Cy5.5 NHS Ester (Non-Sulfonated): High-Performance Near-Infrared Dye for Biomolecule Labeling

Executive Summary: Cy5.5 NHS ester (non-sulfonated) is a near-infrared fluorescent dye optimized for covalent labeling of peptides, proteins, and oligonucleotides containing primary amino groups (APExBIO). The dye exhibits an excitation maximum at 684 nm and emission at 710 nm, supporting deep tissue penetration and low background fluorescence (Kang et al., 2025). The solid reagent is stable for up to 24 months at −20°C protected from light, but is unstable in solution, requiring immediate use after dissolution. Cy5.5 NHS ester (non-sulfonated) has demonstrated high sensitivity for in vivo tumor imaging in xenograft mouse models, with tumor uptake peaking at 30 minutes post-injection (Kang et al., 2025). The use of organic solvents such as DMSO or DMF is required due to its low aqueous solubility.

Biological Rationale

Near-infrared (NIR) fluorescent dyes are essential for non-invasive detection and quantification of biomolecules in living systems. The Cy5.5 NHS ester (non-sulfonated) dye is engineered to label primary amine groups found on lysine residues of proteins, amino-modified oligonucleotides, and peptides. NIR dyes such as Cy5.5 minimize background autofluorescence from biological tissues, enabling clearer imaging of deep-tissue targets (Kang et al., 2025). In tumor biology, optical imaging with NIR probes facilitates the tracking of cancer progression, assessment of therapeutic efficacy, and study of the tumor microenvironment. The human microbiome's interaction with tumor tissue can be visualized using advanced labeling tools, supporting research on bacterial influences on cancer metastasis (Kang et al., 2025).

Mechanism of Action of Cy5.5 NHS Ester (Non-Sulfonated)

Cy5.5 NHS ester (non-sulfonated) contains an N-hydroxysuccinimide (NHS) ester functional group that reacts specifically with primary amines on biomolecules to form stable amide bonds. This covalent labeling can be performed in buffered aqueous solutions containing organic co-solvents (typically DMSO or DMF) due to the dye's low aqueous solubility. The reaction is efficient at pH 7.2–8.5 and proceeds at room temperature. After conjugation, the dye exhibits strong absorption at 684 nm and emits fluorescence at 710 nm. The high extinction coefficient (209,000 M⁻¹cm⁻¹) and moderate quantum yield (0.2) enable sensitive detection. The non-sulfonated variant is preferred for hydrophobic biomolecule labeling and certain in vivo applications where minimal charge is desired (APExBIO).

Evidence & Benchmarks

• Cy5.5 NHS ester (non-sulfonated) produces robust, high-contrast tumor signals in vivo, with maximal uptake at 30 min post-injection and detectable fluorescence for up to 24 hours in xenograft mouse models (Kang et al., 2025).
• Excitation/emission maxima (684/710 nm) support deep tissue imaging with low background in living animals (APExBIO).
• Covalent labeling efficiency is high for proteins, peptides, and oligonucleotides containing primary amines when used with DMSO or DMF as a co-solvent (Cy5.5 NHS Ester: Advanced Near-Infrared Dye for Biomolecule Labeling).
• The solid form is stable for 24 months at −20°C in the dark, but the solution is unstable and must be used immediately (APExBIO).
• Benchmark optical imaging studies report high signal-to-noise ratios for in vivo tumor delineation using Cy5.5 NHS ester–labeled probes (Kang et al., 2025).

Applications, Limits & Misconceptions

Cy5.5 NHS ester (non-sulfonated) is widely used for:

• Labeling proteins, peptides, and oligonucleotides for in vivo and in vitro fluorescence detection (APExBIO).
• Optical imaging of subcutaneous tumors to monitor localization and progression (Kang et al., 2025).
• Tracking biomolecule distribution in neuroscience and translational imaging (Cy5.5 NHS Ester (Non-Sulfonated): Advancing In Vivo Neuro...; this article extends coverage to tumor imaging and workflow integration not detailed in that piece).
• Fluorescent probe development for molecular biology assays and diagnostics (Cy5.5 NHS Ester: Advanced Near-Infrared Dye for Biomolecule Labeling).

Common Pitfalls or Misconceptions

• Not water-soluble: Attempting to dissolve Cy5.5 NHS ester (non-sulfonated) directly in aqueous buffers leads to poor solubility and inefficient labeling. Use organic co-solvents like DMSO or DMF.
• Solution instability: Preparing solutions in advance results in hydrolysis and loss of reactivity. Dissolve immediately before use.
• Non-specific labeling: NHS esters can react with any available primary amine, including those on unintended targets, if not properly controlled.
• Not suitable for live-cell labeling: The hydrophobic, non-sulfonated form may not be optimal for live-cell surface labeling due to membrane permeability issues.
• Light sensitivity: Prolonged light exposure degrades fluorescence; protect the dye and conjugates from light at all stages.

Workflow Integration & Parameters

For efficient labeling, dissolve Cy5.5 NHS ester (non-sulfonated) in DMSO or DMF to a concentration of ≥35.82 mg/mL. Add to the biomolecule in an appropriate buffer (e.g., 50 mM sodium phosphate, pH 7.5–8.0). Typical reactions proceed at room temperature for 30 minutes to 2 hours. Remove excess dye by dialysis or chromatography. For in vivo optical imaging, inject labeled probes into animal models and capture NIR fluorescence using suitable instrumentation. Store the solid dye at −20°C in the dark and use solutions immediately (APExBIO).

Compared to conventional dyes, Cy5.5 NHS ester (non-sulfonated) provides superior penetration depth and minimal background, which is critical for translational studies and advanced molecular imaging workflows. For a practical scenario-driven guide to cell assays, see Reliable Cell Assays with Cy5.5 NHS Ester (Non-Sulfonated); this dossier adds product stability, conjugation specifics, and evidence-based benchmarks.

Conclusion & Outlook

Cy5.5 NHS ester (non-sulfonated), available as SKU A8103 from APExBIO, is a validated, high-performance near-infrared dye for amino group labeling in proteins, peptides, and oligonucleotides. It supports advanced in vivo fluorescence imaging of tumors, with robust optical properties and workflow compatibility. Ongoing research into the tumor microbiome and imaging-driven oncology will further leverage this reagent’s capabilities (Kang et al., 2025). For detailed protocols and broader application domains, refer to Redefining Translational Imaging: Mechanistic and Strategic Perspectives, which offers mechanistic insights and translational guidance beyond the tumor imaging focus of this article.