Cyanine Dye Cy5 Research Articles

Protein Environment and DNA Orientation Affect Protein-Induced Cy3 Fluorescence Enhancement

The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration.

Octreotide Conjugates for Tumor Targeting and Imaging.

Tumor targeting has emerged as an advantageous approach to improving the efficacy and safety of cytotoxic agents or radiolabeled ligands that do not preferentially accumulate in the tumor tissue. The somatostatin receptors (SSTRs) belong to the G-protein-coupled receptor superfamily and they are overexpressed in many neuroendocrine tumors (NETs). SSTRs can be efficiently targeted with octreotide, a cyclic octapeptide that is derived from native somatostatin. The conjugation of cargoes to octreotide represents an attractive approach for effective tumor targeting. In this study, we conjugated octreotide to cryptophycin, which is a highly cytotoxic depsipeptide, through the protease cleavable Val-Cit dipeptide linker using two different self-immolative moieties. The biological activity was investigated in vitro and the self-immolative part largely influenced the stability of the conjugates. Replacement of cryptophycin by the infrared cyanine dye Cy5.5 was exploited to elucidate the tumor targeting properties of the conjugates in vitro and in vivo. The compound efficiently and selectively internalized in cells overexpressing SSTR2 and accumulated in xenografts for a prolonged time. Our results on the in vivo properties indicate that octreotide may serve as an efficient delivery vehicle for tumor targeting.

Dye Encapsulation in Fluorescent Core-Shell Silica Nanoparticles as Probed by Fluorescence Correlation Spectroscopy.

Synthetic advances in the formation of ultrasmall (<10 nm) fluorescent poly(ethylene glycol)-coated (PEGylated) core-shell silica nanoparticles (SNPs), enabling improved particle size and surface chemical property control have led to successful clinical translation of SNPs as diagnostic probes in oncology. Despite the success of such probes, details of the dye incorporation and resulting silica architecture are still poorly understood. Here, we employ afterpulse-corrected fluorescence correlation spectroscopy (FCS) to monitor fast fluorescence fluctuations (lag times <10-5 s) of the negatively charged cyanine dye Cy5 as a probe to study such details for dye encapsulation in 5 nm silica cores of PEGylated core-shell SNPs (C dots). Upon deposition of additional silica shells over the silica core we find that the amplitude of photo-induced cis-trans isomerization decreases, suggesting that the Cy5 dyes are located near or on the surface of the original SNP cores. In combination with time correlated fluorescence decay measurements we deduce radiative and non-radiative rates of the Cy5 dye in these particles. Results demonstrate that FCS is a well-suited tool to investigate aspects of the photophysics of fluorescent nanoparticles, and that conformational changes of cyanine dyes like Cy5 are excellent indicators for the local dye environment within ultrasmall SNPs.

Electrochemiluminescent aptasensor based on resonance energy transfer system between CdTe quantum dots and cyanine dyes for the sensitive detection of Ochratoxin A

In this work, an innovative aptasensor based on electrochemiluminescence resonance energy transfer (ECL-RET) from CdTe quantum dots (QDs) to a cyanine dye (Cy5) fluorophore for the determination of Ochratoxin A (OTA) was fabricated. A strong cathodic ECL emission was obtained by the CdTe QDs modified glassy carbon electrode (GCE). After the immobilization with the capture DNA (cDNA) and the sequential hybridization with the probe DNA-modified Cy5 (pDNA, the aptamer of OTA), the ECL signal enhanced obviously through the ECL-RET. Meanwhile, the spectrum- and distance-related ECL enhancement effect was investigated. When the target OTA was in the presence, the pDNA-Cy5 molecules were released from the electrode surface owing to the specific interaction between OTA and aptamer, resulting in an evident decrease of ECL signal. Under optimal conditions, the developed aptasensor displayed the linear response toward OTA in the wide range of 0.0005–50 ng/mL with a low detection limit of 0.17 pg/mL. With the excellent selectivity, stability and repeatability, the strategy provided an efficient and universal method for the sensitive detection of target in practical application.

Effect of the acrosome reaction on the efficiency of sperm-mediated DNA transfer.

Sperm-mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind exoge- nous DNA and transfer it into oocytes by fertilization. However, SMGT is still undergoing opti- mization to improve its efficiency to produce transgenic animals. The acrosome reaction is neces- sary for spermatozoa to carry the exogenous DNA into oocytes. In this study, the effect of the acrosome reaction on the efficiency of spermatozoa carrying exogenous DNA was evalua- ted. The results showed that the efficiency of the acrosome reaction was significantly higher (p⟨0.05) after incubation with 50 μmol/L progesterone compared to incubation without proges- terone. It was significantly higher (p⟨0.05) in the 20, 40, and 60 min of progesterone treatment groups than in the 0 min treatment group. The spermatozoa were further incubated with cyanine dye Cy5 labeled DNA (Cy5-DNA) for 30 min at 37°C, and positive fluorescence signals were detected after the acrosome reaction was induced by progesterone at concentrations of 0 and 50 μmol/L for 40 min. The percentage of positive Cy5-DNA signals in spermatozoa was 96.61±2.06% and 97.51±2.03% following exposure to 0 and 50 μmol/L progesterone, respective- ly. The percentage of partial spermatozoa heads observed following combination with Cy5-DNA was 39.73±3.03% and 56.88±3.12% following exposure to 0 and 50 μmol/L progesterone, respec- tively. The ratio of positively stained spermatozoa combined with exogenous DNA showed no reduction after the acrosome reaction. These results suggest that the acrosome reaction might not be the key factor affecting the efficiency of SMGT.

Glutathione Triggered Near Infrared Fluorescence Imaging-Guided Chemotherapy by Cyanine Conjugated Polypeptide.

Precise detection of the tumor environment for cancer diagnosis has been strongly demanded for further therapies. Here, a redox-responsive fluorescence switch off/on system PCQ was designed and synthesized conjugated with near-infrared (NIR) cyanine dyes (Cy5.5) and relevant quencher (FQ) in mixed polymeric micelles (PCy and PFQ). The mixed PCQ micelles were prepared with two kinds of polymers with poly(oligo (ethylene glycol) methacrylate) (POEGMA) as the hydrophilic shell, in which fluorescence emission was quenched by fluorescence resonance energy transfer (FRET) effect. The FQ was conjugated with POEGMA by disulfide linkage, which could be broken with a redox environment such as high glutathione (GSH) concentration in tumor cells. After the PCQ micelles got into tumor cells, PFQ blocks in PCQ would be disassembled to recompose PCy micelles. During that process, drugs like doxorubicin (DOX) could be loaded inside and formed PCQ@DOX nanoparticles and then released for accurate NIR bioimaging and drug delivery instantly.

Dendron-Grafted Polylysine-Based Dual-Modal Nanoprobe for Ultra-Early Diagnosis of Pancreatic Precancerosis via Targeting a Urokinase-Type Plasminogen Activator Receptor.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death. Early detection of precancerous pancreatic intraepithelial neoplasia (PanIN) tissues is an urgent challenge to improve the PDAC prognosis. Here, a urokinase-type plasminogen activator receptor (uPAR)-targeted magnetic resonance (MR)/near-infrared fluorescence (NIRF) dual-modal nanoprobe dendron-grafted polylysine (DGL)-U11 for ultra-early detection of pancreatic precancerosis is reported. Because of its good biocompatibility and biodegradability, globular architecture, and well-defined reactive groups, the DGL is chosen as the platform to load with a pancreatic tumor-targeting peptide U11, a magnetic resonance contrast agent Gd3+ -diethylene triamine pentaacetic acid, and a near-infrared fluorescent cyanine dye Cy5.5. The nanoprobe DGL-U11 has several preferable characteristics, such as active peptide targeting to activator receptor, good biocompatibility, dual-modal imaging diagnosis, and well controlled diameter in a range of 15-25 nm. Upon incorporation of the active U11 peptide target to the overexpressed activator receptor uPAR, the targeted nanoprobe DGL-U11 can increase to the earlier PanIN-II stage through in vivo NIRF imaging. Labeled with both MR and NIRF bioimaging reporters, the uPAR-targeted dual-modal nanoprobe is very effective in the targeted imaging of precancerous PanINs and PDAC lesions with high sensitivity and spatial resolution, providing a promising platform to the ultra-early detection of PDAC.

Combination of active targeting, enzyme-triggered release and fluorescent dye into gold nanoclusters for endomicroscopy-guided photothermal/photodynamic therapy to pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating malignancies in patients, and there is an urgent need for an effective treatment method. Herein, we report a novel gold nanocluster-based platform for confocal laser endomicroscopy-guided photothermal therapy (PTT)/photodynamic therapy (PDT) for PDAC, which consists of four components: the PTT-carrier gold nanocluster, an active targeting ligand U11 peptide, a Cathepsin E (CTSE)-sensitive PDT therapy prodrug, and a CTSE-sensitive imaging agent (cyanine dye Cy5.5). Due to the strong coupling among cross-linked gold nanoparticles (AuNPs), the surface plasmon resonance peak of nanoclusters shifts to the near-infrared (NIR) region, thus making the nanoclusters useful in the effective PTT therapy. In the system, the labeling of nanoclusters with U11 peptide can distinctly increase their affinity and accelerate their uptake by pancreatic cancer cells. Cell apoptosis staining demonstrates that, upon incorporation of the uPAR-targeted unit, the antitumor efficacy of CTSE-sensitive nanocluster AuS-U11 is significantly enhanced with respect to that of the non-targeted nanocluster AuS-PEG and the insensitive nanocluster AuC-PEG. Invivo and exvivo optical imaging confirms the high accumulation of AuS-U11 in the in situ pancreatic tumor model. Therapeutic studies further show that the combination of active targeting for tumor tissue, enzyme-triggered drug release of 5-ALA and fluorescent dye Cy5.5 in nanoclusters AuS-U11 could achieve optimal therapeutic efficacy with endomicroscopy-guided photothermal/photodynamic therapy with minimal side effects. As a consequence, the delicate gold nanocluster concept provides a promising strategy to enhance the therapy efficiency in the most challenging PDAC treatment.

Theranostic Polyaminocarboxylate-Cyanine-Transferrin Conjugate for Anticancer Therapy and Near-Infrared Optical Imaging.

Iron chelation therapy has been recognized as a promising antitumor therapeutic strategy. Herein we report a novel theranostic agent for targeted iron chelation therapy and near-infrared (NIR) optical imaging of cancers. The theranostic agent was prepared by incorporation of a polyaminocarboxylate-based cytotoxic chelating agent (N-NE3TA; 7-[2-[(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) and a NIR fluorescent cyanine dye (Cy5.5) onto a tumor-targeting transferrin (Tf). The N-NE3TA-Tf conjugate (without Cy5.5) was characterized and evaluated for antiproliferative activity in HeLa, HT29, and PC3 cancer cells, which have elevated expression levels of the transferrin receptor (TfR). The N-NE3TA-Tf conjugate displayed significant inhibitory activity against all three cancer cell lines. The NIR dye Cy5.5 was then incorporated into N-NE3TA-Tf, and the resulting cytotoxic and fluorescent transferrin conjugate N-NE3TA-Tf-Cy5.5 was shown by microscopy to enter TfR-overexpressing cancer cells. This theranostic conjugate has potential application for dual use in targeted iron chelation cancer therapy and NIR fluorescence imaging.

Harnessing Leaky Modes for Fluorescence Enhancement in Gold-Tipped Silicon Nanowires.

Hybrid structures containing plasmonic and photonic components can enhance light-matter interaction through both photonic and plasmonic modes and potentially their interactions. In this article, we systematically investigate the optical properties of nanoparticle-tipped silicon nanowires (NPTWs) and characterize their ability to enhance the fluorescence signal of two cyanine dyes (Cy3 and Cy5) selectively bound to the gold tip. Although both gold and silicon components contribute to the fluorescence signal enhancement, the experimentally observed strong morphology dependence of the fluorescence emission enhancement is demonstrated to depend on tunable leaky modes of the silicon nanowires. The ability to encode emission fluorescence enhancement in the structure of NPTWs is of high relevance for multiparametric biosensing and imaging.

Single-molecule DNA hybridisation studied by using a modified DNA sequencer: a comparison with surface plasmon resonance data

Current methods for the determination of molecular interactions are widely used in the analytical sciences. To identify new methods, we investigated as a model system the hybridisation of a short 7 nt oligonucleotide labelled with, structurally, very similar cyanine dyes CY3 and DY-547, respectively, to a 34 nt oligonucleotide probe immobilised in a zero-mode waveguide (ZMW) nanostructure. Using a modified commercial off-the-shelf DNA sequencer, we established the principles to measure biomolecular interactions at the single-molecule level. Kinetic data were obtained from trains of fluorescence pulses, allowing the calculation of association and dissociation rate constants (kon, koff). For the 7mer labelled with the positively charged CY3 dye, kon and koff are ~3 larger and ~2 times smaller, respectively, compared with the oligonucleotide labelled with negatively charged DY-547 dye.The effect of neighbouring molecules lacking the 7nt binding sequence on single-molecule rate constants is small. The association rate constants is reduced by only 20–35%. Hybrid dissociation is not affected, since as a consequence of the experimental design, rebinding cannot take place.Results of single-molecule experiments were compared with data obtained from surface plasmon resonance (SPR) performed under comparable conditions. A good correlation for the association rate constants within a factor of 1.5 was found. Dissociation rate constants are smaller by a factor of 2–3 which we interpreted as a result of rebinding to neighbouring probes.Results of SPR measurements tend to systematically underestimate dissociation rate constants. The amount of this deviation depends on the association rate constant and the surface probe density. As a consequence, it is recommended to work at low probe densities to keep this effect small.

Multifunctional gold nanocomposites designed for targeted CT/MR/optical trimodal imaging of human non-small cell lung cancer cells.

Multifunctional gold nanocomposites, which were designed as dendrimer-entrapped gold nanoparticles functionalized with gadolinium, cyanine dye (Cy5.5), and folic acid, were synthesized to be used as the first dendrimer-based clinical nanoprobes for targeted X-ray computed tomography/magnetic resonance/optical trimodal imaging in vitro and in vivo of human non-small cell cancer cells.

Synthesis of TaqMan probes using bisphosphoramidite of cyanine dye Cy3

A method of the synthesis of TaqMan probes with the use of bisphosphoramidite of cyanine dye Cy3 was proposed. The synthesis results in two variants of the probes consisting of either one or two oligonucleotide residues (bearing a quencher at the 3' end) attached to the dye, with the first variant being predominant. The RT-PCR probe of the classical structure bearing one oligonucleotide residue was shown to be more efficient as compared to the probe containing two oligonucleotides.

NON-RADIATIVE ENERGY TRANSFER FROM Sm(III) COMPLEX ON CYANINE DYE Cy5

The optimal conditions for the formation of new chelate Sm(III) with oxyquinoline carboxylic acid derivative (L) and spectral-luminescent characteristics (the maximum wavelength of luminescence, luminescence excitation and lifetime) were determinated. The quenching effect on the luminescence Sm(III)–L complex by cyanine dye Cy5 (fluorescent label of proteins and other biological molecules) was detected. The quenching of chelate complex due to Forster resonance energy transfer of electronic excitation (FRET) from the donor (Sm(III)–L) to the acceptor (Cy5). The intensity of the luminescence of the dye Cy5 in the presence of Sm(III)–L increased in 6 times.The values of the Forster radius (R0 (Sm-L) = 41 A) and the overlap integral of the luminescence energy of the donor with the absorption spectrum of the acceptor (JSm–L= 1.87×1012, M-1cm- 1nm4) calculated from the distribution of the quantum luminescence intensity of the donor, the spectrum and the molar absorption coefficient of the acceptor dye Cy5 possible to characterize as an efficient quencher of luminescence of Sm(III) ions. Sm(III)–L chelate complex is proposed as FRET-donor at first.

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA).

Systemic and Intravitreal Delivery of Dendrimers to Activated Microglia/Macrophage in Ischemia/Reperfusion Mouse Retina.

Microglial activation and associated neuroinflammation play a key role in the pathogenesis of many diseases of the retina, including viral infection, diabetes, and retinal degeneration. Strategies to target activated microglia and macrophages and attenuate inflammation may be valuable in treating these diseases. We seek to develop dendrimer-based formulations that target retinal microglia and macrophages in a pathology-dependent manner, and deliver drugs, either intravenously or intravitreally. Retinal uptake of cyanine dye (Cy5)-conjugated dendrimer (D-Cy5) was assessed in normal and ischemia/reperfusion (I/R) mouse eyes. Microglia/macrophage uptake of the dendrimer was assessed with immunofluorescence using rabbit Iba-1 antibody with Cy3-tagged secondary antibody (microglia/macrophage). Uptake in retina and other organs was quantified using fluorescence spectroscopy. Clearance of D-Cy5 from normal eyes was almost complete by 72 hours after intravitreal injection and 24 hours after intravenous delivery. In eyes with activated microglia after I/R injury, D-Cy5 was retained by activated microglia/macrophage (Iba1+ cells) up to 21 days after intravitreal and intravenous administration. In I/R eyes, the relative retention of intravitreal and intravenous D-Cy5 was comparable, if a 30-fold higher intravenous dose was used. Intravitreal and systemic dendrimers target activated microglia and show qualitatively similar retinal biodistribution when administered by either route. Results provide proof-of-concept insights for developing dendrimer drug formulations as treatment options for retinal diseases associated with microglia or macrophage activation such as age-related macular degeneration, diabetic retinopathy, and retinal degenerations.

A novel "dual-potential" electrochemiluminescence aptasensor array using CdS quantum dots and luminol-gold nanoparticles as labels for simultaneous detection of malachite green and chloramphenicol.

A novel type of "dual-potential" electrochemiluminescence (ECL) aptasensor array was fabricated on a homemade screen-printed carbon electrode (SPCE) for simultaneous detection of malachite green (MG) and chloramphenicol (CAP) in one single assay. The SPCE substrate consisted of a common Ag/AgCl reference electrode, carbon counter electrode and two carbon working electrodes (WE1 and WE2). In the system, CdS quantum dots (QDs) were modified on WE1 as cathode ECL emitters and luminol-gold nanoparticles (L-Au NPs) were modified on WE2 as anode ECL emitters. Then the MG aptamer complementary strand (MG cDNA) and CAP aptamer complementary strand (CAP cDNA) were attached on CdS QDs and L-Au NPs, respectively. The cDNA would hybridize with corresponding aptamer that was respectively tagged with cyanine dye (Cy5) (as quenchers of CdS QDs) and chlorogenic acid (CA) (as quenchers of l-Au NPs) using poly(ethylenimine) (PEI) as a bridging agent. PEI could lead to a large number of quenchers on the aptamer, which increased the quenching efficiency. Upon MG and CAP adding, the targets could induce strand release due to the highly affinity of analytes toward aptamers. Meanwhile, it could release the Cy5 and CA, which recovered cathode ECL of CdS QDs and anode ECL of L-Au NPs simultaneously. This "dual-potential" ECL strategy could be used to detect MG and CAP with the linear ranges of 0.1-100 nM and 0.2-150 nM, with detection limits of 0.03 nM and 0.07 nM (at 3sB), respectively. More importantly, this designed method was successfully applied to determine MG and CAP in real fish samples and held great potential in the food analysis.

Vibrationally Coherent Preparation of the Transition State for Photoisomerization of the Cyanine Dye Cy5 in Water

Femtosecond pump-continuum probe spectroscopy with impulsive excitation was employed to observe coherent wavepacket motions of the cyanine dye Cy5 in water that promote photoisomerization after optical preparation of the first excited singlet state, S1. The chief component in the excited-state vibrational coherence is a resonance Raman-inactive, 273 cm(-1) mode of mixed carbon-carbon bond length alternation and out-of-plane or twisting character. The ultrafast (30 fs) damping of these motions arises from trajectories that irreversibly cross the transition state barrier; after several recurrences to the transition state region, vibrational cooling traps a significant fraction of the excited-state molecules in the planar, Franck-Condon region of the potential energy surface. Motion in the 273 cm(-1) promoting mode is apparently launched by a change in conformation of the conjugated polyene backbone during the first few vibrations of the high-frequency C-C and C═C bond length alternation coordinates that principally contribute to the initial displacement from the Franck-Condon structure. To our knowledge, this work provides the first direct observations of the intramolecular redistribution of excited-state potential energy into reactive motions that are rapidly damped by transition state barrier-crossing events leading to photoisomerization in a conjugated polyene. These results provide insight into the vibrational dynamics that contribute to the photoisomerization of retinal protonated Schiff bases in the rhodopsins and to the formation of intramolecular charge transfer character in carotenoids in photosynthetic light-harvesting proteins.

Electrochemical Modulation of the Fluorescence of Cyanine Dye Cy5

AbstractOrganic fluorescent dyes are widely used in single molecule localization microscopy, where their performances are determined by the photophysical properties. Herein, we utilized a sensitive method to modulate the fluorescence of organic dyes by external potentials using a combination of electrochemical cell and super‐resolution fluorescent microscopy. Cy5 (cyanine dye) was chosen as a model molecule considering its wide application and commercial availability. We applied different potentials on the Au electrode to change the Coulombic charge microenvironment of Cy5. When the electrode potential was adjusted negatively, Cy5 displayed a better photostability. This method is proved effective in adjusting the fluorescence of organic dyes.

The carbamoylmannose moiety of bleomycin mediates selective tumor cell targeting.

Recently, we reported that both bleomycin (BLM) and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Here, we demonstrate that the conjugate of the BLM carbamoylmannose moiety with Cy5** showed tumor cell selective binding and also enhanced cellular uptake in most cancer cell lines. The carbamoyl functionality was required for tumor cell targeting. A dye conjugate prepared from a trivalent cluster of carbamoylmannose exhibited levels of tumor cell binding and internalization significantly greater than those of the simple carbamoylmannose–dye conjugate, consistent with a possible multivalent receptor.