CHAPTER II MOLECULAR IMPRINTED POLYMERS: A...

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


17

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


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.


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


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


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


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




ly attractive in many fields, particularly as selective adsorbents for

phase extraction (SPE)29-32, chromatographic separation33


cheap materials with specific binding sites38. This is achieved by

polymerizing functional and crosslinking monomers in the presence of a


ractions. It is characterized by both the high affinity of

covalent rebinding.

The overall process of forming molecular imprinted

molecular imprinted polymers




ly attractive in many fields, particularly as selective adsorbents for

and chemical


This is achieved by

polymerizing functional and crosslinking monomers in the presence of a



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.


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.


25

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


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.


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.


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


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


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


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


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



high temperatures have a negative impact on the complex stability, which



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

at lower temperatures forms polymers with greater selectivity versus

t elevated temperatures. Commonly used temperature for

. However, the initiation of the polymerization reaction



t on the complex stability, which



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


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.


35

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


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


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


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


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.


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


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



out with a view to overcome this problem, including, an approach for the




Molecular imprinted membrane preparation strategies and

strategies can be envisioned for the preparation of MIM,

sheet shape: (i) Simultaneous formation of


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



ng, an approach for the




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.


43

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


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


45

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


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.


47

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


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.


49

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.


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


51

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.


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


53

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


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


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.


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


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.


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


proposed by Baughman, Zakhidov, and Heer119. It was the use of multiwalled



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



contact imaging of polycrystalline silicon’s rough and hard

Because of the CNT needle-like shape as opposed to the triangular



wear in comparison with silicon probes122. Functionalization is a necessary step

to make nanotubes responsive not only to mechanical load

MIPs recognition


. It was the use of multiwalled



mer materials with nano tubes have improved

. In addition to carbon

nanotube mechanical strength and chemical inertness, another unique property,

arsen et al. reported



contact imaging of polycrystalline silicon’s rough and hard

like shape as opposed to the triangular



. Functionalization is a necessary step

to make nanotubes responsive not only to mechanical loads, but to


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


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


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


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


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


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


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.

References

1. M. V. Polyakov, Adsorption properties and structure of silica gel, Zhur.

Fiz. Khim., 2, 799 (1931).

2. F. H. Dickey, Proceedings of the National Academy of Science of the

United States of America, 35, 227 (1949).

3. R. Curti, U. Colombo, Active sites in stereoselective adsorbents as models

of drug receptors and enzyme active sites, Chim. Ind., 23, 103 (1951).

4. R. Curti, U. Colombo, F. Clerici, Cromatografia con adsorbenti specifici,

Gazz. Chim. Ita., 82, 491 (1952).

5. R. Curti, U. Colombo, Chromatography of stereoisomers with ‘tailor

made’ compounds, J. Am. Chem. Soc., 74, 3961 (1952).

6. C. Pinel, P. Loisil, P. Gallezot, Preparation and utilization of molecularly

imprinted silicas, Adv. Mater., 9, 582 (1997).

7. M. Hunnius, A. Rufinska, W. F. Marier, Selective surface adsorption

versus imprinting in amorphous microporous silicas, Microporous

Mesoporous Mater., 29, 389, (1999).

8. V. Patrikeev, Z. Smirnova, G. Maksimova, Some biological properties of

specifically formed silica, Dokl. Akad. Nauk SSSR, 146, 707, (1962).


66

9. V. Patrikeev, A. Balandin, E. Klabunovskii, J. Mardaszew, G.

Maksimova, Selectivity of an adsorbent produced in the presence of

bacteria with respect to optical isomers, Dokl. Akad. Nauk. SSSR, 132,

850 (1960).

10. G. Wulff, A. Sarhan, The use of polymers with enzyme-analogous

structures for the resolution of racemates, Angew. Chem., Intl. Ed. Engl,

11, 341 (1972).

11. T. Takagishi, I. M. Klotz, Macromolecule-small molecule interactions:

Introduction of additional binding sites in polyethyleneimine by disulfide

cross-linkages, Biopolymers, 11, 483 (1972).

12. J. Sagiv, Organized monolayers by adsorption. III. Irreversible adsorption

and memory effects in skeletonised silane monolayers, Isr. J. Chem., 18,

346 (1979).

13. J. Sagiv, Organized monolayers by adsorption. 1. Formation and

structure of oleophobic mixed monolayers on solid surfaces, J. Am. Chem.

Soc., 102, 92 (1980).

14. S. R. Cohen, R. Naaman, J. Sagiv, Thermally induced disorder in

organized organic monolayers on solid substrates, J. Phys. Chem., 90,

3054 (1986).

15. I. Rubinstein, S. Steinberg, Y. Tor, A. Shanzer, J. Sagiv, Ionic recognition

and selective response in self assembling monolayer membranes on

electrodes, Nature, 332, 426 (1988)

16. T. Kunitake, S. W. Lee, Molecular imprinting in ultrathin titania gel films

via surface sol-gel process, Anal. Chem. Acta, 504, 1 (2004).


67

17. T. R. Ling, Y. Z. Syu, Y. C. Tasi, T. C. Chou, C. C. Liu, Size-selective

recognition of catechol amines by molecular imprinting on silica-alumina

gel, Biosens. Bioelectro., 21, 901 (2005).

18. T. Shiomi, M. Matsai. F. Mizukami, K. Sakaguchi, A method for the

molecular imprinting of hemoglobin on silica surfaces using silanes,

Biomaterials, 26, 5564 (2005).

19. F. Trotta, E. Drioli, C. Baggiani, D. Lacopo, Molecular imprinted

polymeri cmembrane for naringin recognition, J. Membr. Sci., 201, 77

(2002).

20. G. Wulff, The role of binding-site interactions in the molecular imprinting

of polymers, Trends Biotechnol., 11, 85 (1993).

21. K. J. Shea, D. Y. Sasaki, On the control of microenvironment shape of

functionalized network polymers prepared by template polymerization, J.

Am. Chem. Soc., 111, 3442 (1989).

22. A. G. Mayes, M. J. Whitcombe, Synthetic strategies for the generation of

molecularly imprinted organic polymers, Adv. Drug Deliv. Rev., 57, 1742

(2005).

23. R. Arshady, K. Mosbach, Synthesisw of substrate-selective polymers by

host-guest polymerisation, Makromol. Chem., 182, 687 (1981).

24. G. Wulff, Molecular imprinting in cross-linked materials with the aid of

molecular templates: A way towards artificial antibodies, Angew. Chem.,

Int. Ed. Engl., 34, 1812 (1995).

25. K. Mosbach, Molecular imprinting, Trends Biochem. Sci., 19, 9 (1994).

26. B. Okutucu, S. Onal, A. Telefoncu, Noncovalently galactose imprinted

polymer for the recognition of different saccharides, Talanta, 78, 1190

(2009).


68

27. C. Cacho, E. Turiel, A. Martı´n-Esteban, D. Ayala, C. Pe´rez-Conde,

Semi-covalent imprinted polymer using propazine methacrylate as

template molecule for the clean-up of triazines in soil and vegetable

samples, J. Chromatogr., A, 1114, 255 (2006).

28. M. J. Whitcombe, M. E. Rodriguez, P. Villar and E. Vulfson, A new

method for the introduction of recognition site functionality into polymers

prepared by molecular imprinting: Synthesis and characterization of

polymeric receptors for cholesterol, J. Am. Chem. Soc., 117, 7105 (1995).

29. X. Song, J. Li, J. Wang, L. Chen, Quercetin molecularly imprinted

polymers: Preparation, recognition characteristics and properties as

sorbent for solid-phase extraction, Talanta, 80, 694 (2009).

30. B. Rezaei, S. Mallakpour and N. Majidi, Solid-phase molecularly

imprinted pre-concentration and spectrophotometric determination of

isoxicam in pharmaceuticals and human serum, Talanta, 78, 418 (2009).

31. T. Jiang, L. Zhao, B. Chu, Q. Feng, W. Yan, J. Lin, Molecularly

imprinted solid-phase extraction for the selective determination of 17beta-

estradiol in fishery samples with high performance liquid

chromatography, Talanta, 78, 442 (2009).

32. C. Long, Z. Mai, Y. Yang, B. Zhu, X. Xu, L. Lu, X. Zou, Determination

of multi-residue for malachite green, gentian violet and their metabolites

in aquatic products by high-performance liquid chromatography coupled

with molecularly imprinted solid-phase extraction, J. Chromatogr., A,

1216, 2275 (2009).

33. E. Benito-Pen, S. Martins, G. Orellana, M. C. Moreno-Bondi, Water-

compatible molecularly imprinted polymer for the selective recognition of

fluoroquinolone antibiotics in biological samples, Anal. Bioanal. Chem.,

393, 235 (2009).


69

34. T. Alizadeh, M. Zare, M. R. Ganjali, P. Norouzi and B. Tavana, A new

molecularly imprinted polymer (MIP)-based electrochemical sensor for

monitoring 2,4,6-trinitrotoluene (TNT) in natural waters and soil samples,

Biosens. Bioelectron., 25, 1166 (2010) .

35. R. N. Liang, D. A. Song, R. M. Zhang, W. Qin, Potentiometric sensing of

neutral species based on a uniform-sized molecularly imprinted polymer

as a receptor, Angew. Chem., Int. Ed., 49, 2556 (2010).

36. C. Fang, C. Yi, Y. Wang, Y. Cao, X. Liu, Electrochemical sensor based

on molecular imprinting by photo-sensitive polymers, Biosens.

Bioelectron., 24, 3164 (2009).

37. D. Lakshmi, A. Bossi, M. J. Whitcombe, I. Chianella, S. A. Fowler, S.

Subrahmanyam, E. V. Piletska, S. A. Piletsky, Electrochemical sensor for

catechol and dopamine based on a catalytic molecularly imprinted

polymer-conducting polymer hybrid recognition element, Anal. Chem.,

81, 3576 (2009).

38. X. Hu, G. Li, M. Li, J. Huang, Y. Li, Y. Gao, Y. Zhang, Ultrasensitive

specific stimulant assay based on molecularly imprinted photonic

hydrogels, Adv. Funct. Mater., 18, 575 (2008).

39. V. Pichon V, K. Haupt, Affinity separation on molecular imprinted

polymers with special emphasis on solid-phase extraction, J. Liq.

Chromatogr. Relat. Technol., 29, 989 (2006).

40. E. Caro, R. M. Marce´, F. Borrull, P. A. G. Cormack, D. C. Sherrington,

Application of molecularly imprinted polymers to solid-phase extraction

of compounds from environmental and biological samples, Trends. Anal.

Chem., 25, 143 (2006).


70

41. F. G. Tamayo, E. Turiel, A. Martin-Esteban, Molecularly imprinted

polymers for solid-phase extraction and solid-phase microextraction:

Recent developments and future trends, J. Chromatogr. A, 1152, 32

(2007).

42. C. Baggiani, L. Anfossi, C. Giovannoli, Molecular imprinted polymers as

synthetic receptors for the analysis of myco and phyco-toxins, Analyst,

133, 719 (2008).

43. T. Ikegami, T. Mukawa, H. Narial, T. Takeuchi, Bisphenol A-recognition

polymers prepared by covalent molecular imprinting, Analyt. Chim. Acta,

504, 131 (2004).

44. A. Sarbu, S. O. Dima, T. Dobre, G. Florea, E. Bacalum, A. L. Radu, M.

Beda, N. Antohe, Imprinted polymer pearls for molecular recognition of

diosgenin, Scientific Study & Research, IX (3), 391 (2008).

45. M. Ramamoorthy, M. Ulbrich, Molecular imprinting of cellulose acetate-

sulfonated polysulfone blend membranes for Rhodamine B by phase

inversion technique, J. Membr. Sci., 217, 207 (2003).

46. F. Trotta, C. Baggiani, M. P. Luda, A molecular imprinted membrane for

molecular discrimination of tetracycline hydrochloride, J. Membr. Sci.,

254, 13 (2005).

47. C. Baggiani, L. Anfossi, C. Giovannoli, Solid phase extraction of food

contaminants using molecular imprinted polymers, Anal. Chim. Acta,

591, 29 (2007).

48. W. Cummins, P. Duggan, P. Mcloughlin, A comparative study of the

potential of acrylic acid and sol-gel polymers for molecular imprinting,

Anal. Chim. Acta, 542, 52 (2005).


71

49. E. V. Piletska, A. R. Guerreiro, M. J. Whitcombe, S. A. Piletsky, Influence

of the polymerization conditions on the performance of molecularly

imprinted polymers, Macromolecules, 42, 4921 (2009).

50. B. Sellergren, The non-covalent approach to molecular imprinting, in

techniques and instrumentation in analytical chemistry. Molecularly

imprinted polymers. Man-made mimics of antibodies and their

applications in analytical chemistry, B. Sellergren, Editor, Elsevier

Science B.V.: Amsterdam. 113 (2001).

51. A. R. Koohpaei, S. J. Shahtaheri, M. R. Ganjali, A. Rahimi Forushani, F.

Golbabaei, Application of multivariate analysis to the screening of

molecularly imprinted polymers (MIPs) for ametryn, Talanta, 75, 978

(2008).

52. B. Sellergren, R. N. Karmalkar, K. J. Shea, Enantioselective ester

hydrolysis catalyzed by imprinted polymers, J. Org. Chem., 65, 4009

(2000).

53. S. A. Piletsky, E. V. Piletska, A. Bossi, K. Karim, P. Lowe, A. P. F.

Turner, Substitution of antibodies and receptors with molecularly

imprinted polymers in enzyme-linked and fluorescent assays, Biosens.

Bioelectron., 16, 701 (2001).

54. H. Yavuz, R. Say, A. Denizli, Iron removal human plasma based on

molecular recognition using imprinted beads, Mater. Sci. Eng. C., 25, 521

(2005).

55. A. Ersoz, A. Denizli, A. Ozcan, R. Say, Molecular imprinted ligand -

exchage recognition assay of glucose by quartz crystal microbalance,

Biosen. Bioelectron., 20, 2197 (2005).


72

56. S. Piletsky, E. V. Piletska, T. A. Sergeyeva, I. Nicholls, D. Weston, A. P.

F. Turner, Synthesis of biologically active molecules by imprinting

polymerization, Biopolym. Cell, 22, 63 (2006).

57. Haupt, K. and Mosbach, K. Molecular imprinted polymers and their use

in biomimetic sensors, Chemical Reviews. 100(7), 2495 (2000).

58. Y. O. R. Yoshimi, C. Iiyama, K. Sakaib, Gate effect of thin layer of

molecularly-imprinted poly(methacrylic acid-co-ethyleneglycol

dimethacrylate), Sens. Actuators. B, 73, 49 (2001).

59. S. Rodriguez -Mozaz, M. Lopez de Alda, D. Barceló, Advantages and

limitations of on-line solid phase extraction coupled to liquid

chromatography-mass spectrometry technologies verses biosensors for

monitoring of emerging contaminants in water, J. Chromatogra. A, 1152,

97 (2007).

60. S. A. Piletsky, I. Y. Dubey, D. M. Fedoryak, V. P. Kukhar, Substrate

selective polymeric membranes: Selective transfer of nuclic acid

components, Biopolym. Kletka, 6, 55 (1990).

61. F. Trotta, E. Drioli, C. Baggiani, D. Lacopo, Molecular imprinted

polymeric membrane for naringin recognition, J. Membr. Sci. 201, 77

(2002).

62. T. Kobayashi, T. Fukaya, M. Abe, N. Fujii, Phase inversion molecular

imprinting by using template copolymers for high substrate recognition,

Langmuir, 18, 2866 (2002).

63. P. S. Reddy, T. Kobayashi, M. Abe, N. Fujii, Molecular imprinted Nylon-

6 as a recognition material of amino acid, Eur. Polym. J., 38, 521 (2002).


73

64. P. Wang, W. Hu, W. Su, Molecularly imprinted poly(methacrylamide-co-

methacrylic acid) composite membranes for recognition of curcumin,

Anal. Chim. Acta, 615, 54 (2008).

65. N. Hilal, V. Kochkodan, Surface modified microfiltration membrane with

molecularly recognising properties, J. Membr. Sci., 213, 97 (2003).

66. S. A. Piletsky, H. Matuschewski, U. Schedler, A. Wilpert, E. V. Piletska,

T. A. Thiele, M. Ulbricht, Surface functionalization of porous

polypropylene membranes with molecularly imprinted polymers by photo-

graft copolymerization in water, Macromolecules, 33, 3092 (2000).

67. T. A. Sergeyeva, H. Matuschewski, S. A. Piletsky, J. bendig, U. Schedler,

M. Ulbricht, Molecularly imprinted polymer membranes for surface

selective solid phase extraction from water by surface photo-grafting

polymerisation, J. Chromatogr. A, 907, 89 (2001).

68. J. Mathew-Krotz, K. J. Shea, Imprinted polymer membranes for the

selective transport of targeted neutral molecules, J. Am. Chem. Soc., 118,

8154 (1996).

69. T. Kobayashi, H. Y. Wang, T. Fukaya, N. Fujii, In Molecular and Ionic

Recognition with Imprinted Polymers, ACS Symposium Series, Vol. 703,

Bartsch RA, Maeda M (eds). The American Chemical Society:

Washington, DC; 188 (1998).

70. T. Kobayashi, H. Y. Wang, N. Fujii, Molecular imprinting of theophylline

in acrylonitrile-acrylic acid copolymer membrane, Chem. Lett. 927

(1995).

71. H. Y. Wang, T. Kobayashi, N. Fujii, Molecular imprint membranes

prepared by the phase inversion precipitation technique, Langmuir, 12,

4850 (1996).


74

72. H. Y. Wang, T. Kobayashi, T. Fukaya, N. Fujii,. Molecular imprint

membranes prepared by the phase inversion precipitation technique-2:

influence of coagulation temperature in the phase inversion process on the

encoding in polymeric membranes, Langmuir, 13, 5396 (1997).

73. T. Kobayashi, H. Y. Wang, N. Fujii, Molecular imprint membranes of

polyacrylonitrile copolymers with different acrylic acid segments, Anal.

Chim. Acta, 365, 81 (1998).

74. F. Trotta, M. Biasizzo, F. Caldera, Molecularly imprinted membranes,

Membranes, 2, 440 (2012).

75. T. Kobayashi, Y. Murawaki, P. S. Reddy, M. Abe, N. Fujii, Molecular

imprinting of caffeine and its recognition assay by quartz-crystal

microbalance, Anal. Chim. Acta, 435, 141 (2001).

76. H. Y. Wang, T. Kobayashi, N. Fujii, Surface molecular imprinting on

photosensitive dithiocarbamoyl polyacrylonitrile membranes using

photograft polymerization, J. Chem. Technol. Biotechnol., 70, 355 (1997).

77. P. S. Reddy, T. Kobayashi, N. Fujii. Molecular imprinting in hydrogen

bonding networks of polyamide nylon for recognition of amino acids,

Chem. Lett. 28, 293 (1999)

78. P. S. Reddy, T. Kobayashi, M. Abe, N. Fujii, Molecular imprinted Nylon-

6 as a recognition material of amino acids, Eur. Polym. J., 38, 521 (2002).

79. M. Ulbrich, Membrane separations using molecularly imprinted polymers,

J. Chromatogr. B, 804, 113 (2004)

80. A. Conesa, C. Palet, Molecularly imprinted membranes (MIM) for the

enantioseparation of selenoaminoacid compounds, Desalination, 200, 110

(2006)


75

81. T. Kobayashi, H. Y. Wang, N. Fujii, Molecular imprinting of theophiline

in acrylonitrile-acylic acid copolymer membrane, Chem. Lett., 927

(1995).

82. J. M. Krotz, K. J. Shea, Imprinted polymer membranes for the selective

transport of targeted neutral molecules, J. Am. Chem. Soc., 118, 8154

(1996).

83. Y. Yoshikawa, J. Izumi, T. Kitao, S. Koya, Molecular imprinted

polymeric membranes for optical resolution, J. Membr. Sci., 108, 171

(1995).

84. Y. Lu, B. Zhao, Y. Ren, G. Xiao, X. Wang, C. Li, Water-assisted

formation of novel molecularly imprinted polymer membranes with

ordered porous structure, Polymer, 48, 6205 (2007).

85. T. Kobayashi, P. S. Reddy, M. Ohta, M Abe, N. Fujii, Molecularly

imprinted polysulfone membranes having acceptor sites for donor

dibenzofuran as novel membrane adsorbents: Charge transfer interaction

as recognition origin, Chem. Mater., 14, 2499 (2002).

86. M. Ulbricht, R. Malaisamy, Insights into the mechanism of molecular

imprinting by immersion precipitation phase inversion of polymer

blends via a detailed morphology analysis of porous membranes, J. Mater.

Chem., 15, 1487 (2005).

87. J. Liu, Q. Deng,, K. Yang, L. Zhang, Z. Liang, Y. Zhang, Macroporous

molecularly imprinted monolithic polymer columns for protein

recognition by liquid chromatography, J. Sep. Sci., 33, 2757 (2010)

88. R. Malaisamy, M. Ulbricht, Evaluation of molecularly imprinted polymer

blend filtration membranes under solid phase extraction conditions, Sep.

Purif. Technol., 39, 211 (2004).


76

89. H. Y. Wang, S. L. Xia, H. Sun, Y. K. Liu, S. K. Cao, T. Kobayashi,

Molecularly imprinted copolymer membranes functionalized by phase

inversion imprinting for uracil recognition and permselective binding, J.

Chromatogr. B, 804, 127 (2004).

90. F. Tasselli, L. Donato, E. Drioli, Evaluation of molecularly imprinted

membranes based on different acrylic copolymers, J. Membrane Sci., 320,

167 (2008).

91. N. K. Raman, M. T. Anderson, C. J. Brinker, Template based approaches

to the preparation of amorphous, nanoporous silicas, Chem. Mater., 8,

1682 (1996).

92. S. Mann, S. L. Burkett, S. A. Davis, Sol-gel synthesis of organized matter,

Chem. Mater., 9, 2300 (1997).

93. J. Mathew-Krotz, K. J. Shea, Imprinted polymer membranes for the

selective transport of targeted neutral molecules, J Am. Chem. Soc., 118,

8154 (1996).

94. T. A. Sergeyeva, S. A. Piletsky, A. A. Brovko, Conductimetric sensor for

atrazine detection based on molecularly imprinted polymer membranes,

Analyst, 124, 331 (1999).

95. A. Kimaro, L. A. Kelly, G. M. Murray. Molecularly imprinted ionically

permeable membrane for uranyl ion, Chem. Commun., 1282 (2001).

96. M. Yoshikawa, J. Izumi, T. Kitao, Molecular imprinted polymeric

membranes for optical resolution, J. Membr. Sci., 108, 171 (1995).

97. M. Yoshikawa, J. Izumi, T. Kitao. Alternate molecular imprinting: A

facile way to introduce chiral recognition sites, React. Funct. Polym., 42,

93 (1999).


77

98. M. Yoshikawa, T. Ooi, J. Izumi. Alternative molecularly imprinted

membranes from a derivative of natural polymer, cellulose acetate, J.

Appl. Polym. Sci., 72, 493 (1999).

99. M. Yoshikawa, J. Izumi, M. D. Guiver, Recognition and selective

transport of nuclic acid components through molecular imprinted

polymeric membranes, Macromol. Mater. Eng., 286, 52 (2001).

100. Y. Kondo, M. Yoshikawa1, H. Okushita, Molecularly imprinted

polyamide membranes for chiral recognition, Polymer Bulletin, 44, 517

(2000).

101. M. Materska, Quercertin and its derivatives: chemical structure and

bioactivity: A review, Pol. J. Food Nutr. Sci., 58, 407 (2008).

102. P. S. Reddy, T. Kobayashi, N. Fujii. Recognition characteristics of

dibenzofuran by molecularly imprinted polymers made from common

polymers, Eur. Polym. J., 38, 779 (2002).

103. S. Daniel, J. M. Gladis, T. Prasada Rao, Synthesis of imprinted polymer

material with palladium ion nanopores and its analytical application,

Anal. Chim. Acta, 488, 173 (2003).

104. E. Yilmaz, K. Mosbach, K. Haupt, Influence of functional and cross-

linking monomers and the amount of template on the performance of

molecularly imprinted polymers in binding assays, Anal. Commun., 36,

167 (1999).

105. H. Freundlich, Colloid and Capillary Chemistry, Metheun London, 1926

(1926).


78

106. J. Goldstein, D. Newbury, D. Joy, C. Lyman, P. Echlin, E. Lifshin, L.

Sawyer, J. Michael, Scanning Electron Microscopy and X-ray

Microscopy, Kluwer Academic/Plenum, New York, Chapters 1, 9, and 10,

pp. 1-20, 391-449, 453-535 (2003).

107. F. Trotta, C. Baggiani, M. P. Luda, E. Drioli, T. Massari, A molecular

imprinted membrane for molecular discrimination of tetracycline

hydrochloride, J. Membr. Sci., 254, 13 (2005).

108. L. Donato, F. Tasselli, E. Drioli, Molecularly imprinted membranes with

affinity properties for folic acid, Sep. Sci. Technol., 45, 2273 (2010).

109. T. S. C. R. Rebelo, S. A. A. Almeida, J. R. L. Guerreiro, M. C. B. S. M.

Montenegro, M. G. F. Sales, Trimethoprim-selective electrodes with

molecularly imprinted polymers acting as ionophores and potentiometric

transduction on graphite solid-contact, Microchem. J., 98, 21 (2011).

110. M. Yoshikawa, J. Izumi, T. Kitao, S. Sakamoto, Alternative molecularly

imprinted polymeric membranes from a tetrapeptide residue consisting of

D- or L-amino acids, Macromol. Rapid Commun., 18, 761 (1997).

111. M. Yoshikawa, J. Izumi, M. D. Guiver, G. P. Robertson, Recognition and

selective transport of nucleic acid components through molecularly

imprinted polymeric membranes, Macromol. Mater. Eng., 286, 52 (2001).

112. Y. Zhai, Y. Liu, X. Chang, X. Ruan, J. Liu, Metal ion-small molecule

complex imprinted polymer membranes: Preparation and separation

characteristics, React. Funct. Polym., 68, 284 (2008).

113. X. Zhu, Q. Su, J. Cai, J. Yang, Y. Gao, Molecularly imprinted polymer

membranes for substance-selective solid-phase extraction from aqueous

solutions, J. Appl. Polym. Sci., 101, 4468 (2006).


79

114. S. Iijima, Helical microtubules of graphitic carbon, Nature, 354, 56

(1991).

115. S. Iijima, T. Ichihashi, Single-shell carbon nanotubes of 1-nm diameter,

Nature, 363, 603 (1993).

116. G. Pagona, N. Tagmatarchis, Carbon nano tube: Material for medicinal

chemistry and biotechnological applications, Curr. Med. Chem., 13, 1789

(2006).

117. K. Balasubramanian, M. Burghard, Chemically functionalized carbon

nanotubes, Small, 1, 180 (2005).

118. G. Guan, B. Liu, Z. Wang, Z. Zhang, Imprinting of molecular recognition

sites on nanostructures and its applications in chemosensors, Sensors, 8,

8291 (2008).

119. R. H. Baughman, A. A. Zakhidov, W. A. de Heer, Carbon nanotubes –

the route toward applications, Science, 297, 787 (2002).

120. H. Z. Geng, R. Rosen, B. Zheng, H. Shimoda, L. Fleming, J. Liu, O.

Zhou, Fabrication and properties of composites of poly(ethylene oxide)

and functionalized carbon nanotubes, Adv. Mater., 14, 1387 (2002).

121. C. P. Collier, Carbon nanotubes. Properties and applications, edited by

O'Connell, M. J. (Taylor & Francis, Boca Raton, London, New York

(2006).

122. J. Zou, X. Z. Liao, D. J. H. Cockayne, Z. M. Jiang Alternative mechanism

for misfit dislocation generation during high-temperature Ge(Si)/Si(001)

island growth, Appl. Phy. Lett., 81 (2002).

123. D. Pantarotto, C. D. Partidos, R. Graff, J. Hoebeke, J. P. Briand, M. Prato,

A. Bianco, Synthesis, Structural characterization and immunological


80

properties of carbon nanotubes functionalized with peptides, J. Am.

Chem. Soc., 125, 6160 (2003).

124. D. Pantarotto, C. D. Partidos, J. Hoebeke, F. Brown, E. Kramer, J. P.

Briand, S. Muller, M. Prato, A. Bianco: Immunization with peptide-

functionalized carbon nanotubes enhances virus specific neutralizing

antibody responses, Chem. Biol., 10, 961 (2003).

125. B. B. Prasad, R. Madhuri, M. P. Tiwari, P. S. Sharma, Imprinting

molecular recognition sites on multiwalled carbon nanotubes surface for

electrochemical detection of insulin in real samples, Electrochim. Acta,

55, 9146 (2010).

126. M. Zhanga, J. Huanga, P. Yuc, X. Chen, Preparation and characteristics

of protein molecularly imprinted membranes on the surface of multiwalled

carbon nanotubes, Talanta, 81, 162 (2010).

127. S. Kamnw, T. C. Jessop, P. A. Wender, H. Dai, Nanotube molecular

transporters: Internalization of carbon nanotube-protein conjugates into

mammalian cells, J. Am. Chem. Soc., 126, 6850 (2004).

128. C. F. Lopez, S. O. Nielsen, P. B. Moore, M. L. Klein, Understanding

nature’s design for a nanosyringe, Proc. Natl. Acad. Sci., USA, 101, 4431

(2004).

129. D. Cai, J. M. Mataraza, Z. H. Qin, Z. Huang, J. Huang, T. C. Chiles, D.

Carnahan, K. Kempa, Z. Ren, Highly efficient molecular delivery into

mammalian cells using carbon nanotube spearing, Nat. Methods, 2, 449

(2005).

130. P. Cherukuri, S. M. Bachilo, S. H. Litovsky, R. B. Weisman, Near-

infrared fluorescence microscopy of single-walled carbon nanotubes in

phagocitic cells, J. Am. Chem. Soc., 126, 15638 (2004).


81

131. D. Luo, A new solution for improving gene delivery, Trends Biotechnol.,

22, 101 (2004).

132. G. D. Schmidt-Wolf, I. G. S. Wolf, Non-viral and hybrid vectors in

human gene therapy: an update, Trends Mol. Med., 9, 67 (2003).

133. A. Chaudhuri (Ed.): Special issue: Cationic transfection lipids, Curr. Med.

Chem., 10, 1185 (2003).

134. D. A. Dean, D. D. Strong, W. E. Zimmer, Nuclear entry of nonviral

vectors, Gene. Ther., 12, 881 (2005).

135. D. Pantarotto, R. Singh, D. McCarthy, M. Erhardt, J. P. Briand, M. Prato,

K. Kostarelos, A. Bianco, Functionalised carbon nanotubes for plasmid

DNA gene delivery, Angew Chem. Int. Ed. Engl., 43, 5242 (2004).

136. R. Singh, D. Pantarotto, D. McCarthy, O. Chaloin, J. Hoebeke, C. D.

Partidos, J. P. Briand, M. Prato, A. Bianco, K. Kostarelos, Binding and

condensation of plasmid DNA onto functionalized carbon nanotubes:

towards the construction of nanotube-based gene delivery vectors, J. Am.

Chem. Soc., 127, 4388 (2005).

137. Y. Liu, D. C. Wu, W. D. Zhang, X. Jiang, C. B. He, T. S. Chung, S. H.

Goh, K. W. Leong, Polyethylenimine-grafted multiwalled carbon

nanotubes for secure non-covalent immobilization and efficient delivery of

DNA, Angew Chem. Int. Ed. Engl., 44, 4782 (2005).

138. A. Bianco, J. Hoebeke, S. Godefroy, O. Chaloin, D. Pantarotto, J. P.

Briand, S. Muller, M. Prato, C. D. Partidos, Cationic carbon nano tubes

bind to CpG oligodeoxynucleotides and enhance their immunostimulatory

properties, J. Am. Chem. Soc., 127, 58 (2005).


82

139. Q. Lu, J. M. Moore, G. Huang, A. S. Mount, A. M. Rao, L. L. Larcom, P.

C. Ke, RNA polymer translocation with single-walled carbon nanotubes,

Nano Lett., 4, 2473 (2004).

140. T. M. Allen, P. R. Cullis, Drug delivery systems: Entering the main

stream, Science, 303, 1818 (2004).

141. K. Kostarelos, Rational design and engineering of delivery systems for

therapeutics: iomedical exercises in colloid and surface science, Adv.

Colloid Interface Sci., 106, 147 (2003).

142. M. Ferrari, Cancer nanotechnology: opportunities and challenges, Nat.

Rev. Cancer, 5, 161 (2005).

143. W. Zhang, S. R. P. Silva, Application of carbon nanotube in polymer

electrolyte based fuel, Rev. Adv. Mater. Sci., 29, 1 (2011).

144. Z. Yinghuai, A. T. Peng, K. Carpenter, J. A. Maguire, N. S. Hosmane, M.

Takagaki, Substituted carborane-appended water-soluble single-wall

carbon nanotubes: new approach to boron neutron capture therapy drug

delivery, J. Am. Chem. Soc., 127, 9875 (2005).

145. L. Xu, Z. Xu, Molecularly imprinted polymer based on multiwalled

carbon nanotubes for ribavirin recognition, J. Polym. Res., 19, 9942

(2012).

CHAPTER II MOLECULAR IMPRINTED POLYMERS: A...

Documents

Transcript of CHAPTER II MOLECULAR IMPRINTED POLYMERS: A...