| 1. |
- Kyprianou, Dimitris, et al.
(författare)
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New reactive polymer for protein immobilisation on sensor surfaces
- 2009
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Ingår i: Biosensors & bioelectronics. - : Elsevier Science B.V., Amsterdam.. - 0956-5663 .- 1873-4235. ; 24:5, s. 1365-1371
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Tidskriftsartikel (refereegranskat)abstract
- 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 tridimensional (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 biosensors. (C) 2008 Elsevier B.V. All rights reserved.
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| 2. |
- Kyprianou, Dimitris, et al.
(författare)
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The application of polythiol molecules for protein immobilisation on sensor surfaces
- 2010
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Ingår i: Biosensors & bioelectronics. - : Elsevier Science B.V., Amsterdam.. - 0956-5663 .- 1873-4235. ; 25:5, s. 1049-1055
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Tidskriftsartikel (refereegranskat)abstract
- The immobilisation of bio-receptors 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. The development of a protocol for biomolecule immobilisation onto a surface plasmon resonance (SPR) sensor surface using inexpensive polythiol compounds is presented here. The method used here is based on the reaction between primary amines and thioacetal groups, formed upon reaction of o-phthaldialdehyde (OPA) and thiol compounds. The self-assembled thiol monolayers were characterised using contact angle and XPS. The possibility to immobilise proteins on monolayers was assessed by employing BSA as a model protein. For the polythiol layers exhibiting the best performance, a general protocol was optimised suitable for the immobilisation of enzymes and antibodies such as anti-prostate specific antigen (anti-PSA) and anti Salmonella typhimurium. The kinetic data was obtained for PSA binding to anti-PSA and for S. typhimurium cells with a detection limit of 5 x 10(6) cells mL(-1) with minimal non-specific binding of other biomolecules. These findings make this technique a very promising alternative for amine coupling compared to peptide bond formation. Additionally, it offers opportunity for immobilising proteins (even those with low isoelectric point) on neutral polythiol layers without any activation step. (C) 2009 Elsevier B.V. All rights reserved.
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| 3. |
- R. Guerreiro, Antonio, et al.
(författare)
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Influence of continuous magnetic field on the separation of ephedrine enantiomers by molecularly imprinted polymers
- 2008
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Ingår i: Biosensors & bioelectronics. - : Elsevier Science B.V., Amsterdam.. - 0956-5663 .- 1873-4235. ; 23:7, s. 1189-1194
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Tidskriftsartikel (refereegranskat)abstract
- 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 1.55T 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 sites. (c) 2007 Elsevier B.V. All rights reserved.
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| 4. |
- V. Piletska, Elena, et al.
(författare)
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Design of molecular imprinted polymers compatible with aqueous environment
- 2008
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Ingår i: Analytica Chimica Acta. - : Elsevier Science B.V., Amsterdam.. - 0003-2670 .- 1873-4324. ; 607:1, s. 54-60
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Tidskriftsartikel (refereegranskat)abstract
- The main problem of poor water compatibility of molecularly imprinted polymers (MIPs) was addressed in examples describing design of synthetic receptors with high affinity for drugs of abuse. An extensive potentiometric titration of 10 popular functional monomers and corresponding imprinted and Blank polymers was conducted in order to evaluate the subtleties of functional groups ionisation under aqueous conditions. It was found that polymers prepared using 2-trifluoromethacrylic acid (TFMAA) in combination with toluene as porogen possess superior properties which make them suitable for effective template recognition in water. The potential impact of phase separation during polymerisation on formation of high quality imprints has been discussed. Three drugs of abuse such as cocaine, deoxyephedrine and methadone were used as template models in polymer preparation for the practical validation of obtained results. The polymer testing showed that synthesized molecularly imprinted polymers have high affinity and selectivity for corresponding templates in aqueous environment, with imprinting factors of 2.6 for cocaine and 1.4 for methadone and deoxyephedrine. Corresponding Blank polymers were unable to differentiate between analytes, suggesting that imprinting phenomenon was responsible for the recognition properties. (c) 2007 Elsevier B.V. All rights reserved.
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