Analytica Chimica Acta (v.504, #1)

Recent progresses of molecular imprinting in metal oxide matrices were summarized. Application of the surface sol–gel process to mixtures of organic carboxylic acids and titanium alkoxide provides ultrathin layers of titania gel (10–20 nm thick), in which molecule-sized cavities are kept intact upon removal of the organic templates. The imprinted cavity reflects the structural and functional features of the template molecule, and the enantioselective imprinting of dipeptide isomers is observed. Robustness and flexibility of the ultrathin titania layer is demonstrated by the formation of interconnected titania hollow structures. Possible practical applications and unsolved problems of this technique are discussed.
Keywords: Molecular imprinting; Titania; Surface sol–gel process; Ultrathin film; Organic template;

Approaches to the rational design of molecularly imprinted polymers by Matthew P. Davies; Vern De Biasi; David Perrett (7-14).
In our experience the efficient design of molecularly imprinted polymer (MIPs) for novel templates has proved difficult. Following commonly used imprinting protocols, MIPs designed against one template show both a lack of capacity and poor specificity for rebinding either the template or structurally similar analytes. Optimisation methods that involve changing one factor at a time can be laborious.A novel approach for the optimisation of MIPs using chemometrics is described. Sulfonamides, common drug residues in foodstuffs, were used as the model analytes with a methacrylic acid/ethylene glycol dimethacrylate MIP. To avoid the inaccuracies in measurement caused by template bleed a multi-analyte competition rebind assay was developed to select suitable sulfonamides to be used as the template for the MIP, and for the rebind analyte in the chemometric optimisation study. The rebinding efficiencies were monitored by HPLC. The template sulfonamide was selected as sulfamethazine (SMZ), and the rebind analyte as sulfadimethoxine (SDIM). The template:monomer:cross-linker (T:M:X) ratio of the SMZ block MIP was then optimised using a three-level full factorial design to predict a MIP with the highest rebind capacity. On synthesis this was 38.8% for SDIM in a solid phase extraction (SPE) application agreeing with the predication. The factorial design was further utilised to predict an optimum T:M:X ratio for the production of a class specific MIP, capable of binding a range of sulfonamides simultaneously. The predicted optimum T:M:X ratios of (1:10:55) and (1:10:10) were found to be different to commonly used ratios from the MIP literature.
Keywords: Chemometrics; Design of experiments; Molecularly imprinted polymer (MIPs); Solid phase extraction; Sulfonamide;

To date, imprinted polymers in the form of particles are reportedly made by various polymerisation methodologies, each of them developed to suit specific targets. Polymers made by different methods have, so far, never been directly compared and tested under the same conditions to assess their potential rebinding characteristics, and hence the effect of the production method on the performance of the final imprinted material.In this comparative study, polymers with a similar composition were synthesised by bulk, suspension, emulsion, two-step swelling and precipitation polymerisation. The resulting imprinted materials were characterised physically and their capacity to rebind propranolol under identical analytical conditions was assessed from organic and aqueous solutions by radioligand binding. The results showed that, when specific rebinding of ligand was measured in an organic solution (toluene+0.5% acetic acid), the methods compared as follows: precipitation (50%) > suspension (40%) > bulk (35%) > core-shell (15%) > two-step swelling (10%). When specific rebinding from an aqueous solution (sodium citrate 25  mM+0.5% acetic acid + 2% ethanol, pH 4.6) was measured, the results were somewhat different: two-step swelling (20%) ≅ suspension (19%) ≅ bulk (19%) > core-shell (15%) > precipitation (0%).
Keywords: Molecular imprinting; Polymer; Polymerisation; Methods; Particles; Comparison;

Evidence for shape selectivity in non-covalently imprinted polymers by David A Spivak; Ryan Simon; Jason Campbell (23-30).
The existence of shape selectivity in non-covalent molecularly imprinted polymers (MIPs) has been proven using molecular probes. Twelve secondary amines with different sized side chains were imprinted, and enantioselectivity evaluated by HPLC for each amine on each imprinted polymer. Trends in the quantitative structure–binding relationships (QSBR) revealed two major contributions of cavity structure on selectivity afforded by molecularly imprinted polymers. First, sterics play a dominant role in cases where a molecules structure is too big too fit into an imprinted site formed from a smaller template molecule; e.g. on MIPs made with small templates, large analytes give separation factors (α) close to 1.0 (no selectivity), while small analytes give α values of 1.4. Second, molecular structures that are equal to or smaller than those of the template molecule are selected by maximizing Van der Waals interactions within the MIP binding site. Thus, MIPs made with large analytes give α values up to 2.5, while small analytes on the same MIPs give α values closer to 1.1. Template structure also has an effect on MIP enantioselectivity; e.g. branched structures exhibit a 1.7-fold improvement in separation factors (α) by MIPs made for isopropyl versus propyl derivatives, and cyclohexyl versus hexyl derivatives. Full details of these trends are provided in the text.
Keywords: MIP; Shape; Size; Selectivity;

Molecular imprinting using monomers with solid-state polymerization by D Pestov; N Levit; V Maniscalco; B Deveney; G Tepper (31-35).
Molecular imprinting of two diolefinic compounds with solid-state photopolymerization, 2,5-distyrylpyrazine (DSP) and diethyl p-phenylenediacrylate (EPA), was demonstrated. Solid nanoscale particles of the monomer were produced and deposited onto the surface of a surface acoustic wave (SAW) transducer using the technique known as rapid expansion of supercritical solutions (RESS). The particles were polymerized by UV light in the presence of an alkane template vapor. Both imprinted and non-imprinted devices were tested upon exposure to a variety of alkane vapors in the gas phase. The results demonstrate an enhanced sensitivity to vapors at or below the size of the template. A size exclusion mechanism of recognition is proposed.
Keywords: Molecular imprinting; Gas-phase template; Solid-state photopolymerization; Gas-phase recognition;

Acceleration property of a cross-linked polymer catalyst for the hydrolysis of N-dodecanoyl leucine-p-nitrophenyl ester is attributed to the molecularly imprinting of phenyl-1-undecylcarbonylamino-3-methylbutyl phosphonate as the template of transition-state analogue (TSA) and 4-[(3′-methacryloylamino)ethyl]imidazole as the binding site of the polymer catalyst. Equimolar complex of the template of the TSA and the binding site of imidazole-containing monomer through the electrostatic interaction and hydrophobic effect was confirmed by 1 H NMR measurements and the complex was copolymerized with hydrophobic styrene monomer and 10% divinylbenzene cross-linker. A control polymer without the template, but with the cross-linker, was also prepared. After removal of the template, imidazole-containing imprinted polymer gave a ca. two-fold rate-enhancement in comparison to the non-imprinted polymer.
Keywords: Molecularly imprinted polymer; Transition-state analogue; Esterase; Histidine; Amino acid esters;

Adsorption isotherms of a molecular imprinted polymer prepared in the presence of a polymerisable template by Claudio Baggiani; Gianfranco Giraudi; Cristina Giovannoli; Cinzia Tozzi; Laura Anfossi (43-52).
The current opinion about molecular imprinted polymers (MIPs) is that their molecular recognition properties are due to the presence of nanocavities formed during a polymerization process developed in the presence of a template molecule. According to this principle, the shape of these nanocavities is complementary to that of the template and non-covalent interactions are established between the binding site and a single template molecule. Nevertheless, there are some experimental indications that the real molecular recognition mechanism involves clusters of template molecules being packed into the binding site. Recently, it has been proposed that template molecules covalently linked to the binding site can act as nucleation points, enhancing the formation of these molecular clusters.We have tested this hypothesis by studying the adsorption isotherms of polymers prepared by imprinting them with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Three different polymers were considered: P0, prepared without the template, P1, whose template was represented by 2,4,5-T molecules, and P2, whose template was 1/3 constituted by the polymerisable 2-(2,4,5-trichlorophenoxyacetoxy)-ethylmethacrylate (2,4,5-TEMA) and 2/3 by 2,4,5-T. The polymers were prepared by thermoinduced polymerization of template mixtures, 4-vinylpyridine and ethylene dimethacrylate. The crushed polymers were packed into HPLC columns and frontal chromatographic runs were performed by eluting the columns with a mobile phase containing variable amounts of 2,4,5-T.The experimental adsorption isotherms were fitted by using several isotherm models, and the Freundlich–Langmuir model was found to give the best fitting in terms of F-test. All the models considered showed a significant difference between the affinity constant values measured for the polymer P1 and P2, with a higher value for the polymer P2 (for Freundlich–Langmuir model: polymer P1, k=(2.00±0.43)×104  M−1; polymer P2, k=(1.93±0.0535)×105  M−1; ratio P2/P1, 9.65±2.09). Such experimental results support the hypothesis that a polymer prepared with a limited amount of template covalently attached to the binding site shows an increased affinity for the template itself.
Keywords: Molecular imprinting; 2,4,5-Trichlorophenoxyacetic acid; Frontal chromatography; Adsorption isotherm; Affinity constant;

GraphicStereoselective molecularly imprinted polymers (MIPs) have been synthesised via ring-opening metathesis polymerisation, in essentially, quantitative yield. A covalent imprinting strategy was followed during the network formation of the chiral sorbent. Recognition of the substrate however involved non-covalent interactions; a combination of hydrogen bonding and the chiral environment presented by the imprinted cavities. The enantiomeric excess achievable with these new MIPs is solvent dependent and stereoselectivities of up to 20% e.e. (separation factor α=2.2) were found in batch equilibrations.
Keywords: Molecularly imprinted polymers; Ring-opening metathesis polymerisation; Enantioselectivity; Non-covalent interactions;

A comparison of three different methods for the imprinting of small aromatic heterocycles containing only a single nitrogen atom, for the preparation of specific analytical phases, was carried out. A conventional non-covalent approach to the imprinting of pyridine using methacrylic acid as the functional monomer was compared with two sacrificial spacer methods, in which heterocycles were imprinted as covalent template analogues. The results of binding experiments showed that discrimination based on ligand size was possible when polymers were prepared using a silyl ester-based template. The most selective polymer was able to bind pyridine in preference to quinoline or acridine which is opposite to the trend predicted by the pK HB values for the three ligands. Curve fitting of the isotherm for pyridine binding to this polymer to the Langmuir model gave an approximate K d of 1.1±0.1 mM and a binding site concentration of 57±2 mmol g−1. Acridine binding did not show saturation behaviour and was non-specific and cooperative in nature.
Keywords: Imprinted polymers; Molecular recognition; Heterocycles; Templating;

Propofol-imprinted membranes with potential applications in biosensors by Miruna Petcu; Peter N Schaare; Christian J Cook (73-79).
Herein we describe a new thin film imprinted polymer for propofol (active ingredient of a commonly used general intravenous anaesthetic) developed on a membrane support. Propofol binds to this polymer in 2 min and can be removed and tested in 1 min, giving a total assay time of 3 min. The non-specific binding to the polymer is below the detection limit (0.1 μg/ml) and the response rate is below the rate of metabolism of the anaesthetic. Tests performed with this polymer against propofol-spiked blood samples showed good linearity and specificity at clinically relevant concentrations of 1–10 μg/ml and the working range for the system is 0.1–50 μg/ml. The polymer is easily regenerated and can be re-used for subsequent testing (in blood up to 5000 cycles). These results suggest suitability for use in an on-line propofol detection system.
Keywords: Propofol; Biosensor; Molecular imprinting; Anaesthetic;

Selective extraction of antioxidants with molecularly imprinted polymers by O Brüggemann; A Visnjevski; R Burch; P Patel (81-88).
Molecular imprinting technology can be used to generate specific artificial polymeric receptors, i.e., high affinity stationary phases, as already shown for peptides and many other food ingredients. In this work, polymers have been molecularly imprinted with three exemplary antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG). Results of chromatographic evaluations are presented, demonstrating the specificity of the imprinted polymers (MIP) compared to non-specific control polymers (CP). For a BHA imprinted polymer and the respective analyte BHA a separation factor of α=k BHA-MIP′/k CP′=1.12 was found, whereas for the BHT-MIP a higher selectivity was determined with α=k BHT-MIP′/k CP′=1.47. Using the PG selective MIP for PG as analyte a value of α=k PG-MIP′/k CP′=1.24 could be achieved. Furthermore, results of extraction procedures based on MIP and CP phases are presented, demonstrating for instance a higher selectivity of the BHA-MIP in comparison with its CP when using methanol or acetonitrile as analyte solvent, and of the PG-MIP when performing the extraction in acetonitrile.
Keywords: Molecular imprinting; Extraction; Stationary phase; Antioxidants; Chromatography;

Development of trichloroacetic acid sensor based on molecularly imprinted polymer membrane for the screening of complex mixture of haloacetic acids in drinking water by Roongnapa Suedee; Teerapol Srichana; Chutcharin Sangpagai; Chanpa Tunthana; Pikul Vanichapichat (89-100).
This work shows developing conductometric sensor based on molecularly imprinted polymer (MIP) for the screening of complex mixture of haloacetic acids (HAAs) in drinking water. The recognition of the HAAs was achieved by trichloroacetic acid (TCAA)-imprinted polymers synthesised from the copolymerization of 4-vinylpyridine (4-VPD) and ethylene glycol dimethacrylate (EDMA) in the presence of the TCAA template in acetonitrile, either by bulk polymerization (BP) method or by a multi-step swelling polymerization (MSP) method. TCAA-imprinted polymer of both methods was tested for re-binding with the template and its analogs. It was found that these polymers could bind selectively to the template molecule and HAA derivatives. HAA measurements were carried out by the application of the polyvinyl chloride membrane fabricated with TCAA-imprinted polymer on conductometric sensors. The technological parameters (operating frequency, membrane composition, ionic strength and medium pH) for the sensors were identified and optimised in respect to the response to TCAA, using sensor fabricating with BP-based MIP as a model. The selectivity of the sensors constructed with MIPs made by either that of the two imprinting methods was also investigated, which the influence of the method of imprinting on the binding strength and selectivity of the recognition element embedded in sensor was observed. The sensors showed high sensitivity and selectivity for the response toward TCAA, the sensor modified with MSP-based MIP being better. In addition, the sensors, particularly when was constructed with MSP-based MIP exhibited good cross-reactivities with a wide range of HAAs, which is useful for the screening of the group of HAA usually present in chlorinated water in complex mixtures. Thus, the sensor modified with MSP-based MIP was chosen for analytical application. The calibration of this sensor was determined, showing the good linear graphs (R 2>0.970) for HAAs over the concentration range of 25–1000 μg/l and the detection limit of each HAA in the range 0.2–5.0 μg/l. Moreover, the results in real analysis of the sensor indicate the simplicity and reliability of the method. The present work demonstrated that the sensor based on TCAA-imprinted polymer is a fast and sensitive screening method of HAAs in drinking water.
Keywords: Molecularly imprinted polymer; Disinfection by-products; Haloacetic acids; Membranes; Conductometric sensor;

Analysis of NAD(P)+ and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces: by Oleg A. Raitman; Vladimir I. Chegel; Andrei B. Kharitonov; Maya Zayats; Eugenii Katz; Itamar Willner (101-111).
Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD+), β-nicotinamide adenine dinucleotide phosphate NADP+, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD+ or NADP+ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)+ and NAD(P)H cofactors in the concentration range of 1×10−6 to 1×10−3  M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD+ and lactate dehydrogenase using the NADH-imprinted polymer film.
Keywords: Surface plasmon resonance; NAD(P)+ and NAD(P)H cofactors; Imprinted polymers; Sensor;

Development of ion-sensitive field-effect transistor-based sensors for benzylphosphonic acids and thiophenols using molecularly imprinted TiO2 films by Svetlana P Pogorelova; Andrei B Kharitonov; Itamar Willner; Chaim N Sukenik; Hillel Pizem; Thomas Bayer (113-122).
Molecularly imprinted polymeric membranes, containing artificial recognition sites for a number of benzylphosphonic acid derivatives, were prepared by the polymerization of titanium(IV) butoxide in the presence of a titanium(IV) phosphonate complex. Reference polymers were prepared in the same manner but in the absence of the phosphonate template. FTIR spectroscopy was used to follow the formation of a benzylphosphonic acid–Titanium(IV) oxide complex during the imprinting process, and upon the association of the substrate in the imprinted TiO2 thin film. The imprinted polymers were examined as sensing membranes in an ion-sensitive field-effect transistors (ISFETs). The sensors reveal selectivity towards analyzing the imprinted substrates, yet the recognition ability of the sensors strongly depends on the substituents associated with the phosphonic acid structures. The response time of the sensors is ca. 45 s, and the sensors reveal unaffected stability for at least 2 weeks. Also, imprinted TiO2 films for thiophenol, and para-nitrothiophenol were generated on ISFET devices, and the respective substrates were selectively sensed by the functional devices.
Keywords: Imprinted polymers; ISFET; Sensors; Benzylphosphonic acids; Thiophenol; Sol-gel;

Custom synthesis of molecular imprinted polymers for biotechnological application by Sergey Piletsky; Elena Piletska; Kal Karim; Graham Foster; Colton Legge; Anthony Turner (123-130).
A molecularly imprinted polymer (MIP) selective for tylosin was designed and synthesised using a computational method (MIP “dialling”). In re-binding experiments the MIP demonstrated high affinity for tylosin in aqueous solutions and in organic solvents. The synthesised polymer was tested for re-binding with the template and related metabolites such as tylactone, narbomycin and picromycin. The HPLC analysis showed that the computationally designed polymer is specific and capable of separating the template from its structural analogues. The MIP was capable of recovering tylosin from broth samples. The polymer capacity for tylosin was estimated as 6.4 mg/g for MIP, which was suitable for practical application and tylosin recovery from broth samples. Among the advantages of this was the possibility to adsorb tylosin from a complex media with easy removal of oils and other impurities which are present in significant quantities, which can create problems for its chromatographic purification procedure. The MIP “dialling” procedure can have a general significance for the fast preparation of specific adsorbents for biotechnological applications.
Keywords: Tylosin; Computational design; Molecularly imprinted polymer;

Bisphenol A-recognition polymers prepared by covalent molecular imprinting by Takashi Ikegami; Takashi Mukawa; Hiroyuki Nariai; Toshifumi Takeuchi (131-135).
A specific recognition material for bisphenol A (BPA) was prepared by using a covalent imprinting technique. A chloroform solution containing bisphenol A dimethacrylate as a template, ethylene glycol dimethacrylate as a cross-linking agent and 2,2′-azobis(isobutyronitrile) as an initiator was polymerized by UV initiation. When BPA was removed from the resulting polymer by hydrolysis of the ester bonds with aqueous sodium hydroxide, carboxylic acid residues were generated in the polymer. After the polymer was packed into a stainless steel column, retention factors of BPA and related compounds were measured. The imprinted polymer adsorbed BPA and structurally related compounds such as 4,4′-dihydroxybenzophenone, bis(4-hydroxyphenyl)sulfone and 4,4′-dihydroxybiphenyl. A typical association constant (K a) was calculated to be 1.72×105  M−1 by Scatchard analysis. Interestingly, 17α- and 17β-estradiol were also bound to the imprinted polymer (K a=1.68×105  M−1), indicating that the polymer could be used as artificial receptors for screening the compounds having estrogenic action.
Keywords: Bisphenol A; Endocrine disruptors; Covalent imprinting technique; Artificial receptor;

Chiral recognition of octadentate Na+ complex with tetra-armed cyclen by molecularly imprinted polymers by Hiroyuki Kubo; Tomoko Nishimura Player; Satoshi Shinoda; Hiroshi Tsukube; Hiroyuki Nariai; Toshifumi Takeuchi (137-140).
Chiral indanyl substituted tetra-armed cyclens (TAC) formed octadentate complexes with Na+. Since their four side arms stand up and are bundled to form quadruplicated helical structures, they can have Δ- or Λ-types enantiomers based on complex helicity. In this study, TAC-imprinted polymers were prepared using an ion-pair complex of the sodium salt of (S)-indanyl substituted TAC (TAC(S)) and 2-sulfoethyl methacrylate as a template, and ethylene glycol dimethacrylate as a cross-linker. Affinity of the obtained polymers for the TAC Na+ complexes was evaluated chromatographically and the imprinted polymer gave longer retention time for the template than that for its antipode where the separation factor was given to be 1.29–1.45 under the NaOH concentrations of 10–25 mM in the eluent. These results indicate that the imprinted polymer could discriminate helix structures of TAC Na+ complexes.
Keywords: Chiral recognition; Tetra-armed cyclen; Molecular imprinting;

In this work, a molecularly imprinted polymer (MIP) of morphine (MO) was prepared through thermal radical copolymerization of methacrylic acid (MAA) and ethylene glycol dimethacrylate (EDMA) in the presence of MO templates, and a molecularly imprinted sorbent assay (MIA) based on a colorimetric reporter was developed to determine the adsorption isotherm of MO–MIP binding. In practice, the MO-bound MIP was brought into contact with an aqueous mixture of Fe3+ and [Fe(CN)6]3− so that the 3-phenolic group of MO was oxidized and Fe3+ was reduced to Fe2+. As a result, the MO-bound MIP was stained with Prussian blue (PB), which was attributed to the instant co-precipitation of Fe2+ and [Fe(CN)6]3− (K sp=10−40). Accordingly, MO–MIP binding of the blue dye could be detected by visible spectroscopy. In addition, such staining could successfully distinguish MO from codeine. Upon data analyses, a two-site binding isotherm with two dissociation constants of 6.00×10−5 and 1.03×10−3  M was found for MO–MIP binding. MIAs for non-MIP were also performed. In addition, the results of flow-system characterizations and the particle size effect are also described in this paper.
Keywords: Colorimetric detection; Molecularly imprinted polymer (MIP); Molecularly imprinted sorbent assay (MIA); Morphine (MO); Prussian blue (PB);

Application of multivariate analysis to the screening of molecularly imprinted polymers for bisphenol A by F Navarro-Villoslada; Blanca San Vicente; Marı́a C Moreno-Bondi (149-162).
A key issue in the synthesis of molecularly imprinted polymers (MIPs) is the identification and optimisation of the main factors that affect the material structure and its molecular recognition properties. This paper describes the application of an experimental design and multivariate analysis method for the synthesis of bisphenol A (BPA)-selective MIPs. Six factors with a large impact on the MIP synthesis and its analytical performance have been optimised: the amount of template, the type and the percentage of functional and cross-linking monomers, the polymerisation method (i.e. thermal or UV initiation) and the porogenic solvent. The polymers have been prepared in small-scale (mini-MIPs) and, after careful removal of the template, their BPA rebinding capacity has been evaluated and related to the MIP composition. Among the two functional monomers tested, namely 4-vinylpyridine (4-vpy) and methacrylic acid (MAA), the former rendered the best selectivity for BPA analysis. The partial least squares (PLS) models revealed that the photoinitiated polymers with a 1:1 ratio of 4-vinylpyridine to cross-linker (EDMA or TRIM) yield the highest specific binding. Such procedure is time and cost effective and can be used as a general tool in the preparation of MIPs for different analytes.
Keywords: Bisphenol A; Molecularly imprinted polymer; Multivariate analysis; PLS;

The system of polyacrylamide incorporated with methacrylic acid and 2-(dimethylamino)ethyl methacrylate was studied for the possibility of imprinting of lysozyme. The results show that approximately 27% (w/w) of the lysozyme template was not able to be extracted from the molecularly imprinted acrylamide polymers. The amount of the lysozyme template able to be extracted was increased by the addition of methacrylic acid. The molecularly imprinted polymer (MIP), which was prepared with 0.573 M acrylamide, 0.573 M methacrylic acid and 0.573 M 2-(dimethylamino)ethyl methacrylate at a total solution concentration of 20% (w/w), was able to adsorb 83% more lysozyme than the non-imprinted polymer. Selectivity of MIP was also studied.
Keywords: MIP; Polyacrylamide; Lysozyme;

With α-bilirubin as a molecular template, polymerization of methacrylic acid (MAA) was carried out with the aid of the initiator 2,2-azobisisobutyronitrile (AIBN) and the cross-linking agent ethylene glycol dimethylacrylate (EGDMA). Bulk polymerization was successfully carried out so that poly(methacrylic acid-co-ethylene glycol dimethylacrylate) (poly(MAA-EGDMA)) imprinted with α-bilirubin was first developed. UV irradiation polymerization and heated polymerization methods were compared. Effect of different ratios of monomer to EGDMA during the polymerization was also discussed. Proper solvent for better desorption of α-bilirubin from the imprinted poly(MAA-EGDMA) was investigated. In addition, SEM photos were provided for observing the differences between the surfaces of the imprinted poly(MAA-EGDMA) before and after extraction. The corresponding binding results of α-bilirubin imprinted poly(MAA-EGDMA) and non-imprinted poly(MAA-EGDMA) both after extraction were compared. How the pH values during extraction stage affected the binding capacities of the imprinted polymer as well as non-imprinted polymer were also discussed. Similar study and comparison were made for different binding pH values. Different compounds of similar molecular weight were used to show the specific binding of the imprinted polymer for bilirubin. The results further confirmed the successful binding as well as specificity of the imprinted poly(MAA-EGDMA) for α-bilirubin.
Keywords: α-Bilirubin; Poly(MAA-EGDMA); UV irradiation polymerization; Heated polymerization; Binding capacity; Extraction; Specificity;

Biotin-specific synthetic receptors prepared using molecular imprinting by Elena Piletska; Sergey Piletsky; Kal Karim; Ewald Terpetschnig; Anthony Turner (179-183).
The composition of new molecularly imprinted polymers (MIPs) specific for biotin was optimised using molecular modelling software. Three functional monomers: methacrylic acid (MAA), 2-(trifluoromethyl)acrylic acid (TFAA) and 2-acrylamido-2-methylpropanesulfonic acid (AMPSA), which demonstrated the highest binding scores with biotin, were tested on their ability to generate specific binding sites. The imprinted polymers were photografted to the surface of polystyrene microspheres in water. The affinity of the synthetic “receptor” sites was evaluated in binding experiments using horseradish peroxidase-labelled biotin. Good correlation was found between the modelling results and the performance of the materials in the template re-binding study. The dissociation constants for all MIPs were 1.4–16.8 nM, which is sufficient for most analytical applications where biotin is used as a label.
Keywords: Biotin; Computational design; Molecularly imprinted polymer; Photografting polymerisation;

Bioimprinted protein exhibits glutathione peroxidase activity by Junqiu Liu; Kun Zhang; Xiaojun Ren; Guimin Luo; Jiacong Shen (185-189).
A strategy for design of bioimprinted proteins with glutathione peroxidase (GPX) activity has been proposed. The proteins imprinted with a glutathione derivative were converted into selenium-containing proteins by chemical modifying the reactive hydroxyl groups of serines followed by sodium hydrogen selenide displacement. These selenium-containing proteins exhibited remarkable GPX activities and the GPX activities of reduction of H2O2 by glutathione (GSH) were found to be 101–817 U μmol−1, which approaches the activity of a selenium-containing catalytic antibody elicited by a hapten similar to our template. The steady state kinetic study for imprinted protein catalysis revealed Michaelis–Menten kinetics for both H2O2 and GSH, e.g. the pesudo-first-order rate constant k cat (H2O2) and the apparent Michaelis constant K m (H2O2) at 1 mM GSH were calculated to be 784 min−1 and 1.24×10−3  M, respectively, and the apparent second-order rate constant k cat (H2O2)/K m (H2O2) was determined to be 6.33×105  (M min)−1. The kinetics and the template inhibition showed that the strategy might be a remarkably efficient one for generating artificial enzyme with GPX activity.
Keywords: Bioimprining; Glutathione peroxidase; Protein; Catalysis; Selenium;

Molecularly imprinted polymers prepared in aqueous solution selective for [Sar1,Ala8]angiotensin II by Alexandre Rachkov; Minjie Hu; Elena Bulgarevich; Tadashi Matsumoto; Norihiko Minoura (191-197).
Conventional molecular imprinting technology allows the synthesis in organic solvents of molecularly imprinted polymers (MIPs) selective toward relatively low molecular weight compounds. However, synthesis in aqueous media of chemically and mechanically stable MIPs that can recognize biomolecules such as peptides and proteins still is a great challenge. In this article, we report the successful synthesis of peptide-selective MIPs in aqueous solution. HPLC evaluation of these polymers with a water-based mobile phase showed their selectivity for the peptide, [Sar1,Ala8]angiotensin II (SA), that had been used as the template, but not for its parent peptide angiotensin II (AII). The binding capacity and selectivity of our MIPs depended on the ratio of template to functional monomer in the polymerization mixture, as well as on the ionic strength and pH of the chromatographic mobile phase. These MIPs can be used for chromatographic detection of the octapeptide [Sar1,Ala8]angiotensin II in aqueous solution, with a detection limit of 8 pmol and a response that is linear (r 2>0.99) over the concentration range 0.4–20 μM.
Keywords: Molecularly imprinted polymer; Liquid chromatography; Peptides; Molecular recognition; Detection;