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 therapy1-3. 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 emission maximum at 807 nm (10% v/vDMSO/PBS). Little absorption in the visible range accounts for the low autofluorescence, tissue absorbance, and scattering at NIR wavelengths (700-900 nm).
The maleimide of the ICG dye offers the opportunity to develop optimal conjugates. Maleimide active group provides an efficient and convenient way to selectively link ICG dye to sulfhydryl groups (free thiol, R-SH) on various substrates (antibodies, peptides, proteins, oligonucleotides, small molecule drugs etc.) at neutral (physiological) pH without any activation. Maleimides have very low reactivity with amines, alcohols, and phenols (incl. tyrosine and histidine) and do not react with histidine and methionine thus providing very high labeling selectivity.
It has been reported that conjugation to some antibodies may cause decrease in fluorescence intensity of ICG bound to antibody due to the formation of H-dimers4. However, fluorescence is restored after cell binding and internalization, which can be used to design an activatable NIR fluorescent probe. Fluorescence intensity of the unbound ICG-antibody conjugate can be dramatically increased (up to 50-fold) by addition of SDS and β-mercaptoethanol, which diminishes π- π stacking5.