Modulation of FRET efficiency by donor-acceptor ratio in co-condensed fluorophore-silica nanoconjugates

Förster resonance energy transfer (FRET) in nanocomposites is an important step in many applications. In order to maximize the amount of energy transferred from donor, D, to an acceptor, A, it is essential to minimize self-quenching of the donor. In the present work, fluorescein (D) and rhodamine B (A) are covalently bound into silica nanoshells (C-dye-SNS) with varying D:A ratio where the amount of A has been held constant. The total number of dye molecules is varied from 24 to 140 per nanoshell keeping the number of A molecules at ~13 ​± ​2 molecules per particle. In these conditions, FRET is found to be governed by the D:A ratio. An energy transfer process associated with maximum efficiency of 77% with time constant of 130 ps has been observed in these nanoconjugates. Fast fluorescence decays for D and corresponding risetime for A are observed in the present systems, unlike in those with high concentration of D and A (~1000 molecules/nanoshell). Thus, the presently used dye concentrations are found to be more appropriate than the higher concentration used earlier.     

A Novel Visible Range FRET Probe for Monitoring Acid Sphingomyelinase Activity in Living Cells

Activity of acid sphingomyelinase has been implicated in a number of diseases like acute lung injury, sepsis or metastasis of melanoma cells. Here, we present a sphingomyelinase FRET probe based on FAM/BODIPY dyes for real-time monitoring of acid sphingomyelinase. The probe gives rise to a tremendous increase in fluorescence of the fluorescein FRET donor upon cleavage and we show that this is, to a significant part, due to cleavage-associated phase transition, suggesting a more systematic consideration of such effects for future probe development. The probe allows for the first time to monitor relative sphingomyelinase activities of intact living cells by flow cytometry.     

A Dendron-Based Fluorescence Turn-On Probe for Tumor Detection

Specifically amplifying the emission signals of optical probes in tumors is an effective way to improve the tumor-imaging sensitivity and contrast. In this paper, the first case of dendron-based fluorescence turn-on probes mediated by a Förster resonance energy transfer (FRET) mechanism is reported. Dendrons up to the fourth generation with a hydrophilic oligo(ethylene glycol) scaffold are synthesized by a solid-phase synthesis strategy, and show precise and defect-free chemical structures. To construct the fluorescence turn-on probe, one Cy5.5 molecule is conjugated to the focal of a G3 dendron through a robust linkage and eight Black Hole Quencher 3 (BHQ-3) molecules are conjugated to its periphery through a PEG chain bearing a reductively cleavable disulfide linkage. By in vitro and in vivo experiments, it is demonstrated that the fluorescence of the dendron-based probe can be activated effectively and rapidly in the reductive environments of tumor cells and tissues, and the probe thus exhibits amplified tumor signals and weak normal tissue signals. Compared with the reported nanoscale turn-on probes, the dendron-based probe has several significant advantages, such as well-defined chemical structure, precisely controllable fluorophore/quencher conjugation sites and ratio, desirable chemical stability, and reproducible pharmacokinetic and pharmacological profiles, and is very promising in tumor detection.     

Liposomal FRET Assay Identifies Potent Drug-Like Inhibitors of the Ceramide Transport Protein (CERT)

Ceramide transfer protein (CERT) mediates non-vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate-limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non-homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT-mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96-well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.     

高效近红外聚集诱导发光纳米粒子用于生物成像的研究

基于荧光共振能量转移机理（FRET）,利用两亲性聚合物Pluronic F-127共包覆两种聚集诱导发光（Aggregation-induced Emission,AIE）材料TPABDFN和TPE-Me,制备了高效近红外发射TPABDFN/TPE-Me@F127纳米粒子.实验表明,这种聚合物纳米粒子具有很大的斯托克斯位移和较高的荧光量子效率,很好的单分散性、稳定性,以及较好的生物相容性和低的细胞毒性,对HepG2细胞进行荧光生物成像,得到很好的细胞成像效果.     

A Multicolor Large Stokes Shift Fluorogen-Activating RNA Aptamer with Cationic Chromophores

Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogues that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO⁺, which binds to the Chili aptamer with a low-nanomolar K_D. Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO⁺ complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.     

Synthesis and Systematic Evaluation of Dark Resonance Energy Transfer (DRET)‐Based Library and Its Application in Cell Imaging

In this paper, we report a new strategy for constructing a dye library with large Stokes shifts. By coupling a dark donor with BODIPY acceptors of tunable high quantum yield, a novel dark resonance energy transfer (DRET)‐based library, named BNM , has been synthesized. Upon excitation of the dark donor ( BDN ) at 490 nm, the absorbed energy is transferred to the acceptor ( BDM ) with high efficiency, which was tunable in a broad range from 557 nm to 716 nm, with a high quantum yield of up to 0.8. It is noteworthy to mention that the majority of the non‐radiative energy loss of the donor was converted into the acceptor’s fluorescence output with a minimum leak of donor emission. Fluorescence imaging tested in live cells showed that the BNM compounds are cell‐permeable and can also be employed for live‐cell imaging. This is a new library which can be excited through a dark donor allowing for strong fluorescence emission in a wide range of wavelengths. Thus, the BNM library is well suited for high‐throughput screening or multiplex experiments in biological applications by using a single laser excitation source.     

智能型金纳米棒荧光探针的制备及其肿瘤细胞成像研究

开发能特异和灵敏检测基质金属蛋白酶-2(MMP-2)的智能型荧光分子探针,对于癌症的早期精准诊断与治疗至关重要。本文基于MMP-2特异性识别的多肽底物,利用一价铜离子催化的"点击"化学反应将荧光素(FITC)和多肽底物GPLGVRGY相偶联,与SH-PEG-COOH修饰的金纳米棒在EDC和Sulfo-NHS的作用下通过酰胺化反应,制备得到一种新的MMP-2特异性响应的荧光共振能量转移(FRET)金纳米棒探针GNR@FITC。体外重组酶检测实验、MTT实验及细胞共聚焦显微成像结果表明,该探针对MMP-2过表达小鼠乳腺癌细胞4T1表现出较好的特异性和检测灵敏度,具有进一步开发应用于乳腺癌早期诊断的极大潜力。     

Structural and dynamical characterization of unilamellar AOT vesicles in aqueous solutions and their efficacy as potential drug delivery vehicle

Sodium bis(2-ethylhexyl) sulfosuccinate (AOT) is well known to form nanometre sized aqueous droplets in organic solvents and used in several contemporary applications including templates of nanoparticle synthesis. However, the detailed structural characterization of AOT in aqueous media is relatively less attended. Here we have used dynamic light scattering technique for the structural characterization of AOT in aqueous solutions and found to have a monodispersed, unilamellar vesicles (～140 nm diameter). The efficacy of the vesicle to host both charged drugs like H258 (2'-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl)-benzimidazo-2-yl-benzimidazole]), EtBr (ethidium bromide) and hydrophobic drug like DCM (4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran) has also been investigated using F?rster resonance energy transfer. Picosecond resolved and polarization gated spectroscopy have been used to study the solvation dynamics and microviscosity at the surface of the vesicles. We have also performed concentration and temperature dependent studies in order to confirm the stability of the vesicles in aqueous phase. The drug release profile of the vesicles has been studied through in vitro dialysis method. The non-toxic, monodispersed vesicles in aqueous media with a noteworthy stability in wide range of AOT concentration and temperature, capable of hosting drugs of various natures (both hydrophobic and charged) simultaneously for many codelivery applications with controlled drug release profile may find its applications in drug delivery.     

2,6,8‐Trisubstituted 3‐Hydroxychromone Derivatives as Fluorophores for Live‐Cell Imaging

We present the synthesis and photophysical characterisation of a series of structurally diverse, fluorescent 2,6,8‐trisubstituted 3‐hydroxychromone derivatives with high fluorescence quantum yields and molar extinction coefficients. Two of these derivatives ( 9 and 10 a ) have been studied as fluorophores for cellular imaging in HeLa cells and show excellent permeability and promising fluorescence properties in a cellular environment. In addition, we have demonstrated by photophysical characterisation of 3‐isobutyroxychromone derivatives that esterification of the 3‐hydroxyl group results in acceptable and useful fluorescence properties.     

Nanoparticles: Properties,applications and toxicities

This review is provided a detailed overview of the synthesis, properties and applications of nanoparticles (NPs) exist in different forms. NPs are tiny materials having size ranges from 1 to 100 nm. They can be classified into different classes based on their properties, shapes or sizes. The different groups include fullerenes, metal NPs, ceramic NPs, and polymeric NPs. NPs possess unique physical and chemical properties due to their high surface area and nanoscale size. Their optical properties are reported to be dependent on the size, which imparts different colors due to absorption in the visible region. Their reactivity, toughness and other properties are also dependent on their unique size, shape and structure. Due to these characteristics, they are suitable candidates for various commercial and domestic applications, which include catalysis, imaging, medical applications, energy-based research, and environmental applications. Heavy metal NPs of lead, mercury and tin are reported to be so rigid and stable that their degradation is not easily achievable, which can lead to many environmental toxicities.