indocyanine green n succinimidyl ester

ICG-NHS ester

 120.00 765.00

Peptide, protein, and nanoparticle labeling

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Introduction

The non-invasive near-infrared (NIR) fluorescence imaging dye Indocyanine green (ICG) is approved by the United States Food and Drug administration (FDA) for ophthalmologic angiography to determine cardiac output and liver blood flow and function. This dye is also used in cancer patients for the detection of solid tumors, localization of lymphnodes, and for angiography during reconstructive surgery, visualization of retinal and choroidal vasculature, and photodynamic therapy.1-3 In cancer diagnostics and therapeutics, ICG could be used as both an imaging dye and a hyperthermia agent.

ICG is a tricarbocyanine-type dye with NIR-absorbing properties (peak absorption around 800 nm) and little absorption in the visible range thus exhibit low autofluorescence, tissue absorbance, and scatter at NIR wavelengths (700-900 nm).

The succinimidyl esters (NHS) of the ICG dye offer the opportunity to develop optimal conjugates. Succinimidyl ester active groups provide an efficient and convenient way to selectively link ICG dyes to primary amines (R-NH2) on various substrates (Antibodies, peptides, proteins, nucleic-acid, small molecule drugs etc.). Succinimidyl esters have very low reactivity with aromatic amines, alcohols, and phenols, including tyrosine and histidine.

Material Amount Storage Stability
ICG-NHS 1 mg
  • -20° C
  • Desiccate
  • Protect from light
When stored as directed, reactive probes are stable for at least 3 months
5 mg
10 mg

Immediately before use, dissolve the ICG-NHS dyes in anhydrous dimethylformamide (DMF). Once reconstituted, this reactive probe solution is somewhat unstable, especially in presence of moisture that can slowly hydrolyze the succinimidyl ester to the non-reactive carboxylic acid.

The ICG-NHS dye can virtually be conjugated to any primary amine-containing molecule such as peptides, proteins, antibodies, small-molecule drugs… If possible avoid nucleophilic bases, since it will partially degrades ICG core structure to non-NIR-fluorescent by-products.

 

Example of conjugation:

  1. Antibody conjugation: Typical antibody conjugation reactions are carried out in 0.1 M sodium bicarbonate buffer, pH 8.5, at room temperature for 2 hour and protected from light. We recommend trying different molar ratio between antibodies and reactive dyes in order to reach your needs. Labeled antibodies are typically separated from free ICG dye using a gel filtration column, such as SephadexTM G-25, BioGel® P-30, or equivalent. For much larger or smaller proteins, select a gel filtration media with an appropriate molecular weight cut-off or purify by dialysis. Keep labeled antibody at 4°C in PBS. The number of fluorochrome per antibody will vary depending on the molar ratio between antibodies and reactive dyes. An average number of fluorochrome per antibody can be determined by spectrophotometric analysis or/and by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry.

 

For example:

Antibody (250 ug) is diluted in 0.1 M NaHCO3 (pH 8.5) to 2 mg/mL (125 uL) (other buffer could be used such as Na2HPO4 0.1M, pH 8.5). 4 to 8 equivalents of fluorescent ICG-NHS dye (from stock solution in DMSO or DMF at 1 mM) is added to the antibody solution and incubated for 0.5 to 2 hours. After incubation, antibody can be purified by centrifuge filtration using desalting Zeba spin column 7K (0.5 mL) and store in PBS at 4°C. The number of fluorochromes per antibody was determined by spectrophotometric analysis and determined to be approximately 2, 3 ICG dyes per antibody using respectively 4 and 8 molar equivalent of ICG-NHS.

For ICG/Ab calculation, take UV absorbance at 280nm and 800nm and apply following calculation:

(A800/22884)/((A280-A800*0.052)/ε)

Where:

A800: Absorbance at 800nm

A280: Absorbance at 280nm

ε: Molar extinction coefficient of protein (for Antibody ~ 203000 L.mol-1.cm-1)

22884: molar extinction coefficient of ICG in PBS at 800 nm

0.052: Correction factor for ICG

 

  1. Small molecule conjugation: Typical small-molecule conjugation reactions are carried out in dry dimethylformamide (DMF) or dimethylsulfoxide (DMSO) with an organic base such diisopropylethylamine (DIPEA) at room temperature for 1-3 hours and protected from light. Labeled molecules are typically purified by reverse-phase HPLC and appropriate fractions are lyophilized.
  1. Schaafsma B.E. The clinical use of indocyanine green as a near-infrared fluorescent contrast agent for image-guided oncologic surgery. Surg. Oncol. 2011, 104, 323-332.
  2. Saxena V, Sadoqi M, Shao J. Degradation kinetics of indocyanine green in aqueous solution. J Pharm Sci 2003, 92, 2090–7.
  3. Yaseen MA, Yu J, Wong MS, Anvari B. Stability assessment of indocyanine green within dextran-coated mesocapsules by absor- bance spectroscopy. J Biomed Opt 2007, 12, 064031.
Color

Dark green powder

Label

Succinimidyl ester

Product Size

1 kit

Detection Method

Fluorescence, optoacoustic

Excitation Class

Near infrared, NIR

Emission/Excitation(nm):

790/830

Molecular Weight

828.04 g.mol-1

Formula

C49H53N3O7S

Shipping Condition

RT

Regulatory Statement

For Research Use Only. Not for use in diagnostic procedures.

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