A Comprehensive Guide to Cyanine 3: Properties, Binding Mechanisms, and Applications

What is Cyanine 3?

Cyanine 3 (Cy3) is a fluorescent dye that is part of the cyanine family, and it has a bright red glow. Its optical characteristics are an excitation wavelength of about 550 nm and an emission wavelength of about 570 nm. Cy3 has a fluorene ring in the center and nitrogen atoms and many different substituents on the edges. This structure underpins its stable fluorescence and versatility in biological and biomedical applications.

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Catalog	Product Name
ACMA00003098	Sulfo-Cy3.5-SE
ACMA00003089	N-(m-PEG4)-N'-(azide-PEG4)-Cy3
ACMA00003088	Sulfo-Cy3 maleimide
ACMA00003078	Cy3 Amine
ACMA00000425	Sulfo-Cy3-SNAP-Tag
ACMA00000424	N-methyl-N'-methyl-O-(m-PEG4)-O'-(azide-PEG4)-Cy3
ACMA00000165	Cy3 Maleimide
ACM2055138888	diSulfo-Cy3 alkyne
ACM2055138866-1	Sulfo-Cy3-Tetrazine
ACM1895849349-1	Trisulfo-Cy3-Alkyne
ACM146368130	Sulfo Cy3 Carboxylic acids(ethyl)
ACM1251915293	Cy3 Carboxylic acids
ACM1010386625-1	Sulfo-Cy3-YNE

What are the Properties of Cy3?

Cy3 possesses several advantageous chemical and physical properties that make it indispensable in biological and chemical research.

Property	Description
Fluorescence Stability	Exhibits strong photostability under various experimental conditions, reducing photobleaching during imaging.
pH Tolerance	Remains unaffected across a broad pH range, allowing versatility in different experimental setups.
Quantum Yield	Features a high quantum yield, ensuring strong fluorescence intensity for sensitive detection.
Versatile Reactivity	Reactive functional groups such as NHS ester and azide enable covalent conjugation with diverse biomolecules.

In addition, Cy3 derivatives like sulfo-Cy3 exhibit improved solubility and compatibility with aqueous systems, broadening its applicability.

What does Cy3 Bind to?

Derivatives such as Cy3 NHS ester, Cy3 azide and Cy3 carboxylic acid further extend their functionality by introducing various reactive groups. These derivatives enable Cy3 to participate in selective binding reactions for specific experimental requirements.

Cy3 interacts with biomolecules through covalent couplings facilitated by its functional groups, and NHS ester derivatives of Cy3 react efficiently with primary amines in proteins, peptides, and other amine-containing biomolecules. Azide-functionalized Cy3 promotes bioorthogonal reactions, such as click chemistry, for labeling purposes. Derivatives such as Cy3-chitosan bind dyes to biopolymers, enhancing their functional capabilities for targeted applications.

Fig.1 Examples of Cy3 derivatives^[1].

What Applications does Cy3 Have?

Due to Cy3's special nature and binding versatility, it is used in various sciences from cell biology, immunology, molecular biology and materials science.

Fluorescent Labeling and Imaging

Cy3 is a brilliant, highly spectral stable fluorophore that's well-suited to the marking of biomolecules. Cy3-labeled antibodies and proteins in cellular imaging are used in immunocytochemistry and immunohistochemistry to look at the molecular composition of cells and tissues. Cy3 derivatives like Cy3-NHS ester and Cy3-Norbornene are essential for high-resolution imaging and for bringing light into complex biological environments.

Fig.2 Different fluorescent dyes (Cy2-COOH, NHS-Cy3, and NHS-Cy5) were conjugated to dendrimer-PEG-Chol (DPC NPs) to prepare fluorescent probes for plasma membrane imaging and zebrafish visualization^[2].

Molecular Probing and Detection

Cy3 is a common labeling chemical in DNA and RNA in molecular biology, as a probe in nucleic acid hybridization and sequencing. It is also used in fluorescent in situ hybridization (FISH) for the specific detection of genes and thus for diagnosis. Additionally, Cy3 conjugates are employed in studies of protein-protein interactions and protein expression dynamics, advancing our understanding of cellular mechanisms.

Fig.3 Fluorescent "light click" labeling of DNA with Cy3 dye^[3].

Drug Delivery and Therapeutics

The use of Cy3 derivatives (Cy3-Resveratrol, for example) in drug delivery systems that offer fluorescence imaging as well as therapeutic activity is on the rise. These conjugates can target the drug to specific tissue or cell, and because they are fluorescent, they can monitor drug movement and metabolism in real-time. These systems increase therapeutic effectiveness and minimize side effects.

Biomaterial Modification

Cy3 conjugates, including Cy3-chitosan and Cy3-PEG, are employed in material sciences to enhance the functionality of biomaterials. Chitosan-Cy3 conjugates are used in biomedical imaging and drug delivery, while PEGylated Cy3 complexes improve biocompatibility and stability in biological systems. These modifications are pivotal in the development of advanced biosensors and biocompatible implants.

Biosensing and Diagnostics

Cy3 is also central to biosensor design, where it aids the detection of biomarkers, pathogens and toxins. It is highly sensitive and adaptable to many biological matrices, and it could be applied to medical diagnostics and environmental monitoring. Cy3 conjugates are in particular used in ELISA and other immunoassays for the enhancement of sensitivity and specificity.

Conclusion

Cyanine 3 (Cy3) is a highly potent, broad-spectrum fluorescent dye used widely in biomedical studies, molecular biology, and materials science. Its stable fluorescence, wide binding compatibility, and versatility for different experiments make it indispensable to scientific discovery and technological progress. The ongoing development of Cy3 derivatives and conjugates continues to expand its utility, ensuring its pivotal role in both research and applied sciences.

References

1. Gaikwad H., et al. Surface Modification of Erythrocytes with Lipid Anchors: Structure-Activity Relationship for Optimal Membrane Incorporation, in vivo Retention, and Immunocompatibility. Advanced NanoBioMed Research (2022).
2. Xu K-F., et al. Fluorescent dendrimer-based probes for cell membrane imaging: Zebrafish epidermal labeling-based toxicity evaluation. Biosensors and Bioelectronics (2022).
3. Lehmann B., et al. Fluorogenic "photoclick" labelling of DNA using a Cy3 dye. Organic & Biomolecular Chemistry (2018).