Comparison of pyrimethanil-imprinted beads and bulk polymer as stationary phase by non-linear chromatography

A pyrimethanil-imprinted polymer (P1) was prepared by iniferter-mediated photografting a mixture of methacrylic acid and ethylene dimethacrylate onto homemade near-monodispersed chloromethylated polydivinylbenzene beads. The chromatographic behaviour of a column packed with these imprinted beads was compared with another column packed with irregular particles obtained by grinding a bulk pyrimethanil-imprinted polymer (P2). The comparison was made using the kinetic model of non-linear chromatography, studying the elution of the template and of two related substances, cyprodinil and mepanipyrim. Extension of the region of linearity, capacity factors for the template and the related substances, column selectivity, binding site heterogeneity, apparent affinity constant (K) and lumped kinetic association (k_a) and dissociation rate constant (k_d) were studied during a large interval of solute concentration, ranging between 1 and 2000 μg/ml. From the experimental results obtained, in the linearity region of solute concentration column selectivity and binding site heterogeneity remained essentially the same for the two columns, while column capacity (at 20 μg/ml, P1 = 23.1, P2 = 11.5), K (at 20 μg/ml, P1 = 8.3 × 10⁶ M⁻¹, P2 = 2.5 × 10⁶ M⁻¹) and k_a (at 20 μg/ml, P1 = 3.5 μM⁻¹ s⁻¹, P2 = 0.47 μM⁻¹ s⁻¹) significantly increased and k_d (at 20 μg/ml, P1 = 0.42 s⁻¹, P2 = 0.67 s⁻¹) decreased for the column packed with the imprinted beads. These results are consistent with an influence of the polymerisation method on the morphology of the resulting polymer and not on the molecular recognition properties due to the molecular imprinting process.     

New water-soluble iminecalix[4]arene with a deep hydrophobic cavity

We report the synthesis of a new water-soluble iminecalix[4]arene host 4c with a deep hydrophobic cavity. The negatively charged four carboxylate functions on the top of the cavity play a major role in the recognition of charged molecular species. The ¹H NMR titration experiments revealed that host 4c binds with cationic (10-12) and neutral guests (6-9) in water with high binding constants in the order of 10⁴-10⁵ M⁻¹. Cationic guest 9 showed highest binding constant of 2.81 × 10⁵ M⁻¹ with host 4c amongst all tested guests. Selectivity over anionic guests (13-17) is established by the presence of negative charges at the top of the deep hydrophobic cavity, as guests 15 and 17 were not recognized by host 4c. Neutral pyridine derivatives with hydrophobic chains at para positions showed high binding constants of 6.02 × 10⁴-2.23 × 10⁵ M⁻¹. The data obtained for the recognition of the guests by host 4c revealed that the ionic as well as the hydrophobic-hydrophobic interactions are crucial in the molecular recognition in aqueous medium.     

Adsorption isotherms of a molecular imprinted polymer prepared in the presence of a polymerisable template: Indirect evidence of the formation of template clusters in the binding site

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.     

Evaluation of kinetics parameters for poly(l-lactic acid) hydrolysis under high-pressure steam

In order to evaluate more precise kinetics parameters: rate constant k and E_a values for poly(l-lactic acid) hydrolysis, the reaction was carried out under high-pressure steam in a temperature range of 100-130 °C. Molecular weights of hydrolyzates were calculated by the universal calibration method without being influenced by any weight loss. The changes in molecular weight could be successfully explained according to the auto-catalytic hydrolysis mechanism, clearly indicating the critical point. Resulting k and E_a values were estimated as 8.4 × 10⁻⁵-7.2 × 10⁻⁴ s⁻¹ and 87.2 kJ mol⁻¹ with high R² values, respectively. Moreover, to determine the deviation of the parameter values, influences of four factors on the measurements and calculation: (1) use of number-average molecular weight value alone, (2) use of relative molecular weight based on polystyrene standards, (3) weight loss during the hydrolysis, and (4) selection of reaction mechanism were evaluated quantitatively.     

Chromatographic characterisation, under highly aqueous conditions, of a molecularly imprinted polymer binding the herbicide 2,4-dichlorophenoxyacetic acid

The affinity of a 2,4-dichlorophenoxyacetic acid (2,4-D) molecularly imprinted polymer (MIP), which was synthesised directly in an aqueous organic solvent, for its template (2,4-D) was studied and compared with the affinity exhibited by two other reference (control) polymers, NIPA and NIPB, for the same analyte. Zonal chromatography was performed to establish the optimal selectivity, expressed as imprinting factor (IF), under chromatographic conditions more aqueous than those described so far in the literature. Frontal analysis (FA) was performed on columns packed with these polymers, using an optimized mobile phase composed of methanol/phosphate buffer (50/50, v/v), to extract adsorption isotherm data and retrieve binding parameters from the best isotherm model. Surprisingly, the template had comparable and strong affinity for both MIP (K = 3.8 × 10⁴ M⁻¹) and NIPA (K = 1.9 × 10⁴ M⁻¹), although there was a marked difference in the saturation capacities of selective and non-selective sites, as one would expect for an imprinted polymer. NIPB acts as a true control polymer in the sense that it has relatively low affinity for the template (K = 8.0 × 10² M⁻¹). This work provides the first frontal chromatographic characterization of such a polymer in a water-rich environment over a wide concentration range. The significance of this work stems from the fact that the chromatographic approach used is generic and can be applied readily to other analytes, but also because there is an increasing demand for well-characterised imprinted materials that function effectively in aqueous media and are thus well-suited for analytical science applications involving, for example, biofluids and environmental water samples.     

A capacitive immunosensor for detection of cholera toxin

Contamination of food with biological toxins as well as their potential use as weapons of mass destruction has created an urge for rapid and cost effective analytical techniques capable of detecting trace amounts of these toxins. This paper describes the development of a sensitive method for detection of cholera toxin (CT) using a flow-injection capacitive immunosensor based on self-assembled monolayers. The sensing surface consists of monoclonal antibodies against the B subunit of CT (anti-CT), immobilized on a gold transducer. Experimental results show that the immunosensor responded linearly to CT concentrations in the range from 1.0 × 10⁻¹³ to 1.0 × 10⁻¹⁰ M under optimized conditions. The limit of detection (LOD) was 1.0 × 10⁻¹⁴ M. Two more analytical methods were employed for detection of CT using the same antibody namely, sandwich ELISA and surface plasmon resonance (SPR)-based immunosensor. The former had an LOD of 1.2 × 10⁻¹² M and a working range from 3.7 × 10⁻¹¹ to 2.9 × 10⁻¹⁰ M whereas, the later had an LOD of 1.0 × 10⁻¹¹ M and a linearity ranging from 1.0 × 10⁻⁹ to 1.0 × 10⁻⁶ M. These results demonstrate that the developed capacitive immunosensor system has a higher sensitivity than the other two techniques. The binding affinity of CT to the immobilized anti-CT was determined using the SPR-based immunosensor and an association constant (K_A) of 1.4 × 10⁹ M⁻¹ was estimated.     

Hindered, 1,1-disubstituted monomers. Chain transfer to benzene in the radical polymerisation of di-n-butyl itaconate

The transfer constant to benzene in the radical polymerisation of di-n-butyl itaconate, DBI, was measured at 60 °C and found to have the value of 2.2 × 10⁻³. This value is surprisingly high considering that benzene has no readily extractable hydrogen atoms and is usually considered to be a relatively inert solvent in radical polymerisations. The high values of transfer constants in the radical polymerisation of itaconates must be taken into consideration when planning the tailored polymerisation of these monomers.     

Fluorescent sensing of homocysteine by molecular imprinting

The feasibility of biomimetic molecular sensing of homocysteine, an independent risk factor for cardiovascular diseases, was studied. The sensing approach coupled fluorescent derivatization of dl-homocysteine by a thiol-specific fluoro-tagging agent, N-(1-pyrenyl)maleimide, with molecular recognition by a molecularly imprinted polymer (MIP) matrix. The non-covalent MIP was fabricated using the N-(1-pyrenyl)maleimide-dl-homocysteine (PM-H) adduct as template. The PM-H-MIP was found to possess outstanding analyte-specific affinity for PM-H with binding constant, K_B, of 9.28±1.6×10⁵ M⁻¹ and density of recognition sites, B_max, of 11.9±0.8 nmol/g dried MIP. Following in situ fluorescent derivatization, luminescent response of the MIP was found to correlate linearly with concentration of dl-homocysteine in the range corresponding to realistic total homocysteine concentration in blood plasma. Besides being a passive recognition matrix for the binding of the fluoro-tagged analyte, the PM-H-MIP material was found to be able to specifically enhance the rate of derivatization reaction between dl-homocysteine and N-(1-pyrenyl)maleimide. In a sense, the MIP transformed a fluoro-tagging agent, which is generally reactive towards a broad spectrum of thiol-containing species, into a dl-homocysteine-specific derivatizing agent. The mechanism of such analyte-specific enhancement of derivatization rate and its advantages to the biomimetic molecular sensing are discussed.     

A new terpyrrolic analogue of dipyrrolylquinoxalines: an efficient optical-based sensor for anions in organic media

The synthesis and X-ray structural characterization of a new terpyrrolic analog of dipyrrolylquinoxaline (DPQ) is described that contains two pyrrole anion recognition groups bridged by a central 1,2-linked pyrrole. Relative to the ‘parent’ DPQ system, this new terpyrrolic scaffold acts as an improved colorimetric and fluorescent sensor for halide and dihydrogen phosphate anions in organic media. This enhancement is particularly dramatic in the case of H₂PO₄⁻ (studied as its tetrabutylammonium salt); in CH₂Cl₂ it is bound with an affinity constant, K, of 17,500 M⁻¹ by the terpyrrole of this report vs. 80 M⁻¹ in the case of DPQ. The present findings thus support the emerging notion that a range of pyrrole-based anion recognition and sensing systems may be produced by replacing the central quinoxaline spacer in DPQs by other bridging subunits and that these new receptors may have properties that differ dramatically from their DPQ ‘parents’.     

Voltammetric behavior and determination of estrogens at Nafion-modified glassy carbon electrode in the presence of cetyltrimethylammonium bromide

A method is described for the determination of estrogens (estradiol, estrone and estriol) by stripping voltammetry. These estrogens yield one well-defined oxidation peak at the Nafion-modified glassy carbon electrode in the presence of cetyltrimethylammonium bromide (CTAB). The peak current is proportional to the concentration of estradiol in the range from 2.5×10⁻⁸ to 1.5×10⁻⁶ mol/l, and the detection limit is 1×10⁻⁹ mol/l after accumulation of 6 min. The total amounts of estrogens in the blood serums were determined using the voltammetric method, and the average recovery was 106.04%. The mechanism of the oxidation of estradiol was investigated by electrochemical techniques and UV spectra.     

A method for rapid screening of interactions of pharmacologically active compounds with albumin

We determine the association constants for ligand–protein complex formation using the flow injection method. We carry out the measurements at high flow rates (F = 1 mL min⁻¹) of a carrier phase. Therefore, determination of the association constant takes only a few minutes. Injection of 1 nM of the ligand (10 μL of 1 μM concentration of the ligand solution) is sufficient for a single measurement. This method is tested and verified for a number of complexes of selected drugs (cefaclor, etodolac, sulindac) with albumin (BSA). We obtain K = 4.45 × 10³ M⁻¹ for cefaclor, K = 1.00 × 10⁵ M⁻¹ for etodolac and K = 1.03 × 10⁵ M⁻¹ for sulindac in agreement with the literature data. We also determine the association constants of 20 newly synthesized 3β- and 3α-aminotropane derivatives with potential antipsychotic activity – ligands of 5-HT_1A, 5-HT_2A and D₂ receptors with the albumin. Results of the studies reported here indicate that potential antipsychotic drugs bind weakly to the transporter protein (BSA) with K ≈ 10²–10³ M⁻¹. Our method allows measuring K in a wide range of values (10²–10⁹ M⁻¹). This range depends only on the solubility of the ligand and sensitivity of the detector.     

Binding of a hemoregulatory tetrapeptide by a bis-guanidinium crown ether

A synthetic receptor for the molecular recognition of a tetrapeptide in aqueous buffer was obtained by combining a luminescent crown ether with two pyrrole-guanidinium moieties. The compound interacts with ammonium carboxylates of complementary geometry and binds the hemoregulatory peptide Ac-Ser-Asp-Lys-Pro with K=7×10³ M⁻¹ at physiological pH. Shorter fragments and other tetrapeptides show no or significant reduced affinity. The binding of the target peptide to the functionalized crown ether is signalled by an increase of its emission intensity.     

Molecular recognition of polymethoxybenzenes by host molecule comprised of two pyromellitic diimides and two dialkoxynaphthalenes

For the continuing study of the molecular recognition of the π-electron-poor host comprised of two pyromellitic diimides and two dialkoxynaphthalenes, its inclusion with π-electron-rich polymethoxybenzenes has been examined. The UV-vis titration studies indicated following order of the association constants (K_a's) as 1:1 complexes in CHCl₃: 1,3,5-trimethoxybenzene (31.3 M⁻¹)>1,3-dimethoxybenzene (9.2 M⁻¹)>1,2-dimethoxybenzene (6.5 M⁻¹)>1,4-dimethoxybenzenes (2.8 M⁻¹). The X-ray structural analysis of the complexes between the host and dimethoxybenzenes proved the intracavity 1:1 complexes and provided useful information on the structures of the complexes. Not only charge transfer interactions but also other weak interactions such as electrostatic, van der Waals, and the unique hydrogen bonds between the α-hydrogen atoms of the naphthalene and the methoxy oxygen atoms were considered to be responsible for the magnitude of the K_a's. Thus molecular recognition of polymethoxybenzenes has been accomplished by the neutral host using multipoint weak interactions in an organic solvent.     

A flow-injection chemiluminescence method for the determination of some estrogens by enhancement of luminol-hydrogen peroxide-tetrasulfonated manganese phthalocyanine reaction

A novel flow-injection chemiluminescence (FI-CL) method for the determination of estrogens is proposed, based upon its enhancing effect on the CL reaction of luminol with hydrogen peroxide catalyzed by tetrasulfonated manganese phthalocyanine (MnTSPc) in alkaline solution. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of estrone in the range of 1.0 × 10⁻⁷ to 1.0 × 10⁻⁶ mol/l, estradiol in the range of 9.0 × 10⁻⁸ to 1.0 × 10⁻⁶ mol/l and estriol in the range of 3.0 × 10⁻⁷ to 2.0 × 10⁻⁶ mol/l, respectively. The detection limits were 5.1 × 10⁻⁸ mol/l for estrone, 7.2 × 10⁻⁹ mol/l for estradiol and 6.5 × 10⁻⁸ mol/l for estriol with a relative standard deviation of 2.8% for 5.0 × 10⁻⁷ mol/l estrone, 2.4% for 1.0 × 10⁻⁷ mol/l estradiol, and 3.1% for 7.0 × 10⁻⁷ mol/l estriol (n = 11). This method has been applied to the determination of estrogen in pharmaceutical injections and tap water with satisfactory results.     

Characterization of recognition sites for diethyl 4-nitrobenzylphosphonate, an organophosphate pesticide analogue

In a first step towards chemical sensors using molecular imprinted materials, the complexing characteristics of diethyl 4-nitrobenzylphosphonate, an organophosphate pesticide analogue, have been studied. Two molecules have been assessed as potential interacting moieties, specifically a fluoroalcohol and an aromatic acid. The interactions have been first characterized by regular methods, such as ¹H, ³¹P NMR and IR spectroscopy. These showed a stoichiometry 1/1 for both complexes and association constants, respectively, close to 40 ± 10 and 12 ± 2 M⁻¹. In a second step, isothermal titration calorimetry was used and a method was developed to obtain low-association constants. The association constant could be obtained for the fluoroalcohol ligand and was found equal to 63 ± 0.7 M⁻¹. For the acidic molecule, an appropriate model could not be found, preventing the evaluation of this constant.     

Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N-dG adducts by capillary electrophoresis immunoassay

The stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5′-end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (K_b: 3.57 × 10⁸ M⁻¹) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(−)-anti-BPDE-dG displays lowest affinity (K_b: 1.14 ×10⁷ M⁻¹). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90mers (K_b: 6.36 ± 0.54 × 10⁸ M⁻¹) than trans-(−)-anti-BPDE-90mers (K_b: 4.52 ± 0.52 × 10⁸ M⁻¹). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K_b: 3.74 × 10⁸ M⁻¹), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA.     

An organically modified silicate molecularly imprinted solid-phase microextraction device for the determination of polybrominated diphenyl ethers

An organically modified silicate (ORMOSIL) SPME stationary phase molecularly imprinted with BDE-209 has been successfully fabricated by conventional sol-gel technique from phenyltrimethoxysilane and tetraethoxysilane. The thickness of the ORMOSIL-SPME stationary phase, on fused-silica optical fibres, was measured to be ca. 9.5 μm with a volume of ca. 0.12 μL. Rebinding assays and Scatchard analysis revealed that the imprinted ORMOSIL-SPME stationary phase possessed a binding affinity, K_B, of 7.3 ± 1.7 × 10¹⁰ M⁻¹ for BDE-209, with a receptor site density, B_max, of 1.2 × 10⁻³ pmol per SPME device. Besides its molecular template, the ORMOSIL-SPME stationary phase also showed good affinity (log K_B ≥ 9.5) for smaller BDE congeners commonly found in the natural environment. The density of receptor sites within the imprinted matrix for those smaller BDE congeners was even higher than that for BDE-209. This may be attributable to the binding site heterogeneity of the imprinting process that creates deformed binding sites that are suitable for the accommodation of the smaller BDE congeners. Compared to the commercially available polyacrylate and polydimethylsiloxane SPME stationary phases, the imprinted ORMOSIL-SPME devices showed much higher pre-concentration ability towards polybrominated diphenyl ethers (PBDEs), even in direct immersion sampling at room temperature. Coupled with GC-NCI-MS and GC-μECD, the imprinted ORMOSIL-SPME device was able to achieve detection sensitivity of 0.2-3.6 pg mL⁻¹ and 1-8.8 pg mL⁻¹, respectively, for commonly occurring BDE congeners, including medium to high molecular weight PBDEs. The imprinted ORMOSIL-SPME device has been successfully applied to monitor PBDE contents in municipal wastewaters.     

Thiourea-isothiouronium conjugate for strong and selective binding of very hydrophilic H2PO4 anion at the 1,2-dichloroethane-water interface

Potential use of a surfactant-like receptor is demonstrated at the 1,2-dichloroethane-water interface for strong and selective binding of H₂PO₄⁻ over Br⁻ and Cl⁻. The analysis by interfacial tensiometry reveals that the interfacial adsorption of a thiourea-isothiouronium conjugate, BT-C₁, is significantly stabilized by the binding of H₂PO₄⁻ with the adsorption constant of 1.7 × 10⁵ M⁻¹ while the interfacial adsorptivity of this receptor is relatively moderate for Br⁻ (0.81 × 10⁵ M⁻¹) and Cl⁻ (0.63 × 10⁵ M⁻¹). Such complexation-induced interfacial adsorption behaviors of BT-C₁ are discussed as a basis for the development of receptor-based chemical sensors for phosphate anions.     

A molecular-clip-based approach to cofacial zinc-porphyrin complexes

We have synthesized molecular-clip-based cofacial zinc-porphyrin complexes. The complexes are shown to be new efficient receptors for the complexation of 1,4-diazabicyclo[2,2,2]octane, acridinium ions, 1,4,5,8-naphthalene tetracarboxylic dianhydride (NTDA) and 1,2,4,5-benzenetetracarboxylic dianhydride (BTDA). Large binding constants (K_asso) in the range of 2.0 × 10⁴-1.1 × 10⁸ M⁻¹ were obtained for the 1:1 complexes of molecular tweezers (3) and rectangle (8) by inserting the hosts between the two porphyrin rings. The values of K and the spectroscopic results suggest that 3 and 8 serve as effective building blocks to capture the hosts. Semi-empirical calculations show that the guests position themselves within the cleft of the bis-porphyrins.     

Synthesis and evaluation of molecularly imprinted polymers for organotin compounds: a screening method for tributyltin detection in seawater

The environmental impact of some organotin compounds (OTC) has given particulate importance to analytical studies. This paper reports the first attempt to apply the emerging molecular imprinting technology to this field. Several imprinted polymers have been synthesised by the non-covalent free radical approach using sodium methacrylate (NaMA) or 4-vinylpyridine (4-VP) as monomers in the presence of TBT as template molecule in three different polymerisation media (toluene, acetonitrile and methanol/water). The ability of the polymers synthesised to retain and distinguish TBT from its degradation products has been evaluated and optimized. The results clearly showed the presence of cavities within the polymeric matrix allowing specific recognition of TBT. Cross-reactivity from other Sn species (monobutyltin (MBT), dibutyltin (DBT) and inorganic Sn) has also been evaluated. Rapid and direct differentiation of TBT from its main degradation products in seawater was achieved. The analytical characteristics included linearity (0.05-50 μg l⁻¹), a pre-concentration factor of 150, and a quantification limit of 0.04 μg l⁻¹ for 1 l.