Cy5.5 NHS Ester (Non-Sulfonated): Near-Infrared Dye for Biomolecule Labeling and Tumor Imaging

Executive Summary: Cy5.5 NHS ester (non-sulfonated) is a near-infrared (NIR) fluorescent dye for efficient and stable labeling of biomolecules containing primary amino groups (ApexBio). The dye provides strong excitation at 684 nm and emission at 710 nm, minimizing background autofluorescence for in vivo imaging (Kang et al., 2025). Cy5.5 NHS ester is highly soluble in DMSO (≥35.82 mg/mL) but requires organic co-solvents for aqueous labeling (Related article). Its rapid NHS-ester chemistry ensures stable, covalent conjugation to proteins and oligonucleotides, enabling reliable detection in molecular and imaging workflows. The dye is applied successfully in tumor delineation and pharmacokinetics studies, with 24 months’ shelf life at -20°C, protected from light (ApexBio).

Biological Rationale

Near-infrared (NIR) fluorescent dyes are critical tools in modern molecular biology and in vivo imaging. They enable visualization of biological processes with high specificity and minimal background. Cy5.5 NHS ester (non-sulfonated) is designed for covalent labeling of amino groups (primarily lysine residues and N-termini) in peptides, proteins, and oligonucleotides (ApexBio). The NIR spectral window (650–900 nm) substantially reduces interference from tissue autofluorescence, allowing for deep tissue penetration and higher signal-to-noise ratios in live animal imaging (Kang et al., 2025). Tumor imaging relies on these properties to delineate malignant tissue with clarity during research and preclinical studies. The NHS ester reactive group offers site-selective, rapid, and efficient modification of biomolecules, making Cy5.5 NHS ester a key reagent for targeted imaging, pharmacokinetic assays, and molecular diagnostics (Related article).

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

Cy5.5 NHS ester (non-sulfonated) contains a reactive N-hydroxysuccinimide (NHS) ester moiety. NHS esters selectively react with primary amines on biomolecules under mildly basic conditions (typically pH 7.5–8.5), forming a stable amide linkage. This reaction is rapid (minutes to hours, depending on conditions) and is highly specific to lysine side chains and terminal amines. Cy5.5 is a near-infrared cyanine dye with an excitation maximum at 684 nm and emission maximum at 710 nm, allowing for deep-tissue imaging. The dye is non-sulfonated, resulting in hydrophobicity and high solubility in organic solvents such as DMSO (≥35.82 mg/mL) and DMF. Poor aqueous solubility necessitates pre-dissolution in organic solvent, followed by labeling in buffered aqueous media containing the biomolecule. After conjugation, the resulting dye-labeled product exhibits robust fluorescence, stable amide bonds, and compatibility with a wide range of detection platforms (ApexBio).

Evidence & Benchmarks

• Cy5.5 NHS ester (non-sulfonated) forms stable amide bonds with primary amines in peptides, proteins, and nucleic acids under pH 7.5–8.5 and reaction times of 30–120 min (ApexBio).
• Excitation and emission maxima are 684 nm and 710 nm, respectively, verified in aqueous buffer after conjugation (Cy5.5 NHS Ester article).
• In vivo optical imaging studies using Cy5.5-labeled probes enable clear tumor delineation and tracking of tumor-associated bacteria, as demonstrated in mouse models (Kang et al., 2025).
• The product remains stable as a solid for at least 24 months at -20°C in the dark, but is unstable in solution and should be freshly prepared (ApexBio).
• Solubility in DMSO is ≥35.82 mg/mL, but in water is negligible, necessitating co-solvent use for biomolecule labeling (ApexBio).

Applications, Limits & Misconceptions

Cy5.5 NHS ester (non-sulfonated) is widely used in the following areas:

• Fluorescent labeling of proteins: Enables sensitive detection in western blots, ELISA, and immunofluorescence (ApexBio).
• Molecular imaging: Utilized in preclinical in vivo imaging of tumors, including detection and tracking of tumor-associated bacteria (Kang et al., 2025).
• Pharmacokinetic studies: Facilitates real-time monitoring of labeled drug distribution and clearance (Related article).
• Bio-conjugation research: Provides robust amide chemistry for stable conjugates in diagnostics and research assays.

Common Pitfalls or Misconceptions

• Misconception: Cy5.5 NHS ester (non-sulfonated) is water-soluble.
  Fact: It is poorly soluble in aqueous solutions and requires organic co-solvent pre-dissolution (ApexBio).
• Misconception: The dye is stable in solution for extended periods.
  Fact: The NHS ester is hydrolytically unstable; solutions must be freshly prepared before use.
• Misconception: All biomolecules are equally labeled.
  Fact: Only molecules with accessible primary amines (lysines, N-termini) are labeled.
• Misconception: Near-infrared dyes are interchangeable.
  Fact: Cy5.5 has unique spectral properties (684/710 nm) and is not directly equivalent to Cy5 or other NIR dyes.
• Misconception: The product is suitable for live-cell labeling without optimization.
  Fact: Cell viability and labeling efficiency must be validated empirically for each application.

For a detailed discussion of labeling protocols and spectral properties, see this resource; this article extends those findings by integrating new in vivo tumor imaging benchmarks and stability data.

Workflow Integration & Parameters

• Preparation: Dissolve Cy5.5 NHS ester (non-sulfonated) in anhydrous DMSO or DMF to make a concentrated stock solution (e.g., 10 mM).
• Labeling reaction: Add the dye solution to the biomolecule in a buffered aqueous solution (pH 7.5–8.5) at 4°C–25°C. Typical reaction time: 30–120 min.
• Quenching/unreacted dye removal: Purify labeled biomolecules by gel filtration, dialysis, or spin columns to remove free dye.
• Storage: Store solid Cy5.5 NHS ester at -20°C in the dark; avoid repeated freeze-thaw cycles. Labeled products are typically stable for weeks at 4°C, protected from light.
• Detection: Excite at 684 nm; collect emission at 710 nm on appropriate imaging equipment.

Refer to the Cy5.5 NHS ester (non-sulfonated) product page for detailed protocols and troubleshooting tips.

Conclusion & Outlook

Cy5.5 NHS ester (non-sulfonated) is a validated, high-performance near-infrared dye for amino group labeling in biomolecules, with proven utility in tumor imaging and molecular diagnostics (Kang et al., 2025). Its robust chemistry and spectral properties make it a standard for sensitive and specific in vivo and in vitro detection. Ongoing research continues to expand its applications in microbiome-tumor interactions and targeted therapeutic monitoring. Users should observe solvent compatibility and labeling specificity limits for optimal results. For further reading, this article clarifies and updates protocol details and imaging benchmarks compared to previous summaries.