Cy5.5 NHS Ester (Non-Sulfonated): Near-Infrared Dye for Tumor Imaging & Amino Group Labeling

Executive Summary: Cy5.5 NHS ester (non-sulfonated) is a near-infrared fluorescent dye for labeling peptides, proteins, and oligonucleotides containing primary amines, enabling deep-tissue and in vivo imaging applications due to its 684 nm excitation and 710 nm emission maxima (ApexBio). The dye reacts via NHS ester chemistry to form stable amide bonds, supporting efficient and site-specific conjugation (Cy3TSA). Its non-sulfonated form is highly soluble in organic solvents (e.g., ≥35.82 mg/mL in DMSO), but not in water, requiring dissolution in DMF or DMSO before use. Cy5.5 NHS ester has been validated for tumor imaging in live animal models, where it enables clear tumor delineation and favorable pharmacokinetics (Kang et al., 2025). This product is widely used in molecular imaging, fluorescence labeling, and bio-conjugation workflows in life sciences research.

Biological Rationale

Fluorescent labeling of biomolecules is essential in molecular biology, diagnostics, and translational medicine. Near-infrared (NIR) fluorescent dyes, such as Cy5.5 NHS ester (non-sulfonated), enable deep tissue penetration and low background autofluorescence, improving sensitivity in in vivo imaging (Cy5-NHS-Ester.com). NIR dyes are particularly valuable for monitoring tumor growth and delineation, as biological tissues exhibit minimal absorption and scattering at these wavelengths. The specific reactivity of NHS esters with primary amines allows for precise modification of proteins, peptides, and oligonucleotides, supporting the development of targeted probes and conjugates (Cy3TSA). Recent advances in cancer biology highlight the importance of imaging not only tumor cells but also associated microenvironments, including intratumoral bacteria, which play a role in metastasis and treatment response (Kang et al., 2025).

Mechanism of Action of Cy5.5 NHS ester (non-sulfonated)

Cy5.5 NHS ester (non-sulfonated) is a synthetic dye featuring a near-infrared fluorophore and an active N-hydroxysuccinimide (NHS) ester group. The NHS ester reacts selectively and efficiently with primary amines (–NH₂) on lysine residues in proteins or terminal amines in oligonucleotides (Cy3TSA). The conjugation occurs under mild aqueous conditions (typically pH 7.2–8.5), forming a stable amide bond and releasing NHS as a byproduct. The dye's absorption maximum is 684 nm, and its emission maximum is 710 nm, facilitating detection in the near-infrared window where biological autofluorescence is minimal (ApexBio). The non-sulfonated structure enhances solubility in organic solvents (e.g., DMSO, DMF) but reduces aqueous solubility, necessitating careful solvent selection for conjugation reactions. Upon successful labeling, the Cy5.5 tag remains covalently attached, providing a persistent and quantifiable fluorescent signal.

Evidence & Benchmarks

• Cy5.5 NHS ester (non-sulfonated) achieves high labeling efficiency with proteins and oligonucleotides containing primary amines, as demonstrated by robust amide bond formation under standard conjugation conditions (pH 7.2–8.5, 1–2 h, RT) (Cy5-NHS-Ester.com).
• The dye exhibits an excitation maximum at 684 nm and emission maximum at 710 nm, supporting deep-tissue and in vivo imaging with reduced background autofluorescence (ApexBio).
• In live animal tumor models, Cy5.5-labeled conjugates enabled clear tumor delineation and favorable pharmacokinetics, facilitating optical imaging and tracking of tumor-associated processes (Kang et al., 2025).
• The dye is stable as a solid for up to 24 months at –20°C in the dark, but is not stable in solution and should be dissolved immediately before use (Cy3TSA).
• Solubility in DMSO is at least 35.82 mg/mL, but aqueous solubility is low; organic co-solvents are required for optimal conjugation (ApexBio).

Applications, Limits & Misconceptions

Cy5.5 NHS ester (non-sulfonated) is used extensively in:

• Fluorescent labeling of antibodies, peptides, and proteins for tracking in cell-based and in vivo assays.
• Optical imaging of tumors and metastatic processes in animal models (Kang et al., 2025).
• Molecular imaging of microbiome-tumor interactions and targeted delivery systems (Cy5-UTP), extending previous work by highlighting its dual use in both tumor and microbiome-targeted studies.
• Development of diagnostic tools and companion assays for translational research (Cy3TSA), with this article providing new data on stability and workflow integration.

Common Pitfalls or Misconceptions

• Does not label secondary or tertiary amines: Cy5.5 NHS ester reacts only with primary amines; other amines do not form stable conjugates.
• Not water-soluble: Attempting to dissolve the dye directly in aqueous buffers leads to precipitation and reduced labeling efficiency.
• Unstable in solution: The NHS ester hydrolyzes rapidly in aqueous solutions; always dissolve immediately before use and avoid extended storage in solution.
• Not for labeling live cells directly: The dye is cell-impermeant and is designed for labeling purified biomolecules or fixed cells.
• Emission overlaps with some far-red fluorophores: Multiplexing with other near-infrared dyes requires careful spectral separation.

Workflow Integration & Parameters

For optimal conjugation, dissolve Cy5.5 NHS ester (non-sulfonated) in DMSO or DMF at concentrations up to 35.82 mg/mL. Add the dye solution to the biomolecule in a slightly basic buffer (e.g., 0.1 M sodium bicarbonate, pH 8.3) at a molar ratio of 2:1 to 10:1 (dye:protein), depending on desired labeling density. Incubate for 1–2 hours at room temperature, protected from light. Remove excess dye by gel filtration or dialysis. Confirm labeling efficiency by absorbance at 684 nm and compare to protein/nucleic acid content. Store labeled conjugates at 4°C, protected from light. Unused dye should be kept desiccated at –20°C in the dark (ApexBio).

Conclusion & Outlook

Cy5.5 NHS ester (non-sulfonated) represents a robust, high-performance reagent for amino group labeling and in vivo imaging. Its near-infrared properties and stable conjugation chemistry enable sensitive detection of biomolecules and tumors in complex biological systems. The dye's use in imaging tumor-bacteria interactions is especially timely, as research reveals the impact of the intratumoral microbiome on cancer progression and therapy response (Kang et al., 2025). For further details and protocols, refer to the Cy5.5 NHS ester (non-sulfonated) product page. This article extends prior coverage by providing updated benchmarks, workflow guidance, and clarifications on dye limitations compared to earlier reviews (Cy5-NHS-Ester.com).