Browsing by Author "Piletska, Elena V."
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Item Open Access Biotin–specific synthetic receptors prepared using molecular imprinti(Elsevier Science B.V., Amsterdam., 2004-02-16T00:00:00Z) Piletska, Elena V.; Piletsky, Sergey A.; Karim, K.; Terpetschnig, E.; Turner, Anthony P. F.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-methyl-propanesulfonic 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. A good correlation was found between the modelling results and the performance of the materials in the template rebinding 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.Item Open Access Controlled release of the herbicide simazine from computationally designed molecularly imprinted polymers(Elsevier Science B.V., Amsterdam., 2005-11-02T00:00:00Z) Piletska, Elena V.; Turner, Nicholas W.; Turner, Anthony P. F.; Piletsky, Sergey A.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 discusseItem Open Access Custom synthesis of molecular imprinted polymers for biotechnological application: preparation of a polymer selective for tylosin(Elsevier Science B.V., Amsterdam., 2004-02-16T00:00:00Z) Piletsky, Sergey A.; Piletska, Elena V.; Karim, K.; Foster, G.; Legge, C.; Turner, Anthony P. F.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 appliItem Open Access Development of the custom polymeric materials specific for aflatoxin B1 and ochratoxin A for application with the ToxiQuant T1 sensor tool(Elsevier Science B.V., Amsterdam., 2010-04-16T00:00:00Z) Piletska, Elena V.; Karim, K.; Coker, R.; Piletsky, Sergey A.Two polymers were computationally designed with affinity to two of the most abundant mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) for application in the ToxiQuant T1 System. The principle of quantification of AFB1 and OTA using the ToxiQuant T1 instrument comprised of a fluorimetric analysis of mycotoxins adsorbed on the polymer upon exposure to UV light. High affinity of the developed resins allowed the adsorption of both toxins as discrete bands on the top of the cartridge with detection limit as low as 1 ng quantity of mycotoxins.Item Open Access Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA - development of a novel assay for vancomycin(ACS American Chemical Society, 2013-09-03T00:00:00Z) Chianella, Iva; Guerreiro, Antonio R.; Moczko, Ewa; Caygill, J. S.; Piletska, Elena V.; Perez De Vargas Sansalvador, Isabel M.; Whitcombe, Michael J.; Piletsky, Sergey A.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 ELISAItem Open Access Does size matter? Study of performance of pseudo-ELISAs based on molecularly imprinted polymer nanoparticles prepared for analytes of different sizes(Royal Society of Chemistry, 2016-01-18) Cáceres, C.; Canfarotta, F.; Chianella, Iva; Pereira, E.; Moczko, Ewa; Esen, C.; Guerreiro, Antonio R.; Piletska, Elena V.; Whitcombe, Michael J.; Piletsky, Sergey A.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.Item Open Access Influence of continuous magnetic field on the performance of molecularly imprinted polymers(Elsevier Science B.V., Amsterdam., 2008-02-28T00:00:00Z) Guerreiro, Antonio R.; Korkhov, Vadim; Mijangos, Irene; Piletska, Elena V.; Rodins, Juris; Turner, Anthony P. F.; Piletsky, Sergey A.A set of polymers was imprinted with (−)-ephedrine using UV initiation, under the influence of a constant external magnetic field with intensities ranging from 0 to 1.55 T. Synthesised materials were characterised by X-ray crystallography, infrared spectroscopy, swelling and surface area. Recognition properties were assessed by the ability to discriminate between (+) and (−)- ephedrine and by Scatchard analyses on chromatographic mode. It was shown that polymer morphology and recognition properties are affected by the magnetic field. This resulted in considerable improvements in the chromatographic resolution of ephedrine enantiomers by materials synthesised under the influence of magnetic field. Apparently the magnetic field improved the ordering of the polymer structure and facilitated the formation of more uniform imprinting sItem Open Access Influence of initiator and different polymerisation conditions on performance of molecularly imprinted polymers(Elsevier Science B.V., Amsterdam., 2006-09-15T00:00:00Z) Mijangos, Irene; Navarro-Villoslada, Fernando; Guerreiro, Antonio R.; Piletska, Elena V.; Chianella, Iva; Karim, Kal; Turner, Anthony P. F.; Piletsky, Sergey A.A set of polymers was imprinted with (−)-ephedrine using two different initiators. A chemometrics approach was used to optimise experiments aimed at analysis of the interplay of parameters such as polymerisation time, temperature and percentage of initiator. The results presented demonstrate the importance of keeping the right balance between these various parameters of polymerisation conditions. It is shown that enhancing one single parameter such as polymer rigidity does not necessarily improve polymer performance. In general it could be concluded that MIPs should be synthesised over a long period of time using low concentration of initiator and low temperature. The best selectivity was achieved for polymers prepared by photo-initiation with 2,2-dimethoxy-2-phenylacetophenone as initiatotorItem Open Access Interactions between heavy metals and photosynthetic materials studied by optical techniques(Elsevier Science B.V., Amsterdam., 2009-11-01T00:00:00Z) Ventrella, A.; Catucci, L.; Piletska, Elena V.; Piletsky, Sergey A.; Agostiano, A.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.Item Open Access New reactive polymer for protein immobilisation on sensor surfaces(Elsevier Science B.V., Amsterdam., 2009-01-01T00:00:00Z) Kyprianou, Dimitris; Guerreiro, Antonio R.; Chianella, Iva; Piletska, Elena V.; Fowler, Steven A.; Karim, Kal; Whitcombe, Michael J.; Turner, Anthony P. F.; Piletsky, Sergey A.Immobilisation of biorecognition elements on transducer surfaces is a key step in the development of biosensors. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. A novel protocol for the covalent immobilisation of biomolecules containing primary amines using an inexpensive and simple polymer is presented. This tri-dimensional (3D) network leads to a random immobilisation of antibodies on the polymer and ensures the availability of a high percentage of antibody binding sites. The reactivity of the polymer is based on the reaction between primary amines and thioacetal groups included in the polymer network. These functional groups (thioacetal) do not need any further activation in order to react with proteins, making it attractive for sensor fabrication. The novel polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on a metal transducer surface. For demonstration purposes the polymer was immobilised on Au Biacore chips. The resulting polymer layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. A general protocol suitable for the immobilisation of bovine serum albumin (BSA), enzymes and antibodies such as polyclonal anti-microcystin-LR antibody and monoclonal anti-prostate specific antigen (anti-PSA) antibody was then optimised. The affinity characteristics of developed immunosensors were investigated in reaction with microcystin-LR, and PSA. The calculated detection limit for analytes depended on the properties of antibodies. The detection limit for microcystin-LR was 10 ng mL−1 and for PSA 0.01 ng mL−1. The non-specific binding of analytes to synthesised polymers was very low. The polymer-coated chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. These findings make this new polymer very promising for the development of low-cost, easy to prepare and sensitive biosensItem Open Access A new reactive polymer suitable for covalent immobilisation and monitoring of primary amines(Elsevier Science B.V., Amsterdam., 2001-04-01T00:00:00Z) Piletska, Elena V.; Piletsky, Sergey A.; Subrahmanyam, S.; Turner, Anthony P. F.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.Item Open Access Novel linear polymers able to inhibit bacterial quorum sensing(Wiley, 2015-01-28) Cavaleiro, Eliana; Duarte, Ana Sofia; Esteves, Ana Cristina; Correia, António; Whitcombe, Michael J.; Piletska, Elena V.; Piletsky, Sergey A.; Chianella, IvaBacterial phenotypes, such as biofilm formation, antibiotic resistance and virulence expression, are associated with quorum sensing. Quorum sensing is a density-dependent regulatory system of gene expression controlled by specific signal molecules, such as N-acyl homoserine lactones (AHLs), produced and released by bacteria. This study reports the development of linear polymers capable to attenuate quorum sensing by adsorption of AHLs. Linear polymers were synthesized using MMA as backbone monomer and methacrylic acid and itaconic acid as functional monomers. Two different quorum sensing-controlled phenotypes, Vibrio fischeri bioluminescence and Aeromonas hydrophila biofilm formation, were evaluated to test the polymers' efficiency. Results showed that both phenotypes were significantly affected by the polymers, with the itaconic acid-containing material being more effective than the methacrylic acid one. The polymer inhibitory effects were reverted by the addition of lactones, confirming attenuation of quorum sensing through sequestration of signal molecules. The polymers also showed no cytotoxicity when tested using a mammalian cell line.Item Open Access Photochemical polymerization of thiophene derivatives in aqueous solution(Royal Society of Chemistry, 2004-08-20T00:00:00Z) Piletsky, Sergey A.; Piletska, Elena V.; Karim, K.; Davis, Frank; Higson, Seamus P. J.; Turner, Anthony P. F.A novel method of photochemical polymerization of thiophene derivatives in aqueous solution catalyzed by potassium dichromate and initiated by illumination is described.Item Open Access Selection of imprinted nanoparticles by affinity chromatography(Elsevier Science B.V., Amsterdam., 2009-04-15T00:00:00Z) Guerreiro, Antonio R.; Chianella, Iva; Piletska, Elena V.; Whitcombe, Michael J.; Piletsky, Sergey A.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 template.Item Open Access The stabilisation of receptor structure in low cross-linked MIPs by an immobilised template(Royal Society of Chemistry, 2009-01-21T00:00:00Z) Garcinuno, Rosa M.; Chianella, Iva; Guerreiro, Antonio R.; Mijangos, Irene; Piletska, Elena V.; Whitcombe, Michael J.; Piletsky, Sergey A.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 enhancing sensor signals.Item Open Access A sulfur-sulfur cross-linked polymer synthesized from a polymerizable dithiocarbamate as a source of dormant radicals(John Wiley & Sons, Ltd, 2010-06-01T00:00:00Z) Garcia Con, Luis Miguel; Whitcombe, Michael J.; Piletska, Elena V.; Piletsky, Sergey A.Item Open Access Towards the development of multisensor for drugs of abuse based on molecular imprinted polymers.(Elsevier Science B.V., Amsterdam., 2005-06-22T00:00:00Z) Piletska, Elena V.; Romero-Guerra, M.; Chianella, Iva; Karim, K.; Turner, Anthony P. F.; Piletsky, Sergey A.The synthetic receptors for cocaine, deoxyephedrine, methadone and morphine were computationally designed and produced using molecular imprinting. The structure and energy of the molecular complexes were analysed by computational techniques. The possible structures of the binding sites in the synthetic receptors have been compared with those of corresponding natural receptors. The composition of imprinted polymers was optimised to allow adequate performance under the same experimental conditions. All selected molecular imprinting polymers (MIPs) demonstrated stronger affinity in comparison with corresponding blank polymers resulting in imprinted factors (I) equal to 1.2 (cocaine), 2.5 (deoxyephedrine), 3.5 (methadone) and 3 (morphine) which suggested that the specific binding site for each molecule was successfully created. The polymers studied possessed good selectivity and affinity towards their templates and could be recommended for the integration with sensor devices. From a practical point of view, especially for multisensor requirements, the synthetic receptors based on imprinted polymers could be superior to natural receptors due to their stability, robustness and compatibility with automation processes required for sensor fabrication.