Browsing by Author "Guerreiro, Antonio R."
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Item Open Access Automatic solid-phase synthesis of molecularly imprinted nanoparticles (MIP NPs)(Cranfield University, 2012-11) Poma, Alessandro; Piletsky, Sergey A.; Guerreiro, Antonio R.Molecularly Imprinted Polymers (MIPs) are potential generic alternatives to antibodies in diagnostics and separations. To compete with biomolecules in these technological niches, MIPs need to share the characteristics of antibodies (solubility, size, specificity and affinity) whilst maintaining the advantages of MIPs (low cost, short development time and high stability). For this reason the interest in preparing MIPs as nanoparticles (MIP NPs) has increased exponentially in the last decade. Cont/d.Item Open Access Conductance based sensing and analysis of soluble phosphates in wastewater(Elsevier Science B.V., Amsterdam., 2014-02-15T00:00:00Z) Warwick, C.; Guerreiro, Antonio R.; Gomez-Caballero, Alberto; Wood, Elizabeth; Kitson, James; Robinson, James; Soares, AnaThe current standard method used for measuring soluble phosphate in environmental water samples is based on a colourimetric approach, developed in the early 1960s. In order to provide an alternative, label free sensing solution, a molecularly imprinted polymer (MIP) was designed to function as a phosphate receptor. A combination of functional monomer (N-allylthiourea), cross-linker and monomer/template ratios were optimised in order to maximise the binding capacity for phosphate. When produced in membrane format, the MIP's ability to produce a reversible change in conductance in the presence of phosphate was explored for fabrication of a sensor which was able to selectively detect the presence of phosphate compared to sulphate, nitrate and chloride. In wastewater samples the sensor had a limit of detection of 0.16 mg P/l, and a linear range between 0.66 and 8 mg P/l. This is below the minimum monitoring level (1 mg P/l) as required by current legislation for wastewater discharges, making the sensor as developed promising for direct quantification of phosphate in environmental monitoring applications.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 Introducing MINA-The Molecularly Imprinted Nanoparticles Assay(Wiley, 2014-02-06) Shutov, Roman V.; Guerreiro, Antonio R.; Moczko, Ewa; Perez De Vargas Sansalvador, Isabel M.; Chianella, Iva; Whitcombe, Michael J.; Piletsky, Sergey A.A new ELISA‐ (enzyme‐linked immunosorbent assay)‐like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid‐phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays.Item Open Access A molecular imprinted polymer based sensor for measuring phosphate in wastewater samples(IWA Publishing, 2014-01-31) Warwick, Christopher; Guerreiro, Antonio R.; Wood, Elizabeth; Kitson, James; Robinson, James; Soares, AnaPhosphate detection in water samples is still completed using colorimetric standard methods, which have a number of disadvantages, to such as being time consuming, requiring filtration, a number of different reagents, frequent calibration and proper disposal of waste chemicals generated. Hence, a simple cost effective analytical method and instrumentation is highly desirable to aid the optimisation of treatment processes and assist the water industry in their efforts to comply with stringent regulations such as the EU's Water Framework Directive. A sensor based on molecular imprinted polymer (MIP) and a conductance transducer was developed for direct and label-free detection of phosphate in water. The sensor was able to measure the presence of phosphate in wastewater samples with good reproducibility, a linear range of 0.66-8 mg P L-1 and a lower detection limit of 0.16 mg P L-1. The sensor was further tested to measure phosphate concentrations in unfiltered field samples such as domestic wastewater treatment influent and river water and demonstrated a close correlation with reference measurements. The phosphate MIP sensor offers a way forward as either a handheld sensor for use in the field, or as a potential solution for remote, continuous monitoring of phosphate,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 Removal of heavy metals using different polymer matrixes as support for bacterial immobilisation(Elsevier Science B.V., Amsterdam., 2011-07-01T00:00:00Z) Pires, Carlos; Marques, Ana P. G. C.; Guerreiro, Antonio R.; Magan, Naresh; Castro, Paula M. L.Great attention is focused on the microbial treatment of metal contaminated environments. Three bacterial strains, 1C2, 1ZP4 and EC30, belonging to genera Cupriavidus, Sphingobacterium and Alcaligenes, respectively, showing high tolerance to Zn and Cd, up to concentrations of 1000ppm, were isolated from a contaminated area in Northern Portugal. Their contribution to Zn and Cd removal from aqueous streams using immobilised alginate, pectate and a synthetic cross-linked polymer was assessed. In most cases, matrices with immobilised bacteria showed better metal removal than the non-inoculated material alone. For the immobilisation with all the polymers, 1C2 was the strain that increased the removal of Zn the most, whereas EC30 was the most promising for Cd removal, especially when combined with the synthetic polymer with up to a ca. 11-fold increase in metal removal when compared to the polymer alone. Removal of individual metals from binary mixtures showed that there was differential immobilisation. There was greater removal of Cd than Zn (removals up to 40% higher than those showed for Zn). The results show that metal contaminated environments constitute a reservoir of microorganisms resistant/tolerant to heavy metals that have the capacity to be exploited in bioremediation strategies.Capsule immobilisation of bacteria in the naturally occurring alginate and pectate and in a synthetic cross-linked polymer increased the Zn and Cd removal abilities from single and binary contaminated waters; the applications with the synthetic polymer were the most promising for Cd and Zn removal in single and binary mixtures.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 Selective vancomycin detection using optical fibre long period gratings functionalised with molecularly imprinted polymer nanoparticles(Royal Society of Chemistry, 2014-02-25) Korposh, Sergiy; Chianella, Iva; Guerreiro, Antonio R.; Caygill, J. S.; Piletsky, Sergey A.; James, Stephen W.; Tatam, Ralph P.An optical fibre long period grating (LPG) sensor modified with molecularly imprinted polymer nanoparticles (nanoMIPs) for the specific detection of antibiotics is presented. The operation of the sensor is based on the measurement of changes in refractive index induced by the interaction of nanoMIPs deposited onto the cladding of the LPG with free vancomycin (VA). The binding of nanoMIPs to vancomycin was characterised by a binding constant of 4.3 ± 0.1 × 10−8 M. The lowest concentration of analyte measured by the fibre sensor was 10 nM. In addition, the sensor exhibited selectivity, as much smaller responses were obtained for high concentrations (∼700 μM) of other commonly prescribed antibiotics such as amoxicillin, bleomycin and gentamicin. In addition, the response of the sensor was characterised in a complex matrix, porcine plasma, spiked with 10 μM of VA.Item Open Access Solid-phase synthesis of electroactive nanoparticles of molecularly imprinted polymers. A novel platform for indirect electrochemical sensing applications(Elsevier, 2016-01-29) Mazzotta, Elisabetta; Turco, Antonio; Chianella, Iva; Guerreiro, Antonio R.; Piletsky, Sergey A.; Malitesta, CosiminoElectroactive nanoparticles of molecularly imprinted polymers (MIP NPs) specific for a non-electroactive template (i.e., the antibiotic vancomycin) were for the first time synthesized by solid-phase synthesis adding two ferrocene-derivative monomers (namely, vinylferrocene and ferrocenylmethyl methacrylate) in different amount to polymerization mixture. MIP NPs were characterized by dynamic light scattering and by cyclic voltammetry studies. This latter allowed identifying the synthetic conditions determining the highest MIP NP electroactivity. The content of electrochemical label was verified by X-Ray Photoelectron Spectroscopy, which provided an estimation of the amount of ferrocene moieties in nanoparticle structure. In the attempt to apply MIP NPs for sensing applications, nanoparticles were anchored to a Nafion modified electrode by a simple self-assembly process and the indirect electrochemical detection of vancomycin was allowed by the change of ferrocene group redox properties upon the exposure to vancomycin. The observed behavior is believed to be due to hindering of the electron transfer process of the ferrocene redox sites within nanoparticles by their interaction with non-electroactive vancomycin. A novel sensing platform is thus developed by directly anchoring to the electrode surface an electroactive probe integrated within the imprinted polymer thus allowing the selective, easy and rapid electrochemical detection of non-electroactive target molecules.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.