Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated Quantum Dots for BRET-Coupled Near-Infrared Imaging of Epidermal Growth Factor Receptors

Abstract

For the highly sensitive near-infrared (NIR) optical detection of epidermal growth factor receptors (EGFRs) expressed on cancer cells, bioluminescence resonance energy transfer (BRET) coupled NIR quantum dots (QDs) are prepared by direct conjugation of his-tagged Renilla luciferase (RLuc) recombinant protein (HisRLuc·GB1) to glutathione-coated CdSeTe/CdS QDs (GSH-QDs). The recombinant protein has two functional groups consisting of a luciferase enzyme and an immunoglobulin binding domain (GB1) of protein G. Recombinant protein (HisRLuc·GB1) conjugated QDs (GB1·RLuc-QDs) show BRET-coupled NIR emission, which results from energy transfer from luciferin to QDs with a high BRET efficiency of ca. 50%. Since the GB1·RLuc-QDs have the GB1 domain at their surface, the QDs have an ability to bind the Fc moiety of immunoglobulin G (IgG). The resulting IgG bound QDs can be used as a molecular imaging probe with NIR fluorescence and BRET-coupled NIR emission. For NIR optical detection of EGFRs on cancer cells, we conjugated anti-EGFR monoclonal antibody to the GB1·RLuc-QDs. Herein, we show that the detection sensitivity of EGFRs by BRET-coupled NIR emission of GB1·RLuc-QDs is at least three times higher than that of the NIR fluorescence of the QDs. The conjugates between anti-EGFR antibody and GB1·RLuc-QDs make it possible to perform BRET-based highly sensitive NIR imaging of EGFRs in living cells.

Shortwave-infrared (SWIR) fluorescence molecular imaging using indocyanine green–antibody conjugates for the optical diagnostics of cancerous tumours

“…At three days post-injection, most of ICG-Herceptin accumulated to the breast tumour, leading to intense uorescence from the tumour. To conrm the active accumulation of ICG-Herceptin to the breast tumour, a control experiment using ICG-Normal IgG 33 was performed. In this case, signicant SWIR uorescence was not observed from the breast tumour compared to the case of an ICG-Herceptin injected mouse (the right images in Fig.…”

Bioconjugation of IgG Secondary Antibodies to Polymer Dots for Multicolor Subcellular Imaging

“…Multiplexed visualization of subcellular structures with fluorescent labels has become a routine procedure in cell biology and medical diagnostics. − To improve the imaging quality, the fluorophore conjugates should emit bright signals and exhibit specific labeling with minimal nonspecific binding in the subcellular environment. − Because the fluorescence brightness and stability of conventional organic dyes are not sufficient, the development of new fluorescent probes has attracted considerable attention in the interdisciplinary field of chemistry and biomedicine. − Fluorescent proteins provide the ability to visualize and track molecules in living cells, thus heralding a new era in cell biology. − Besides, fluorescent nanoparticles can overcome the disadvantages of dyes by offering strong and stable fluorescence . For example, semiconductor quantum dots show improved brightness and photostability as compared to organic dye. , Upconversion nanoparticles exhibit a background-free and stable nonblinking feature for imaging and single-particle tracking .…”

Bioluminescence-Based Energy Transfer Using Semiconductor Quantum Dots as Acceptors

“…The higher signal-to-noise ratio from the BRET-sensitized NIR QD emission compared to standard fluorescent dye labeled Annexin V probe again proved the potential of these conjugates as an optical imaging tool. The same research group applied a similar strategy for imaging of cancer cells by targeting the overexpression of a transmembrane protein called epidermal growth factor receptor (EGFR) which is commonly found in many cancer cells [46]. The targeting molecule was an EGFR specific antibody, which was linked to QD-bound Luc via an antibody binding domain genetically fused to Luc during its expression.…”