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    • Cy5.5 NHS Ester (Non-Sulfonated): Atomic Evidence for NIR...

    2026-03-02

    Cy5.5 NHS Ester (Non-Sulfonated): Atomic Evidence for NIR Biomolecule Labeling

    Executive Summary: Cy5.5 NHS ester (non-sulfonated) is a near-infrared fluorescent dye optimized for high-specificity labeling of amino groups in biomolecules (APExBIO). The dye exhibits an excitation maximum at 684 nm and an emission maximum at 710 nm, enabling deep-tissue imaging with minimal autofluorescence (cy5tsa.com). Its high extinction coefficient (209,000 M⁻¹cm⁻¹) and moderate quantum yield (0.2) support sensitive detection in protein, peptide, and oligonucleotide conjugation. In vivo studies demonstrate that Cy5.5 NHS ester enables optical imaging of subcutaneous tumors, with peak uptake at 30 minutes and signal persistence for up to 24 hours (Li et al., 2025). The product, supplied by APExBIO, maintains 24-month shelf stability at -20°C and must be protected from light for optimal activity (APExBIO).

    Biological Rationale

    Near-infrared (NIR) fluorescent dyes offer low background signal and deep tissue penetration in biological imaging. Cy5.5 NHS ester (non-sulfonated) is specifically engineered for labeling amino groups on proteins, peptides, and oligonucleotides, leveraging the stability and reactivity of the NHS ester functional group. Labeling with Cy5.5 NHS ester allows real-time, non-invasive visualization of biomolecules in live cells and animal models. This capability is crucial for tracking molecular dynamics, imaging tumors, and monitoring therapeutic distribution (cy5tsa.com). The NIR emission window (700–900 nm) minimizes overlap with tissue autofluorescence and maximizes signal-to-noise ratio in in vivo applications (cy5-hydrazide.com). The efficient labeling chemistry ensures covalent attachment to accessible primary amines, preserving biomolecule function for downstream assays.

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

    Cy5.5 NHS ester contains an N-hydroxysuccinimide (NHS) ester moiety, which selectively reacts with primary amines. The labeling reaction is initiated by dissolving the dye in an organic solvent such as DMSO or DMF. The activated dye is then introduced to the target biomolecule in a suitable aqueous buffer, typically at pH 7.5–8.5. Under these conditions, the NHS ester forms a stable amide bond with the amino group, resulting in covalent conjugation. The process is efficient in organic/aqueous co-solvent systems due to the dye's low water solubility and high solubility in DMSO (≥35.82 mg/mL). The fluorophore's extended conjugated system absorbs strongly at 684 nm and emits at 710 nm, enabling detection by standard NIR fluorescence platforms. The dye's moderate quantum yield (0.2) and high extinction coefficient ensure robust signal output.

    Evidence & Benchmarks

    • Cy5.5 NHS ester (non-sulfonated) exhibits an excitation maximum at 684 nm and emission maximum at 710 nm under standard buffer conditions (pH 7.4, 25°C) (APExBIO).
    • The extinction coefficient is 209,000 M⁻¹cm⁻¹, supporting high sensitivity in fluorescence detection workflows (cy5maleimide.com).
    • Quantum yield is measured at 0.2 in phosphate-buffered saline (PBS), balancing brightness with reduced photobleaching (cy5tsa.com).
    • Solubility in DMSO is ≥35.82 mg/mL, with low aqueous solubility, necessitating organic co-solvents for effective conjugation (APExBIO).
    • In vivo optical imaging using Cy5.5 NHS ester-labeled probes enables detection of subcutaneous tumor xenografts in mice, with maximal tumor localization at 30 minutes and detectable signal up to 24 hours post-injection (Li et al., https://doi.org/10.1002/adfm.202518001).
    • The dye is stable for up to 24 months at -20°C when protected from light, but is unstable in solution and should be freshly prepared (APExBIO).
    • Labeling efficiency is highest for proteins and peptides with accessible lysine residues; plasmid DNA can also be labeled efficaciously under optimized conditions (cy5-hydrazide.com).

    Applications, Limits & Misconceptions

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

    • Near-infrared fluorescence imaging of tumors and organs in small animal models.
    • Protein and peptide labeling for in vitro and in vivo assays.
    • Oligonucleotide and plasmid DNA labeling for molecular tracking.
    • Enabling dual-modality imaging and theranostic approaches in nanomedicine (Li et al., 2025).

    This article expands on the practical chemistry and in vivo data provided in Cy5.5 NHS Ester (Non-Sulfonated): Benchmarks for Near-Infrared Imaging, by detailing specific storage, solubility, and workflow parameters for reproducible results.

    Compared to Cy5.5 NHS Ester (Non-Sulfonated): NIR Fluorophore for Next-Generation Imaging, this article provides updated in vivo imaging benchmarks and clarifies the boundaries of labeling efficiency in complex biological matrices.

    Common Pitfalls or Misconceptions

    • Cy5.5 NHS ester (non-sulfonated) is not water-soluble; attempts to dissolve directly in aqueous buffers lead to precipitation and poor labeling efficiency.
    • Storage of the dye in solution, especially at room temperature or with light exposure, leads to rapid hydrolysis and loss of labeling activity.
    • Conjugation to biomolecules lacking accessible primary amines (e.g., highly glycosylated proteins) is inefficient or fails.
    • The dye's emission signal is not suitable for standard (visible-range) fluorescence instruments; NIR-capable detectors are required.
    • It cannot be used for live animal imaging without prior conjugation to a targeting biomolecule; free dye is rapidly cleared and non-specific.

    Workflow Integration & Parameters

    For optimal labeling, Cy5.5 NHS ester (non-sulfonated) should be dissolved in dry DMSO or DMF immediately before use. Conjugation reactions are typically performed at pH 7.5–8.5 in phosphate or bicarbonate buffer, with a dye-to-biomolecule molar ratio of 5:1 to 20:1 depending on application. Reactions are usually incubated at room temperature for 1–2 hours. Excess dye is removed by dialysis or size-exclusion chromatography. Labeled products should be stored at 4°C, protected from light. For in vivo tumor imaging, labeled probes are typically injected intravenously in animal models, with imaging conducted at specified time points (e.g., 30 min, 2 h, 24 h) using NIR fluorescence platforms. The Cy5.5 NHS ester (non-sulfonated) A8103 kit from APExBIO provides comprehensive documentation and technical support for these workflows.

    Conclusion & Outlook

    Cy5.5 NHS ester (non-sulfonated) remains a benchmark NIR fluorophore for amino group labeling in molecular biology and in vivo imaging. Its unique spectral properties, robust labeling chemistry, and validated applications in tumor imaging support its ongoing use in advanced biomedical research. Future developments may further optimize its photostability and expand compatibility with emerging bioorthogonal labeling workflows. For additional mechanistic details and emerging applications, see Cy5.5 NHS Ester (Non-Sulfonated): Redefining Tumor Imaging, which this article extends by synthesizing new in vivo evidence and workflow parameters.