常见的荧光探针及其在活性氧检测中的应用

荧光探针具有高灵敏度、高活性,在物质成分测定及反应进程监测中得到了广泛的应用。常见的荧光探针根据荧光团的不同可以分为香豆素类、氟硼荧染料、菁染料、萘酰亚胺类、荧光素和罗丹明类。活性氧(Reactive Oxygen Species, ROS)与各种细胞过程有关,主要包括转录因子的激活、基因表达、细胞增殖和死亡。由于活性氧的寿命短、活性高,且会与蛋白质、DNA、脂质膜等迅速反应,给其检测工作带来了困难。荧光探针作为一种高灵敏度的检测方法,在活性氧的检测工作中起到了重要作用。在介绍了不同荧光探针的基础上,着重对检测活性氧的荧光探针进行了详细的分类与描述。     

生物标示用3H—吲哚菁染料

随着生物技术和荧光标示技术的飞速发展，3H0吲哚菁染料已成为在DNA，蛋白质及核酸等分析检测中使用的主要荧光探针。其中代表性化合物Cy3.、Cy5.作为新一代商品化荧光标示剂在生物芯片、分子信标等方面得到了重要应用。本文综述了该类碳菁染料在生物领域中取得的重大应用进展，并探讨了其结构与性能之间的关系。     

能够识别多种离子的香豆素类染料探针分子

发展环境水样和生物体内各种金属离子、阴离子的定量检测方法对于避免离子中毒事故和研究金属离子的生物活性具有重要意义。香豆素类染料分子化学性质稳定，光物理性能优秀，非常适合用于开发具有离子识别能力的荧光传感体系。本文围绕香豆素分子的结构设计，从“能够识别多种离子的香豆素探针分子”和“基于香豆素染料分子的金属离子阵列传感器”2个研究方向，讨论了香豆素荧光染料探针分子在同步识别多种金属离子和阴离子应用领域的研究进展。可以看出，无论是开发具有多个识别位点的特异性探针分子，还是基于模式识别原理的阵列传感器，香豆素类染料分子在多种离子识别领域均展现出光明的应用前景。     

生物标示用3H-吲哚菁染料

随着生物技术和荧光标示技术的飞速发展,3H-吲哚菁染料已成为在DNA、蛋白质及核酸等分析检测中使用的主要荧光探针。其中代表性化合物Cy3.、Cy5.作为新一代商品化荧光标示剂在生物芯片、分子信标等方面得到了重要应用。本文综述了该类碳菁染料在生物领域中取得的重大应用进展、并探讨了其结构与性能之间的关系。     

基于罗丹明类荧光探针的研究进展

罗丹明是以氧杂蒽为母体的碱性呫吨染料,因其优良的光学性能如吸收系数大、光稳定性好和荧光量子产率高等,常常被选作荧光母体用于金属离子荧光探针的设计和制备。主要对罗丹明类荧光探针在铜离子、汞离子、铁离子检测中的应用进行归纳总结,对其结构特征、作用机理、检测水平和应用范围等进行了详细的比较分析。最后,提出了此类荧光探针目前存在的问题和今后的发展趋势。     

萘酰亚胺类功能材料应用研究进展

简述了1,8-萘酰亚胺及其衍生物的合成方法,着重介绍其近年在荧光增白剂、荧光染料和其他功能材料上的应用研究进展,特别是在生物医用材料和荧光传感器等领域的应用。在生物医用材料方面的应用主要包括抗肿瘤药物、DNA荧光探针和荧光分子开关;而荧光传感器领域的应用则主要包括阴离子传感器和阳离子传感器。最后,展望了此类功能材料的发展趋势。     

萘酰亚胺类功能材料应用研究进展

简述了1,8-萘酰亚胺及其衍生物的合成方法,着重介绍其近年在荧光增白剂、荧光染料和其他功能材料上的应用研究进展,特别是在生物医用材料和荧光传感器等领域的应用.在生物医用材料方面的应用主要包括抗肿瘤药物、DNA荧光探针和荧光分子开关;而荧光传感器领域的应用则主要包括阴离子传感器和阳离子传感器.最后,展望了此类功能材料的发展趋势.     

萘酰亚胺类功能材料应用研究进展

简述了1,8-萘酰亚胺及其衍生物的合成方法,着重介绍其近年在荧光增白剂、荧光染料和其他功能材料上的应用研究进展,特别是在生物医用材料和荧光传感器等领域的应用.在生物医用材料方面的应用主要包括抗肿瘤药物、DNA荧光探针和荧光分子开关;而荧光传感器领域的应用则主要包括阴离子传感器和阳离子传感器.最后,展望了此类功能材料的发展趋势.     

水溶性二氟化硼-二吡咯甲烷(BODIPY)类荧光染料研究进展

简要介绍了二氟化硼-二吡咯甲烷(BODIPY)荧光染料的基本结构、优异的光物理和光化学性能以及自身存在的溶解性差等缺陷,重点综述了近年来各种水溶性BODIPY类荧光染料衍生物的合成及其在水环境中作为荧光探针的应用研究成果,并提出通过对BODIPY母体染料结构进行化学修饰,开发研制出许多具有不同性能的BODIPY衍生物并研究其应用将成为国内外十分活跃的研究课题。     

适用于纺织荧光染色的染料及其应用研究进展

荧光染料凭借发射强度高、色彩鲜艳、荧光强烈等特点，在纺织领域吸引了许多染料专家们的目光，是一类非常具有应用前景的功能性染料。该文按染料结构分类的方式综述了从上世纪至今商品化分散、酸性和活性荧光染料以及新型1, 8-萘酰亚胺类、香豆素类、半花菁类和其它类新型荧光染料及其衍生物在纺织染色领域的研究进展，最后提出适用于纺织荧光染色的染料在纺织领域未来的发展趋势。     

Stacked Thiazole Orange Dyes in DNA Capable of Switching Emissive Behavior in Response to Structural Transitions

Functional nucleic acids with the capability of generating fluorescence in response to hybridization events, microenvironment or structural changes are valuable as structural probes and chemical sensors. We now demonstrate the enzyme-assisted preparation of nucleic acids possessing multiple thiazole orange (TO) dyes and their fluorescent behavior, that show a spectral change from the typical monomer emission to the excimer-type red-shifted emission. We found that the fluorescent response and emission wavelength of the TO dyes were dependent on both the state of the DNA structure (single- or double-stranded DNA) and the arrangement of the TO dyes. We showed that the fluorescent behavior of the TO dyes can be applied for the detection of RNA molecules, suggesting that our approach for preparing the fluorescent nucleic acids functionalized with multiple TO dyes could be useful to design a fluorescence bioimaging and detection technique of biomolecules.     

Fluorescent amino-substituted squaraine probes for bovine serum albumin

The synthesis and spectroscopic properties of fluorescent amino-substituted squaraine dyes have been presented. The interaction between bovine serum albumin (BSA) and fluorescence probes derived from squaric acid was studied by UV-vis and fluorescence spectroscopies. The Benesi–Hildebrand, Lehrer, and Stern–Volmer equations were used to present the influence of BSA concentration on the binding constant for the process of association with squaraine dyes, as well as the effect of the concentration of fluorescence probes on the quenching process. It was also shown that the interaction between BSA and squaraine dyes is spontaneous. The number of binding sites to bovine serum albumin for a series of squaraine dyes has also been calculated.     

Fluorescent pH probes based on boron dipyrromethene dyes

Two novel fluorescent pH probes based on 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes were configured as a “fluorophore-spacer-receptor” system. Their absorption and fluorescence properties were investigated in various solvents; in aqueous solution, based on their photo-induced electron transfer, the probes exhibited 3-fold (pH 8.70–4.93) and 14-fold (pH 9.02–3.29) fluorescence enhancement upon increase in the acidity of the solution, respectively. The probes can be used as fluorescent pH probes which are excitable with visible light; the pK_a values of the probes were determined from the fluorescence changes.     

Novel,N-ethyl-2-styrylquinolinum iodides as fluorophores for monitoring of polymerization process,Part I

A series of of p-substituted 2-styrylquinolinium iodides were prepared by the condensation of N-ethyl-2-methylquinolinium salts with p-substituted benzaldehydes. The spectroscopic properties of the styryl quinolinium dyes are characterized in organic solvents of varying polarities. The electronic absorption and fluorescence emission spectra of the dyes demonstrate their high sensitivity to the nature of substituents introduced into the aromatic ring. The dyes were investigated as fluorescent probes for monitoring the progress of the photochemically initiated free-radical polymerization of a mixture of 2-ethyl-2-(hydroxymethyl)-1,3-propanediol triacrylate and 1-methyl-2-pyrrolidinone. During the course of the polymerization an increase in the fluorescence intensity of the dyes by at least one order of magnitude was recorded; a feature which renders the dyes as good fluorescent probes for such polymerization reactions. The term “probe sensitivity” has been defined and appears in the range from 0.08 to 11 for the styryl dyes.     

Fluorescent hybridization probes for sensitive and selective DNA and RNA detection

We outline the different approaches taken by our group in the design of fluorescent hybridization sensors. Molecular beacons (MBs) and binary probes (BPs) using two dyes (2d-MB and 2d-BP, respectively) have been synthesized; these sensors serve as switches in emission upon binding to target biomolecules, such as DNA. These sensors allow for ratiometric fluorescence detection of polynucleotides (PNs) by visualization of the probes when bound to a target PN. Additionally, three-dye MBs (3d-MB) and BPs (3d-BP) have been developed, where an energy-transfer cascade is employed to decrease the overlap between the fluorophore emission spectra, resulting in a low direct excitation of the acceptor fluorophore. Pyrene-based MB (Py-MB) and BP (Py-BP), which possess the advantage of long fluorescence lifetimes, have also been synthesized. Time-resolved fluorescence spectra (TRES) can be used to discriminate between short-lived background fluorescence and long-lived fluorescence of the pyrene probes. This technique was demonstrated by time-resolving the signal of a Py-BP from the background fluorescence in Aplysia californica cell extracts.     

Bright fluorescent dsDNA probes: novel polycationic asymmetric monomethine cyanine dyes based on thiazolopyridine‐quinolinium chromophore

Seven tri‐ and tetracationic monomeric and homodimeric monomethine cyanine dyes based on thiazolo[4,5‐b]pyridinium and quinolinium end groups were synthesised and characterised. The dyes were tested as fluorescent DNA intercalating probes to apply in DNA gel electrophoresis. The DNA samples stained with all dyes from the series demonstrated bright fluorescent signals. DNA fragments were successfully visualised under orange and green filters as well as under standard UV transillumination. Two of the studied dyes revealed higher sensitivity to DNA when compared with the commercial dimeric cyanine dye TOTO‐1. Their sensitivity reached that of the commercial dimeric cyanine dye YOYO‐1, but the emission was shifted to longer wavelengths. These qualities make the dyes suitable to apply in a wide range of medical and scientific analytical methods.     

Fluorescence resonance energy transfer as a probe for G-quartet formation by a telomeric repeat

The secondary structure of guanine-rich oligodeoxynucleotides has been investigated with fluorescent probes. Intramolecular folding of a telomeric oligonucleotide into a quadruplex led to fluorescence resonance energy transfer (FRET) between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5' and 3' ends of the DNA, respectively. Depending on oligonucleotide length, quenching efficiency varied between 0.45 and 0.72 at 20 degrees C. The conjugation of the dyes to the oligonucleotide had a limited, but significant, influence on the thermodynamics of G-quartet formation. Intramolecular folding was demonstrated from the concentration independence of fluorescence resonance energy transfer over a wide concentration range. Folding of the oligonucleotide was confirmed by UV absorption, UV melting, and circular dichroism experiments. The folding of the G-quartet could be followed at concentrations as low as 100 pM. Fluorescence resonance energy transfer can thus be used to reveal the formation of multistranded DNA structures.     

Bivalent Display of Dicysteine on Peptide Nucleic Acids for Homogenous DNA/RNA Detection through in Situ Fluorescence Labelling

Fluorogenic probes that signal the presence of specific DNA or RNA sequences are key enabling tools for molecular disease diagnosis and imaging studies. Usually, at least one fluorophore is attached through covalent bonding to an oligonucleotide probe. However, the additional conjugation step increases costs. Here we introduce a method that avoids the requirement for the preparation of fluorescence‐labelled oligonucleotides and provides the opportunity to alter the fluorogenic reporter dye without resynthesis. The method is based on adjacent hybridization of two dicysteine‐containing peptide nucleic acid (PNA) probes to form a bipartite tetracysteine motif that binds profluorescent bisarsenical dyes such as FIAsH, ReAsH or CrAsH. Binding is accompanied by strong increases in fluorescence emission (with response factors of up to 80‐fold and high brightness up to 50 mL mol^?1 cm^?1). The detection system provides sub‐nanomolar limits of detection and allows discrimination of single nucleotide variations through more than 20‐fold changes in fluorescence intensity. To demonstrate its usefulness, the FIAsH‐based readout of the bivalent CysCys‐PNA display was interfaced with a rolling‐circle amplification (RCA) assay used to detect disease‐associated microRNA let‐7a.     

New Fluorescent Nanoparticles for Ultrasensitive Detection of Nucleic Acids by Optical Methods

For decades the detection of nucleic acids and their interactions at low abundances has been a challenging task that has thus far been solved by enzymatic target amplification. In this work we aimed at developing efficient tools for amplification‐free nucleic acid detection, which resulted in the synthesis of new fluorescent nanoparticles. Here, the fluorescent nanoparticles were made by simple and inexpensive radical emulsion polymerization of butyl acrylate in the presence of fluorescent dyes and additional functionalization reagents. This provided ultra‐bright macrofluorophores of 9–84 nm mean diameter, modified with additional alkyne and amino groups for bioconjugation. By using click and NHS chemistries, the new nanoparticles were attached to target‐specific DNA probes that were used in fluorimetry and fluorescence microscopy. Overall, these fluorescent nanoparticles and their oligonucleotide derivatives have higher photostability, brighter fluorescence and hence dramatically lower limits of target detection than the individual organic dyes. These properties make them useful in approaches directed towards ultrasensitive detection of nucleic acids, in particular for imaging and in vitro diagnostics of DNA.     

Spectroscopic study of cyanine dyes interacting with the biopolymer,DNA

Dyes intercalated into DNA strands or bound to grooves show fluorescence intensity changes and aggregate formation depending on the conditions. In order to establish some empirical rules concerning dye intercalation, spectroscopic studies for the effects of DNA on several series of cyanine dyes with different aromatic rings, conjugated chain length and alkyl substituents were made. Absorption spectra, fluorescence intensity and circular dichroism spectra showed strong dependence on the species of dyes. Combination of preceding studies and these present results indicates that cyanine dyes tend to intercalate into DNA strand if their polymethine bridge was composed of only one carbon. For molecules with the longer chains irregular aggregates were formed by small amounts of DNA, which transformed into complexes composed of multiple dye and DNA strands. These results would serve as a useful guideline for designing of optical functional materials and devices utilizing DNA complex.