Affibody as an alternative bioreceptor for biosensors

Antibodies and aptamers are the two most commonly used bioreceptor types in biosensor development. Antibodies are large, Y-shaped proteins (~150 kDa) composed of heavy and light chains linked by disulfide bonds and generally contain glycosylated groups. Antibodies are produced by the immune system's response to specific antigens and are highly specific. However, antibodies have several limitations, such as poor stability to heat and temperature fluctuations, a relatively short shelf life and limited modification options that can reduce binding affinity [64]. Alternatively, aptamers are developed as synthetic bioreceptors consisting of small, single-stranded DNA or RNA molecules (6 to 30 kDa) obtained through an in vitro selection process using the SELEX method. Aptamers offer advantages such as good thermal stability, resistance to temperature variations, a long shelf life and ease of chemical modification without reducing binding affinity. However, aptamers also have disadvantages, particularly their susceptibility to degradation by nuclease enzymes in the blood [52,64,65].

Due to the limited availability of antibodies and aptamers, affibody has emerged as a superior alternative bioreceptor for biosensor applications. Affibody is an alternative binding protein derived from the Z domain of Staphylococcal protein A [64]. It is a small protein (~7 kDa) composed of 58 amino acids arranged in an alpha-helical structure, as shown in Figure 2 [66].

Figure 2.: Structure of an affibody scaffold. Asterisks mark the segments where randomized amino acids are located. Reproduced from [66] with copyright permission

Affibody acts as an immune-independent affinity molecule capable of targeting a wide variety of proteins [22]. Due to its alpha-helical composition, affibody lacks disulfide bonds, allowing its use in intracellular applications and enabling production in simple organisms such as prokaryotes rather than animal-based systems required for antibody synthesis [67]. A comparison of affibody, antibody and aptamer bioreceptors for biosensing applications is shown in Table 2.

Characteristics | Structure | Synthesis process | Other properties | Reference
Affibody | Small protein molecule (~7 kDa) consisting of 58 amino acids in alpha-helical form without disulfide bonds | Recombinant expression or SPPS | High solubility and stability under various conditions, strong binding affinity | [21,22]
Antibody | Y-shaped protein molecule (150 kDa) consisting of a chain of amino acids having heavy and light chains, disulfide bonds and glycosylation groups | Produced by the immune system in response to foreign substances | High specificity, short shelf life, not heat stable, temperature-resistant, limited modifications before losing binding affinity | [64]
Aptamer | short single-stranded DNA or RNA molecules (6 to 30 kDa) in the form of Secondary structures (loops, hairpins, stems) [22-24]. | SELEX | Long shelf life, heat-stable, temperature-resistant, modifiable without decreasing binding affinity, easily degraded by enzymes in the blood | [19,68,69]