Browsing by Author "Piletsky, Sergey A."
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Item Open Access Analysis of skin tissues spatial fluorescence distribution by the Monte Carlo simulation(Iop Publishing Ltd, 2003-07-21T00:00:00Z) Churmakov, D. Y.; Meglinski, I. V.; Piletsky, Sergey A.; Greenhalgh, D. A.A novel Monte Carlo technique of simulation of spatial fluorescence distribution within the human skin is presented. The computational model of skin takes into account the spatial distribution of fluorophores, which would arise due to the structure of collagen fibres, compared to the epidermis and stratum corneum where the distribution of fluorophores is assumed to be homogeneous. The results of simulation suggest that distribution of auto-fluorescence is significantly suppressed in the near-infrared spectral region, whereas the spatial distribution of fluorescence sources within a sensor layer embedded in the epidermis is localized at an ‘effective’ deItem Open Access Analytical technology for cleaning verification and analysis of drug purity in pharmaceutical production.(Cranfield University, 2008-02) Corrigan, Damion K.; Piletsky, Sergey A.; McCrossen, SeanProduction of pharmaceuticals is a complex process which goes beyond the synthetic reactions undertaken to produce the final drug product. In multi purpose manufacturing facilities the equipment used in the processes must be proven to be sufficiently free of residue from the previous compound so that safe manufacture of the next compound can commence. Cross contamination can pose serious health risks so cleaning verification is a process of extreme importance. Pharmaceutical products may contain impurities that originate from the synthetic stages of production, the starting materials or from in situ reactions taking place in the final drug. Some of these impurities may be genotoxic. Genotoxic impurities are a class of impurities for which awareness is currently growing in the pharmaceutical industry. Traditional analytical chemistry methods such as HPLC are currently employed for the detection and quantification of cleaning residues and genotoxic impurities. These methods can be extremely time consuming. The aims of this project are to investigate swab sampling in cleaning verification, to develop alternative analytical methods which speed up and enhance knowledge of the cleaning verification process and to begin the development of an assay system for commonly occurring genotoxic impurities.Item Open Access Attenuation of quorum sensing using computationally designed polymers(Cranfield University, 2010-05) Stavroulakis, Georgios; Piletsky, Sergey A.; Tothill, Ibtisam E.; Piletsky, E.; Robinson, G.It is generally accepted that the majority of Gram-negative and Gram-positive bacteria communicate via production and sensing of small signal molecules, autoinducers. The ability of bacteria to sense their population density is termed quorum sensing (QS). Quorum sensing controls certain phenotypic traits, particularly virulence factors and biofilm formation. In this project a new solution for the attenuation of quorum sensing which involves selective sequestering of the signal molecules using rationally designed synthetic polymers was explored. Cont/d.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 Biomimetic sensors for HbA1c(Cranfield University, 2010-07) Biela, Anna; Piletsky, Sergey A.; Turner, Anthony P. F.Diabetes mellitus is a growing health problem worldwide. Suitable long-term control and management of this disease are enabled by determination of glycated haemoglobin (HbA1c) in blood. The results are given as %HbA1c of total haemoglobin. Presently available tests vary in cost and convenience and there is an identified need to introduce improved equipment for self-monitoring. This dissertation focuses on fast and straightforward detection of glycated haemoglobin (HbA1c) using cyclic voltammetry and chronoamperometry. Haemoglobin was determined by monitoring its reaction with potassium ferricyanide on screen printed electrodes at an oxidative potential +500 mV. A working electrode was modified with carbon nanotubes to enhance electron transfer. A calibration curve was linear in a range from 0.83 to 83 mg/mL. Another innovative approach to detecting haemoglobin using its enzymatic activity was also developed. Detection of haemoglobin was performed with hydroquinone and hydrogen peroxide on screen printed electrodes at a potential -400 mV in a Flow Injection Analysis system (FIA). Cont/d.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 Catalytic molecularly imprinted polymer membranes: Development of the biomimetic sensor for phenols detection(Elsevier Science B.V., Amsterdam., 2010-02-05T00:00:00Z) Sergeyeva, T. A.; Slinchenko, O. A.; Gorbach, L. A.; Matyushov, V. F.; Brovko, O. O.; Piletsky, Sergey A.; Sergeeva, L. M.; Elska, G. V.Portable biomimetic sensor devices for the express control of phenols content in water were developed. The synthetic binding sites mimicking active site of the enzyme tyrosinase were formed in the structure of free-standing molecularly imprinted polymer membranes. Molecularly imprinted polymer membranes with the catalytic activity were obtained by co-polymerization of the complex Cu (II)–catechol–urocanic acid ethyl ester with (tri)ethyleneglycoldimethacrylate, and oligourethaneacrylate. Addition of the elastic component oligourethaneacrylate provided formation of the highly cross-linked polymer with the catalytic activity in a form of thin, flexible, and mechanically stable membrane. High accessibility of the artificial catalytic sites for the interaction with the analyzed phenol molecules was achieved due to addition of linear polymer (polyethyleneglycol Mw 20,000) to the initial monomer mixture before the polymerization. As a result, typical semi-interpenetrating polymer networks (semi-IPNs) were formed. The cross-linked component of the semi-IPN was represented by the highly cross-linked catalytic molecularly imprinted polymer, while the linear one was represented by polyethyleneglycol Mw 20,000. Extraction of the linear polymer from the fully formed semi-IPN resulted in formation of large pores in the membranes’ structure. Concentration of phenols in the analyzed samples was detected using universal portable device oxymeter with the oxygen electrode in a close contact with the catalytic molecularly imprinted polymer membrane as a transducer. The detection limit of phenols detection using the developed sensor system based on polymers–biomimics with the optimized composition comprised 0.063 mM, while the linear range of the sensor comprised 0.063–1 mM. The working characteristics of the portable sensor devices were investigated. Storage stability of sensor systems at room temperature comprised 12 months (87%). As compared to traditional methods of phenols detection the developed sensor system is characterized by simplicity of operation, compactness, andItem Open Access Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts(Elsevier Science B.V., Amsterdam., 2010-02-05T00:00:00Z) Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A.; Concheiro, Angel; Alvarez-Lorenzo, CarmenThis work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA·HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross- linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA·HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g−1 (i.e., 170 mg g−1) under flow (1 mL min−1) of 0.2 mM sodium cholate sItem 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 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 Design of molecularly imprinted polymers for sensors and solid phase extraction(Cranfield University, 2002-04) Subrahmanyam, Sreenath; Turner, Anthony P. F.; Piletsky, Sergey A.This thesis presents broadly the applications of molecularly imprinted polymers in sensors and solid phase extraction. Sensors for creatine and creatinine have been reported using a novel method of rational design of molecularly imprinted polymers (MIPs), and solid phase extraction of aflatoxin-B 1 has also been described in the thesis. A method for the selective detection of creataine and creatinine is reported in this thesis, which is based on the reaction between polymerised hemithioacetal, formed by allyl mercaptan, o-phthalic aldehyde, and primary amine leading to the formation of fluorescent isoindole complex. This method was demonstrated for the detection of creatine using creatine-imprinted MIPs. Since MIPs created using traditional methods were unable to differentiate between creatine and creatinine, a new approach to the rational design of a MIP selective for creatinine was developed using computer simulation. A virtual library of functional monomers was assigned and screened against the target molecule, creatinine, using molecular modeling software. The monomers giving the highest binding score were further tested using simulated annealing in order to mimic the complexation of the functional monomers with template in the monomer mixture. The result of this simulation gave an optimised MIP composition. The computationally designed polymer demonstrated superior selectivity in comparison to the polymer prepared using traditional approach, a detection limit of 25 μM and good stability. The 'Bite-and- Switch' approach combined with molecular imprinting can be used for the design of assays and sensors, selective for amino containing substances. MEP for the selective binding properties for aflatoxin-B 1 was prepared using the computational approach. The results obtained demonstrate that the MISPE offers a simple, convenient and a rapid methodology for solid phase extraction of aflatoxin-B 1 even at very low concentrations of 2 ppb. The commercially available C-18 cartridges were able to recover only about 52% of aflatoxin-B 1 at concentrations of 2 ppb when compared with almost complete recovery by the MIP. We have proved here that, MIPs as a solid phase extraction materials offer important and practical advantages with respect to other solid phase extraction methodologies.Item 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 Development of affinity sensors for Microcystin-LR based on a computationally designed molecularly imprinted polymer(Cranfield University, 2003-01) Chianella, Iva; Piletsky, Sergey A.; Chen, B.; Tothill, Ibtisam E.In this work the development of affinity sensors for the detection of microcystin-LR based on a computationally designed artificial receptor is presented. Microcystin-LR is a cyclic heptapeptide hepatotoxin produced by Cyanobacteria (aquatic organisms also known as blue-green algae), which during blooms period can release toxins in water. Clinical signs of hepatotoxicosis have been observed in domestic animals and livestock and recently also in humans. At present, analysis of these toxins is achieved largely using conventional, time consuming and expensive techniques such as chromatographic methods (HPLC, TLC) and immunoassay. Therefore, the necessity of an easy and inexpensive method of analysis such a biosensor is becoming urgent. In this work an artificial receptor for microcystin-LR was synthesised using a combined approach of molecular imprinting and computer modelling. A computer-aided rational design was applied to study microcystin-LRlmonomers interactions in order to find an optimal composition for the synthesis of the receptor. The optimised composition, suggested by computer modelling, consisted in 1 mol of2-acrylamido-2-methyl-propanesulfonic acid and 6 mol ofurocanic acid ethyl ester for 1 mol of template. This monomer composition was then used to synthesise a molecularly imprinted polymer (MIP) and an enzyme- linked competitive assay was developed to characterise the computational receptor. In the assay, computational MIP was able both to detect 0.1 ~g rl of microcystin-LR and to distinguish the analyte among analogues such as microcystin-YR, microcystin-RR and nodularin. The computationally designed receptor was then used as a sensing element for the construction of sensor devices. A MIP-based piezoelectric sensor, capable of detecting 35 ~g rl of toxin in water, was developed. In order to improve the system sensitivity, the computational polymer was also used as a material in solid-phase extraction (SPE) for samples pre-concentration. The receptor was able to pre-concentrate up to 1,000 fold tap water samples spiked with only 1 J.1g rl of toxin. By combining MIP-based SPE and piezoelectric sensor an improved system with a minimum detectable concentration of toxin of 0.35 ~g rl was achieved. Encouraging preliminary results were also obtained in developing a MIP-based electrochemical sensor.Item Open Access Development Of Novel Matrices For Biomolecule Immobilisation On Sensor Surfaces(Cranfield University, 2010) Kyprianou, Dimitris; Chianella, Iva; Piletsky, Sergey A.The development of a novel protocol for the covalent immobilisation of biomolecules containing primary amines using either polythiol compounds or novel, inexpensive and simple polymers is presented in this thesis. When developing biosensors, the method used for the immobilisation of the sensing elements is very important. The immobilisation needs to be fast, cheap and most importantly should not affect the biorecognition activity of the immobilised receptor. The chemistry used for the immobilisation is based on the well known reaction between primary amines and thioacetal groups, formed upon reaction of o-phthaldialdehyde (OPA) and thiol compounds. Initially the possibility to use this chemistry to immobilise receptors and develop biosensors was proved using commercially available polythiol compounds. Such compounds can be irreversibly adsorbed, creating self-assembling monolayers (SAMs), on noble metal transducer surfaces. These SAMs were immobilised on Biacore surface plasmon resonance (SPR) gold chips and then used to study kinetic of biomolecules interactions and to detect cells. A general protocol suitable for the immobilisation of enzymes and antibodies such as anti-prostate specific antigen (anti-PSA) and anti-Salmonella typhimurium antibody was optimised. Kinetic data were obtained for PSA binding to anti-PSA antibody and they were compared to the results obtained using commercially available Biacore chips, CM1. For Salmonella typhimurium cells, a detection limit of 5 × 106 cells ml-1 with minimal non-specific binding of other biomolecules was obtained. An interesting capability shown by these SAMs, in contrast with commercially available chips, was the opportunity to immobilise any proteins, even those with very low or high isoelectric points, pI. In addition protein immobilisation was achieved with a simple step, without requirement of any activation. These findings make this immobilisation technique a very promising alternative to peptide bond formation for amine coupling. Even though, the developed SAMs showed to be useful for certain type of applications (kinetic study and detection of very large analyte), it was clear that due to a combination of factors (e.g. limited and steric hindrance), they were not suitable for the development of biosensors good enough for practical applications. Therefore to overcome the drawbacks shown by polythiol SAMs, a novel 3-D polymer was developed. The main advantage of this polymer is the tridimensional (3D) network, which, after immobilisation, ensures the availability of a high percentage of receptor binding sites. As the polythiol SAMs, also the 3-D polymer contains thioacetal groups, which do not need any activation to react with primary amines in proteins. The novel 3-D polymer also contains thiol derivative groups (disulphide groups or thioethers) that promote self-assembling on metal surfaces. As before, the polymer was immobilised on SPR gold chips and the resulting layer was characterised using contact angle meter, atomic force microscopy (AFM) and ellipsometry. Contact angle demonstrated that the immobilisation of polymer on sensor surface produced a relatively hydrophobic surface. The thickness of polymer layer was determined by applying ellipsometry, whereas AFM showed the change of surface roughness after polymer attachment. A general protocol suitable for the immobilisation of BSA, enzymes and antibodies such as polyclonal anti-microcystin-LR 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 the antibodies. The detection limit for microcystin-LR was 10 ng ml and for PSA 0.05 ng ml. The 3-D polymer chips were stored for up to 2 months without any noticeable deterioration in their ability to react with proteins. The performance of 3-D polymer chips were also compared with commercially available Biacore chips, as CM5. The main advantages were found to be the low cost, the possibility to immobilise biomolecules at physiological pH (pH 7.4), the lack of any activation step for biomolecules immobilisation and the opportunity to immobilise proteins with very different pI (also very low pI). Despite the successful detection of PSA achieved in buffer (detection limit 0.05 ng ml-1) using 3-D polymer chips, the detection of proteins in serum resulted to be very challenging due to the complex nature of the matrix, which contains a high content of many different compounds. Different techniques were applied in order to reduce the non specific adsorption of serum on 3-D polymer sensors with antibodies immobilised on the surface. Satisfactory results were finally obtained by including the surfactant P20 into the measuring system. The detection of PSA in serum using 3-D polymer sensors, however, became possible only by switching from a direct detection to a ‘sandwich detection’. In this sandwich format, after injecting samples of PSA (prepared both in buffer or 20% serum) onto a specific antibody (capture-Ab, C-Ab) immobilised on the 3-D polymer surface, the analytical signal is recorded by injecting a second specific Ab (detection-Ab, prepared in PBS), which recognises a different epitope of the antigen. With this format, the analytical signal is recorded in absence of any complex matrix, avoiding interference from non specific adsorption. The detection limit for PSA, obtained using the sandwich immunosensor (developed on 3-D polymer chips) was 0.1 ng ml-1 in buffer and 5 ng ml-1 in 20% serum, which is very close to the sensitivity necessary for detection of the prostate biomarker in real samples. Therefore this study has demonstrated the opportunity to apply the novel 3-D polymer for development of biosensors suitable for applications in real samples.Item 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 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 Immunosensor for okadaic acid using quartz crystal microbalance(Elsevier Science B.V., Amsterdam., 2002-10-23T00:00:00Z) Tang, Alice X. J.; Pravda, Miloslav; Guilbault, George G.; Piletsky, Sergey A.; Turner, Anthony P. F.An immunosensor for the determination of okadaic acid (OA) using a quartz crystal microbalance (QCM) was developed and optimised in standard solutions. Several coupling techniques, protein A, protein G and polyethylenimine (PEI) with glutaraldehyde (GA) cross-linking, were investigated for the determination of okadaic acid and a very good result was obtained with PEI coupling. With the PEI coupling method, the optimisation of incubation time for the activation of PEI on the crystal surface using GA, the effect of the dilution factor of OA-BSA conjugate and the amount of antibody on crystal frequency were studied. Different molar ratios (4:1, 14:1, 30:1) of OA to bovine serum albumin (BSA) for the conjugation were examined and the results using ELISA and a QCM showed that a ratio of 14:1 was slightly better than the other two. The strong attachment of the cross-linked complex to the gold surface resulted in an excellent storage lifetime of 38 days. However, the detection limit (1.9 µg/ml) and the sensitivity of the sensor were not satisfactory. Significant improvement of the performance of the device was obtained by incorporating an antibody-BSA hydrogel. Initial results showed that the minimum amount of analyte detectable and the sensitivity of the device were improved by 524 and 80 fold, respectively
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