Bioconjugation and stabilisation of biomolecules in biosensors

Abstract

Suitable bioconjugation strategies and stabilisation of biomolecules on electrodes is essential for the development of novel and commercially viable biosensors. In the present review, the functional groups that comprise the selectable targets for practical bioconjugation methods are discussed. We focus on describing the most common immobilisation techniques used in biosensor construction, which are classified into irreversible and reversible methods. Concerning the stability of proteins, the two main types of stability may be defined as (i) storage or shelf stability, and (ii) operational stability. Both types of stability are explained, as well as the introduction of an electrophoretic technique for predicting protein-polymer interactions. In addition, solution and dry stabilisation as well as stabilisation using the covalent immobilisation of proteins are discussed including possible factors that influence stability. Finally, the integration of nanomaterials, such as magnetic particles, with protein immobilisation is discussed in relation to protein stability studies.

Keywords: bioconjugation; bioreceptor immobilisation; electrochemical biosensors; magnetic particles; protein stability; screen-printed electrodes.

Figure 1.. Irreversible immobilisation methods

Schematic representation of some irreversible methods: (a) covalent binding through primary amines, (b) cross-linking using carbodi-imide, and (c) entrapment on beads or fibres and micro-encapsulation.

Figure 2.. Reversible immobilisation methods

Schematic representation of some reversible methods: (a) adsorption, physical and ionic binding, (b) bioaffinity with strept(avidin)/biotin and Protein A/G, (c) chelation or metal binding, and (d) disulfide bonds.

Figure 3.. Stabilisation by protein immobilisation

Schematic representation of (a) magnetic particles, (b) activated with functional groups, and (c) conjugated to biological molecules [32]; (d) solution stability of antibody-modified magnetic particles over 3 months at 32°C using stabilisers from Applied Enzyme Technology Ltd. Activity tested by optical magneto-immunoassay using 15 ng/ml PSA, 0.1 mg/ml anti-PSA antibody-modified magnetic particles (Abcam, ab10184) and 1 μg/ml horseradish peroxidase (HRP)-labelled antibody (Abcam, ab24466).