Molecularly imprinted polymers in pseudoimmunoassay

Immunoassays are a class of analytical techniques based on the selective affinity of a biological antibody for its antigen. Competitive binding assays, of which the radioimmunoassay (RIA) was the first example, are based on the competition between analyte and a labelled probe for a limited number of binding sites. Molecularly imprinted polymers (MIPs) have been shown to be suitable replacements for biological antibodies in such techniques. Molecularly imprinted sorbent assays (MIAs) similar to RIA have been developed for a range of analytes of clinical and environmental interest. Limits of detection and selectivities of such assays are often similar to those using biological antibodies. Some assays have been used for measurements directly in biological fluids. The field is reviewed and it is shown that some perceived disadvantages of MIPs do not hinder their application in competitive binding assays: many MIAs have been demonstrated in aqueous solvents, and it has been shown that the quantity of template required to prepare imprinted polymers can be drastically reduced, and that binding site heterogeneity is not a problem as long as the sites which bind the probe most strongly are selective. Finally, recent developments including assays in microtitre plates, the use of enzyme-labelled probes, flow-injection assays and a scintillation proximity MIA are discussed.     

Molecularly imprinted polymers: synthesis and characterisation

This short review aims to present, in clear English, a summary of the principal synthetic considerations pertaining to good practice in the polymerisation aspects of molecular imprinting, and is primarily aimed at researchers familiar with molecular imprinting methods but with little or no prior experience in polymer synthesis. It is our hope that this will facilitate researchers to plan their own syntheses of molecular imprints in a more logical and structured fashion, and to begin to appreciate the limitations of the present synthetic approaches in this molecularly complex area, as well as the scope for rationally designing improved imprinted materials in the future.     

Molecularly imprinted polymers for the determination of a pharmaceutical development compound in plasma using 96-well MISPE technology

The use of molecularly imprinted polymers (MIPs) as sorbents for the solid phase extraction (SPE) of a pharmaceutical compound in development, prior to quantitative analysis was investigated. Three MIPs were synthesised using a structural analogue as the template molecule. Each polymer was prepared with different monomers and porogens. The MIPs were then tested for their performance both in organic and aqueous environments, the final aim being to load plasma directly onto the polymers. At an early development stage, there is a limited amount of compound available. Due to this limitation, reducing the amount of template required for imprinting was investigated. A MIP capable of extracting the analyte directly from plasma was produced. The specificity of the polymer allowed the method to be validated at a lower sensitivity than a more conventional SPE assay. For the first time, MIPs were packed into 96-well blocks enabling high throughput analysis. The analytical method was fully validated for imprecision and inaccuracy down to 4 ng/ml in plasma.     

Molecularly imprinted polymer formats for capillary electrochromatography

The research aimed towards the adaptation of molecularly imprinted polymers (MIPs) to the capillary format and the use of these highly selective matrices for capillary electrochromatography (CEC) is reviewed in this article. The MIP is prepared by incorporation of a template molecule into a polymerization protocol. After polymerization and extraction of the template from the resulting polymer a highly selective material with recognition cavities complementary to the template in size, shape and chemical functionality is obtained. MIPs have been used as recognition elements in several different analytical techniques. In combination with CEC a novel separation system with a unique selectivity towards a predetermined target (the template) is achieved. The merge of molecular imprinting technology (MIT) and CEC have introduced several interesting polymer formats, due to the adaptation of the MIP to the miniaturized capillary format. The polymer formats can be classified according to their preparation protocols and appearance into three conceptually different categories, i.e. the monolith, the coating and the nanoparticles. The preparation protocols, characteristics and applications of these formats will be discussed.     

Separation and screening of compounds of biological origin using molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) represent a new class of materials possessing high selectivity and affinity for the target molecule. Since their discovery in 1972, molecularly imprinted polymers have attracted considerable interest from bio- and chemical laboratories to pharmaceutical institutes. They have been utilized as sensors, catalysts, sorbents for solid-phase extraction, stationary phase for liquid chromatography, mimics of enzymes, receptors and antibodies. Among which, the application of molecularly imprinted polymers for molecular recognition-based separation and screening of compounds has undergone rapid extension and received much attention in recent years. This article mainly focuses on the separation and screening of certain pharmacophoric compounds of interests from biological origin using molecular imprinting technology. Examples of extraction and recognition of active components as anti-tumors or anti-Hepatitis C virus inhibitors from Chinese traditional herbs using molecularly imprinting technology are particularized in this article. Comparison between the screening effect based on MIPs and that based on antibodies is also represented. Consequently the merits and demerits of these two technologies are highlighted.     

Synthesis and evaluation of molecularly imprinted polymers for enalapril and lisinopril, two synthetic peptide anti-hypertensive drugs

Molecularly imprinted polymers (MIPs) for the recognition of enalapril and lisinopril were prepared using 4-vinylpyridine as the functional monomer. Following thermal polymerisation the resulting materials were crushed, ground and sieved. First generation MIPs were produced in protic polar porogenic solvents (mixture of methanol (MeOH) and acetonitrile (ACN)). These MIPs were used and validated as sorbents for solid phase extraction and binding assays. Second generation MIPs were produced with polar aprotic porogenic solvent (DMSO). These polymers were packed in HPLC columns in order to investigate their molecular recognition properties in a dynamic mode. The study of the mobile phase composition included two major parameters: organic modifier content and pH value. Retention factors illustrate selective binding of the template from the imprinted polymers, compared to structurally related compounds.     

Performance analysis of molecularly imprinted polymers for carboxylate and aminophosphate templates using commercially available basic functional monomers

A survey of commercially available amine-based monomers for binding and selectivity of carboxylate and phosphonic acid templates has revealed that the best selectivity is found for the pyridine-based monomers, while the highest affinity was found for 2-(dimethylamino)ethyl methacrylate (2-DEMA, 1). In fact, a more general finding is that selectivity is higher for aromatic amine-based monomers even though affinity remains higher for aliphatic amine-based monomers. An attempt to combine the optimal properties of these two classes of amine monomers, i.e. 2-vinylpyridine (2-VPY, 2), and 2-DEMA by using both simultaneously in a single imprinted polymer resulted in an MIP whose properties were dominated by the aliphatic amine-based monomer 2-DEMA. A controversy between the two commercially available vinylpyridine monomers, 2-VPY and 4-vinylpyridine (4-VPY, 3), was investigated, revealing that neither monomer is generally better for molecular imprinting; rather, the choice of 2-VPY or 4-VPY is template specific (although the preponderance of data tends to frequently favor 4-VPY). Phosphonic acid templates proved to be less successful as templates for molecular imprinting versus carboxylate functionalized templates, although binding was obtained and shown to be controllable via an ion-exchange process.     

Grafting of molecularly imprinted polymers on iniferter-modified carbon nanotube

Molecularly imprinted polymers (MIPs) were grafted on iniferter-modified carbon nanotube (CNT). Tween 20 was first immobilized on CNT by hydrophobic interactions. The hydroxyl-functionalized CNT was modified by silanisation with 3-chloropropyl trimethoxysilane. The iniferter groups were then introduced by reacting the CNT-bound chloropropyl groups with sodium N,N-diethyldithiocarbamate. UV light-initiated copolymerization of ethylene glycol dimethacrylate (crosslinking agent) and methacrylic acid (functional monomer) resulted in grafting of MIP on CNT for theophylline as a model template. MIPs grafted on CNT were characterized with elemental analysis, scanning electron microscopy, and thermogravimetric analysis. The theophylline-imprinted polymer on CNT showed higher binding capacity for theophylline than non-imprinted polymer on CNT and selectivity for theophylline over caffeine and theobromine (similar structure molecules). The data of theophylline and caffeine binding into the theophylline-imprinted polymer correlated well with the Scatchard plot. These MIPs on CNT can potentially be applied to probe materials in biosensor system based on CNT field effect transistor.     

Molecularly imprinted polymer-assisted sample clean-up of ochratoxin A from red wine: merits and limitations

A new analytical method for the determination of the carcinogenic mycotoxin ochratoxin A (OTA) in red wines has been developed involving a two-dimensional solid-phase extraction (SPE) clean-up protocol on C18-silica and a target-selective molecularly imprinted polymer (MIP). Prior removal of the interfering acidic matrix compounds by C18 solid-phase extraction was crucial for a successful clean-up as direct sample loading onto the MIP led to poor recoveries. The combined solid-phase extraction protocol afforded extracts suitable for sensitive ochratoxin A quantification by HPLC-fluorescence detection. Preliminary validation of the method performance with spiked (0.033-1.0 ng OTA/ml) and commercial red wines provided recoveries >90% and < 10%, with limit of detection (LOD) and limit of quantification (LOQ) of 0.01 and 0.033 ng/ml. However, a similarly favorable performance characteristics was observed in control experiments in which the MIP was replaced by the corresponding non-imprinted polymer (NIP). These findings provide evidence that under the employed experimental conditions specific analyte binding to imprinted binding sites plays a minor role in selective OTA retention. In the framework of this study, other problems inherent to MIP-based solid-phase extraction have been addressed. These include the reproducible preparation of MIP materials with consistent molecular recognition characteristics, the potential for repeated use of MIP, unfavorable polymer swelling in application-relevant solvents, potential sample contamination by template bleeding, and slow analyte binding kinetics.     

Retentivity and enantioselectivity of uniformly sized molecularly imprinted polymers for d-chlorpheniramine and -brompheniramine in hydro-organic mobile phases

Uniformly sized molecularly imprinted polymers (MIPs) for d-chlorpheniramine (CP) and -brompheniramine (BP) have been prepared by a multi-step swelling and polymerization method using methacrylic acid (MAA) or 2-(trifluoromethyl)acrylic acid (TFMAA) and ethylene glycol dimethacrylate (EDMA) as a functional monomer and cross-linker, respectively. The retentive and enantioselective properties of CP, BP and their structurally related compounds on the MIPs were evaluated using hydro-organic mobile phases. CP and BP enantiomers were retained the most as a monovalent cation on MAA-co-EDMA polymers and a divalent cation on TFMAA-co-EDMA polymers. Ion exchange and hydrophobic interactions could mainly work for the retention and enantioseparation of CP and BP on both MAA-co-EDMA and TFMAA-co-EDMA polymers in hydro-organic mobile phases. Though the respective MIPs gave the highest enantioselectivity for the template molecule, cross-reactivity for CP and BP was observed with them.     

Characterization of the heterogeneous binding site affinity distributions in molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) are polymers that can be tailored with affinity and selectivity for a molecule of interest. Offsetting the low cost and ease of preparation of MIPs is the presence of binding sites that vary widely in affinity and selectivity. Presented is a review of methods that take into account binding site heterogeneity when calculating the binding properties of MIPs. These include the bi-Langmuir, Freundlich, and Langmuir-Freundlich binding models. These methods yield a measure of heterogeneity in the form of binding site affinity distributions and the heterogeneity index. Recent developments have made these methods surprisingly easy to use while also yielding more accurate measures of the binding properties of MIPs. These have allowed for easier comparison and optimization of MIPs. Heterogeneous binding models have also led to a better understanding of the imprinting process and of the advantages and limitations of MIPs in chromatographic and sensor applications.     

Retention mechanism of analytes in the solid-phase extraction process using molecularly imprinted polymers. Application to the extraction of triazines from complex matrices

The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3 MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 microg/mL. Environmental variables were not forensically significant in determining the population distribution. HVA was not found to be a reliable indicator of dopamine or levodopa administration.     

Development of molecularly imprinted polymers for the binding of nitrofurantoin

Novel molecularly imprinted polymers (MIPs) for the recognition of nitrofurantoin (NFT) were prepared by photoinitiated polymerisation in polar solvent using 2,6-bis(methacrylamido) pyridine (BMP) as the functional monomer and carboxyphenyl aminohydantoin (CPAH) as the analogue of the template. The binding constants of the complex between BMP and nitrofurantoin or CPAH in DMSO were determined with 1H NMR titration to be 630 ± 104 and 830 ± 146 M⁻¹, respectively. To study the influence of the functional monomer, two polymer compositions were prepared containing the template, the functional monomer and the crosslinker in the molar ratio 1:1:12 for MIP1 and 1:4:20 for MIP2, respectively. The imprinting factor at saturation concentration of nitrofurantoin, which is the ratio of the amount bound to the MIP and the non-imprinted control polymer (NIP), was determined to be 2.47 for MIP1 and 2.49 for MIP2. The cross reactivity of the imprinted polymers seems to be determined by the ability to form hydrogen bonds to the functional monomer while the shape of the molecule has no real influence.     

Molecularly imprinted polymer films for reflectometric interference spectroscopic sensors

Reflectometric interference spectroscopic measurements were performed on molecularly imprinted polymer (MIP) films with the herbicide atrazine as the template molecule. A conventional imprinting protocol was used relying on non-covalent interactions between the functional monomers and the template. The MIPs were deposited on glass transducers by two different methods: spin-coating followed by in situ polymerization of thin films of monomers containing a sacrificial polymeric porogen, and autoassembly of MIP nanoparticles with the aid of an associative linear polymer. Reproducible results were obtained upon measurements of atrazine solutions in toluene with both films. Atrazine concentrations as low as 1.7 ppm could be detected with the autoassembled particle film. No or very little analyte adsorption was observed onto non-imprinted control films made by spin-coating and by particle assembly, respectively. We believe that these MIP layers in combination with the general reflectrometric transduction scheme could be a versatile sensing tool for the detection of environmentally important and other analytes.     

Molecularly imprinted polymer-based optical immunosensors

Molecularly imprinted polymers (MIPs) are artificial antibodies for a target molecule. The review focuses mainly on mechanistic steps involved in forming MIPs and the role of co-monomers and porogen. In addition, the electronic transition between different energy levels is explained with the help of the Jablonski diagram. Diverse receptor and target molecules for anchoring artificial MIPs are discussed, accentuating the synergetic effects obtained. The binding efficiency, selectivity, and sensitivity of various optical sensors are discussed intensively. In addition to this, we focused on synthesis, physical forms, characterization techniques, and microorganism detection of imprinted polymers. A brief investigation on the use of MIPs in cancer diagnosis is also included, and attention is extended to the important challenges faced in using imprinted polymers.     

Molecularly imprinted composite cryogels for hemoglobin depletion from human blood

A molecularly imprinted composite cryogel (MICC) was prepared for depletion of hemoglobin from human blood prior to use in proteome applications. Poly(hydroxyethyl methacrylate) based MICC was prepared with high gel fraction yields up to 90%, and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, swelling studies, flow dynamics and surface area measurements. MICC exhibited a high binding capacity and selectivity for hemoglobin in the presence of immunoglobulin G, albumin and myoglobin. MICC column was successfully applied in fast protein liquid chromatography system for selective depletion of hemoglobin for human blood. The depletion ratio was highly increased by embedding microspheres into the cryogel (93.2%). Finally, MICC can be reused many times with no apparent decrease in hemoglobin adsorption capacity. Copyright © 2014 John Wiley & Sons, Ltd.     

Amino-acid containing metallomonomers copolymerized into porous organic polymers: applicability to allylic alkylation catalysis

This paper reports the synthesis of a polymerizable dppe-derivative with eight pendent styrenyl units linked to the dppe core through four tyrosine residues. This diphosphine ligand was utilized in the synthesis of a series of P₂PdX₂ complexes (X₂=(R)-BINOL, (S)-BINOL, Cl₂ and π-1,3-Ph₂-allyl⁺). The compounds were used as comonomers for the synthesis of porous organic polymers (poly EDMA). Molecular imprinting effects on X₂-ligand removal were investigated. Overall, the presence of a cavity of significant size was found to be beneficial to the rate of the allylic alkylation reaction, however, chiral BINOL shaped cavities did not influence the enantio-selectivity of the reaction.     

Evaluation of molecularly imprinted solid-phase extraction for a 1,2:3,4-diepoxybutane adduct to valine

A molecularly imprinted polymer, MIP, was prepared and evaluated as SPE sorbent for a cyclicized adduct formed to N-terminal valine (Pyr-Val) in hemoglobin from 1,2:3,4-diepoxybutane (DEB). This metabolite plays an important role in the carcinogenesis of 1,3-butadiene. The hydrazide of Pyr-Val, formed after hydrazinolysis of hemoglobin, as well as necessary standards was synthesized. The MIP was prepared from methacrylic acid with a structure analogue to the investigated adduct as template and the method was developed for aqueous conditions. Selective desorption was achieved when the sample was washed with water after loading in 10% acetonitrile. The primary interaction with the binding sites in the imprints was most likely of ionic character. Quantification of the Pyr-Val adduct was performed with LC/ESI-MS/MS, yielding an instrumental LOD of 150 pg injected amount.     

Characterization of QCM sensor surfaces coated with molecularly imprinted nanoparticles

Molecularly imprinted polymers (MIPs) are gaining great interest as tailor-made recognition materials for the development of biomimetic sensors. Various approaches have been adopted to interface MIPs with different transducers, including the use of pre-made imprinted particles and the in situ preparation of thin polymer layers directly on transducer surfaces. In this work we functionalized quartz crystal microbalance (QCM) sensor crystals by coating the sensing surfaces with pre-made molecularly imprinted nanoparticles. The nanoparticles were immobilized on the QCM transducers by physical entrapment in a thin poly(ethylene terephthalate) (PET) layer that was spin-coated on the transducer surface. By controlling the deposition conditions, it was possible to gain a high nanoparticle loading in a stable PET layer, allowing the recognition sites in nanoparticles to be easily accessed by the test analytes. In this work, different sensor surfaces were studied by micro-profilometry and atomic force microscopy and the functionality was evaluated using quartz crystal microbalance with dissipation (QCM-D). The molecular recognition capability of the sensors were also confirmed using radioligand binding analysis by testing their response to the presence of the test compounds, (R)- and (S)-propranolol in aqueous buffer.     

Molecularly imprinted polymers coupled to matrix assisted laser desorption ionization mass spectrometry for femtomoles detection of cardiac troponin I peptides

Molecularly imprinted polymers (MIPs) were combined to MALDI‐TOF‐MS to evaluate a selective enrichment (SE) method for the determination of clinically relevant biomarkers from complex biological samples. The concept was proven with the myocardial injury marker Troponin I (cTnI). In a first part, MIP materials entailed for the recognition of cTnI epitopes (three peptides selected) were prepared and characterized in dimensions (0.7–2μm), dissociation constants (58–817 nM), kinetics of binding (5–60 min), binding capacity (ca. 1.5 µg/mg polymer), imprinting factors (3 > IF > 5) and selectivity for the peptide epitope. Then, the MIPs, incubated with cTnI peptides and spotted on the target with the DHB matrix, were assayed for the desorption of the peptides in MALDI‐TOF‐MS. The measured detection limit was ca. 300 femtomols. Finally, the MIP‐SE MALDI‐TOF‐MS was tested for its ability to enrich in the cTnI peptides from a complex sample, mimic of serum (i.e. 81 peptides of digested albumin). The MIP‐SE MALDI‐TOF‐MS successfully enriched in cTnI peptides from the complex sample proving the technique could offer a flexible platform to prepare entailed materials suitable for diagnostic purposes. Copyright © 2015 John Wiley & Sons, Ltd.