*4.1. Microcystins*

Microcystins (MCs), a group of toxins produced by a number of cyanobacteria species, are monocyclic heptapeptides with the general structure cyclo(D)-Ala-X-(D)-erythro-b-methyliso-Asp-Y-Adda-(D)-iso-Glu-N-meth-yldehydro-Ala (X and Y represent L-amino acids). They are the most common cyano-toxins [138–142]. The unusual Adda amino acid, unique to MCs, is responsible for the toxicity of the molecule. There are more than 100 known variants of MCs, which are found in a wide variety of aquatic environments, in particular, eutrophic waters. Exposure to MCs via consumption of poisoned drinking-water or eating contaminated fish can cause permanent multiple organ injuries, developmental effects, reproductive effects and cancer. Therefore, it is important to develop highly sensitive methods for on-site monitoring of MCs. In addition, as the most potent congener, the Microcystin-LR (MC-LR) is commonly used to evaluate the toxicological data on the effects of MCs. The maximum tolerance limit of MC-LR concentration is 1 μg L−<sup>1</sup> in different water sources by the WHO provisional guideline. Electrochemical biosensors, including 2D nanomaterial-based amperometric immunosensors, impedimetric aptasensors, and PEC aptasensors, have been extensively employed to detect MCs/MC-LR [143–159]. Li et al. have fabricated an electrochemical immunosensor based on GO-AuNP nanocomposites for MC-LR detection in water samples though layer-by-layer alternate electrodeposition of GO and chloroauric acid (HAuCl4) on the GCE surface for 20 cycles [147]. The GO-AuNP-decorated GCE was then modified by the conducting polymer (poly(2,5-di-(2-thienyl)-1-pyrrole-1-(p-benzoicacid)) and 1-iso-butyl-3-methylimidazolium bis(tri-fluoromethane-sulfonyl) imide ionic liquid (IL). A polyclonal antibody of MC-LR was immobilized on the electrode by the conventional EDC/NHS reaction. The GO-AuNP nanocomposites enhance electron transfer of Fe(CN)6 3−/4− to the electrode while the IL acts as the stabilizer of the antibody. The as-developed electrochemical immunosensor has good repeatability (e.g., RSD = 1.2%) and long-term stability (e.g., retain 95% activity over a 20 weeks storage period), and can detect MC-LR in water samples with a very low LOD of 3.7 × 10−<sup>17</sup> mol L−1. Recently, He et al. synthesized a kind of magnetic rGO nanocomposite (Fe3O4@PDA/RGO) for constructing a MC-LR electrochemical immunosensor by using the hydrothermal treatment of Fe3O4 nanocluster@Polydopamine core@shell nanoparticles (Fe3O4@PDA) with GO (as shown in Figure 8) [153]. Due to its surface area and easy separation, the Fe3O4@PDA/RGO clearly enhances the antigen immobilization ability of the electrode. Then, a secondary-antibody and circularization DNA template were conjugated on gold nanorods (AuNRs) for recognizing the captured MC-LR-antibody pair on the Fe3O4@PDA/RGO-modified electrode surface and rolling circle amplification. Because the rolling circle amplification strategy can generate massive repeated DNA sequences, the signal of the immunosensor is greatly enhanced by

hybridization of electrochemical active probes with the repeated DNA sequences. Under the optimal conditions, the as-developed immunosensor has good detection performance including a wide linear range (from 0.01 mg L−<sup>1</sup> to 50 mg <sup>L</sup>−1) and a low LOD (0.007 mg <sup>L</sup>−1), which can be employed to detect MC-LR in real samples (e.g., river water). A series of PEC aptasensor-based various GO/rGO nanocomposites have been developed for sensitively detecting MC-LR since the PEC method has been considered to be a more sensitive technique, ascribed to the combination of electrochemical and optical techniques [149,151,157]. For instance, Du et al. developed a PEC aptasensing platform based on AgI-nitrogen-doped graphene (AgI-NG) nanocomposites as photo-cathodes and a MC-LR aptamer as the recognition unit [157]. The PEC aptasensor has a LOD of 3.7 × 10−<sup>17</sup> mol <sup>L</sup>−1, which can be employed to determine MC-LR in inaquatic products (e.g., fish extracts). As a graphene analogue, the MoS2 nanosheet is also expected to serve as an excellent functional material for development of electrochemical biosensors. As shown in Figure 9, Pang et al. constructed an enzyme-free electrochemical immunosensor for detecting MC-LR based on a unique competitive detection scheme using MoS2 nanosheets/BSA-stabilized gold nanocluster (MoS2/AuNCs) nanocomposites and Au core/Pt shell nanoparticles (Au@PtNPs) [155]. Due to its large surface area and excellent biocompatibility, the MoS2/AuNCs nanocomposite was employed as a platform for improving the biological activity and immobilizing amount of antibody on the electrode surface. The as-developed enzyme-free electrochemical immunosensor has good stability (e.g., 92% of the initial level remained after being stored at 4 ◦C for four weeks), and exhibits a wide linear range of 1.0 ng L−1–1.0 mg L−<sup>1</sup> with a LOD of 0.3 ng L−1. The practicability of the as-developed immunosensor has been demonstrated by detection of MC-LR in various water samples including tap water, lake water, and river water. The MC-LR amounts in these water samples detected by the immunosensor are consistent with those determined by the conventional ELISA method. Very recently, Liu et al. developed an electrochemical aptasensor for sensitive and selective determination of microcystin-LR by using a dual signal amplification system consisting of a ternary nanocomposite and HRP [159]. The ternary nanocomposites were prepared by depositing AuNPs on the MoS2 nanosheets covered with TiO2 nanobeads (TiONBs). The MoS2 nanosheet-modified TiONBs provide a large surface area for e fficiently immobilizing AuNPs and thiolated MC-LR aptamers. Due to the combination of good electron transfer and high catalytic capability of the ternary composite, the aptasensor has a wide dynamic range from 0.005 to 30 nmol L−<sup>1</sup> and a LOD of 0.002 nmol L−1.

**Figure 8.** Schematic representation of ( **A**) the preparation of Ab2-AuNR-cirDNA, (**B**) the formation of magnetic graphene composite, and ( **C**) the construction process of the proposed MC-LR immunosensor (adapted from He et al. 2017 [153], Copyright 2017 The Royal Society of Chemistry and reproduced with permission).

**Figure 9.** Schematic representation of the preparation and detection principle of the MC-LR immunosensor (adapted from Pang et al. 2018 [155], Copyright 2018 Elsevier B.V. and reproduced with permission).
