Browsing by Author "Whitcombe, Michael J."
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Item Open Access Deposition of functionalized polymer layers in surface plasmon resonance immunosensors by in-situ polymerization in the evanescent wave field(Elsevier Science B.V., Amsterdam., 2009-01-01T00:00:00Z) Chegel, Vladimir; Whitcombe, Michael J.; Turner, Nicholas W.; Piletsky, Sergey A.Traditionally, the integration of sensing gel layers in surface plasmon resonance (SPR) is achieved via “bulk” methods, such as precipitation, spin- coating or in-situ polymerization onto the total surface of the sensor chip, combined with covalent attachment of the antibody or receptor to the gel surface. This is wasteful in terms of materials as the sensing only occurs at the point of resonance interrogated by the laser. By isolating the sensing materials (antibodies, enzymes, aptamers, polymers, MIPs, etc.) to this exact spot a more efficient use of these recognition elements will be achieved. Here we present a method for the in-situ formation of polymers, using the energy of the evanescent wave field on the surface of an SPR device, specifically localized at the point of interrogation. Using the photo-initiator couple of methylene blue (sensitizing dye) and sodium p-toluenesulfinate (reducing agent) we polymerized a mixture of N,N-methylene-bis-acrylamide and methacrylic acid in water at the focal point of SPR. No polymerization was seen in solution or at any other sites on the sensor surface. Varying parameters such as monomer concentration and exposure time allowed precise control over the polymer thickness (from 20–200 nm). Standard coupling with 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide was used for the immobilization of protein G which was used to bind IgG in a typical biosensor format. This model system demonstrated the characteristic performance for this type of immunosensor, validating our deposition mItem Open Access Development of advanced nanosized molecularly inprinted polymers via surface-initiatied 'living' radical polymerisation(Cranfield University, 2012-01) Ivanova-Mitseva, Petya K.; Piletsky, Sergey A.; Whitcombe, Michael J.; Piletsky, E.Surface-initiated photo-iniferter mediated controlled polymerisation was used as a technique for the development of advanced and smart materials. Molecularly imprinted polymer (MIP) shell nanoparticles (NPs) were synthesised in this way from PAMAM dendrimers, used as a graftable core, in 2 min irradiation time. Surprisingly the so-synthetised NPs were around 200 nm and had a cubic shape. Cont/d.Item Open Access Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA - development of a novel assay for vancomycin(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 Dormant radical technology synthesis of materials and potential applications(Cranfield University, 2011-11) Garcia Con, Luis Miguel; Whitcombe, Michael J.; Piletsky, Sergey A.This research was focused on the study of the polymer dormant radical systems, species containing free radical structures that have longer lifetimes and greater stability than radicals in general. In order to understand the nature and reactivity of the dormant radicals, polymeric systems capable of producing dormant free radicals were synthesised. In addition, the use of these novel polymeric materials in a range of applications were studied. Those applications exploited the nature of the dormant radical groups and included controlled modifications in the polymeric structure, heterogeneous catalysis and chromatographic separations.Item Open Access Effect of the cross-linker on the general performance and temperature dependent behaviour of a molecularly imprinted polymer catalyst of a Diels-Alder reaction(2011-11-01T00:00:00Z) Henschel, H.; Kirsch, N.; Hedin-Dahlström, J.; Whitcombe, Michael J.; Wikman, S.; Nicholls, I. A.Here we present a series of molecularly imprinted polymers capable of catalysing the Diels-Alder reaction between benzyl 1,3-butadienylcarbamate (1) and N,N- dimethyl acrylamide (2). The polymer systems studied here demonstrated an unusual cross-linker and temperature dependent behaviour, namely that polymer catalysis of the Diels-Alder reaction was lower at elevated temperature, in contrast to the solution reaction. Furthermore, not only was the catalytic activity significantly influenced by the choice of cross-linker, but in a similar fashion also the extent of the temperature effect, indicating a close relationship between catalysis and the observed inhibition. Molecular dynamics simulations of both the polymer systems studied were used to provide insight into the molecular background of transition state stabilisation, and differences in properties of the systems based on different cross-linkers.Item 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 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 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 Patterned gallium surfaces as molecular mirrors(Elsevier Science B.V., Amsterdam., 2007-09-30T00:00:00Z) Bossi, Alessandra; Rivetti, Claudio; Mangiarotti, Laura; Whitcombe, Michael J.; Turner, Anthony P. F.; Piletsky, Sergey A.An entirely new means of printing molecular information on a planar film, involving casting nanoscale impressions of the template protein molecules in molten gallium, is presented here for the first time. The metallic imprints not only replicate the shape and size of the proteins used as template. They also show specific binding for the template species. Such a simple approach to the creation of antibody-like properties in metallic mirrors can lead to applications in separations, microfluidic devices, and the development of new optical and electronic sensors, and will be of interest to chemists, materials scientists, analytical specialists, and electronic engineers.Item Open Access The rational development of molecularly imprinted polymer-based sensors for protein detection.(Royal Society of Chemistry, 2011-02-22T00:00:00Z) Whitcombe, Michael J.; Chianella, Iva; Larcombe, Lee; Piletsky, Sergey A.; Noble, James; Porter, Robert; Horgan, AdrianThe detection of specific proteins as biomarkers of disease, health status, environmental monitoring, food quality, control of fermenters and civil defence purposes means that biosensors for these targets will become increasingly more important. Among the technologies used for building specific recognition properties, molecularly imprinted polymers (MIPs) are attracting much attention. In this critical review we describe many methods used for imprinting recognition for protein targets in polymers and their incorporation with a number of transducer platforms with the aim of identifying the most promising approaches for the preparation of MIP-based protein sensors (277 references).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 Synthesis of controlled polymeric cross-linked coatings via iniferter polymerisation in the presence of tetraethyl thiuram disulphide chain terminator(Elsevier Science B.V., Amsterdam., 2010-05-15T00:00:00Z) Bossi, Alessandra; Whitcombe, Michael J.; Uludag, Yildiz; Fowler, S.; Chianella, Iva; Subrahmanyam, S.; Sanchez, I.; Piletsky, Sergey A.A “grafting from” approach has been used for controlled deposition of cross- linked polymers by living radical polymerisation. Borosilicate glass was modified with N,N-diethylaminodithiocarbamoylpropyl(trimethoxy)silane, in order to confine the iniferter reactive groups solely at its surface, then placed in solution with monomers and cross-linker. The polymerisation was initiated by UV irradiation. Formation of the cross-linked polymers was studied in terms of time course of the reaction, type of monomers incorporated and influence of oxygen. Grafted surfaces were characterised by AFM, FT-IR, ellipsometry and contact angle measurements. The ability to control the grafted layer improved dramatically when the chain terminator agent, N,N-N′,N′-tetraethyl thiuram disulphide (TED) was added. Upon irradiation TED increases the concentration of passive capping radicals and decreases the possibility of recombination of active macro-radicals, thus prolonging their lifetime. In the absence of TED the thickness of produced coatings was below 10 nm. TED added at different concentrations assisted in the formation of grafted layers of 10–130 nm thickness. Iniferter chemistry in the presence of TED can be used for growing nanometre-scale polymer layers on solid supports. It constitutes a robust general platform for controlled grafting and offer a general solution to address the needs of surface derivatisation in sensors tItem Open Access The use of a quartz crystal microbalance as an analytical tool to monitor particle/surface and particle/particle interactions under dry ambient and pressurized conditions: a study using common inhaler components(Royal Society of Chemistry, 2016-11-24) Turner, Nicholas W.; Bloxham, M.; Piletsky, Sergey A.; Whitcombe, Michael J.; Chianella, IvaMetered dose inhalers (MDI) and multidose powder inhalers (MPDI) are commonly used for the treatment of chronic obstructive pulmonary diseases and asthma. Currently, analytical tools to monitor particle/particle and particle/surface interaction within MDI and MPDI at the macro-scale do not exist. A simple tool capable of measuring such interactions would ultimately enable quality control of MDI and MDPI, producing remarkable benefits for the pharmaceutical industry and the users of inhalers. In this paper, we have investigated whether a quartz crystal microbalance (QCM) could become such a tool. A QCM was used to measure particle/particle and particle/surface interactions on the macroscale, by additions of small amounts of MDPI components, in the powder form into a gas stream. The subsequent interactions with materials on the surface of the QCM sensor were analyzed. Following this, the sensor was used to measure fluticasone propionate, a typical MDI active ingredient, in a pressurized gas system to assess its interactions with different surfaces under conditions mimicking the manufacturing process. In both types of experiments the QCM was capable of discriminating interactions of different components and surfaces. The results have demonstrated that the QCM is a suitable platform for monitoring macro-scale interactions and could possibly become a tool for quality control of inhalers.