一个具有大Stokes位移的苯并噻唑类pH荧光探针

本文通过乙烯基将作为荧光团的苯并噻唑与作为H+受体的4-吡啶基桥联构筑了一个基于分子内电荷转移机制的pH荧光探针BTP2。研究表明该探针的Stokes位移为237 nm,远大于相应2-吡啶基类似物BTP1。滴定实验表明该探针的荧光在pH3.80至5.50之间随pH值增大而增强,且不受其他金属离子的干扰,具有检测胞内酸性细胞器pH的良好前景。探针pKa为4.72,略高于BTP1。4-吡啶基连接导致的更大的Stokes位移表明调节吡啶连接位置可以实现对该类探针分子Stokes位移的调控。     

Supramolecular Fluorescence Resonance Energy Transfer in Nucleobase‐Modified Fluorogenic RNA Aptamers

RNA aptamers form compact tertiary structures and bind their ligands in specific binding sites. Fluorescence‐based strategies reveal information on structure and dynamics of RNA aptamers. Herein, we report the incorporation of the universal emissive nucleobase analog 4‐cyanoindole into the fluorogenic RNA aptamer Chili, and its application as a donor for supramolecular FRET to the bound ligands DMHBI⁺ or DMHBO⁺. The photophysical properties of the new nucleobase–ligand‐FRET pair revealed structural restraints for the overall RNA aptamer organization and identified nucleotide positions suitable for FRET‐based readout of ligand binding. This strategy is generally suitable for binding‐site mapping and may also be applied for responsive aptamer devices.     

Reductive Charge Transfer through an RNA Aptamer

The transfer of charges through double helical DNA is a very well investigated bioelectric phenomenon. RNA, on the contrary, has been less studied in this regard. The few available data report on charge transfer through RNA duplex structures mainly composed of homonucleotide sequences. In the light of the RNA world scenarios, it is an interesting question, if charge transfer can be coupled with RNA function. Functional RNAs however, contain versatile structural motifs. Therefore, electron transport also through non-Watson–Crick base-paired regions might be required. We here demonstrate distance-dependent reductive charge transfer through RNA duplexes and through the non-Watson–Crick base-paired region of an RNA aptamer.     

Bleaching-resistant,Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.     

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.     

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series**

The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.     

A comparative physicochemical study of unsymmetrical indium phthalocyanines in the presence of magnetic nanoparticles or quantum dots

Asymmetric indium phthalocyanine (3, containing an NH₂ group) was conjugated (via an amide bond) to magnetic nanoparticle (MNP) functionalized with carboxylic acid or glutathione-capped CdTe/ZnSe/ZnO quantum dots to form 3-MNPs or 3-QDs. Techniques such as time-resolved fluorescence measurements, transmission electron microscopy, XPS, elemental analysis, FTIR, NMR (¹H, ¹³C, and cozy), electronic spectroscopy, as well as mass spectroscopy were employed to characterize 3 and its nanoconjugates. The phthalocyanine conjugated to quantum dot (3-QDs) possesses the lowest Ф_pd higher Ф_? and Ф_T as well as longer triplet lifetimes compares to 3-MNPs and free phthalocyanine.     

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.     

Characterization of the Self-Assembly of Glutathione Stabilized Cadmium Selenide–Zinc Sulfide Quantum Dots with a Cyanine5-Labeled Peptide by Capillary Electrophoresis and Fluorescence

Capillary electrophoresis with fluorescence detection was used to characterize the self-assembly of cadmium selenide–zinc sulfide quantum dots and a cyanine5-labeled peptide (cyanine5-EAAAAAHHHHHH). The self-assembly was driven by metal-affinity forces to allow characterization by Förster resonance energy transfer. The Förster resonance energy transfer signal increased with the ratio of cyanine5-EAAAAAHHHHHH to quantum dots and reached a plateau at a ratio of 32:1. This method illustrates the application of quantum dot-based bioanalysis.     

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.     

Multienzyme detection and in-situ monitoring of enzyme activity by bending CE using quantum dots-based polypeptide substrate

Multienzyme detection and monitoring enzyme activity in situ are significant for the disease to diagnose. This study aims to develop a quantum dots (QDs)-based nanoprobe Cyanine5-DDDLEVLFQFPGLVPRGSGGHHHHHH-QDs (Cy5-LEVLVP-QD), which is able to detect two enzymes inside a bent capillary using CE. Cy5-LEVLVP and QDs were allowed to bind with each other through metal affinity interaction and then injected the Cy5-LEVLVP-QD complex into a capillary with different bends, followed by related enzyme that can cleave the Cy5-LEVLVP peptide. The fluorescence of Cy5 was excited by QDs due to Förster resonance energy transfer. By monitoring the peaks produced by the original Cy5-LEVLVP-QD complex and a significant fluorescence change, sensitive analysis of two different enzymes was conducted. Therefore, the novel approach of using capillaries with semicircular bends could prove particularly useful for enzyme investigating in disease.     

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.     

Composite Mesoporous Silica Nanoparticles with Dual-color Afterglow for Cross-correlation-based Living Cell Imaging

Room temperature phosphorescence (RTP) materials are characterized with emission after removing the excitation source. Such long-lived emission feature possesses great potential in biological fluorescence imaging because it enables a way regarding temporal dimension for separating the interference of autofluorescence and common noises typically encountered in conventional fluorescence imaging. Herein, we constructed a new type of mesoporous silica nanoparticles (MSNs)-based composite nanoparticles (NPs) with dual-color long-lived emission, namely millisecond-level green phosphorescence and sub-millisecond-level delayed red fluorescence by encapsulating a typical RTP dye and Rhodamine dye in the cavities of the MSNs with the former acting as energy donor (D) while the latter as acceptor (A). Benefiting from the close D-A proximity, energy match between the donor and the acceptor and the optimized D/A ratio in the composite NPs, efficient triplet-to-singlet Förster resonance energy transfer (TS-FRET) in the NPs occurred upon exciting the donor, which enabled dual-color long-lived emission. The preliminary results of dual-color correlation imaging of live cells based on such emission feature unequivocally verified the unique ability of such NPs for distinguishing the false positive generated by common emitters with single-color emission feature.     

一种大斯托克斯位移的红光发射荧光探针选择性检测次氯酸

设计、合成了一种基于巴比妥酸衍生物的具有 D-π-A结构的光学探针 3。该探针能够作为一种高度灵敏和选择性的次氯酸指示剂, 快速实现对次氯酸的比色和荧光信号(开-关)的双响应(约 15 s)。推测的响应机制是 ClO^-与 C=C之间发生了亲电加成和氧化裂解反应, 导致探针的 D-π-A 结构遭到破坏, 从而阻断了其分子内电荷转移(intramolecular charge transfer, ICT)进程。探针只需一步即可合成, 同时具有红光发射(628 nm)和较大的斯托克斯位移(158 nm), 检测限(limit of detection, LOD)低至14 nmol·L^-1。此外, 探针还表现出低细胞毒性, 并成功应用于活细胞成像。     

一种大斯托克斯位移的红光发射荧光探针选择性检测次氯酸

设计、合成了一种基于巴比妥酸衍生物的具有D-π-A结构的光学探针3。该探针能够作为一种高度灵敏和选择性的次氯酸指示剂,快速实现对次氯酸的比色和荧光信号(开-关)的双响应(约15 s)。推测的响应机制是ClO^-与C=C之间发生了亲电加成和氧化裂解反应,导致探针的D-π-A结构遭到破坏,从而阻断了其分子内电荷转移(intramolecular charge transfer,ICT)进程。探针只需一步即可合成,同时具有红光发射(628 nm)和较大的斯托克斯位移(158 nm),检测限(limit of detection,LOD)低至14 nmol·L^-1。此外,探针还表现出低细胞毒性,并成功应用于活细胞成像。     

A Red to Near‐IR Fluorogen: Aggregation‐Induced Emission,Large Stokes Shift,High Solid Efficiency and Application in Cell‐Imaging

A tetraphenylethene (TPE) derivative modified with the strong electron acceptor 2‐dicyano‐methylene‐3‐cyano‐4,5,5‐trimethyl‐2,5‐dihydrofuran (TCF) was obtained in high yield by a simple two‐step reaction. The resultant TPE‐TCF showed evident aggregation‐induced emission (AIE) features and pronounced solvatochromic behavior. Changing the solvent from apolar cyclohexane to highly polar acetonitrile, the emission peak shifted from 560 to 680 nm (120 nm redshift). In an acetonitrile solution and in the solid powder, the Stokes shifts are as large as 230 and 190 nm, respectively. The solid film emits red to near‐IR (red‐NIR) fluorescence with an emission peak at 670 nm and a quantum efficiency of 24.8 %. Taking the advantages of red‐NIR emission and high efficiency, nanoparticles (NPs) of TPE‐TCF were fabricated by using tat‐modified 1,2‐distearoylsn‐glycero‐3‐phosphor‐ethanol‐amine‐N‐[methoxy‐(polyethyl‐eneglycol)‐2000] as the encapsulation matrix. The obtained NPs showed perfect membrane penetrability and high fluorescent imaging quality of cell cytoplasm. Upon co‐incubation with 4,6‐diamidino‐2‐phenylindole (DAPI) in the presence of tritons, the capsulated TPE‐TCF nanoparticles could enter into the nucleus and displayed similar staining properties to those of DAPI.     

Molecular aptamers for real-time protein-protein interaction study

Protein-protein interactions play critical roles in cellular functions, but current techniques for real-time study of these interactions are limited. We report the real-time monitoring of protein-protein interactions without labeling either of the two interacting proteins; this procedure poses minimum effects on the binding properties of the proteins. Our strategy uses a protein/aptamer complex to probe the interactions in a competitive assay where the binding of an aptamer to its target protein is altered by a second protein that interacts with the target protein. Two signal transduction strategies, fluorescence resonance energy transfer (FRET) and fluorescence anisotropy, have been designed to study the interactions of human alpha-thrombin with different proteins by using two aptamers specific for two binding sites on alpha-thrombin. Our method has been shown to be simple and effective, does not require labeling of proteins, makes use of easily obtainable aptamers, provides detailed protein-protein interaction information and has excellent sensitivity for protein detection and protein-protein interaction studies. The FRET and the fluorescent anisotropy approaches complement each other in providing insight into the kinetics, mechanisms, binding sites and binding dynamics of the interacting proteins.     

具有大斯托克位移的荧光探针分子

用荧光法来监视多个生理参数时,需要几个不同的荧光探针分子.这些探针分子要被同一波长激发,但是具有明显分离的、不同的发射波长.目前,大多数荧光探针只有小的斯托克位移(50-90 nm),从而限制了它们在多个物质同时检测上的应用.在这项工作中,我们提出了一个新的分子探针设计:受体-荧光分子1-间隔-荧光分子2(简称RFSF...     

Human Serum Albumin Labelling with a New BODIPY Dye Having a Large Stokes Shift

BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. These are widely used as chemosensors, laser materials, and molecular probes. At the same time, BODIPY dyes have small or moderate Stokes shifts like most other fluorophores. Large Stokes shifts are preferred for fluorophores because of higher sensitivity of such probes and sensors. The new boron containing BODIPY dye was designed and synthesized. We succeeded to perform an annulation of pyrrole ring with coumarin heterocyclic system and achieved a remarkable difference in absorption and emission maximum of obtained fluorophore up to 100 nm. This BODIPY dye was equipped with linker arm and was functionalized with a maleimide residue specifically reactive towards thiol groups of proteins. BODIPY residue equipped with a suitable targeting protein core can be used as a suitable imaging probe and agent for Boron Neutron Capture Therapy (BNCT). As the most abundant protein with a variety of physiological functions, human serum albumin (HSA) has been used extensively for the delivery and improvement of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare albumin-based multimodal constructions. The released sulfhydryl groups of the homocysteine functional handle in thiolactone modified HSA were labeled with BODIPY dye to prepare a labeled albumin-BODIPY dye conjugate confirmed by MALDI-TOF-MS, UV-vis, and fluorescent emission spectra. Cytotoxicity of the resulting conjugate was investigated. This study is the basis for a novel BODIPY dye-albumin theranostic for BNCT. The results provide further impetus to develop derivatives of HSA for delivery of boron to cancer cells.