Anti-Cy3 / Cy5 monoclonal antibody, clone EA08

Cyanine is a class of fluorescent dyes with excellent fluorescent properties and is one of the many fluorescent groups of interest for constructing fluorescent probes. It also has excellent biocompatibility and low toxicity to biological samples.

The chemical structure of classical cyanine is characterized by two nitrogen atoms independently embedded in a heterocyclic ring with 𝜋-conjugated polymethylene chains connecting the two nitrogen atoms. The heterocyclic structure may be pyrrole, imidazole, thiazole, benzothiazole, pyridine or quinoline. Variations of nitrogen substituents on polymethyl chains and heterocycles lead to the structural diversity of cyanine derivatives. Bridged polymethylene chains are conjugated chains formed by positively charged nitrogen atoms at the end of sp2 hybridized carbon atoms through π-electron displacement. Cyanine can also be subdivided according to the number of bridging methyl chains into apocyanine (directly linked without any methyl groups), monomethyl anthocyanins, trimethyl anthocyanins, pentamethyl anthocyanins (dicarboxyanthocyanins), and heptamethyl anthocyanins (tricarboxyanthocyanins).

Figure 1. Representative structures of cyanine fluorophores
(Source: Bilici K, et al. 2021)

Traditional cyanine-based chemosensors bind analyte-sensitive molecules to a cyanine scaffold and are the tool of choice for the detection of small molecules such as reactive oxygen species, reactive nitrogen species, biothiols and heavy metal ions. The primary mechanism of this class of fluorescent probe detection modes is an analyte-initiated reaction that alters the chemical structure of the cyanine while producing a spectral change that either couples the cyanine to the detection target for localization. Many enzymes and biomolecules are difficult to detect by enzyme-catalyzed reactions, but they are closely related to the onset, progression, and prognosis of a variety of diseases, and thus the selective detection of these targets can not only facilitate basic research but also generate clinical benefits. The combination of cyanine with targeted warheads provides a powerful visualization tool for targeted fluorescence imaging of these biomolecules, and in the case of proteins, although proteins have different functions, they all share a similar targeting strategy, i.e., targeting warheads ranging from traditional antibodies, small molecules, and peptides, to emerging aptamers and non-immunoglobulin protein scaffolds. In terms of nucleic acids, the corresponding targeting warheads mainly include oligonucleotides and their synthetic analogs (PNAs, peptide nucleic acids). Since targeting capability is achieved through the interaction between the targeting warhead and the biomolecule target, it is important to ensure that the cyanine coupling does not interfere with the binding affinity of the warhead. To this end, it is necessary to study the biological properties of the target, especially the structure and dynamics of the target-warhead complexes at the molecular level, in order to provide guidance for the selection of target warheads, the design of conjugation sites and conjugation chemistry.

Figure 2. Available targeting warheads that were reported to conjugate with cyanine fluorophores for targeted fluorescence imaging of proteins and nucleic acids
(Source: Li Y, et al. 2020)

Anti-Cyanine3 / Cyanine5 monoclonal antibody