*4.2. Antibody*

Immunoassay is based on detecting antigen–antibody conjugates or excessive other reagents (e.g., enzyme-labelled second antibody). Furthermore, it can be divided into competitive mode and non-competitive mode based on whether the analyte competes for a restricted number of binding antibody sites with the labelled analyte (e.g., indirect competitive immunoassay) or not (e.g., sandwich format) [61]. Immunoassays can also be classified as homogeneous and heterogeneous assays. Antibodies and antigens move freely from a complex immune situation to the solution phase in a homogenous format. However, it can be seen differently in a heterogeneous structure where the antibodies (or sometimes antigens) can be immobilized on a solid support to form the complex. Both types have been widely investigated, but homogeneous assays benefit from the possibility of multiplexing the complex format and separations are fast.

In contrast, the heterogeneous structure takes advantage of the elevated ratio of surface area to volume, which provides an additional higher sensitivity. Electrochemical immune sensors exhibit high sensitivity and selectivity compared to redox detection, which is extremely important in detecting various pesticides to decrease their mutual interference. The detection principle is mainly based on the current or impedance changes induced by antibody–antigen interaction, including chronoamperometry (CA) and electrochemical impedance spectroscopy (EIS).

A non-competitive immunoassay combined the with magneto-electrochemical immune sensors. It was developed to detect herbicide atrazine, one of the most used pesticides globally [62]. It is based on the recombinant M13 phage particles that bear a molecule named peptide. It is recognized explicitly as the immune complex of atrazine with an anti-atrazine monoclonal antibody. However, it is worth mentioning that each phage bore thousands of HRP Molecules, indicating the increased activity of pyrocatechol oxidation in the presence of hydrogen peroxide (H2O2). The phage anti-immunocomplex electrochemical immunosensor (PhAIEI) had dominant features, which provided a 200-fold improvement in sensitivity and a 10-fold wide linear working range compared with previous work with the same monoclonal antibody and anti-immunocomplex peptide. By chronoamperometry (CA), the fabricated PhAIEI was successfully applied in untreated river samples with excellent recoveries.

The leucomalachite green and malachite green in the water from a fish farm were detected by a BSA-decorated gold nanocluster (BSA-AuNC) with antibody composite film using the electrochemical impedance spectroscopy (EIS) method. The film was modified by a glassy carbon electrode (GCE). The modification was carried out due to the potential hazards to the human immune system and the human reproductive system [63]. Moreover, the BSA-AuNCs interface's stability was improved via a diazotization method, and the antibody against leucomalachite green was chemically connected with the interface under the optimum conditions. After two weeks, the EIS immunosensor showed acceptable repeatability and stability with a negligible impedance reduction. A low LOD of 0.03 ng/mL was also obtained and compared with the ELISA method.

Azri et al., have developed an ultrasensitive electrochemical immunosensor for the detection of aflatoxin B1 (AFB1) based on an indirect competitive enzyme-linked immunosorbent assay (ELISA) to study the antigen–antibody interaction and optimize the optimum parameters of the assay [55]. The immunosensor demonstrated an excellent duplicability (RSD of 9%), and the response was logarithmic, where the detection range of 50–10,000 pM of IMD under the optimal conditions. The sensor was developed with the combination of BSA-labelled antigen and enzymatic tags. Compared with standard analytical methods, the developed sensor demonstrated a more comprehensive lower detection limit and a comprehensive range of responses which satisfied the detection requirements considering the European Union legislation. Saravanan et al., proposed a simple, disposable, and low-cost, paper-based immunosensor to detect bacteria in water [64]. The screen-printed fabrication technique was used for printing a conductive carbon electrode onto a commercial hydrophobic paper. Carboxyl groups were utilized for functionalization with the lectin Concanavalin A, which was covalently immobilized as the selective coating for biorecognition element for interacting with mono- and oligosaccharides. A linear calibration curve was developed for bacterial concentrations ranging from 103~106 CFU mL<sup>−</sup>1, with the projected lower detection limit of 1.9 × <sup>10</sup><sup>3</sup> CFU mL<sup>−</sup>1.

Immunomagnetic assays with the introduction of magnetic beads (MBs) are particularly effective for enhancing the analytical performance. A huge surface area allows them to be utilized in immobilization of biomolecules, such as enzymes, DNA, and antibodies. Chemical and physical stability, low toxicity, and high biocompatibility make them suitable for the immobilization of biomolecules. Efficient dispersing ability enables them to shorten the reaction time between dissolved species and biomolecules [54]. The electrochemical glyphosate (N-(phosphonomethyl)glycine) biosensor has been developed with a disposable screen-printed electrochemical cell and applied to the analysis of spiked beer samples based on the competitive assay, as is shown in Figure 1c [54]. With tetramethylbenzidine (TMB) as the enzymatic substrate, the affinity reaction's scope has been achieved by monitoring the current (A) due to reducing the enzymatic effect. The concentration range was found as 0–10,000 ng, where the detection limit was 5 ng/L and the quantification limit was 30 ng/L. An indirect competitive ELISA was exhibited in Figure 1d [55], competition occurred between aflatoxin B1-bovine serum albumin (AFB1–BSA) and free AFB1 (in peanut sample and standard) for the binding site of a fixed amount of anti-AFB1 antibody on the multiwalled carbon nanotubes/chitosan/screen-printed carbon electrode (MWCNTs/CS/SPCE). Figure 1e showed the effect of various blocking agents on background reading by eight percent skimmed milk, one percent BSA, casein, protein-free, and superblock [55].
