Fluorophore Conjugation Research Articles

Recent advances in near-infrared fluorescence-guided imaging surgery using indocyanine green.

Near-infrared (NIR) fluorescence imaging has better tissue penetration, allowing for the effective rejection of excitation light and detection deep inside organs. Indocyanine green (ICG) generates NIR fluorescence after illumination by an NIR ray, enabling real-time intraoperative visualization of superficial lymphatic channels and vessels transcutaneously. The HyperEye Medical System (HEMS) can simultaneously detect NIR rays under room light to provide color imaging, which enables visualization under bright light. Thus, NIR fluorescence imaging using ICG can provide for excellent diagnostic accuracy in detecting sentinel lymph nodes in cancer and microvascular circulation in various ischemic diseases, to assist us with intraoperative decision making. Including HEMS in this system could further improve the sentinel lymph node mapping and intraoperative identification of blood supply in reconstructive organs and ischemic diseases, making it more attractive than conventional imaging. Moreover, the development of new laparoscopic imaging systems equipped with NIR will allow fluorescence-guided surgery in a minimally invasive setting. Future directions, including the conjugation of NIR fluorophores to target specific cancer markers might be realistic technology with diagnostic and therapeutic benefits.

Quantum dot/antibody conjugates for in vivo cytometric imaging in mice

Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates that have an appropriate size for long circulation and diffusion and show virtually no nonspecific binding to cells/serum while binding to cells of interest with high specificity. In addition, these conjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, we show that this can be achieved using compact (∼15 nm in hydrodynamic diameter) and biocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine-norbornene cycloaddition. Our QD immunoconstructs were used for in vivo single-cell labeling in bone marrow. The intravital imaging studies using a chronic calvarial bone window showed that our QD-Ab conjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells (Sca1(+)c-Kit(+) cells). This in vivo cytometric technique may be useful in a wide range of structural and functional imaging to study the interactions between cells and between a cell and its environment in intact and diseased tissues.

Genetic encoding of unnatural amino acids for labeling proteins.

The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labeling proteins with any probe. Here we describe the genetic encoding of dienophile-bearing unnatural amino acids into proteins expressed in Escherichia coli and mammalian cells using the pyrrolysyl-tRNA synthetase/tRNACUA pair and its variants. We describe the rapid fluorogenic labeling of proteins containing these unnatural amino acids in vitro, in E. coli, and in live mammalian cells with tetrazine-fluorophore conjugates in a bioorthogonal Diels-Alder reaction with inverse electron demand. These approaches have been extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on the labeling and imaging field.

Exploring a 2-naphthoic acid template for the structure-based design of P2Y14 receptor antagonist molecular probes.

The P2Y14 receptor (P2Y14R), one of eight P2Y G protein-coupled receptors (GPCR), is involved in inflammatory, endocrine, and hypoxic processes and is an attractive pharmaceutical target. The goal of this research is to develop high-affinity P2Y14R fluorescent probes based on the potent and highly selective antagonist 4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid (6, PPTN). A model of hP2Y14R based on recent hP2Y12R X-ray structures together with simulated antagonist docking suggested that the piperidine ring is suitable for fluorophore conjugation while preserving affinity. Chain-elongated alkynyl or amino derivatives of 6 for click or amide coupling were synthesized, and their antagonist activities were measured in hP2Y14R-expressing CHO cells. Moreover, a new Alexa Fluor 488 (AF488) containing derivative 30 (MRS4174, Ki = 80 pM) exhibited exceptionally high affinity, as compared to 13 nM for the alkyne precursor 22. A flow cytometry assay employing 30 as a fluorescent probe was used to quantify specific binding to P2Y14R. Known P2Y receptor ligands inhibited binding of 30 with properties consistent with their previously established receptor selectivities and affinities. These results illustrate that potency in this series of 2-naphthoic acid derivatives can be preserved by chain functionalization, leading to highly potent fluorescent molecular probes for P2Y14R. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands. This approach demonstrates the predictive power of GPCR homology modeling and the relevance of newly determined X-ray structures to GPCR medicinal chemistry.

Toehold-mediated internal control to probe the near-field interaction between the metallic nanoparticle and the fluorophore.

Metallic nanoparticles (MNPs) are known to alter the emission of vicinal fluorophores through the near-field interaction, leading to either fluorescence quenching or enhancement. Much ambiguity remains in the experimental outcome of such a near-field interaction, particularly for bulk colloidal solution. It is hypothesized that the strong far-field interference from the inner filter effect of the MNPs could mask the true near-field MNP-fluorophore interaction significantly. Thus, in this work, a reliable internal control capable of decoupling the near-field interaction from far-field interference is established by the use of the DNA toehold concept to mediate the in situ assembly and disassembly of the MNP-fluorophore conjugate. A model gold nanoparticle (AuNP)-Cy3 system is used to investigate our proposed toehold-mediated internal control system. The maximum fluorescence enhancement is obtained for large-sized AuNP (58 nm) separated from Cy3 at an intermediate distance of 6.8 nm, while fluorescence quenching is observed for smaller-sized AuNP (11 nm and 23 nm), which is in agreement with the theoretical values reported in the literature. This work shows that the toehold-mediated internal control design can serve as a central system for evaluating the near-field interaction of other MNP-fluorophore combinations and facilitate the rational design of specific MNP-fluorophore systems for various applications.

The photolytic activity of poly-arginine cell penetrating peptides conjugated to carboxy-tetramethylrhodamine is modulated by arginine residue content and fluorophore conjugation site.

Upon light irradiation, Fluorophore-cell-penetrating peptide (Fl-CPP) conjugates can disrupt the integrity of biological membranes. This activity can in turn be used to photoinduce the disruption of endocytic organelles and promote the delivery of entrapped macromolecules such as proteins or RNAs into live cells. Recent mechanistic studies have shown that ROS production by the fluorophore and a latent lytic ability of CPPs act in synergy to elicit photolysis. However, how the structure of fluorophore-CPP conjugates impacts this synergistic activity remains unclear. Herein, using red blood cells (RBCs) as a model of biological membranes, we show that the number of arginine residues in a CPP as well as the position of fluorophore with respect to the CPP dramatically affect the photolytic activity of a fluorophore-CPP conjugate. These factors should therefore be considered for the development of effective photoinducible delivery agents.

Morphing hydrogel patterns by thermo-reversible fluorescence switching.

Stimuli responsive surfaces that show reversible fluorescence switching behavior in response to temperature changes were fabricated. Oligo(ethylene glycol) methacrylate thermoresponsive polymers with amine end-groups were prepared by atom transfer radical polymerization (ATRP). The polymers were patterned on silicon surfaces by electron beam (e-beam) lithography, followed by conjugation of self-quenching fluorophores. Fluorophore conjugated hydrogel thin films were bright when the gels were swollen; upon temperature-induced collapse of the gels, self-quenching of the fluorophores led to significant attenuation of fluorescence. Importantly, the fluorescence was regained when the temperature was cooled. The fluorescence switching behavior of the hydrogels for up to ten cycles was investigated and the swelling-collapse was verified by atomic force microscopy. Morphing surfaces that change shape several times upon increase in temperature were obtained by patterning multiple stimuli responsive polymers.

Practical Labeling Methodology for Choline‐Derived Lipids and Applications in Live Cell Fluorescence Imaging

Lipids of the plasma membrane participate in a variety of biological processes, and methods to probe their function and cellular location are essential to understanding biochemical mechanisms. Previous reports have established that phosphocholine-containing lipids can be labeled by alkyne groups through metabolic incorporation. Herein, we have tested alkyne, azide and ketone-containing derivatives of choline as metabolic labels of choline-containing lipids in cells. We also show that 17-octadecynoic acid can be used as a complementary metabolic label for lipid acyl chains. We provide methods for the synthesis of cyanine-based dyes that are reactive with alkyne, azide and ketone metabolic labels. Using an improved method for fluorophore conjugation to azide or alkyne-modified lipids by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), we apply this methodology in cells. Lipid-labeled cell membranes were then interrogated using flow cytometry and fluorescence microscopy. Furthermore, we explored the utility of this labeling strategy for use in live cell experiments. We demonstrate measurements of lipid dynamics (lateral mobility) by fluorescence photobleaching recovery (FPR). In addition, we show that adhesion of cells to specific surfaces can be accomplished by chemically linking membrane lipids to a functionalized surface. The strategies described provide robust methods for introducing bioorthogonal labels into native lipids.

ChemInform Abstract: Picazoplatin, an Azide‐Containing Platinum(II) Derivative for Target Analysis by Click Chemistry.

AbstractThe new title compound (I) readily binds DNA and RNA oligonucleotides and undergoes facile post‐labeling click reactions to alkyne—fluorophore conjugates.

Synthesis of triazole linked fluorescent amino acid and carbohydrate bio-conjugates: a highly sensitive and skeleton selective multi-responsive chemosensor for Cu(ii) and Pb(ii)/Hg(ii) ions

A set of triazole-based chromogenic and fluorescent chemosensors with amino acid/carbohydrate–fluorophore conjugates have been designed and synthesized. The metal cation-sensing properties of glycine–anthracene, C24H24O4N4 (3), glycine–pyrene C26H24O4N4 (4), glucose–anthracene, C32H33O10N3 (5) and glucose–pyrene, C34H33O10N3 (6) bio-conjugates have been studied systematically. The significant changes in their absorption spectra are accompanied by a strong color change from light yellow to brown for 3 and 4 and colorless to greenish blue for 5 and 6. Receptors 3 and 4 have potential in the “naked eye” detection of Cu2+ and 5 and 6 for Pb2+/Hg2+ ion. The receptors 3 and 4 show fluorescence diminution following Cu2+ coordination within the limit of detection at 0.89 parts per billion (ppb) and this is unprecedented, whereas the receptors 5 and 6 present drastic fluorescence quenching upon addition of Hg2+ and Pb2+ within the limit of detection at 4 and 2 ppb respectively. Interestingly, their fluorescence and colorimetric responses are preserved in the presence of water that can be used for the selective colorimetric detection of these ions in aqueous environments. Along with the spectroscopic data, combined 1H NMR titration of the complexes and the DFT studies suggest the proposed coordination modes.

Detection of vascular endothelial growth factor in colon cancer xenografts using bevacizumab based near infrared fluorophore conjugate.

BackgroundThe aim of this study was to develop the near infrared fluorescence (NIRF)-based imaging agent for the visualization of vascular endothelial growth factor (VEGF) in colon cancer. AlexaFluor 750 conjugating with bevacizumab, and injected intravenously into nude mice bearing VEGF over-expressing HT29 human colorectal cancer. Optical imaging was performed at 15 min, 24 h and 48 h post injection. Immunofluorescences staining of the tumor sections were performed. HT29 colorectal cancer xenografts were clearly visualized with bevacizumab-AlexaFluor 750.ResultsEx vivo analysis showed 2.1 ± 0.4%, 37.6 ± 6.3% and 38.5 ± 6.2% injected dose/g accumulated in the tumors at 15 min, 24 h and 48 h respectively. Tumor uptake was significantly decreased in pretreated with excess of bevacizumab (p = 0.002). Immunofluorescence analysis showed strong staining of anti-CD 31 antibody around the blood vessels. Anti-VEGF-A and bevacizumab showed heterogeneous expression throughout the tumor.ConclusionsCurrent study successfully detected the VEGF expression in HT29 colorectal cancer xenografts, signifying as a potential agent for non-invasive imaging of VEGF expression, which may be applied in clinical practice.

Rigid tetrazine fluorophore conjugates with fluorogenic properties in the inverse electron demand Diels-Alder reaction.

1,2,4,5-Tetrazine fluorophore derivatives with structurally rigid molecular designs were synthesized using Sonogashira and Stille cross-coupling as well as copper-catalyzed azide-alkyne cycloaddition. The synthesized bichromophoric systems exhibit low fluorescence quantum yields due to quenching by the tetrazine. The extent of fluorescence quenching observed for those systems was shown to depend on the distance between the fluorophore and the tetrazine. The decreased fluorescence is "turned on" by conversion of the tetrazine in the inverse electron demand Diels-Alder cycloaddition. Time resolved spectroscopy indicated resonance energy transfer between BODIPY and the tetrazine as the underlying quenching mechanism. The synthesized conjugates were successfully applied in protein labeling experiments.

NIR fluorescence labelled carbon nano-onions: synthesis, analysis and cellular imaging.

The preparation of novel NIR fluorescent carbon based nanomaterials, consisting of boron difluoride azadipyrromethene fluorophores covalently attached to carbon nano-onions, is demonstrated. In addition, the analysis of the new nanomaterial is presented. The fluorescent nano-derivative properties are customized such that their emission can be reversibly on/off modulated in response to pH, which is demonstrated in solution and in cells. The in vitro imaging of HeLa Kyoto cells is carried out and the cellular uptake of the carbon nano-onion NIR fluorophore conjugates is verified.

Fluorescent probes of the isoxazole–dihydropyridine scaffold: MDR-1 binding and homology model

Isoxazole-1,4-dihydropyridines (IDHPs) were tethered to fluorescent moieties using double activation via a lanthanide assisted Weinreb amidation. IDHP–fluorophore conjugate 3c exhibits the highest binding to date for IDHPs at the multidrug-resistance transporter (MDR-1), and IDHP–fluorophore conjugates 3c and 7 distribute selectively in SH-SY5Y cells. A homology model for IDHP binding at MDR-1 is presented which represents our current working hypothesis.

Fluorescently labeled chimeric anti‐CEA antibody improves detection and resection of human colon cancer in a patient‐derived orthotopic xenograft (PDOX) nude mouse model

The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model.

Fluorescein Derivatives in Intravital Fluorescence Imaging

Intravital fluorescence microscopy enables the direct imaging of fluorophores in vivo and advanced techniques such as fluorescence lifetime imaging (FLIM) enable the simultaneous detection of multiple fluorophores. Consequently, it is now possible to record distribution and metabolism of a chemical in vivo and to optimise the delivery of fluorophores in vivo. Recent clinical applications with fluorescein and other intravital fluorescent stains have occurred in neurosurgery, dermatology [including photodynamic therapy (PDT)] and endomicroscopy. Potential uses have been identified in periodontal disease, skin graft and cancer surgery. Animal studies have demonstrated that diseased tissue can be specifically stained with fluorophore conjugates. This review focuses on the fluorescein derived fluorophores in common clinical use and provides examples of novel applications from studies in tissue samples.

598 Chimeric Antibodies to CEA Improve Detection of Human Colon Cancer in Orthotopic Mouse Models

Positive surgical margins after colorectal cancer surgery are strong predictors for higher local recurrence rates and poor overall survival. Currently, no real-time, reliable detection assays for positive surgical margins at the time of surgery exist. We have previously shown improved detection and resection of primary pancreatic cancer with a mouse-derived fluorophoreconjugated antibody against the tumor antigen CEA in open laparotomies in mouse models. The aim of this study was to demonstrate improved sensitivity and specificity of a new chimerized antibody against CEA in detection of CEA-expressing colon cancer for improved resection in xenograft mouse models. Mouse models of human colon cancer were established with fragments of a CEA-expressing patient colon tumor. Two to four weeks after implantation, mice were randomized to fluorescence-guided surgery (FGS) or bright-field surgery (BS). Mice in the FGS group received tail vein injections of the chimeric anti-CEA-Alexa488 antibody 24 hours prior to resection. Preand postoperative images were obtained to assess for completeness of resection. Mice were then followed for 6 months postoperatively to assess for recurrence and overall survival. At termination, all tumor lesions were harvested and evaluated histologically. The chimeric antibody was also tested on frozen tumor and normal tissue arrays comparing it to the mouse antibody. The chimeric antibody directed against CEA demonstrated improved sensitivity and specificity in labeling CEA-expressing tumor compared to the mouse antibody. The fluorophore conjugation efficiency to the chimeric CEA antibody (Chi-Ab) was 2-fold higher than the mouse CEA antibody (M-Ab). On frozen tumor tissue arrays, the signal intensity of the Chi-Ab was significantly brighter compared to the M-Ab indicating improved binding to tumor tissue. For colon, pancreas and lung tumor samples, the signal intensity with the Chi-Ab was 94.1, 85.3, and 106.1, respectively compared to 72.5, 60.1, and 80.5 for M-Ab (p ,0.001). The Chi-Ab demonstrated improved specificity as evident by the lower signal intensity in normal human tissue samples compared to M-Ab (normal colon tissue: 4.3 vs 5.4; normal pancreas tissue: 1.5 vs 2.7) indicating decreased binding of Chi-Ab. The chimeric CEA antibody was also accurate in labeling human colon cancer in mouse xenografts enabling improved detection of tumor margins for more effective resection, increasing the R0 resection rate from 86% to 96%. The chimeric form of our fluorophore-conjugated CEA antibody has more effective labeling of human CEA-expressing cancer in tissue arrays and in our xenograft mouse models of human colon cancer. The improved sensitivity and specificity of the chimeric fluorophoreconjugated antibody is clinically translatable.

A review of the synthetic strategies for the development of BODIPY dyes for conjugation with proteins.

Conjugation of fluorophores to proteins is fundamental to several biotechnological applications and in the last decades boron-dipyrromethene (BODIPY) dyes started to be used in this field. BODIPYs are dipyrrin derivatives complexed to difluoroboryl that possesses several attractive photphysical properties. Herein we reviewed the synthetic methodologies published by academic and non-academic research groups aiming the obtainment of reactive BODIPY derivatives for conjugation with proteins.

ChemInform Abstract: Organic Solid‐State Fluorescence: Strategies for Generating Switchable and Tunable Fluorescent Materials

AbstractReview: 210 refs.

A Hexylchloride-Based Catch-and-Release System for Chemical Proteomic Applications

Bioorthogonal ligation methods that allow the selective conjugation of fluorophores or biotin to proteins and small molecule probes that contain inert chemical handles are an important component of many chemical proteomic strategies. Here, we present a new catch-and-release enrichment strategy that utilizes a hexylchloride group as a bioorthogonal chemical handle. Proteins and small molecules that contain a hexylchloride tag can be efficiently captured by an immobilized version of the self-labeling protein HaloTag. Furthermore, by using a HaloTag fusion protein that contains a protease cleavage site, captured proteins can be selectively eluted under mild conditions. We demonstrate the utility of the hexylchloride-based catch-and-release strategy by enriching protein kinases that are covalently and noncovalently bound to ATP-binding site-directed probes from mammalian cell lysates. Our catch-and-release system creates new possibilities for profiling enzyme families and for the identification of the cellular targets of bioactive small molecules.