Probing hydroxyl radicals and their imaging in living cells by use of FAM-DNA-Au nanoparticles

The incorporation of gold nanoparticles (Au NPs) as quencher modules in fluorescent probes for DNA damage caused by intracellular hydroxyl radicals (HO*) is reported. Au NPs of 15 nm diameter were decorated with DNA oligomers terminating in thiol functions in their 3' positions and possessing 5' fluorophore modifications. The Au NPs, which have high extinction coefficients, functioned as excellent fluorescent quenchers in the fluorophore-Au NP composites. FRET is switched off as a factor of HO*-induced strand breakage in the single-stranded DNAs, restoring the fluorescence of the quenched fluorophores, which can be followed by spectrofluorimetry. In vitro assays with HO*-generating Fenton reagent demonstrated increases in fluorescence intensity with a linear range from 8.0 nM to 1.0 microM and a detection limit as low as 2.4 nM. Confocal microscopic imaging of macrophages and HepG2 revealed that the probe is cell-permeable and intracellular HO*-responsive. The unique combination of good selectivity and high sensitivity establishes the potential value of the probe for facilitating investigations of HO*-mediated cellular homeostasis and injury.     

Dye-modified nanochannel materials for photoelectronic and optical devices

Artificial photonic antenna systems have been realised by incorporating organic dyes into zeolite L. The size and aspect ratio of the cylindrically shaped zeolite crystals can be tuned over a wide range, adding to the versatility of this host material. A 600 nm sized crystal, for example, consists of about 96 000 one-dimensional channels oriented parallel to the cylinder axis. Geometrical constraints imposed by the host structure lead to supramolecular organisation of the guests, allowing high concentrations of non- or only very weakly interacting dye molecules. A special twist is added to these systems by plugging the channel openings with a second type of fluorescent dye, a so-called stopcock molecule. The two types of molecules are precisely tuned to each other; the stopcocks are able to accept excitation energy from the dyes in the channels, but cannot pass it back. The supramolecular organisation of dyes in the zeolite channels corresponds to a first stage of organisation, allowing light-harvesting within the volume of a cylindrical crystal and radiationless energy transport to either the cylinder ends or centre. The second stage of organisation represents the coupling to an external acceptor or donor stopcock fluorophore at the channel entrances, which can then trap or inject electronic excitation energy. The third stage of organisation is realised by interfacing the material to an external device through a stopcock intermediate. We observed that electronic-excitation-energy transfer in dye-zeolite L materials occurs mainly along the channel axis and we have shown that macroscopically organised materials can be prepared. The new materials offer unique possibilities as building blocks for optical, electro-optical and sensing devices.     

Synthesis, characterization, and application of cationic water-soluble oligofluorenes in DNA-hybridization detection

A simple and efficient approach was developed for the synthesis of a series of cationic water-soluble oligofluorenes up to a chain length of a heptamer. Bromoalkyl-substituted fluorenyl boronic esters as the key intermediates were synthesized by using a modified Miyaura reaction. With an increasing number of repeat units (trimer to hexamer), the size-specific oligomers have shown redshifts in both the absorption and emission maxima. The emission maximum reaches the limit for the hexamer in both water and buffer solution. The quantum yields of the oligomers decreased with increased oligomer size in water. Both fluorescence quenching of the oligomers by 9,10-anthraquinone-2,6-disulfonate and the fluorescence resonance energy transfer experiments with the oligomers as the donor and fluorescein (Fl)-labeled double-stranded DNA (dsDNA-Fl) as the acceptor revealed the chain-length-dependent behavior. The Stern-Volmer quenching constant increased with the molecular size, whereas the highest donor-sensitized Fl emission was observed for the hexamer. These size-specific oligomers also served as a model to study the structure-property relationships for cationic polyfluorenes.     

Quantifying interactions between cell receptors and adhesion ligand-modified polymers in solution

Specific interactions between cells and cell-interactive polymers in solution were investigated by the fluorescence resonance energy transfer (FRET) technique and rheological measurements. The green fluorescence emission was dramatically reduced when rhodamine-stained cells were mixed with a fluorescein-labeled RGD-alginate solution, compared with those mixed with no RGD-containing alginate solution, which indicated an occurrence of FRET and existence of specific interactions between the cells and the polymers in solution. Rheological measurements also confirmed the formation of ordered structures of cell/polymer mixtures, caused by specific cell-polymer interactions. The FRET method was able to provide a useful means of investigating cell-polymer interactions, both in a qualitative and quantitative manner, and this approach to monitoring and controlling specific interactions between cells and polymers could be useful in the design and tailoring of polymeric carriers for cells, as well as for biological drugs, especially for tissue engineering applications.     

Fluorescence detection techniques for protein kinase assay

Traditional methods for protein kinase (PK) assay are mainly based on use of ³²P-labeled adenosine triphosphate (ATP); applications of such methods are, however, hampered by radioactive waste and short half-life of ³²P-labeled ATP. Therefore non-radioactive methods, such as fluorescence detection techniques are good alternative. In this review, we describe the principles of four fluorescence techniques (fluorescence intensity endpoint measurement, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and fluorescence lifetime imaging) and provide an overview of applications of these fluorescence detection techniques in protein kinase assay, underlining their relative advantages and limitations. Research trends in this field are also highlighted. Figure Schematic representation of kinase assay based on direct fluorescence polarization measurements. The fluorescent peptide, on phosphorylation by kinase, binds to a phosphospecific antibody, which leads to a high FP value     

A Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol‐containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide‐α‐thioester and an N‐terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160‐fold) in its fluorescence ratio (I₄₅₈/I₆₀₃). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.     

荧光增强型共轭聚电解质的合成及对肝素的双通道检测

通过Suzuki偶联反应制备了含有四苯基乙烯和苯并噻二唑2种结构单元的阳离子型共轭聚电解质P1,并通过核磁共振氢谱确定了2种结构单元的比例为0.803:0.197,与投料比基本一致.当在P1的水溶液中加入不良溶剂THF时,在紫外光照下可以观察到其溶液颜色由土黄色(无聚集态)转变为橙黄色(聚集态).利用P1在不同聚集态下的荧光强度和发光颜色变化,可以实现对肝素的双通道检测.当肝素逐渐滴加到P1的水溶液中,P1的荧光发射强度随着肝素浓度的增加而线性增加,且其最大发射峰峰位发生线性红移,正负电荷饱和时强度达到最大且波长不再移动,该性质可作为双通道检测信号来标定肝素的浓度,从而提高肝素浓度检测的准确性.动态激光光散射的测试以及肝素滴定紫外图谱的变化结果表明,肝素与P1作用时聚集诱导发光和荧光共振能量转移两种作用共存,从而导致了荧光强度的增强,且伴随最大发射波长的红移.