiators commonly used for molecular imprinting
Initiator name Structure
2,2’-Azobis(isobutyronitrile)
(AIBN) N NN
N
2,2’-Azobis(dimethylvaleronitrile)
(ABDV) NN
C
N
C
N
2,2’-Azobis(2-methylisobutyronitrile)
(AMBN) N N
C
NC
N
vi) Molar ratio between template and monomer
Generally, the molar ratio between template and monomer in the
synthesis of imprinted polymers affect the affinity and imprinting efficiency of
molecular imprinted polymers. Lower molar ratios induce less binding sites in
polymers due to fewer template - monomer complexes, but over-high ones
produce higher non-specific binding capacity, diminishing the binding
selectivity. So, in order to gain high imprinting efficiency, the molar ratio of
templates to monomers should be optimized.
vii) Polymerization conditions
Several studies hav
polymers at lower temperatures forms polymers with greater selectivity versus
polymers made at elevated temperatures. Commonly used temperature for
polymerisation is 60°C. However, the initiation of the polymerization reaction
was very fast and therefore hard to control at this temperature and resulted in
low reproducibility of molecular imprinted polymer. Furthermore, the relatively
high temperatures have a negative impac
reduced the reproducibility of the monolithic stationary phases and produced
high column pressure drops. Thus, relatively low temperatures with a
Figure II. 1. An example of bulk synthesis method for dissolution of monomer and template in a solvent (2) free radical polymerization initiated with an azo initiator and a cross-linker agent in a water bath (3) crushing the block polymer into fine particles and (4) removal of template fromthe polymer matrix using Soxhlet apparatus
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers:
33
Polymerization conditions
Several studies have shown that polymerization of molecular imprinted
at lower temperatures forms polymers with greater selectivity versus
t elevated temperatures. Commonly used temperature for
. However, the initiation of the polymerization reaction
was very fast and therefore hard to control at this temperature and resulted in
low reproducibility of molecular imprinted polymer. Furthermore, the relatively
high temperatures have a negative impact on the complex stability, which
reduced the reproducibility of the monolithic stationary phases and produced
high column pressure drops. Thus, relatively low temperatures with a
An example of bulk synthesis method for MIP production (1) dissolution of monomer and template in a solvent (2) free radical polymerization initiated with an azo initiator and a
linker agent in a water bath (3) crushing the block polymer into fine particles and (4) removal of template fromthe polymer matrix using Soxhlet apparatus
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
molecular imprinted
at lower temperatures forms polymers with greater selectivity versus
t elevated temperatures. Commonly used temperature for
. However, the initiation of the polymerization reaction
was very fast and therefore hard to control at this temperature and resulted in
low reproducibility of molecular imprinted polymer. Furthermore, the relatively
t on the complex stability, which
reduced the reproducibility of the monolithic stationary phases and produced
high column pressure drops. Thus, relatively low temperatures with a
MIP production (1) dissolution of monomer and template in a solvent (2) free radical polymerization initiated with an azo initiator and a
linker agent in a water bath (3) crushing the block polymer into fine particles and (4) removal of template from
Chapter 2Chapter 2Chapter 2Chapter 2
34
prolonged reaction time were selected in order to yield a more reproducible
polymerization. Where complexation is driven by hydrogen bonding then lower
polymerization temperatures are preferred, and under such circumstances
photochemically active initiators may well be preferred as these can operate
efficiently at low temperatures.
A typical example of the synthesis of molecular imprinted polymer
using bulk polymerisation method with the above mentioned components such
as template, monomer, initiator and crosslinking agent are given in Fig. II.1.
2.4. Applications of molecularly imprinted polymers
Molecular imprinted polymers are selective solid surfaces that can
theoretically be used as substitutes for proteins such as antibodies and cell-
surface receptors. The imprinted cavities may also be chemically reactive and
therefore the molecular imprinted polymers may function like an enzyme.
Catalytic molecular imprinted polymers have been prepared by imprinting an
analogue of the transition state of a reaction because the transition state itself is
unstable and is likely to decompose before the polymerisation is complete. The
efficiency of catalytic molecular imprinted polymers is not as good as natural
enzymes since they are finely tuned bio molecules which have evolved high
catalytic activity in their natural medium. Only modest rate enhancements have
been achieved with catalytic molecular imprinted polymers52. Many variables
are involved in polymerisation and assessing molecular imprinted polymers.
Therefore extensive optimisation studies may be required to achieve the
efficiency of a natural enzyme. However compared to proteins, molecular
imprinted polymers are more stable in extreme temperatures and organic
solvents. Therefore molecular imprinted polymers may be used in applications
in which enzymes tend to degrade, such as in organic synthesis.
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Natural antibodies have been used in enzyme-linked immune sorbent
assays in which an enzyme-linked optical change (fluorescence or colorimetric)
occurs when the target analyte is bound by the antibody. However, because
proteins are easily denatured by organic solvents, this limits the scope of
analytes which can be determined through this method. Therefore molecular
imprinted polymers could be developed as substitutes for natural receptors.
Molecular imprinted polymers specific for atrazine and epinephrine could be
used as a substitute for antibodies in an immunoassay53.
A drawback is that often molecular imprinted polymers do not perform
well in the presence of water which can be found in biological or environmental
samples. The polarity of water can interfere with interactions between the target
analyte and the imprinted sites. This would have a greater effect if specificity
relied on non-covalent interactions such as hydrogen bonds but would be less of
a problem for covalent imprinted polymers (if the formation of the reversible
covalent bond was not disrupted by the presence of water). Molecular imprinted
polymers that work in aqueous systems have employed strong non-covalent
interactions such as metal-ion chelation54.
Molecular imprinted polymer sensors have been created which
selectively re-bound glucose from aqueous carbonate buffer. Glucose formed a
complex with the copper (II) complex which had been polymerised into the
polymer structure55.
Receptor type molecular imprinted polymers, to lesser extent catalytic
imprinted polymers, can be incorporated into a biomimetic sensor56. These
mimic the role of a biological receptor/enzyme in a biosensor57. The binding
event can be coupled to piezoelectric, optical or electrochemical transduction
methods. An interesting detection method uses the “gate effect” observed in
some molecular imprinted polymers. The binding of the template to the polymer
Chapter 2Chapter 2Chapter 2Chapter 2
36
causes the polymer structure to change which allows the passage of analytes
through the polymer56. Theophylline molecular imprinted polymers coupled to
an electrochemical cell had an increased anodic current compared to the non-
imprinted polymer when it re-bound theophylline. This suggested that binding
of the template increased the permeability of the imprinted polymers58. It was
also shown by atomic force spectroscopy that the surface of the molecular
imprinted polymers became rough in the presence of theophylline which
supported the idea that the polymer changed configuration and became more
permeable.
The recognition capacity of molecular imprinted polymers can also be
used as separation matrices. Molecular imprinted polymers have been prepared
for trapping biological and environmental analytes by solid phase extraction
(SPE). Analogue imprinting plays an important role in the success of creating a
MIP-SPE because template extraction is seldom 100% efficient. Theoretically
the molecular imprinted polymers can recognise the target analyte because it
has a similar shape and functional group orientation to the template. It was
observed by gas chromatography that template leaked from molecular imprinted
polymers created using an analogue of sameridine. Molecular imprinted
polymers have better selectivity compared to common sorbents like alkyl
bonded silica. Therefore co-extraction of undesired compounds from a complex
environmental or biological sample is minimised. Molecular imprinted
polymers can be used to separate a pair of enantiomers. This is influenced by
the shape of the imprint, the spatial distribution of the functional groups and the
number of interactions between the template and functional monomer.
Enantiomeric pairs may be resolved because they exhibit multiple points of
interaction with the functional monomer. In other cases enantiomers could be
resolved even if they only had one point of interaction. Molecular imprinted
polymers are also cheaper and easier to produce in comparison to immune
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37
sorbents. They are also more stable and have a higher load capacity. However
site heterogeneity and low mass transfer in some imprinted polymers may limit
the performance of the polymers in certain applications59.
2.5. Limitations of molecular imprinted polymers prepared by bulk
polymerisation method
Although the bulk imprinted polymers prepared by conventional
methods exhibit high selectivity, some disadvantages were also suffered, such
as the heterogeneous distribution of the binding sites, embedding of most
binding sites, and poor site accessibility for template molecule (Fig. II.2). To
resolve these problems, scientists have made efforts to prepare membrane
structured imprinted polymers. The thin layers of membranes were prepared by
Figure II.2. Schematic representation of the limitations of bulk polymerisation method
Chapter 2Chapter 2Chapter 2Chapter 2
38
phase inversion methods. The binding cavities in the membranes can effectively
improve the accessibility of template molecules and they also exhibited
excellent recognizing, separating, catalyzing, and bio-sensing properties.
2.6. Molecularly imprinted membranes
Interest in membranes has been increasing in various fields of science
and technology due to the recognition that membranes play an indispensable
role in the solving of basic problems confronted by the present world, such as
resource, energy, information, environment, artificial organs, and so forth.
Supposing permeation across the membrane to be interpreted by a solution-
diffusion mechanism, the flux and selectivity are thought to be governed by
both diffusivity and solubility. The former depends on the size of the permeant
and/or its structure and, therefore, the range of diffusivity is intrinsically
limited. On the other hand, solubility might give naught to infinity, depending
on its chemical nature and the combination of substrate and membrane
materials. Solubility thus has the potential to be changed from zero to infinity.
Regarding membrane separation techniques which is already been applied in
many industrial fields, separation is mainly attained by the difference in size of
the molecules separated or by that in ion dissociation constant. In order to
improve the separation ability of synthetic membranes, it is necessary to
introduce recognition sites, which discriminate between the target molecules
and others, and to incorporate target molecules into the membrane. The
separation membranes, such as carriers, channels, or transporters in biological
or cell membranes play an important role for the transport of specific materials.
Such a molecular recognition structure, which also recognizes a specific
molecule, is introduced into synthetic polymeric membranes, and such synthetic
polymeric membranes may show increased membrane performance. However,
it might require a great deal of effort and time to prepare such molecular
recognition compounds. Although these compounds are introduced into
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39
polymeric membranes, they do not always show the same recognition ability
when they are freely available in solution. Molecularly imprinted membrane
(MIM), first studied by Piletsky et al. offered us a new approach to selectively
recognize the molecules in complicated systems60. The main methods to prepare
molecular imprinted membranes include phase inversion in the presence of
template molecules61-64, surface imprinting65-67 and in situ polymerization by
bulk polymerization68.
2.6.A. Imprinted non cross-linked membranes formed by precipitation by
phase-inversion
Kobayashi et al. in 1995, developed a method to form membranes by
solubilizing a linear polymer with template in DMSO, casting this on a plate
and adding water to precipitate the polymer. Such membranes have not been
studied in diffusive permeation experiments, perhaps because they have too
high porosity. Kobayashi reviewed his work in 199869. Although not formally
involving ‘‘covalent assembly’’, this approach fulfils our definition of
molecular imprinting in all other respects, and has thus been included in this
review.
Theophylline was imprinted in this way in poly(acrylonitrile-co-acrylic
acid) to give a free-standing membrane of 100 µm thickness which was
employed as an adsorbent in batch-binding and filtration/solid-phase extraction
experiments in water where it adsorbed more theophylline than caffeine70-73.
Subsequently poly(acrylonitrile-co-methacrylic acid) was also used to imprint
theophylline and batch-binding studies on the membrane reported74.
Poly(acrylonitrile-co-styrene) and poly(acrylonitrile-co-vinylpyridine) were
used to imprint caffeine75. Membranes were used in batch-binding studies and
membranes cast on quartz crystals were used in sensor experiments. In each
case selective adsorption of caffeine from aqueous solution being described.
Chapter 2Chapter 2Chapter 2Chapter 2
40
A polymer membrane prepared by phase-inversion was grafted with a
thin layer of acrylic acid-co-ethylenebis(acrylamide), polymerized in water in
the presence of theophylline76. L-Glutamine was imprinted in Nylon-6 using
formic acid as the solvent and water as the precipitant and the resulting 100 mm
thick membrane shown to adsorb L-glutamine selectively from water in batch-
binding experiments77. Subsequently the free-standing membrane was
employed in aqueous filtration/solid-phase extraction, and membranes on quartz
crystals were used in an aqueous sensor experiment78.
Recently, the preparation, morphology and diffusive permeability of
molecular imprinted membranes have aroused increasing attention79,80. The
ability of molecular imprinted membranes to change their diffusive permeability
automatically by responding to the presence of template molecules is the most
interesting phenomenon. Molecular imprinted membranes may be applicable as
novel separation devices, chemical sensors, drug delivery systems with
molecular recognition and biomimetic membranes. However, it is desirable to
increase the selectivity of these membranes to make them suitable for practical
applications. In order to achieve this aim it is necessary to improve the
understanding of the basic nature and recognition mechanism of molecular
imprinted membranes by preparing with different functional monomers.
The development of synthetic membranes having molecular imprinting
properties is an important approach for future functional separation materials,
although little is known about a class of membranes made of molecular
imprinted polymers81-83 (Scheme II.3). In such cases a phase inversion process
of the co-polymer was applied to encode information of the template molecule.
Scheme II.3. Development of molecular imprinted polymer and membranes
2.6.B. Molecular imprinted membrane preparation strategies and
structures
Three main strategies can be envisioned for the preparation of MIM,
with a three-dimensional
molecular imprinted polymer structure and membrane morphology, (ii)
sequential approach - preparation of membranes from pr
“conventional” molecular imprinted membranes, i.e., particles, and (iii)
sequential approach - preparation of molecular imprinted membranes on or in
support membranes with suited morphology and the cross
In most studies p
recognition sites are distributed in the bulk polymer phase, so their accessibility
is limited, giving low membrane performance. Many studies have been carried
out with a view to overcome this problem, includi
production of molecular imprinted membranes with an ordered porous structure.
The highly ordered porosity is produced by evaporating a polymer solution in a
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers:
41
Scheme II.3. Development of molecular imprinted polymer and membranes
Molecular imprinted membrane preparation strategies and
strategies can be envisioned for the preparation of MIM,
dimensional and flat-sheet shape: (i) Simultaneous formation of
molecular imprinted polymer structure and membrane morphology, (ii)
preparation of membranes from previously synthesized
“conventional” molecular imprinted membranes, i.e., particles, and (iii)
preparation of molecular imprinted membranes on or in
support membranes with suited morphology and the cross-section.
In most studies performed on molecular imprinted membranes, the
recognition sites are distributed in the bulk polymer phase, so their accessibility
is limited, giving low membrane performance. Many studies have been carried
out with a view to overcome this problem, including, an approach for the
production of molecular imprinted membranes with an ordered porous structure.
The highly ordered porosity is produced by evaporating a polymer solution in a
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
Molecular imprinted membrane preparation strategies and
strategies can be envisioned for the preparation of MIM,
sheet shape: (i) Simultaneous formation of
molecular imprinted polymer structure and membrane morphology, (ii)
eviously synthesized
“conventional” molecular imprinted membranes, i.e., particles, and (iii) non-
preparation of molecular imprinted membranes on or in
erformed on molecular imprinted membranes, the
recognition sites are distributed in the bulk polymer phase, so their accessibility
is limited, giving low membrane performance. Many studies have been carried
ng, an approach for the
production of molecular imprinted membranes with an ordered porous structure.
The highly ordered porosity is produced by evaporating a polymer solution in a
Chapter 2Chapter 2Chapter 2Chapter 2
42
volatile solvent under controlled humidity. The development of this technique
was proposed also by Lu et al.84, to gain ordered porosity from random
poly(styrene-co-acrylonitrile) using THF as a solvent using the water-assisted
method in the presence of template. SEM analysis showed the highly ordered
and regular pore structure of the molecular imprinted membrane surface and
transported it with good efficiency. This could be attributed to the porous
structures of the molecular imprinted membranes, because the ordered porous
structures on the surface and in the cross section allow the accessibility of
recognition sites, thus the molecular imprinted membrane showed the highest
transport rate toward the template molecule.
However, the first proposal of an easy technique for obtaining efficient
MIMs with non-covalent bonds was made by Kobayashi and co-workers in
199581 using the phase inversion technique to form membranes and
subsequently used by some authors85-89. As far as membrane technology is
concerned, one of the most common polymeric membranes used for molecular
recognition is polyacrylonitrile and its co-polymers. Tasselli et al.90 published a
study on the binding capacity of a polyacrylonitrile membrane, varying the
amount and the type of the functional monomers (itaconic acid, acrylic acid,
acrylamide), using the phase inversion technique in a polar solvent.
Another way is, to synthesis a molecularly imprinted membrane
employing supercritical carbon dioxide as an antisolvent, thereby inducing the
phase separation of the polymer solution. Membrane preparation employing
supercritical carbon dioxide is similar to conventional immersion precipitation
of polymers, but achieves better results.
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2.6.C. Preparation of self-supported MIM - simultaneous formation of
molecular imprinted polymer structure and membrane shape
Self-supported flat-sheet membranes should be at least 10 µm thick in
order to have sufficient stability. Hence, for simultaneous molecular imprinted
membrane preparation, control of film thickness, e.g., by solution casting or
using moulds, is essential. Also, when established molecular imprinting
polymer synthesis protocols are to be applied, the “synchronization” of
imprinting and film solidification are of critical importance for molecular
imprinted membrane shape, structure and function.
i) Sol-gel processes towards inorganic or inorganic/organic hybrid materials
After the first demonstration of molecular imprinting by the synthesis of
silica networks through a sol-gel process, imprinting attempts with purely
inorganic materials have been very much focussed onto creating well-defined
micropores using templates, thus also preparing inorganic membranes91,92.
However, inorganic imprinted membranes with molecular recognition function
have not yet been reported.
ii) In situ crosslinking polymerization
Free-standing, but brittle membranes were prepared by thermally
initiated cross-linking co-polymerization of one of the “standard” monomer
mixtures (methacrylic acid/ethylenegycoldimethacylate) for molecular
imprinting93. Scanning electron microscopic studies revealed a regular porous
structure built up by 50 to 100 nm diameter nodules. Imprinted membranes with
a thickness between 60 and 120 µm could be prepared94 using an oligourethane-
acrylate macro monomer. Another polymer was prepared by crosslinking co-
polymerization of styrene monomers followed by leaching of a polyester
present in the reaction mixture95. Based on Scanning electron microscopy and
Chapter 2Chapter 2Chapter 2Chapter 2
44
permeation data, it was speculated that “trans-membrane channels” had been
obtained, induced by the removable macromolecular pore former.
iii) Polymer solution phase inversion (Alternative imprinting)
Phase inversion (PI), the main approach towards technical polymeric
membranes, can also be applied for molecular imprinting, in which
solidification of a polymer is taken place instead of an in situ polymerization.
Yoshikawa et al have used polystyrene resins with peptide recognition groups,
in a blend with a matrix polymer, for molecular imprinted membrane formation
via a “dry PI” process96-99. The permeability was much higher for the molecular
imprinted membrane as compared with the blank membranes. Kobayashi et al.
have used functional acrylate copolymers for a “wet PI” process yielding
asymmetric porous molecular imprinted membrane100. Recently, the polymer
selection for “wet PI imprinting” has been extended to most of the commonly
used membrane materials, like, cellulose acetate, polyamide, polyacrylonitrile
and polysulfone101,102. The formation of porous molecular imprinted membrane
from a compatible blend of a matrix polymer for adjusting a permanent pore
structure and a functional polymer for providing binding groups103. Considering
the limitations faced with the conventional in situ crosslinking polymerisation
approach towards molecular imprinted membrane materials, it is remarkable,
that most molecular imprinted membrane prepared via “alternative imprinting”
had at least acceptable binding performance in aqueous media. However, such
molecular imprinted membrane lost their “template memory” when exposed to
a too organic environment where swelling and chain rearrangement seemed to
“erase” the imprinted information97.
In conclusion, for all simultaneous preparations, the limited accessibility
of imprinted sites due to a random distribution inside and on the surface of the
bulk polymer phase remains a major unsolved problem. Thus, the advantage of
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membrane preparation technologies to provide well defined pore structures is
not yet fully exploited for obtaining self supported micro and macroporous
molecular imprinted membrane.
2.7. Composition of molecular imprinted membranes
i) Templates
The binding strength of the polymer as well as the fidelity in the
recognition depends on the number and type of interaction sites, the template
shape, and the monomer template rigidity. Templates offering multiple sites of
interactions for the functional monomer are likely to yield binding sites of
higher specificity and affinity for the template. A notable increase in affinity
and selectivity was obtained with an increase in the basicity of the templates.
The shape and size of the template may in some cases be sufficient to create
steric complementarity for efficient discrimination between two molecules.
Templates that possess conformational rigidity that can fit in the cavity
of the polymer with minimal change in conformation will increase the affinity
and selectivity in the recognition. This is due to the fact that templates that fit
perfectly into the site will involve minimal loss in entropy due to
conformational changes in the site as well as in the template after binding. In
addition to the type of template, the ratio of the template to the functional
monomer has been known to play a key role in the selectivity and sensitivity in
the imprinted polymers when the possible interactions involved inside the
matrix are taken into account. The optimum ratio has to be determined for each
individual template.
ii) Monomers
The type of functional monomer used for producing a useful imprinted
polymer is very important, as it is the component that is principally involved in
forming an effective chemical bond with the print molecule. The functional
Chapter 2Chapter 2Chapter 2Chapter 2
46
monomer must strongly interact with the template to achieve a high yield of
imprinted binding sites and allow the maximum number of complementary
interactions to be developed in the polymeric matrix. In general, analytes
containing basic functional groups are best imprinted with monomers
containing acidic functional groups and vice versa. Better recognition ability
has been achieved in some cases with polymeric combinations of two or more
functional monomers (giving ter-polymers or higher) compared with
recognition observed with the corresponding copolymers. The success of this
process depends on the kind of template and the relative strength of the template
and the complexes of functional monomer as compared with their interaction
with one another.
iii) Porogen
The choice of the porogenic solvent is critical in most molecular
imprinting procedures. Porogens govern the strength of non-covalent
interactions and influence the polymer morphology such as inner surface area
and average pore size. The solvent used in the polymer formation should be
non-polar as possible in order to maximize the strength of hydrogen and ionic
interactions between the print molecule and the monomer while allowing rapid
dissolution of the print molecule.
The recognition ability of the molecular imprinted membrane depends
on the type of solvent used in the rebinding step. In general, better recognition
ability is obtained with non-polar solvents. The morphology is also affected by
swelling when exposed to different kinds of porogens. It is generally observed
that the choice of recognition solvent should be more or less identical to the
imprinting solvent in order to avoid any swelling problems, which will affect
the recognition of the polymer.
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iv) Temperature
Molecular imprinted membrane prepared at low temperatures using a
photo-initiator exhibited higher enantiomer separation capabilities. This fact
was attributed to the stability of the monomer/imprint complexes due to the
more favourable entropy, leading to well defined imprints in the resultant
membrane.
2. 8. Polymer evaluation and characterisation
i) Discrete distribution models: Langmuir isotherm
The simplest and most frequently used approach in adsorption studies is
the Langmuir isotherm (LI) as shown in equation (1). Haupt et al. used
Langmuir isotherm to determine the affinity of a series of theophylline
imprinted polymers104. It is then assumed that once template molecules occupy
a site, no further adsorption can take place at that site. Therefore, in theory a
saturation value is reached beyond which no further sorption can take place.
This value allows the calculation of the surface binding capacity.
The Langmuir model can be expressed as
�� =����
��� (1)
where Ce is the equilibrium concentration in mmol/g and ‘qe’ is the amount of
template bound at equilibrium. ‘Qo’ is the binding capacity and ‘b’ is the
binding energy.
ii) Continuous distribution model: Freundlich isotherm
The Freundlich isotherm (FI) is the most easily applied model as it
consists of two fitting parameters ‘a’ and ‘m’ as shown in equation. The
empirical form of Freundlich isotherm105 has been widely used for modelling
Chapter 2Chapter 2Chapter 2Chapter 2
48
heterogeneous surface. Freundlich isotherm describes the relationship of the
concentration of the bound (B) and free (F) guest molecules as:
B = aFm (2)
The two fitting parameters ‘a’ and ‘m’ yield the measure of physical
binding parameters, where ‘a’ is related to the median association constant
Ko = a1/m , and the second fitting parameter ‘m’ is the heterogeneity index. The
value can vary from zero to one, with one being homogeneous and values
approaching zero being increasingly heterogeneous. To determine the suitability
of the Freundlich isotherm in accessing the binding behaviour of molecular
imprinted polymer the experimental binding isotherm is plotted in log B verses
log F format.
iii) Infrared spectroscopy (IR)
In IR spectroscopy infrared radiation is focused on the sample. When
the frequency of the IR radiation is equal to the specific vibration of the sample
molecules, the molecules absorb the radiation. The IR radiation passing through
the sample is detected, and the obtained spectrum shows the changes in infrared
radiation intensity as a function of frequency. Usually, the positions of the IR
absorption bands are presented in the spectrum as wave numbers, which are
directly proportional to frequency. The intensity of the absorption band depends
on the change in dipole moment of the molecule caused by the absorption.
Thus, functional groups containing polar bond, can easily be detected with IR
while analysis of groups containing non-polar bonds is much more difficult. IR
spectroscopy enables both qualitative and quantitative analysis, and it is
applicable for both inorganic and organic membrane samples. Infrared
spectroscopy is often utilized in the determination of the chemical composition
of membrane samples and in the localization of different compounds on the
sample surface.
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iv) Scanning electron microscopy (SEM)
In a scanning electron microscope a fine beam of electrons scans the
membrane surface. This causes several kinds of interactions generating different
signals, of which secondary electrons and back scattered electrons are used in
the image forming106. Secondary electron images can be used to get an idea
about the size, geometry and distribution of pores on the surface of the
membranes. Due to the large depth of field, the SEM images visualize the
membrane surface morphology three-dimensionally106. SEM analysis showed
the highly ordered and regular pore structure of the molecular imprinted
membrane surface and the cross-section. Permeation experimentation results
showed that the molecular imprinted membranes recognized the template
molecule effectively and transported it with good efficiency. This could be
attributed to the porous structures of the molecular imprinted membranes,
because the ordered porous structures on the surface and in the cross section
allow the accessibility of recognition sites, thus the molecular imprinted
membrane showed the highest transport rate toward the template molecule.
(v) X-ray diffraction (XRD)
In X-ray diffraction method, X-rays which are known to be the light of
extremely short wave length could be used to investigate the internal structure
of the polymer. X- ray diffraction studies making use of the Bragg’s equation
gives information about the crystallanity, chemical combination and
interpretation of patterns in a particular imprinted polymer. The Bragg’s
equation (3) is given as,
nλλλλ = 2d sinθθθθ (3)
where ‘n’ is the order of reflection, ‘λ’ is the wavelength of X-ray used in A°,
‘d’ is the inter planar spacing and ‘θ’ is the glancing angle.
Chapter 2Chapter 2Chapter 2Chapter 2
50
2.9. Application of molecular imprinted membranes
The unique feature of molecular imprinted membrane is the interplay of
selective binding and permeation of molecules, making them potentially
superior to state of the art. Synthetic separation membranes are already applied
in various industries. Receptor and transport properties of microporous
molecular imprinted membranes can based on template-specific binding sites in
trans-membrane pores serve as fixed carriers for “facilitated” transport.
Furthermore, template binding in microporous molecular imprinted membrane
can lead to a “gate-effect” which either increases or decreases membrane
permeability. Alternatively, molecular imprinted membrane can also function as
absorbers, leading to a retardation of template transport followed by
breakthrough, once the binding sites have been saturated. In the last decade, the
“proof-of-feasibility” has been shown for all types of molecular imprinted
membranes. However, significantly advanced preparation methods, preferably
towards composite membranes, and a much more detailed structural
characterization will be necessary in order to be able to rationally design
selective molecular imprinted membrane. In general, molecular imprinted
membrane could serve as model systems for cellular trans membrane transport
and natural receptors. Applications in sensors could be immediately derived
from those models. However, an ultimate aim in membrane technology, the
combination of molecular recognition and sieving in high performance
membranes for challenging separation applications will be realized with
advanced molecular imprinted membranes.
The use of molecularly imprinted membranes for the discrimination of
enantiomers is most promising in separation technology because such
separations can be performed in a continuous process, unlike conventional
crystallization or chromatographic methods. Imprinted membranes are also easy
to scale up and do not require a great deal of energy. Because of these
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advantages, imprinted membranes have been used in various industries
including water treatment, medicine purification and food processing.
The application potential for imprinted membranes will be based on the
success of their further development, driven by tackling those problems which
cannot be solved by state of the art separation membranes. Of course,
separations using improved or novel membranes must then still be compared
with other unit operations.
i) Separations based on imprinted membranes
The vision of a tailored and truly molecule selective separation for a
wide range of target molecules is the strongest motivation for the development
of imprinted membranes. Molecular imprinted membranes for chiral separations
may, are the first examples for practical applications. In comparison with
chromatography, the upscaling of a membrane separation should be much
easier. Such success could serve as a “door opener” for other imprinted
membranes. Molecular imprinted membranes with a microporous barrier
enabling facilitated trans-membrane transport using imprinted sites would
enable continuous separations. “Gate imprinted membranes” could be
developed towards environment sensitive or switchable membranes. All
technical areas with pure or purified special target molecules as products will in
the future benefit from such novel imprinted membranes separations.
Alternatively, imprinted membranes absorbers will be used mainly for isolation
and removal of small molecular fractions, especially from a large volume. Such
imprinted membranes will be applied instead of conventional absorbers or in
combination with other membrane separation steps, especially in water
treatment and in food and pharmacy industries.
Chapter 2Chapter 2Chapter 2Chapter 2
52
ii) Pharmaceutical and food applications
Trotta et al. suggested that using poly(acrylic acid-co-acrylonitrile) for
the production of membranes resulted in an asymmetric pore structure, prepared
by phase inversion technique107. The membranes containing the antibiotic
tetracycline hydrochloride template were prepared using the same method, but
adding the required amount of the template molecule (2 wt%).
Chloramphenicol, tetracycline hydrochloride analogue, was used to test the
selectivity of the imprinted membrane. The resulting membrane shows
molecular recognition properties for the highly water-soluble tetracycline
hydrochloride. About 140 µg (0.29 µmol) of tetracycline hydrochloride were
retained per gram of imprinted membrane. More generally, it is possible to
recognize several bio-molecules in solution selectively. Molecular imprinted
membranes of biotechnological interest were obtained either by the coagulation
or modification of molecular imprinted membranes introducing imprinted nano
particles. It is observed that membranes of poly(acrylonitrile-co-acrylic acid)
imprinted with uric acid, a marker for several diseases, such as gout, showed
good recognition capacity and selectivity towards the template (the detection of
uric acid was 2.4 times higher than theophylline). The selectivity of this device
was 1.96 times higher than that of albumin. In some cases, the recognition
properties of methacrylic acid-co-acrylic acid membranes were improved by
loading imprinted cross-linked methyl methacrylic acid-methacrylic acid nano
spheres. In this way, different membranes were obtained for application in the
biomedical field or for various biotechnological uses, on account of their bio-
mimetic behaviour. It is also suggested that preparing new polymeric systems
through imprinted polymer for potential application in extra corporeal blood
purification. Membranes produced using the phase inversion methods were
prepared to remove low density lipoproteins and cholesterol from plasma
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employing the model compounds phosphatidylcholine and α-amylase as target
molecules.
Donato et al. suggested extracting folic acid; a constituent of the vitamin
B group, from aqueous solutions, using a novel procedure based on the
membrane separation process employing molecular imprinted membranes
prepared using the phase inversion technique108. The molecular imprinted
membranes were made with poly(acrylonitrile-co-acrylamide) and folic acid as
the template molecule. In the field of antibiotics, Rebelo et al. published a study
on molecular imprinted membranes, in which they described the preparation of
new molecular imprinted membranes based ion selective electrodes109. The
polymeric sensor was synthesized with methacrylic acid and 2-vinylpyridine as
functional monomers, including the template molecule. The sensing material
was dispersed in a polyvinylchloride matrix and plasticized with o-nitrophenyl
octyl ether.
iii) Polymer membranes for chiral recognition of amino acids and nucleic
acids
In 1997, Yoshikawa et al. presented alternative molecularly imprinted
polymeric membranes prepared from a polystyrene resin bearing D- or L-amino
acids110. Steric effects interaction between the carboxyl group in the print
molecule and the amino group in the tetrapeptide residues are considered as
important factors and electrodialysis of the racemic amino acid solution shows
that perm selectivity directly reflects its adsorption selectivity. The membrane
containing tetrapeptide residues of L-amino acids and imprinted by an L-amino
acid derivative, recognized the L-isomer over the D-isomer.
Molecular imprinting technology is also very useful for studying
nucleotides. Yoshikawa et al used 9-ethyladenine as a print molecule and
investigated the recognition and selective transport of adenosine and guanosine
Chapter 2Chapter 2Chapter 2Chapter 2
54
mixtures111. The printed polymers were polystyrene, cellulose acetate and
polysulfone. The imprinted membranes synthesized in this way recognized/
adsorbed adenosine instead to guanosine. However, guanosine was preferably
permeated over adenosine, probably because of the relatively high affinity
between adenosine and membrane.
iv) Metal ion separation
Due to the biological and environmental impact of metal ions, the
development of new methods for selective separation, purification and
determination of these compounds are of continuing interest. A new approach
was proposed for preparing a metal ion-imprinted polymer membrane through
in situ polymerization using the Zn(II)-(2,2'-bipyridyl) complex as the template,
4-vinylpyridine as the monomer112. The imprinted membranes revealed higher
selective adsorption and permeation for the template than the control non-
imprinted membranes. Selective permeation of Zn(II) over Cu(II) was observed.
Cross-linked chitosan presented lower adsorption capability because of amino
groups. Imprinting with Ag(I) overcame this problem. Competitive removals of
Ag(I)/Cu(II) and Ag(I)/Ni(II) from mixtures were also studied. The non-
imprinted membranes are selective for Cu(II) and Ni(II). Chitosan Imprinted
membranes showed relative selectivity coefficients for Ag(I)/Cu(II) and
Ag(I)/Ni(II) 9 and 10.7 times higher than the non-imprinted membrane,
respectively. In this way, the imprinted membranes are good for selective silver
removal in a solution containing interfering ions such as Cu(II) and Ni(II).
v) Separation of herbicides, pesticides, organic pollutants
Zhu et al. prepared a novel thin layer composite molecular imprinted
membrane selective for monocrotophos pesticide by means of in situ
polymerization of methacrylic acid with EGDMA as cross-linker in Nylon-6,
introducing specific binding sites into the membrane whilst maintaining its pore
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
55
structure113. Membrane selectivity was evaluated in filtration experiments using
three other organophosphorus pesticides (mevinphos, phosphamidon and
omethoate). The composite molecular imprinted membrane had low binding
affinity for the other pesticides in comparison to the good sorption of the
template. In another way presented a study on 2,4-dichlorophenoxy acetic acid
imprinted in polypyrrole polymers onto a carbon glass electrode. By
performing cyclic voltammetry, it was possible to establish that the device made
thus can conspicuously improve the sensitivity and selectivity of 2,4-
dichlorophenoxy acetic acid analysis, as well as potentially having good
repeatability.
We can also develop a portable bio-mimetic sensor device for the
specific control of phenol content in water. The synthetic structure reproduced
the active site of the enzyme tyrosinase in molecularly imprinted polymer
membranes. Those membranes with a catalytic activity were obtained by co-
polymerizing the Cu(II)-catechol-urocanic acid ethyl ester complex with
triethyleneglycoldimethacrylate, adding the elastic component oligourethane
acrylate. This procedure led to the creation of a thin, flexible, and mechanically
stable highly cross-linked polymer membrane with catalytic activity.
Investigation of the pH influence demonstrated that pH dependence peaked at
neutral pH values. The oxidation of the catechol is inhibited at pH ≤ 5. In order
to examine the selectivity of the new sensor system, catechol analogs like
phenol, 4-nitrophenol, 1,2,3-trihydroxybenzol, 2-methoxyphenol, m-diphenol,
p-diphenol, bisphenol A, 1,2-naphthalenediol, and 1,4-naphthalenediol were
added to the electrochemical cell. Unlike conventional biosensor devices made
with mushroom tyrosinase that recognize different phenolic compounds, the
sensor system developed had high selectivity. It gave catalytic oxidation of o-
diphenols and no response was observed with their structural analogs.
Chapter 2Chapter 2Chapter 2Chapter 2
56
2.10. Molecular imprinted polymers on carbon nanotube
However, some limitations for the molecular imprinted polymers are
seen such as, a heterogeneous distribution of binding sites in the network
polymer, poor site accessibility for template molecules, and slow kinetic
binding times, are endured with application of the imprinted polymers. Also, the
main drawback of molecular imprinted polymer applications in electrochemical
techniques is a lower conductivity. Now a days, multiwalled carbon nanotubes
(MWCNT) are considered for their high electrical and thermal conductivity
properties. Because of their unique characteristics in a variety of applications,
multiwalled carbon nanotubes have successfully been used to detect proteins,
tumour markers, and some drugs. Also, MWCNTs can be an outstanding option
as a support material to overcome the previously discussed problems that are
encountered with the use of molecular imprinted polymers. Through the
formation of the molecular imprinted polymer on the surface of MWCNTs, the
accessibility of the analyte to binding sites can be improved, and the binding
time can be reduced. In an effort to improve molecular imprinted polymer
properties, they can be immobilized as nano layer recognition sites on
MWCNTs. The main aim of this work was the direct nano layer preparation and
characterization of molecularly imprinted polymers on multiwalled carbon
nanotubes (MIPCNTs) for progesterone and testosterone as templates that could
exhibit better molecular recognition properties.
Carbon nanotubes (CNTs) describe a family of nano materials made up
entirely of carbon. First carbon nanotubes, which were observed and described
by Iijima, had walls built from two to fifty graphene sheets, and so they came to
be called multiwalled carbon nanotubes (MWCNT) 114. Structurally MWCNTs
consist of multiple layers of graphite superimposed and rolled in on them to
form a tubular shape. Later, singlewalled nanotubes (SWCNT) were
discovered115. Carbon nanotubes can be stretched as sheets of graphite rolled
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57
into seamless cylinders. Depending on the number of sheets, these nanotubes
are called single- or multiwalled carbon nanotubes. Carbon nanotubes can be
metallic and semiconducting114. Moreover, MWCNTs are polymers of pure
carbon and can be reacted and manipulated using the rich chemistry of carbon.
This provides opportunity to modify the structure and to optimise solubility and
dispersion, allowing innovative applications in materials, electronics, chemical
processing and energy management. On the other hand, the chemical structure
of carbon nanotubes does not allow them to react or make complexes with many
chemical elements. Therefore, functionalization of carbon nanotubes becomes a
very important field of chemistry. Functional, i.e. enriched CNTs, with some
other physico-chemical properties, offer new possibilities in technological
applications of this nano materials116-117. The carbon nanotube unique properties
make it desirable for different applications. For most of these applications
nanotubes require functionalization, such as changing some of the graphite
properties to make nanotubes soluble in different media, or attaching different
groups or even inorganic particles for future utilization of modified nanotubes.
A particular kind of CNTs-polymer composites is represented by CNTs-
MIPs composites, in which the polymer part is a molecularly imprinted polymer
(Scheme II.4). CNTs impart electrical conductivity to molecular imprinted
polymers, while molecular imprinting on these one-dimensional nanostructures
will endow the nanotubes with molecular recognition functions, further
expanding their application fields118. The introduced MWCNTs exhibited
noticeable enhancement on the sensitivity of the molecular imprinted polymer
sensor, meanwhile, the molecularly imprinted film displayed high sensitivity
and excellent selectivity for the target molecule.
Chapter 2Chapter 2Chapter 2Chapter 2
Scheme II. 4. Schematic representation of MWCNTsprocess.
2.10.A. Applications of functionalized carbon nanotubes
One of the first applications with multiwalled carbon nano tubes was
proposed by Baughman, Zakhidov, and Heer
carbon nano tubes as reinforcement or as electrically conductive components in
polymer composite materials. Due to the nano tube unique properties and light
mass, the resulting poly
mechanical strength and electrical conductivity
nanotube mechanical strength and chemical inertness, another unique pro
such as high flexibility
comparison of CNT probes with commercial etched silicon probes. The
researchers reported that they did not observe any degradation of resolution
during intermittent-contact imaging of polycrystalline silicon’s rough and hard
surfaces. Because of the CNT needle
shaped silicon probe, the CNT could scan and show detailed morphologies, not
seen with regular probes. Samples scanned with CNT probes showed negligible
wear in comparison with silicon prob
to make nanotubes responsive not only to mechanical load
58
Schematic representation of MWCNTs-MIPs recognition process.
Applications of functionalized carbon nanotubes
One of the first applications with multiwalled carbon nano tubes was
proposed by Baughman, Zakhidov, and Heer119. It was the use of multiwalled
carbon nano tubes as reinforcement or as electrically conductive components in
polymer composite materials. Due to the nano tube unique properties and light
mass, the resulting polymer materials with nano tubes have improved
mechanical strength and electrical conductivity120. In addition to carbon
nanotube mechanical strength and chemical inertness, another unique pro
such as high flexibility is characteristic of the CNTs121. Larsen
comparison of CNT probes with commercial etched silicon probes. The
researchers reported that they did not observe any degradation of resolution
contact imaging of polycrystalline silicon’s rough and hard
Because of the CNT needle-like shape as opposed to the triangular
shaped silicon probe, the CNT could scan and show detailed morphologies, not
seen with regular probes. Samples scanned with CNT probes showed negligible
wear in comparison with silicon probes122. Functionalization is a necessary step
to make nanotubes responsive not only to mechanical load
MIPs recognition
One of the first applications with multiwalled carbon nano tubes was
. It was the use of multiwalled
carbon nano tubes as reinforcement or as electrically conductive components in
polymer composite materials. Due to the nano tube unique properties and light
mer materials with nano tubes have improved
. In addition to carbon
nanotube mechanical strength and chemical inertness, another unique property,
arsen et al. reported
comparison of CNT probes with commercial etched silicon probes. The
researchers reported that they did not observe any degradation of resolution
contact imaging of polycrystalline silicon’s rough and hard
like shape as opposed to the triangular
shaped silicon probe, the CNT could scan and show detailed morphologies, not
seen with regular probes. Samples scanned with CNT probes showed negligible
. Functionalization is a necessary step
to make nanotubes responsive not only to mechanical loads, but to
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
59
electromagnetic forces as well. Magnetic nanotubes, for example, are attractive
for use in polymer composites with aligned paramagnetic needles or as
magnetic stirrers in micro fluidic and nanofluidic devices. Functionalization of
the outer surface of carbon nanotubes enriches the carbon nanotubes with
additional properties, such us solubility and compatibility with different
materials, thus making them attractive for composites and functional
suspensions and colloids useful in different aspects of our life. Current or short-
term applications are often based on the use of MWCNTs as a superior
replacement of electrically conductive carbon blacks.
i) Peptide delivery by carbon nanotubes
Pantarotto et al. studied the application of CNT as a template for
presenting bioactive peptides to the immune system123. For this purpose, a β-cell
epitope of the foot-and mouth disease virus (FMDV) was covalently attached to
the amine groups present on CNT, using a bifunctional linker. The peptides
around the CNT adopt the appropriate secondary structure for recognition by
specific monoclonal and polyclonal antibodies. The immunogenic features of
peptide CNT conjugates were subsequently assessed in vivo124. Immunisation
of mice with FMDV peptide nanotube conjugates elicited high antibody
responses as compared with the free peptide. These antibodies were peptide-
specific since antibodies against CNT were not detected. In addition, the
antibodies displayed virus neutralising ability. The use of CNT as potential
novel vaccine delivery tools was validated by interaction with the
complement125. The complement is that part of the human immune system
composed of a series of proteins responsible for recognising, opsonising,
clearing and killing pathogens, apoptotic or necrotic cells and foreign materials.
Salvador-Morales et al. showed that pristine CNT activate the complement
following both the classical and the alternative way by selective adsorption of
Chapter 2Chapter 2Chapter 2Chapter 2
60
some of its proteins125. This might support the enhancement of antibody
response following immunisation with peptide-CNT conjugates.
ii) Cellular uptake of carbon nanotubes
An important characteristic of functionalised-MWCNT is their high
propensity to cross cell membranes126,127. CNT labelled with a fluorescent agent
were easily internalised and could be tracked into the cytoplasm or the nucleus
of fibroblasts using epifluorescence and confocal microscopy126. The
mechanism of uptake of this type of functionalised-CNT appears to be passive
and endocytosis-independent. Incubation with cells in the presence of
endocytosis inhibitors did not influence the cell penetration ability of
functionalised-CNT. Furthermore functionalised-CNT showed similar
behaviour when incubation with the cells was carried out at low temperatures.
Cellular uptake was confirmed by Dai and colleagues127 who in later studies
used oxidised CNT to covalently link fluorescein or biotin, allowing for a
biotin-avidin complex formation with fluorescent streptavidin. Again the
nanotubes were observed inside the cells. In this case, the protein-CNT
conjugates were found in endosomes, suggesting an uptake pathway via
endocytosis. Functionalised water soluble CNT were incubated with HeLa cells.
The cells were subsequently embedded into an epoxy resin that was sliced using
a diamond microtome.
Some tubes were also identified at the cell membrane during the process
of translocation. The conformation of CNT perpendicular to the plasma
membrane during uptake suggested a mechanism similar to nano needles, which
perforate and diffuse through the lipid bilayer of plasma membrane without
inducing cell death. Dynamic simulation studies have shown that amphiphilic
nanotubes can theoretically migrate through artificial lipid bilayers via a similar
mechanism128. Nano penetration was also recently suggested by Cai et al. who
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61
proposed an efficient in vitro delivery technique called nanotube spearing129.
MCF-7 breast cancer cells were grown on a substrate and incubated with
magnetic CNT. A rotating magnetic field first drove the nanotubes to spear the
cells. In a subsequent step, a static field pulled the tubes into the cells. On the
basis of SEM images, it seems that the tubes cross the cell membrane like tiny
needles. Another efficient way to observe CNT intra cellularly was developed
by Weismann et al., who used near-infrared fluorescence130. They showed that
macrophage cells could ingest significant amounts of nanotubes without
apparent toxic effects. Therefore, there is mounting evidence that
functionalised-CNT are capable of efficient cellular uptake by a mechanism that
has not yet been clearly identified. However, the nature of the functional group
at the CNT surface seems to play a determinant role in the mechanism of
interaction with cells.
iii) Nucleic acid delivery by carbon nanotubes
Ammonium-functionalised CNT were tested for their ability to form
supramolecular complexes with nucleic acids via electrostatic interactions.
Many cationic systems are being investigated for the delivery of nucleic acids to
cells131-133. Their common goal is to enhance gene transfer and expression,
because plasmid DNA alone penetrates into cells and reaches their nucleus with
considerable difficulty134. Similar to other families of non-viral vectors (i.e.
liposomes, cationic polymers, micro particles and nano particles), the
macromolecular cationic nature of the functionalised-CNT has been exploited to
condense plasmid DNA135,136. To explore the potential of CNT as gene transfer
vectors, plasmid DNA expressing β-galactosidase was adsorbed on
functionalised-CNT carrying ammonium groups. Both single- and multiwalled
cationic CNT are able to form stable complexes, characterised by electron
microscopy, surface plasmon resonance, electrophoresis and fluorescence dye
exclusion136. Following formation of the complexes, gene transfer experiments
Chapter 2Chapter 2Chapter 2Chapter 2
62
showed a clear effect of functionalised-CNT on the expression of β-
galactosidase135. Five to ten times higher levels of gene expression than that of
DNA alone were obtained. More recently, the efficiency of DNA transfer using
functionalised-CNT was increased by covalent modification of the external
walls of the tubes with polyethyleneimine137. Polyethyleneimine grafted
MWCNT complexes and delivered plasmid DNA to different cell types;
however, the measured levels of luciferase expression were similar to that of
polyethyleneimine alone. Using a similar approach, we demonstrated that
cationic carbon nanotubes are able to condense short oligodeoxy nucleotide
sequences and improve their immune stimulating activity138.
CNT were also used to deliver non-encoding RNA polymers into
cells139. SWCNT condensed RNA by non-specific binding. The hybrids showed
negligible toxicity as found by monitoring cell growth. It is evident that CNT
can form stable supramolecular assemblies with nucleic acids, thus opening the
way to diverse applications including gene therapy, genetic vaccination and
immune potentiation enhancement.
iv) Drug delivery with carbon nanotubes
The search for new and effective drug delivery systems is a fundamental
issue of continuous interest140. A drug delivery system is generally designed to
improve the pharmacological and therapeutic profile of a drug molecule141. The
ability of functionalised-CNT to penetrate into the cells offers the potential of
using functionalised-CNT as vehicles for the delivery of small drug
molecules126,127. However, the use of functionalised-CNT for the delivery of
anticancer, antibacterial or antiviral agents has not yet been fully ascertained.
The development of delivery systems able to carry one or more therapeutic
agents with recognition capacity, optical signals for imaging and/or specific
targeting is of fundamental advantage, for example in the treatment of cancer
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63
and different types of infectious diseases142. For this purpose, we have
developed a new strategy for the multiple functionalization of CNT with
different types of molecules143. A fluorescent probe for tracking the cellular
uptake of the material and an antibiotic moiety as the active molecule were
covalently linked to CNT. MWCNT were functionalised with amphotericin B
and fluorescein.
The antibiotic linked to the nanotubes was easily internalised into
mammalian cells without toxic effects in comparison with the antibiotic
incubated alone. In addition, amphotericin B bound to CNT preserved its high
antifungal activity against a broad range of pathogens, including Candida
albicans, Cryptococcus neoformans and Candida parapsilosis. In an alternative
approach by a different group, SWCNT have been functionalised with
substituted carborane cages to develop a new delivery system for an efficient
boron neutron capture therapy144. These types of water soluble CNT were aimed
at the treatment of cancer cells. Indeed, these studies showed that some specific
tissues contained carborane following intravenous administration of the CNT
conjugate and, more interestingly, that carborane was concentrated mainly at
the tumour site. Another class of carbon nano materials similar to CNT have
also been used for drug delivery145. Singlewalled carbon nano horns are nano
structured spherical aggregates of graphitic tubes. It is found that
dexamethasone could be adsorbed in large amounts onto oxidised nanohorns
and maintains its biological integrity after being liberated. This was confirmed
by activation of glucocorticoid response in mouse bone marrow cells and
induction of alkaline phosphatase in mouse osteoblasts.
2.11. Aim of current research
The goal of the present work is to develop a molecularly imprinted
polymer against small, poorly functionalised compounds. The model class of
Chapter 2Chapter 2Chapter 2Chapter 2
64
compounds chosen are hormones. In this study progesterone and testosterone
(Fig. II.3) were chosen because of their low molecular mass and their average
chemical functionality. They also possess a distinct molecular shape, as discussed
before and can contribute to the imprinting effect. Cholesterol was also chosen as
a template because it has a similar shape to both progesterone and testosterone.
However it contains a hydroxyl group which may help to form stronger
interactions with the functional monomer. The recognition mechanism appeared
to be dominated by hydrogen bond formation with the functional monomers
because structural analogues that did not have a hydroxyl group gave a poor
response. Those molecular imprinted polymers bound progesterone specifically
and showed poor cross-reactivity against testosterone, which was expected
because it has a different molecular structure and functional group.
O
O
H
H
H
ProgesteroneO
OH
H
H
H
Testosterone
Figure II.3. Templates used in the present study
The option to modify molecular imprinted polymers after
polymerisation is analogous to the post-translational modification of hormones
and increases the repertoire of techniques to create molecular imprinted
polymers that are better suited for their final application. The chosen hormone
would have to be miscible with the functional monomer, crosslinking monomer
and initiator. From the perspective of conventional molecular imprinting theory,
the alkene group in hormone may permanently incorporate the molecule into the
polymer structure. The molecular imprinted polymer showed a higher affinity
Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: Molecular Imprinted Polymers: A ReviewA ReviewA ReviewA Review
65
for the template compared to a molecular imprinted polymer prepared without
the polymerisable template. These were based on comparing the affinity
constants calculated from different adsorption isotherm models. If the binding
capacity of the molecular imprinted polymer was correlated to the number of
high affinity sites then a molecular imprinted polymer with fewer high affinity
sites would bind to less analyte and vice versa.
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