2-1 Endocrine disrupted chemicals
2-1-1 Introduction
Recently, scientists have found that emerging environmental contaminant have high potential to cause disruption of endocrine systems and affect the metabolism and abnormal sexual development in aquatic organisms or wild life in very small concentration. Those were called endocrine disrupting chemicals (EDCs). Actually, the Organization of Economic and Cooperate Development define EDCs as ”an exogenous substance or mixture that alters the function(s) of the endocrine systems and consequently causes adverse health effect in an intact organism, or its progeny or (sub) pollution”.21 EDCs also called environmental hormones and wide variety of chemical compounds have been found to be capable of disrupting the normal endocrine functions. Table 2-1 shows the list of EDCs including pesticides, surfactants, dioxins, polycyclic aromatic hydrocarbons, synthetic and natural hormones, and heavy metal.
EDCs are toxic and carcinogenic, and exist in the environment for a long time by their stability and bioaccumulation. The effects associated with the presence of EDCs in the environment are: (1) feminization of male fishes, (2) toxic to the reproductive system and development in mammals, fishes, and birds, (3) eggshell thinning in birds of prey, and (4) causing irreversible damage to the aquatic life. Moreover, the EDCs can lead some adverse effects in human health and the function of the endocrine system by binding to nuclear receptor. The effects of EDCs in human beings reported so far have been (1) low sperm counts, (2) increase of the incidence of breast, testicular and prostate cancers, (3) early puberty, (4) carcinogenic and impotence at low levels, and (5) the endometriosis.7, 21 Therefore, EDCs are great concern because of their potential in altering the normal endocrine
function and physiological status of organism.
Table 2- 1. List of some chemical compounds assorted as EDCs.21
No. EDC class Compound detected Use/origin
1. Phthalates Butylbenzylphthalate, di-(2-ethylhexyl)phthalate and di-n-butylphthalate
They are found in detergents, resins, some addictives and monomers used in the production of plastics 2. Pesticides Dichlorodiphenyltrichloroethane,
deltamethrin, carbofuran, atrazine, vinclozolin, carbendazim and tributylin
Extensively used in agriculture, insecticides, herbicides and fungicides are included in this class
3. Organotin compounds
Tributyltin and triphenyltin Compounds used in antifouling paints on ships
4. Alkylphenols (surfactants)
Nonylphenol, etoxylate,
octylphenol, octylphenol etoxylate
They are used during the production of phenol resins, plastic additives, and emulsifiers in agricultural or industrial applications
They can be produced during the incineration of chlorinated aromatic compounds, paper and in the production of PVC plastic
No. EDC class Compound detected Use/origin
6. Bisphenols Bisphenol A It is used in the manufacture of polymers, flame retardants and rubber chemicals
7. Parabens Methyl, ethyl, propyl and butylparabens
Compounds used as
preservatives in most cosmetics, personal care products been used as coolants and lubricants in transformers, capacitors, and other electrical equipment. Although they are no longer being used, they are present in some old installation 9. Polycyclic
processes of coal, oil, and wood 10. Brominated
Compounds used in many products including furniture, of oral contraceptives as well as steroids used for substitution therapy during menopause
No. EDC class Compound detected Use/origin
12. Phytoestrogens Daidzen and genistein, matairesol, enterodiol and enterolactone
Natural substances found in many food plants such as grains, cereals, vegetables, fruits and others
13. Natural hormones
Estrone, 17β-estradiol, oestriol Estrogens naturally and daily excreted in the human urine and animals
14. Heavy metals Cadmium, mercury and lead Industrial mining and metallurgy
2-1-2 Natural hormones
Among the various EDCs, natural and synthetic hormones are more interesting due to their high estrogenic potency. The natural hormones of EDCs produced by animal’s endocrine system have frequently found in wastewater effluents and rivers.22 Generally, estrone, estradiol, and oestriol are crucial natural hormones, since they are synthesized and excreted by the ovary every day. Upon entering human body, they can be toxic and carcinogenic even at trace concentration levels, and cause harmful effects such as inhibition of egg implantation and impotence.1, 2 For example, the feminization of male fish occurs when reached E2 at 1-10 ng/L.3
2-1-2 Analytical methods
The most commonly used methods for E2 determination were including immune technique and instrumental analysis. Immunoassay assay including enzyme-linked immunosorbent assay and radioimmunoassay can be employed for multianalyte screening of a series of analytes because of ease use, relatively simple process, and fairly good
contaminants or low stability in hash environment. The analytical instrument such as high-performance liquid chromatography and gas chromatography coupled with mass spectrometry and optical detectors currently have a good sensitivity and the ability to evaluate many pollutants in complicated mixture. Nevertheless, they are costly investment, fussy procedure, and use of large amounts of organic solvents and environmental samples.3, 6,
7 The commercial column for analysis is expensive and less specific.
2-2 General strategy of molecularly imprinted method
2-2-1 Concept
Molecular imprinted polymers (MIPs) remained a potential method for the design and development of new materials with improved molecular recognition capabilities have been realized in recent years.23 The first concept of MIPs method is considered as the
“lock-and-key” analogy with the action of a substrate and an enzyme, which proposed by Fischer in 1984. The complementary to the shape of the substrate was imprinted by the enzyme’s active site, so that the substrate fits like a key into the specific lock of the enzyme.
Following the concept, the molecularly imprinted polymer retains a “molecular memory” of template. We named it ”molecularly imprinted polymers” similar with lock and key.24, 25
Figure 2-1 shows the schematic representation of the molecular imprinting procedure.
First of all, the functional monomers, template, and cross-linker will self-assembly. Then, a pre-polymerization process with all species will spontaneously occur when the initiator present. Subsequent polymerization, the functional monomers-template complexes are held in position by the formation of a rigid polymeric matrix. Finally, removal of template using solvent extraction or combustion, the materials will be leaving a complementary cavity with the recognition ability in material.26 Therefore, the imprinting protocol yields a bulk material with specific recognition site for many applications.
Figure 2- 1. Illustration of molecular imprinting process.
Synthetic polymer with molecular imprinting process is a protocol which based on the polymerization process in the presence of the imprint molecule, sometimes used target analyte or a molecule closely related to the target analyte. The cleavage of template creates the specific binding sites, which determining the selectivity and sensitivity. Actually, the created binding sites in MIPs can be classified into different types (Figure 2-2). Site A and Site B were expected specific recognition sites in MIPs. Virtually, site A with meso- and macro-pores (> 20 Å) is more accessible by analyte compared to site B with the smaller micro-pores (< 20 Å) where the mass transfer is slow. So, the porosity and large specific surface areas with accessible meso- and macro-pores are favored for transporting and diffusion in MIPs. However, some unfavorable conditions emerge in the final materials including inaccessible site (site C) and numbers of non-specific binding sites (site F). Their show low selectivity and sensitivity due to lack of specific cavities. In addition, template self-association is appeared (site D), if adding excess of template in preparation process, which also reduced the selectivity. Another unexpected types in MIPs are number of templates remained in materials after extraction process (site G) and induced binding site without deep cavity (site E).27 Therefore, the cavities of MIPs have great influence on selectivity.
Figure 2- 2. Types of binding site in MIPs.27
2-2-2 Imprinting methods
Essentially, there are two imprinting methodologies to fabricate the MIPs. The methods of imprinting are according to interactions between functional monomers and template complex. Generally, the imprinting methods divide into two types including covalent and non-covalent interaction. Figure 2-3 displays the ordinary preparation of the covalent and non-covalent imprinting process.26 Two of the procedures will facilitate produce the high network imprinted polymers which are rigidity and insoluble.
Figure 2- 3. The representation of the covalent and non-covalent imprinting process.26
The classical methods of covalent imprinting were approached via covalent binding such as boronic acid, diol, aldehyde, or amino groups within/inside cavity before the pre-polymerization.25 The functional monomers residues are only appeared in binding site, which may reduce non-specific interactions and degree of heterogeneity.28, 29 Although the stronger covalent binding is facile to generate the rigidity and homogeneity of binding site matrix, the subsequent extraction of template is more difficult to reach.26
In the simpler non-covalent imprinting, the interactions between template and functional monomers uses weak and easily reversible non-covalent bonds including hydrogen bond, electrostatic attraction, hydrophobic interaction, and electrostatic interaction. Unlike those used in covalent imprinting, the fragile interaction is facilitated extraction or elution in the following procedure and easily obtained. In addition, it generally offers much more variety of functionality in MIPs binding site. However, the low yield of functional high-affinity receptor site and non-specific single point interaction were undesirable conditions for applications.29 The comparison of covalent and non-covalent imprinting process lists in Table 2-2.
Table 2- 2. The comparison of the covalent and non-covalent imprinting.
Terms Covalent Non-covalent
Pre-polymerization Need for some degree of synthetic chemistry
No need
Extraction Hard to remove the template Easy to remove the template Rebinding rate Slow kinetic Fast kinetic
Binding sites Homogeneous of receptor sites Heterogeneous of receptor sites Advantages 1. Stoichiometric nature help
to lower non-specific interaction.
2. High affinity receptor sites are easy produced.
1. The wide range of functional groups can be targeted.
2. Simplicity of the preparation process.
Disadvantages 1. More complex process.
2. A few types of functional groups can be used.
3. Lower template recovery.
1. Apparent non-specific binding.
2. Low yield of functional high-affinity receptor sites.
2-2-3 Synthesis
Different applications of the MIPs depended on their properties. The properties including format types, particle size, and samples required particular characteristic varied with different synthesis methods. So far, variety procedures for MIPs preparation have been developed. Organic-MIPs often formed by (1) bulk polymerization, (2) suspension polymerization, (3) precipitation polymerization, and (4) emulsion core-shell polymerization.
The other method for inorganic or organic-inorganic hybrid MIPs is sol-gel method.
The bulk polymerization is first method to synthesis organic MIPs and vast employed due to the simplicity and university. First, template and functional groups dissolve in
adequate solvent. Then, template-functional monomers complex will present by covalent or non-covalent interaction when self-assemblely occurred. After cross-linker and initiator added, the above mixture solution underwent irradiation or heat derived to polymerize bulk monolithic format. The network of MIPs produced by cross-linker will preserve the template-functional monomers complex. Following, the template extracted by suitable extraction apparatus. In the end, they pulverized with a mortar and pestle for following sieve, and then preparation process finished. On the other hand, the solvent is used to create porosity polymers. The whole process involves some problems. There is represent less than 50% of the content loss in polymers during the serious of synthesizing imprinted polymers.30 In addition, irregular particle created by grinding cause liable problem when packed into a column or coating the surface of transducer. On the other hand, the heterogeneity is produced under lack of control polymerization.
Another means for preparing MIPs is suspension polymerization. The suspension polymerization is occurred in organic solvent. The feature of the method is the surfactant involved the polymerization process to disperse unique particle which considered tiny bulk polymerization. Regarding precipitation polymerization, the mechanism is similar with suspension polymerization without surfactants. The precipitation of polymeric chain in the form of particles is based on the phase separation of solvent because they growth more and more insoluble in medium. The particle size in precipitation polymerization is around submicron (0.3-10 μm). There is no need stabilizer in this case because the rigidity obtained from cross-linker. This approach yields uniform size particles and the morphology is easily controlled. The core-shell particles are obtained by two steps process. The first step is emulsion polymerization. Seed latex can be prepared in this step. The second step is mixed with other monomers before the polymerization. The size, morphology, chemical properties can easy control in this method.31 The other method for inorganic or
2-3-4 Template
One of the many attractive features of the molecular imprinted protocol is wide variety of print molecule which had successfully been used in recent year. The imprinted of small organic molecules (e.g., amino acid, glucose, insecticides, nicotine, proteins and steroids) have successfully established for the preparation of selective recognition network, but the larger molecule structure employed in imprinted method is still a challenge.26, 30 A larger molecules is obstacle to the imprinted process and do not liable to create well-defined binding cavities. Furthermore, the structure of larger molecule do not easy penetrate the matrix for reoccupation of binding sites.30 In the ideal polymerization with compatibility, templates choose must be chemically inert in preparation process. Thus, the alternative imprinted process may be adjusted if the template will participate or unstable under polymerization conditions (e.g. elevated temperature for free radical polymerization or UV irradiation).32
2-3-5 Functional monomers
A functional monomer with functional groups plays an important role for producing a recognize site by formation of covalent or non-covalent interactions with the template.6 The functional monomers-template complex has to preserve in the polymerization process, and make sure of the functional monomers participates in formation of network. In that way, the complex can be fixed in matrix. Since the desirable template-functional monomers interactions (covalent and non-covalent bonds) were created in recognition sites, it is very helpful to define the applicable and congruous functionality monomers for the template and cross-linkers.29 Subsequently, removal of the imprint molecule leads to the cavities with matching size and shape to the analyte in polymers. Therefore, the polymer rebinding the target analyte is high selectivity and sensitivity through complementary shape and size to the initial template. In rebinding process, functional groups within the cavities will generate covalent and non-covalent interactions to analyte. Therefore, the functional monomers not
only favor generation of cavities but also benefit to rebinding. The functional monomers normally are used in excess relative to the moles of template, and excess are preferring to non-specific binding.32
Typical functional monomers used carboxylic acids, sulphonic acids, and amino acids for non-covalent interactions; boronate ester, ketone, and aldehyde for covalent interactions;
an iminodiacetic acid is commonly used for metal chelating interaction; and silanes are used in polysiloxane-based strategy. The majority of organic MIPs are based on both of functional groups of acrylate or vinyl monomers. In organic MIPs system, methacrylic acid is widely used function monomers due to the facile form of hydrogen bonds with variety target molecules. Generally, evaluation of the binding properties exhibits that hydrogen binding does certainty an important role in the molecular recognition in popular acrylic-based polymers during rebinding the target molecule. Figure 2-4 reveals some common functional groups for non-covalent and covalent MIPs.
(a)
(b)
Figure 2- 4. Various functional monomers commonly used for non-covalent molecular imprinting (a) and for covalent molecular imprinting (b).25
2-3-6 Cross-linkers
In final imprinted polymers, the very high degree (70-98%) cross-linkers are necessary for fulfilling three dimensional structures. Therefore, the cross-linkers of imprinted polymers provide materials some features. First of all, the cross-linker is controlling the morphology and porosity of material matrix. It is determining the diffusion of analyte into materials. Secondly, it serves to fix the recognition site in network and stabilizes. Finally, it participates in adequate mechanical stability of network.32 Owing to the formation of
network which is high polymeric nature via cross-link, MIPs with innately stable and robustness have been capable of chemical and thermal stable in hash environment. Besides, it is notable the interaction of cross-linkers and functional monomers should ensure smooth incorporation in polymerization. Appropriate ratios of cross-linker and functional monomers are crucial for maintaining recognitions specificity of a material. The less level of cross-linkers induce the lower binding specificity, due to the functional groups are not sufficiently fixed by cross-linkers. Conversely, the high levels cross-linkers reduce the loading capacity, and the extraction of template may also be hindered.10
Generally, divinylbenzene is used as cross-linker of styrene or acrylic-based MIPs and pentaerythritol triacrylate or pentaerythritol tetraacrylate is used for peptide-based MIPs.
The other common crossl-inkers such as ethylene glycol dimethacrylate and trimethylolpropane trimethacrylate are utilized in many imprinting process.32 The chemical structure of various well-known cross-links commonly used for molecularly imprinted are shown in Figure 2-5.
Figure 2- 5. Cross-linked monomers used to synthesis MIPs.25
2-3-7 Porogens
The solvent, referred to as porogen, used in the imprinting step is required not only to bring all the components in the polymerization but also responsible to generate a highly porous structure that raising the efficacious extraction and rebinding.26 The porosity of materials was determined by the type of solvent in the polymerization. The phase separation between the growth polymers and solvent enhances the porosity structure.33 Phase separate later occurred in higher solubility porogen in the polymerization will tend to produce small pore and higher specific surface areas of materials. In addition, porogen with lower solubility induced phase separate early, which provides more large pore and lower specific surface areas of materials.32 Increasing the porogens content in the polymerization increases the pore volume of materials. Beside, the use of more polar solvents such as acetic acid or methanol will tend to dissociate the molecule interaction such as hydrogen bonding or bridging of ionic salt between template and monomers, and imprinting is less sfficient.10 So, low solvent polarity including chloroform and benzene is desirable when the template and functional monomers interaction employed by non-covalent interaction.30
2-3 The inorganic - MIPs
2-3-1 Sol-gel process
The sol-gel process is a convenient and versatile method of preparing transparent optical glass or other ceramic material at ambient environment. It is also enabling entrapment of numerous organic, organometallic and biological molecules within network by sol-gel derived process in laboratory. In general, sol-gel materials have several advantages for example (a) compatible with organic and inorganic reagents, (b) physical chemical and thermal stable relates to organic polymers, (c) optically transparent and suitable for spectroscopic measurement.18 In addition, sol-gel process is facile to produce in a wide variety of forms: spherical shaped powder, thin film coating, inorganic membrane, or
extremely porous aerogel materials. The schematic of sol-gel process and various products is shown in Figure 2-6.
Figure 2- 6. The schematic of sol-gel process and various products.18
The basic sol-gel reaction involving three steps: hydrolysis, condensation, and polymerizations. Figure 2-7 illustrates typical sol-gel process including hydrolysis, condensation and polymerization toward silica. In practice, three of processes illustrate the aggregation process from colloidal suspension solution (sol) to gel phase. They are included hydrolysis of precursors, condensation between hydroxyl groups, and finally gelation to gel phase when the metal alkoxide is mixed with water and a mutual solvent in presence of acid or base catalyst.18, 25
Figure 2- 7. Typical chemical reaction in sol-gel process toward silica.18
In hydrolysis step, the precursor hydrolysis with water results hydroxylated products and corresponding byproducts (alcohol or water). Following step is condensation between an alkoxide groups and a silane groups or two silane groups yield siloxan and byproduct of
In hydrolysis step, the precursor hydrolysis with water results hydroxylated products and corresponding byproducts (alcohol or water). Following step is condensation between an alkoxide groups and a silane groups or two silane groups yield siloxan and byproduct of