Elsevier

Biosensors and Bioelectronics

Volume 50, 15 December 2013, Pages 460-471
Biosensors and Bioelectronics

Site-directed antibody immobilization techniques for immunosensors

https://doi.org/10.1016/j.bios.2013.06.060Get rights and content

Highlights

  • Antibody orientation on the surface is a pivotal step in immunosensor development.

  • Three most common antibody site-directed immobilization strategies are reviewed.

  • Immobilization via Fc binding proteins, oxidized oligosaccharides and sulfhydryl groups of the hinge region.

  • The principles, advantages, and arising problems of these techniques are discussed.

  • Comparison of different antibody immobilization strategies is presented.

Abstract

Immunosensor sensitivity, regenerability, and stability directly depend on the type of antibodies used for the immunosensor design, quantity of immobilized molecules, remaining activity upon immobilization, and proper orientation on the sensing interface. Although sensor surfaces prepared with antibodies immobilized in a random manner yield satisfactory results, site-directed immobilization of the sensing molecules significantly improves the immunosensor sensitivity, especially when planar supports are employed. This review focuses on the three most conventional site-directed antibody immobilization techniques used in immunosensor design. One strategy of immobilizing antibodies on the sensor surface is via affinity interactions with a pre-formed layer of the Fc binding proteins, e.g., protein A, protein G, Fc region specific antibodies or various recombinant proteins. Another immobilization strategy is based on the use of chemically or genetically engineered antibody fragments that can be attached to the sensor surface covered in gold or self-assembled monolayer via the sulfhydryl groups present in the hinge region. The third most common strategy is antibody immobilization via an oxidized oligosaccharide moiety present in the Fc region of the antibody. The principles, advantages, applications, and arising problems of these most often applied immobilization techniques are reviewed.

Introduction

Despite the long-lasting interest and multiple efforts in immunosensor design, few examples of commercialization have been reported (Deacon et al., 1991, Ruano-Lopez et al., 2009, Johnson et al., 2011), the most widely known immunosensor being the pregnancy test for the detection of human chorionic gonadotropin (hCG). At first glance, this might look surprising as immunosensors seem to be a promising and attractive technique, offering high specificity due to the use of very specific immune molecules, non-destructive approach to sample, simple operation, uncomplicated sample preparation and, most importantly, high sensitivity, especially when different signal transducers have recently been combined to yield low limits of detection (Ramanaviciene et al., 2012). However, in order to achieve the characteristics mentioned above, some important issues have to be resolved because the sensitivity, stability and longevity of an immunosensor for the most part depends on the amount of the immobilized immune molecules on the surface, their conformational stability, remaining activity after the immobilization procedure and their orientation on the sensor surface, especially considering immunoglobulins, which are asymmetrical molecules with their recognition sites taking different positions in space following different immobilization procedures, and thus leading to hindered interactions with the analyte. Schematic representation of an antibody molecule is depicted in Fig. 1.

Although both antigen and antibody can be immobilized on the sensor surface (Lu et al., 1996, Rao et al., 1998, Jung et al., 2008a, Holford et al., 2012, Zeng et al., 2012), in this review we will mainly focus on site-directed antibody immobilization. It is a very attractive technique, especially in clinical applications as it is simple, precise, allows direct analyte detection, and could be used in cases when the immune response is minimal.

There are two main approaches that can be used in antibody-based sensor surface preparation: random and site-directed antibody immobilization. The main principles of these approaches are presented in Fig. 2. This review is mainly centered on site-directed antibody immobilization on planar supports. Despite many publications reporting the benefit of the site-directed antibody orientation on flat surfaces, it has been suggested that on three dimensional supports different antibody orientations result in only minor differences of specific activity (Johnsson et al., 1995). In contrast, the significance of antibody orientation on a three dimensional support has been shown in another publication (Patel and Andrien, 2010). Thus it is extremely difficult to evaluate the role of antibody orientation on irregular surfaces. However, certain studies investigating this problem have been included in the review for illustrative purposes. On the other hand, planar supports are of a smaller surface area, so the influence of antibody density and steric effects are very explicit and in this case site-directed antibody immobilization is a valuable tool for adjusting these characteristics that should definitely be considered. Different supports have been reported to be used for immunosensor design, for example, noble metals, especially gold (Hafaiedh et al., 2013), glass (Tedeschi et al., 2003, Zhao et al., 2006), silicon (Yakovleva et al., 2002, Dhanekar and Jain, 2013), silicon nitride (Caballero et al., 2012, Kurihara et al., 2013), indium–tin oxide (Bandodkar et al., 2010). The immobilization support is usually determined by the method used for signal transducer and can be modified for different purposes.

The simplest sensor preparation technique is based on random adsorption of antibody molecules on the sensor surface. Although adsorption does not require the use of multiple materials and complex reactions, it also results in serious disadvantages, such as denaturation of proteins, very low stability and random protein orientation (Buijs et al., 1996, Hlady and Buijs, 1996, Wiseman and Frank, 2011).

The most commonly used technique is, however, antibody amine coupling to the sensor surface previously modified with different coatings that allow biomolecule immobilization, such as, self assembled monolayers, dextran or various polymers (Zhou et al., 2006, Kyprianou et al., 2013). Niemeyer's group developed strategy based on protein conjugation to oligonucleotides, for further hybridization and immobilization on a surface (Niemeyer et al., 1994). Such an approach has been successfully used for hybrid molecule cleavage and regeneration on a sensor (Bombera et al., 2012).

A self assembled monolayer (SAM) is a layer formed of n-carbon atom alkyl chains with certain functional groups, in many cases a group enabling the molecules to attach to the sensor surface and a carboxyl group that later can be used for the formation of a peptide bond with amino groups randomly scattered on the antibody molecule surface. Different variants of SAMs are available, not only modified with different functional groups for various immobilization strategies but also exhibiting disparate responses to non-specific binding (Silin et al., 1997, Stigter et al., 2005). Direct antibody immobilization via SAMs has multiple advantages, such as a well-known, tested and quite simple immobilization technique, stability and reusability of immunosensors due to covalent bonds. However, a critical drawback is the relatively small sensitivity caused by random antibody orientation and subsequent decreased availability of antibody active sites to antigen in comparison to site-directed antibody immobilization methods (Tsai and Pai, 2009, Kausaite-Minkstimiene et al., 2010), although in some cases very efficient random immobilization based immunosensors have been reported (Billah et al., 2008, Mattos et al., 2012).

In order to avoid random antibody immobilization and improve antigen binding site availability, site-directed antibody immobilization methods are being constantly developed, e. g., incubation of the antibody with its antigen prior to immobilization on a mixed self-assembled monolayer, so that the active sites would remain protected from the active groups of the support (Yoon et al., 2011), cyclic voltammetry assisted coupling of the hydroxyl groups of the oligosaccharide moieties present on antibodies to the cyano groups of the poly-(2-cyano-ethylpyrrole) (Um et al., 2011), the use of UV irradiation to break the disulfide bridges upon adsorption by nearby tryptophan residues, this way freeing the sulfhydryl groups that can be directly coupled to the gold surface in an controlled manner (Della Ventura et al., 2011), calixarene derivatives able to orient an antibody in a site-directed manner (Oh et al., 2005), and fusion proteins, such as pentamerized bispecific antibodies (decabodies) (Hussack et al., 2009), ZZ-alkaline phosphatase-His fusion protein exhibiting Fc binding (Yang et al., 2013) or cutinase-single chain antibody fusion proteins that allow the oriented immobilization of cutinase to phosphonate ligands (Kwon et al., 2004) to name a few. However, the most popular and widely employed site-directed antibody immobilization techniques are immobilization via Fc binding proteins, via antibody fragments and via oxidized oligosaccharide moieties. These antibody immobilization methods, their advantages and problems, possible solutions, comparison among different techniques and new approaches will be reviewed in this publication. Since the authors tried to relate the advantages and disadvantages of the methods with the immobilization mechanism, a clearer and a more compact comparison of the discussed techniques is presented in Table 1.

Section snippets

Site-directed immobilization via proteins binding the Fc region of immunoglobulins

Proteins A and G have been firstly and most widely used in affinity chromatography, especially for antibody purification (Aybay and Imir, 2000, Burckstummer et al., 2006, Evazalipour et al., 2012), but numerous uses of these proteins in immunosensor design have also been reported. By binding the Fc region of an immunoglobulin these proteins immobilize immunoglobulins on the surface in a site-directed manner with antigen-binding regions directed towards the analyte (Fig. 3A) (Sauer-Eriksson et

Comparison of different antibody immobilization strategies

Since all site-directed antibody immobilization methods described above seem promising, it is interesting to investigate what is the relation between the randomly oriented antibodies and immobilized in a site-directed manner and to compare the results obtained by different site-directed immobilization strategies. However, not only different approaches to evaluating antigen binding of the immobilized ligands are presented in publications reviewed in this chapter, e.g., sensitivity, kinetic

Conclusions

Despite constantly appearing new trends in oriented antibody immobilization, most published immunosensor applications are still based on the more conventional techniques, i.e., the Fc binding proteins, antibody fragments and immobilization via the oxidized oligosaccharide moiety in some cases additionally improved by certain modifications (e.g., biotin and avidin/streptavidin or signal molecule tagging). From the overview of literature it seems that neither of these methods is significantly

Acknowledgements

This research is funded by a grant (No. MIP-059/2012) from the Research Council of Lithuania.

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