A new fluorescent and colorimetric probe for Cu2+ in live cells

Based on a change in structure between spirocyclic (non-fluorescent) and ring-open (fluorescent) forms of rhodamine-based dyes, a new fluorescent and colorimetric Cu(2+) probe was designed and synthesized. Upon treatment with Cu(2+), the weakly fluorescent probe exhibited a strong fluorescence response with high selectivity. In addition, the turn-on fluorescent probe upon the addition of Cu(2+) was applied in live cell imaging.     

基于罗丹明染料的金属阳离子荧光探针

罗丹明是以氧杂蒽为母体的碱性呫吨染料,具有优良的光学性质,如延伸到可见光区的吸收及荧光、高的荧光量子产率及大的摩尔吸光系数等,使其成为制备荧光探针的理想生色团。本文综述了近年来用于检测金属阳离子的罗丹明类荧光探针的研究进展,特别是对基于螺酰胺环“关-开”机理、荧光共振能量转移(FRET)机理和光诱导电子转移(PET)机理的罗丹明类铜离子、汞离子、铁离子荧光探针进行了系统的阐述,包括结构特征、检测水平和应用范围。最后提出了这类荧光探针面临的问题与发展趋势。     

A fluorescent "turn-on" probe for the dual-channel detection of Hg(II) and Mg(II) and its application of imaging in living cells

A novel rhodamine-based fluorescent chemosensor (RND) was synthesized that contains two independent fluorophores and acts as a very sensitive, selective and reversible off-on probe for Hg(II). Importantly, this newly developed sensing system exhibited different fluorescent responses toward Hg(II) and Mg(II) at 589 nm and 523 nm, respectively. RND also displayed detectable color change upon treatment with Hg(II). Results from confocal laser scanning microscopy experiments demonstrated that this chemosensor is cell permeable and can be used as a fluorescent probe for monitoring Hg(II) or Mg(II) in living cells. This probe can also indirectly detect glutathione (GSH) and cysteine (Cys) with good linear relationships.     

Fe(III)- and Hg(II)-selective dual channel fluorescence of a rhodamine-azacrown ether conjugate

A rhodamine-azacrown ether conjugate (1) demonstrates Fe(III)-selective green fluorescence, while showing Hg(II)-selective orange fluorescence. This is the first example of rhodamine-based fluorescent probe that shows dual channel fluorescence for two different metal cations.     

Solid-phase extraction of mercury(II) with magnetic core-shell nanoparticles, followed by its determination with a rhodamine-based fluorescent probe

Magnetic Fe₃O₄@SiO₂ core shell nanoparticles containing diphenylcarbazide in the shell were utilized for solid phase extraction of Hg(II) from aqueous solutions. The Hg(II) loaded nanoparticles were then separated by applying an external magnetic field. Adsorbed Hg(II) was desorbed and its concentration determined with a rhodamine-based fluorescent probe. The calibration graph for Hg(II) is linear in the 60 nM to 7.0 μM concentration range, and the detection limit is at 23 nM. The method was applied, with satisfying results, to the determination of Hg(II) in industrial waste water.

官能化罗丹明基金属阳离子荧光探针

重金属离子对环境和生物体具有极强的生理毒性,故高选择性和高灵敏度的离子检测荧光探针的研究有着重要意义。荧光分子探针在表达分子间识别行为以及复杂的环境、生命体系的内状态信息方面具有优异的性能,已广泛深入的用于构建新型功能性探针分子。本文综述了近年来基于罗丹明的金属阳离子如Hg₂₊、Cu₂₊、Fe₃₊等荧光分子探针的研究进展,包括探针的结构特征、检测机理、检测水平,更重要的是其在环境检测、生物成像、分子器件等方面的新应用,并对荧光探针所面临的问题和发展前景做了分析。     

新型2-吡啶苄基胺类罗丹明基活性小分子探针的合成

以罗丹明B和乙二胺为原料,通过缩合反应制得罗丹明酰乙二胺(1, Rhb);然后以无水THF作溶剂,化合物1与三聚氯嗪发生亲核取代反应,得到罗丹明酰乙二胺-三聚氯嗪化合物(2, Rhbc);最后在氮气氛围下,化合物2与N,N-二甲基吡啶胺反应合成了目标产物双(2 吡啶苄基) 胺罗丹明基小分子探针(3, Rhbd),其结构经¹H NMR、 IR、 HR-MS和元素分析表征。该探针可以识别Fe³⁺,检出限为30 nM。     

Duplex probes: a new approach for the detection of specific nucleic acids in homogenous assays

A novel method for the detection of specific nucleic acids in homogenous solution was developed. The method is based on the use of duplex probes in which fluorescent donor and quencher labeled on either oligonucleotide are held in close proximity, so that fluorescence is quenched. Amplification of the target sequence results in the cleavage of the probe and the resulting fluorescence can be detected. The fluorescent data analysis demonstrated that the duplex probes can specifically recognize the presence of target, and a significantly higher lever of relative fluorescent signal than TaqMan probes is obtainable. Combined with real-time PCR instruments, the assay can be used to quantify the input target molecules. As few as five copies of initial target molecules can be detected, and a large dynamic linear ranger (five orders of magnitude) is obtained.     

一种Fe3+荧光探针的合成及其检测性能研究

金属离子的高效检测对于人类健康和环境保护极其重要。本文通过对甲基苯磺酸催化下3-脱氢莽草酸甲酯与若丹明6G乙二胺缩合的芳构化反应合成了一种检测Fe~(3+)的荧光探针分子Rh M606。该探针分子在乙醇溶液中特异性地与Fe~(3+)络合,导致若丹明内酰胺开环,在553nm处出现强的荧光发射峰。该荧光探针对Fe~(3+)具有良好的选择性,可定量检测水中的Fe~(3+),检测限为3. 93μmol/L。     

Fluorescence Correlation Spectroscopy Study of Probe Diffusion in Poly(vinyl alcohol) Solutions and Gels

In this study, we demonstrate how the diffusion of probe particles in aqueous poly(vinyl alcohol) (PVA) solutions and gels is affected by: (i) the presence of cross-links, (ii) the cross-link density, (iii) the polymer concentration. We apply fluorescence correlation spectroscopy (FCS) to measure the diffusion time of a rhodamine-based fluorescent particle (TAMRA) and TAMRA-labeled dextran in PVA solutions and gels prepared at various polymer concentrations (1% to 8.6% w/v) and cross-link densities (1/400 to 1/50 cross-link monomers per PVA monomers). The measurements indicate that the probe particles are slowed down with increasing polymer concentration and with increasing cross-link density. Also, FCS can detect differences in the diffusion times measured in “fresh” and “aged” PVA solutions. We find that FCS provides a quantitative measure of network inhomogeneities.     

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.     

Morpholine-Substituted Rhodamine Analogue with Multi-Configurational Switches for Optical Sensing of pH Gradient under Extreme Acidic Environments

Superior pH-responsive molecules are required for the development of functional materials applicable to advanced molecular technologies. Despite having been widely developed, many rhodamine-based pH-responsive molecules exhibit a single configurational switch for “turn-on”. Herein, we report a new type of rhodamine-based pH-responsive molecule with multi-configurational switches displaying stable two-step structural and color conversion in response to pH. This rhodamine analogue could be successfully applied to optical sensing of pH gradient under extreme acidic environments both in solution and on hydrogel through high-contrast color change. We demonstrated that this multi-responsive character enabled optical memory of different pH information.     

Steric and stereoscopic disulfide construction for cross-linkage via N-dithiophthalimides

Disulfide bonds are a significant motif in life and drug-delivery systems. In particular, steric hindrance and stereoscopic disulfide linkers are closely associated with the stability of antibody–drug conjugates, which affects the potency, selectivity, and pharmacokinetics of drugs. However, limited availability and diversity of tertiary thiols impede the construction of steric and stereoscopic disulfides for cross-linkage in biochemistry and pharmaceuticals. Through modulating the mask effect of disulfurating reagents, we develop a facile and robust strategy for construction of diverse steric and stereoscopic disulfides via N-dithiophthalimides. The practical cross-linkage of biomolecules including amino acids, saccharides, and nucleosides with different drugs and fluorescent molecules is successfully established through hindered disulfide linkers.

A series of steric and stereoscopic disulfides are constructed with N-dithiophthalimides, enabling the cross-linkage of biomolecules, drugs and fluorescent molecules.     

Fluorescence turn-on chemodosimeter for rapid detection of mercury (II) ions in aqueous solution and blood from mice with toxicosis

The heavy metal mercury (Hg) is a threat to the health of people and wildlife in many environments. Among various chemical forms, Hg²⁺ salts are usually more toxic than their counterparts because of their greater solubility in water; thus, they are more readily absorbed from the gastrointestinal tract into circulation. Therefore, new chemical receptors for detecting Hg²⁺ ions in circulation are needed. In this study, we developed a rhodamine-based turn-on fluorescence probe to monitor Hg²⁺ in aqueous solution and in blood of mice with toxicosis. The chemodosimeter responds to Hg²⁺ ions stoichiometrically, rapidly, and irreversibly at room temperature as a result of a chemical reaction that produces strongly fluorescent oxadiazole. The new fluorescent probe shows good fluorescence response, with high sensitivity and selectivity, toward Hg²⁺ ions in aqueous solution and in blood from mice with toxicosis and facilitates the naked-eye detection of Hg²⁺ ions.     

Parallel Detection of Different DNA Sequences on One Gold Electrode

Motivated by the potential of electrochemical techniques to analyze hybridization events fast and in a simple and cost‐effective way we present here a detection system allowing a parallel electrochemical DNA analysis. For this purpose different probe DNA strands have been immobilized on one electrode. By the use of two different target DNA sequences, both marked with the redox active methylene blue, we can show that hybridization with the complementary probe sh“NA strands can occur without steric hindrance. Each target has been recognized down to 3nM with a very high specificity of the sensor. In addition, we can detect two different ssDNA targets labeled with different redox active molecules, methylene blue and ferrocene, on one sensor surface simultaneously.     

A rhodamine-based Hg2+-selective fluorescent probe in aqueous solution

A new rhodamine-based Hg²⁺-selective fluorescent probe (I) was designed and synthesized. Compound I displays excellent selective and sensitive response to Hg²⁺ over other transition metal ions in neutral aqueous solutions. I itself is a colorless, nonfluorescent compound. Upon addition of Hg²⁺ to its solution, the thiosemicarbazide moiety of I undergoes an irreversible desulfurization reaction to form the corresponding 1,3,4-oxadiazole (II), a colorful and fluorescent product, causing instantaneous development of visible color and strong fluorescence emission. Based on this mechanism, a fluorogenic probe for Hg²⁺ was developed. The fluorescence increases linearly with the Hg²⁺ concentration up to 0.8 μmol L⁻¹ with the detection limit of 9.4 nmol L⁻ (3σ).     

Design and synthesis of novel rhodamine-based chemodosimeters derived from [2.2]paracyclophane and their application in detection of Hg2+ion

For a better insight into the spectroscopic properties of [2.2]paracyclophane in fluorescent probes, a novel rhodamine-based chemodosimeter bearing [2.2]paracyclophane 4a has been designed and synthesized. The probe 4a exhibits a highly selective and sensitive response to Hg²⁺ over other transition metal ions in aqueous solution. Its detection limit is determined to be 77 nM. The significant changes in the fluorescence color could be used for the naked-eye detection. Furthermore, the probe 4a shows good membrane permeability and can be applied to detect intracellular Hg²⁺ in human lung adenocarcinoma cells (A549 cells). The crystal structure and spectral properties of its congener 4b that contains one 12-bromo [2.2]paracyclophane group and rhodamine moiety are also investigated for a comparison.     

Water-soluble through-bond energy transfer cassettes for intracellular imaging

A special, water-soluble, fluorescent probe 1 was designed. This consisted of a fluorescein-based component to harvest irradiation at 488 nm and a rhodamine-based part designed to emit it at a significantly longer wavelength. This cassette was used to label an illustrative protein called ACBP. Evidence was accumulated to support the assertion that ACBP-1 bound its native ligand with a binding constant similar to that of the unlabeled protein, and retained its secondary structure (CD). ACBP-1 was imported into cells using the Chariot peptide. Confocal images proved that some ACBP-1 localized into the nucleus (as expected) and, most significantly, it could be visualized more effectively by irradiating at the donor (fluorescein-like) part of the cassette, than the acceptor (rhodamine-like) part. Overall, this study demonstrates that cassettes of this kind can label a protein without significantly perturbing its function or secondary structure and they can be visualized effectively via irradiation of the donor and observation of the acceptor fluorescence.     

Rhodamine-based highly sensitive colorimetric off-on fluorescent chemosensor for Hg2+ in aqueous solution and for live cell imaging

A novel rhodamine-based highly sensitive and selective colorimetric off-on fluorescent chemosensor for Hg(2+) ions is designed and prepared by using the well-known thiospirolactam rhodamine chromophore and furfural hydrazone as signal-reporting groups. The photophysical characterization and Hg(2+)-binding properties of sensor RS1 in neutral N, N-dimethylformamide (DMF) aqueous solution are also investigated. The signal change of the chemosensor is based on a specific metal ion induced reversible ring-opening mechanism of the rhodamine spirolactam. The response of the chemosensor for Hg(2+) ions is instantaneous and reversible. And it successfully exhibits a remarkably "turn on" response toward Hg(2+) over other metal ions (even those that exist in high concentration). Moreover, this sensor is applied for in vivo imaging in Rat Schwann cells to confirm that RS1 can be used as a fluorescent probe for monitoring Hg(2+) in living cells with satisfying results, which further demonstrates its value of practical applications in environmental and biological systems.     

Aminoxy-linked rhodamine hydroxamate as fluorescent chemosensor for Fe in aqueous media

A novel feric ion-selective rhodamine-based fluorescent chemosensor, which contains a bis-aminoxy chain moiety, has been developed. The multi-dentate binding site of rhodamine fluorophore shows selective detection of ferric iron over other biologically important metal ions in aqueous media and also shows 1:1 binding stoichiometry.