Selection of imprinted nanoparticles by affinity chromatography

Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (Kd 6.6 × 10−8) M and were also able to discriminate between related functional analogues of the templ

Substitution of antibodies and receptors with molecularly imprinted polymers in enzyme-linked and fluorescent assays

A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid, 3- thiopheneboronic acid and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in ELISA

The stabilisation of receptor structure in low cross-linked MIPs by an immobilised template

In molecularly imprinted polymers (MIPs) a high level of cross-linking is usually important for preserving the receptor structure. We propose here an alternative approach for stabilising binding sites, which involves the use of an immobilised template. The idea is based on the assumption that an immobilised template will ‘‘hold’’ polymeric chains and complementary functionalities together, preventing the collapsing of the binding sites. To test this postulate, a range of polymers was prepared using polymerisable (2,4-diamino-6- (methacryloyloxy)ethyl-1,3,5-triazine) and non-polymerisable (or extractable) (2,4-diamino-6-methyl-1,3,5-triazine) templates, methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker. The level of cross- linking was varied from 12 to 80%. Polymerisations were performed in acetonitrile using UV initiation. Binding properties of the synthesised materials were characterised both by HPLC and equilibrium batch binding experiments followed by HPLC-MS or UV-visible detection. The adsorption isotherms of polymers were obtained and fitted to the Langmuir model to calculate dissociation constant, Kd, and concentration of binding sites for each material. The results strongly indicate that the presence of an immobilised template improves the affinity of MIPs containing low percentages of cross- linker. The low cross-linked MIPs synthesised with a polymerisable template also retain a reasonable degree of selectivity. Low crosslinked MIPs with such binding characteristics would be useful for the creation of new types of optical and electrochemical sensors, where induced fit or the ‘‘gate effect’’ could be used more effectively for generating and enhancin

Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA - development of a novel assay for vancomycin

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop ELISA type assays is presented here for the first time. NanoMIPs were synthesized by a solid phase approach with immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering and electron microscopy. Immobilization, blocking and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a HRP-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was three orders of magnitude better than a previously described ELISA based on antibodies. In these experiments nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELIS

Does size matter? Study of performance of pseudo-ELISAs based on molecularly imprinted polymer nanoparticles prepared for analytes of different sizes

The aim of this work is to evaluate whether the size of the analyte used as template for the synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) can affect their performance in pseudo-enzyme linked immunosorbent assays (pseudo-ELISAs). Successful demonstration of a nanoMIPs-based pseudo-ELISA for vancomycin (1449.3 g mol) was demonstrated earlier. In the present investigation, the following analytes were selected: horseradish peroxidase (HRP, 44 kDa), cytochrome C (Cyt C, 12 kDa) biotin (244.31 g mol) and melamine (126.12 g mol). NanoMIPs with a similar composition for all analytes were synthesised by persulfate-initiated polymerisation in water. In addition, core-shell nanoMIPs coated with polyethylene glycol (PEG) and imprinted for melamine were produced in organics and tested. The polymerisation of the nanoparticles was done using a solid-phase approach with the correspondent template immobilised on glass beads. The performance of the nanoMIPs used as replacement for antibodies in direct pseudo-ELISA (for the enzymes) and competitive pseudo-ELISA for the smaller analytes was investigated. For the competitive mode we rely on competition for the binding to the nanoparticles between free analyte and corresponding analyte-HRP conjugate. The results revealed that the best performances were obtained for nanoMIPs synthesised in aqueous media for the larger analytes. In addition, this approach was successful for biotin but completely failed for the smallest template melamine. This problem was solved using nanoMIP prepared by UV polymerisation in an organic media with a PEG shell. This study demonstrates that the preparation of nanoMIP by solid-phase approach can produce material with high affinity and potential to replace antibodies in ELISA tests for both large and small analytes. This makes this technology versatile and applicable to practically any target analyte and diagnostic field

A new reactive polymer suitable for covalent immobilisation and monitoring of primary amines

A new polymer able to react with primary amines was synthesised from allyl mercaptan, o-phtalic dialdehyde and ethylene glycol dimethacrylate by radical polymerisation. Reactive thioacetale formed by allyl mercaptan and dialdehyde can bind primary amino groups without additional pre-activation forming fluorescent isoindole complex. It gives a great opportunity to monitor binding and loading of the amino compounds on the reactive surface. The reactive polymer is found to be an effective matrix for immobilisation of the proteins and other amino containing compounds in affinity chromatography and could be used for their detection in solution

Interactions between heavy metals and photosynthetic materials studied by optical techniques

In this work studies on rapid inhibitory interactions between heavy metals and photosynthetic materials at different organization levels were carried out by optical assay techniques, investigating the possibility of applications in the heavy metal detection field. Spinach chloroplasts, thylakoids and Photosystem II proteins were employed as biotools in combination with colorimetric assays based on dichlorophenol indophenole (DCIP) photoreduction and on fluorescence emission techniques. It was found that copper and mercury demonstrated a strong and rapid photosynthetic activity inhibition, that varied from proteins to membranes, while other metals like nickel, cobalt and manganese produced only slight inhibition effects on all tested photosynthetic materials. By emission measurements, only copper was found to rapidly influence the photosynthetic material signals. These findings give interesting information about the rapid effects of heavy metals on isolated photosynthetic samples, and are in addition to the literature data concerning the effects of growth in heavy metal enriched media

Controlled release of the herbicide simazine from computationally designed molecularly imprinted polymers

The present study describes the development of materials suitable for environmental control of algae. Molecularly imprinted polymers (MIPs) were used as simazine carriers able to provide the controlled release of simazine into water. Three polymers were designed using computational modelling. The selection of methacrylic acid (MA) and hydroxyethyl methacrylate (HEM) as functional monomers was based on results obtained using the Leapfrog™ algorithm. A cross- linked polymer made without functional monomers was also prepared and tested as a control. The release of simazine from all three polymers was studied. It was shown that the presence of functional monomers is important for polymer affinity and for controlled release of herbicide. The speed of release of herbicide correlated with the calculated binding characteristics. The high-affinity MA- based polymer released 2% and the low-affinity HEM-based polymer released 27% of the template over 25 days. The kinetics of simazine release from HEM-based polymer show that total saturation of an aqueous environment could be achieved over a period of 3 weeks and this corresponds to the maximal simazine solubility in water. The possible use of these types of polymers in the field of controlled release is discuss