Current Conjugation Methods for Immunosensors

Abstract

Recent advances in the development of immunosensors using polymeric nanomaterials and nanoparticles have enabled a wide range of new functions and applications in diagnostic and prognostic research. One fundamental challenge that all immunosensors must overcome is to provide the specificity of target molecular recognition by immobilizing antibodies, antibody fragments, and/or other peptides or oligonucleotide molecules that are capable of antigen recognition on a compact device surface. This review presents progress in the application of immobilization strategies including the classical adsorption process, affinity attachment, random cross-linking and specific covalent linking. The choice of immobilization methods and its impact on biosensor performance in terms of capture molecule loading, orientation, stability and capture efficiency are also discussed in this review.

Keywords: immobilization methods; immunosensors; polymeric nanomaterials.

Figure 1

Overall structure of Ab and alternative recombinant binder scaffolds used in biosensors. (A) IgG2a monoclonal antibody with two heavy chains colored in green and two light chains colored in blue (PDB:1IGT) [18]; (B) The green fluorescent protein (GFP)-VHH (PDB: 3OGO) [19]; (C) DARPin against tubulin beta chain (PDB: 4DUI) [20]; (D) anti-fluorescein ScFv (PDB: 1X9Q) [21]; (E) human epidermal growth factor receptor 2 (HER2) binding affibody (PDB: 2KZJ) [22]; (F) 5-hydroxytryptophan aptmer (PDB: 5KPY) [23]. We used PyMol to generate all the structures in this figure.

Figure 2

Functional groups on Abs used for conjugation and the result of random and oriented immobilization onto surfaces.

Figure 3

Ab immobilization scheme. (A) EDC/NHS coupling of Ab surface amine to carboxyl and (B) carboxyl groups to amine groups; (C) Sulfhydryl-reactive chemical group coupling to Ab surface thiol groups; (D) Reduction of antibody disulfides to reactive thiols for gold substrates binding; (E) Reduction of antibody disulfides for site specific pyridazinedone coupling; and (F) Oxidation of sugar chains for reactive aldehyde groups.

Figure 4

Oriented immobilization scheme for VHHs. (A) C-terminal N₃ group introduced by artificial amino acid incorporation followed by conversion to biotin group for streptavidin binding; and (B) C-terminal N₃ group attached via sortase mediated transpeptidation followed by site specific attachment on DBCO modified surface.

Figure 5

ScFv and gold-binding peptide dual-expression system in E. coli [60].