2.2.1  Antibodies and antibody fragments
Antibodies and antibody fragments are among the most commonly utilized biorecognition elements for pathogen detection using electrochemical biosensors. Biosensors employing antibody-based biorecognition elements are commonly referred to as immunosensors. Given antibodies exhibit high selectivity and binding affinity for target species and can be generated for a wide range of infectious agents, antibodies are the gold-standard biorecognition element for pathogen detection. Antibodies contain recognition sites that selectively bind to antigens through a specific region of the antigen, referred to as an epitope (Patris et al. 2016). Antibodies can be labeled with fluorescent or enzymatic tags, which leads to the designation of the approach as label-based. While label-based approaches present measurement constraints associated with the use of additional reagents and processing steps (Cooper, 2009; Sang et al. 2016), antibody labeling may also alter the binding affinity to the antigen, which could affect the biosensor's selectivity. A detailed discussion of label-based biosensing approaches for pathogen detection has been reported elsewhere (Ahmed et al. 2014; Alahi and Mukhopadhyay, 2017; Bozal-Palabiyik et al. 2018; Leonard et al. 2003). A list of recent label-based approaches for pathogen detection using electrochemical biosensors, however, is provided in Table 2.
While both monoclonal and polyclonal antibodies enable the selective detection of pathogens (Patris et al. 2016), they vary in terms of production method, selectivity, and binding affinity. Monoclonal antibodies are produced by hybridoma technology (Birch and Racher, 2006; James and Bell, 1987). Thus, monoclonal antibodies are highly selective and bind to a single epitope, making them less vulnerable to cross-reactivity. While monoclonal antibodies tend to have a higher degree of selectivity, they are more expensive and take longer to develop than polyclonal antibodies. Polyclonal antibodies are produced by separation of immunoglobulin proteins from the blood of an infected host (Birch and Racher, 2006). Polyclonal antibodies target different epitopes on a single antigen. While polyclonal antibodies exhibit increased variability between batches, they are relatively less expensive to produce than monoclonal antibodies and facilitate robust measurements in various settings (Byrne et al. 2009). Drawbacks to antibody use include high cost and stability challenges, such as the need for low-temperature storage. As shown in Table 1, Table 2, both monoclonal and polyclonal antibodies are used as biorecognition elements for pathogen detection. For assays involving secondary binding steps, monoclonal antibodies typically serve as the primary biorecognition element and are immobilized on the electrode, while polyclonal antibodies serve as the secondary biorecognition element and often facilitate target labeling. For assays that do not require secondary binding steps, polyclonal antibodies are also commonly used as immobilized biorecognition elements for pathogen detection. For example, Pandey et al. immobilized monoclonal anti-E. coli on a composite nanostructured electrode to detect E. coli across a wide dynamic range of 10 to 108 CFU/mL with a LOD of 3.8 CFU/mL (Pandey et al. 2017). Wu et al. used polyclonal anti-E. coli for detection of E. coli via amperometry that exhibited a LOD of 5 × 103 CFU/mL (Wu et al. 2016). Lin et al. used monoclonal antibodies for detection of avian influenza virus H5N1 in chicken swabs across a dynamic range of 2- 1 to 24 hemagglutination units (HAU)/50 μL using EIS and the ferri/ferrocyanide (Fe(CN)6 3 - /4-) couple as a redox probe (Lin et al. 2015). Luka et al. detected Cryptosporidium parvum (C. parvum) with a LOD of 40 cells/mm2 via capacitive sensing and Fe(CN)6 3 - /4- (Luka et al. 2019).
Antibody fragments, such as single-chain variable fragments (scFvs), offer selectivity similar to antibodies, but they have the advantage of achieving relatively higher packing densities on electrode surfaces due to their relatively smaller size. For example, half-antibody fragments have been shown to improve biosensor sensitivity without the loss of selectivity, which warrants further investigation of reduced antibodies as biorecognition elements for pathogen detection applications (Sharma and Mutharasan, 2013). In addition to scFvs, Fabs, re-engineered IgGs, and dimers can also potentially be used as biorecognition elements for pathogen detection (Byrne et al. 2009).