New reactive polymer for protein immobilisation on sensor surfaces

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 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 ngmL(-1) and for PSA 0.01 ngmL(-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.

Development of Non-Labeled QCM Biosensor for the Detection of <i>β</i>-Galactosidase: A Comparative Study of Gold and Polystyrene Nanoparticles

“…Nanofilms on transducer surfaces have the advantage of allowing easy miniaturization and creation of novel functional interfaces. Selfassembled monolayer of compounds containing thiol groups have shown to be an attractive mean to chemically modify surfaces for various applications like controlling surface wettability, structuring surfaces, binding of species such as metal ions, nanoparticles, and biomolecules [8][9][10][11].…”

Impact of ligand density on the optimization of ion‐exchange membrane chromatography for viral vector purification

“…The three new sensor chips employed here were customized to expose different ligand densities: a SAM on a bare gold sensor surface, MUA-DEAE; a 30 nm long dextran layer chip, CM3-DEAE (Kyprianou et al, 2009); and a 100 nm long dextran layer chip, CM5-DEAE (Wofsy and Goldstein, 2002;Yu et al, 2004). All three chips were covalently immobilized with DEAE ligands.…”

“…For CM3-DEAE and CM5-DEAE, DEAE ligands were covalently immobilized to either a CM3 sensor chip or a CM5 sensor chip (all Biacore/GE Healthcare), which contains a dextran extension layer of 30 nm (Kyprianou et al, 2009) or 100 nm (Wofsy and Goldstein, 2002;Yu et al, 2004), respectively, over the sensor surface with available carboxyl groups for coupling chemistry.…”

The application of polythiol molecules for protein immobilisation on sensor surfaces

“…Covalent immobilisation includes amino coupling Piletska et al, 2001), aldehyde coupling (Abraham et al, 1995) and thiol coupling methods . The covalent attachment can also occur on gold surfaces modified with polymers such as carboxydextran matrix and thioacetal matrix (Kyprianou et al, 2009) or self-assembled monolayers (Nuzzo and Allara, 1983). The selection of the immobilisation procedure is a critical point for the development of a successful sensor.…”