The natural complementary interactions of molecular species have intrigued scientists for generations. The aim to mimic these interactions has evolved with the use of molecular imprinted polymers. Molecular imprinting is a technology that is used widely to obtain robust, stable and cheap materials with specific binding sites. The technique introduces desired properties into synthetic polymers using appropriate templates. This is achieved by forming a complex between a target or template molecule and functional monomers in the presence of a suitable solvent leading to polymerisation. By removing the target molecule, complementary binding sites are obtained with very high specificity for the functional groups of the target molecule. These complementary cavities are capable of selectively recognising and binding the imprint species in solution by intermolecular forces.(Bui, Merlier et al. 2010) Molecularly imprinted polymers (MIPs) are used in several applications such as chromatography, solid phase extraction, catalysis, immunoassays and sensors.(Alexander, Andersson et al. 2006)
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Figure 1. Formation of MIPs complementary cavities by removal of template.(Biotage )
Biomimetic receptors such as MIPs show significant potential for a recognition element in a biosensor. Expensive biological receptors such as antibodies can be replaced using these inexpensive synthetic MIPs. A general biosensor contains biological receptors such as antibodies or enzymes that are immobilized to the surface of the transducer element. Once binding of the analyte to the biological receptor occurs the transducer will generate a signal that can be read. Antibodies and enzymes have proven to be excellent molecules for this use in biosensors, with hundreds of commercial biosensors incorporating this technology into the device. However limited stability, difficult storage and expense of these biological receptors have shown to be problematic. Researchers now depend on cheap synthetic replacements such as MIPs.(Hillberg, Brain et al. 2005)
The objective of this project is to use molecular imprinted polymers as a recognition technology for determining if Adrosterone, Etiocholanolone, testosterone, 5α-androstane-3α 17βdiol or 5β-androstane-3α 17βdiol present in a urine sample. MIPs have already been synthesized for testosterone and epitestosterone; however, from studying the chemical structure and orientation of these five molecules previously, the molecules have the same basic structure where only R groups and stereochemistry differs. Therefore the proposed idea is that theoretically using this core base structure as a target for molecular imprinting, a polymer can synthesized that will detect all five molecules as all will bind due to the similarity of chemical structures.
History of Molecular Imprinted Polymers
Interest in MIPs has grown dramatically in the past several decades and they have been exploited for numerous applications. Although the interest in this technique is relatively new, the concept behind the technique is date back to 1972 when Wulff and Klotz first reported the imprinting technology using target molecules during the polymerisation process which in turn were recognised by the synthesised imprinted polymers. However the original idea was first described by Polyakov in 1931. Polyakov performed his experiment that involved polymerising sodium silicate in water and after a two week period a target was added to the polymer. After the silica was allowed dry for 20-30 days the additive was removed by extensive washing steps. Upon completing this, Polyakov saw that a memory for the target was apparent in the polymer.(Alexander, Andersson et al. 2006)
The most common way to produce polymers in today's world is free radial polymerisation due to a tolerance for a large variety of functional groups and template structures. This method is exploited hugely on a commercial scale to produce plastics. The reaction can be performed under mild conditions and produce high yield with cheap monomers. Free radical polymerisation involves three steps, initiation, chain growth or propagation and termination. Initiation involves a free radical being created by the initiator molecule which in turn is transferred to the monomer leading to chain growth. During the chain growth step the radical initiator continues to attack the monomer until the monomer is exhausted and termination occurs.(Cormack, Elorza 2004, Parisi 2012)
Figure 2. Free radical polymerisation steps.
Two forms of MIPs have been produce, covalent imprinted polymers and non-covalent imprinted polymers. These two synthetic polymers differ only by the bonds formed between the target molecule and functional monomer. Covalent imprinted was first described by Wulff in 1972, this method involved covalently binding the target molecule to the functional monomer before polymerization took place. To remove the template from the matrix post polymerisation the covalent bonds between the template and polymer must be cleaved. This is achieved by Soxhlet extraction or treated with appropriate reagents.(Parisi 2012) After this method was described by Wulff, Mosback et al described the non-covalent imprinting method by which the target molecule and functional monomers form a complex by self assembly. Non-covalent imprinting remains the most common method used in MIP fabrication.(van Nostrum 2005) The synthesis is achieved by simply mixing the target molecule with an appropriate functional monomer and solvent. The target can subsequently be removed from the polymer after synthesis by washing steps. The recombination of the template by the MIPs exploits the non-covalent interactions achieved via this method.(Parisi 2012)
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The affinity of the binding sites for the target molecule created during polymerisation fluctuates depending on the type of polymerisation performed. Non-covalently bonded MIPs have generally weaker binding affinity than those prepared with by the covalent method as the hydrogen bonding, hydrophobic, electrostatic and π-π interactions between the target molecule and functional monomers are used significantly for binding.(Parisi 2012)
Molecular Imprinting Polymerisation Process
The target molecule is of most importance when preparing MIPs. However not all target molecules are compatible for templating. The desired properties of a target molecule include, chemically inert during polymerisation process, if template prove to react in the polymerisation reaction or is unstable under the reaction conditions alternative imprinting processes must be pursued. The main property a target should have is non polymerisable groups or a group that has potential to inhibit the free radical polymerisation process. Only when the target is studied for these desired properties is can be considered for templating.(Cormack, Elorza 2004)
Functional monomers play an important role for binding interaction in the imprinted binding sites. Evidently the functionality of the target and the functionality of the monomer must be complementary to maximise the imprinting effect. The cross-linkers also play a significant role. The cross-linker is the controlling agent for the morphology of the polymer matrix as well as stabilising the imprinted binding sites and the overall mechanical stability of the polymer. Generally cross-linkers are used in excess to produce porous networks and to generate materials with mechanical stability. A number of cross-linkers suitable for molecular imprinting are commercially available included cross-linkers that simultaneously act as a functional monomer for simplistic preparation.(Cormack, Elorza 2004)
Initiator molecules must also be carefully chosen to be adequate for free radical polymerisation with target molecule and functional monomer. Initiators with low polymerisation temperatures are preferred to obtain the stability of the target molecule. Once these four components of the reaction have been studied and chosen they are brought into a solution with a suitable solvent. The solvent also has the important function to create macroporous polymers. The level of the solvent can be exploited to control the morphology of the total pore volume in the resulting polymer. Thus using thermodynamically good solvents result in polymers consisting of well developed porous structures and highly specific surface areas. Pore volume is directionally proportional to solvent volume.(Cormack, Elorza 2004) In order to obtain high performing MIPs the temperature of polymerisation is another factor to be considered. High temperature results in the formation of unwanted species due to equilibrium driven away from the template-functional monomer complex. Because of these various strategies have been proposed to obtain stable pre-polymerisation complex by decreasing the overall kinetic energy of the system. An example of the is UV induced polymerisation (Parisi 2012)
Biomimetic receptors such as MIPs previously discussed show significant potential for a recognition element in a biosensor. For detection of binding a number of transducer elements have been described for use with MIPs, Electrochemical sensors, Optical detection, Acoustic sensors (Piezoeletric detection) and other techniques.(Fuchs, Soppera et al. 2012) For our device the detection method must be me minimisable. This can be easily done with electrochemical sensors.
For the past decade remarkable progress has been made for the integration of MIPs and electrochemical sensors. With the use of conductometirc measurements and MIP nanomaterials electrochemical sensors with excellent selectivity and simplicity have been obtained. For electrochemical sensing polyprrole (PPy) is the most broadly used conducting polymer. Molecular imprinting technology and electrochemically synthesized polymers were firstly introduced by Hutchins and Bachas.(Hutchins 1995) A nitrate potentiometric sensor was developed by electrosynthesising PPy in NaNO3 solution. The nitrate was not removed from the solution post synthesis. High selectivity was seen when other anions failed to replace the nitrogen. It must be noted that this approach by Hutchins and Bachas was limited to charged polymers and templates.(Malitesta 2011) In conducting polymers the delocalization of charges along the polymer chains induces the formation of states in the gap, polarons and bipolarons, which are involved in charge transport. This charge allows the polymer to have metal-like conductivity.(Pardieu, Cheap et al. 2009)
Anodic oxidation of suitable electroactive functional monomers is the most common method used to prepare conducting polymers. The mechanism of electropolymerisation which results in conducting polymers is described in numerous ways in literature and is not fully understood. However in simplified terms the reaction involves an electroactive monomer such as PPy undergoing both chemical and electrode reaction steps to produce a conducting polymer matrix.(Pardieu, Cheap et al. 2009, Malitesta, Losito et al. 1999)
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Introducing an electrochemical sensor to a MIP net work involves this electropolymerisation. This method has proven to be easily obtained and provide a sensitive layer with high precision on electrode surface. Obtaining high precision is particularly important for multisensory production. Electro chemical sensors work by monitoring the current at a fixed voltage. This is known as amperometry. Amperometric sensors dominate the biosensor market due to their desirable properties such as ease of production and inexpensiveness. The signal relies on the rate of mass transfer to the electrode surface. The most common method is to measure the decrease in O2 tension versus the hydrogen peroxide production. In some cases a mediator molecule is exploited and its oxidation potential determined. (Piletsky, Turner 2002)
Potentiometric sensors are another type of sensor wildly used for MIP detection. These sensors measure the electrical potential of an electrode when no current is flowing. The signal is measured as the potential difference between the working electrode and the reference electrode. Ion-selective electrodes have been the most commonly used in this type of transducer.(Piletsky, Turner 2002) A major advantage to this type of transducer is that the template isn't required to be removed from the membrane to create electrical potential. Conductometric sensors work on the principle of measuring the time dependence of the change in conductivity caused by the binding of the recognition element to its complementary analyte. Impedance is the total electrical resistance to the flow of an alternating current being passed through a matrix such as a polymer membrane.(Piletsky, Turner 2002)
The intrinsic synthesis of these MIPs works as a disadvantage when applying detectors to the matrix. The cross-linking necessary to obtain high specificity in the polymers results in hard, fragile materials that can't be manipulated easily. This has been addressed by added a plasticiser to the compound. Oligourethane has proven to be successful and produces thin polymers with high stability that can which can be directly integrated with conductometric sensors. Integrating MIPs onto a transducer surface is done by electropolymerisation.(Malitesta 2011, Piletsky, Turner 2002)
Another promising form of sensing for combining with MIPs is acoustic sensor. These sensors work on the principle of detecting changes in the propagation, velocity or frequency of the acoustic wave which is generated upon analyte binding. Piezoelectric quartz crystals are the most common form of transducers for acoustic sensing. The piezoelectric quartz sensor measures the minute changes of mass generated by the binding of the analyte on a sensing reagent which is located on the quartz crystal. (Arenas, Ebarvia et al. 2010, Haupt, Noworyta et al. 1999) Coating a quartz crystal can be achieved via direct synthesis of the polymer onto the surface of the device. This is usually performed by placing the synthesis mixture on a gold film and a quartz crystal on top of this to make a sandwich structure. Polymerisation can be photoinitiated by a UV lamp which results in the polymer formation between the gold and quartz layer. Target molecules can be removed from the crystal post synthesis by soaking it in methanol overnight.
The detection method for the device proposed in the project must be minimisable. Therefore Optical detection must be avoided as it can't be minimised easily. Electrochemical is the preferred method of detection due to its ease of production and inexpensiveness.
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