IRDye 800CW-Human serum albumin

Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive alternative to radionuclide imaging in small animals or with probes in close proximity of the target in humans (4, 5). Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment. It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumor cells and endoscopic imaging of the pancreas and colon. The sentinel lymph node (SLN) is considered to be the first lymph node to receive lymphatic flow from tumor sites and therefore, will contain metastatic tumor cells (6). SLN mapping has been used in diagnosis of metastasis of solid tumors (7). Radical lymph node dissection is performed in patients with malignant cells in the SLNs. Presently, SLN mapping is performed by a combination of radioactive tracer and blue dye, which require a radiologist. The current procedure is also time-consuming and requiring a steep learning curve. NIR quantum dots (QDs) has been studied in SLN mapping in small animals (4, 8). However, there are only limited studies of long-term toxicity of QDs in animals (9). IRDye CW800-CW is an indocyanine-type NIR fluorophore with peak absorption at 775 nm, and peak excitation emission at 796 nm. It provides a quantum yield of 9% with an extinction coefficient of 242,000 Mcm. It has a molecular weight of 962 Da. Human serum albumin (HSA) has been successfully conjugated with IRDye 800CW to form IRDye 800CW-HSA (HSA800) for non-invasive NIR mapping of SLNs in small animals (10-12).